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1.  Hippo coactivator YAP1 upregulates SOX9 and endows stem-like properties to esophageal cancer cells 
Cancer research  2014;74(15):4170-4182.
Cancer stem cells are proposed to initiate and maintain tumor growth. Deregulation of normal stem cell signaling may lead to the generation of cancer stem cells (CSCs); however, the molecular determinants of this process remain poorly understood. Here we show that the transcriptional co-activator YAP1 is a major determinant of CSC properties in non-transformed cells and in esophageal cancer cells by direct upregulation of SOX9. YAP1 regulates the transcription of SOX9 through a conserved TEAD binding site in the SOX9 promoter. Expression of exogenous YAP1 in vitro or inhibition of its upstream negative regulators in vivo results in elevated SOX9 expression accompanied by the acquisition of CSCs properties. Conversely, shRNA-mediated knockdown of YAP1 or SOX9 in transformed cells attenuates CSC phenotypes in vitro and tumorigenecity in vivo. The small molecule inhibitor of YAP1, Verteporfin (VP) significantly blocks CSCs properties in cells with high YAP1 and a high proportion of ALDH1+. Our findings identify YAP1 driven SOX9 expression is a critical event in acquisition of CSC properties, suggesting that YAP1 inhibition may offer an effective means of therapeutically targeting the CSC population.
PMCID: PMC4136429  PMID: 24906622
Hippo; YAP1; SOX9; Cancer stem cells and Esophageal Cancer
2.  Design of a study to determine the impact of insecticide resistance on malaria vector control: a multi-country investigation 
Malaria Journal  2015;14:282.
Progress in reducing the malaria disease burden through the substantial scale up of insecticide-based vector control in recent years could be reversed by the widespread emergence of insecticide resistance. The impact of insecticide resistance on the protective effectiveness of insecticide-treated nets (ITN) and indoor residual spraying (IRS) is not known. A multi-country study was undertaken in Sudan, Kenya, India, Cameroon and Benin to quantify the potential loss of epidemiological effectiveness of ITNs and IRS due to decreased susceptibility of malaria vectors to insecticides. The design of the study is described in this paper.
Malaria disease incidence rates by active case detection in cohorts of children, and indicators of insecticide resistance in local vectors were monitored in each of approximately 300 separate locations (clusters) with high coverage of malaria vector control over multiple malaria seasons. Phenotypic and genotypic resistance was assessed annually. In two countries, Sudan and India, clusters were randomly assigned to receive universal coverage of ITNs only, or universal coverage of ITNs combined with high coverage of IRS. Association between malaria incidence and insecticide resistance, and protective effectiveness of vector control methods and insecticide resistance were estimated, respectively.
Cohorts have been set up in all five countries, and phenotypic resistance data have been collected in all clusters. In Sudan, Kenya, Cameroon and Benin data collection is due to be completed in 2015. In India data collection will be completed in 2016.
The paper discusses challenges faced in the design and execution of the study, the analysis plan, the strengths and weaknesses, and the possible alternatives to the chosen study design.
PMCID: PMC4508808  PMID: 26194648
3.  Effect of Short-Term Enzymatic Treatment on Cell Migration and Cartilage Regeneration: In Vitro Organ Culture of Bovine Articular Cartilage 
Tissue Engineering. Part A  2014;20(13-14):1807-1814.
Depending on the damage extent and adjacent tissue condition in traumatic cartilage injury, it is possible to heal the tissue by resident cells. Unlike autologous chondrocyte implantation, short-term enzymatic treatment is an effective single-step procedure without extra cell expansion. Moreover, this method has been shown to significantly increase cellularity in lesion edges, resulting in enhanced integration and interfacial strength. We hypothesize that the locally digested extracellular matrix by treatment allows effortless cell migration from the adjacent tissue. Full-thickness cartilage discs and osteochondral explants were prepared from mature bovine stifle joints. These specimens were treated with collagenase in a culture medium. Two concentrations, 0.25 and 0.5 mg/mL, were used with various treating time of 10, 30, and 180 min. The cartilages were subsequently washed and cultured with fibrin hydrogel. The effect of enzymatic treatment on cell migration was apparent in both experiments of the cartilage disc and full-thickness cartilage defect model. In the disc culture, the treatment resulted in an approximately three to four times higher number of migrated cells than nontreated control. In short-term collagenase-treated groups, the proteoglycan (PG) loss was localized in the edge of tissue with minimal cell death. The treatment also accelerated cell migration in the full-thickness cartilage defects and some cells differentiated into chondrocytes with the deposit of PG. Gene expression results could support the characteristics of migrated cells, which had migratory ability and chondrogenic differentiation potential with overexpression of collagen type I and II, respectively. Based on these results, short-term enzymatic treatment, which can accelerate cell migration into traumatically injured cartilage, has great potential for clinical application.
PMCID: PMC4086788  PMID: 24428547
4.  Respiratory medicine and research at McGill University: A historical perspective 
The history of respiratory medicine and research at McGill University (Montreal, Quebec) is tightly linked with the growth of academic medicine within its teaching hospitals. Dr Jonathan Meakins, a McGill medical graduate, was recruited to the Royal Victoria Hospital in 1924; as McGill’s first full-time clinical professor and Physician-in-Chief at the Royal Victoria Hospital. His focus on respiratory medicine led to the publication of his first book, Respiratory Function in Disease, in 1925. Meakins moved clinical laboratories from the Department of Pathology and placed them within the hospital. As such, he was responsible for the development of hospital-based research.
