Physiological processes are regulated by a diverse array of neuropeptides that coordinate organ systems. The neuropeptides, many of which act through G protein–coupled receptors, affect the levels of cyclic nucleotides (cAMP and cGMP) and Ca2+ in target tissues. In this perspective, their roles in molting, osmoregulation, metabolite utilization, and cardiovascular function are highlighted. In decapod crustaceans, inhibitory neuropeptides (molt-inihibiting hormone and crustacean hyperglycemic hormone) suppress the molting gland through cAMP- and cGMP-mediated signaling. In insects, the complex movements during ecdysis are controlled by ecdysis-triggering hormone and a cascade of downstream neuropeptides. Adipokinetic/hypertrehalosemic/hyperprolinemic hormones mobilize energy stores in response to increased locomotory activity. Crustacean cardioacceleratory (cardioactive) peptide, proctolin, and FMRFamide-related peptides act on the heart, accessory pulsatile organs, and excurrent ostia to control hemolymph distribution to tissues. The osmoregulatory challenge of blood gorging in Rhodnius prolixus requires the coordinated release of serotonin and diuretic and antidiuretic hormones acting on the midgut and Malpighian tubules. These studies illustrate how multiple neuropeptides allow for flexibility in response to physiological challenges.
Structural analysis of the catalytic module of Caldicellulosiruptor bescii family 3 pectate lyase shows that this new structure is very similar to the previously solved structure of a family 3 pectate lyase from Bacillus sp. strain KSM-P15.
A 1.5 Å resolution X-ray structure of the catalytic module of Caldicellulosiruptor bescii family 3 pectate lyase is reported (PDB entry 3t9g). The resulting structure was refined to an R factor of 0.143 and an R
free of 0.178. Structural analysis shows that this new structure is very similar to the previously solved structure of a family 3 pectate lyase from Bacillus sp. strain KSM-P15 (PDB entry 1ee6), with a root-mean-square deviation of 0.93 Å and a sequence identity of 53%. This structural similarity is significant considering that C. bescii is a hyperthermophile and Bacillus sp. is a mesophile.
pectate lyases; PL3; Caldicellulosiruptor bescii
Dyslipidemia from highly active antiretroviral therapy (HAART) use has been reported to be less severe among persons with HIV and hepatitis C (HCV) compared to those with HIV monoinfection. However, the effect on lipoprotein ratios is less clear. The total cholesterol/high-density lipoprotein ratio (TC/HDL-C ratio) is a robust measure of cardiovascular disease (CVD) risk but has not been examined in the context of HIV/HCV-coinfected patients. We compared the TC/HDL-C ratio before HAART initiation and after at least 6 months on HAART between patients monoinfected with HIV and coinfected with HIV and HCV. Pre- and post-HAART TC, HDL-C, and non-HDL-C were also assessed. Although TC, HDL-C, and non-HDL-C significantly increased after HAART initiation in both HIV and HIV/HCV patients, the TC/HDL-C ratio did not. In addition, although the pre- and post-HAART TC, HDL-C, non-HDL-C, and TC/HDL-C ratio were significantly different between HIV and HIV/HCV patients, the magnitude in the change from pre- to post-HAART was not significantly different between infection groups. These results persisted after controlling for age, sex, race, current pharmacotherapy for lipoproteins, body mass index, and current CD4 cell count. The magnitude of change in the TC/HDL-C ratio after HAART initiation is not significantly different between HIV and HIV/HCV patients, suggesting subsequent CVD risk in HIV/HCV patients may be greater than currently appreciated.
To assess the safety of duloxetine with regards to bleeding-related events in patients who concomitantly did, versus did not, use nonsteroidal anti-inflammatory drugs (NSAIDs), including aspirin.
Safety data from all placebo-controlled trials of duloxetine conducted between December 1993 and December 2010, and post-marketing reports from duloxetine-treated patients in the US Food and Drug Administration Adverse Event Reporting System (FAERS), were searched for bleeding-related treatment-emergent adverse events (TEAEs). The percentage of patients with bleeding-related TEAEs was summarized and compared between treatment groups in all the placebo-controlled studies. Differences between NSAID user and non-user subgroups from clinical trial data were analyzed by a logistic regression model that included therapy, NSAID use, and therapy-by-NSAID subgroup interaction. In addition, to determine if higher duloxetine doses are associated with an increased incidence of bleeding-related TEAEs, and whether the use of concomitant NSAIDs might influence the dose effect if one exists, placebo-controlled clinical trials with duloxetine fixed doses of 60 mg, 120 mg, and placebo were analyzed. Also, the incidence of bleeding-related TEAEs reported for duloxetine alone was compared with the incidence in patients treated with duloxetine and concomitant NSAIDs. Finally, the number of bleeding-related cases reported for duloxetine in the FAERS database was compared with the numbers reported for all other drugs.
