BACKGROUND AND OBJECTIVES:
The 2010 Affordable Care Act mandates that health insurance companies make those up to age 26 eligible for their parents’ policies. Thirty-four states previously enacted similar laws. The authors sought to examine the impact on access to care of state laws extending eligibility of parents’ insurance to young adults.
By using a difference-in-differences analysis, we examined the 2002–2004 and 2008–2009 Behavior Risk Factor Surveillance System to compare 3 states enacting laws in 2005 or 2006 with 17 states that have not enacted laws on 4 outcomes: self-reported health insurance coverage, identification of a personal physician/clinician, physical exam from a physician within the past 2 years, and forgoing care in the past year due to cost.
For each outcome there was differential improvement among states enacting laws compared with states without laws. Health insurance differentially increased 0.2% (95% confidence interval [CI], −3.8% to 4.2%), from 67.6% to 68.1% pre-post in states enacting laws and from 68.5% to 68.7% in states without. Personal physician/clinician identification differentially increased 0.9% (95% CI −3.1% to 5.0%), from 62.4% to 65.5% in states enacting laws and from 58.0% to 60.2% in states without. Recent physical exams differentially increased significantly 4.6% (95% CI, 0%–9.2%), from 77.3% to 81.2% in states enacting laws and from 76.2% to 75.5% in states without. Forgone care due to cost differentially decreased significantly 3.9% (95% CI, −0.3% to −7.5%), from 20.4% to 18.2% in states enacting laws and from 17.8% to 19.4% in states without.
States that expanded eligibility to parents’ insurance in 2005 or 2006 experienced improvements in access to care among young adults.
parental insurance; state laws; Affordable Care Act
We have used sinusoidal and band limited Gaussian noise stimuli along with information measures to characterize the linear and non-linear responses of morpho-physiologically identified posterior canal (PC) afferents and to examine the relationship between mutual information rate and other physiological parameters Our major findings are: 1) spike generation in most PC afferents is effectively a stochastic renewal process, and spontaneous discharges are fully characterized by their first order statistics; 2) a regular discharge, as measured by normalized coefficient of variation (cv*), reduces intrinsic noise in afferent discharges at frequencies below the mean firing rate; 3) coherence and mutual information rates, calculated from responses to band limited Gaussian noise, are jointly determined by gain and intrinsic noise (discharge regularity), the two major determinants of signal to noise ratio in the afferent response; 4) measures of optimal non-linear encoding were only moderately greater than optimal linear encoding, indicating that linear stimulus encoding is limited primarily by internal noise rather than by non-linearities; 5) a leaky integrate and fire model reproduces these results and supports the suggestion that the combination of high discharge regularity and high discharge rates serves to extend the linear encoding range of afferents to higher frequencies. These results provide a framework for future assessments of afferent encoding of signals generated during natural head movements and for comparison with coding strategies used by other sensory systems.
Turtle; Vestibular; Information; Afferent; Model; Dynamics
Monoclonal antibodies are successful biologics in treating a variety of diseases, including the prevention or treatment of viral infections. CL184 is a 1:1 combination of two human monoclonal IgG1 antibodies (CR57 and CR4098) against rabies virus, produced in the PER.C6 human cell line. The two antibodies are developed as replacements of human rabies immune globulin (HRIG) and equine rabies immune globulin (ERIG) in postexposure prophylaxis (PEP). The rapid fluorescent focus inhibition test (RFFIT) is a cell-based virus neutralization assay which is usually performed to determine the biological potency of a vaccine and to measure the levels of protection against rabies in humans and animals. In order to confirm the suitability of this assay as a pharmacodynamic assay, we conducted a validation using both HRIG- and CL184-spiked serum samples and sera from vaccinated donors. The validation results met all analytical acceptance criteria and showed that HRIG and CL184 serum concentrations can be compared. Stability experiments showed that serum samples were stable in various suboptimal conditions but that rabies virus should be handled swiftly once thawed. We concluded that the assay is suitable for the measurement of polyclonal and monoclonal rabies neutralizing antibodies in clinical serum samples.
