DNA and histone modifications exhibit noticeable impacts on gene expression 1 . Being the most prevalent internal modification in mRNA, N 6 -Methyladenosine (m 6 A) mRNA modification emerges as an important post-transcriptional mechanism of gene regulation 2 - 4 and plays critical roles in various normal and pathological bioprocesses 5 - 12 . However, how m 6 A is precisely and dynamically deposited in the transcriptome remains elusive. Here we report that H3K36me3 histone modification, a marker for transcription elongation, globally guides m 6 A modification. We found that m 6 A modifications enrich in the vicinity of H3K36me3 peaks, and are reduced globally when cellular H3K36me3 is depleted. Mechanistically, H3K36me3 is recognized and bound directly by METTL14, a critical component of the m 6 A methyltransferase complex (MTC), which in turn facilitates the binding of the m 6 A MTC to adjacent RNA polymerase II, and thereby delivering the m 6 A MTC to actively transcribed nascent RNAs to deposit m 6 A co-transcriptionally. In mouse embryonic stem cells, phenocopying Mettl14 silencing, H3K36me3 depletion also induces m 6 A reduction transcriptome-wide and in pluripotency transcripts, resulting in increased cell stemness. Collectively, our studies reveal the critical roles of H3K36me3 and METTL14 in determining precise and dynamic m 6 A deposition in mRNA, and uncover another layer of gene expression regulation involving crosstalk between histone modification and RNA methylation.

Strategies that enhance the function of T cells are critical for immunotherapy. One negative regulator of T-cell activity is ligand PD-L1, which is expressed on dentritic cells (DCs) or some tumor cells, and functions through binding of programmed death-1 (PD-1) receptor on activated T cells. Here we described for the first time a non-viral mediated approach to reprogram primary human T cells by disruption of PD-1. We showed that the gene knockout of PD-1 by electroporation of plasmids encoding sgRNA and Cas9 was technically feasible. The disruption of inhibitory checkpoint gene PD-1 resulted in significant reduction of PD-1 expression but didn’t affect the viability of primary human T cells during the prolonged in vitro culture. Cellular immune response of the gene modified T cells was characterized by up-regulated IFN-γ production and enhanced cytotoxicity. These results suggest that we have demonstrated an approach for efficient checkpoint inhibitor disruption in T cells, providing a new strategy for targeting checkpoint inhibitors, which could potentialy be useful to improve the efficacy of T-cell based adoptive therapies.

METHODS. Two different pipelines of neoantigen identification were established in this study: (a) Clinical-grade targeted sequencing was performed in patients with refractory solid tumor, and mutant peptides with high variant allele frequency and predicted high HLA-binding affinity were synthesized de novo. (b) An inventory-shared neoantigen peptide library of common solid tumors was constructed, and patients' hotspot mutations were matched to the neoantigen peptide library. The candidate neoepitopes were identified by recalling memory T cell responses in vitro. Subsequently, neoantigen-loaded dendritic cell vaccines and neoantigen-reactive T cells were generated for personalized immunotherapy in 6 patients. RESULTS. Immunogenic neoepitopes were recognized by autologous T cells in 3 of 4 patients who used the de novo synthesis mode and in 6 of 13 patients who used the shared neoantigen peptide library. A metastatic thymoma patient achieved a complete and durable response beyond 29 months after treatment. Immune-related partial response was observed in another patient with metastatic pancreatic cancer. The remaining 4 patients achieved prolonged stabilization of disease with a median progression-free survival of 8.6 months. CONCLUSION. The current study provides feasible pipelines for neoantigen identification. Implementing these strategies to individually tailor neoantigens could facilitate neoantigen-based translational immunotherapy research.