Across duloxetine clinical trials, there was a significantly greater incidence of bleeding-related TEAEs in duloxetine- versus placebo-treated patients overall and also in those patients who did not take concomitant NSAIDS, but no significant difference was seen among those patients who did take concomitant NSAIDS. There was no significant difference in the incidence of bleeding-related TEAEs in the subset of patients treated with duloxetine 120 mg once daily versus those treated with 60 mg once daily regardless of concomitant NSAID use. The combination of duloxetine and NSAIDs was associated with a statistically significantly higher incidence of bleeding-related TEAEs compared with duloxetine alone. A similarly higher incidence of bleeding-related TEAEs was seen in patients treated with placebo and concomitant NSAIDs compared with placebo alone. Bleeding-related TEAEs reported in the FAERS database were disproportionally more frequent for duloxetine taken with NSAIDs compared with the full FAERS background, but there was no difference in the reporting of bleeding-related TEAEs when the cases reported for duloxetine taken with NSAIDs were compared against the cases reported for NSAIDs alone.
Concomitant use of NSAIDs was associated with a higher incidence of bleeding-related TEAEs in clinical trials regardless of whether patients were taking duloxetine or placebo; bleeding-related TEAEs did not appear to increase along with duloxetine dose regardless of NSAID use. In spontaneously reported post-marketing data, the combination of duloxetine and NSAID use was not associated with an increased reporting of bleeding-related events when compared to NSAID use alone.
antidepressant; gastrointestinal bleeding; NSAID; aspirin
Motor impairment and travel time have been shown to be important barriers to recruitment for Parkinson's disease (PD) clinical trials. This study determined whether use of Internet-based video communication for study visits would improve likelihood of participating in PD clinical trials.
Subjects and Methods:
University of Utah PD clinic patients were invited to complete a survey asking if they would be willing to participate in a hypothetical research study under four different scenarios. McNemar's test was used to test the hypothesis that remote assessments would improve willingness to participate.
Willingness to participate was 101/113 (87%) in the standard scenario. Willingness to participate was highest (93%; p=0.046) with most visits occurring via telemedicine at a local clinic, followed by some visits occurring via telemedicine at a local clinic (91%; p=0.157). Willingness to participate was lower with some (80%; p=0.008) or most (82%; p=0.071) visits occurring by home telemonitoring.
Use of telemedicine may be an acceptable means to improve participation in clinical trials. This would need to be confirmed with the use of a larger-scale inquiry involving rural populations. Future research should assess subject or caregiver comfort and trainability with respect to computer-based technology in the home and systems barriers for wider implementation of telemedicine in neurology.
Parkinson's disease; clinical trials; telemedicine; telemonitoring; neurodegenerative disease
Iron is an essential element for the hyperthermophilic archaeon Pyrococcus furiosus, and many of its iron-containing enzymes have been characterized. How iron assimilation is regulated, however, is unknown. The genome sequence contains genes encoding two putative iron-responsive transcription factors, DtxR and Fur. Global transcriptional profiles of the dtxR deletion mutant (ΔDTXR) and the parent strain under iron-sufficient and iron-limited conditions indicated that DtxR represses the expression of the genes encoding two putative iron transporters, Ftr1 and FeoAB, under iron-sufficient conditions. Under iron limitation, DtxR represses expression of the gene encoding the iron-containing enzyme aldehyde ferredoxin oxidoreductase and a putative ABC-type transporter. Analysis of the dtxR gene sequence indicated an incorrectly predicted translation start site, and the corrected full-length DtxR protein, in contrast to the truncated version, specifically bound to the promoters of ftr1 and feoAB, confirming its role as a transcription regulator. Expression of the gene encoding Ftr1 was dramatically upregulated by iron limitation, but no phenotype was observed for the ΔFTR1 deletion mutant under iron-limited conditions. The intracellular iron concentrations of ΔFTR1 and the parent strain were similar, suggesting that under the conditions tested, Ftr1 is not an essential iron transporter despite its response to iron. In contrast to DtxR, the Fur protein appears not to be a functional regulator in P. furiosus, since it did not bind to the promoters of any of the iron-regulated genes and the deletion mutant (ΔFUR) revealed no transcriptional responses to iron availability. DtxR is therefore the key iron-responsive transcriptional regulator in P. furiosus.
