cIn many organisms, the geometry of encounter of haploid germ cells is arbitrary. In Saccharomyces cerevisiae, the resulting zygotes have been seen to bud asymmetrically in several directions as they produce diploid progeny. What mechanisms account for the choice of direction, and do the mechanisms directing polarity change over time? Distinct subgroups of cortical "landmark" proteins guide budding by haploid versus diploid cells, both of which require the Bud1/Rsr1 GTPase to link landmarks to actin. We observed that as mating pairs of haploid cells form zygotes, bud site specification progresses through three phases. The first phase follows disassembly and limited scattering of proteins that concentrated at the zone of cell contact, followed by their reassembly to produce a large medial bud. Bud1 is not required for medial placement of the initial bud. The second phase produces a contiguous bud(s) and depends on axial landmarks. As the titer of the Axl1 landmark diminishes, the third phase ultimately redirects budding toward terminal sites and is promoted by bipolar landmarks. Thus, following the initial random encounter that specifies medial budding, sequential spatial choices are orchestrated by the titer of a single cortical determinant that determines whether successive buds will be contiguous to their predecessors.

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1 . S. cerevisiae zygotes result from the fusion of haploid cells of distinct mating type (MATa, MATalpha) and give rise to corresponding stable diploids that successively generate as many as 20 diploid progeny as a result of their strikingly asymmetric mitotic divisions 2 . Zygote formation is orchestrated by a complex sequence of events: In this process, soluble mating factors bind to cognate receptors, triggering receptor-mediated signaling cascades that facilitate interruption of the cell cycle and culminate in cell-cell fusion. Zygotes may be considered a model for progenitor or stem cell function.Although much has been learned about the formation of zygotes and although zygotes have been used to investigate cell-molecular questions of general significance, almost all studies have made use of mating mixtures in which zygotes are intermixed with a majority population of haploid cells [3][4][5][6][7][8] . Many aspects of the biochemistry of zygote formation and the continuing life of the zygote therefore remain uninvestigated.Reports of purification of yeast zygotes describe protocols based on their sedimentation properties 9 ; however, this sedimentation-based procedure did not yield nearly 90% purity in our hands. Moreover, it has the disadvantage that cells are exposed to hypertonic sorbitol. We therefore have developed a versatile purification procedure. For this purpose, pairs of haploid cells expressing red or green fluorescent proteins were co-incubated to allow zygote formation, harvested at various times, and the resulting zygotes were purified using a flow cytometrybased sorting protocol. This technique provides a convenient visual assessment of purity and maturation. The average purity of the fraction is approximately 90%. According to the timing of harvest, zygotes of varying degrees of maturity can be recovered. The purified samples provide a convenient point of departure for "-omic" studies, for recovery of initial progeny, and for systematic investigation of this progenitor cell. Video LinkThe video component of this article can be found at

Background Patients with isocitrate dehydrogenase (IDH) mutant gliomas have been associated with longer survival time than those that are IDH wildtype. Previous studies have shown the prognostic value of O-6-methylguanine-DNA methyltransferase (MGMT) promoter methylation for glioblastoma multiforme (GBM), which are predominantly IDH wildtype. Little is known of the prognostic value of MGMT methylation status for IDH mutant gliomas. Methods We retrospectively identified IDH mutant gliomas patients between 2011 and 2020 that were tested for MGMT promoter methylation. We generated Kaplan-Meier estimator curves and performed Cox proportional hazard models for overall survival (OS) and progression-free survival (PFS) to compare the outcomes of MGMT promoter methylated versus MGMT unmethylated patients. Results Of 419 IDH mutant gliomas with MGMT promoter methylation testing, we identified 54 GBM, 223 astrocytoma, and 142 oligodendroglioma. 62.3% patients had MGMT methylated tumors while 37.7% were MGMT unmethylated. On Kaplan-Meier analysis, median OS for all MGMT methylated patients was 17.7 years and for unmethylated patients 14.6 years. Median PFS for all MGMT methylated patients was 7.0 years and for unmethylated patients 5.2 years. During univariate subgroup analysis, MGMT methylation is only prognostic for GBM in OS and PFS and anaplastic oligodendroglioma for OS. In multivariate analysis, MGMT unmethylated GBM patients carry a higher risk of dying (HR 7.72, 95% CI 2.10-28.33) and recurrence (HR 3.85, 95% CI 1.35-10.96). Conclusions MGMT promoter methylation is associated with better OS and PFS for IDH mutant GBM. MGMT promoter methylation testing for other IDH mutant glioma subtypes may not provide additional information on prognostication.