Dr Ronald Christie was recruited as a postdoctoral fellow by Meakins in the early 1930s. After his fellowship, he returned to Britain but came back to McGill from St Bartholomew’s Hospital (London, United Kingdom) to become Chair of the Department of Medicine in 1955; he occupied the post for 10 years. He published extensively on the mechanical properties of the lung in common diseases such as emphysema and heart failure.
Dr David Bates was among Dr Christie’s notable recruits; Bates in turn, recruited Drs Maurice McGregor, Margaret Becklake, William Thurlbeck, Joseph Milic-Emili, Nicholas Anthonisen, Charles Bryan and Peter Macklem. Bates published extensively in the area of respiratory physiology and, with Macklem and Christie, coauthored the book Respiratory Function in Disease, which integrated physiology into the analysis of disease.
Dr JA Peter Paré joined the attending staff of the Royal Victoria Hospital and the Royal Edward Laurentian Hospital in 1949. A consummate clinician and teacher, he worked closely with Dr Robert Fraser, the Chair of Radiology, to write the reference text Diagnosis of Diseases of the Chest. This was a sentinel contribution in its focus on radiographic findings as the foundation for a systematic approach to diagnosis, and the correlation of these findings with pathological and clinical observations.
Dr Margaret Becklake immigrated to Montreal from South Africa in 1957. Her research focused on occupational lung disease. She established the respiratory epidemiology research unit at McGill. She was renowned for her insistence on the importance of a clearly stated, relevant research question and for her clarity and insight.
Dr William Thurlbeck, another South African, had developed an interest in emphysema and chronic bronchitis and applied a structure-function approach in collaboration with Peter Macklem and other respirologists. As chief of the Royal Victoria autopsy service, he used pathological specimens to develop a semiquantitative grading system of gross emphysema severity. He promoted the use of morphometry to quantify structural abnormalities.
Dr James Hogg studied the functional consequences of pathological processes for lung function during his PhD studies under the joint supervision of Drs Macklem and Thurlbeck. His contributions to understanding the structural basis for chronic obstructive pulmonary disease (COPD) are numerous, reflecting his transdisciplinary knowledge of respiratory pathology and physiology. He trained other outstanding investigators such as Peter Paré Jr, with whom he founded the Pulmonary Research Laboratory in St Paul’s Hospital in Vancouver (British Columbia) in 1977.
A signal event in the evolution of respiratory research at McGill was the construction of the Meakins-Christie Laboratories in 1972. These laboratories were directed by Dr Peter Macklem, a trainee of Dr Becklake’s. The research within the laboratory initially focused on respiratory mechanics, gas distribution within the lung and the contribution of airways of different sizes to the overall mechanical behaviour of the lungs. The effects of cigarette smoking on lung dysfunction, mechanisms of possible loss of lung elastic recoil in asthma and control of bronchomotor tone were all additional areas of active investigation. Dr Macklem pioneered the study of the physiological consequences of small airway pathology.
Dr Joseph Milic-Emili succeeded Dr Macklem as director of the Meakins-Christie Laboratories in 1979. Milic-Emili was renowned for his work on ventilation distribution and the assessment of pleural pressure. He led the development of convenient tools for the assessment of respiratory drive. He clarified the physiological basis for carbon dioxide retention in patients with COPD placed on high inspired oxygen concentrations.
Another area that captured many investigators’ attention in the 1980s was the notion of respiratory failure as a consequence of respiratory muscle fatigue. Dr Charalambos (‘Charis’) Roussos made seminal contributions in this field. These studies triggered a long-lasting interest in respiratory muscle training, in rehabilitation, and in noninvasive mechanical ventilation for acute and chronic respiratory failure.
Dr Ludwig Engel obtained his PhD under the supervision of Peter Macklem and established himself in the area of ventilation distribution in health and in bronchoconstriction and the mechanics of breathing in asthma; he trained many investigators including one of the authors, Dr Jim Martin, who succeeded Milic-Emili as director of the Meakins Christie Laboratories from 1993 to 2008. Dr Martin developed small animal models of allergic asthma, and adopted a recruitment strategy that diversified the research programs at the Meakins Christie Laboratories.
Dr Manuel Cosio built on earlier work with Macklem and Hogg in his development of key structure-function studies of COPD. He was instrumental in recruiting a new generation of young investigators with interests in sleep medicine and neuromuscular diseases.
The 1970s and 1980s also witnessed the emergence of a topnotch respiratory division at the Montreal General Hospital, in large part reflecting the leadership of Dr Neil Colman, later a lead author of the revised Fraser and Paré textbook. At the Montreal General, areas of particular clinical strength and investigation included asthma, occupational and immunological lung diseases.
In 1989, the Meakins Christie Laboratories relocated to its current site on Rue St Urbain, adjacent to the Montreal Chest Institute. Dr Qutayba Hamid, on faculty at the Brompton Hospital, joined the Meakins-Christie Labs in 1994. In addition to an outstanding career in the area of the immunopathology of human asthma, he broadened the array of techniques routinely applied at the labs and has ably led the Meakins-Christie Labs from 2008 to the present.
