Kar4 is required for the normal pattern of meiotic gene expression.

Kar4p, the yeast homolog of the mammalian methyltransferase subunit METTL14, is required for efficient mRNA m6A methylation, which regulates meiotic entry. Kar4p is also required for a second seemingly non-catalytic function during meiosis. Overexpression of the early meiotic transcription factor, IME1, can bypass the requirement for Kar4p in meiotic entry but the additional overexpression of the translational regulator, RIM4, is required to permit sporulation in kar4Δ/Δ. Using microarray analysis and RNA sequencing, we sought to determine the impact of removing Kar4p and consequently mRNA methylation on the early meiotic transcriptome in a strain background (S288c) that is sensitive to the loss of early meiotic regulators. We found that kar4Δ/Δ mutants have a largely wild type transcriptional profile with the exception of two groups of genes that show delayed and reduced expression: (1) a set of Ime1p-dependent early genes as well as IME1, and (2) a set of late genes dependent on the mid-meiotic transcription factor, Ndt80p. The early gene expression defect is likely the result of the loss of mRNA methylation and is rescued by overexpressing IME1, but the late defect is only suppressed by overexpression of both IME1 and RIM4. The requirement for RIM4 led us to predict that the non-catalytic function of Kar4p, like methyltransferase complex orthologs in other systems, may function at the level of translation. Mass spectrometry analysis identified several genes involved in meiotic recombination with strongly reduced protein levels, but with little to no reduction in transcript levels in kar4Δ/Δ after IME1 overexpression. The low levels of these proteins were rescued by overexpression of RIM4 and IME1, but not by the overexpression of IME1 alone. These data expand our understanding of the role of Kar4p in regulating meiosis and provide key insights into a potential mechanism of Kar4p's later meiotic function that is independent of mRNA methylation.

Kar4, the yeast homolog of METTL14, is required for mRNA m6A methylation and meiosis.

KAR4, the yeast homolog of the mammalian mRNA N6A-methyltransferase complex component METTL14, is required for two disparate developmental programs in Saccharomyces cerevisiae: mating and meiosis. To understand KAR4's role in yeast mating and meiosis, we used a genetic screen to isolate 25 function-specific mutant alleles, which map to non-overlapping surfaces on a predicted structure of the Kar4 protein (Kar4p). Most of the mating-specific alleles (Mat-) abolish Kar4p's interaction with the transcription factor Ste12p, indicating that Kar4p's mating function is through Ste12p. In yeast, the mRNA methyltransferase complex was previously defined as comprising Ime4p (Kar4p's paralog and the homolog of mammalian METTL3), Mum2p (homolog of mammalian WTAP), and Slz1p (MIS), but not Kar4p. During meiosis, Kar4p interacts with Ime4p, Mum2p, and Slz1p. Moreover, cells lacking Kar4p have highly reduced levels of mRNA methylation during meiosis indicating that Kar4p is a key member of the methyltransferase complex, as it is in humans. Analysis of kar4Δ/Δ and 7 meiosis-specific alleles (Mei-) revealed that Kar4p is required early in meiosis, before initiation of S-phase and meiotic recombination. High copy expression of the meiotic transcriptional activator IME1 rescued the defect of these Mei- alleles. Surprisingly, Kar4p was also found to be required at a second step for the completion of meiosis and sporulation. Over-expression of IME1 in kar4Δ/Δ permits pre-meiotic S-phase, but most cells remained arrested with a monopolar spindle. Analysis of the function-specific mutants revealed that roughly half became blocked after premeiotic DNA synthesis and did not sporulate (Spo-). Loss of Kar4p's Spo function was suppressed by overexpression of RIM4, a meiotic translational regulator. Overexpression of IME1 and RIM4 together allowed sporulation of kar4Δ/Δ cells. Taken together, these data suggest that Kar4p regulates meiosis at multiple steps, presumably reflecting requirements for methylation in different stages of meiotic gene expression.

Validation and refinement of the "Airman Comprehensive Assessment": Evaluating competency proficiencies of enlisted members.

