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1.  Survival and growth patterns of white spruce (Picea glauca [Moench] Voss) rangewide provenances and their implications for climate change adaptation 
Ecology and Evolution  2014;4(12):2360-2374.
Intraspecific assisted migration (ISAM) through seed transfer during artificial forest regeneration has been suggested as an adaptation strategy to enhance forest resilience and productivity under future climate. In this study, we assessed the risks and benefits of ISAM in white spruce based on long-term and multilocation, rangewide provenance test data. Our results indicate that the adaptive capacity and growth potential of white spruce varied considerably among 245 range-wide provenances sampled across North America; however, the results revealed that local populations could be outperformed by nonlocal ones. Provenances originating from south-central Ontario and southwestern Québec, Canada, close to the southern edge of the species' natural distribution, demonstrated superior growth in more northerly environments compared with local populations and performed much better than populations from western Canada and Alaska, United States. During the 19–28 years between planting and measurement, the southern provenances have not been more susceptible to freezing damage compared with local populations, indicating they have the potential to be used now for the reforestation of more northerly planting sites; based on changing temperature, these seed sources potentially could maintain or increase white spruce productivity at or above historical levels at northern sites. A universal response function (URF), which uses climatic variables to predict provenance performance across field trials, indicated a relatively weak relationship between provenance performance and the climate at provenance origin. Consequently, the URF from this study did not provide information useful to ISAM. The ecological and economic importance of conserving white spruce genetic resources in south-central Ontario and southwestern Québec for use in ISAM is discussed.
doi:10.1002/ece3.1100
PMCID: PMC4203285  PMID: 25360273
Assisted migration; genetic conservation; genetic gain; geographic genetic variation; local adaptation; universal response function
2.  Novel DNA methyltransferase-1 (DNMT1) depleting anticancer nucleosides, 4′-thio-2′-deoxycytidine and 5-aza-4′-thio-2′-deoxycytidine 
Purpose
Currently approved DNA hypomethylating nucleosides elicit their effects in part by depleting DNA methyltransferase I (DNMT1). However, their low response rates and adverse effects continue to drive the discovery of newer DNMT1 depleting agents. Herein, we identified two novel 2′-deoxycytidine (dCyd) analogs, 4′-thio-2′-deoxycytidine (T-dCyd) and 5-aza-4′-thio-2′-deoxycytidine (aza-T-dCyd) that potently deplete DNMT1 in both in vitro and in vivo models of cancer and concomitantly inhibit tumor growth.
Methods
DNMT1 protein levels in in vitro and in vivo cancer models were determined by Western blotting and antitumor efficacy was evaluated using xenografts. Effects on CpG methylation were evaluated using methylation-specific PCR. T-dCyd metabolism was evaluated using radiolabeled substrate.
Results
T-dCyd markedly depleted DNMT1 in CCRF-CEM and Kg1a leukemia and nCI-H23 lung carcinoma cell lines, while it was ineffective in the HCt-116 colon or IgrOV-1 ovarian tumor lines. On the other hand, aza-T-dCyd potently depleted DNMT1 in all of these lines indicating that dCyd analogs with minor structural dissimilarities induce different DNMT1 turnover mechanisms. Although T-dCyd was deaminated to 4′-thio-2′-deoxyuridine, very little was converted to 4′-thio-thymidine nucleotides, suggesting that inhibition of thymidylate synthase would be minimal with 4′-thio dCyd analogs. Both T-dCyd and aza-T-dCyd also depleted DNMT1 in human tumor xenografts and markedly reduced in vivo tumor growth. Interestingly, the selectivity index of aza-T-dCyd was at least tenfold greater than that of decitabine.
Conclusions
Collectively, these data show that 4′-thio modified dCyd analogs, such as T-dCyd or aza-T-dCyd, could be a new source of clinically effective DNMT1 depleting anticancer compounds with less toxicity.
doi:10.1007/s00280-014-2503-z
PMCID: PMC4194194  PMID: 24908436
DNMT1; DNA methyltransferase; Decitabine; azacytidine; Zebularine; Deoxycytidine
3.  Approaches to studying and manipulating the enteric microbiome to improve autism symptoms 
Microbial Ecology in Health and Disease  2015;26:10.3402/mehd.v26.26878.
There is a growing body of scientific evidence that the health of the microbiome (the trillions of microbes that inhabit the human host) plays an important role in maintaining the health of the host and that disruptions in the microbiome may play a role in certain disease processes. An increasing number of research studies have provided evidence that the composition of the gut (enteric) microbiome (GM) in at least a subset of individuals with autism spectrum disorder (ASD) deviates from what is usually observed in typically developing individuals. There are several lines of research that suggest that specific changes in the GM could be causative or highly associated with driving core and associated ASD symptoms, pathology, and comorbidities which include gastrointestinal symptoms, although it is also a possibility that these changes, in whole or in part, could be a consequence of underlying pathophysiological features associated with ASD. However, if the GM truly plays a causative role in ASD, then the manipulation of the GM could potentially be leveraged as a therapeutic approach to improve ASD symptoms and/or comorbidities, including gastrointestinal symptoms. One approach to investigating this possibility in greater detail includes a highly controlled clinical trial in which the GM is systematically manipulated to determine its significance in individuals with ASD. To outline the important issues that would be required to design such a study, a group of clinicians, research scientists, and parents of children with ASD participated in an interdisciplinary daylong workshop as an extension of the 1st International Symposium on the Microbiome in Health and Disease with a Special Focus on Autism (www.microbiome-autism.com). The group considered several aspects of designing clinical studies, including clinical trial design, treatments that could potentially be used in a clinical trial, appropriate ASD participants for the clinical trial, behavioral and cognitive assessments, important biomarkers, safety concerns, and ethical considerations. Overall, the group not only felt that this was a promising area of research for the ASD population and a promising avenue for potential treatment but also felt that further basic and translational research was needed to clarify the clinical utility of such treatments and to elucidate possible mechanisms responsible for a clinical response, so that new treatments and approaches may be discovered and/or fostered in the future.