The Meakins Christie Laboratories have had a remarkable track record that continues to this day. The basis for its enduring success is not immediately clear but it has almost certainly been linked to the balance of MD and PhD scientists that brought perspective and rigour. The diverse disciplines and research programs also facilitated adaptation to changing external research priorities.
The late 1990s and the early 21st century also saw the flourishing of the Respiratory Epidemiology Unit, under the leadership of Drs Pierre Ernst, Dick Menzies and Jean Bourbeau. It moved from McGill University to the Montreal Chest Institute in 2004. This paved the way for expanded clinical and translational research programs in COPD, tuberculosis, asthma, respiratory sleep disorders and other pulmonary diseases. The faculty now comprises respiratory clinician-researchers and PhD scientists with expertise in epidemiological methods and biostatistics.
Respiratory physiology and medicine at McGill benefitted from a strong start through the influence of Meakins and Christie, and a tight linkage between clinical observation and physiological research. The subsequent recruitment of talented and creative faculty members with absolute dedication to academic medicine continued the legacy. No matter how significant the scientific contributions of the individuals themselves, their most important impact resulted from the training of a large cohort of other gifted physicians and graduate students. Some of these are further described in the accompanying full-length online article.
PMCID: PMC4324519  PMID: 25664457
6.  PTSD and Sexual Orientation: An Examination of Criterion A1 and Non-Criterion A1 Events 
This large-scale cross-sectional study compared posttraumatic stress disorder (PTSD) prevalence among White, Black, and Latino lesbian, gay and bisexual individuals (LGBs; n = 382) and compared them with heterosexual individuals (n = 126). Building on previous research, we relaxed the criteria of the Diagnostic and Statistical Manual of Mental Disorders (4th ed.; DSM–IV; American Psychiatric Association, 1994), allowing non-Criterion A1 events such as ending a relationship, unemployment, homelessness, and separation from parents to qualify, and we assessed differences in PTSD prevalence between standard DSM–IV criteria and the relaxed criteria. Findings revealed that participants reporting a non-Criterion A1 event were more likely than those reporting a Criterion A1 event to have symptoms diagnosable as PTSD. There was no significant difference in either DSM–IV or relaxed Criterion A1 PTSD prevalence between lesbian and gay, and heterosexual individuals or between bisexual and heterosexual individuals. Compared with White LGBs, Black and Latino LGBs had higher prevalence of PTSD with the relaxed Criterion A1 definition, but this was statistically significant only for Latinos.
PMCID: PMC4477838  PMID: 26113955
Criterion A1; PTSD; gay; lesbian; bisexual
Ultrasound in medicine & biology  2014;40(6):1177-1186.
Low-intensity pulsed ultrasound (LIPUS) has been frequently studied for its beneficial effects on the repair of injured articular cartilage. Here, we hypothesized that these effects are due to stimulation of chondrogenic progenitor cell (CPC) migration toward injured areas in cartilage through focal adhesion kinase (FAK) activation. CPC chemotaxis in bluntly impacted osteochondral explants was examined by confocal microscopy and migratory activity of cultured CPCs was measured in trans-well and monolayer scratch assays. FAK activation by LIPUS was analyzed in cultured CPCs by western blot. LIPUS effects were compared with the effects of two known chemotactic factors; formylated-methionine peptides (fMLF), and high-mobility group box 1 (HMGB1) protein. LIPUS significantly enhanced CPC migration on explants and in cell culture assays. Phosphorylation of FAK at the kinase domain (Tyr 576/577) was maximized by 5 minute exposure to LIPUS at a dose of 27.5 mW/cm2 and at a frequency of 3.5 MHz. Treatment with fMLF, but not HMBG1 enhanced FAK activation to a degree similar to LIPUS, but neither fMLF nor HMGB1 enhanced the LIPUS effect. LIPUS-induced CPC migration was blocked by suppressing FAK phosphorylation with a Src family kinases (SFKs) inhibitor that blocks FAK phosphorylation. Our results imply that LIPUS might be utilized to promote cartilage healing by inducing the migration of CPCs to injured sites, which could delay or prevent the onset of post-traumatic osteoarthritis (PTOA).
PMCID: PMC4034572  PMID: 24612644
Low-intensity pulsed ultrasound (LIPUS); Articular Cartilage; Post-traumatic osteoarthritis (PTOA); Focal adhesion kinase (FAK); Cell migration
Post-traumatic osteoarthritis (PTOA) is characterized by progressive cartilage degeneration in injured joints. Since fibronectin fragments (Fn-fs) degrade cartilage mainly through up-regulating matrix metalloproteinases (MMPs) and pro-inflammatory cytokines, we hypothesized that Fn-fs play a key role in PTOA by promoting chondrolysis in and around injured cartilage. To test this hypothesis, we profiled the catabolic events focusing on fibronectin fragmentation and proteinase expression in bovine osteochondral explants following a single blunt impact on cartilage with a drop tower device which created partial-thickness tissue damage. Injured and control explants were cultured for up to 14 days. The presence of Fn-fs, MMPs (-1, -3, -13), ADAMTS-5 in culture media and in cartilage was determined with immunoblotting. The daily proteoglycan (PG) depletion of cartilage matrix was assessed with DMMB assay. The effect of explant-conditioned media on chondrocytes was also examined with immunoblotting. Impacted cartilage released significantly higher amount of native Fn, three chondrolytic Fn-fs and PG than non-impacted controls did. Those increases coincided with up-regulation of MMP-3 both in conditioned media and in impacted cartilage. These findings support our hypothesis that PTOA may be propelled by Fn-fs which act as catabolic mediators through up-regulating cartilage-damaging proteinases.
