Promoters are key players in gene regulation. They receive signals from various sources (e.g. cell surface receptors) and control the level of transcription initiation, which largely determines gene expression. In vertebrates, transcription start sites and surrounding regulatory elements are often poorly defined. To support promoter analysis, we present CORG , a framework for studying upstream regions including untranslated exons (5' UTR).
The automated annotation of promoter regions integrates information of two kinds. First, statistically significant cross-species conservation within upstream regions of orthologous genes is detected. Pairwise as well as multiple sequence comparisons are computed. Second, binding site descriptions (position-weight matrices) are employed to predict conserved regulatory elements with a novel approach. Assembled EST sequences and verified transcription start sites are incorporated to distinguish exonic from other sequences.
As of now, we have included 5 species in our analysis pipeline (man, mouse, rat, fugu and zebrafish). We characterized promoter regions of 16,127 groups of orthologous genes. All data are presented in an intuitive way via our web site. Users are free to export data for single genes or access larger data sets via our DAS server . The benefits of our framework are exemplarily shown in the context of phylogenetic profiling of transcription factor binding sites and detection of microRNAs close to transcription start sites of our gene set.
The CORG platform is a versatile tool to support analyses of gene regulation in vertebrate promoter regions. Applications for CORG cover a broad range from studying evolution of DNA binding sites and promoter constitution to the discovery of new regulatory sequence elements (e.g. microRNAs and binding sites).
Pigment epithelium-derived factor (PEDF) is an endogenously produced glycoprotein with a spectrum of biological roles across diverse pathologies. Recent research has focused on the biochemical properties of PEDF and its associated receptors. This review discusses the recent developments in PEDF biochemistry and how this new knowledge will help progress our understanding of PEDF as a molecular mediator for anti-angiogenesis and -tumorigenesis. Additionally, pathophysiological roles for PEDF in healing and tissue homeostasis are being revealed and our enhanced understanding of the interactions between PEDF and its receptors may yet prove useful in propelling PEDF towards clinical application.
Pigment epithelium-derived factor; structure; receptor; biochemistry; healing; repair
Although 3DCRT is the worldwide standard for the treatment of esophageal cancers, IMRT improves dose conformality and reduces radiation exposure to normal tissues. We hypothesized that the dosimetric advantages of IMRT should translate to substantive benefits in clinical outcomes compared to 3DCRT.
Methods and Materials
Analysis was performed on 676 nonrandomized patients (3DCRT=413, IMRT=263) with stage Ib-IVa (AJCC 2002) esophageal cancers treated with chemoradiation at a single institution from 1998–2008. An inverse probability of treatment weighting (IPW) and inclusion of propensity score (treatment probability) as a covariate were used to compare overall survival (OS) time, time to local failure, and time to distant metastasis, while accounting for effects of other clinically relevant covariates. Propensity scores were estimated using logistic regression.
A fitted multivariate inverse probability weighted (IPW)-adjusted Cox model showed that OS time was significantly associated with several well-known prognostic factors, along with radiation modality (IMRT vs 3DCRT, HR=0.72, p<0.001). Compared to IMRT, 3DCRT patients had a significantly greater risk of dying (72.6% vs 52.9%, IPW log rank test: p<0.0001) and for local-regional recurrence (LRR) (p=0.0038). There was no difference in cancer-specific mortality (Gray’s test, p=0.86), or distant metastasis (p=0.99) between the two groups. An increased cumulative incidence of cardiac deaths was seen in the 3DCRT group (p=0.049), but most deaths were undocumented (5 year estimate: 11.7% in 3DCRT vs 5.4% in IMRT, Gray’s test, p=0.0029).
Overall survival, locoregional control, and non-cancer related deaths were significantly better for IMRT compared to 3DCRT. Although these results need confirmation, IMRT should be considered for the treatment of esophageal cancer.
IMRT; 3D-conformal radiation therapy; chemoradiation; esophageal cancer; propensity score
Mentorship influences career planning, academic productivity, professional satisfaction, and most notably, the pursuit of academic medicine careers. Little is known about the role of mentoring in recruiting Black/African American and Hispanic/Latino residents into academia. The objective of this study was to assess the influence of mentoring on academic medicine career choice among a cohort of racially and ethnically diverse residents.