The purpose of this review is to summarize the research to date on vitamin K status in chronic kidney disease (CKD). This review includes a summary of the data available on vitamin K status in patients across the spectrum of CKD as well as the link between vitamin K deficiency in CKD and bone dynamics, including mineralization and demineralization, as well as ectopic mineralization. It also describes two current clinical trials that are underway evaluating vitamin K treatment in CKD patients. These data may inform future clinical practice in this population.
vitamin K; chronic kidney disease; renal insufficiency; vitamin K-dependent proteins
Vertebral deformities often occur in patients who recall no trauma, and display no evident fracture on radiographs. We hypothesise that vertebral deformity can occur by a gradual creep mechanism which is accelerated following minor damage. “Creep” is continuous deformation under constant load.
Materials and methods
Forty-five thoracolumbar spine motion segments were tested from cadavers aged 42–92 years. Vertebral body areal BMD was measured using DXA. Specimens were compressed at 1 kN for 30 min, while creep in each vertebral body was measured using an optical MacReflex system. After 30 min recovery, each specimen was subjected to a controlled overload event which caused minor damage to one of its vertebrae. The creep test was then repeated.
Vertebral body creep was measurable in specimens with BMD <0.5 g/cm2. Creep was greater anteriorly than posteriorly (p < 0.001), so that vertebrae gradually developed a wedge deformity. Compressive overload reduced specimen height by 2.24 mm (STD 0.77 mm), and increased vertebral body creep by 800 % (anteriorly), 1,000 % (centrally) and 600 % (posteriorly). In 34 vertebrae with complete before-and-after data, anterior wedging occurring during the 1st creep test averaged 0.07° (STD 0.17°), and in the 2nd test (after minor damage) it averaged 0.79° (STD 1.03°). The increase was highly significant (P < 0.001). Vertebral body wedging during the 2nd creep test was proportional to the severity of damage, as quantified by specimen height loss during the overload event (r2 = 0.51, p < 0.001, n = 34).
Minor damage to an old vertebral body, even if it is barely discernible on radiographs, can accelerate creep to such an extent that it makes a substantial contribution to vertebral deformity.
Vertebral deformity; Fracture; Creep; BMD; Cadaveric; Damage
Osteoarthritis (OA) is the most common joint disease characterised by degradation of articular cartilage and bone remodelling. For almost a decade chondrocyte apoptosis has been investigated as a possible mechanism of cartilage damage in OA, but its precise role in initiation and/or progression of OA remains to the determined. The aim of this study is to determine the role of chondrocyte apoptosis in spontaneous animal models of OA. Right tibias from six male Dunkin Hartley (DH) and Bristol Strain 2 (BS2) guinea pigs were collected at 10, 16, 24 and 30 weeks of age. Fresh-frozen sections of tibial epiphysis were microscopically scored for OA, and immunostained with caspase-3 and TUNEL for apoptotic chondrocytes. The DH strain had more pronounced cartilage damage than BS2, especially at 30 weeks. At this time point, the apoptotic chondrocytes were largely confined to the deep zone of articular cartilage (AC) with a greater percentage in the medial side of DH than BS2 (DH: 5.7%, 95% CI: 4.2–7.2), BS2: 4.8%, 95% CI: 3.8–5.8), p > 0.05). DH had a significant progression of chondrocyte death between 24 to 30 weeks during which time significant changes were observed in AC fibrillation, proteoglycan depletion and overall microscopic OA score. A strong correlation (p ≤ 0.01) was found between chondrocyte apoptosis and AC fibrillation (r = 0.3), cellularity (r = 0.4) and overall microscopic OA scores (r = 0.4). Overall, the rate of progression in OA and apoptosis over the study period was greater in the DH (versus BS2) and the medial AC (versus lateral). Chondrocyte apoptosis was higher at the later stage of OA development when the cartilage matrix was hypocellular and highly fibrillated, suggesting that chondrocyte apoptosis is a late event in OA.
