Distinct checkpoint and homolog biorientation pathways regulate meiosis I in Drosophila oocytes.

Mitosis and meiosis have two mechanisms for regulating the accuracy of chromosome segregation: error correction and the spindle assembly checkpoint (SAC). We have investigated the function of several checkpoint proteins in meiosis I of Drosophila oocytes. Evidence of a SAC response by several of these proteins is found upon depolymerization of microtubules by colchicine. However, unattached kinetochores or errors in biorientation of homologous chromosomes does not induce a SAC response. Furthermore, the metaphase I arrest does not depend on SAC genes, suggesting the APC is inhibited even if the SAC is silenced. Two SAC proteins, ROD of the ROD-ZW10-Zwilch (RZZ) complex and MPS1, are also required for the biorientation of homologous chromosomes during meiosis I, suggesting an error correction function. Both proteins aid in preventing or correcting erroneous attachments and depend on SPC105R for localization to the kinetochore. We have defined a region of SPC105R, amino acids 123-473, that is required for ROD localization and biorientation of homologous chromosomes at meiosis I. Surprisingly, ROD removal, or "streaming", is independent of the dynein adaptor Spindly and is not linked to the stabilization of end-on attachments. Instead, meiotic RZZ streaming appears to depend on cell cycle stage and may be regulated independently of kinetochore attachment or biorientation status. We also show that dynein adaptor Spindly is also required for biorientation at meiosis I, and surprisingly, the direction of RZZ streaming.

mTORC1 activity oscillates throughout the cell cycle, promoting mitotic entry and differentially influencing autophagy induction.

Mechanistic Target of Rapamycin Complex 1 (mTORC1) is a master metabolic regulator that is active in nearly all proliferating eukaryotic cells; however, it is unclear whether mTORC1 activity changes throughout the cell cycle. We find that mTORC1 activity oscillates from lowest in mitosis/G1 to highest in S/G2. The interphase oscillation is mediated through the TSC complex but is independent of major known regulatory inputs, including Akt and Mek/Erk signaling. By contrast, suppression of mTORC1 activity in mitosis does not require the TSC complex. mTORC1 has long been known to promote progression through G1. We find that mTORC1 also promotes progression through S and G2 and is important for satisfying the Chk1/Wee1-dependent G2/M checkpoint to allow entry into mitosis. We also find that low mTORC1 activity in G1 sensitizes cells to autophagy induction in response to partial mTORC1 inhibition or reduced nutrient levels. Together, these findings demonstrate that mTORC1 is differentially regulated throughout the cell cycle, with important phase-specific consequences for proliferating cells.

Meiosis-specific functions of kinetochore protein SPC105R required for chromosome segregation in Drosophila oocytes.

The reductional division of meiosis I requires the separation of chromosome pairs towards opposite poles. We have previously implicated the outer kinetochore protein SPC105R/KNL1 in driving meiosis I chromosome segregation through lateral attachments to microtubules and coorientation of sister centromeres. To identify the domains of SPC105R that are critical for meiotic chromosome segregation, an RNAi-resistant gene expression system was developed. We found that the SPC105R C-terminal domain (aa 1284-1960) is necessary and sufficient for recruiting NDC80 to the kinetochore and building the outer kinetochore. Furthermore, the C-terminal domain recruits BUBR1, which in turn recruits the cohesion protection proteins MEI-S332 and PP2A. Of the remaining 1283 amino acids, we found the first 473 are most important for meiosis. The first 123 amino acids of the N-terminal half of SPC105R contain the conserved SLRK and RISF motifs that are targets of PP1 and Aurora B kinase and are most important for regulating the stability of microtubule attachments and maintaining metaphase I arrest. The region between amino acids 124 and 473 are required for lateral microtubule attachments and biorientation of homologues, which are critical for accurate chromosome segregation in meiosis I.

Meiosis-specific functions of kinetochore protein SPC105R required for chromosome segregation in Drosophila oocytes.