15However, in some other pathological conditions, including congestive heart failure, hypercholesterolemia, ischemiareperfusion, and restenosis after coronary angioplasty, the NO-mediated relaxant response is impaired, whereas the EDHFmediated relaxant response is increased as a compensation. 15Abstract-The small conductance and intermediate conductance Ca 2+-activated K + channels are known to be involved in the endothelium-dependent hyperpolarization. Ca 2+ entry into endothelial cells stimulates these channels, causing membrane hyperpolarization in endothelial cells and underlying smooth muscle cells. In the present study, with the use of coimmunoprecipitation and double immunolabeling methods, we demonstrated a physical interaction of transient receptor potential vanilloid 4 (TRPV4) with K Ca 2.3 in rat mesenteric artery endothelial cells. Acetylcholine and 4α-PDD mainly acted through TRPV4-K Ca 2.3 pathway to induce smooth muscle hyperpolarization and vascular relaxation. K Ca 3.1 was also involved in the process but at a much lesser degree than that of K Ca 2.3. Stimulating TRPV4-K Ca 2.3 signaling pathway also increased local blood flow in mesenteric beds and reduced systemic blood pressure in anesthetized rats. In streptozotocin-induced diabetic rats, the expression levels of TRPV4 and K Ca 2.3 were reduced, which could be an underlying reason for the dysfunction of endothelium-dependent hyperpolarization in these animals. These results demonstrated an important physiological and pathological role of TRPV4-K Ca 2. However, the molecular mechanism of altered EDHF responses in these disease states is poorly understood.In the present study, we identified a previously unknown physical association between TRPV4 and K Ca 2.3 and uncovered the functional role of this TRPV4-K Ca 2.3 signaling pathway in smooth muscle hyperpolarization and relaxation. We also found that reduced expression levels of TRPV4 and K Ca 2.3 could be an underlying mechanism for EDHF dysfunction in diabetic rats. Materials and MethodsSee the Methods section in the online-only Data Supplement for details. Cell Preparation and CultureAll animal experiments were conducted in accordance with the regulation of the US National Institute of Health (NIH) Guide for the Care and Use of Laboratory Animals. Primary mesenteric artery endothelial cells (MAECs) were isolated from male Sprague-Dawley rats, cultured for 3 to 5 days, and used for experiments without passage. Immunostaining, Immunoprecipitation, and ImmunoblotsDouble immunolabeling was performed in rat MAECs using anti-TRPV4 antibody together with either anti-K Ca 2.3 or anti-K Ca 3.1 antibody. Artery sections were stained with either anti-TRPV4 or anti-K Ca 2.3 antibody. Immunoprecipitation and immunoblots were performed following the protocol described elsewhere with slight modifications. . The membrane potentials of MAECs were also measured using perforated wholecell patch clamp with an EPC-9 amplifier. High-impedance sharp microelectrodes were used to measure smooth muscle cell membran...

Abstractobjective There is a high burden of both diabetes (DM) and tuberculosis (TB) in China, and as DM increases the risk of TB and adversely affects TB treatment outcomes, there is a need for bidirectional screening of the two diseases. How this is best performed is not well determined. In this pilot project in China, we aimed to assess the feasibility and results of screening DM patients for TB within the routine healthcare setting of five DM clinics.method Agreement on how to screen, monitor and record was reached in May 2011 at a national stakeholders meeting, and training was carried out for staff in the five clinics in July 2011. Implementation started in September 2011, and we report on 7 months of activities up to 31 March 2012. DM patients were screened for TB at each clinic attendance using a symptom-based enquiry, and those positive to any symptom were referred for TB investigations.results In the three quarters, 72% of 3174 patients, 79% of 7196 patients and 68% of 4972 patients were recorded as having been screened for TB, resulting in 7 patients found who were already known to have TB, 92 with a positive TB symptom screen and 48 of these newly diagnosed with TB as a result of referral and investigation. All patients except one were started on anti-TB treatment. TB case notification rates in screened DM patients were several times higher than those of the general population, were highest for the five sites combined in the final quarter (774 ⁄ 100 000) and were highest in one of the five clinics in the final quarter (804 ⁄ 100 000) where there was intensive in-house training, special assignment of staff for screening and colocation of services.conclusion This pilot project shows that it is feasible to carry out screening of DM patients for TB resulting in high detection rates of TB. This has major public health and patient-related implications.

ABSTRACT

The lack of efficient antigen and activator delivery systems, as well as the restricted migration of dendritic cells (DCs) to secondary lymph organs, dramatically limits DC-based adoptive immunotherapy. We selected two spherical gold nanoparticle (AuNP)-based vehicles of optimal size for activator and antigen delivery. Their combination (termed the NanoAu-Cocktail) was associated with the dual targeting of CpG oligonucleotides (CpG-ODNs) and an OVA peptide (OVAp) to DC subcellular compartments, inducing enhanced antigen cross-presentation, upregulated expression of costimulatory molecules and elevated secretion of T helper1 cytokines. We demonstrated that the intravenously transfused NanoAu-Cocktail pulsed DCs showed dramatically improved in vivo homing ability to lymphoid tissues and were settled in T cell area. Especially, by tissue-distribution analysis, we found that more than 60% of lymphoid tissues-homing DCs accumulated in liver-draining lymph nodes (LLNs). The improved homing ability of NanoAu-Cocktail pulsed DCs was associated with the high expression of chemokine receptor 7 (CCR7) and rearrangement of the cytoskeletons. In addition, by antigen-specific tetramers detection, NanoAu-Cocktail pulsed DCs were proved able to elicit strong antigen-specific CD8+ T cell responses, which provided enhanced protection from viral invasions. This study highlights the importance of codelivering antigen/adjuvant using different sized gold nanoparticles to improve DC homing and therapy.

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