Objectives: To investigate the effect of dexamethasone on triggering receptor expressed on myeloid cells-1 (TREM-1).
Methods: Wild-type and tumor necrosis factor (TNF−/−) mice were pre-treated with saline, dexamethasone, or hydrocortisone and exposed to a lethal infection of Pseudomonas aeruginosa. Mortality and TREM-1 on neutrophil membranes was measured after sacrifice. U937 human monocytic cells were stimulated with lipopolysaccharide (LPS) or heat-killed P. aeruginosa without or with dexamethasone or hydrocortisone, and cell-surface TREM-1 and soluble TREM-1 (sTREM-1) were quantified. Expression of TREM-1 and sTREM-1 was also studied in LPS-stimulated U937 cells incubated in the absence or presence of TNFα or anti-TNFα antibody.
Results: Pre-treatment with dexamethasone, but not hydrocortisone, prolonged animal survival. Mice pre-treated with dexamethasone showed decreased expression of TREM-1 on neutrophils. In U937 cells, LPS or heat-killed P. aeruginosa induced the expression of TREM-1 and the release of sTREM-1. U937 TREM-1 and sTREM-1 were decreased upon addition of dexamethasone but not hydrocortisone. The suppressive effect of dexamethasone was enhanced in the presence of exogenous TNFα and lost in the presence of anti-TNFα antibody. In TNF−/− mice, dexamethasone suppression of mortality and TREM-1 neutrophil expression was lost. Gene expression of TREM-1 in U937 monocytes was decreased after treatment with dexamethasone.
Conclusion: TREM-1/sTREM-1 is a novel site of action of dexamethasone. This action is associated with down-regulation of gene expression and is mediated by TNFα.
TREM-1; TNFα; monocytes; Pseudomonas aeruginosa; dexamethasone
We tested the hypothesis that dopamine-dependent motor learning mechanism underlies the long-duration response to levodopa in Parkinson disease (PD) based on our studies in a mouse model. By data-mining the motor task performance in dominant and nondominant hands of the subjects in a double-blind randomized trial of levodopa therapy, the effects of activity and dopamine therapy were examined.
We data-mined the Earlier versus Later Levodopa Therapy in Parkinson's Disease (ELLDOPA) study published in 2005 and performed statistical analysis comparing the effects of levodopa and dominance of handedness over 42 weeks.
The mean change in finger-tapping counts from baseline before the initiation of therapy to predose at 9 weeks and 40 weeks increased more in the dominant compared to nondominant hand in levodopa-treated subjects in a dose-dependent fashion. There was no significant difference in dominant vs nondominant hands in the placebo group. The short-duration response assessed by the difference of postdose performance compared to predose performance at the same visit did not show any significant difference between dominant vs nondominant hands.
Active use of the dominant hand and dopamine replacement therapy produces synergistic effect on long-lasting motor task performance during “off” medication state. Such effect was confined to dopamine-responsive symptoms and not seen in dopamine-resistant symptoms such as gait and balance. We propose that long-lasting motor learning facilitated by activity and dopamine is a form of disease modification that is often seen in trials of medications that have symptomatic effects.
Atherosclerotic cardiovascular disease is the leading cause of death in insulin-resistant (type 2) diabetes. Vascular endothelial dysfunction paves the way for atherosclerosis through impaired nitric oxide availability, inflammation, and generation of superoxide. Surprisingly, we show that ablation of the three genes encoding isoforms of transcription factor FoxO in endothelial cells prevents atherosclerosis in Low-density lipoprotein receptor knockout mice by reversing these sub-phenotypes. Paradoxically, the atheroprotective effect of FoxO deletion is associated with a marked decrease of insulin-dependent Akt phosphorylation in endothelial cells, owing to reduced FoxO-dependent expression of the insulin receptor adaptor proteins, Irs1 and Irs2. These findings support a model in which FoxO is the shared effector of multiple atherogenic pathways in endothelial cells. FoxO ablation lowers the threshold of Akt activity required for protection from atherosclerosis. The data demonstrate that FoxO inhibition in endothelial cells has the potential to mediate wide-ranging therapeutic benefits for diabetes-associated cardiovascular disease.