The reductional division of meiosis I requires the separation of chromosome pairs towards opposite poles. We have previously implicated the outer kinetochore protein SPC105R/KNL1 in driving meiosis I chromosome segregation through lateral attachments to microtubules and co-orientation of sister centromeres. To identify the domains of SPC105R that are critical for meiotic chromosome segregation, an RNAi-resistant gene expression system was developed. We found that SPC105R's C-terminal domain (aa 1284-1960) is necessary and sufficient for recruiting NDC80 to the kinetochore and building the outer kinetochore. Furthermore, the C-terminal domain recruits BUBR1, which in turn recruits the cohesion protection proteins MEI-S332 and PP2A. Of the remaining 1283 amino acids, we found the first 473 are most important for meiosis. The first 123 amino acids of the N-terminal half of SPC105R contain the conserved SLRK and RISF motifs that are targets of PP1 and Aurora B kinase and are most important for regulating the stability of microtubule attachments and maintaining metaphase I arrest. The region between amino acids 124 and 473 are required for two activities that are critical for accurate chromosome segregation in meiosis I, lateral microtubule attachments and bi-orientation of homologs.

mTORC1 activity oscillates throughout the cell cycle promoting mitotic entry and differentially influencing autophagy induction.

Mechanistic Target of Rapamycin Complex 1 (mTORC1) is a master metabolic regulator that stimulates anabolic cell growth while suppressing catabolic processes such as autophagy. mTORC1 is active in most, if not all, proliferating eukaryotic cells. However, it remains unclear whether and how mTORC1 activity changes from one cell cycle phase to another. Here we tracked mTORC1 activity through the complete cell cycle and uncover oscillations in its activity. We find that mTORC1 activity peaks in S and G2, and is lowest in mitosis and G1. We further demonstrate that multiple mechanisms are involved in controlling this oscillation. The interphase oscillation is mediated through the TSC complex, an upstream negative regulator of mTORC1, but is independent of major known regulatory inputs to the TSC complex, including Akt, Mek/Erk, and CDK4/6 signaling. By contrast, suppression of mTORC1 activity in mitosis does not require the TSC complex, and instead involves CDK1-dependent control of the subcellular localization of mTORC1 itself. Functionally, we find that in addition to its well-established role in promoting progression through G1, mTORC1 also promotes progression through S and G2, and is important for satisfying the Wee1- and Chk1- dependent G2/M checkpoint to allow entry into mitosis. We also find that low mTORC1 activity in G1 sensitizes cells to autophagy induction in response to partial mTORC1 inhibition or reduced nutrient levels. Together these findings demonstrate that mTORC1 is differentially regulated throughout the cell cycle, with important phase-specific functional consequences in proliferating cells.

A Bifunctional PARP-HDAC Inhibitor with Activity in Ewing Sarcoma.

PURPOSE: Histone deacetylase (HDAC) inhibition has been shown to induce pharmacologic "BRCAness" in cancer cells with proficient DNA repair activity. This provides a rationale for exploring combination treatments with HDAC and PARP inhibition in cancer types that are insensitive to single-agent PARP inhibitors (PARPi). Here, we report the concept and characterization of a novel bifunctional PARPi (kt-3283) with dual activity toward PARP1/2 and HDAC enzymes in Ewing sarcoma cells. EXPERIMENTAL DESIGN: Inhibition of PARP1/2 and HDAC was measured using PARP1/2, HDAC activity, and PAR formation assays. Cytotoxicity was assessed by IncuCyte live cell imaging, CellTiter-Glo, and spheroid assays. Cell-cycle profiles were determined using propidium iodide staining and flow cytometry. DNA damage was examined by γH2AX expression and comet assay. Inhibition of metastatic potential by kt-3283 was evaluated via ex vivo pulmonary metastasis assay (PuMA). RESULTS: Compared with FDA-approved PARP (olaparib) and HDAC (vorinostat) inhibitors, kt-3283 displayed enhanced cytotoxicity in Ewing sarcoma models. The kt-3283-induced cytotoxicity was associated with strong S and G2-M cell-cycle arrest in nanomolar concentration range and elevated DNA damage as assessed by γH2AX tracking and comet assays. In three-dimensional spheroid models of Ewing sarcoma, kt-3283 showed efficacy in lower concentrations than olaparib and vorinostat, and kt-3283 inhibited colonization of Ewing sarcoma cells in the ex vivo PuMA model. CONCLUSIONS: Our data demonstrate the preclinical justification for studying the benefit of dual PARP and HDAC inhibition in the treatment of Ewing sarcoma in a clinical trial and provides proof-of-concept for a bifunctional single-molecule therapeutic strategy.