chondrocyte apoptosis; osteoarthritis; animal models; disease progression; caspase-3
Pre-conception or gestationally-induced diabetes increases morbidities and elevates long-term cardiovascular disease risks in women and their children. Spontaneously hyperglycemic (d)-NOD/ShiLtJ females, a type 1 diabetes model, develop bradycardia and hypotension after midpregnancy compared with normoglycemic, age and gestation day (gd)-matched controls (c-NOD). We hypothesized that onset of the placental circulation at gd9–10 and rapid fetal growth from gd14 correlate with aberrant hemodynamic outcomes in d-NOD females. To develop further gestational time course correlations between maternal cardiac and renal parameters, high-frequency ultrasonography was applied to virgin and gd8–16 d- and c-NODs. Cardiac output and left ventricular (LV) mass increased in c- but not d-NODs. Ultrasound and postmortem histopathology showed overall greater LV dilation in d- than c-NOD mice in mid-late gestation. These changes suggest blunted remodeling and altered functional adaptation of d-NOD hearts. Umbilical cord ultrasounds revealed lower fetal heart rates from gd12 and lower umbilical flow velocities at gd14 and 16 in d- versus c-NOD pregnancies. From gd14–16, d-NOD fetal losses exceeded those of c-NOD. Similar aberrant responses in human diabetic pregnancies may elevate postpartum maternal and child cardiovascular risk, particularly if mothers lack adequate prenatal care or have poor glycemic control over gestation.
PMID: 23636813 CAMSID: cams2944
Pregnancy; diabetes; NOD mouse; cardiovascular system; cardiac adaptations
To assess internal medicine (IM) and surgery program directors’ views of the likely effects of the 2011 Accreditation Council for Graduate Medical Education duty hours regulations.
In fall 2010, investigators surveyed IM and surgery program directors, assessing their views of the likely impact of the 2011 duty hours standards on learning environment, workload, education opportunities, program administration, and patient outcomes.
Of 381 IM program directors, 287 (75.3%) responded; of 225 surgery program directors, 118 (52.4%) responded. Significantly more surgeons than internists indicated that the new regulations would likely negatively impact learning climate, including faculty morale and residents’ relationships (P < 0.001). Most leaders in both specialties (80.8% IM, 80.2% surgery) felt that the regulations would likely increase faculty workload (P = .73). Both IM (82.2%) and surgery (96.6%) leaders most often rated, of all education opportunities, first-year resident clinical experience to be adversely affected (P < .001). Respondents from both specialties indicated that they will hire more nonphysician/midlevel providers (59.5% IM, 89.0% surgery, P < .001) and use more nonteaching services (66.8% IM, 70.1% surgery, P = .81). Respondents expect patient safety (45.1% IM, 76.9% surgery, P < .001) and continuity of care (83.6% IM across all training levels, 97.5% surgery regarding first-year residents) to decrease.
IM and surgery program directors agree that the 2011 duty hours regulations will likely negatively affect the quality of the learning environment, workload, education opportunities, program administration, and patient outcomes. Careful evaluation of actual impact is important.
Though a third of amphibian species worldwide are thought to be imperiled, existing assessments simply categorize extinction risk, providing little information on the rate of population losses. We conducted the first analysis of the rate of change in the probability that amphibians occupy ponds and other comparable habitat features across the United States. We found that overall occupancy by amphibians declined 3.7% annually from 2002 to 2011. Species that are Red-listed by the International Union for Conservation of Nature (IUCN) declined an average of 11.6% annually. All subsets of data examined had a declining trend including species in the IUCN Least Concern category. This analysis suggests that amphibian declines may be more widespread and severe than previously realized.
Many aspects of both grip function and tactile perception depend on complex frictional interactions occurring in the contact zone of the finger pad, which is the subject of the current review. While it is well established that friction plays a crucial role in grip function, its exact contribution for discriminatory touch involving the sliding of a finger pad is more elusive. For texture discrimination, it is clear that vibrotaction plays an important role in the discriminatory mechanisms. Among other factors, friction impacts the nature of the vibrations generated by the relative movement of the fingertip skin against a probed object. Friction also has a major influence on the perceived tactile pleasantness of a surface. The contact mechanics of a finger pad is governed by the fingerprint ridges and the sweat that is exuded from pores located on these ridges. Counterintuitively, the coefficient of friction can increase by an order of magnitude in a period of tens of seconds when in contact with an impermeably smooth surface, such as glass. In contrast, the value will decrease for a porous surface, such as paper. The increase in friction is attributed to an occlusion mechanism and can be described by first-order kinetics. Surprisingly, the sensitivity of the coefficient of friction to the normal load and sliding velocity is comparatively of second order, yet these dependencies provide the main basis of theoretical models which, to-date, largely ignore the time evolution of the frictional dynamics. One well-known effect on taction is the possibility of inducing stick–slip if the friction decreases with increasing sliding velocity. Moreover, the initial slip of a finger pad occurs by the propagation of an annulus of failure from the perimeter of the contact zone and this phenomenon could be important in tactile perception and grip function.