A strategic convenience sample of U.S. residents attending national professional conferences between March and July 2010; residents completed a quantitative survey and a subset participated in focus groups.
Of the 250 residents, 183 (73%) completed surveys and 48 participated in focus groups. Thirty-eight percent of residents were white, 31% Black/African American, 17% Asian/other, and 14% Hispanic/Latino. Most respondents (93%) reported that mentorship was important for entering academia, and 70% reported having sufficient mentorship to pursue academic careers. Three themes about mentorship emerged from focus groups: (1) qualities of successful mentorship models; (2) perceived benefits of mentorship; and (3) the value of racial/ethnic and gender concordance. Residents preferred mentors they selected rather than ones assigned to them, and expressed concern about faculty using checklists. Black/African American, Hispanic/Latino, and female residents described actively seeking out mentors of the same race/ethnicity and gender, but expressed difficulty finding such mentors. Lack of racial/ethnic concordance was perceived as an obstacle for minority mentees, requiring explanation of the context and nuances of their perspectives and situations to non-minority mentors.
The majority of residents in this study reported having access to mentors. However, data show that the lack of diverse faculty mentors may impede diverse residents’ satisfaction and benefit from mentorship relationships compared to white residents. These findings are important for residency programs striving to enhance resident mentorship and for institutions working to diversify their faculty and staff to achieve institutional excellence.
Residents; Postgraduate; Academic careers; Career choice; Diversity; Minority; Mentorship; Mentoring; Medical education
Sorting nexin 27 (SNX27), a brain-enriched PDZ domain protein, regulates endocytic sorting and trafficking. Here, we show that Snx27−/− mice exhibit severe neuronal deficits in the hippocampus and cortex. While Snx27+/− mice exhibit grossly normal neuroanatomy, we find defects in synaptic function, learning and memory, and a reduction in ionotropic glutamate receptors (NMDARs and AMPARs). SNX27 interacts with these receptors through its PDZ domain, regulating their recycling to the plasma membrane. We demonstrate a concomitant reduction of SNX27 and C/EBPβ in Down syndrome brains and identify C/EBPβ as a transcription factor for SNX27. Down syndrome causes over-expression of miR-155, a chromosome 21-encoded microRNA that negatively regulates C/EBPβ, thereby reducing SNX27 and resulting in synaptic dysfunction. Up-regulating SNX27 in the hippocampus of Down syndrome mice rescues synaptic and cognitive deficits. Our identification of the role of SNX27 in synaptic function establishes a novel molecular mechanism of Down syndrome pathogenesis.
Exceedingly little experimental research exists on the popular recreational drug mephedrone (4-methylmethcathinone) despite clinical reports concerning its behavioral and cardiovascular toxicity.
To characterize mephedrone preclinically by examining its capacity to: 1) serve as a discriminative stimulus, 2) disrupt the acquisition of response sequences, and 3) disrupt mean arterial pressure (MAP) and heart rate (HR).
Methods and Results
In one group of subjects that reliably discriminated 3.2 mg/kg of mephedrone from saline (n=9), substitution tests indicated that stimulants (cocaine, MDMA and methamphetamine) more closely approximated the mephedrone discriminative stimulus than non-stimulants (fenfluramine, morphine, and phencyclidine), although none fully substituted. In a second group (n=6), mephedrone (0.56–10 mg/kg, i.p.) dose-dependently decreased response rate and increased errors in both components of a procedure in which subjects either acquired a new response sequence each session (repeated acquisition) or completed the same response sequence each session (performance). Finally, in a third group (n=12), radio telemetry probes were used to measure the changes in MAP and HR elicited by mephedrone and then compared them to a known stimulant, methamphetamine. In these studies, mephedrone (0.01–9 mg/kg, i.v.) elicited increases in MAP and HR that were very similar to those elicited by methamphetamine (0.01–9 mg/kg, i.v.). The tachycardia and pressor responses to mephedrone (3 mg/kg) were blocked by the β-blocker atenolol (1 mg/kg, i.v.) and the α1, α2-blocker phentolamine (3 mg/kg, i.v.), respectively.