Beyond proficiency on occupationally specific tasks, the U.S. Air Force expects members to develop proficiency on institutionally valued "soft skill" competencies (e.g., Teamwork, Communication, and Initiative) throughout their careers. As such, all E1-E6 members are annually evaluated using Behaviorally Anchored Rating Scales (BARS) designed to measure such competencies. Despite mandated use, these Airman Comprehensive Assessment (ACA) scales previously have not been empirically evaluated. To address this gap, we surveyed Air Force supervisors, using a criterion-related sampling methodology to validate the behavioral anchors for each scale. Supervisors identified two subordinates of the same rank/career field who they viewed as having (a) high potential for future success in an Air Force career or, alternately, (b) lower potential for future career success and rated each subordinate on the individual behaviors that comprise the 12 scales. ACA items were intermixed with scale items previously identified as distinguishing top performers in civilian organizations. Results demonstrate scale reliability and generally validate the ACA competency scales as stronger differentiators of supervisor-rated career potential than competency scales developed for civilian organizations. We provide recommendations for re-calibration of scale anchors based on the relative percentage of high vs. low potential members that demonstrate each behavior, and suggest changes to improve correspondence between measured competency proficiency and supervisor-rated career potential.

Development of a situational judgment test to supplement current US air force measures of officership.

Aptitude requirements for US Air Force officer commissioning include completion of a college degree and minimum scores on the Air Force Officer Qualifying Test (AFOQT) Verbal and Quantitative composites. Although the AFOQT has demonstrated predictive validity for officer training, the Air Force has striven to improve predictive validity and diversity. To this end, a Situational judgment Test (SJT) was added to the AFOQT in 2015. SJT development was consistent with recommendations to broaden the competencies assessed by the AFOQT with the goal of providing incremental validity, while reducing adverse impact for historically underrepresented groups. To ensure content validity and realism, SJT development was based on competencies identified in a large-scale analysis of officership and input from junior officers in scenario and response generation and scoring. Psychometric evaluations have affirmed its potential benefits for inclusion on the AFOQT. An initial study showed the SJT to be perceived as highly face valid regardless of whether it was presented as a paper-and-pencil test (with narrative or scripted scenarios) or in a video-based format. Preliminary studies demonstrated criterion-related validity within small USAF samples, and a larger Army cadet sample. Additionally, operational administration of the SJT since 2015 has demonstrated its potential for improving diversity (i.e., reduced adverse impact relative to the AFOQT Verbal and Quantitative composites). Predictive validation studies with larger Air Force officer accession samples are ongoing to assess the incremental validity of the SJT beyond current AFOQT composites for predicting important outcomes across accession sources.

Implementing and evaluating a high-resolution diode array for patient-specific quality assurance of robotic brain stereotactic radiosurgery/radiotherapy.

The purpose of the study was to introduce and evaluate a high-resolution diode array for patient-specific quality assurance (PSQA) of CyberKnife brain stereotactic radiosurgery (SRS) and stereotactic radiotherapy (SRT). Thirty-three intracranial plans were retrospectively delivered on the SRS MapCHECK using fixed cone, Iris, and multileaf collimator (MLC). The plans were selected to cover a range of sites from large tumor bed, single/multiple small brain metastases (METs) to trigeminal neuralgia. Fiducial tracking using the four fiducials embedded around the detector plane was used as image guidance. Results were analyzed before and after registration based on absolute dose gamma criterion of 1 mm distance-to-agreement and 0.5%-3% dose-difference. Overall, the gamma passing rates (1 mm and 3% criterion) before registration for all the patients were above 90% for all three treatment modalities (96.8 ± 3.5%, the lowest passing rate of 90.4%), and were improved after registration (99.3 ± 1.5%). When tighter criteria (1 mm and 2%) were applied, the gamma passing rates after registration for all the cases dropped to 97.3 ± 3.2%. For trigeminal neuralgia cases, we applied 1 mm and 0.5% criterion and the passing rates dropped from 100 ± 0.0% to 98.5 ± 2.0%. The mean delivery time was 33.4 ± 11.7 min, 24.0 ± 4.9 min, and 17.1 ± 2.6 min for the fixed cone, Iris, and MLC, respectively. With superior gamma passing rates and reasonable quality assurance (QA) time, we believe the SRS MapCHECK could be a good option for routine PSQA for CyberKnife SRS/SRT.