contact area; contact mechanics; human skin; occlusion; stick–slip; tribology
Crenarchaeotal genomes encode the 3-hydroxypropionate/4-hydroxybutyrate (3-HP/4-HB) cycle for carbon dioxide fixation. Of the 13 enzymes putatively comprising the cycle, several of them, including methylmalonyl-coenzyme A (CoA) epimerase (MCE) and methylmalonyl-CoA mutase (MCM), which convert (S)-methylmalonyl-CoA to succinyl-CoA, have not been confirmed and characterized biochemically. In the genome of Metallosphaera sedula (optimal temperature [Topt], 73°C), the gene encoding MCE (Msed_0639) is adjacent to that encoding the catalytic subunit of MCM-α (Msed_0638), while the gene for the coenzyme B12-binding subunit of MCM (MCM-β) is located remotely (Msed_2055). The expression of all three genes was significantly upregulated under autotrophic compared to heterotrophic growth conditions, implying a role in CO2 fixation. Recombinant forms of MCE and MCM were produced in Escherichia coli; soluble, active MCM was produced only if MCM-α and MCM-β were coexpressed. MCE is a homodimer and MCM is a heterotetramer (α2β2) with specific activities of 218 and 2.2 μmol/min/mg, respectively, at 75°C. The heterotetrameric MCM differs from the homo- or heterodimeric orthologs in other organisms. MCE was activated by divalent cations (Ni2+, Co2+, and Mg2+), and the predicted metal binding/active sites were identified through sequence alignments with less-thermophilic MCEs. The conserved coenzyme B12-binding motif (DXHXXG-SXL-GG) was identified in M. sedula MCM-β. The two enzymes together catalyzed the two-step conversion of (S)-methylmalonyl-CoA to succinyl-CoA, consistent with their proposed role in the 3-HP/4-HB cycle. Based on the highly conserved occurrence of single copies of MCE and MCM in Sulfolobaceae genomes, the M. sedula enzymes are likely to be representatives of these enzymes in the 3-HP/4-HB cycle in crenarchaeal thermoacidophiles.
The vital nature of metal uptake and balance in biology is evident in the highly evolved strategies to facilitate metal homeostasis in all three domains of life. Several decades of study on metals and metalloproteins have revealed numerous essential bio-metal functions. Recent advances in mass spectrometry, x-ray scattering/absorption, and proteomics have exposed a much broader usage of metals in biology than expected. Even elements such as uranium, arsenic, and lead are implicated in biological processes as part of an emerging and expansive view of bio-metals. Here we discuss opportunities and challenges for established and newer approaches to study metalloproteins with a focus on technologies that promise to rapidly expand our knowledge of metalloproteins and metal functions in biology.
metallomics; metalloproteomics; metalloproteins; biometals
Whole-body and thoracic ionizing radiation exposure are associated with increased cardiovascular disease (CVD) risk. In atomic bomb survivors, radiation dose is also associated with increased hypertension incidence, suggesting that radiation dose may be associated with chronic renal failure (CRF), thus explaining part of the mechanism for increased CVD. Multivariate Poisson regression was used to evaluate the association of radiation dose with various definitions of chronic kidney disease (CKD) mortality in the Life Span Study (LSS) of atomic bomb survivors. A secondary analysis was performed using a subsample for whom self-reported information on hypertension and diabetes, the two biggest risk factors for CRF, had been collected. We found a significant association between radiation dose and only our broadest definition of CRF among the full cohort. A quadratic dose excess relative risk model [ERR/Gy2 = 0.091 (95% CI: 0.05, 0.198)] fit minimally better than a linear model. Within the subsample, association was also observed only with the broadest CRF definition [ERR/Gy2 = 0.15 (95% CI: 0.02, 0.32)]. Adjustment for hypertension and diabetes improved model fit but did not substantially change the ERR/Gy2 estimate, which was 0.17 (95% CI: 0.04, 0.35). We found a significant quadratic dose relationship between radiation dose and possible chronic renal disease mortality that is similar in shape to that observed between radiation and incidence of hypertension in this population. Our results suggest that renal dysfunction could be part of the mechanism causing increased CVD risk after whole-body irradiation, a hypothesis that deserves further study.