Mephedrone produces behavioral and cardiovascular responses that are similar to other stimulants; however, differences from the classical stimulants were also apparent.
mephedrone; bath salts; drug discrimination; learning; blood pressure; heart rate; stimulants
Diverse pathological changes occur in the white matter (WM) in patients with schizophrenia. Various microstructural alterations including a reduction in axonal number or diameter, reduced myelination or poor coherence of fibers could account for these changes. Abnormal integrity of macromolecules such as myelin (‘dysmyelination’) can be studied by applying multiple modalities of WM imaging such as diffusion tensor (DTI) and magnetization transfer imaging (MTI) in parallel. Using ultra-high field (7 Tesla) MTI in 17 clinically stable patients with schizophrenia and 20 controls, we evaluated the voxel-wise distribution of macromolecular WM abnormalities. Patients had a significant reduction in magnetization transfer ratio (MTR) in WM adjacent to visual processing regions and inferior temporal cortex (Cohen’s d=1.54). Among the regions showing MTR reduction, a concurrent reduction in fractional anisotropy occurs proximal to the lingual gyrus. Multiple regression analysis revealed that the degree of Fractional Anisotropy (FA) reduction in the putatively ‘dysmyelinated’ regions in patients predicted impaired processing speed (β=0.74;p=0.003), a core cognitive dysfunction in schizophrenia. In controls, MTR/FA in the occipito-temporal regions were not associated with processing speed. Our findings suggest that dysmyelination in visual processing regions is present in patients with schizophrenia with greatest cognitive and functional impairment. Combined DTI/MTI deficits in the occipito-temporal region may be an important variable when considering potential treatment targets for improving cognitive function in schizophrenia.
myelination; magnetization transfer; visual processing; occipital; schizophrenia
C/EBPβ is an important regulator of oncogene-induced senescence (OIS). Here, we show that C/EBPγ, a heterodimeric partner of C/EBPβ whose biological functions are not well understood, inhibits cellular senescence. Cebpg−/− mouse embryonic fibroblasts (MEFs) proliferated poorly, entered senescence prematurely, and expressed a proinflammatory gene signature, including elevated levels of senescence-associated secretory phenotype (SASP) genes whose induction by oncogenic stress requires C/EBPβ. The senescence-suppressing activity of C/EBPγ required its ability to heterodimerize with C/EBPβ. Covalently linked C/EBPβ homodimers (β∼β) inhibited the proliferation and tumorigenicity of RasV12-transformed NIH 3T3 cells, activated SASP gene expression, and recruited the CBP coactivator in a Ras-dependent manner, whereas γ∼β heterodimers lacked these capabilities and efficiently rescued proliferation of Cebpg−/− MEFs. C/EBPβ depletion partially restored growth of C/EBPγ-deficient cells, indicating that the increased levels of C/EBPβ homodimers in Cebpg−/− MEFs inhibit proliferation. The proliferative functions of C/EBPγ are not restricted to fibroblasts, as hematopoietic progenitors from Cebpg−/− bone marrow also displayed impaired growth. Furthermore, high CEBPG expression correlated with poorer clinical prognoses in several human cancers, and C/EBPγ depletion decreased proliferation and induced senescence in lung tumor cells. Our findings demonstrate that C/EBPγ neutralizes the cytostatic activity of C/EBPβ through heterodimerization, which prevents senescence and suppresses basal transcription of SASP genes.
Immune control of Mycobacterium tuberculosis depends on interferon γ (IFN-γ)–producing CD4+ lymphocytes. Previous studies have shown that T cells from patients with tuberculosis produce less IFN-γ, compared with healthy donors, in response to mycobacterial antigens, although IFN-γ responses to mitogens are preserved. In this work, we found that M. tuberculosis–induced IFN-γ production by human T cells correlated with phosphorylation of the mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase (ERK), and p38. Moreover, the majority of IFN-γ–producing T cells expressed signaling lymphocyte activation molecule (SLAM), and SLAM activation further increased ERK phosphorylation. Interestingly, patients with tuberculosis had delayed activation of ERK and p38, and this was most marked in patients with the poorest IFN-γ responses (ie, low responders). Besides, SLAM signaling failed to phosphorylate ERK in low responders. Our findings suggest that activation of p38 and ERK, in part through SLAM, mediates T-cell IFN-γ production in response to M. tuberculosis, a pathway that is defective in patients with tuberculosis.