A mechanism for the coordination of proliferation and differentiation by spatial regulation of Fus2p in budding yeast.

Yeast cells induce the genes required for mating prior to the completion of mitosis. To ensure proper cell cycle progression prior to mating differentiation, a key cytoplasmic regulator of cell fusion, Fus2p, is sequestered in the nucleus by cyclin-dependent kinase (Cdk). In response to pheromone signaling, the mitogen-activated protein kinase Fus3p phosphorylates Ser 84 in Fus2p to drive nuclear export. We found that Fus3p becomes active and phosphorylates S84 as early as S phase, raising the question of how Cdk prevents inappropriate activation of Fus2p. Countering Fus3p, Cdk and a p21-activated kinase, Cla4p, maintain Fus2p's nuclear localization by phosphorylating Ser 67, which drives nuclear import and inhibits nuclear export. When Cdk and Cla4p activities drop after cell division, Fus3p promotes Fus2p export both via S84 phosphorylation and by down-regulating S67 phosphorylation. Thus, potential premature activation of Fus2p in mitosis is prevented by cell cycle-dependent phosphorylation that overrides the mating pheromone-induced phosphorylation that drives nuclear export.

Tailored Adaptive Personality Assessment System (TAPAS) as an indicator for counterproductive work behavior: Comparing validity in applicant, honest, and directed faking conditions.

The predictive validity of the Tailored Adaptive Personality Assessment System (TAPAS), the U.S. Army's first computer-adaptive personality test incorporating multidimensional pairwise preference items, has been demonstrated for training performance in both the Army and Air Force. While the unique TAPAS format has been described as more resistant to applicant faking than traditional self-report personality measures, evidence regarding the magnitude of applicant score distortion on TAPAS, and how such distortion (if present) may affect reliability and validity, has been limited. To address this gap, the present study compared operational TAPAS scores of Air Force enlisted recruits (administered pre-accession to applicants) to their post-accession retest scores under honest and directed faking ("fake good") conditions (based on re-administration of TAPAS during Basic Military Training). Data are presented on the relationship of applicant pre-accession scores to their retest scores under honest conditions (a form of test-retest reliability) and the magnitude of mean score differences in applicant, honest, and directed faking conditions is documented. Further, the validity of the TAPAS as an indicator for counterproductive work behaviors (CWB) was evaluated. Results indicate that TAPAS scores are relatively stable over time and the TAPAS methodology appears to reduce score distortion. In addition, the results suggest that the validities of the TAPAS scores as CWB correlates are comparable across honest and directed faking testing conditions and generally in line with those found for traditional Likert-type self-report Big Five measures.

Emergent Soft Lithographic Tools for the Fabrication of Functional Polymeric Microstructures.

Polymeric microstructures (PMs) are useful to a broad range of technologies applicable to, for example, sensing, energy storage, and soft robotics. Due to the diverse application space of PMs, many techniques (e. g., photolithography, 3D printing, micromilling, etc.) have been developed to fabricate these structures. Stemming from their generality and unique capabilities, the tools encompassed by soft lithography (e. g., replica molding, microcontact printing, etc.), which use soft elastomeric materials as masters in the fabrication of PMs, are particularly relevant. By taking advantage of the characteristics of elastomeric masters, particularly their mechanical and chemical properties, soft lithography has enabled the use of non-planar substrates and relatively inexpensive equipment in the generation of many types of PMs, redefining existing communities and creating new ones. Traditionally, these elastomeric masters have been produced from relief patterns fabricated using photolithography; however, recent efforts have led to the emergence of new methods that make use of masters that are self-forming, dynamic in their geometric and chemical properties, 3D in architecture, and/or sacrificial (i. e., easily removed/released using phase changes). These "next generation" soft lithographic masters include self-assembled liquid droplets, microscale balloons, templates derived from natural materials, and hierarchically microstructured surfaces. The new methods of fabrication supported by these unique masters enable access to numerous varieties of PMs (e. g., those with hierarchical microstructures, overhanging features, and 3D architectures) that would not be possible following established methods of soft lithography. This review explores these emergent soft lithographic methods, addressing their operational principles and the application space they can impact.

