Evaluation of the inhibitory effects of itraconazole on letermovir.

AIMS: Letermovir, a cytomegalovirus (CMV) DNA terminase complex inhibitor, is a substrate of ABCB1 (P-glycoprotein; P-gp), organic anion transporting polypeptide (OATP)1B1/3, UDP-glucuronosyltransferase (UGT)1A1, UGT1A3 and possibly ABCG2 (breast cancer resistance protein; BCRP). A study was conducted to evaluate the effects of itraconazole, a prototypic ABCB1/ABCG2 inhibitor, on letermovir pharmacokinetics (PK) and the effects of letermovir on itraconazole PK. METHODS: In an open-label, fixed-sequence study in 14 healthy participants, 200 mg oral itraconazole was administered once daily for 4 days. Following a 10-day washout, 480 mg oral letermovir was administered once daily for 14 days (Days 1-14) and then coadministered with 200 mg itraconazole once daily for 4 days (Days 15-18). Intensive PK sampling was performed for letermovir and itraconazole. PK and safety were evaluated. RESULTS: Letermovir geometric mean ratio (GMR; 90% confidence interval [CI]) for area under the concentration-time curve from time 0 to 24 h (AUC0-24 ) was 1.33 (1.17, 1.51) and for maximum concentration (Cmax ) was 1.21 (1.05, 1.39) following administration with/without itraconazole. Itraconazole GMR (90% CI) for AUC0-24 was 0.76 (0.71, 0.81) and for Cmax was 0.84 (0.76, 0.92) following administration with/without letermovir. Coadministration of letermovir with itraconazole was generally well tolerated. CONCLUSIONS: The increase in letermovir exposure with coadministration of itraconazole is likely predominantly due to inhibition of intestinal ABCB1 and potentially ABCG2 transport. The mechanism for the decrease in itraconazole exposure is unknown. The modest changes in letermovir and itraconazole PK are not considered clinically meaningful.

Preparing fourth year medical students to care for patients with opioid use disorder: how this training affects their intention to seek addiction care opportunities during residency.

BACKGROUND & OBJECTIVES: In 2021, the USA recorded 100,000 annual deaths from drug overdose, representing the most frequent cause of death in adults under age 55. The integration of care for substance use disorders (SUDs) into undergraduate medical education is not well established. It is unclear whether a short course on management of opioid use disorder (OUD) offered to fourth year medical students could increase graduating students' knowledge and preparedness to treat these disorders. METHODS: We designed a 2-hour interactive case-based session on patient care for OUD and delivered it virtually as part of a Transition to Residency course. A retrospective pre-/post-test assessment instrument determined the impact of this session on students' perceived knowledge, confidence, and intention to seek further educational opportunities for OUD. RESULTS: Of 144 participants, 58 students (40.3%) completed the retrospective pre-/post- survey. There were statistically significant improvements in perceived knowledge and attitudes on the 12-item survey. The largest gains in perceived knowledge on a 5-point scale occurred in the categories regarding buprenorphine induction (pre 2.9; post 4.22; p < 0.001), managing inpatient opioid withdrawal (pre 2.84; post 4.27; p < 0.001), and the role of methadone in treating withdrawal (pre 3.16; post 4.29; p < 0.001). All (n = 58) survey respondents would recommend the training to a colleague and felt that the session would benefit their professional practice. Over 90% (93.1%) of respondents planned on seeking additional SUD learning opportunities during residency. CONCLUSIONS: A 2-hour interactive case-based teaching session delivered to medical students improved perceived knowledge, attitudes, and future interest in obtaining education around OUD. As the opioid epidemic shows no sign of abating, we would advocate for the inclusion SUD education as part of Transition to Residency courses.

Safety and immunogenicity of intramuscular, single-dose V590 (rVSV-SARS-CoV-2 Vaccine) in healthy adults: Results from a phase 1 randomised, double-blind, placebo-controlled, dose-ranging trial.