PMCID: PMC4034576  PMID: 24610678
cartilage; fibronectin fragments; impact; matrix metalloproteinase-3; proteoglycan
9.  Insights into the Recognition, Binding and Reactivity of Catalytic Metallodrugs Targeting Stem Loop IIb of Hepatitis C IRES RNA 
ChemMedChem  2014;9(6):1275-1285.
Complex Cu-GGHYrFK-amide (1-Cu) was previously reported as a novel metallotherapeutic that catalytically inactivates stem loop IIb of the Hepatitis C Virus (HCV) Internal Ribosomal Entry Site (IRES) RNA and demonstrates significant antiviral activity in a cellular HCV replicon assay. Herein are described additional studies focused on understanding the cleavage mechanism, as well as the relationship of catalyst configuration to structural recognition and site-selective cleavage of the structured RNA motif. These are advanced by use of a combination of MALDI-TOF mass spectrometry, melting temperature determination, and computational analysis to develop a structural model for binding and reactivity toward SLIIb of the IRES RNA. In addition, the binding, reactivity, and structural chemistry of the all d-amino acid form of this metallopeptide, complex 2-Cu, is reported and compared to complex 1-Cu. In vitro RNA binding and cleavage assays for complex 2-Cu show a KD of 76 ± 3 nM, and Michaelis-Menten parameters of kcat of 0.14 ± 0.01 min−1 and KM of 7.9 ± 1.2 µM, with a turnover number exceeding 40. In a luciferase-based cellular replicon assay Cu-GGhyrfk-amide shows activity similar to the parent peptide, complex 1-Cu, with IC50 of 1.9 ± 0.4 µM and cytotoxicity exceeding 100 µM. RT-PCR experiments confirm a significant reduction in HCV RNA levels in replicon assays for up to nine days when treated with complex 1-Cu in three day dosing increments. This study shows the influence that the α-carbon stereocenter has for this the new class of compounds, while detailed mass spectrometry and computational analysis provide new insights into the mechanisms of recognition, binding, and reactivity.
PMCID: PMC4163017  PMID: 24756921
HCV IRES RNA; copper ATCUN motif; RNA cleavage; stem-loop; catalytic; metallodrug
10.  Actin cytoskeletal remodeling with protrusion formation is essential for heart regeneration in Hippo-deficient mice 
Science signaling  2015;8(375):ra41.
The mammalian heart regenerates poorly, and damage commonly leads to heart failure. Hippo signaling is an evolutionarily conserved kinase cascade that regulates organ size during development and prevents adult mammalian cardiomyocyte regeneration by inhibiting the transcriptional coactivator Yap, which also responds to mechanical signaling in cultured cells to promote cell proliferation. To identify Yap target genes that are activated during cardiomyocyte renewal and regeneration, we performed Yap chromatin immunoprecipitation sequencing (ChIP-Seq) and mRNA expression profiling in Hippo signaling-deficient mouse hearts. We found that Yap directly regulated genes encoding cell cycle progression proteins, as well as genes encoding proteins that promote F-actin polymerization and that link the actin cytoskeleton to the extracellular matrix. Included in the latter group were components of the dystrophin glycoprotein complex (DGC), a large molecular complex that, when defective, results in muscular dystrophy in humans. Cardiomyocytes near scar tissue of injured Hippo signaling-deficient mouse hearts showed cellular protrusions suggestive of cytoskeletal remodeling. The hearts of mdx mutant mice, which lack functional dystrophin and are a model for muscular dystrophy, showed impaired regeneration and cytoskeleton remodeling, but normal cardiomyocyte proliferation after injury. Our data showed that, in addition to genes encoding cell cycle progression proteins, Yap regulated genes that enhance cytoskeletal remodeling Thus, blocking the Hippo pathway input to Yap may tip the balance so that Yap responds to the mechanical changes associated with heart injury to promote repair.
PMCID: PMC4442128  PMID: 25943351
11.  Temporoparietal Headache as the Initial Presenting Symptom of a Massive Aortic Dissection 
Aortic dissection is a life-threatening medical emergency often presenting with severe chest pain and acute hemodynamic compromise. The presentation of aortic dissection can sometimes be different thus leading to a challenge in prompt diagnosis and treatment as demonstrated by the following presentation and discussion. We present a case of a 71-year-old male who presented to the emergency department with complaints of left sided temporoparietal headache and was eventually diagnosed with a thoracic aortic dissection involving the ascending aorta and descending aorta, with an intramural hematoma in the descending aorta. This case illustrates the importance of keeping in mind aortic dissection as a differential diagnosis in patients with acute onset headaches in which any intracranial source of headache is not found.
PMCID: PMC4438158  PMID: 26064702
12.  Macro advances in microRNAs and myocardial regeneration 
Current opinion in cardiology  2014;29(3):207-213.