Application of an in Vitro Blood-Brain Barrier Model in the Selection of Experimental Drug Candidates for the Treatment of Huntington's Disease.

Huntington's disease (HD) is a neurodegenerative disease caused by polyglutamine expansion in the huntingtin protein. For drug candidates targeting HD, the ability to cross the blood-brain barrier (BBB) and reach the site of action in the central nervous system (CNS) is crucial for achieving pharmacological activity. To assess the permeability of selected compounds across the BBB, we utilized a two-dimensional model composed of primary porcine brain endothelial cells and rat astrocytes. Our objective was to use this in vitro model to rank and prioritize compounds for in vivo pharmacokinetic and brain penetration studies. The model was first characterized using a set of validation markers chosen based on their functional importance at the BBB. It was shown to fulfill the major BBB characteristics, including functional tight junctions, high transendothelial electrical resistance, expression, and activity of influx and efflux transporters. The in vitro permeability of 54 structurally diverse known compounds was determined and shown to have a good correlation with the in situ brain perfusion data in rodents. We used this model to investigate the BBB permeability of a series of new HD compounds from different chemical classes, and we found a good correlation with in vivo brain permeation, demonstrating the usefulness of the in vitro model for optimizing CNS drug properties and for guiding the selection of lead compounds in a drug discovery setting.

Ground Same Intratransport Efficacy as Air for Acute Aortic Diseases.

INTRODUCTION: Patients with acute aortic diseases (AAoD) usually require transfer to tertiary centers for possible surgical care, for which intratransport management represents important continuing spectrum of care. There is little information comparing intratransport efficacy of air (ART) vs ground transport (GRT), nor how effectively they manage these patients' pain. Our study aims to compare how effective ART and GRT manage patients' intratransport HR, pressure. METHODS: Charts were reviewed of adult patients interhospital transferred to a quaternary academic center (UMMC) between 01/01/2011 and 09/30/2015. Outcomes were percentages of patients achieving target hemodynamic parameters, mortality. RESULTS: We analyzed 226 patients, 58 (26%) transported by Air and 102 (45%) type A dissection. Ground transport was associated with higher percentage of patients with target HR 60-80 bpm comparing to ART (58% vs 43%, 95% CI 0.3-0.99). Both ART and GRT were associated with similar frequencies of patients achieving target SBP and adequate pain control. Time intervals from transfer request to surgery, and mortality were similar for both types of transport. CONCLUSION: Ground transport teams were more successful at achieving predefined target heart rate than Air transport. Intra-transport management of other vital signs and pain were equally effectively between both Air and Ground transport.

Influence of the bud neck on nuclear envelope fission in Saccharomyces cerevisiae.

Studies have shown that nuclear envelope fission (karyokinesis) in budding yeast depends on cytokinesis, but not distinguished whether this was a direct requirement, indirect, because of cell cycle arrest, or due to bud neck-localized proteins impacting both processes. To determine the requirements for karyokinesis, we examined mutants conditionally defective for bud emergence and/or nuclear migration. The common mutant phenotype was completion of the nuclear division cycle within the mother cell, but karyokinesis did not occur. In the cdc24 swe1 mutant, at the non-permissive temperature, multiple nuclei accumulated within the unbudded cell, with connected nuclear envelopes. Upon return to the permissive temperature, the cdc24 swe1 mutant initiated bud emergence, but only the nucleus spanning the neck underwent fission suggesting that the bud neck region is important for fission initiation. The neck may be critical for either mechanical reasons, as the contractile ring might facilitate fission, or for regulatory reasons, as the site of a protein network regulating nuclear envelope fission, mitotic exit, and cytokinesis. We also found that 77-85% of pairs of septin mutant nuclei completed nuclear envelope fission. In addition, 27% of myo1Δ mutant nuclei completed karyokinesis. These data suggested that fission is not dependent on mechanical contraction at the bud neck, but was instead controlled by regulatory proteins there.

Are Prescription Opioids Driving the Opioid Crisis? Assumptions vs Facts.