Tuberculosis; signaling; CREB; MAPK; IFN-γ
CD1d is a nonclassical Ag-presenting molecule that presents glycolipid Ags to NKT cells that are involved in immune defense and tumor rejection. It also plays a role in immunoregulatory functions in the epidermis. The mechanisms controlling the expression of CD1d are not well understood. Therefore, we cloned the CD1d gene promoter and characterized its activities in primary human keratinocytes and other cell lines of epithelial origin. We found that a CCAAT box in the CD1d promoter is required for its expression in keratinocytes. We show here that transcription factor C/EBP-β binds to the CCAAT box in the CD1d promoter in vitro and in vivo. Consistent with these observations, deletion of the gene encoding for C/EBP-β caused a loss of CD1d expression. The in vivo regulation of CD1d has significant implications for the pathologic mechanisms of certain immunologic skin diseases in which NKT cells play a role, such as allergic contact dermatitis and psoriasis. Together, these data show a central role for C/EBP-β in regulating CD1d transcription.
Abiraterone acetate, an androgen biosynthesis inhibitor, improves overall survival (OS) in metastatic castration-resistant prostate cancer (mCRPC) post-chemotherapy. Many mCRPC patients never receive chemotherapy and thus cannot benefit from abiraterone acetate; we evaluated this agent in mCRPC patients who had not received chemotherapy.
In this double-blind study, 1088 patients were randomized 1:1 to abiraterone acetate (1000 mg) plus prednisone (5 mg twice daily) or placebo plus prednisone. Co-primary end points were radiographic progression-free survival (rPFS) and OS. Secondary end points measured clinically relevant landmarks of mCRPC progression. Patient-reported outcomes included pain progression and quality of life.
The study was unblinded after a planned interim analysis (IA) at 43% of OS events. Treatment with abiraterone acetate-prednisone resulted in a 57% reduction in the risk of radiographic progression or death (hazard ratio [HR], 0.43; 95% confidence interval [CI]: 0.35 to 0.52; P<0.001; 13% OS events IA) and an estimated 25% decrease in the risk of death (HR, 0.75; 95% CI: 0.61 to 0.93; P=0.009; 43% OS events IA). Secondary end points supported superiority of abiraterone acetate-prednisone: time to cytotoxic chemotherapy initiation, opiate use for cancer-related pain, prostate-specific antigen progression (all P<0.001) and performance status deterioration (P=0.005). Self-reported time to pain progression and patient functional status degradation favored abiraterone acetate-prednisone (P=0.05 and P=0.003). Grade 3/4 mineralocorticoid-related adverse events and liver function test abnormalities were more common with abiraterone acetate-prednisone.
Abiraterone acetate produces OS and rPFS benefits, as well as significant delays in clinical deterioration and initiation of chemotherapy, in mCRPC.
Abiraterone acetate; prednisone; metastatic castration-resistant prostate cancer; androgen; CYP17
Malnutrition is a significant clinical problem in infants with biliary atresia. The natural history of poor growth and its potential association with early transplantation or death in children with biliary atresia was determined. Serial weight- and length-for-age z-scores were computed as part of a retrospective study of 100 infants who underwent hepatoportoenterostomy (HPE) for biliary atresia at 9 U.S. pediatric centers between 1997 and 2000. Poor outcome was defined as transplantation or death by 24 months of age (n = 46) and good outcome was defined as survival with native liver at 24 months of age with total serum bilirubin less than 6 mg/dL (n = 54). Growth velocity was significantly slower in the poor outcome group compared to the good outcome group (P < 0.001 for both weight and length). Mean weight z-scores were significantly lower by 6 months after HPE in the poor outcome group (−2.1 ± 1.4) compared to the good outcome group (−1.2 ± 1.4) (P < 0.001). In a subgroup with total bilirubin between 2 and 6 mg/dL at 3 months after HPE (n = 28), the weight z-scores at 3 months after HPE were significantly lower in the poor outcome group (−2.0 ± 1.2) compared to the good outcome group (−1.0 ± 1.2) (P = 0.04) despite similar bilirubin concentrations.
Growth failure after HPE was associated with transplantation or death by 24 months of age. The combination of intermediate bilirubin concentrations and poor mean weight z-scores 3 months after HPE was also associated with poor clinical outcome.
Postoperative pain after total knee arthroplasty remains one of the most important challenges facing patients undergoing this surgery. Providing a balance of adequate analgesia while limiting the functional impact of regional anesthesia and minimizing opioid side effects is critical to minimize adverse events and improve patient satisfaction.