Neurotransmission involves the exo-endocytic cycling of synaptic vesicle (SV) membranes. Endocytic membrane retrieval and clathrin-mediated SV reformation require curvature-sensing and membrane-bending BAR domain proteins such as endophilin A. While their ability to sense and stabilize curved membranes facilitates membrane recruitment of BAR domain proteins, the precise mechanisms by which they are targeted to specific sites of SV recycling has remained unclear. Here, we demonstrate that the multi-domain scaffold intersectin 1 directly associates with endophilin A to facilitate vesicle uncoating at synapses. Knockout mice deficient in intersectin 1 accumulate clathrin-coated vesicles at synapses, a phenotype akin to loss of endophilin function. Intersectin 1/endophilin A1 complex formation is mediated by direct binding of the SH3B domain of intersectin to a non-canonical site on the SH3 domain of endophilin A1. Consistent with this, intersectin-binding defective mutant endophilin A1 fails to rescue clathrin accumulation at neuronal synapses derived from endophilin A1-3 triple knockout (TKO) mice. Our data support a model in which intersectin aids endophilin A recruitment to sites of clathrin-mediated SV recycling, thereby facilitating vesicle uncoating.
endophilin; intersectin; neurotransmission; SH3 domains; synaptic vesicle recycling
To perform construct validation of the population-based Dietary Inflammatory Index (DII) using dietary data from two different dietary assessments and serum high-sensitivity C-reactive protein (hs-CRP) as the construct validator.
Using data derived from (i) three 24 h dietary recalls (24HR) at baseline and at the end of each subsequent quarter (i.e. up to fifteen over a year) and (ii) a 7 d dietary recall (7DDR) measured at baseline and then quarterly, regression analyses were conducted to test the effect of the DII score on serum hs-CRP as dichotomous (≤3mg/l, >3mg/l), while controlling for important potential confounders.
Existing data from the Seasonal Variation of Blood Cholesterol Study (SEASONS), a longitudinal observational study of healthy participants recruited in Worcester, MA, USA and participants were followed for 1 year.
Participants who had at least one hs-CRP measurement over her/his 1-year participation (n 495 for 24HR, n 559 for 7DDR).
Higher DII scores were associated with values of hs-CRP >3 mg/l (OR = 1·08; 95% CI 1·01, 1·16, P = 0·035 for the 24HR; and OR = 1·10; 95% CI 1·02, 1·19, P = 0·015 for the 7DDR).
The population-based DII was associated with interval changes in hs-CRP using both the 24HR and 7DDR. The success of this first-of-a-kind attempt at relating individuals’ intakes of inflammation-modulating foods using this refined DII, and the finding that there is virtually no drop-off in predictive capability using a structured questionnaire in comparison to the 24HR standard, sets the stage for use of the DII in a wide variety of other epidemiological and clinical studies.
Diet; Inflammation; C-reactive protein; Adults; Predictive ability
Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis, and new therapies are needed. RG7787 is a novel low-immunogenic anti-mesothelin recombinant immunotoxin (RIT), engineered to overcome the limitations of SS1P, a RIT now in clinical trials. In vitro activity was evaluated on five established PDAC cell lines (KLM-1, AsPC-1, BxPC-3, Panc 3.014 and PK-1) and on PDAC cells directly established from a patient tumor (GUMC108). RG7787 had subnanomolar IC50s in most cell lines, and was significantly more active than SS1P in GUMC108, KLM-1 and Panc 3.014 cells. GUMC108 was most sensitive, with RG7787 killing >99% of the cells. In a subcutaneous KLM-1 xenograft mouse model, two cycles of 3 × 2.5 mg/kg RG7787 QOD combined with two cycles of 1 × 50 mg/kg paclitaxel induced near-complete responses, with all tumors regressing below 5 mm3 within 30 days after therapy was initiated (>95% decrease) and no significant growth increase for at least another 3 weeks. RG7787 alone gave limited but significant regressions and paclitaxel by itself arrested tumor growth. Quantifying the uptake of Alexa647-labeled RG7787 in tumors showed that the RIT reached only 45% of KLM-1 cells, accounting in part for the limited responses. Paclitaxel did not improve RG7787 uptake, which thus cannot explain the beneficial effect of the combination therapy. In conclusion, RG7787 has high cytotoxic activity on PDAC cell lines as well as on primary patient cells. In vivo, this novel RIT gives durable near-complete tumor responses when combined with paclitaxel. RG7787 merits further evaluation for the treatment of PDAC.