Objective: Sharp increases in opioid prescriptions, and associated increases in overdose deaths in the 2000s, evoked widespread calls to change perceptions of opioid analgesics. Medical literature discussions of opioid analgesics began emphasizing patient and public health hazards. Repetitive exposure to this information may influence physician assumptions. While highly consequential to patients with pain whose function and quality of life may benefit from opioid analgesics, current assumptions about prescription opioid analgesics, including their role in the ongoing opioid overdose epidemic, have not been scrutinized. Methods: Information was obtained by searching PubMed, governmental agency websites, and conference proceedings. Results: Opioid analgesic prescribing and associated overdose deaths both peaked around 2011 and are in long-term decline; the sharp overdose increase recorded in 2014 was driven by illicit fentanyl and heroin. Nonmethadone prescription opioid analgesic deaths, in the absence of co-ingested benzodiazepines, alcohol, or other central nervous system/respiratory depressants, are infrequent. Within five years of initial prescription opioid misuse, 3.6% initiate heroin use. The United States consumes 80% of the world opioid supply, but opioid access is nonexistent for 80% and severely restricted for 4.1% of the global population. Conclusions: Many current assumptions about opioid analgesics are ill-founded. Illicit fentanyl and heroin, not opioid prescribing, now fuel the current opioid overdose epidemic. National discussion has often neglected the potentially devastating effects of uncontrolled chronic pain. Opioid analgesic prescribing and related overdoses are in decline, at great cost to patients with pain who have benefited or may benefit from, but cannot access, opioid analgesic therapy.

Stable Pseudohyphal Growth in Budding Yeast Induced by Synergism between Septin Defects and Altered MAP-kinase Signaling.

Upon nutrient limitation, budding yeasts like Saccharomyces cerevisiae can be induced to adopt alternate filament-like growth patterns called diploid pseudohyphal or invasive haploid growth. Here, we report a novel constitutive pseudohyphal growth state, sharing some characteristics with classic forms of filamentous growth, but differing in crucial aspects of morphology, growth conditions and genetic regulation. The constitutive pseudohyphal state is observed in fus3 mutants containing various septin assembly defects, which we refer to as sadF growth (septin assembly defect induced filamentation) to distinguish it from classic filamentation pathways. Similar to other filamentous states, sadF cultures comprise aggregated chains of highly elongated cells. Unlike the classic pathways, sadF growth occurs in liquid rich media, requiring neither starvation nor the key pseudohyphal proteins, Flo8p and Flo11p. Moreover sadF growth occurs in haploid strains of S288C genetic background, which normally cannot undergo pseudohyphal growth. The sadF cells undergo highly polarized bud growth during prolonged G2 delays dependent on Swe1p. They contain septin structures distinct from classical pseudo-hyphae and FM4-64 labeling at actively growing tips similar to the Spitzenkörper observed in true hyphal growth. The sadF growth state is induced by synergism between Kss1p-dependent signaling and septin assembly defects; mild disruption of mitotic septins activates Kss1p-dependent gene expression, which exacerbates the septin defects, leading to hyper-activation of Kss1p. Unlike classical pseudo-hyphal growth, sadF signaling requires Ste5, Ste4 and Ste18, the scaffold protein and G-protein ß and γ subunits from the pheromone response pathway, respectively. A swe1 mutation largely abolished signaling, breaking the positive feedback that leads to amplification of sadF signaling. Taken together, our findings show that budding yeast can access a stable constitutive pseudohyphal growth state with very few genetic and regulatory changes.

Quantification of beam steering with an ionization chamber array.

Routine quality assurance for linear accelerators (linacs) usually involves verification of beam steering with a water scanning system. We established a beam steering procedure that uses a 2D ionization chamber array (ICA) and verified the equivalence of beam symmetry between the ICA and a water scanning system. The ICA calibration accuracy, reproducibility and stability were evaluated and the uncertainty in the measurement of beam symmetry due to the array calibration was examined. Forty-five photon beams and 80 electron beams across 7 Varian C-series and 4 TrueBeam linacs were steered in the radial and transverse directions using an ICA. After beam steering, profiles were re-measured using the ICA and in-water using a 3D Scanner (3DS). Beam symmetries measured with the ICA and 3DS were compared by (a) calculating the difference in point-by-point symmetry, (b) plotting the histogram distribution of the symmetry differences, and (c) comparing ICA and 3DS differences with their respective Varian symmetry protocol analysis. Array calibrations from five different occurrences (2012 to 2016) over six different beams reproduced within 0.5%. The uncertainty in beam symmetry was less than 0.5% due to the uncertainties in the array calibration. After all beams were steered using the ICA, the point-by-point symmetry differences between ICA and 3DS at the off-axis positions of 20% and 80% of field size for all beam profiles indicated that 95% of point-by-point symmetry comparisons agreed within 0.7%, and 100% agreed within 1.0%; after steering with the ICA 97.8% of photon beam profiles (88 of 90) and 97.5% of electron beam profiles (156 of 160) had symmetry within 1% when measured with the 3DS. All photon and electron beam profiles had symmetry within 1.1% and 1.2%, respectively, for profiles measured with the 3DS. Our data demonstrate that a calibrated ICA can be used to steer photon and electron beams achieving beam symmetry within 1% when re-measured with a 3D water scanning system.

