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Prostate cancer is the second most common malignancy in men worldwide and consists of a mixture of tumor and non-tumor cell types. To characterize the prostate cancer tumor microenvironment, we perform single-cell RNA-sequencing on prostate biopsies, prostatectomy specimens, and patient-derived organoids from localized prostate cancer patients. We uncover heterogeneous cellular states in prostate epithelial cells marked by high androgen signaling states that are enriched in prostate cancer and identify a population of tumor-associated club cells that may be associated with prostate carcinogenesis. ERG -negative tumor cells, compared to ERG -positive cells, demonstrate shared heterogeneity with surrounding luminal epithelial cells and appear to give rise to common tumor microenvironment responses. Finally, we show that prostate epithelial organoids harbor tumor-associated epithelial cell states and are enriched with distinct cell types and states from their parent tissues. Our results provide diagnostically relevant insights and advance our understanding of the cellular states associated with prostate carcinogenesis. The changes that prostate cancer (PCa) induces in its microenvironment are not fully understood. Here the authors use single-cell RNA-seq and organoids to characterise how the microenvironment responds to PCa, and also identify tumour-associated epithelial cell states and club cells.

Two broadly known characteristics of germ cells in many organisms are their development as a ‘cyst’ of interconnected cells and their high sensitivity to DNA damage. Here we provide evidence that in the Drosophila testis, connectivity serves as a mechanism that confers to spermatogonia a high sensitivity to DNA damage. We show that all spermatogonia within a cyst die synchronously even when only a subset of them exhibit detectable DNA damage. Mutants of the fusome, an organelle that is known to facilitate intracyst communication, compromise synchronous spermatogonial death and reduces overall germ cell death. Our data indicate that a death-promoting signal is shared within the cyst, leading to death of the entire cyst. Taken together, we propose that intercellular connectivity supported by the fusome uniquely increases the sensitivity of the germline to DNA damage, thereby protecting the integrity of gamete genomes that are passed on to the next generation.

Histologic variant (HV) subtypes of bladder cancer are clinically aggressive tumors that are more resistant to standard therapy compared to conventional urothelial carcinoma (UC). Little is known about the transcriptional programs that account for their biological differences. Here we show using single cell analysis that HVs harbor a tumor cell state characterized by expression of MUC16 (CA125), MUC4 , and KRT24 . This cell state is enriched in metastases, predicted to be highly resistant to chemotherapy, and linked with poor survival. We also find enriched expression of TM4SF1 , a transmembrane protein, in HV tumor cells. Chimeric antigen receptor (CAR) T cells engineered against TM4SF1 protein demonstrated in vitro and in vivo activity against bladder cancer cell lines in a TM4SF1 expression-dependent manner, highlighting its potential as a therapeutic target. Single cell analysis of histologic variant bladder tumors detects a shared CA125+ tumor cell state associated with aggressive clinical features and reveals enriched expression of TM4SF1, a membrane protein that can be targeted with CAR T cells.

rDNA loci, composed of hundreds of tandemly duplicated arrays of rRNA genes, are known to be among the most unstable genetic elements due to their repetitive nature. rDNA instability underlies aging (replicative senescence) in yeast cells, however, its contribution to the aging of multicellular organisms is poorly understood. In this study, we investigate the dynamics of rDNA loci during aging in the Drosophila male germline stem cell (GSC) lineage, and show that rDNA copy number decreases during aging. Our study further reveals that this age-dependent decrease in rDNA copy number is heritable from generation to generation, yet GSCs in young animals that inherited reduced rDNA copy number are capable of recovering normal rDNA copy number. Based on these findings, we propose that rDNA loci are dynamic genetic elements, where rDNA copy number changes dynamically yet is maintained through a recovery mechanism in the germline.