BACKGROUND: Vaccines against COVID-19 are needed to overcome challenges associated with mitigating the global pandemic. We report the safety and immunogenicity of V590, a live recombinant vesicular stomatitis virus-based COVID-19 vaccine candidate. METHODS: In this placebo-controlled, double-blind, three-part phase 1 study, healthy adults were randomised to receive a single intramuscular dose of vaccine or placebo. In Part 1, younger (18-54 years) and, in Part 2, older (≥55 years) adults seronegative for SARS-CoV-2 nucleocapsid received one of four V590 dose levels (5.00 × 105; 2.40 × 106; 1.15 × 107; or 5.55 × 107 plaque-forming units [pfu]) or placebo. In Part 3, a single V590 dose level (5.55 × 107 pfu) or placebo was administered to younger SARS-CoV-2 seropositive adults. Primary endpoints included adverse events (AEs) and for Parts 1 and 2 anti-SARS-CoV-2 serum neutralising antibody responses measured by 50% plaque reduction neutralisation (PRNT50) assay at Day 28. Registration NCT04569786 [P001-02]. FINDINGS: 232 participants were randomised and 219 completed the study. In seronegative participants, anti-SARS-CoV-2 spike-specific antibody responses to V590 were low and comparable to placebo across the lower dose levels. At the highest dose level (5.55 × 107 pfu), anti-SARS-CoV-2 spike-specific PRNT50 was 2.3-fold higher than placebo. The most frequently reported AEs were injection-site pain (38.4%), headache (15.1%) and fatigue (13.4%). INTERPRETATION: V590 was generally well-tolerated. However, Day 28 anti-SARS-Cov-2 spike-specific antibody responses in seronegative participants following a single intramuscular administration of V590 were not sufficient to warrant continued development. FUNDING: The study was funded by Merck Sharp & Dohme LLC., a subsidiary of Merck & Co., Inc., Rahway, NJ, USA.

Differences in perceived clinical knowledge uptake among health profession students and licensed clinicians receiving buprenorphine waiver training in Oregon.

Background: Addressing the opioid crisis requires an understanding of how to train both health professional students and practicing clinicians on medications for opioid use disorder (mOUD). We designed a robust evaluation instrument to assess the impact of training on perceived clinical knowledge in these different categories of learners. Methods: We enrolled 3rd and 4th year medical, physician assistant (PA), and nurse practitioner (NP) students, as well as practicing PAs, NPs, and physicians to undertake the Drug Addiction Treatment Act (DATA) Waiver Training for mOUD. We designed and implemented a cross-sectional survey to assess perceived change in clinical knowledge as a result of training in opioid use disorder and satisfaction with training. Results: Twenty-one MD/DO and 45 NP/PA students, and 24 practicing MD/DO and 27 NP/PAs completed the survey. Among health professional students (n = 66) and practicing clinicians (n =51), perceived clinical knowledge scores increased significantly (p < 0.001) for all 13 variables. Program evaluation scores for the buprenorphine waiver training were high with no statistical differences between students and practicing clinicians. Overall, the majority of participants indicated they would recommend the training to a colleague (Students' score = 4.84; practicing clinician scores = 4.53; scale = strongly disagree = 1 to strongly agree = 5). Conclusions: Our novel instrument allowed us to determine that the implementation of buprenorphine waiver trainings for health professional students and practicing clinicians leads to significant increases in perceived knowledge, interest and confidence in diagnosing and treating OUD. Although the buprenorphine waiver can now be obtained without training, many waivered providers still do not prescribe buprenorphine; integrating training into medical, NP, and PA curriculum for students and offering the training to practicing clinicians may increase confidence and uptake of mOUD.

Low-dose Buprenorphine Initiation in Hospitalized Adults With Opioid Use Disorder: A Retrospective Cohort Analysis.

OBJECTIVES: Patients with opioid use disorder (OUD) can initiate buprenorphine without requiring a withdrawal period through a low-dose (sometimes referred to as "micro-induction") approach. Although there is growing interest in low-dose buprenorphine initiation, current evidence is limited to case reports and small case series. METHODS: We performed a retrospective cohort study of patients with OUD seen by a hospital-based addiction medicine consult service who underwent low-dose buprenorphine initiation starting during hospital admission. We then integrated our practice-based experiences with results from the existing literature to create practice considerations. RESULTS: Sixty-eight individuals underwent 72 low-dose buprenorphine initiations between July 2019 and July 2020. Reasons for low-dose versus standard buprenorphine initiation included co-occurring pain (91.7%), patient anxiety around the possibility of withdrawal (69.4%), history of precipitated withdrawal (9.7%), opioid withdrawal intolerance (6.9%), and other reason/not specified (18.1%). Of the 72 low-dose buprenorphine initiations, 50 (69.4%) were completed in the hospital, 9 (12.5%) transitioned to complete as an outpatient, and 13 (18.1%) were terminated early. We apply our experiences and findings from literature to recommendations for varied clinical scenarios, including acute illness, co-occurring pain, opioid withdrawal intolerance, transition from high dose methadone to buprenorphine, history of precipitated withdrawal, and rapid hospital discharge. We share a standard low-dose initiation protocol with potential modifications based on above scenarios. CONCLUSIONS: Low-dose buprenorphine initiation offers a well-tolerated and versatile approach for hospitalized patients with OUD. We share lessons from our experiences and the literature, and provide practical considerations for providers.