immunotoxin; KLM-1; mesothelin; pancreatic cancer
SLC4 transporters are membrane proteins that in general mediate the coupled transport of bicarbonate (carbonate) and share amino acid sequence homology. These proteins differ as to whether they also transport Na+ and/or Cl−, in addition to their charge transport stoichiometry, membrane targeting, substrate affinities, developmental expression, regulatory motifs, and protein-protein interactions. These differences account in part for the fact that functionally, SLC4 transporters have various physiological roles in mammals including transepithelial bicarbonate transport, intracellular pH regulation, transport of Na+ and/or Cl−, and possibly water. Bicarbonate transport is not unique to the SLC4 family since the structurally unrelated SLC26 family has at least three proteins that mediate Cl−-HCO3− exchange. The present review focuses on the first of the sodium-dependent SLC4 transporters that was identified whose structure has been most extensively studied: the electrogenic Na+-base cotransporter NBCe1. Mutations in NBCe1 cause proximal renal tubular acidosis (pRTA) with neurologic and ophthalmologic extrarenal manifestations. Recent studies have characterized important structure-function properties of the transporter and how they are perturbed as a result of mutations that cause pRTA. It has become increasingly apparent that the structure of NBCe1 differs in several key features from the SLC4 Cl−-HCO3− exchanger AE1 whose structural properties have been well-studied. In this review, the structure-function properties and regulation of NBCe1 will be highlighted and its role in health and disease will be reviewed in detail.
SLC4A4; carbonate; bicarbonate; renal tubular acidosis; transport; acid-base; pH
Background: Although acute treatments have been shown to be effective in treating early-onset depression, only one-third or thereabouts reach a remission within 3 months. Unfortunately, delayed time to remission in early-onset depression leads to poorer therapeutic outcomes. Clearly, there is a need to identify, diagnose, and provide effective treatment of a depressed patient quickly. A sophisticated understanding of depression subscales and their change over time with treatment could enhance pathways to individualized treatment approaches for childhood depression.
Objective: Previous studies have found that the clinician-measured instrument, Children's Depression Rating Scale-Revised (CDRS-R) measures multiple subscales (or components) of depression. The aim of this study was to see how these subscales may change over the course of a 12-week study. This knowledge will help determine if dimensions/subscales of childhood depression (paralleling the adult literature) using the subscales derived from factor analysis procedure is useful.
Methods: We examined two clinical trials in which youth (n=234) with major depressive disorder (MDD) were treated openly with fluoxetine for eight sessions spread over 12 weeks. The CDRS-R was completed based on clinician interviews with parent and child at each session. Classical test theory and component analysis with associated parallel analysis (oblique rotation) were conducted on each week's scores.
Results: Although more factors were needed for the baseline and first two therapy sessions, a two-factor solution sufficed thereafter. Depressed facial affect, listless speech, and hypoactivity best defined Factor I, whereas sleep problems, appetite disturbance, physical symptoms, irritability, guilt, and weeping best defined Factor II. All other symptoms cross-loaded almost equally on the two factors. The scale's reliability (internal consistency) improved from baseline to exit sessions (α=0.65–0.91). As a result, the clinicians' assessments of the various symptoms became more highly related to one another. This caused the first eigenvalue to increase from 3.24 to 7.38 and the variance explained to increase (%) from 19% to 43% over sessions. These two factors may reflect 1) clinician-observed signs and 2) reported symptoms of depression.