Surface Molding of Microscale Hydrogels with Microactuation Functionality.

This work describes the fabrication of numerous hydrogel microstructures (µ-gels) via a process called "surface molding." Chemically patterned elastomeric-assembly substrates were used to organize and manipulate the geometry of liquid prepolymer microdroplets, which, following photo-initiated crosslinking, maintained the desired morphology. By adjusting the state of strain during the crosslinking process, a continua of structures could be created using one pattern. These arrays of µ-gels have stimuli-responsive properties that are directly applicable to actuation where the basis shape and array geometry of the µ-gels can be used to rationally generate microactuators with programmed motions. As a method, "surface molding," represents a powerful addition to the soft-lithographic toolset that can be readily applied to the simultaneous synthesis of large numbers of geometrically and functionally distinct polymeric microstructures.

A comparison of methods for monitoring photon beam energy constancy.

In extension of a previous study, we compared several photon beam energy metrics to determine which was the most sensitive to energy change; in addition to those, we accounted for both the sensitivity of each metric and the uncertainty in determining that metric for both traditional flattening filter (FF) beams (4, 6, 8, and 10 MV) and for flattening filter-free (FFF) beams (6 and 10 MV) on a Varian TrueBeam. We examined changes in these energy metrics when photon energies were changed to ± 5% and ± 10% from their nominal energies: 1) an attenuation-based metric (the percent depth dose at 10 cm depth, PDD(10)) and, 2) profile-based metrics, including flatness (Flat) and off-axis ratios (OARs) measured on the orthogonal axes or on the diagonals (diagonal normalized flatness, FDN). Profile-based metrics were measured near dmax and also near 10 cm depth in water (using a 3D scanner) and with ioniza-tion chamber array (ICA). PDD(10) was measured only in water. Changes in PDD, OAR, and FDN were nearly linear to the changes in the bend magnet current (BMI) over the range from -10% to +10% for both FF and FFF beams: a ± 10% change in energy resulted in a ± 1.5% change in PDD(10) for both FF and FFF beams, and changes in OAR and FDN were > 3.0% for FF beams and > 2.2% for FFF beams. The uncertainty in determining PDD(10) was estimated to be 0.15% and that for OAR and FDN about 0.07%. This resulted in minimally detectable changes in energy of 2.5% for PDD(10) and 0.5% for OAR and FDN. We found that the OAR- or FDN- based metrics were the best for detecting energy changes for both FF and FFF beams. The ability of the OAR-based metrics determined with a water scanner to detect energy changes was equivalent to that using an ionization chamber array. We recommend that OAR be measured either on the orthogonal axes or the diagonals, using an ionization chamber array near the depth of maximum dose, as a sensitive and efficient way to confirm stability of photon beam energy.

Heparanase induces inflammatory cell recruitment in vivo by promoting adhesion to vascular endothelium.

