Single-cell RNA sequencing of Trypanosoma brucei from tsetse salivary glands unveils metacyclogenesis and identifies potential transmission blocking antigens.

Tsetse-transmitted African trypanosomes must develop into mammalian-infectious metacyclic cells in the fly's salivary glands (SGs) before transmission to a new host. The molecular mechanisms that underlie this developmental process, known as metacyclogenesis, are poorly understood. Blocking the few metacyclic parasites deposited in saliva from further development in the mammal could prevent disease. To obtain an in-depth perspective of metacyclogenesis, we performed single-cell RNA sequencing (scRNA-seq) from a pool of 2,045 parasites collected from infected tsetse SGs. Our data revealed three major cell clusters that represent the epimastigote, and pre- and mature metacyclic trypanosome developmental stages. Individual cell level data also confirm that the metacyclic pool is diverse, and that each parasite expresses only one of the unique metacyclic variant surface glycoprotein (mVSG) coat protein transcripts identified. Further clustering of cells revealed a dynamic transcriptomic and metabolic landscape reflective of a developmental program leading to infectious metacyclic forms preadapted to survive in the mammalian host environment. We describe the expression profile of proteins that regulate gene expression and that potentially play a role in metacyclogenesis. We also report on a family of nonvariant surface proteins (Fam10) and demonstrate surface localization of one member (named SGM1.7) on mature metacyclic parasites. Vaccination of mice with recombinant SGM1.7 reduced parasitemia early in the infection. Future studies are warranted to investigate Fam10 family proteins as potential trypanosome transmission blocking vaccine antigens. Our experimental approach is translationally relevant for developing strategies to prevent other insect saliva-transmitted parasites from infecting and causing disease in mammalian hosts.

Adopting an external focus of attention improves sprinting performance in low-skilled sprinters.

For more than 10 years, researchers have investigated how the focusing of conscious attention influences motor skill execution. This line of investigation has consistently demonstrated that directing attention externally rather than internally improves motor skill learning and performance. The purpose of this study was to test the prediction that participants completing a 20-m sprint would run significantly faster when using an external focus of attention rather than an internal or no-focus of attention. Participants were college-aged volunteers (N = 84; 42 women, 42 men; mean age = 20.32, SD = 1.73 years) with no prior sprint training. This study used a counterbalanced within-participant design. Each participant completed 3 days of testing, with each day utilizing a different focus of attention (i.e. internal, external, or control). Running times were collected automatically using infrared timing gates. Data were analyzed using a 1-way repeated measures analysis of variance (ANOVA). The results of the ANOVA revealed a significant main effect for condition, F (1, 83) = 6565.3, p ≤ 0.001. Follow-up analysis indicated that the trials completed in the external focus condition (mean = 3.75 seconds, SD = 0.43) were significantly faster than trials completed in the internal (mean = 3.87 seconds, SD = 0.64) and control conditions (mean = 3.87 seconds, SD = 0.45). The analysis also indicated that the control and internal conditions were not significantly different. The results of this study extend the findings of previous research and demonstrate sprinting performance can be improved by using an external focus of attention.

Dendritic Cells Enhance Polyfunctionality of Adoptively Transferred T Cells That Target Cytomegalovirus in Glioblastoma.

Median survival for glioblastoma (GBM) remains <15 months. Human cytomegalovirus (CMV) antigens have been identified in GBM but not normal brain, providing an unparalleled opportunity to subvert CMV antigens as tumor-specific immunotherapy targets. A recent trial in recurrent GBM patients demonstrated the potential clinical benefit of adoptive T-cell therapy (ATCT) of CMV phosphoprotein 65 (pp65)-specific T cells. However, ex vivo analyses from this study found no change in the capacity of CMV pp65-specific T cells to gain multiple effector functions or polyfunctionality, which has been associated with superior antitumor efficacy. Previous studies have shown that dendritic cells (DC) could further enhance tumor-specific CD8+ T-cell polyfunctionality in vivo when administered as a vaccine. Therefore, we hypothesized that vaccination with CMV pp65 RNA-loaded DCs would enhance the frequency of polyfunctional CMV pp65-specific CD8+ T cells after ATCT. Here, we report prospective results of a pilot trial in which 22 patients with newly diagnosed GBM were initially enrolled, of which 17 patients were randomized to receive CMV pp65-specific T cells with CMV-DC vaccination (CMV-ATCT-DC) or saline (CMV-ATCT-saline). Patients who received CMV-ATCT-DC vaccination experienced a significant increase in the overall frequencies of IFNγ+, TNFα+, and CCL3+ polyfunctional, CMV-specific CD8+ T cells. These increases in polyfunctional CMV-specific CD8+ T cells correlated (R = 0.7371, P = 0.0369) with overall survival, although we cannot conclude this was causally related. Our data implicate polyfunctional T-cell responses as a potential biomarker for effective antitumor immunotherapy and support a formal assessment of this combination approach in a larger randomized study.Significance: A randomized pilot trial in patients with GBM implicates polyfunctional T-cell responses as a biomarker for effective antitumor immunotherapy. Cancer Res; 78(1); 256-64. ©2017 AACR.