Conclusions: Factor analysis of CDRS-R data in a single session consistently generates a complex and difficult to interpret structure of at least three factors. This makes it very difficult to understand what these factors measure. However, when gathered over additional sessions, the CDRS-R structure tends to simplify to two factors. The reasons for this simplification are as yet unclear and in need of further study.
Borrelia burgdorferi sensu lato is a group of spirochetes belonging to the genus Borrelia in the family of Spirochaetaceae. The spirochete is transmitted between reservoirs and hosts by ticks of the family Ixodidae. Infection with B. burgdorferi in humans causes Lyme disease or Lyme borreliosis. Currently, 20 Lyme disease-associated Borrelia species and more than 20 relapsing fever-associated Borrelia species have been described. Identification and differentiation of different Borrelia species and strains is largely dependent on analyses of their genetic characteristics. A variety of molecular techniques have been described for Borrelia isolate speciation, molecular epidemiology, and pathogenicity studies. In this unit, we focus on three basic protocols, PCR-RFLP-based typing of the rrs-rrlA and rrfA-rrlB ribosomal spacer, ospC typing, and MLST. These protocols can be employed alone or in combination for characterization of B. burgdorferi isolates or directly on uncultivated organisms in ticks, mammalian host reservoirs, and human clinical specimens.
spirochetes; molecular typing; OspC; MLST; Borrelia burgdorferi; Lyme disease
Immunotoxins are a novel class of antibody-based therapeutics currently in clinical development. This review of the field will help physicians better inform patients about the potential benefits and toxicities of these experimental treatments.
Immunotoxins are a novel class of antibody-conjugated therapeutics currently in clinical development for a variety of malignancies. They consist of an antibody-based targeting domain fused to a bacterial toxin payload for cell killing. Immunotoxins kill cells by inhibiting protein synthesis, a unique mechanism of action that is toxic to both dividing and nondividing cells. Recent advances in the design and administration of immunotoxins are overcoming historical challenges in the field, leading to renewed interest in these therapeutics.
Antibody conjugate; Recombinant immunotoxin; Vascular leak syndrome; Antidrug antibody
Dysphagia is common in Parkinson’s disease (PD) and causes significant morbidity and mortality. PD dysphagia has usually been explained as dysfunction of central motor control, much like other motor symptoms that are characteristic of the disease. However, PD dysphagia does not correlate with severity of motor symptoms nor does it respond to motor therapies. It is known that PD patients have sensory deficits in the pharynx, and that impaired sensation may contribute to dysphagia. However, the underlying cause of the pharyngeal sensory deficits in PD is not known. We hypothesized that PD dysphagia with sensory deficits may be due to degeneration of the sensory nerve terminals in the upper aerodigestive tract (UAT). We have previously shown that Lewy-type synucleinopathy (LTS) is present in the main pharyngeal sensory nerves of PD patients, but not in controls. In this study, the sensory terminals in UAT mucosa were studied to discern the presence and distribution of LTS. Whole-mount specimens (tongue-pharynx-larynx-upper esophagus) were obtained from 10 deceased human subjects with clinically diagnosed and neuropathologically confirmed PD (five with dysphagia and five without) and four age-matched healthy controls. Samples were taken from six sites and immunostained for phosphorylated α-synuclein (PAS). The results showed the presence of PAS-immunoreactive (PAS-ir) axons in all the PD subjects and in none of the controls. Notably, PD patients with dysphagia had more PAS-ir axons in the regions that are critical for initiating the swallowing reflex. These findings suggest that Lewy pathology affects mucosal sensory axons in specific regions of the UAT and may be related to PD dysphagia.