An update of the salmon louse (Lepeophtheirus salmonis) reference genome assembly.

Salmon lice have plagued the salmon farming industry and have negatively impacted salmon populations in the wild. In response, researchers have generated high density genetic maps, genome assemblies, transcriptomes, and whole-genome resequencing data to better understand this parasite. In this study, we used long-read sequencing technology to update the previous genome assemblies of Atlantic Ocean salmon lice with a more contiguous assembly and a more comprehensive gene catalog of Pacific Ocean salmon lice. We were also able to further characterize genomic features previously identified from other studies by using published resequenced genomes of 25 Atlantic and 15 Pacific salmon lice. One example was further characterizing the ZW sex chromosomes. For both the Atlantic and Pacific Ocean salmon lice subspecies, we found that the female W-chromosome is only a small fraction of the Z-chromosome and that the vast majority of the W and Z-chromosome do not contain conserved regions (i.e. pseudoautosomal regions). However, conserved orthologous protein sequences can still be identified between the W- and Z-chromosomes.

The pink salmon genome: Uncovering the genomic consequences of a two-year life cycle.

Pink salmon (Oncorhynchus gorbuscha) adults are the smallest of the five Pacific salmon native to the western Pacific Ocean. Pink salmon are also the most abundant of these species and account for a large proportion of the commercial value of the salmon fishery worldwide. A two-year life history of pink salmon generates temporally isolated populations that spawn either in even-years or odd-years. To uncover the influence of this genetic isolation, reference genome assemblies were generated for each year-class and whole genome re-sequencing data was collected from salmon of both year-classes. The salmon were sampled from six Canadian rivers and one Japanese river. At multiple centromeres we identified peaks of Fst between year-classes that were millions of base-pairs long. The largest Fst peak was also associated with a million base-pair chromosomal polymorphism found in the odd-year genome near a centromere. These Fst peaks may be the result of a centromere drive or a combination of reduced recombination and genetic drift, and they could influence speciation. Other regions of the genome influenced by odd-year and even-year temporal isolation and tentatively under selection were mostly associated with genes related to immune function, organ development/maintenance, and behaviour.

Radiologic presentation of chronic granulomatous prostatitis mimicking locally advanced prostate adenocarcinoma.

We present a case of nonspecific granulomatous prostatitis (GP), a clinical mimic of prostate adenocarcinoma. A 54-year-old man presented with lower urinary tract symptoms and raised prostate-specific antigen. Magnetic resonance imaging showed features consistent with prostate cancer, including low T2-signal intensity in the peripheral and transition zones with signs of extracapsular extension. Diffusion-weighted imaging showed high-signal intensity, with low apparent diffusion coefficient values, whereas dynamic contrast enhancement demonstrated a type 3 washout curve, similar to that found in prostate cancer. Transperineal sector-guided prostate biopsy confirmed nonspecific GP, and the patient was treated conservatively. We discuss and compare nonspecific, chronic GP as a radiologic mimic of prostate adenocarcinoma patient.

Clinical outcomes in patients with nonfunctioning pituitary adenomas managed conservatively.