Acute and Chronic Effects of Rifampin on Letermovir Suggest Transporter Inhibition and Induction Contribute to Letermovir Pharmacokinetics.

Rifampin has acute inhibitory and chronic inductive effects that can cause complex drug-drug interactions. Rifampin inhibits transporters including organic-anion-transporting polypeptide (OATP)1B and P-glycoprotein (P-gp), and induces enzymes and transporters including cytochrome P450 3A, UDP-glucuronosyltransferase (UGT)1A, and P-gp. This study aimed to separate inhibitory and inductive effects of rifampin on letermovir disposition and elimination (indicated for cytomegalovirus prophylaxis in hematopoietic stem cell transplant recipients). Letermovir is a substrate of UGT1A1/3, P-gp, and OATP1B, with its clearance primarily mediated by OATP1B. Letermovir (single-dose) administered with rifampin (single-dose) resulted in increased letermovir exposure through transporter inhibition. Chronic coadministration with rifampin (inhibition plus potential OATP1B induction) resulted in modestly decreased letermovir exposure vs. letermovir alone. Letermovir administered 24 hours after the last rifampin dose (potential OATP1B induction) resulted in markedly decreased letermovir exposure. These data suggest rifampin may induce transporters that clear letermovir; the modestly reduced letermovir exposure with chronic rifampin coadministration likely reflects the net effect of inhibition and induction. OATP1B endogenous biomarkers coproporphyrin (CP) I and glycochenodeoxycholic acid-sulfate (GCDCA-S) were also analyzed; their exposures increased after single-dose rifampin plus letermovir, consistent with OATP1B inhibition and prior reports of inhibition by rifampin alone. CP I and GCDCA-S exposures were substantially reduced with letermovir administered 24 hours after the last dose of rifampin vs. letermovir plus chronic rifampin coadministration. This study suggests that OATP1B induction may contribute to reduced letermovir exposure after chronic rifampin administration, although given the complexity of letermovir disposition alternative mechanisms are not fully excluded.

Buprenorphine Microdose Induction for the Management of Prescription Opioid Dependence.

Prescription opioid dependence remains a major source of morbidity and mortality in the United States. Patients previously on high-dose opioids may poorly tolerate opioid tapers. Current guidelines support the use of buprenorphine therapy in opioid-tapering protocols, even among patients without a diagnosis of opioid use disorder. Buprenorphine microinduction protocols can be used to transition patients to buprenorphine therapy without opioid withdrawal. From November 2019 to April 2020, we transitioned 8 patients on high-dose prescribed opioids for pain to sublingual buprenorphine-naloxone using a microdose protocol without any evidence of precipitated withdrawal. Six of these patients remain on buprenorphine-naloxone and report improved analgesia. Because of its simplicity, the buprenorphine microinduction protocol can be easily adapted for telemedicine and may help to prevent unnecessary clinic visits and opioid-related admissions in the setting of social distancing regulations during the coronavirus 2019 pandemic.

Dual Plasmepsin-Targeting Antimalarial Agents Disrupt Multiple Stages of the Malaria Parasite Life Cycle.

Artemisin combination therapy (ACT) is the main treatment option for malaria, which is caused by the intracellular parasite Plasmodium. However, increased resistance to ACT highlights the importance of finding new drugs. Recently, the aspartic proteases Plasmepsin IX and X (PMIX and PMX) were identified as promising drug targets. In this study, we describe dual inhibitors of PMIX and PMX, including WM382, that block multiple stages of the Plasmodium life cycle. We demonstrate that PMX is a master modulator of merozoite invasion and direct maturation of proteins required for invasion, parasite development, and egress. Oral administration of WM382 cured mice of P. berghei and prevented blood infection from the liver. In addition, WM382 was efficacious against P. falciparum asexual infection in humanized mice and prevented transmission to mosquitoes. Selection of resistant P. falciparum in vitro was not achievable. Together, these show that dual PMIX and PMX inhibitors are promising candidates for malaria treatment and prevention.