We asked whether bupivacaine delivered through an elastomeric device decreases the (1) patients’ perception of pain after TKA; (2) narcotic consumption; and (3) narcotic-related side effects as compared with a placebo.
In this prospective, double-blind, placebo-controlled study, all patients received standardized regional anesthesia, a preemptive and multimodal analgesic protocol, and a continuous intraarticular infusion at 5 mL/hour through an elastomeric infusion pump. The patients were randomized to receive either an infusion pump filled with (1) 300 mL of 0.5% bupivacaine, the experimental group (n = 75); or (2) 300 mL of 0.9% normal saline solution, the control group (n = 75). Data concerning postoperative pain levels through a visual analog scale, postoperative opioid consumption, opioid-related side effects, and complications were collected and analyzed.
Patients in the experimental group receiving the bupivacaine reported a reduction in pain levels in highest, lowest, and current visual analog scale scores compared with the placebo group on the first postoperative day and highest visual analog scale score on postoperative Day 2 along with a 33% reduction in opioid consumption on postoperative Day 2 and a 54% reduction on postoperative Day 3.
In patients undergoing TKA, continuous intraarticular analgesia provided an effective adjunct for pain relief in the immediate postoperative period without the disadvantages encountered with other analgesic methods.
Level of Evidence
Level I, therapeutic study. See Guidelines for Authors for a complete description of levels of evidence.
Human rhinoviruses (HRVs) cause common colds, and the recently discovered HRV-C is increasingly associated with lower respiratory illness among populations such as children and asthmatic patients.
To determine how HRV-C is associated with respiratory illness and to evaluate changes in prevalence and species over 2 decades.
A prospective study of children younger than 5 years was performed at the Vanderbilt Vaccine Clinic over a 21-year period. Nasal-wash specimens from children presenting with upper or lower respiratory illness at acute care visits were tested for HRV and HRV-positives genotyped. Demographic and clinical features were compared between children with or without HRV, and with different HRV species.
HRV was detected in 190 of 527 (36%) specimens from a population of 2009 children from 1982 through 2003. Of these, 36% were HRV-C. Age (P = .039) and month of illness (P <.001) were associated with HRV infection and HRV species. HRV-C was significantly associated with lower respiratory illness, compared with HRV-A (P = .014). HRV-A and HRV-C prevalence fluctuated throughout the 21-year period; HRV-C was more prevalent during winter (P = .058).
HRV-C is not a new virus but has been significantly associated with childhood lower respiratory illness in this population for several decades. Temporal changes in virus prevalence occur, and season may predict virus species. Our findings have implications for diagnostic, preventive, and treatment strategies due to the variation in disease season and severity based on species of HRV infection.
Rhinovirus; HRV-C; children; season; lower respiratory illness
Typical oncology practice often includes not only an initial, frontline treatment, but also subsequent treatments given if the initial treatment fails. The physician chooses a treatment at each stage based on the patient’s baseline covariates and history of previous treatments and outcomes. Such sequentially adaptive medical decision-making processes are known as dynamic treatment regimes, treatment policies, or multi-stage adaptive treatment strategies. Conventional analyses in terms of frontline treatments that ignore subsequent treatments may be misleading, because they actually are an evaluation of more than front-line treatment effects on outcome. We are motivated by data from a randomized trial of four combination chemotherapies given as frontline treatments to patients with acute leukemia. Most patients in the trial also received a second-line treatment, chosen adaptively and subjectively rather than by randomization, either because the initial treatment was ineffective or the patient’s cancer later recurred. We evaluate effects on overall survival time of the 16 two-stage strategies that actually were used. Our methods include a likelihood-based regression approach in which the transition times of all possible multi-stage outcome paths are modeled, and estimating equations with inverse probability of treatment weighting to correct for bias. While the two approaches give different numerical estimates of mean survival time, they lead to the same substantive conclusions when comparing the two-stage regimes.
Causal inference; Clinical trial; Dynamic treatment regime; Treatment policy
To evaluate the accuracy and consistency of a gradient-based PET segmentation method, GRADIENT, as compared to manual (MANUAL) and constant threshold (THRESHOLD) methods.