Purpose of review
Myocardial injury and disease often results in heart failure, the leading cause of death worldwide. To achieve myocardial regeneration and foster development of efficient therapeutics for cardiac injury, it is essential to uncover molecular mechanisms that will promote myocardial regeneration. In this review, we examine the latest progress made in elucidation of the roles of small non coding RNAs called microRNAs (miRs) in myocardial regeneration.
Recent findings
Promising progress has been made in studying cardiac regeneration. Several miRs, which includes miR-590, miR-199a, miR-17-92 cluster, miR-199a-214 cluster, miR-34a, and miR-15 family, have been recently shown to play an essential role in myocardial regeneration by regulating different processes during cardiac repair, including cell death, proliferation and metabolism. For example, miR-590 promotes cardiac regeneration through activating cardiomyocytes proliferation, while miR-34a inhibits cardiac repair through inducing apoptosis.
These recent findings shed new light on our understanding of myocardial regeneration and suggest potential novel therapeutic targets to treat cardiac disease.
PMCID: PMC4286387  PMID: 24625819
MicroRNA; cardiomyocyte; cardiac injury; regeneration
Focal adhesions are transmembrane protein complexes that attach chondrocytes to the pericellular cartilage matrix and in turn, are linked to intracellular organelles via cytoskeleton. We previously found that excessive compression of articular cartilage leads to cytoskeleton-dependent chondrocyte death. Here we tested the hypothesis that this process also requires integrin activation and signaling via focal adhesion kinase (FAK) and Src family kinase (SFK). Osteochondral explants were treated with FAK and SFK inhibitors (FAKi, SFKi respectively) for 2 hours and then subjected to a death-inducing impact load. Chondrocyte viability was assessed by confocal microscopy immediately and at 24 hours post-impact. With no treatment immediate post-impact viability was 59%. Treatment with 10μM SFKi, 10μM or 100μM FAKi improved viability to 80%, 77%, and 82% respectively (p<0.05). After 24 hours viability declined to 34% in controls, 48% with 10μM SFKi, 45% with 10μM FAKi, and 56% with 100μM FAKi (p<0.01) treatment. These results confirmed that most of the acute chondrocyte mortality was FAK- and SFK-dependent, which implicates integrin-cytoskeleton interactions in the death signaling pathway. Together with previous findings, these data support the hypothesis that the excessive tissue strains accompanying impact loading induce death via a pathway initiated by strain on cell adhesion receptors.
PMCID: PMC4034578  PMID: 24249698
Articular cartilage; Chondrocytes; Focal adhesions; Integrins; Posttraumatic osteoarthritis (PTOA)
14.  A Validated Model of the Pro- and Anti-Inflammatory Cytokine Balancing Act in Articular Cartilage Lesion Formation 
Traumatic injuries of articular cartilage result in the formation of a cartilage lesion and contribute to cartilage degeneration and the risk of osteoarthritis (OA). A better understanding of the framework for the formation of a cartilage lesion formation would be helpful in therapy development. Toward this end, we present an age and space-structured model of articular cartilage lesion formation after a single blunt impact. This model modifies the reaction-diffusion-delay models in Graham et al. (2012) (single impact) and Wang et al. (2014) (cyclic loading), focusing on the “balancing act” between pro- and anti-inflammatory cytokines. Age structure is introduced to replace the delay terms for cell transitions used in these earlier models; we find age structured models to be more flexible in representing the underlying biological system and more tractable computationally. Numerical results show a successful capture of chondrocyte behavior and chemical activities associated with the cartilage lesion after the initial injury; experimental validation of our computational results is presented. We anticipate that our in silico model of cartilage damage from a single blunt impact can be used to provide information that may not be easily obtained through in in vivo or in vitro studies.
PMCID: PMC4354422  PMID: 25806365
articular cartilage; structured model; lesion formation and abatement; EPO; IL-6
15.  Corrigendum: “A Validated Model of the Pro- and Anti-Inflammatory Cytokine Balancing Act in Articular Cartilage Lesion Formation” 
PMCID: PMC4443297  PMID: 26075201
articular cartilage; structured model; lesion formation and abatement; EPO; IL-6
16.  Knockout of SRC-1 and SRC-3 in Mice Decreases Cardiomyocyte Proliferation and Causes a Noncompaction Cardiomyopathy Phenotype 
Noncompaction cardiomyopathy (NCC) is a congenital heart disease that causes ventricular dysfunction and high mortality rate in children. The mechanisms responsible for NCC are still unknown. The steroid receptor coactivator-1 (SRC-1) and SRC-3 are transcriptional coactivators for nuclear hormone receptors and certain other transcription factors that regulate many genes in development and organ function. However, the roles of SRC-1/3 in heart morphogenesis, function and NCC occurrence are unknown. This study aims to examine the spatial and temporal expression patterns of SRC-1/3 in the heart and investigate the specific roles of SRC-1/3 in heart development, function and NCC occurrence. Immunochemical analysis detected SRC-1/3 expressions in the proliferating cardiomyocytes of mouse heart at prenatal and neonatal stages, while these expressions disappeared within two weeks after birth. Through generating and characterizing mouse lines with global or cardiomyocyte-specific knockouts of SRC-1/3, we found ablation of SRC-1/3 in the myocardial lineage resulted in prominent trabeculae, deep intertrabecular recesses and thin ventricular wall and septum. These developmental defects caused a failure of trabecular compaction, decreased internal ventricular dimension, reduced cardiac ejection fraction and output and led to a high rate of postnatal mortality. Collectively, these structural and functional abnormalities closely simulate the phenotype of NCC patients. Further molecular analysis of cardiomyocytes in vivo and in vitro revealed that SRC-1/3 directly up-regulate cyclin E2, cyclin B1 and myocardin to promote cardiomyocyte proliferation and differentiation. In conclusion, SRC-1/3 are required for cardiomyocyte proliferation and differentiation at earlier developmental stages, and their dysfunction causes NCC-like abnormalities in the hearts of newborn and adult mice.