Low levels of initiation, engagement, and retention in substance use disorder treatment including pharmacotherapy among HIV-infected and uninfected veterans.

BACKGROUND: Substance use disorders (SUDs) are common in healthcare settings and contribute to poor outcomes, particularly in patients living with HIV. We assessed initiation, engagement, and retention in SUD treatment and pharmacotherapy following an index SUD episode in a national sample of HIV-infected and uninfected patients receiving care in the Department of Veterans Affairs (VA) healthcare system. METHODS: We used electronic national VA data (years 2000-2015) from 52,995 HIV-infected and 111,229 age-, race-, gender-, and region-matched uninfected patients. We defined index SUD episodes as outpatient visits or inpatient/residential admissions with associated primary or secondary ICD-9 codes for substance use in patients without SUD-related services or pharmacotherapy in the preceding 5 months. RESULTS: Overall, 57,428 (35%) patients had at least 1 index SUD episode. HIV-infected patients were more likely than uninfected controls to have at least one index SUD episode (35.7% vs. 34.6%; p < .001). Rates of initiation, engagement, and retention in SUD treatment after the index SUD episode were <17% for both groups. In adjusted models, HIV-infected patients were more likely than uninfected patients to be retained in SUD treatment at 6 months (Odds Ratio 1.10; 95% Confidence Interval 1.04-1.16). SUD pharmacotherapy initiation and engagement was uncommon in both HIV-infected and uninfected patients. CONCLUSIONS: In this national VA sample, initiation of SUD treatment and pharmacotherapy were uncommon for both HIV-infected and uninfected patients. Interventions to improve initiation, engagement, and retention in the full range of services, including SUD pharmacotherapy, are warranted for all patients with SUD in the VA.

Predictors of timely opioid agonist treatment initiation among veterans with and without HIV.

BACKGROUND: Opioid use disorder (OUD) is prevalent among people with HIV (PWH). Opioid agonist therapy (OAT) is the most effective treatment for OUD and is associated with improved health outcomes, but is often not initiated. To inform clinical practice, we identified factors predictive of OAT initiation among patients with and without HIV. METHODS: We identified 19,698 new clinical encounters of OUD between 2000 and 2012 in the Veterans Aging Cohort Study (VACS), a national observational cohort of PWH and matched uninfected controls. Mixed effects models examined factors predictive of OAT initiation within 30-days of a new OUD clinical encounter. RESULTS: 4.9% of both PWH and uninfected patients initiated OAT within 30 days of a new OUD clinical encounter. In adjusted models, participants with a psychiatric diagnosis (aOR = 0.54, 95% CI 0.47 - 0.62), PWH (aOR = 0.79, 95% CI 0.68-0.92), and rural residence (aOR = 0.56, 95% CI 0.39-0.78) had a lower likelihood of any OAT initiation, while African-American patients (aOR = 1.60, 95% CI 1.34-1.92), those with an alcohol related diagnosis (aOR = 1.76, 95% CI 1.48-2.08), diagnosis year 2005-2008 relative to 2000-2004 (aOR = 1.24, 95% CI 1.05-1.45), and patients with HCV (aOR = 1.50, 95% CI 1.27-1.77) had a greater likelihood of initiating any OAT within 30 days. Predictive factors were similar in the total sample and PWH only models. CONCLUSIONS: PWH were less likely to receive timely OAT initiation than demographically similar uninfected patients. Given the health benefits of such treatment, the low rate of OAT initiation warrants focused efforts in both PWH and uninfected populations.

The Malaria Parasite Cyclin H Homolog PfCyc1 Is Required for Efficient Cytokinesis in Blood-Stage Plasmodium falciparum.