Methods and Materials
Contouring accuracy was evaluated with sphere phantoms and clinically realistic Monte Carlo PET phantoms of the thorax. The sphere phantoms were 10–37 mm in diameter and were acquired at 5 institutions emulating clinical conditions. One institution also acquired a sphere phantom with multiple source-to-background ratios (SBR) of 2:1, 5:1, 10:1, 20:1, and 70:1. One observer segmented (contoured) each sphere with GRADIENT and THRESHOLD from 25–50% at 5% increments. Subsequently, seven physicians segmented lessions (7–264ml) from 25 digital thorax phantoms using GRADIENT, THRESHOLD, and MANUAL.
For spheres < 20 mm in diameter, GRADIENT was the most accurate with a mean absolute %error in diameter of 8.15% (10.2%SD) compared to 49.2% (51.1%SD) for 45% THRESHOLD (p < 0.005). For larger spheres the methods were statistically equivalent. For varying SBR, GRADIENT was the most accurate for spheres > 20 mm, (p < 0.065) and < 20 mm (p < 0.015).
For digital thorax phantoms, GRADIENT was the most accurate, (p-value < 0.01), with a mean absolute %error in volume of 10.99% (11.9%SD) followed by 25% THRESHOLD at 17.5% (29.4%SD), and MANUAL, at 19.5% (17.2%SD). GRADIENT had the least systematic bias, 4 with a mean %error in volume of −0.05% (16.2%SD) compared with 25% THRESHOLD at - 2.1% (34.2%SD) and MANUAL at −16.3% (20.2%SD) (p-value < 0.01). Inter-observer variability was reduced using GRADIENT compared to both 25% THRESHOLD and MANUAL (p-value < 0.01, Levene's Test).
GRADIENT was the most accurate and consistent technique for target volume contouring. GRADIENT was also the most robust for varying imaging conditions. GRADIENT has the potential to play an important role for tumor delineation in radiation therapy planning and response assessment.
PET scan; lung cancer; tumor segmentation; radiation therapy planning
Background. Light exposure to the eye can influence different physiological functions, for example, the suprachiasmatic nucleus (SCN). By affecting the autonomic nervous system, the SCN may influence the heart rate variability (HRV). Standardized colored light exposure alters HRV but the results are inconsistent. In this study we investigated the effects of nonstandardized red light (approx. 640 nm) and blue (approx. 480 nm) light (approx. 50 lx) on cardiorespiratory coordination and HRV. Methods. 17 healthy subjects (7 males, age: 26.5 ± 6.2 years) were exposed to the following sequence (10 minutes each): daylight-red light-daylight-blue light-daylight. Red and blue lights were created by daylight passing through colored glass panes. Spectral measures of HRV (LF: low frequency, HF: high frequency oscillations, and sympathovagal balance LF/HF) and measures of cardiorespiratory coordination (HRR: heart respiration ratio, PCR: phase coordination ratio) were analyzed. Results. The LF component increased and the HF component decreased after red light. Consequently, LF/HF increased after red light. Furthermore, during red light HRR and PCR confined to 4 : 1, that is, 4 heartbeats during one respiratory cycle. Conclusion. Nonstandardized red and blue lights are able to alter the autonomic control reflected by HRV as well as cardiorespiratory coordination.
The striated muscle–specific actin-binding proteins Xin and Xirp2 are identified as novel ligands of the SH3 domains of the thin filament ruler nebulin and nebulette. The interaction is spatially restricted to structures associated with myofibril development or remodeling, indicating a role for these proteins in myofibril assembly and repair.
The Xin actin-binding repeat–containing proteins Xin and XIRP2 are exclusively expressed in striated muscle cells, where they are believed to play an important role in development. In adult muscle, both proteins are concentrated at attachment sites of myofibrils to the membrane. In contrast, during development they are localized to immature myofibrils together with their binding partner, filamin C, indicating an involvement of both proteins in myofibril assembly. We identify the SH3 domains of nebulin and nebulette as novel ligands of proline-rich regions of Xin and XIRP2. Precise binding motifs are mapped and shown to bind both SH3 domains with micromolar affinity. Cocrystallization of the nebulette SH3 domain with the interacting XIRP2 peptide PPPTLPKPKLPKH reveals selective interactions that conform to class II SH3 domain–binding peptides. Bimolecular fluorescence complementation experiments in cultured muscle cells indicate a temporally restricted interaction of Xin-repeat proteins with nebulin/nebulette during early stages of myofibril development that is lost upon further maturation. In mature myofibrils, this interaction is limited to longitudinally oriented structures associated with myofibril development and remodeling. These data provide new insights into the role of Xin actin-binding repeat–containing proteins (together with their interaction partners) in myofibril assembly and after muscle damage.