CONTEXT: The natural history and the optimum management of patients with nonfunctioning pituitary adenomas (NFPAs) are unclear. OBJECTIVE: Our objective was to characterize the natural history of patients with NFPAs managed conservatively. DESIGN AND PATIENTS: We conducted a retrospective analysis of patients presenting to a tertiary referral centre between 1986 and 2009. Patients with pituitary adenomas and no clinical or biochemical evidence of hormonal hypersecretion were included. Those presenting with apoplexy or a radiological follow-up period of less than 1 year were excluded. The pituitary imaging for all patients was re-examined by two neuroradiologists in consensus. OUTCOME MEASURES: The outcome measures were change in tumour size and pituitary hormone function. RESULTS: Sixty-six patients were managed conservatively for a mean follow-up period of 4·3 years (range: 1-14·7). Forty-seven (71%) had a macroadenoma, and nineteen (29%) had a microadenoma. Tumour size decreased or remained stable in 40% of macroadenomas and 47% of microadenomas. The median annual growth rate of enlarging macroadenomas and microadenomas was 1·0 mm/year and 0·4 mm/year, respectively. The median annual growth rate of macroadenomas was significantly higher than that of microadenomas (P < 0·01). Sixty-eight percentage of patients with a macroadenoma had pituitary hormone deficiency in one or more axes, compared to 42% of those with a microadenoma. CONCLUSION: Patients with NFPAs without optic chiasm compression can be managed conservatively. All patients need pituitary function assessment, irrespective of tumour size. These findings provide clinically relevant data for the management of patients with NFPAs.

Peripheral nerve field stimulation for the management of localized chronic intractable back pain: results from a randomized controlled study.

OBJECTIVES: Improved device technology has caused a renewed interest in peripheral nerve field stimulation (PNfS). This study sought to obtain preliminary estimates of the safety and efficacy of PNfS in patients with localized chronic intractable pain of the back. MATERIALS AND METHODS: This Institutional Review Board-approved, prospective, randomized, controlled, crossover study consisted of two phases. During phase I, patients rotated through four stimulation groups (minimal, subthreshold, low frequency, and standard stimulation). If a 50% reduction in pain was achieved during any of the three active stimulation groups (responder), the patient proceeded to phase II, which began with implant of the permanent system and lasted 52 weeks. The primary endpoint was a reduction in pain, assessed by the visual analog scale (VAS). Analysis of variance, including the effects of patient, treatment, and study period, was used for phase I results. Phase II results were analyzed by paired t-tests. RESULTS: A total of 44 patients were enrolled at five sites. Of these patients, 32 were implanted with a trial system and 30 completed phase I. During phase I, there were significant differences in mean VAS scores between minimal stimulation and subthreshold stimulation (p = 0.003), low frequency stimulation (p < 0.001), and standard stimulation (p < 0.001). Twenty-four patients were classified as responders to the therapy, and 23 patients received permanent system placement. Significant differences in VAS scores were observed between baseline and all follow-up visits during phase II (p < 0.001) CONCLUSIONS: The results provide evidence to support safety and effectiveness of PNfS as an aid in the management of chronic, localized back pain.

Should TIA patients be hospitalized or referred to a same-day clinic?: a decision analysis.

OBJECTIVE: For patients presenting with TIA, a previous study concluded that hospitalization is cost-effective compared to discharge without treatment from the emergency department. We performed a cost-effectiveness analysis of hospitalization vs urgent clinic evaluation following TIA. METHODS: Among a cohort of TIA patients, we created a decision tree model to compare the decision to hospitalize or refer to urgent-access specialty clinic. We estimated probabilities, utilities, and direct costs from the available literature and calculated incremental cost-effectiveness ratio (ICER). We assumed equal access to standard medical treatments between the 2 approaches; however, we estimated higher tissue plasminogen activator (tPA) utilization among hospitalized patients. We performed sensitivity analyses to assess all assumptions in our model. RESULTS: In patients with TIA aged 65-74 years, hospitalization yielded additional 0.00026 quality-adjusted life-years (QALYs) at 1 year, but at an additional cost of $5,573 per patient compared to urgent clinic evaluation (ICER = $21,434,615/QALY). Over 30 years, the ICER was $3,473,125/QALY. These results were not sensitive to varying 48-hour stroke risk, length of stay, tPA utilization rate, QALYs saved per tPA treatment, and hospitalization and clinic costs, and cost saved per tPA treatment. CONCLUSION: Despite increased access to tPA in the hospital, we found that hospitalization is not cost-effective compared to same-day clinic evaluation following TIA. A very small fraction of patients benefits from hospitalization if urgent-access TIA clinics are available. The widespread development of urgent-access TIA clinics is warranted.