All well-studied eukaryotic cell cycles are driven by cyclins, which activate cyclin-dependent kinases (CDKs), and these protein kinase complexes are viable drug targets. The regulatory control of the Plasmodium falciparum cell division cycle remains poorly understood, and the roles of the various CDKs and cyclins remain unclear. The P. falciparum genome contains multiple CDKs, but surprisingly, it does not contain any sequence-identifiable G1-, S-, or M-phase cyclins. We demonstrate that P. falciparum Cyc1 (PfCyc1) complements a G1 cyclin-depleted Saccharomyces cerevisiae strain and confirm that other identified malaria parasite cyclins do not complement this strain. PfCyc1, which has the highest sequence similarity to the conserved cyclin H, cannot complement a temperature-sensitive yeast cyclin H mutant. Coimmunoprecipitation of PfCyc1 from P. falciparum parasites identifies PfMAT1 and PfMRK as specific interaction partners and does not identify PfPK5 or other CDKs. We then generate an endogenous conditional allele of PfCyc1 in blood-stage P. falciparum using a destabilization domain (DD) approach and find that PfCyc1 is essential for blood-stage proliferation. PfCyc1 knockdown does not impede nuclear division, but it prevents proper cytokinesis. Thus, we demonstrate that PfCyc1 has a functional divergence from bioinformatic predictions, suggesting that the malaria parasite cell division cycle has evolved to use evolutionarily conserved proteins in functionally novel ways.IMPORTANCE Human infection by the eukaryotic parasite Plasmodium falciparum causes malaria. Most well-studied eukaryotic cell cycles are driven by cyclins, which activate cyclin-dependent kinases (CDKs) to promote essential cell division processes. Remarkably, there are no identifiable cyclins that are predicted to control the cell cycle in the malaria parasite genome. Thus, our knowledge regarding the basic mechanisms of the malaria parasite cell cycle remains unsatisfactory. We demonstrate that P. falciparum Cyc1 (PfCyc1), a transcriptional cyclin homolog, complements a cell cycle cyclin-deficient yeast strain but not a transcriptional cyclin-deficient strain. We show that PfCyc1 forms a complex in the parasite with PfMRK and the P. falciparum MAT1 homolog. PfCyc1 is essential and nonredundant in blood-stage P. falciparum PfCyc1 knockdown causes a stage-specific arrest after nuclear division, demonstrating morphologically aberrant cytokinesis. This work demonstrates a conserved PfCyc1/PfMAT1/PfMRK complex in malaria and suggests that it functions as a schizont stage-specific regulator of the P. falciparum life cycle.

An APC/C-Cdh1 Biosensor Reveals the Dynamics of Cdh1 Inactivation at the G1/S Transition.

B-type cyclin-dependent kinase activity must be turned off for mitotic exit and G1 stabilization. B-type cyclin degradation is mediated by the anaphase-promoting complex/cyclosome (APC/C); during and after mitotic exit, APC/C is dependent on Cdh1. Cdh1 is in turn phosphorylated and inactivated by cyclin-CDK at the Start transition of the new cell cycle. We developed a biosensor to assess the cell cycle dynamics of APC/C-Cdh1. Nuclear exit of the G1 transcriptional repressor Whi5 is a known marker of Start; APC/C-Cdh1 is inactivated 12 min after Whi5 nuclear exit with little measurable cell-to-cell timing variability. Multiple phosphorylation sites on Cdh1 act in a redundant manner to repress its activity. Reducing the number of phosphorylation sites on Cdh1 can to some extent be tolerated for cell viability, but it increases variability in timing of APC/C-Cdh1 inactivation. Mutants with minimal subsets of phosphorylation sites required for viability exhibit striking stochasticity in multiple responses including budding, nuclear division, and APC/C-Cdh1 activity itself. Multiple cyclin-CDK complexes, as well as the stoichiometric inhibitor Acm1, contribute to APC/C-Cdh1 inactivation; this redundant control is likely to promote rapid and reliable APC/C-Cdh1 inactivation immediately following the Start transition.

An essential malaria protein defines the architecture of blood-stage and transmission-stage parasites.

Blood-stage replication of the human malaria parasite Plasmodium falciparum occurs via schizogony, wherein daughter parasites are formed by a specialized cytokinesis known as segmentation. Here we identify a parasite protein, which we name P. falciparum Merozoite Organizing Protein (PfMOP), as essential for cytokinesis of blood-stage parasites. We show that, following PfMOP knockdown, parasites undergo incomplete segmentation resulting in a residual agglomerate of partially divided cells. While organelles develop normally, the structural scaffold of daughter parasites, the inner membrane complex (IMC), fails to form in this agglomerate causing flawed segmentation. In PfMOP-deficient gametocytes, the IMC formation defect causes maturation arrest with aberrant morphology and death. Our results provide insight into the mechanisms of replication and maturation of malaria parasites.

Self-reported oral health needs and dental-care seeking behavior among women who use methamphetamine.