The transient leaf assay in Nicotiana benthamiana is widely used in plant sciences, with one application being the rapid assembly of complex multigene pathways that produce new fatty acid profiles. This rapid and facile assay would be further improved if it were possible to simultaneously overexpress transgenes while accurately silencing endogenes. Here, we report a draft genome resource for N. benthamiana spanning over 75% of the 3.1 Gb haploid genome. This resource revealed a two-member NbFAD2 family, NbFAD2.1 and NbFAD2.2, and quantitative RT-PCR (qRT-PCR) confirmed their expression in leaves. FAD2 activities were silenced using hairpin RNAi as monitored by qRT-PCR and biochemical assays. Silencing of endogenous FAD2 activities was combined with overexpression of transgenes via the use of the alternative viral silencing-suppressor protein, V2, from Tomato yellow leaf curl virus. We show that V2 permits maximal overexpression of transgenes but, crucially, also allows hairpin RNAi to operate unimpeded. To illustrate the efficacy of the V2-based leaf assay system, endogenous lipids were shunted from the desaturation of 18∶1 to elongation reactions beginning with 18∶1 as substrate. These V2-based leaf assays produced ∼50% more elongated fatty acid products than p19-based assays. Analyses of small RNA populations generated from hairpin RNAi against NbFAD2 confirm that the siRNA population is dominated by 21 and 22 nt species derived from the hairpin. Collectively, these new tools expand the range of uses and possibilities for metabolic engineering in transient leaf assays.
Epithelioid trophoblastic tumor (ETT) is a rare gestational trophoblastic tumor. Cases of ETT present as abnormal vaginal bleeding in women of reproductive age, with low human chorionic gonadotropin (hCG) levels. ETT can be a sequela of any gestational event and can present in both intrauterine and extrauterine sites. Metastasis and death have been reported. We present a case of a 44-year-old female incidentally diagnosed with ETT following laparoscopic-assisted vaginal hysterectomy. Postoperative evaluation for metastatic disease was negative. The patient has been closely followed and remains disease free 8 months postoperatively. ETT presents a diagnostic challenge due to its rarity and histologic resemblance to other pathologies. ETT is relatively chemoresistant and managed surgically. Misdiagnosis delays effective treatment and affects survival.
The present article reports on the successful management of a large flail chest with traumatic pulmonary herniation in a patient who could not be weaned from mechanical ventilation following a course of conservative management. Surgical intervention involved open reduction and internal fixation with tubular plates to stabilize the flail segment, followed by a pectoralis major myocutaneous flap to repair the chest wall defect. Following surgical intervention, the patient was able to be weaned from mechanical ventilation and showed remarkable improvement in pulmonary function parameters. To the authors’ knowledge, the present report is the first to describe the use of open reduction and internal fixation of the chest wall and flap reconstruction to treat lung herniation with a flail chest segment.
Flail chest; Pectoralis major myocutaneous flap; Pulmonary herniation
Two new 2,2′-bipyridine (bpy) based ligands with ancillary BODIPY chromophores attached at the 4 and 4′-positions were prepared and characterized, which vary in the substitution pattern about the BODIPY periphery by either excluding (BB1) or including (BB2) a β-alkyl substituent. Both absorb strongly throughout the visible region and are strongly emissive. The basic photophysics and electrochemical properties of BB1 and BB2 are comparable to those of the BODIPY monomers on which they are based. The solid-state structures and electronic structure calculations both indicate that there is negligible electronic communication between the BODIPY moieties and the intervening bpy spacers. Electrogenerated chemiluminescence spectra of the two Bpy-BODIPY derivatives are similar to their recorded fluorescence profiles and are strongly influenced by substituents on the BODIPY chromophores. These 2,2′-bipyridine derivatives represent a new set of ligands that should find utility in applications including light-harvesting, photocatalysis, and molecular electronics.
BODIPY; bipyridine; electrochemistry; photophysics; electrogenerated chemiluminescence
Simultaneous electroencephalograph-functional magnetic resonance imaging (EEG-fMRI ) recording has become an important tool for investigating spatiotemporal properties of brain events, such as epilepsy, evoked brain responses, and changes in brain rhythms. Reduction of noise in EEG signals during fMRI recording is crucial for acquiring high-quality EEG-fMRI data. The main source of the noise includes the gradient artifact, the radio frequency (RF) pulse artifact, and the cardiac pulse artifact. Since the RF pulse artifact is relatively small in amplitude, little attention has been paid to this artifact, and its origin is not well understood. However, the amplitude of the RF pulse artifact fluctuates randomly even if a very high EEG sampling rate is used, making it more salient than the gradient artifact after postprocessing for noise removal. In this paper, we investigate the cause of the RF pulse artifact in EEG systems that use carbon wires.