PMCID: PMC4515817  PMID: 26221073
knockout mice; heart diseases; myocyte proliferation; cardiac output; nuclear receptor coactivator
17.  The surgical learning curve and accuracy of minimally invasive lumbar pedicle screw placement using CT based computer-assisted navigation plus continuous electromyography monitoring – a retrospective review of 627 screws in 150 patients 
This study retrospectively assessed the accuracy of placement of lumbar pedicle screws placed by a single surgeon using a minimally-invasive, intra-operative CT-based computer navigated technique in combination with continuous electromyography (EMG) monitoring. The rates of incorrectly positioned screws were reviewed in the context of the surgeon's experience and learning curve.
Data was retrospectively reviewed from all consecutive minimally invasive lumbar fusions performed by the primary author over a period of over 4 years from April 2008 until October 2012. All cases that had utilized computer-assisted intra-operative CT-based image guidance and continuous EMG monitoring to guide percutaneous pedicle screw placement were analysed for the rates of malposition of the pedicle screws. Pedicle screw malposition was defined as having occurred if the screw trajectory was adjusted intraoperatively due to positive EMG responses, or due to breach of the pedicle cortex by more than 2mm on intraoperative CT imaging performed at the end of the instrumentation procedure. Further analysis of the data was undertaken to determine if the rates of malposition changed with the surgeon's experience with the technique.
Six hundred and twenty-seven pedicle screws were placed in one hundred and fifty patients. The overall rate of intraoperative malposition and subsequent adjustment of pedicle screw placement was 3.8% (24 of 627 screws). Screw malposition was detected by intraoperative CT imaging. Warning of potential screw misplacement was provided by use of the EMG monitoring. With increased experience with the technique, rates of intraoperative pedicle screw malposition were found to decrease from 5.1% of screws in the first fifty patients, to 2.0% in the last 50 patients. Only one screw was suboptimally placed at the end of surgery, which did not result in a neurological deficit.
The use of CT-based computer-assisted navigation in combination with continuous EMG monitoring during percutaneous transpedicular screw placement results in very low rates of malposition and neural injury that compare favourably with previously reported rates. Pedicle screw placement accuracy continues to improve as the surgeon becomes more experienced with the technique.
PMCID: PMC4325487  PMID: 25694919
minimally invasive spine surgery; lumbar fusion; Pedicle screw; Spinal navigation; learning curve
18.  Vitamin D Supplementation Suppresses Hypoxia-Stimulated Placental Cytokine Secretion, Hypertension and CD4+ T Cell Stimulation in Response to Placental Ischemia 
To investigate a role of Vitamin D in the pathogenesis of preeclampsia (PE), and to discern any potential benefits of Vitamin D supplementation on hypertension in the RUPP rat model of PE.
Study design
Blood and placentas from normal pregnancies (NP) and PE were collected following elective cesarean delivery without evidence of infection. Circulating Vitamin D was extracted by HPLC and measured via mass spectrometry. Media for placenta explants was supplemented with Vitamin D and exposed to hypoxic (1% O2) or normoxic (6% O2) conditions for 24 hours. ELISAs were performed on media and normalized to total protein to determine cytokine secretion. RUPP rats were supplemented with vitamin D by oral gavage, and blood pressure (MAP) and pup weights were measured in NP and RUPP rats with or without Vitamin D supplementation. Flow cytometry was used to evaluate CD4+ Tcells in control RUPP rats and RUPP rats treated with Vitamin D.
Inflammatory cytokine secretion was higher (p<0.05) while the anti-inflammatory cytokine, IL-10, was significantly lower in the media of PE placentas compared to NP (p=0.005). Vitamin D supplementation decreased hypoxia stimulated pro-inflammatory cytokine secretion (p=0.003) in the media of PE placentas. Vitamin D decreased MAP and circulating CD4+ T cells in the RUPP rat model of PE (p<0.05).
Vitamin D supplementation may be useful in the treatment or prevention of hypertensive disorders in pregnancy.