Alpha-synuclein histopathology; Dysphagia; Lewy-type synucleinopathy; Parkinson’s disease; Peripheral sensory nerves; Upper aerodigestive tract
Non-adherence to safer sex and non-adherence to ART can each have adverse health consequences for HIV-infected individuals and their sex partners, but little is known about the association of these behaviors with each other. This “dual risk” has potential negative public health consequences since non-adherence can lead to the development of resistant virus that can then be transmitted to sex partners.
Among participants in the Multi-site Adherence Collaboration in HIV (MACH14) we examined, at study baseline, the association between the frequency of unprotected sex (assessed by self-report) and ART adherence (assessed by Medication Event Monitoring System, Aardex) among the sexually active participants in the five studies (N=459) that collected sexual risk behavior. The bivariate association between sexual risk behaviors and ART adherence was assessed by Pearson correlations; subsequently ANOVAs were used to evaluate the role of demographic characteristics, depression and substance use in explaining the “dual risk” outcome (sexual risk and non-adherence).
Among participants who had been sexually active, more unprotected anal/vaginal sex was weakly associated with poorer ART adherence (r = −.12, p=0.01 for the overall sample). Further analysis showed this association was driven by the heterosexual men in the sample (r = −.29, p<0.001), and was significant only for this group, and not for gay/bisexual men or for women (heterosexual and homosexual). Neither substance use nor depression accounted for the association between sexual risk and ART adherence.
HIV-infected heterosexual men who are having difficulty adhering to ART are also more likely to engage in risky sexual behaviors and therefore may benefit from counseling about these risk behaviors. We must identify procedures to screen for these risk behaviors and develop interventions, appropriately tailored to specific populations and identified risk factors, that can be integrated into routine clinical care for people living with HIV. This will become increasingly important in the context of wider access to treatment globally, including new recommendations for ART initiation earlier in a patients’ disease course (e.g., “Test and Treat” paradigms).
ART adherence; sexual risk behavior; test-and-treat; depression; substance use
The ability of high density lipoprotein (HDL) particles to accept cholesterol from peripheral cells such as lipid-laden macrophages and to transport cholesterol to the liver for catabolism and excretion in a process termed reverse cholesterol transport (RCT) is believed to underlie the beneficial cardiovascular effects of elevated HDL. The liver X receptors (LXRα and LXRβ) regulate RCT by controlling the efflux of cholesterol from macrophages to HDL and the excretion, catabolism and absorption of cholesterol in the liver and intestine. Importantly, treatment with LXR agonists increases RCT and decreases atherosclerosis in animal models. Nevertheless, LXRs are expressed in multiple tissues involved in RCT and their tissue specific contributions to RCT are still not well defined.
Approach and Results
Utilizing tissue-specific LXR deletions together with in vitro and in vivo assays of cholesterol efflux and fecal cholesterol excretion we demonstrate that macrophage LXR activity is neither necessary nor sufficient for LXR agonist-stimulated RCT. In contrast, the ability of LXR agonists primarily acting in the intestine to increase HDL mass and HDL function appears to underlie the ability of LXR agonists to stimulate RCT in vivo.
We demonstrate that activation of LXR in macrophages makes little or no contribution to LXR agonist-stimulated RCT. Unexpectedly our studies suggest that the ability of macrophages to efflux cholesterol to HDL in vivo is not regulated by macrophage activity but is primarily determined by the quantity and functional activity of HDL.
Reverse cholesterol transport; Liver X receptors; Macrophage; HDL; Atherosclerosis
To explore the early childhood pulmonary outcomes of infants who participated in the NICHD SUPPORT Trial, using a factorial design that randomized extremely preterm infants to lower vs. higher oxygen saturation targets and delivery room CPAP vs. intubation/surfactant, found no significant difference in the primary composite outcome of death or BPD.
The Breathing Outcomes Study, a prospective secondary to SUPPORT, assessed respiratory morbidity at 6 month intervals from hospital discharge to 18–22 months corrected age (CA). Two pre-specified primary outcomes, wheezing more than twice per week during the worst 2 week period and cough longer than 3 days without a cold were compared between each randomized intervention.