Carbon; electroencephalography; electromagnetic radiative interference; magnetic resonance imaging (MRI)
We sought to determine the incidence of necrotizing enterocolitis (NEC) and spontaneous intestinal perforation (SIP) in surviving extremely low-birth-weight (ELBW, <1000 g birth weight) infants and to establish the impact of NEC on outcomes by hospital discharge and at 18 to 22 months adjusted age in a large, contemporary, population-based practice.
Hospital outcome data for all ELBW infants born in the greater Cincinnati region from 1998 to 2009 were extracted from the National Institute of Child Health Neonatal Research Network Database. Neurodevelopmental outcome at 18 to 22 months was assessed using Bayley Scales of Infant Development-II scores for Mental Developmental Index and Psychomotor Developmental Index. Multivariable logistic regression was used and adjusted odds ratios reported to control for confounders.
From 1998 to 2009, ELBW infants accounted for 0.5% of the 352 176 live-born infants in greater Cincinnati. The incidence of NEC was 12%, with a 50% case-fatality rate. Death before discharge, morbid complications of prematurity and neurodevelopmental impairment were all increased among infants diagnosed with NEC. Infants with surgical NEC and SIP had a higher incidence of death, but long-term neurodevelopmental outcomes were not different comparing surviving ELBW infants with medical NEC, surgical NEC and SIP.
Although ELBW infants comprise a very small proportion of live-born infants, those who develop NEC and SIP are at an increased risk for death, morbid complications of prematurity and neurodevelopmental impairment. No significant differences in neurodevelopmental outcomes were observed between the medical and surgical NEC and SIP groups.
necrotizing enterocolitis; extremely low-birth-weight; neurodevelopmental outcome; Bayley scales of infant development
Cochleates are phospholipid-calcium precipitates derived from the interaction of anionic lipid vesicles with divalent cations. Proteoliposomes from bacteria may also be used as a source of negatively charged components, to induce calcium-cochleate formation. In this study, proteoliposomes from V. cholerae O1 (PLc) (sized 160.7±1.6 nm) were transformed into larger (16.3±4.6 µm) cochleate-like structures (named Adjuvant Finlay Cochleate 2, AFCo2) and evaluated by electron microscopy (EM). Measurements from transmission EM (TEM) showed the structures had a similar size to that previously reported using light microscopy, while observations from scanning electron microscopy (SEM) indicated that the structures were multilayered and of cochleate-like formation. The edges of the AFCo2 structures appeared to have spaces that allowed penetration of negative stain or Ovalbumin labeled with Texas Red (OVA-TR) observed by epi-fluorescence microscopy. In addition, freeze fracture electron microscopy confirmed that the AFCo2 structures consisted of multiple overlapping layers, which corresponds to previous descriptions of cochleates. TEM also showed that small vesicles co-existed with the larger cochleate structures, and in vitro treatment with a calcium chelator caused the AFCo2 to unfold and reassemble into small proteoliposome-like structures. Using OVA as a model antigen, we demonstrated the potential loading capacity of a heterologous antigen and in vivo studies showed that with simple admixing and administration via intragastric and intranasal routes AFCo2 provided enhanced adjuvant properties compared with PLc.
Malignant transformation of a mature cystic teratoma (MCT) is an infrequent, often asymptomatic event. We report the first example of a struma ovarii with a focus of follicular variant of papillary thyroid carcinoma (a), mucinous adenocarcinoma (b), and strumal carcinoid tumor (c)—all three arising in one mature cystic teratoma of the ovary. From our reviews, we found limited data to guide management when these malignant foci occur within an MCT. Consideration should be given to thyroidectomy followed by total-body scanning and serum studies for foci of thyroid carcinoma and adjuvant therapy with thyroidectomy and radioablation if residual disease is identified (a). Additionally, extrapolating from data for mucinous adenocarcinomas, consideration could be given to adjuvant chemotherapy after appropriate staging (b). Strumal carcinoid tumors should be treated as tumors of low malignant potential. Observation is appropriate if after complete staging, no invasive implants are noted (c).