PMCID: PMC4235666  PMID: 25414911
Vitamin D; Hypertension; Preeclampsia; Cytokines
19.  CD4+ T Cells Play a Critical Role in Mediating Hypertension in Response to Placental Ischemia 
Similar to preeclamptic women, hypertension in the chronic Reduced Uterine Perfusion Pressure Rat Model Of Preeclampsia (RUPP) is associated with increased CD4+ T cells, cytokines, sFlt-1 and agonistic autoantibodies to the AngII receptor (AT1-AA). We examined the effect inhibition of T cell co-stimulation in RUPP rats treated with (A) (abatacept, 250 mg/kg, infused i.v. at gestation day 13), on hypertension and sFlt-1, TNF-α and AT1-AA. RUPP surgical procedure was performed on day 14. On day 19 MAP increased from 94+2 mmHg in Normal Pregnant (NP) to 123 ± 3 mmHg in RUPP control rats. This response was attenuated by Abatacept, MAP was 104 ± 2 mmHg in RUPP ± A, and 96 ± 2 mmHg NP ± A. Percent circulating CD4+ T cells were 66 ± 3% in RUPPs compared to 55 ± 3% NP rats (p<0.04) but were normalized in RUPP ± A rats (54 ± 3%). The twofold increase in TNF alpha seen in RUPPs (277 ± 47 pg/ml) was decreased to 80 ± 18 pg/ml in RUPP+A. Placental sFlt-1 was reduced 70 % to 151 ± 28 in RUPP ± A compared 488 ± 61 pg/ml in RUPP (p<0.001). AT1-AA decreased from 20 ± 0.8 bpm in control RUPP to 6 ± 0.7 bpm in RUPP ± A. We next determined the effect of RUPP in causing hypertension in pregnant T cell deficient rats by examining MAP in NP (123 ± 5 mmHg) and RUPP athymic nude rats (123 ± 7 mmHg). In the absence of T cells, hypertension in response to placental ischemia was completely abolished. Collectively these data indicate that CD4+ Tcells in response to placental ischemia play an important role in the pathophysiology of hypertension associated with preeclampsia.
PMCID: PMC4231445  PMID: 25401050
Hypertension; Preeclampsia; Placental ischemic insult
20.  Endothelin-1, oxidative stress and endogenous ANGII: mechanisms of AT1-AA-enhanced Renal and Blood Pressure Response during pregnancy 
Hypertension  2013;62(5):10.1161/HYPERTENSIONAHA.113.01648.
Hypertension during preeclampsia is associated with increased maternal vascular sensitivity to angiotensin II (ANGII). This study was designed to determine mechanisms whereby agonistic autoantibodies to the ANGII type I receptor (AT1-AA) enhance blood pressure (MAP) and renal vascular sensitivity to ANGII during pregnancy. First, we examined MAP and renal artery resistance index (RARI) in response to chronic administration of ANGII or AT1-AA or AT1-AA+ANGII in pregnant rats compared to control pregnant rats. In order to examine mechanisms of heightened sensitivity in response to AT1-AA during pregnancy we examined the role of endogenous ANGII in AT1-AA infused pregnant rats, Endothelin-1 and oxidative stress in AT1-AA+ANGII treated rats. Chronic ANGII increased MAP from 95 +/−2 in NP rats to 115 +/−2 mmHg. Chronic AT1-AA increased MAP to 118+/−1 mmHg in NP rats which further increased to 123+/−2 with AT1-AA+ANGII. Increasing ANGII from (10−11-10−8) decreased Af-Art diameter 15-20% but sharply decreased Af-Art diameter 60% in AT1-AA pretreated vessels. RARI increased from 0.67 in NP rats to 0.70 with AT1-AA infusion, which was exacerbated to 0.74 in AT1-AA + ANGII infused rats. AT1-AA-induced hypertension decreased with Enalapril but was not attenuated. Both tissue ET-1 and ROS increased with AT1-AA+ANGII compared to AT1-AA alone and blockade of either of these pathways had significant effects on MAP or RARI. These data support the hypothesis that AT1-AA, via activation of ET-1 and oxidative stress and interaction with endogenous ANGII, are important mechanisms whereby MAP and renal vascular responses are enhanced during preeclampsia.
PMCID: PMC3845356  PMID: 24041954
Angiotensin II; AT1-AA; Preeclampsia
22.  The Roles of Mechanical Stresses in the Pathogenesis of Osteoarthritis 
Cartilage  2013;4(4):286-294.
Excessive joint surface loadings, either single (acute impact event) or repetitive (cumulative contact stress), can cause the clinical syndrome of osteoarthritis (OA). Despite advances in treatment of injured joints, the risk of OA following joint injuries has not decreased in the past 50 years. Cumulative excessive articular surface contact stress that leads to OA results from posttraumatic joint incongruity and instability, and joint dysplasia, but may also cause OA in patients without known joint abnormalities. In vitro investigations show that excessive articular cartilage loading triggers release of reactive oxygen species (ROS) from mitochondria, and that these ROS cause chondrocyte death and matrix degradation. Preventing release of ROS or inhibiting their effects preserves chondrocytes and their matrix. Fibronectin fragments released from articular cartilage subjected to excessive loads also stimulate matrix degradation; inhibition of molecular pathways initiated by these fragments prevents this effect. Additionally, injured chondrocytes release alarmins that activate chondroprogentior cells in vitro that propogate and migrate to regions of damaged cartilage. These cells also release chemokines and cytokines that may contribute to inflammation that causes progressive cartilage loss. Distraction and motion of osteoarthritic human ankles can promote joint remodeling, decrease pain, and improve joint function in patients with end-stage posttraumatic OA. These advances in understanding of how altering mechanical stresses can lead to remodeling of osteoarthritic joints and how excessive stress causes loss of articular cartilage, including identification of mechanically induced mediators of cartilage loss, provide the basis for new biologic and mechanical approaches to the prevention and treatment of OA.
