Rerouting trafficking circuits through posttranslational SNARE modifications.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are membrane-associated trafficking proteins that confer identity to lipid membranes and facilitate membrane fusion. These functions are achieved through the complexing of Q-SNAREs with a specific cognate target R-SNARE, leading to the fusion of their associated membranes. These SNARE complexes then dissociate so that the Q-SNAREs and R-SNAREs can repeat this cycle. Whilst the basic function of SNAREs has been long appreciated, it is becoming increasingly clear that the cell can control the localisation and function of SNARE proteins through posttranslational modifications (PTMs), such as phosphorylation and ubiquitylation. Whilst numerous proteomic methods have shown that SNARE proteins are subject to these modifications, little is known about how these modifications regulate SNARE function. However, it is clear that these PTMs provide cells with an incredible functional plasticity; SNARE PTMs enable cells to respond to an ever-changing extracellular environment through the rerouting of membrane traffic. In this Review, we summarise key findings regarding SNARE regulation by PTMs and discuss how these modifications reprogramme membrane trafficking pathways.

Detection of Low-Frequency Epitope-Specific T Cells in Blood of Healthy Individuals according to an Optimized In Vitro Amplification System.

Detection and amplification of epitope-specific T cells hold great promise for diagnosis and therapy of cancer patients. Currently, measurement and retrieval of epitope-specific T cells is hampered by limited availability of patients' biomaterials and lack of sensitive and easy-to-implement T cell priming and expansion. We have developed an in vitro T cell amplification system starting from healthy donor blood and tested different subsets and ratios of autologous T cells and APCs as well as the resting period between amplification cycles. We demonstrated in 10 different donors significantly enhanced frequency of T cells specific for MelanA/HLA-A2, which relied on coculturing of naive T cells and CD11c+ dendritic cells in a 1:1 ratio followed by three weekly amplification cycles using the effluent of the naive T cell sort as APCs, a 24-h rest period prior to every reamplification cycle, and IFN-γ production as a readout for epitope-specific T cells. Using this system, MelanA/HLA-A2-specific T cells were enriched by 200-fold, measuring up to 20-60% of all T cells. We extended this system to enrich NY-ESO-1/HLA-A2- and BMLF-1/HLA-A2-specific T cells, examples of a cancer germline Ag and an oncoviral Ag differing in their ability to bind to HLA-A2 and the presence of specific T cells in the naive and, in case of BMLF-1, also the Ag-experienced repertoire. Collectively, we have developed a sensitive and easy-to-implement in vitro T cell amplification method to enrich epitope-specific T cells that is expected to facilitate research and clinical utility regarding T cell diagnosis and treatments.

Splicing neoantigen discovery with SNAF reveals shared targets for cancer immunotherapy.

Immunotherapy has emerged as a crucial strategy to combat cancer by "reprogramming" a patient's own immune system. Although immunotherapy is typically reserved for patients with a high mutational burden, neoantigens produced from posttranscriptional regulation may provide an untapped reservoir of common immunogenic targets for new targeted therapies. To comprehensively define tumor-specific and likely immunogenic neoantigens from patient RNA-Seq, we developed Splicing Neo Antigen Finder (SNAF), an easy-to-use and open-source computational workflow to predict splicing-derived immunogenic MHC-bound peptides (T cell antigen) and unannotated transmembrane proteins with altered extracellular epitopes (B cell antigen). This workflow uses a highly accurate deep learning strategy for immunogenicity prediction (DeepImmuno) in conjunction with new algorithms to rank the tumor specificity of neoantigens (BayesTS) and to predict regulators of mis-splicing (RNA-SPRINT). T cell antigens from SNAF were frequently evidenced as HLA-presented peptides from mass spectrometry (MS) and predict response to immunotherapy in melanoma. Splicing neoantigen burden was attributed to coordinated splicing factor dysregulation. Shared splicing neoantigens were found in up to 90% of patients with melanoma, correlated to overall survival in multiple cancer cohorts, induced T cell reactivity, and were characterized by distinct cells of origin and amino acid preferences. In addition to T cell neoantigens, our B cell focused pipeline (SNAF-B) identified a new class of tumor-specific extracellular neoepitopes, which we termed ExNeoEpitopes. ExNeoEpitope full-length mRNA predictions were tumor specific and were validated using long-read isoform sequencing and in vitro transmembrane localization assays. Therefore, our systematic identification of splicing neoantigens revealed potential shared targets for therapy in heterogeneous cancers.

Atypical cofilin signaling drives dendritic cell migration through the extracellular matrix via nuclear deformation.