Similar multicellular structures can evolve within the same organism that may have different evolutionary histories, be controlled by different regulatory pathways, and play similar but nonidentical roles. In the human fungal pathogen Candida albicans, a quite extraordinary example of this has occurred. Depending upon the configuration of the mating type locus (a/α versus a/a or α/α), C. albicans forms alternative biofilms that appear similar morphologically, but exhibit dramatically different characteristics and are regulated by distinctly different signal transduction pathways. Biofilms formed by a/α cells are impermeable to molecules in the size range of 300 Da to 140 kDa, are poorly penetrated by human polymorphonuclear leukocytes (PMNs), and are resistant to antifungals. In contrast, a/a or α/α biofilms are permeable to molecules in this size range, are readily penetrated by PMNs, and are susceptible to antifungals. By mutational analyses, a/α biofilms are demonstrated to be regulated by the Ras1/cAMP pathway that includes Ras1→Cdc35→cAMP(Pde2-|)→Tpk2(Tpk1)→Efg1→Tec1→Bcr1, and a/a biofilms by the MAP kinase pathway that includes Mfα→Ste2→ (Ste4, Ste18, Cag1)→Ste11→Hst7→Cek2(Cek1)→Tec1. These observations suggest the hypothesis that while the upstream portion of the newly evolved pathway regulating a/a and α/α cell biofilms was derived intact from the upstream portion of the conserved pheromone-regulated pathway for mating, the downstream portion was derived through modification of the downstream portion of the conserved pathway for a/α biofilm formation. C. albicans therefore forms two alternative biofilms depending upon mating configuration.

Among the hemiascomycetes, only Candida albicans must switch from the white phenotype to the opaque phenotype to mate. In the recent evolution of this transition, mating-incompetent white cells acquired a unique response to mating pheromone, resulting in the formation of a white cell biofilm that facilitates mating. All of the upstream components of the white cell response pathway so far analyzed have been shown to be derived from the ancestral pathway involved in mating, except for the mitogen-activated protein (MAP) kinase scaffold protein, which had not been identified. Here, through binding and mutational studies, it is demonstrated that in both the opaque and the white cell pheromone responses, Cst5 is the scaffold protein, supporting the evolutionary scenario proposed. Although Cst5 plays the same role in tethering the MAP kinases as Ste5 does in Saccharomyces cerevisiae , Cst5 is approximately one-third the size and has only one rather than four phosphorylation sites involved in activation and cytoplasmic relocalization. IMPORTANCE Candida albicans must switch from white to opaque to mate. Opaque cells then release pheromone, which not only induces cells to mate but also in a unique fashion induces mating-incompetent white cells to form biofilms that facilitate opaque cell mating. All of the tested upstream components of the newly evolved white cell pheromone response pathway, from the receptor to the mitogen-activated protein (MAP) kinase cascade, are the same as those of the conserved opaque cell response pathway. One key element, however, remained unidentified, the scaffold protein for the kinase cascade. Here, we demonstrate that Cst5, a homolog of the Saccharomyces cerevisiae scaffold protein Ste5, functions as the scaffold protein in both the opaque and the white cell pheromone responses. Pheromone induces Cst5 phosphorylation, which is involved in activation and cytoplasmic localization of Cst5. However, Cst5 contains only one phosphorylation site, not four as in the S. cerevisiae ortholog Ste5. These results support the hypothesis that the entire upper portion of the newly evolved white cell pheromone response pathway is derived from the conserved pheromone response pathway in the mating process. Candida albicans must switch from white to opaque to mate. Opaque cells then release pheromone, which not only induces cells to mate but also in a unique fashion induces mating-incompetent white cells to form biofilms that facilitate opaque cell mating. All of the tested upstream components of the newly evolved white cell pheromone response pathway, from the receptor to the mitogen-activated protein (MAP) kinase cascade, are the same as those of the conserved opaque cell response pathway. One key element, however, remained unidentified, the scaffold protein for the kinase cascade. Here, we demonstrate that Cst5, a homolog of the Saccharomyces cerevisiae scaffold protein Ste5, functions as the scaffold protein in both the opaque and the white cell pheromone responses. Pheromone induces Cst5 phosphorylation, which is involved in activation and cytoplasmic localization of Cst5. However, Cst5 contains only one phosphorylation site, not four as in the S. cerevisiae ortholog Ste5. These results support the hypothesis that the entire upper portion of the newly evolved white cell pheromone response pathway is derived from the conserved pheromone response pathway in the mating process.