This study describes the prevalence of self-reported oral health needs and dental care-seeking behavior among women who use methamphetamine, using respondent-driven sampling in San Francisco, California, from 2007 to 2009 (N = 322). The sample had a high prevalence of self-reported dental needs; however, a low proportion of those reporting needs sought care. In bivariate analysis, the preferred route of methamphetamine use and frequency were not associated with self-reported dental needs. Over 90% of the sample used illicit substances in addition to methamphetamine, which may limit our ability to detect an association between methamphetamine use and oral health needs.

Health and oral health care needs and health care-seeking behavior among homeless injection drug users in San Francisco.

Few existing studies have examined health and oral health needs and treatment-seeking behavior among the homeless and injection drug users (IDUs). This paper describes the prevalence and correlates of health and oral health care needs and treatment-seeking behaviors in homeless IDUs recruited in San Francisco, California, from 2003 to 2005 (N = 340). We examined sociodemographic characteristics, drug use patterns, HIV status via oral fluid testing, physical health using the Short Form 12 Physical Component Score, self-reported needs for physical and oral health care, and the self-reported frequency of seeking medical and oral health care. The sample had a lower health status as compared to the general population and reported a frequent need for physical and oral health care. In bivariate analysis, being in methadone treatment was associated with care-seeking behavior. In addition, being enrolled in Medi-Cal, California's state Medicaid program, was associated with greater odds of seeking physical and oral health care. Methamphetamine use was not associated with higher odds of needing oral health care as compared to people who reported using other illicit drugs. Homeless IDUs in San Francisco have a large burden of unmet health and oral health needs. Recent cuts in Medi-Cal's adult dental coverage may result in a greater burden of oral health care which will need to be provided by emergency departments and neighborhood dental clinics.

Regulated degradation of the APC coactivator Cdc20.

BACKGROUND: Cdc20 is a highly conserved activator of the anaphase-promoting complex (APC), promoting cell-cycle-regulated ubiquitination and proteolysis of a number of critical cell-cycle-regulatory targets including securin and mitotic cyclins. APC-Cdc20 activity is tightly regulated, and this regulation is likely important for accurate cell cycle control. One significant component of Cdc20 regulation is thought to be Cdc20 proteolysis. However, published literature suggests different mechanisms and requirements for Cdc20 proteolysis. The degree to which Cdc20 proteolysis is cell-cycle regulated, the dependence of Cdc20 proteolysis on Cdc20 destruction boxes (recognition sequences for APC-mediated ubiqutination, either by Cdc20 or by the related Cdh1 APC activator), and the need for APC itself for Cdc20 proteolysis all have been disputed to varying extents. In animals, Cdc20 proteolysis is thought to be mediated by Cdh1, contributing an intrinsic order of APC activation by Cdc20 and then by Cdh1. One report suggests a Cdh1 requirement for Cdc20 proteolysis in budding yeast; this idea has not been tested further. RESULTS: We characterized Cdc20 proteolysis using Cdc20 expressed from its endogenous locus; previous studies generally employed strongly overexpressed Cdc20, which can cause significant artifacts. We analyzed Cdc20 proteolysis with or without mutations in previously identified destruction box sequences, using varying methods of cell cycle synchronization, and in the presence or absence of Cdh1. Cdc20 instability is only partially dependent on destruction boxes. A much stronger dependence on Cdh1 for Cdc20 proteolysis was observed, but Cdh1-independent proteolysis was also clearly observed. Cdc20 proteolysis independent of both destruction boxes and Cdh1 was especially detectable around the G1/S transition; Cdh1-dependent proteolysis was most notable in late mitosis and G1. CONCLUSIONS: Cdc20 proteolysis is under complex control, with different systems operating at different points in the cell cycle. This complexity is likely to explain apparent conflicts in previously published literature on this subject. A major mode of control of Cdc20 proteolysis occurs in late mitosis/early G1 and is Cdh1-dependent, as in animal cells; this mode may contribute to the known sequential activation of the APC by Cdc20 followed by Cdh1. An independent mode of Cdc20 proteolysis, independent of destruction boxes and Cdh1, occurs at G1/S; we do not know the mechanism or function of this mode of proteolysis, but speculate that it may contribute to sharpening and restricting activation of APC-Cdc20 to early mitosis.

Requirements and reasons for effective inhibition of the anaphase promoting complex activator CDH1.