Intracellular delivery of nanocarriers (NC) is controlled by their design and target cell phenotype, microenvironment, and functional status. Endothelial cells (EC) lining the vascular lumen represent an important target for drug delivery. Endothelium in vivo is constantly or intermittently (as, for example, during ischemia-reperfusion) exposed to blood flow, which influences NC–EC interactions by changing NC transport properties, and by direct mechanical effects upon EC mechanisms involved in NC binding and uptake. EC do not internalize antibodies to marker glycoprotein PECAM(CD31), yet internalize multivalent NC coated with PECAM antibodies (anti-PECAM/NC) via a noncanonical endocytic pathway distantly related to macropinocytosis. Here we studied the effects of flow on EC uptake of anti-PECAM/NC spheres (∼180 nm diameter). EC adaptation to chronic flow, manifested by cellular alignment with flow direction and formation of actin stress fibers, inhibited anti-PECAM/NC endocytosis consistent with lower rates of anti-PECAM/NC endocytosis in vivo in arterial compared to capillary vessels. Acute induction of actin stress fibers by thrombin also inhibited anti-PECAM/NC endocytosis, demonstrating that formation of actin stress fibers impedes EC endocytic machinery. In contrast, acute flow without stress fiber formation, stimulated anti-PECAM/NC endocytosis. Anti-PECAM/NC endocytosis did not correlate with the number of cell-bound particles under flow or static conditions. PECAM cytosolic tail deletion and disruption of cholesterol-rich plasmalemma domains abrogated anti-PECAM/NC endocytosis stimulation by acute flow, suggesting complex regulation of a flow-sensitive endocytic pathway in EC. The studies demonstrate the importance of the local flow microenvironment for NC uptake by the endothelium and suggest that cell culture models of nanoparticle uptake should reflect the microenvironment and phenotype of the target cells.
intracellular delivery; endothelial cells; vascular immunotargeting; cell adhesion molecules; endocytosis; fluid shear stress
Bacteria utilize a large multi-protein chemosensory array to sense attractants and repellents in their environment. The array is a hexagonal lattice formed from three core proteins: a transmembrane receptor, the His kinase CheA, and the adaptor protein CheW. The resulting, highly networked array architecture yields several advantages including strong positive cooperativity in the attractant response and rapid signal transduction through the preformed, integrated signalling circuit. Moreover, when isolated from cells or reconstituted in isolated bacterial membranes, the array possesses extreme kinetic stability termed ultra-stability (Erbse & Falke (2009) Biochemistry 48:6975-87) and is the most long-lived multi-protein enzyme complex described to date. The isolated array retains kinase activity, attractant regulation and its bound core proteins for days or more at 22° C. The present work quantitates this ultra-stability and investigates its origin. The results demonstrate that arrays consist of two major components: (i) a quasi-stable component with a lifetime of 1–2 days that decays due to slow proteolysis of CheA kinase in the lattice, and (ii) a truly ultra-stable component with a lifetime of ~20 days that is substantially more protected from proteolysis. Following proteolysis of the quasi-stable component the apparent positive cooperativity of the array increases, arguing the quasi-stable component is not as cooperative as the ultra-stable component. Introduction of structural defects into the array by coupling a bulky probe to a subset of receptors reveals that modification of only 2% of the receptor population is sufficient to abolish ultra-stability, supporting the hypothesis that the ultra-stable component requires a high level of array spatial order. Overall, the findings are consistent with a model in which the quasi- and ultra-stable components arise from distinct regions of the array, such that the ultra-stable regions possess more extensive, better-ordered, multi-valent interconnectivities between core components, thereby yielding extraordinary stability and cooperativity. Furthermore, the findings indicate that the chemosensory array is a promising platform for the development of ultra-stable biosensors.
protein complex; protein array; protein stability; sensory transduction; chemoreceptor; histidine-kinase; CheA
Lower airway abnormalities are common in patients with primary ciliary dyskinesia (PCD), a pediatric syndrome that results from structural or functional defects in motile cilia. Patients can suffer from recurrent bacterial infection in the lung, bronchiectasis, and respiratory distress in addition to chronic sinusitis, otitis media, infertility, and laterality defects. However, surprisingly little is known about the pulmonary phenotype of mouse models of this disorder.