This paper experimentally investigates the rheology of dense granular flow through itssolid-like to fluid-like transition. Between the well-established flow regimes – quasi-static and grain-inertial – the physical description of the transition remains elusive. Our experiment uses a top-rotating torsional shear cell capable of ± 1 μm accuracy in height and 5 decades (10−3 − 100 rad s−1) in rotation rate. The data on beach sand shows that shear and normal stresses exhibit an inverse rate-dependence under a controlledvolume environment in the transitional regime, while in the limiting regimes the results are in agreement with previous work. Theshear-weakening stresses illustrate a previouslyunknown ‘dip’ with increasingshear rate. Under a controlled-pressure environment, however, the shear-compacting volume-fraction ‘peaks’ with increasing shear-rate. We combine these results from both configurations to infer a constitutive law based on a rate-invariant granular fluid compressibility. The formulation provides an equation-of-state for dynamic granular systems, with state variables of pressure, strain rate and free-volume-fraction. Fitting parameters from independent constant-volume and constant-pressure data shows good agreement in validating our model. Moreover, the degree of grain jaggedness is essential to the rate-dependence within the transitional regime. The results on the solid–fluid transitionmay elucidate the evolution of granular flow anisotropies.

Subordinating a multivariate Lévy process, the subordinate, with a univariate subordinator gives rise to a pathwise construction of a new Lévy process, provided the subordinator and the subordinate are independent processes. The variance-gamma model in finance was generated accordingly from a Brownian motion and a gamma process. Alternatively, multivariate subordination can be used to create Lévy processes, but this requires the subordinate to have independent components. In this paper, we show that there exists another operation acting on pairs (T, X) of Lévy processes which creates a Lévy process X ☉ T. Here, T is a subordinator, but X is an arbitrary Lévy process with possibly dependent components. We show that this method is an extension of both univariate and multivariate subordination and provide two applications. We illustrate our methods giving a weak formulation of the variance-α-gamma process that exhibits a wider range of dependence than using traditional subordination. Also, the variance generalised gamma convolution class of Lévy processes formed by subordinating Brownian motion with Thorin subordinators is further extended using weak subordination.

Evolution is thought to drive the progression of populations, conferring advantages through alterations in structure and form. However, the corollary to this theory would dictate that the things that do not change, those which are evolutionarily conserved, would imply some importance to their existence. Though many examples of this type of conserved phenomena have been observed, the underlying purposes for their existence remain poorly explored or understood.Two ubiquitous features of germ cells in nearly all metazoans are their development as a cyst of interconnected cells and the relative sensitivity of the germline to DNA damage. We show that in the Drosophila male germline, cysts of interconnected spermatogonia always die in unison even when only a subset of the cells within display cytologically detectable DNA damage. Our experiments showed that this all-or-none germ cell death depends upon the connectivity between members of a cyst, and is likely based on mitochondrial signals originating from the damaged cells. Interestingly, the relative sensitivity of spermatogonia at any given stage of development correlated to the number of interconnected germ cells contained within the cyst, suggesting that degree of connectivity dictates the robustness with which spermatogonia induce germ cell death in response to insult. This created a model where we propose that perhaps one reason germ cell connectivity has been so strongly conserved is to confer a robust quality control mechanism to germ cells, ensuring the fidelity of genomes that are passed on to the next generation.Another general feature of nearly all eukaryotic genomes is the organization of the ribosomal RNA genes into highly-transcribed cistrons and large tandem arrays known as the rDNA. In yeast, instability of the rDNA due to its arrangement has been shown to play a central role in replicative aging, but little was known about its role in higher eukaryotes. We speculated that if similar instability would manifest during aging of multicellular organisms, it would be likely to occur in long-lived, mitotically-active tissue stem cells. We show that germline stem cells exhibit ectopic activation of normally-silent rDNA loci during aging, and germ cells experience a dramatic reduction of rRNA gene copy number on the actively transcribed array. Furthermore, these phenotypes present in the old parents (from abnormal rDNA activation to reduced gene copy number) are heritable and able to be observed in the subsequent generation. Thus our work suggests that there may be a conserved role for dynamicity of the rDNA and rDNA instability during aging.The work contained in this dissertation attempts to reconcile recurring themes observed in evolution by leveraging the powerful tools of the Drosophila model system. The results in the first part offer one possible explanation for why pre-meiotic germ cells are connected to one another, linking it to their well-documented sensitivity to DNA damage and speculating that the driving force behind it all is a desire to increase genomic quality control for gametes. The second half of this dissertation looks at a unique genomic element shared by most eukaryotes, the rDNA, and suggests that this remarkable conservation underlies a more fundamental role in replicative aging in multicellular organisms.