Anaphase promoting complex (APC)-Cdh1 targets multiple mitotic proteins for degradation upon exit from mitosis into G1; inhibitory phosphorylation of Cdh1 by cyclin-dependent kinase (CDK) and Polo kinase has been proposed to prevent the premature degradation of substrates in the ensuing cell cycle. Here, we demonstrate essentiality of CDK phosphorylation of Cdh1 in Saccharomyces cerevisiae by exact endogenous gene replacement of CDH1 with CDK-unphosphorylatable CDH1-m11; in contrast, neither Cdh1 polo kinase sites nor polo interaction motifs are required. CDH1-m11 cells arrest in the first cycle with replicated DNA and sustained polarized growth; most cells have monopolar spindles. Blocking proteolysis of the Cin8 kinesin in CDH1-m11 cells does not promote spindle pole body (SPB) separation. In contrast, expression of undegradable mitotic cyclin results in both SPB separation and the restoration of isotropic growth. A minority of CDH1-m11 cells arrest with short bipolar spindles that fail to progress to anaphase; this can be accounted for by a failure to accumulate Cdc20 and consequent failure to cleave cohesin. Bipolar spindle assembly in CDH1-m11 cells is strikingly sensitive to gene dosage of the stoichiometric Cdh1 inhibitor ACM1. Thus, different spindle-regulatory pathways have distinct sensitivities to Cdh1, and ACM1 may buffer essential CDK phosphorylation of Cdh1.

Building community partnerships: case studies of Community Advisory Boards at research sites in Peru, Zimbabwe, and Thailand.

BACKGROUND: Differences in resources, knowledge, and infrastructure between countries initiating and countries hosting HIV prevention research trials frequently yield ethical dilemmas. Community Advisory Boards (CABs) have emerged as one strategy for establishing partnerships between researchers and host communities to promote community consultation in socially sensitive research. PURPOSE: To understand the evolution of CABs and community partnerships at international research sites conducting HIV prevention trials. METHODS: Three research sites of the HIV Prevention Trials Network (HPTN) were selected to include geographical representation and diverse populations at risk for HIV/AIDS - in Lima, Peru; Chitungwiza, Zimbabwe; and Chiang Mai, Thailand. Data collection included review of secondary data, including academic publications and site-specific progress reports; observations at the research sites; face-to-face interviews with CAB members, research staff, and other key informants; and focus groups with study participants. Rapid assessment techniques were used for data analysis. RESULTS: Two of the three CABs developed new strategies for community representation in response to new studies. All three CABs expanded their original function and became advocates for broader community interests beyond HIV prevention. The participation and input of community representatives, in response to critical incidents that occurred at the sites over the past five years, helped to solidify partnerships between researchers and communities. LIMITATIONS: Rapid Assessment is an exploratory methodology designed to provide an understanding of a situation based on the integration of multiple data sources, collected within a short period of time, without a formal examination of transcribed and coded data. Case studies, as a method, are meant to draw out what can be learned from a single case but are not, in the scientific sense, generalizable. CONCLUSIONS: In developing countries, CABs can be dynamic entities that enhance the HIV research process, assist in responding to issues involving research ethics, and prepare communities for HIV research.

A chromatin immunoprecipitation screen reveals protein kinase Cbeta as a direct RUNX1 target gene.

RUNX1 (also known as AML1) is a DNA-binding transcription factor that functions as a tumor suppressor and developmental determinant in hematopoietic cells. Target promoters have been identified primarily through the use of differential expression strategies and candidate gene approaches but not biochemical screens. Using a chromatin immunoprecipitation screen, we identified protein kinase Cbeta as a direct RUNX1 target gene and demonstrate that endogenous RUNX1 binds the chromatinized protein kinase Cbeta promoter of U937 cells. A phylogenetically conserved RUNX1-binding site within the PKCbeta promoter binds RUNX1 in electrophoretic mobility shift analyses and confers RUNX1 responsiveness on a heterologous promoter. Changes in RUNX1 activity affect endogenous protein kinase Cbeta expression, and a dominant-negative form of RUNX1 protects U937 cells from apoptotic stimuli previously shown to be dependent on protein kinase Cbeta. This protection can be reversed by the ectopic expression of protein kinase Cbeta. Together these findings demonstrate that protein kinase Cbeta is a direct, downstream target of RUNX1 and links RUNX1 to a myeloid apoptotic pathway.