Throughout its elongation, the new flagellum of the procyclic form of the African trypanosome Trypanosoma brucei is tethered at its tip to the lateral aspect of the old flagellum. This phenomenon provides a cytotactic mechanism for influencing inheritance of cellular pattern. Here, we show that this tethering is produced via a discrete, mobile transmembrane junction – the flagella connector. Light and electron microscopy reveal that the flagella connector links the extending microtubules at the tip of the new flagellum to the lateral aspect of three of the doublet microtubules in the old flagellar axoneme. Two sets of filaments connect the microtubules to three plates on the inner faces of the old and new flagellar membranes. Three differentiated areas of old and new flagellar membranes are then juxtaposed and connected by a central interstitial core of electron-dense material. The flagella connector is formed early in flagellum extension and is removed at the end of cytokinesis, but the exact timing of the latter event is slightly variable. The flagella connector represents a novel form of cellular junction that is both dynamic and mobile.

gamma-tubulin is an essential part of a multiprotein complex that nucleates the minus end of microtubules. Although the function of gamma-tubulin in nucleating cytoplasmic and mitotic microtubules from organizing centers such as the centrosome and spindle pole body is well documented, its role in microtubule nucleation in the eukaryotic flagellum is unclear. Here, we have used Trypanosoma brucei to investigate possible functions of gamma-tubulin in the formation of the 9 + 2 flagellum axoneme. T. brucei possesses a single flagellum and forms a new flagellum during each cell cycle. We have used an inducible RNA interference (RNAi) approach to ablate expression of gamma-tubulin, and, after induction, we observe that the new flagellum is still formed but is paralyzed, while the old flagellum is unaffected. Electron microscopy reveals that the paralyzed flagellum lacks central pair microtubules but that the outer doublet microtubules are formed correctly. These differences in microtubule nucleation mechanisms during flagellum growth provide insights into spatial and temporal regulation of gamma-tubulin-dependent processes within cells and explanations for the organization and evolution of axonemal structures such as the 9 + 0 axonemes of sensory cells and primary cilia.

BackgroundProstate cancer (PCa) has been under investigation as a target for antigen-specific immunotherapies in metastatic disease settings for the last two decades leading to a licensure of the first therapeutic cancer vaccine, Sipuleucel-T, in 2010. However, neither Sipuleucel-T nor other experimental PCa vaccines that emerged later induce strong T-cell immunity.MethodsIn this first-in-man study, VANCE, we evaluated a novel vaccination platform based on two replication-deficient viruses, chimpanzee adenovirus (ChAd) and MVA (Modified Vaccinia Ankara), targeting the oncofetal self-antigen 5T4 in early stage PCa. Forty patients, either newly diagnosed with early-stage PCa and scheduled for radical prostatectomy or patients with stable disease on an active surveillance protocol, were recruited to the study to assess the vaccine safety and T-cell immunogenicity. Secondary and exploratory endpoints included immune infiltration into the prostate, prostate-specific antigen (PSA) change, and assessment of phenotype and functionality of antigen-specific T cells.ResultsThe vaccine had an excellent safety profile. Vaccination-induced 5T4-specific T-cell responses were measured in blood by ex vivo IFN-γ ELISpot and were detected in the majority of patients with a mean level in responders of 198 spot-forming cells per million peripheral blood mononuclear cells. Flow cytometry analysis demonstrated the presence of both CD8+ and CD4+ polyfunctional 5T4-specific T cells in the circulation. 5T4-reactive tumor-infiltrating lymphocytes were isolated from post-treatment prostate tissue. Some of the patients had a transient PSA rise 2–8 weeks following vaccination, possibly indicating an inflammatory response in the target organ.ConclusionsAn excellent safety profile and T-cell responses elicited in the circulation and also detected in the prostate gland support the evaluation of the ChAdOx1-MVA 5T4 vaccine in efficacy trials. It remains to be seen if this vaccination strategy generates immune responses of sufficient magnitude to mediate clinical efficacy and whether it can be effective in late-stage PCa settings, as a monotherapy in advanced disease or as part of multi-modality PCa therapy. To address these questions, the phase I/II trial, ADVANCE, is currently recruiting patients with intermediate-risk PCa, and patients with advanced metastatic castration-resistant PCa, to receive this vaccine in combination with nivolumab.Trial registrationThe trial was registered with the U.S. National Institutes of Health (NIH) Clinical Trials Registry (ClinicalTrials.gov identifier NCT02390063).

African Americans (AA) have a higher incidence of multiple myeloma (MM) than White patients. Mortality is also higher in AA compared toWhite patients. AA more commonly have IgH translocations t(11;14) and t(14;16), compared to Caucasians. We sought to characterize the demographic representation in MM clinical trials and to evaluate outcomes based on race and ethnicity. We conducted a pooled analysis of all trials submitted to the United States (U.S) Food and Drug Administration (FDA) to support approval of a MM therapeutic between 2006 and 2019. Demographic characteristics were analyzed descriptively. An age-adjusted stratified Cox regression model was used to evaluate the relationship between time-to-event outcomes and race and ethnicity. Nineteen global trials comprising 10,157 patients were pooled.White, Asian, and Black patients comprised 84%, 7%, and 4% of the dataset, respectively. Hispanic patients comprised 4%. The age-adjusted overall survival (OS) hazard ratio [HR] for Black compared to White patientss was 0.89 (95% confidence interval [CI], 0.75 to 1.05). The age-adjusted HR for U.S. Black versus U.S. White patients, was 0.82 (95% CI, 0.66 to 1.02). For Rest of World (RoW) Black versus RoW White patients, HR was 1.31( 95% CI, 0.97 to 1.77). Black and Hispanic patients were underrepresented in the trials supporting approval of MM drugs. Black patients were primarily enrolled in the U.S. Outcomes in U.S. patients were more favorable compared to patients in the RoW. Given the higher incidence of MM in AA and different disease characteristics, efforts should be made to improve representation of AA in MM clinical trials.