To mount an adaptive immune response, dendritic cells must migrate to lymph nodes to present antigens to T cells. Critical to 3D migration is the nucleus, which is the size-limiting barrier for migration through the extracellular matrix. Here, we show that inflammatory activation of dendritic cells leads to the nucleus becoming spherically deformed and enables dendritic cells to overcome the typical 2- to 3-µm diameter limit for 3D migration through gaps in the extracellular matrix. We show that the nuclear shape change is partially attained through reduced cell adhesion, whereas improved 3D migration is achieved through reprogramming of the actin cytoskeleton. Specifically, our data point to a model whereby the phosphorylation of cofilin-1 at serine 41 drives the assembly of a cofilin-actomyosin ring proximal to the nucleus and enhances migration through 3D collagen gels. In summary, these data describe signaling events through which dendritic cells deform their nucleus and enhance their migratory capacity.

Purification of primary human placental leukocytes to study maternal-fetal interactions.

Decidual leukocytes play key roles in maternal-fetal tolerance and immunity. Here, we present detailed methods to purify, culture, and functionally analyze human placental dNK, dTreg, dTem, and dMɸ from decidua parietalis, the maternal part of the placental membranes; decidua basalis, the maternal part of the placenta; and placental villi. These sites have high clinical relevance in the development of villitis and chorioamnionitis. This allows in-depth phenotypic and functional investigation of placental immune populations and their interactions with extravillous trophoblasts. For complete details on the use and execution of this protocol, please refer to Ikumi et al.,1 Tilburgs et al.,2 Salvany-Celades et al.,3 Crespo et al.,4 van der Zwan et al.5.

Transcriptomics of Epstein-Barr virus aids to the classification of T-cell evasion in nasopharyngeal carcinoma.

Epstein-Barr virus (EBV) contributes to oncogenesis and immune evasion in nasopharyngeal carcinoma (NPC). At present, an aggregated, higher-level view on the impact of EBV genes toward the immune microenvironment of NPC is lacking. To this end, we have interrogated tumor-derived RNA sequences of 106 treatment-naive NPC patients for 98 EBV transcripts, and captured the presence of 10 different immune cell populations as well as 23 different modes of T-cell evasion. We discovered 3 clusters of EBV genes that each associate with distinct immunophenotypes of NPC. Cluster 1 associated with gene sets related to immune cell recruitment, such as those encoding for chemoattractants and their receptors. Cluster 2 associated with antigen processing and presentation, such as interferon-related genes, whereas cluster 3 associated with presence of M1-like macrophages, absence of CD4+ T cells, and oncogenic pathways, such as the nuclear factor kappa light-chain enhancer of activated B-cell pathway. We discuss these 3 EBV clusters regarding their potential for stratification for T-cell immunity in NPC together with the next steps needed to validate such therapeutic value.

Antigen-specific decidual CD8+ T cells include distinct effector memory and tissue-resident memory cells.

Maternal decidual CD8+ T cells must integrate the antithetical demands of providing immunity to infection while maintaining immune tolerance for fetal and placental antigens. Human decidual CD8+ T cells were shown to be highly differentiated memory T cells with mixed signatures of dysfunction, activation, and effector function. However, no information is present on how specificity for microbial or fetal antigens relates to their function or dysfunction. In addition, a key question, whether decidual CD8+ T cells include unique tissue-resident memory T cells (Trm) or also effector memory T cell (Tem) types shared with peripheral blood populations, is unknown. Here, high-dimensional flow cytometry of decidual and blood CD8+ T cells identified 2 Tem populations shared in blood and decidua and 9 functionally distinct Trm clusters uniquely found in decidua. Interestingly, fetus- and virus-specific decidual CD8+ Trm cells had similar features of inhibition and cytotoxicity, with no significant differences in their expression of activation, inhibitory, and cytotoxic molecules, suggesting that not all fetus-specific CD8+ T cell responses are suppressed at the maternal-fetal interface. Understanding how decidual CD8+ T cell specificity relates to their function and tissue residency is crucial in advancing understanding of their contribution to placental inflammation and control of congenital infections.

Frequency of Peripheral CD8+ T Cells Expressing Chemo-Attractant Receptors CCR1, 4 and 5 Increases in NPC Patients with EBV Clearance upon Radiotherapy.