Extremely thermophilic bacteria of the genus Caldicellulosiruptor utilize carbohydrate components of plant cell walls, including cellulose and hemicellulose, facilitated by a diverse set of glycoside hydrolases (GHs). From a biofuel perspective, this capability is crucial for deconstruction of plant biomass into fermentable sugars. While all species from the genus grow on xylan and acid-pretreated switchgrass, growth on crystalline cellulose is variable. The basis for this variability was examined using microbiological, genomic, and proteomic analyses of eight globally diverse Caldicellulosiruptor species. The open Caldicellulosiruptor pangenome (4,009 open reading frames [ORFs]) encodes 106 GHs, representing 43 GH families, but only 26 GHs from 17 families are included in the core (noncellulosic) genome (1,543 ORFs). Differentiating the strongly cellulolytic Caldicellulosiruptor species from the others is a specific genomic locus that encodes multidomain cellulases from GH families 9 and 48, which are associated with cellulose-binding modules. This locus also encodes a novel adhesin associated with type IV pili, which was identified in the exoproteome bound to crystalline cellulose. Taking into account the core genomes, pangenomes, and individual genomes, the ancestral Caldicellulosiruptor was likely cellulolytic and evolved, in some cases, into species that lost the ability to degrade crystalline cellulose while maintaining the capacity to hydrolyze amorphous cellulose and hemicellulose.
The model archaeon Pyrococcus furiosus grows optimally near 100°C on carbohydrates and peptides. Its genome sequence (NCBI) was determined 12 years ago. A genetically tractable strain, COM1, was very recently reported, and here we describe its genome sequence. Of 1,909,827 bp in size, it is 1,571 bp longer (0.1%) than the reference NCBI sequence. The COM1 genome contains numerous chromosomal rearrangements, deletions, and single base changes. COM1 also has 45 full or partial insertion sequences (ISs) compared to 35 in the reference NCBI strain, and these have resulted in the direct deletion or insertional inactivation of 13 genes. Another seven genes were affected by chromosomal deletions and are predicted to be nonfunctional. In addition, the amino acid sequences of another 102 of the 2,134 predicted gene products are different in COM1. These changes potentially impact various cellular functions, including carbohydrate, peptide, and nucleotide metabolism; DNA repair; CRISPR-associated defense; transcriptional regulation; membrane transport; and growth at 72°C. For example, the IS-mediated inactivation of riboflavin synthase in COM1 resulted in a riboflavin requirement for growth. Nevertheless, COM1 grew on cellobiose, malto-oligosaccharides, and peptides in complex and minimal media at 98 and 72°C to the same extent as did both its parent strain and a new culture collection strain (DSMZ 3638). This was in spite of COM1 lacking several metabolic enzymes, including nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase and beta-glucosidase. The P. furiosus genome is therefore of high plasticity, and the availability of the COM1 sequence will be critical for the future studies of this model hyperthermophile.
Copper (Cu) is an important enzyme co-factor that is also extremely toxic at high intracellular concentrations, making active efflux mechanisms essential for preventing Cu accumulation. Here, we have investigated the mechanistic role of metallochaperones in regulating Cu efflux. We have constructed a computational model of Cu trafficking and efflux based on systems analysis of the Cu stress response of Halobacterium salinarum. We have validated several model predictions via assays of transcriptional dynamics and intracellular Cu levels, discovering a completely novel function for metallochaperones. We demonstrate that in addition to trafficking Cu ions, metallochaperones also function as buffers to modulate the transcriptional responsiveness and efficacy of Cu efflux. This buffering function of metallochaperones ultimately sets the upper limit for intracellular Cu levels and provides a mechanistic explanation for previously observed Cu metallochaperone mutation phenotypes.