The gene transfer agent (RcGTA) of Rhodobacter capsulatus is the model for a family of novel bacteriophage-related genetic elements that carry out lateral transfer of essentially random host DNA. Genuine and putative gene transfer agents have been discovered in diverse genera and are becoming recognized as potentially an important source of genetic exchange and microbial evolution in the oceans. Despite being discovered over 30 years ago, little is known about many essential aspects of RcGTA biology. Here, we validate the use of direct fluorescence reporter constructs, which express the red fluorescent protein mCherry in R. capsulatus. A construct containing the RcGTA promoter fused to mCherry was used to examine the single-cell expression profiles of wild type and RcGTA overproducer R. capsulatus populations, under different growth conditions and growth phases. The majority of RcGTA production clearly arises from a small, distinct sub-set of the population in the wild type strain and a larger sub-set in the overproducer. The most likely RcGTA release mechanism concomitant with this expression pattern is host cell lysis and we present direct evidence for the release of an intracellular enzyme accompanying RcGTA release. RcGTA ORF s is annotated as a ‘cell wall peptidase’ but we rule out a role in host lysis and propose an alternative function as a key contributor to RcGTA invasion of a target cell during infection.
How phenylephrine and ephedrine treatments affect global and regional haemodynamics is of major clinical relevance. Cerebral tissue oxygen saturation ()-guided management may improve postoperative outcome. The physiological variables responsible for changes induced by phenylephrine and ephedrine bolus treatment in anaesthetized patients need to be defined.
A randomized two-treatment cross-over trial was conducted: one bolus dose of phenylephrine (100–200 µg) and one bolus dose of ephedrine (5–20 mg) were given to 29 ASA I–III patients anaesthetized with propofol and remifentanil. , mean arterial pressure (MAP), cardiac output (CO), and other physiological variables were recorded before and after treatments. The associations of changes were analysed using linear-mixed models.
The CO decreased significantly after phenylephrine treatment [▵CO=−2.1 (1.4) litre min−1, P<0.001], but was preserved after ephedrine treatment [▵CO=0.5 (1.4) litre min−1, P>0.05]. The was significantly decreased after phenylephrine treatment [▵=−3.2 (3.0)%, P<0.01] but preserved after ephedrine treatment [▵=0.04 (1.9)%, P>0.05]. CO was identified to have the most significant association with (P<0.001). After taking CO into consideration, the other physiological variables, including MAP, were not significantly associated with (P>0.05).
Associated with changes in CO, decreased after phenylephrine treatment, but remained unchanged after ephedrine treatment. The significant correlation between CO and implies a cause–effect relationship between global and regional haemodynamics.
cardiac output; cerebral tissue oxygen saturation; ephedrine; mean arterial pressure; phenylephrine
Patients with chronic liver disease are at an increased risk of developing transfusion-associated acute lung injury (TRALI) from plasma containing blood products. Similarly, red blood cell transfusions have been associated with post-operative and nosocomial infections in surgical and critical care populations. Patients undergoing liver transplantation receive a large amount of cellular and plasma containing blood products, but it is presently unclear which blood components are associated with these post-operative complications.
A retrospective cohort study of 525 consecutive liver transplant patients revealed a peri-operative TRALI incidence of 1.3% (7/525), 95%CI [0.6%–2.7%], associated with an increased hospital mortality (28.6% (2/7) vs. 2.9% (15/518), p=0.02) and intensive care unit (ICU) length of stay (2 days, [1–11] vs. 0 days [0–2], 0.03). Only high plasma containing blood products (plasma and platelets) were associated with the development of TRALI. A total of 14.3% (74/525) of patients developed a post-operative infection which was also associated with an increased in-hospital mortality (10.8% (8/74) vs. 2.0% (9/451), p < 0.01) and prolonged length of stay. Multivariate logistic regression identified the number of red blood cell units transfused (adj OR 1.08 95%CI [1.02–1.14], p<0.01), the presence of peri-operative renal dysfunction and re-operation to be significantly associated with post-operative infection.