PMCID: PMC4109888  PMID: 25067995
posttraumatic osteoarthritis; joint injury; mechanical loading of joints; joint instability; alarmins
23.  Integration of L-R Pitx2 transcription and Wnt signaling drives asymmetric gut morphogenesis via Daam2 
Developmental cell  2013;26(6):629-644.
A critical aspect of gut morphogenesis is initiation of a leftward tilt. Failure to do so leads to gut malrotation and volvulus. The direction of tilt is specified by asymmetric cell behaviors within the dorsal mesentery (DM), which suspends the gut tube, and is downstream of Pitx2, the key transcription factor responsible for the transfer of left-right (L-R) information from early gastrulation to morphogenesis. Although Pitx2 is a master regulator of L-R organ development, its cellular targets that drive asymmetric morphogenesis are not known. Using laser microdissection and targeted gene misexpression in the chicken DM, we show that Pitx2-specific effectors mediate Wnt signaling to activate the formin Daam2, a key Wnt effector and itself a Pitx2 target, linking actin dynamics to cadherin-based junctions, to ultimately generate asymmetric cell behaviors. Our work highlights how integration of two conserved cascades may be the ultimate force through which Pitx2 sculpts L-R organs.
PMCID: PMC3965270  PMID: 24091014
24.  Somatic mosaicism and allele complexity induced by CRISPR/Cas9 RNA injections in mouse zygotes 
Developmental biology  2014;393(1):3-9.
Tyrosinase is the rate-limiting enzyme for the production of melanin pigmentation. In the mouse and other animals, homozygous null mutations in the Tyrosinase gene (Tyr) result in the absence of pigmentation, i.e. albinism. Here we used the CRISPR/Cas9 system to generate mono- and bi-allelic null mutations in the Tyr locus by zygote injection of two single-guide and Cas9 RNAs. Injection into C57BL/6N wild-type embryos resulted in one completely albino founder carrying two different Tyr mutations. In addition, three pigmentation mosaics and fully pigmented littermates were obtained that transmitted new mutant Tyr alleles to progeny in test crosses with albinos. Injection into Tyr heterozygous (B6CBAF1/J × FVB/NJ) zygotes resulted in the generation of numerous albinos and also mice with a graded range of albino mosaicism. Deep sequencing revealed that the majority of the albinos and the mosaics had more than two new mutant alleles. These visual phenotypes and molecular genotypes highlight the somatic mosaicism and allele complexity in founders that occurs for targeted genes during CRISPR/Cas9-mediated mutagenesis by zygote injection in mice.
PMCID: PMC4166609  PMID: 24984260
Gene targeting; Pigmentation; Knockout; Mosaic; Genome editing; Melanocyte
25.  Progesterone supplementation attenuates hypertension and the autoantibody to the angiotensin II type I receptor in response to elevated interleukin-6 during pregnancy 
Preeclampsia is a multisystem disorder recognized as hypertension with proteinuria developing >20 weeks’ gestation. Preeclampsia is associated with chronic immune activation characterized by increased T and B lymphocytes, cytokines, and antibodies activating the angiotensin II type I receptor (AT1-AA). Hypertension in response to elevated interleukin (IL)-6 during pregnancy occurs with increased renin activity and AT1-AA, and reduced kidney function.
We aim to determine whether 17-alpha-hydroxyprogesterone caproate (17-OHPC), progesterone, improved inflammatory pathways during elevated IL-6 in pregnant rats. IL-6 (5 ng/d) was infused via miniosmotic pumps into normal pregnant (NP) rats beginning on day 14 of gestation and 17-OHPC (3.32 mg/kg) was diluted in normal saline and injected on day 18. Blood pressure (mean arterial pressure [MAP]) determination and serum collection were performed on day 19 of gestation.
MAP in NP was 100 ± 3 mm Hg, which increased with IL-6 to 112 ± 4 mm Hg (P < .05). Pregnant rats given 17-OHPC alone had a MAP of 99 ± 3 mm Hg and MAP increased to 103 ± 2 mm Hg in IL-6OHPC. AT1-AA was 1.2 ± 0.5 bpm in NP rats, increased to 17±9 bpm with IL-6 infusion but administration of 17-OHPC significantly blunted AT1-AA to 4 ± 0.8 bpm in NP6OHPC. Total circulating nitrate/nitrite was significantly decreased and placental Ser1177-phosporylated-eNOS/eNOS was lowered with IL-6 infusion. Supplementation of 17-OHPC significantly improved placental Ser1177-phosporylated-eNOS/eNOS however, circulating nitrate/nitrite was unchanged with 17-OHPC supplementation.
This study illustrates that 17-OHPC attenuated hypertension, decreased AT1-AA activity, and improved placental nitric oxide in response to elevated IL-6 during pregnancy and could lend hope to a new potential therapeutic for preeclampsia.
PMCID: PMC4117731  PMID: 24548847
cytokines; hypertension; inflammation; nitric oxide; pregnancy; progesterone; renin angiotensin system

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