2065 Background: Gliomas, which constitute the majority of primary brain cancers in adults, are assigned to a particular grade based on histological and molecular criteria set forth by the World Health Organization (WHO). A subset of initially low grade glioma patients transition to a higher grade at recurrence through a process known as malignant transformation (MT). Despite its significant implications on clinical prognosis, MT remains a poorly understood phenomenon in regards to its incidence rates, effects on survival, and potential prognostic factors. Our study aims to elucidate MT in patients that possess a mutation in the isocitrate dehydrogenase ( IDH) gene, one of the most common genetic alterations in gliomas. Methods: All known IDH mutant glioma patients, seen at UCLA between 1986 and 2022, who received at least 1 repeat resection for presumed recurrent disease were included. Based on surgical pathology reports, patients were categorized into MT and non-transforming progression groups, and further stratified by diagnosis in compliance with the 2016 WHO Classification of Tumors of the Central Nervous System. Relevant clinical information, including patient demographics, survival data, tumor microscopic descriptions, and magnetic resonance imaging, was accessed via electronic medical records. Kaplan Meier analyses were conducted to evaluate the predictive power of various clinical, pathological, and radiological markers. Results: Of 724 total IDH mutant patients screened, 253 received a second surgery and were thus incorporated in this study. Among the 196 patients with lower grade pathologies capable of MT, 129 (65.8%) progressed to a higher grade at recurrence while 71 (36.2%) did not. By diagnosis, the incidence rates of MT for initial grade 2 astrocytomas, grade 3 astrocytomas, and grade 2 oligodendrogliomas were determined to be 74.7%, 48.9%, and 66.1%, respectively. MT was associated with worse overall survival and post-recurrence survival compared to non-transformation in astrocytomas, a trend not seen in oligodendrogliomas. Across all relevant diagnoses, a subset of 36 MT patients did not receive treatment in the interval between initial and recurrent surgery, demonstrating the existence of spontaneously occurring MT. Furthermore, consolidating data from pathology and MRI reports revealed that a greater extent of abnormal molecular characteristics at initial diagnosis and earlier post-operative contrast enhancement may predict MT. Conclusions: These results highlight the distinct nature of gliomas that undergo MT, particularly in tumors of astrocytic differentiation. Considering its adverse impact on clinical outcome, understanding and anticipating this phenomenon is instrumental for the determination of optimal treatment among glioma patients.

Background Tumor surveillance of IDH mutant gliomas is accomplished via serial contrast MRI. When new contrast enhancement (CEnew) is detected during post-surgical surveillance, clinicians must assess whether CEnew indicates pseudoprogression (PsP) or tumor progression (TP). PsP has been better studied in IDH wild-type glioblastoma but has not been well-characterized in IDH mutant gliomas. We conducted a retrospective study evaluating the incidence, predictors, natural history, and survival of PsP patients in a large cohort of IDH mutant glioma patients treated at a single institution. Methods We identified 587 IDH mutant glioma patients treated at UCLA. We directly inspected MRI images and radiology reports to identify CEnew and categorized CEnew into TP or PsP using MRI or histopathology. Results 56% of patients developed CEnew (326/587); of these, 92/326 patients (28% of CEnew; 16% of all) developed PsP and 179/326 (55%) developed TP. All PsP patients had prior radiation, chemotherapy, or chemoradiotherapy. PsP was associated with longer OS versus TP patients and similar OS versus no CEnew. PsP differs from TP based on earlier time of onset (median 5.8 vs. 17.4 months from treatment, p<0.0001) and MRI features that include punctate enhancement and enhancement location. Conclusion PsP patients represented 28% of CEnew patients and 16% of all patients; PsP patients demonstrated superior outcomes to TP patients, and equivalent survival to patients without CEnew. PsP persists for <1 year, occurs after treatment, and differs from TP based on time of onset and radiographic features. Poor outcomes after CEnew are driven by TP.