Radiotherapy (RT) is the standard-of-care for Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC), where the post-RT clearance of plasma EBV DNA is prognostic. Currently, it is not known whether the post-RT clearance of plasma EBV DNA is related to the presence of circulating T-cell subsets. Blood samples from NPC patients were used to assess the frequency of T-cell subsets relating to differentiation, co-signaling and chemotaxis. Patients with undetectable versus detectable plasma EBV DNA levels post-RT were categorized as clearers vs. non-clearers. Clearers had a lower frequency of PD1+CD8+ T cells as well as CXCR3+CD8+ T cells during RT compared to non-clearers. Clearers exclusively showed a temporal increase in chemo-attractant receptors CCR1, 4 and/or 5, expressing CD8+ T cells upon RT. The increase in CCR-expressing CD8+ T cells was accompanied by a drop in naïve CD8+ T cells and an increase in OX40+CD8+ T cells. Upon stratifying these patients based on clinical outcome, the dynamics of CCR-expressing CD8+ T cells were in concordance with the non-recurrence of NPC. In a second cohort, non-recurrence associated with higher quantities of circulating CCL14 and CCL15. Collectively, our findings relate plasma EBV DNA clearance post-RT to T-cell chemotaxis, which requires validation in larger cohorts.

Cancer-Associated Fibroblasts as Players in Cancer Development and Progression and Their Role in Targeted Radionuclide Imaging and Therapy.

Cancer Associated Fibroblasts (CAFs) form a major component of the tumour microenvironment, they have a complex origin and execute diverse functions in tumour development and progression. As such, CAFs constitute an attractive target for novel therapeutic interventions that will aid both diagnosis and treatment of various cancers. There are, however, a few limitations in reaching successful translation of CAF targeted interventions from bench to bedside. Several approaches targeting CAFs have been investigated so far and a few CAF-targeting tracers have successfully been developed and applied. This includes tracers targeting Fibroblast Activation Protein (FAP) on CAFs. A number of FAP-targeting tracers have shown great promise in the clinic. In this review, we summarize our current knowledge of the functional heterogeneity and biology of CAFs in cancer. Moreover, we highlight the latest developments towards theranostic applications that will help tumour characterization, radioligand therapy and staging in cancers with a distinct CAF population.

Effects of topical tranexamic acid during open reduction and internal fixation of acetabular fractures: A retrospective study.

OBJECTIVE: The aim of this study was to assess the effect of topical tranexamic acid on blood loss and transfusion rates in acetabular fracture surgery. METHODS: The medical records of 61 patients who underwent open reduction and internal fixation for acetabular fracture between 2012 and 2015 were retrospectively reviewed. The patients were divided into two groups: Group I consisted of 31 patients (19 men and 12 women, mean age: 52 ± 19 years) who received intraoperatively a topical tranexamic acid solution of 3 g and Group 2 consisted of 30 control patients (17 men and 13 women, mean age: 48 ± 24 years) who received only 0.9% saline solution. The groups were compared based on their intraoperative blood loss, Postoperative drain output at 24 and 48 h, and postoperative hemoglobin levels, and transfusion rates. RESULTS: The mean intraoperative blood loss was 410 ± 100 ml in Group 1, compared to 570 ml ± 160 ml of the control group (p < 0.05). The postoperative drain output after 24 h was 210 ± 70 ml in Group 1 compared to 330 ± 90 ml of the control group (p < 0.05). The drain output at 48 h was (50 ± 20 ml) in group 1 compared to 90 ± 40 ml of the control group (p < 0.05). The transfusion rate was significantly low group 1 (42%) than the control group (97%). Hemoglobin drop was again significantly less in tranexamic acid group (2.1 ± 1.1) than the control group (3.2 ± 1.3). The nadir postoperative hemoglobin was higher in the Group 1 (10.4 ± 1.5) than the control group (9.2 ± 1.3). CONCLUSION: Topical administration of tranexamic acid reduces intraoperative and postoperative blood loss in acetabular fracture surgery, decreasing transfusion rates. LEVEL OF EVIDENCE: Level III, Therapeutic Study.

The Majority of Corona Mortis Are Small Calibre Venous Blood Vessels: A Cadaveric Study of North Indians.