Copper (Cu) toxicity is a problem of medical, agricultural, and environmental significance. Cu toxicity severely inhibits growth of plant roots significantly affecting their morphology; Cu overload also accounts for some of the most common metal-metabolism abnormalities and neuropsychiatric problems including Wilson's and Menkes diseases. There is a large body of literature on how Cu enters and exits the cell; the kinetic and structural details of Cu translocation between trafficking, sensing, metabolic, and pumping proteins; and phenotypes associated with defects in metalloregulatory and efflux functions. Although the role of metallochaperones in Cu-cytotoxicity has been poorly studied, it has been observed that in animals deletion of metallochaperones results in elevated intracellular Cu levels along with overexpression of the P1-type ATPase efflux pump, ultimately causing malformation with high mortality. These observations are mechanistically explained by a predictive model of the Cu circuit in Halobacterium salinarum, which serves as an excellent model system for Cu trafficking and regulation in organisms with multiple chaperones. Constructed through iterative modeling and experimentation, this model accurately recapitulates known dynamical properties of the Cu circuit and predicts that intracellular Cu-buffering emerges as a consequence of the interplay of paralogous metallochaperones that traffic and allocate Cu to distinct targets.
Next-generation sequencing (NGS) has transformed genetic research and is poised to revolutionize clinical diagnosis. However, the vast amount of data and inevitable discovery of incidental findings require novel analytic approaches. We therefore implemented for the first time a strategy that utilizes an a priori structured framework and a conservative threshold for selecting clinically relevant incidental findings.
We categorized 2016 genes linked with Mendelian diseases into “bins” based on clinical utility and validity, and used a computational algorithm to analyze 80 whole genome sequences in order to explore the use of such an approach in a simulated real-world setting.
The algorithm effectively reduced the number of variants requiring human review and identified incidental variants with likely clinical relevance. Incorporation of the Human Gene Mutation Database (HGMD) improved the yield for missense mutations, but also revealed that a substantial proportion of purported disease-causing mutations were misleading.
This approach is adaptable to any clinically relevant bin structure, scalable to the demands of a clinical laboratory workflow, and flexible with respect to advances in genomics. We anticipate that application of this strategy will facilitate pre-test informed consent, laboratory analysis, and post-test return of results in a clinical context.
Whole genome sequencing; whole exome sequencing; clinical informatics; incidental findings; secondary findings
The internal pudendal arteries are the key resistance vessels controlling the peripheral circulatory component of sexual responses in both male and females. Previous studies in the male rat demonstrated that this vessel has markedly heightened susceptibility to vascular damage compared to other vessels in the body. Evidence suggests that the female may also be susceptible to vascular pathologies contributing to sexual dysfunction.
To characterize the anatomical, morphological and functional properties of the pudendal artery in female rats.
The pelvic arteries in young Sprague-Dawley female rats were dissected to generate a composite representation of the vascular gross anatomy. Morphometry was performed on perfusion-fixed pudendal arteries whereas others were mounted in a wire myograph to assess responses to vasoactive drugs. These measures were contrasted with a previous study examining male rats.
Main Outcomes Measured
Gross anatomy, lumen diameter, wall thickness, cross sectional area and contractile responses in the internal pudendal artery.
The gross anatomy of the pudendal artery in female rats appears to parallel that found in male rats; acting as the primary feeder vessel of the clitoral, labial and vaginal tissue. Compared to the male rat, the female pudendal artery has a smaller lumen diameter (169±5.7 vs 303±13.8um), wall thickness (14±0.7 vs 47±2.2um) and cross-sectional area (8±0.4 vs 52±3.4×103 μm2). These structural differences also translate into a decreased contractile capacity of the pudendal arteries from female rats vs. males (8.1±2.7 vs 20±1.4mN).
Although the gross anatomical features of the vasculature tree supplying the genital tissue in male and female rats appears to have similarities, the tissue specific properties of the vessel itself has a very different structure-function balance. We hypothesize this discordance likely reflects the very different sex-specific roles of this vessel in regulating blood flow during arousal.
We recently reported the isolation of a mutant of Pyrococcus furiosus, COM1, that is naturally and efficiently competent for DNA uptake. While we do not know the exact nature of this mutation, the combined transformation and recombination frequencies of this strain allow marker replacement by direct selection using linear DNA. In testing the limits of its recombination efficiency, we discovered that marker replacement was possible with as few as 40 nucleotides of flanking homology to the target region. We utilized this ability to design a strategy for selection of constructed deletions using PCR products with subsequent excision, or “pop-out,” of the selected marker. We used this method to construct a “markerless” deletion of the trpAB locus in the GLW101 (COM1 ΔpyrF) background to generate a strain (JFW02) that is a tight tryptophan auxotroph, providing a genetic background with two auxotrophic markers for further strain construction. The utility of trpAB as a selectable marker was demonstrated using prototrophic selection of plasmids and genomic DNA containing the wild-type trpAB alleles. A deletion of radB was also constructed that, surprisingly, had no obvious effect on either recombination or transformation, suggesting that its gene product is not involved in the COM1 phenotype. Attempts to construct a radA deletion mutation were unsuccessful, suggesting that this may be an essential gene. The ease and speed of this procedure will facilitate the construction of strains with multiple genetic changes and allow the construction of mutants with deletions of virtually any nonessential gene.