The pulmonary phenotype of two mouse models of PCD, nm1054 and bgh, which lack Pcdp1 and Spef2, respectively, was investigated by histological and immunohistochemical analysis. In addition, both models were challenged with Streptococcus pneumoniae, a common respiratory pathogen found in the lungs of PCD patients. Histopathological analyses reveal no detectable cellular, developmental, or inflammatory abnormalities in the lower airway of either PCD model. However, exposure to S. pneumoniae results in a markedly enhanced inflammatory response in both models. Based on analysis of inflammatory cells in bronchoalveolar lavage fluid and flow cytometric analysis of cytokines in the lung, the bgh model shows a particularly dramatic lymphocytic response by 3 days post-infection compared to the nm1054 model or wild type animals.
Defects in ciliary motility result in a severe response to pulmonary infection. The PCD models nm1054 and bgh are distinct and clinically relevant models for future studies investigating the role of mucociliary clearance in host defense.
Cilia; Lung; Primary ciliary dyskinesia; Streptococcus pneumoniae
The renal disorder C3 glomerulopathy with dense deposit disease (C3G-DDD) pattern results from complement dysfunction and primarily affects children and young adults. There is no effective treatment, and patients often progress to end-stage renal failure. A small fraction of C3G-DDD cases linked to factor H or C3 gene mutations as well as autoantibodies have been reported. Here, we examined an index family with 2 patients with C3G-DDD and identified a chromosomal deletion in the complement factor H–related (CFHR) gene cluster. This deletion resulted in expression of a hybrid CFHR2-CFHR5 plasma protein. The recombinant hybrid protein stabilized the C3 convertase and reduced factor H–mediated convertase decay. One patient was refractory to plasma replacement and exchange therapy, as evidenced by the hybrid protein quickly returning to pretreatment plasma levels. Subsequently, complement inhibitors were tested on serum from the patient for their ability to block activity of CFHR2-CFHR5. Soluble CR1 restored defective C3 convertase regulation; however, neither eculizumab nor tagged compstatin had any effect. Our findings provide insight into the importance of CFHR proteins for C3 convertase regulation and identify a genetic variation in the CFHR gene cluster that promotes C3G-DDD. Monitoring copy number and sequence variations in the CFHR gene cluster in C3G-DDD and kidney patients with C3G-DDD variations will help guide treatment strategies.
A previous study at the GHESKIO HIV clinic confirmed that highly active antiretroviral therapy (HAART) prophylaxis reduced mother-to-child transmission (MTCT) and infant mortality in Haiti. This analysis looks at maternal outcomes in this cohort after delivery.
Records of 508 HIV-positive Haitian women who delivered between1999-2005 were analyzed. We examined mortality, loss to follow-up, time to death or HAART initiation, and time of decline of CD4 count to350 cells/microliter.
170 women reached a CD4≤200 or developed clinical AIDS and were started on long-term HAART. The median CD4 count at HAART initiation was 178 (IQR 106-227). CD4 decline was stratified by CD4 at delivery to project the mean months to a CD4 of 350. With an initial CD4=350-499 cells/microliter it was 19 months (95% CI 14 - 28) while with a CD4>500 cells/microliter it was 71 months (95% CI 59 - 88). At study close 257 women remained in follow-up with loss to follow up three times less in those on HAART (3.2/100 person-years) than those not on HAART (9.8/100 person-years).
The threshold for starting treatment was often missed in HIV-infected women after delivery. Success of follow-up of women after delivery was favorably influenced by being on HAART. Women with high (>500) initial CD4 counts had a protracted time (5-7 years) before they reach a threshold CD4 count, in contrast to those with CD4<500 cells/μL. Strategies for post-partum treatment of women should be informed by the speed with which they are likely to progress.
Haiti; PMTCT; HAART
Rhodonellum psychrophilum GCM71T, isolated from the cold and alkaline submarine ikaite columns in the Ikka Fjord in Greenland, displays optimal growth at 5 to 10°C and pH 10. Here, we report the draft genome sequence of this strain, which may provide insight into the mechanisms of adaptation to these extreme conditions.