PURPOSE: Corona mortis is an abnormal arterial or venous anastomosis between the external iliac and the obturator system of vessels and may cause significant hemorrhage during pelvi-acetabular fracture surgeries, hernia repair and laparoscopic gynecological procedures. Previous studies have estimated a prevalence of corona mortis between 34% and 70%. This cadaveric study was conducted to estimate the prevalence of corona mortis in the North Indian population. MATERIALS AND METHODS: Twelve cadavers (24 hemipelvises; 11 males and 1 female) with a mean age of 68 (range, 54-82) years were included in this study. RESULTS: Corona mortis was observed in 14 hemipelvises (58.3%). A total of 19 (79.2%) vascular anastomoses of diameter greater than 1 mm were observed; 5 hemipelvises (20.8%) had corona mortis on the right side, 9 hemipelvises (37.5%) on the left side and bilateral in 5 (41.7%) cases. Two hemipelvises (8.3%) had an arterial connection. An aberrant obturator artery was observed in 1 (4.2%) hemipelvis. A venous connection was found in 14 specimens (58.3% of hemipelvises). The average distance of the connecting vein from the symphysis pubis was 41 (35-70) mm. A vessel diameter of greater than 4 mm was observed in 4/24 (16.7%) of hemipelvises. CONCLUSION: The frequency of venous corona mortis was higher than arterial corona mortis and the majority (83.3%) were small calibre (<4 mm). The presentation pattern and the number of arterial or venous anastomoses were different in the majority of hemipelvises and dissimilar in both hemipelvises of the same cadaver in the majority of cases.

Oligonucleotide microarrays with stem-loop probes: enhancing the hybridization of nucleic acids for sensitive analysis.

We have demonstrated that the dynamics of nucleic acid hybridization in microarrays depend on the physical structure of immobilized probes. We have immobilized oligonucleotide-3'-phosphates with and without stem-loop structure on epoxylated glass surface, followed by hybridization under different conditions, viz., hybridization buffer, pH condition, temperature and ionic strength. In a comparative study, we have established that array constructed using probes with stem-loop structure displayed approximately 2.2 times higher hybridization signals than the probes without it. The stem-loop DNA array format is simple and flexible in design and thus potentially useful in various DNA diagnostic tests.

Choice of polymer matrix, its functionalization and estimation of functional group density for preparation of biochips.

Oligonucleotide microarray has become an important and powerful tool for various genomic analyses, where, unlike conventional methods, one can identify simultaneously a large number of targets in a given sample. Postsynthesis immobilization, the most widely used method, involves the noncovalent and covalent fixing of suitably modified oligonucleotides on the solid supports. Among the various functional groups aminoalkyl, hydroxyalkyl, mercaptoalkyl, aldehyde, epoxy, and carboxyl the most frequently used functional groups on the polymeric surfaces. Because glass and polypropylene, the most widely used materials, are nonporous in nature, the functional groups density on the surface remains very low. In order to know the exact concentration of a ligand to be immobilized, it is essential to estimate the accessible functional groups on these surfaces. For this purpose, sensitive methods are required to estimate exact density of available functional groups on the surfaces. Apart from physical methods, a number of sensitive chemical methods, by making use of high extinction coefficient of 4,4'-dimethoxytrityl cation (epsilon(498) = 70,000 L mol-1 cm-1), have been reported to estimate accessible functional groups on the glass based polymer supports. In this chapter, use of these reagents for spectrophotometric determination of functional group density on glass microslides and polypropylene film has been discussed.

Spectrophotometric estimation of functional groups on microslides for preparation of biochips.

A universal reagent 1-O-(4,4'-dimethoxytrityl)-6-aminohexanol (DTAH) is described for the estimation of surface-bound functionalities (epoxy, aldehyde, and carboxyl) required for preparation of oligonucleotide arrays (biochips). The method involves the reaction of universal reagent DTAH with surface-bound functionality under microwaves for 10 min, followed by washings to remove the excess reagent. In the subsequent step, a weighed amount of DTAH-treated surface is exposed to acid to liberate 4,4'-dimethoxytrityl cation, which is measured at 505 nm to determine the functional group loading on the surface.

A new synthetic protocol for labeled oligonucleotides, using a chemically cleavable universal linker.

A two-step general method for labeling of synthetic oligonucleotides is described. The protocol employs a cleavable universal linker, 5'-O-(4,4'-dimethoxytrityl)-3'-O-benzoyl-2'-O-(2-cyanoethyl-N,N-diisopropyl)-uridine phosphoramidite, to effect coupling to polymer-bound oligonucleotide chains. Sequentially, coupling with commercially available phosphoramidite reagent of an appropriate label (Biotin, HEX etc.) in an automated DNA synthesizer is carried out. The labeled oligomers, obtained after cleavage and deprotection reactions, are analyzed on RP-HPLC. A distinctive feature of this protocol is the recovery of free oligomers from their labeled analogs under mild conditions. The oligomers obtained are comparable to the corresponding standard oligonucleotides (HPLC).

Universal reusable polymer support for oligonucleotide synthesis.

A new efficient reusable universal polymer support for oligonucleotide synthesis, based on a non-ammoniacal cleavable linker, is described. Twenty six cycles of oligonucleotide synthesis have been carried out without compromising the quality of the fully deprotected oligonucleotides.