The American Board of Internal Medicine Certification Examination (ABIM-CE) is one of several methods used to assess medical knowledge, an Accreditation Council for Graduate Medical Education (ACGME) core competency for graduating internal medicine residents. With recent changes in graduate medical education program directors and internal medicine residents are seeking evidence to guide decisions regarding residency elective choices. Prior studies have shown that formalized elective curricula improve subspecialty ABIM-CE scores. The primary aim of this study was to evaluate whether the number of subspecialty elective exposures or the specific subspecialties which residents complete electives in impact ABIM-CE scores.
ABIM-CE scores, elective exposures and demographic characteristics were collected for MedStar Georgetown University Hospital internal medicine residents who were first-time takers of the ABIM-CE in 2006–2010 (n=152). Elective exposures were defined as a two-week period assigned to the respective subspecialty. ABIM-CE score was analyzed using the difference between the ABIM-CE score and the standardized passing score (delta-SPS). Subspecialty scores were analyzed using percentage of correct responses. Data was analyzed using GraphPad Prism version 5.00 for Windows.
Paired elective exposure and ABIM-CE scores were available in 131 residents. There was no linear correlation between ABIM-CE mean delta-SPS and the total number of electives or the number of unique elective exposures. Residents with ≤14 elective exposures had higher ABIM-CE mean delta-SPS than those with ≥15 elective exposures (143.4 compared to 129.7, p=0.051). Repeated electives in individual subspecialties were not associated with significant difference in mean ABIM-CE delta-SPS.
This study did not demonstrate significant positive associations between individual subspecialty elective exposures and ABIM-CE mean delta-SPS score. Residents with ≤14 elective exposures had higher ABIM-CE mean delta-SPS than those with ≥15 elective exposures suggesting there may be an “ideal” number of elective exposures that supports improved ABIM-CE performance. Repeated elective exposures in an individual specialty did not correlate with overall or subspecialty ABIM-CE performance.
Resident education; Gender; Elective; Subspecialty; Graduate medical education
Atherosclerosis developed during premenopausal years predicts postmenopausal atherosclerosis burden. The objective of this study was to determine the effects of dietary soy protein isolate (SPI) and social status on atherogenesis and arterial gene expression in a premenopausal monkey model.
Socially housed premenopausal cynomolgus macaques (n = 84) were fed an atherogenic diet deriving protein from casein/lactalbumin or SPI (containing 1.88 mg isoflavones/g). After 36 months of diet consumption, iliac artery biopsies were assessed for atherosclerosis and expression of mRNA transcripts related to inflammation, macrophage and T-cell content, and estrogen receptors (ERs).
SPI reduced plaque size (P < 0.05), total plasma cholesterol, non–high-density lipoprotein cholesterol (HDLc), and the total plasma cholesterol/HDLc ratio (all P < 0.003), while increasing triglycerides (P < 0.006) and HDLc (P < 0.0001). Arterial mRNA for CD68 (P < 0.001), CD3 (P < 0.02), and CD4 (P < 0.001) and inflammatory markers monocyte chemotactic protein-1, intercellular adhesion molecule-1, and interleukin-6 (all P < 0.0001) were also lower in the group receiving SPI. For most outcomes, this effect remained even after adjustments for plaque size and plasma lipid concentrations. Arterial ER-α was inversely associated with atherosclerosis (P < 0.02) and increased with SPI (P < 0.001). Subordinate monkeys had lower ER-β (P < 0.02) and higher interleukin-6 (P < 0.05) transcripts but did not differ from dominant monkeys in extent of atherosclerosis (P > 0.9).
Premenopausal consumption of SPI had plasma lipid–independent beneficial effects on the pathobiological processes involved in atherosclerotic plaque development, thus potentially establishing the basis for reduced postmenopausal complications. Dominant social status provided similar, albeit less extensive, benefits in risk markers.
Isoflavones; Inflammation; Nuclear factor-κB; Social status; Cynomolgus macaques