Heparanase (HPSE1) is known to be involved in mechanisms of metastatic tumor cell migration. This enzyme selectively cleaves heparan sulfate proteoglycans (HSPG), which are ubiquitously expressed in mammals and are known to be involved in regulating the activity of an array of inflammatory mediators. In the present study, we have investigated the effects of human recombinant heparanase, the inactive precursor of this enzyme (proheparanase) and enzymatically inactivated heparanase, on inflammatory cell recruitment in the rat and on human leukocyte-endothelial adhesion in vitro. Intraperitoneal injection of heparanase (500 µg) induced a significant inflammatory cell infiltrate in the rat, as assessed by peritoneal lavage 4 h later. Intravital microscopy of the mesenteric microcirculation of anesthetized rats showed an increase in rolling and adherent cells in postcapillary venules that was sensitive to heparin, a nonselective inhibitor of heparanase activity. In vitro, heparanase augmented the adhesion of human neutrophils and mononuclear cells to human umbilical vein endothelial cells in a concentration-dependent manner. Proheparanase had similar effects to the active enzyme both with respect to leukocyte accumulation in the peritoneal cavity and adhesion in vitro. However, heat-inactivated heparanase induced cell adhesion in vitro but was without effect in vivo. Together, these data indicate a role for heparanase in inflammatory cell trafficking in vivo that appears to require enzymatic activity.

Kar4 is Required for the Normal Pattern of Meiotic Gene Expression.

Kar4p, the yeast homolog of the mammalian methyltransferase subunit METTL14, is required for the initiation of meiosis and has at least two distinct functions in regulating the meiotic program. Cells lacking Kar4p can be driven to sporulate by co-overexpressing the master meiotic transcription factor, IME1 , and the translational regulator, RIM4 , suggesting that Kar4p functions at both the transcriptional and translational level to regulate meiosis. Using microarray analysis and RNA sequencing, we found that kar4 Δ/Δ mutants have a largely wild type transcriptional profile with the exception of two groups of genes that show delayed and reduced expression: (1) a set of Ime1p-dependent early genes as well as IME1 , and (2) a set of late genes dependent on the mid-meiotic transcription factor, Ndt80p. The early gene expression defect is rescued by overexpressing IME1 , but the late defect is only suppressed by overexpression of both IME1 and RIM4 . Mass spectrometry analysis identified several genes involved in meiotic recombination with strongly reduced protein levels, but with little to no reduction in transcript levels in kar4 Δ/Δ after IME1 overexpression. The low levels of these proteins were rescued by overexpression of RIM4 and IME1 , but not by the overexpression of IME1 alone. These data expand our understanding of the role of Kar4p in regulating meiosis and provide key insights into a potential mechanism of Kar4p's later meiotic function that is independent of mRNA methylation. Author Summary: Kar4p is required at two stages during meiosis. Cells lacking Kar4p have a severe loss of mRNA methylation and arrest early in the meiotic program, failing to undergo either pre-meiotic DNA synthesis or meiotic recombination. The early block is rescued by overexpression of the meiotic transcription factor, IME1 . The kar4 Δ/Δ cells show delayed and reduced expression of a set of Ime1p-dependent genes expressed early in meiosis as well as a set of later genes that are largely Ndt80p-dependent. Overexpression of IME1 rescues the expression defect of these early genes and expedites the meiotic program in the wild type S288C strain background. However, IME1 overexpression is not sufficient to facilitate sporulation in kar4 Δ/Δ. Completion of meiosis and sporulation requires the additional overexpression of a translational regulator, RIM4 . Analysis of kar4 Δ/Δ's proteome during meiosis with IME1 overexpression revealed that proteins important for meiotic recombination have reduced levels that cannot be explained by equivalent reductions in transcript abundance. IME1 overexpression by itself rescues the defect associated with a catalytic mutant of Ime4p, implying that the early defect reflects mRNA methylation. The residual defects in protein levels likely reflect the loss of a non-catalytic function of Kar4p, and the methylation complex, which requires overexpression of RIM4 to suppress.

Vir1p, the Yeast Homolog of Virilizer, is Required for mRNA m 6 A Methylation and Meiosis.