One or more interviews were completed for 918 of 922 eligible infants. The incidence of wheezing and cough were 47.9% and 31.0%, respectively, and did not differ between study arms of either randomized intervention. Infants randomized to lower vs. higher oxygen saturation targets had similar risks of death or respiratory morbidities (except for croup, treatment with oxygen or diuretics at home). Infants randomized to CPAP vs. intubation/surfactant had fewer episodes of wheezing without a cold (28.9% vs. 36.5%, p<0.05), respiratory illnesses diagnosed by a doctor (47.7% vs. 55.2%, p<0.05) and physician or emergency room visits for breathing problems (68.0% vs. 72.9%, p<0.05) by 18–22 months CA.
Treatment with early CPAP rather than intubation/surfactant is associated with less respiratory morbidity by 18–22 months CA. Longitudinal assessment of pulmonary morbidity is necessary to fully evaluate the potential benefits of respiratory interventions for neonates.
Bronchopulmonary Dysplasia; Infant, Newborn; Infant, Low Birth Weight; Infant, Extremely Low Birth Weight; Infant, Premature; Infant, Extremely Low Gestational Age; Infant mortality; Respiratory morbidity; Intensive care, neonatal; Hospital Readmission; Oximetry; Randomized controlled trial; Retinopathy of prematurity (ROP); Continuous Positive Airway Pressure; Intubation, endotracheal; Pulmonary surfactants/therapeutic use; Oxygen inhalation therapy/methods; Oxygen administration & dosage; Follow-up studies
Thyroid hormones modulate every component of the cardiovascular system necessary for normal cardiovascular development and function. When cardiovascular disease is present, thyroid function tests are characteristically indicated to determine if overt thyroid disorders or even subclinical dysfunction exists. As hypothyroidism, hypertension and cardiovascular disease all increase with advancing age monitoring of TSH, the most sensitive test for hypothyroidism, is important in this expanding segment of our population. A better understanding of the impact of thyroid hormonal status on cardiovascular physiology will enable health care providers to make decisions regarding thyroid hormone evaluation and therapy in concert with evaluating and treating hypertension and cardiovascular disease. The goal of this review is to access contemporary understanding of the effects of thyroid hormones on normal cardiovascular function and the potential role of overt and subclinical hypothyroidism and hyperthyroidism in a variety of cardiovascular diseases.
Thyroid dysfunction; cardiac output; heart failure; peripheral vascular function; atrial fibrillation; coronary artery disease
India has large PWID (persons who inject drugs) population estimated at 177,000. PWIDs are at high risk for HIV, Hepatitis B (HBV) and Hepatitis C (HCV) infections. We report the prevalence of HIV, HBV and HCV infections and correlates of HIV-HCV co-infection among male PWIDs in Delhi.
3748 male PWIDs were recruited for a longitudinal HIV incidence study. Participants were tested for HBV and HCV infections at their first follow-up visit (FV1) using serum HBV-surface antigen, and HCV-antibody tests followed by HCV RNA PCR, respectively. All PWIDs who were HIV-negative at enrollment, were re-tested for HIV at FV1. Multinomial logistic regression was employed to identify predictors of HIV, HCV and HIV-HCV co-infection.
Overall prevalence of HIV, HBV and HCV among 2,292 participants tested at FV1 was 25.9 %, 9.7 % and 53.7 %, respectively. 6.4 % of the participants had HIV mono-infection, 34.1 % had HCV mono-infection, and 19.6 % had HIV-HCV co-infection. 26 % of HIV-positive participants without HCV were HBsAg positive.