Patients undergoing liver transplantation are at high risk of developing post-operative complications from blood transfusion. Plasma containing blood products were associated with the development of TRALI while red blood cells were associated with the development of post-operative infection in a dose dependent manner.
transfusion-related acute lung injury; nosocomial infection; Pulmonary Edema; Blood Component Transfusion; Liver Cirrhosis
The discovery of molecules that bind tightly and selectively to desired proteins continues to drive innovation at the interface of chemistry and biology. This paper describes the binding of human insulin by the synthetic receptor cucurbituril (Q7) in vitro. Isothermal titration calorimetry and fluorescence spectroscopy experiments show that Q7 binds to insulin with an equilibrium association constant of 1.5 × 106 M-1 and with 50-100-fold selectivity versus proteins that are much larger but lack an N-terminal aromatic residue, and >1000-fold selectivity versus an insulin variant lacking the N-terminal phenylalanine (Phe) residue. The crystal structure of the Q7•insulin complex shows that binding occurs at the N-terminal Phe residue and that the N-terminus unfolds to enable binding. These findings suggest that site-selective recognition is based on the properties inherent to a protein terminus, including the unique chemical epitope presented by the terminal residue and the greater freedom of the terminus to unfold, like the end of a ball of string, to accommodate binding. Insulin recognition was predicted accurately from studies on short peptides and exemplifies an approach to protein recognition by targeting the terminus.
Cone photoreceptor cyclic nucleotide-gated (CNG) channel is essential for central and color vision and visual acuity. Mutations in the cone channel subunits CNGA3 and CNGB3 are linked to achromatopsia and progressive cone dystrophy in humans. Over 50 mutations have been identified in the CNGA3 subunit. The R277C and R283W substitutions are among the most frequently occurring mutations. This study investigated the defects of these two mutations using a heterologous expression system. The wild type and mutant CNGA3 were expressed in HEK293 cells, the channel’s expression and cellular localization were examined by immunoblotting and immunofluorecences labeling, and activity of the channel was evaluated by ratiometric [Ca2+]i measurements and by electrophysiological recordings. By using this model system we observed dysfunction of the mutant channels. Co-expression of the mutant channel with the wild type subunit did not affect the wild type channel’s activity. Immunoflurescence labeling showed apparent cytosol aggregation of the immunoreactivity in cells expressing the mutants. Thus these disease-causing mutations appear to induce loss of function by impairing the channel cellular trafficking and plasma membrane targeting. Therapeutic supplementation of the wild type transgene may help correct the visual disorders caused by these two mutations.
The task of an organism to extract information about the external environment from sensory signals is based entirely on the analysis of ongoing afferent spike activity provided by the sense organs. We investigate the processing of auditory stimuli by an acoustic interneuron of insects. In contrast to most previous work we do this by using stimuli and neurophysiological recordings directly in the nocturnal tropical rainforest, where the insect communicates. Different from typical recordings in sound proof laboratories, strong environmental noise from multiple sound sources interferes with the perception of acoustic signals in these realistic scenarios. We apply a recently developed unsupervised machine learning algorithm based on probabilistic inference to find frequently occurring firing patterns in the response of the acoustic interneuron. We can thus ask how much information the central nervous system of the receiver can extract from bursts without ever being told which type and which variants of bursts are characteristic for particular stimuli. Our results show that the reliability of burst coding in the time domain is so high that identical stimuli lead to extremely similar spike pattern responses, even for different preparations on different dates, and even if one of the preparations is recorded outdoors and the other one in the sound proof lab. Simultaneous recordings in two preparations exposed to the same acoustic environment reveal that characteristics of burst patterns are largely preserved among individuals of the same species. Our study shows that burst coding can provide a reliable mechanism for acoustic insects to classify and discriminate signals under very noisy real-world conditions. This gives new insights into the neural mechanisms potentially used by bushcrickets to discriminate conspecific songs from sounds of predators in similar carrier frequency bands.
We present a calculation of the microwave field distribution in a magic angle spinning (MAS) probe utilized in dynamic nuclear polarization (DNP) experiments. The microwave magnetic field (B1S) profile was obtained from simulations performed with the High Frequency Structure Simulator (HFSS) software suite, using a model that includes the launching antenna, the outer Kel-F stator housing coated with Ag, the RF coil, and the 4 mm diameter sapphire rotor containing the sample. The predicted average B1S field is 13µT/W1/2, where S denotes the electron spin. For a routinely achievable input power of 5 W the corresponding value is γ SB1S = 0.84 MHz. The calculations provide insights into the coupling of the microwave power to the sample, including reflections from the RF coil and diffraction of the power transmitted through the coil. The variation of enhancement with rotor wall thickness was also successfully simulated. A second, simplified calculation was performed using a single pass model based on Gaussian beam propagation and Fresnel diffraction. This model provided additional physical insight and was in good agreement with the full HFSS simulation. These calculations indicate approaches to increasing the coupling of the microwave power to the sample, including the use of a converging lens and fine adjustment of the spacing of the windings of the RF coil. The present results should prove useful in optimizing the coupling of microwave power to the sample in future DNP experiments. Finally, the results of the simulation were used to predict the cross effect DNP enhancement (ε) vs. ω1S/(2π) for a sample of 13C-urea dissolved in a 60:40 glycerol/water mixture containing the polarizing agent TOTAPOL; very good agreement was obtained between theory and experiment.