N 6 -Methyladenosine (m 6 A) is one of the most abundant modifications found on eukaryotic mRNAs. mRNA methylation regulates a host of biological processes including meiosis, a specialized diploid cell division program that results in the formation of haploid cells (gametes). During budding yeast meiosis, m 6 A levels peak early, before the initiation of the meiotic divisions. High-throughput studies and work from our lab showed that Ygl036wp, a previously uncharacterized protein interacts with Kar4p, a meiotic protein required for mRNA m 6 A-methylation. Ygl036wp has no discernable domains except for several intrinsically disordered regions. However, protein folding prediction tools showed that Ygl036wp folds like VIRMA/Virilizer/VIR, which is involved in mRNA m 6 A-methylation in higher eukaryotes. In addition, Ygl036wp has several conserved motifs shared with VIRMA/Virilizer/VIR proteins. Accordingly, we propose to call the gene VIR1 for budding yeast ortholog of VIR MA/Virilizer/VIR 1 . In support, Vir1p interacts with all other members of the yeast methyltransferase complex and is required for mRNA m 6 A methylation and meiosis. Vir1p is required for the stability of proteins comprising the methyltransferase complex, suggesting that Vir1p acts as a scaffold to stabilize the complex. The vir1 Δ/Δ mutant is defective for premeiotic S-phase, which is suppressed by overexpression of the early meiotic transcription factor IME1; additional overexpression of the translational regulator RIM4 is required for sporulation. Consistent with IME1 suppression, vir1 Δ/Δ exhibits a defect in the abundance of IME1 mRNA, as well as transcripts within Ime1p's regulon. Suppression by IME1 revealed a defect in the expression of the middle meiotic transcription factor, Ndt80p (and genes in its regulon), which is rescued by additional overexpression of RIM4 . Together, these data suggest that Vir1p is required for cells to initiate the meiotic program and for progression through the meiotic divisions and spore formation. Author Summary: Ygl036wp is a previously uncharacterized protein that we propose to name Vir1p (budding yeast ortholog of VIR MA/Virilizer/VIR 1 ). Work from our lab and others initially found an interaction between Vir1p and members of the yeast mRNA methyltransferase complex (Kar4p and Mum2p). We found that Vir1p interacts with all known members of the methyltransferase complex and is required for mRNA methylation. Vir1p is required early in meiosis; vir1 Δ/Δ mutants arrest due to the reduced expression of Ime1p. Lower levels of Ime1p cause severe disruption to the meiotic transcriptome in vir1 Δ/Δ. The vir1 Δ/Δ meiotic defect can be partially suppressed by the overexpression of IME1 ; full suppression requires overexpression of both IME1 and RIM4 . Using recent advances in protein folding predictions, we found that Vir1p is a remote homolog of VIRMA/Virilizer/VIR and shares conserved motifs with the protein from other organisms. Vir1p, like VIRMA/Virilizer/VIR, stabilizes the methyltransferase complex.

Vir1p, the yeast homolog of virilizer, is required for mRNA m6A methylation and meiosis.

N6-Methyladenosine (m6A) is among the most abundant modifications of eukaryotic mRNAs. mRNA methylation regulates many biological processes including playing an essential role in meiosis. During meiosis in the budding yeast, Saccharomyces cerevisiae, m6A levels peak early, before the initiation of the meiotic divisions. High-throughput studies suggested, and this work confirms that the uncharacterized protein Ygl036wp interacts with Kar4p, a component of the mRNA m6A-methyltransferase complex. Protein structure programs predict that Ygl036wp folds like VIRMA/Virilizer/VIR, which is involved in mRNA m6A-methylation in higher eukaryotes. In addition, Ygl036wp contains conserved motifs shared with VIRMA/Virilizer/VIR. Accordingly, we propose the name VIR1 for budding yeast ortholog of VIRMA/Virilizer/VIR 1. Vir1p interacts with all other members of the yeast methyltransferase complex and is itself required for mRNA m6A methylation and meiosis. In the absence of Vir1p proteins comprising the methyltransferase complex become unstable, suggesting that Vir1p acts as a scaffold for the complex. The vir1Δ/Δ mutant is defective for the premeiotic S-phase, which is suppressed by overexpression of the early meiotic transcription factor IME1; additional overexpression of the translational regulator RIM4 is required for sporulation. The vir1Δ/Δ mutant exhibits reduced levels of IME1 mRNA, as well as transcripts within Ime1p's regulon. Suppression by IME1 revealed an additional defect in the expression of the middle meiotic transcription factor, Ndt80p (and genes in its regulon), which is rescued by overexpression of RIM4. Together, these data suggest that Vir1p is required for cells to initiate the meiotic program and for progression through the meiotic divisions and spore formation.