In the regression model, having practiced at least one risky injection in the past month (relative risk ratio (RRR): 1.38; 95 % CI: 1.01-1.89) and not knowing his own HIV status (RRR: 1.65, 95 % CI: 1.25-2.17) were independent predictors for HIV-HCV co-infection. Longer duration of drug injections was associated with a higher likelihood of HCV mono-infection (2–5 years RRR: 2.13; 6–10 years RRR: 2.74; ≥11 years RRR: 3.14) and HIV-HCV co-infection (2–5 years RRR: 5.14; 6–10 years RRR: 8.53; >11 years RRR: 8.03). Higher frequency of injection days/month was associated with a higher likelihood of HCV mono-infection (≤10 days/month RRR: 1.61; 11–20 days/month RRR: 3.15; 21–30 days/month RRR: 3.47) and HIV-HCV co-infections (≤10 days/month RRR: 2.26; 11–20 days/month RRR: 3.46; 21–30 days/month RRR: 4.83).
We report a high prevalence of HIV, HCV and HIV-HCV co-infection among male PWIDs in Delhi. A tenth of the participants were HBsAg positive. Targeted Intervention programs should make HBV/HCV testing, prevention and care more accessible for PWIDs.
HIV; Hepatitis B; Hepatitis C; HIV-HCV co-infection; People Who Inject Drugs (PWID); India
Despite the tremendous hurdles presented by the complexity of the liver’s structure and function, advances in liver physiology, stem cell biology and reprogramming, and the engineering of tissues and devices are accelerating the development of cell-based therapies for treating liver disease and liver failure. This State of the Art Review discusses both the near and long-term prospects for such cell-based therapies and the unique challenges for clinical translation.
The search for a homologous template is a fundamental, yet largely uncharacterized, reaction in DNA double-strand break repair. Two reports now demonstrate that broken chromosomes increase their movement and explore large volumes of nuclear space searching for a homologous template. Break mobility requires resection and recombination enzymes, as well as damage-checkpoint components.
DNA double-strand breaks (DSB) can arise during DNA replication, or after exposure to DNA-damaging agents, and their correct repair is fundamental for cell survival and genomic stability. Here, we show that the Smc5–Smc6 complex is recruited to DSBs de novo to support their repair by homologous recombination between sister chromatids. In addition, we demonstrate that Smc5–Smc6 is necessary to suppress gross chromosomal rearrangements. Our findings show that the Smc5–Smc6 complex is essential for genome stability as it promotes repair of DSBs by error-free sister-chromatid recombination (SCR), thereby suppressing inappropriate non-sister recombination events.
A single double-strand break (DSB) induced by HO endonuclease triggers both repair by homologous recombination and activation of the Mec1-dependent DNA damage checkpoint in budding yeast1–6. Here we report that DNA damage checkpoint activation by a DSB requires the cyclin-dependent kinase CDK1 (Cdc28) in budding yeast. CDK1 is also required for DSB-induced homologous recombination at any cell cycle stage. Inhibition of homologous recombination by using an analogue-sensitive CDK1 protein7,8 results in a compensatory increase in non-homologous end joining. CDK1 is required for efficient 5′ to 3′ resection of DSB ends and for the recruitment of both the single-stranded DNA-binding complex, RPA, and the Rad51 recombination protein. In contrast, Mre11 protein, part of the MRX complex, accumulates at unresected DSB ends. CDK1 is not required when the DNA damage checkpoint is initiated by lesions that are processed by nucleotide excision repair. Maintenance of the DSB-induced checkpoint requires continuing CDK1 activity that ensures continuing end resection. CDK1 is also important for a later step in homologous recombination, after strand invasion and before the initiation of new DNA synthesis.