Dynamic Nuclear Polarization; Instrumentation; Cryogenic MAS
We report the dynamic nuclear polarization of 1H spins in magic angle spinning spectra recorded at 5 T and 84K via the solid effect using Mn2+ and Gd3+ complexes as polarizing agents. We show that the magnitude of the enhancements can be directly related to the effective linewidth of the central (MS = −1/2 →+1/2) EPR transition. Using a Gd3+ complex with a narrow central transition EPR linewidth of 29 MHz, a maximum enhancement of ~13 is observed, which is comparable to previous results on the narrow linewidth trityl radical.
High-grade osteosarcoma is an aggressive tumor most commonly affecting adolescents. The early age of onset might suggest genetic predisposition; however, the vast majority of the tumors are sporadic. Early onset, most often lack of a predisposing condition or lesion, only infrequent (<2%) prevalence of inheritance, extensive genomic instability, and a wide histological heterogeneity are just few factors to mention that make osteosarcoma difficult to study. Therefore, it is sensible to design and use models representative of the human disease. Here we summarize multiple osteosarcoma models established in vitro and in vivo, comment on their utilities, and highlight newest achievements, such as the use of zebrafish embryos. We conclude that to gain a better understanding of osteosarcoma, simplification of this extremely complex tumor is needed. Therefore, we parse the osteosarcoma problem into parts and propose adequate models to study them each separately. A better understanding of osteosarcoma provides opportunities for discovering and assaying novel effective treatment strategies.
“Sometimes the model is more interesting than the original disease”
PJ Hoedemaeker (1937–2007).
Community-acquired respiratory distress syndrome toxin (CARDS TX) from M. pneumoniae was cloned, expressed, purified and crystallized. A diffraction data set from a native CARDS TX crystal was obtained at 2.2 Å resolution.
Community-acquired respiratory distress syndrome toxin (CARDS TX) is a 591-amino-acid protein with ADP-ribosyltransferase and vacuolating activities that damages the cells lining the respiratory tracts of patients infected with the bacterial pathogen Mycoplasma pneumoniae. Crystals of CARDS TX were grown in space group C2, with unit-cell parameters a = 191.4, b = 107.4, c = 222.1 Å, β = 90.6°. A complete 2.2 Å data set was obtained from a single CARDS TX crystal.
CARDS TX; community-acquired respiratory distress syndrome; Mycoplasma pneumoniae; ADP-ribosyltransferases; ADP-ribosylation
We employ a Hodgkin-Huxley type model of basolateral ionic currents in bullfrog saccular hair cells to study the genesis of spontaneous voltage oscillations and their role in shaping the response of the hair cell to external mechanical stimuli. Consistent with recent experimental reports, we find that the spontaneous dynamics of the model can be categorized using conductance parameters of calcium activated potassium, inward rectifier potassium, and mechano-electrical transduction ionic currents. The model is demonstrated to exhibit a broad spectrum of autonomous rhythmic activity, including periodic and quasiperiodic oscillations with two independent frequencies as well as various regular and chaotic bursting patterns. Complex patterns of spontaneous oscillations in the model emerge at small values of the conductance of Ca2+ activated potassium currents. These patterns are significantly affected by thermal fluctuations of the mechano-electrical transduction current. We show that self-sustained regular voltage oscillations lead to enhanced and sharply tuned sensitivity of the hair cell to weak mechanical periodic stimuli. While regimes of chaotic oscillations are argued to result in poor tuning to sinusoidal driving, chaotically oscillating cells do provide a high sensitivity to low-frequency variations of external stimuli.