Very few gene conversions in mitotic cells are associated with crossovers, suggesting that these events are regulated. This may be important for the maintenance of genetic stability. We have analyzed the relationship between homologous recombination and crossing-over in haploid budding yeast and identified factors involved in the regulation of crossover outcomes. Gene conversions unaccompanied by a crossover appear 30 min before conversions accompanied by exchange, indicating that there are two different repair mechanisms in mitotic cells. Crossovers are rare (5%), but deleting the BLM/WRN homolog, SGS1, or the SRS2 helicase increases crossovers 2- to 3-fold. Overexpressing SRS2 nearly eliminates crossovers, whereas overexpression of RAD51 in srs2Δ cells almost completely eliminates the noncrossover recombination pathway. We suggest Sgs1 and its associated topoisomerase Top3 remove double Holliday junction intermediates from a crossover-producing repair pathway, thereby reducing crossovers. Srs2 promotes the noncrossover synthesis-dependent strand-annealing (SDSA) pathway, apparently by regulating Rad51 binding during strand exchange.
In response to DNA double-strand breaks (DSBs), eukaryotic cells rapidly phosphorylate histone H2A isoform H2AX at a C-terminal serine (to form γ-H2AX) and accumulate repair proteins at or near DSBs. To date, these events have been defined primarily at the resolution of light microscopes, and the relationship between γ-H2AX formation and repair protein recruitment remains to be defined.
We report here the first molecular-level characterization of regional chromatin changes that accompany a DSB formed by the HO endonuclease in Saccharomyces cerevisiae. Break induction provoked rapid γ-H2AX formation and equally rapid recruitment of the Mre11 repair protein. γ-H2AX formation was efficiently promoted by both Tel1p and Mec1p, the yeast ATM and ATR homologs; in G1-arrested cells, most γ-H2AX formation was dependent on Tel1 and Mre11. γ-H2AX formed in a large (ca. 50 kb) region surrounding the DSB. Remarkably, very little γ-H2AX could be detected in chromatin within 1–2 kb of the break. In contrast, this region contains almost all the Mre11p and other repair proteins that bind as a result of the break.
Both Mec1p and Tel1p can respond to a DSB, with distinct roles for these checkpoint kinases at different phases of the cell cycle. Part of this response involves histone phosphorylation over large chromosomal domains; however, the distinct distributions of γ-H2AX and repair proteins near DSBs indicate that localization of repair proteins to breaks is not likely to be the main function of this histone modification.
Antiretroviral treatment for HIV-infection before immunologic decline (early ART) and pre-exposure chemoprophylaxis (PrEP) can prevent HIV transmission, but routine adoption of these practices by clinicians has been limited.
Between September and December 2013, healthcare practitioners affiliated with a regional AIDS Education and Training Center in New England were invited to complete online surveys assessing knowledge, beliefs and practices regarding early ART and PrEP. Multivariable models were utilized to determine characteristics associated with prescribing intentions and practices.
Surveys were completed by 184 practitioners. Respondent median age was 44 years, 58% were female, and 82% were white. Among ART-prescribing clinicians (61% of the entire sample), 64% were aware that HIV treatment guidelines from the Department of Health and Human Services recommended early ART, and 69% indicated they would prescribe ART to all HIV-infected patients irrespective of immunologic status. However, 77% of ART-prescribing clinicians would defer ART for patients not ready to initiate treatment. Three-fourths of all respondents were aware of guidance from the U.S. Centers for Disease Control and Prevention recommending PrEP provision, 19% had prescribed PrEP, and 58% of clinicians who had not prescribed PrEP anticipated future prescribing. Practitioners expressed theoretical concerns and perceived practical barriers to prescribing early ART and PrEP. Clinicians with higher percentages of HIV-infected patients (aOR 1.16 per 10% increase in proportion of patients with HIV-infection, 95% CI 1.01–1.34) and infectious diseases specialists (versus primary care physicians; aOR 3.32, 95% CI 0.98–11.2) were more likely to report intentions to prescribe early ART. Higher percentage of HIV-infected patients was also associated with having prescribed PrEP (aOR 1.19, 95% CI 1.06–1.34), whereas female gender (aOR 0.26, 95% CI 0.10–0.71) was associated with having not prescribed PrEP.
These findings suggest many clinicians have shifted towards routinely recommending early ART, but not PrEP, so interventions to facilitate PrEP provision are needed.
Health care disparities; HIV; physician-patient communication; medication adherence