5α-reductase 1 (5αR1) and 5α-reductase 2 (5αR2) convert testosterone into the more potent androgen dihydrotestosterone. 5αR2 is the main isoenzyme in normal prostate tissue; however, most prostate tumors have increased 5αR1 and decreased 5αR2 expression. Previously, finasteride (5αR2 inhibitor) treatment begun 3 weeks post-tumor implantation had no effect on Dunning R3327-H rat prostate tumor growth. We believe the tumor compensated for finasteride treatment by increasing tumor 5αR1 expression or activity. We hypothesize that finasteride treatment would not significantly alter tumor growth even if begun before tumor implantation, whereas dutasteride (5αR1 and 5αR2 inhibitor) treatment would decrease tumor growth regardless of whether treatment was initiated before or after tumor implantation.
Sixty 8-week-old male nude mice were randomized to Control, Pre- and Post-Finasteride, and Pre- and Post-Dutasteride (83.3 mg drug/kg diet) diet groups. Pre- and post-groups began their treatment diets 1–2 weeks prior to or 3 weeks after subcutaneous injection of 1×105 WPE1-NA22 human prostate cancer cells, respectively. Tumors were allowed to grow for 22 weeks; tumor areas, body weights, and food intakes were measured weekly. At study's conclusion, prostate and seminal vesicle weights were significantly decreased in all treatment groups versus the control; dutasteride intake significantly decreased seminal vesicle weights compared to finasteride intake. No differences were measured in final tumor areas or tumor weights between groups, likely due to poor tumor growth. In follow-up studies, proliferation of WPE1-NA22 prostate cancer cells and parent line RWPE-1 prostate epithelial cells were unaltered by treatment with testosterone, dihydrotestosterone, or mibolerone, suggesting that these cell lines are not androgen-sensitive.
The lack of response of WPE1-NA22 prostate cancer cells to androgen treatment may explain the inadequate tumor growth observed. Additional studies are needed to determine whether finasteride and dutasteride are effective in decreasing prostate cancer development/growth.
Pro-coagulant and pro-inflammatory intramyocardial (micro)vasculature plays an important role in acute myocardial infarction (AMI). Currently, inhibition of serine protease dipeptidyl peptidase 4 (DPP4) receives a lot of interest as an anti-hyperglycemic therapy in type 2 diabetes patients. However, DPP4 also possesses anti-thrombotic properties and may behave as an immobilized anti-coagulant on endothelial cells. Here, we studied the expression and activity of endothelial DPP4 in human myocardial infarction in relation to a prothrombogenic endothelial phenotype. Using (immuno)histochemistry, DPP4 expression and activity were found on the endothelium of intramyocardial blood vessels in autopsied control hearts (n = 9). Within the infarction area of AMI patients (n = 73), this DPP4 expression and activity were significantly decreased, coinciding with an increase in Tissue Factor expression. In primary human umbilical vein endothelial cells (HUVECs), Western blot analysis and digital imaging fluorescence microscopy revealed that DPP4 expression was strongly decreased after metabolic inhibition, also coinciding with Tissue Factor upregulation. Interestingly, inhibition of DPP4 activity with diprotin A also enhanced the amount of Tissue Factor encountered and induced the adherence of platelets under flow conditions. Ischemia induces loss of coronary microvascular endothelial DPP4 expression and increased Tissue Factor expression in AMI as well as in vitro in HUVECs. Our data suggest that the loss of DPP4 activity affects the anti-thrombogenic nature of the endothelium.
DPP4; Acute myocardial infarction; Tissue Factor; ROS; Coagulation; Platelet adhesion
A comparison between the efficiency of recombinase-mediated cassette exchange (RMCE) reactions catalyzed in Escherichia coli by the site-specific recombinases Flp of yeast and Int of coliphage HK022 has revealed that an Flp-catalyzed RMCE reaction is more efficient than an Int-HK022 catalyzed reaction. In contrast, an RMCE reaction with 1 pair of frt sites and 1 pair of att sites catalyzed in the presence of both recombinases is very inefficient. However, the same reaction catalyzed by each recombinase individually supplied in a sequential order is very efficient, regardless of the order. Atomic force microscopy images of Flp with its DNA substrates show that only 1 pair of recombination sites forms a synaptic complex with the recombinase. The results suggest that the RMCE reaction is sequential.
Site-specific recombination; Flp recombinase; Coliphage HK022; Integrase; Recombinase-mediated cassette exchange; Atomic force microscopy