doi:10.3402/mehd.v26.26878
PMCID: PMC4425814  PMID: 25956237
autism spectrum disorder; clinical trials; Clostridia; fecal microbiota transplantation (FMT); microbiome; mitochondria; probiotic; short chain fatty acids; vancomycin; gastrointestinal
4.  Increased Biodiversity in the Environment Improves the Humoral Response of Rats 
PLoS ONE  2015;10(4):e0120255.
Previous studies have compared the immune systems of wild and of laboratory rodents in an effort to determine how laboratory rodents differ from their naturally occurring relatives. This comparison serves as an indicator of what sorts of changes might exist between modern humans living in Western culture compared to our hunter-gatherer ancestors. However, immunological experiments on wild-caught animals are difficult and potentially confounded by increased levels of stress in the captive animals. In this study, the humoral immune responses of laboratory rats in a traditional laboratory environment and in an environment with enriched biodiversity were examined following immunization with a panel of antigens. Biodiversity enrichment included colonization of the laboratory animals with helminths and co-housing the laboratory animals with wild-caught rats. Increased biodiversity did not apparently affect the IgE response to peanut antigens following immunization with those antigens. However, animals housed in the enriched biodiversity setting demonstrated an increased mean humoral response to T-independent and T-dependent antigens and increased levels of “natural” antibodies directed at a xenogeneic protein and at an autologous tissue extract that were not used as immunogens.
doi:10.1371/journal.pone.0120255
PMCID: PMC4390306  PMID: 25853852
5.  Cromolyn ameliorates acute and chronic injury in a rat lung transplant model 
BACKGROUND
Mast cells have been associated with obliterative bronchiolitis (OB) in human pulmonary allografts, although their role in the development of OB remains unknown.
METHODS
In this study, we evaluated the role of mast cells in pulmonary allograft rejection using an orthotopic rat pulmonary allograft model that utilizes chronic aspiration of gastric fluid to reliably obtain OB. Pulmonary allograft recipients (n = 35) received chronic aspiration of gastric fluid with (n = 10) and without (n = 16) treatment with a mast cell membrane stabilizer, cromolyn sodium, or chronic aspiration with normal saline (n = 9) as a control.
RESULTS
The acute graft injury associated with long ischemic time in the model (6 hours total ischemic time; typical acute graft injury rate ~30%) was apparently blocked by cromolyn, because peri-operative mortality associated with the acute graft injury was not observed in any of the animals receiving cromolyn (p = 0.045). Further, the rats receiving cromolyn developed significantly fewer OB lesions than those treated with gastric fluid alone (p < 0.001), with a mean reduction of 46% of the airways affected.
CONCLUSIONS
These findings provide impetus for further studies aimed at elucidating the effects of cromolyn and the role of mast cells in pulmonary allotransplantation.
doi:10.1016/j.healun.2014.03.004
PMCID: PMC4336160  PMID: 24768366
mast cells; aspiration; pulmonary allograft; gastric fluid; obliterative bronchiolitis
6.  A model for the induction of autism in the ecosystem of the human body: the anatomy of a modern pandemic? 
Microbial Ecology in Health and Disease  2015;26:10.3402/mehd.v26.26253.
Background
The field of autism research is currently divided based on a fundamental question regarding the nature of autism: Some are convinced that autism is a pandemic of modern culture, with environmental factors at the roots. Others are convinced that the disease is not pandemic in nature, but rather that it has been with humanity for millennia, with its biological and neurological underpinnings just now being understood.
Objective
In this review, two lines of reasoning are examined which suggest that autism is indeed a pandemic of modern culture. First, given the widely appreciated derailment of immune function by modern culture, evidence that autism is strongly associated with aberrant immune function is examined. Second, evidence is reviewed indicating that autism is associated with ‘triggers’ that are, for the most part, a construct of modern culture. In light of this reasoning, current epidemiological evidence regarding the incidence of autism, including the role of changing awareness and diagnostic criteria, is examined. Finally, the potential role of the microbial flora (the microbiome) in the pathogenesis of autism is discussed, with the view that the microbial flora is a subset of the life associated with the human body, and that the entire human biome, including both the microbial flora and the fauna, has been radically destabilized by modern culture.
Conclusions
It is suggested that the unequivocal way to resolve the debate regarding the pandemic nature of autism is to perform an experiment: monitor the prevalence of autism after normalizing immune function in a Western population using readily available approaches that address the well-known factors underlying the immune dysfunction in that population.
doi:10.3402/mehd.v26.26253
PMCID: PMC4310853  PMID: 25634608
microbiome; fauna; autism; pandemic
7.  An assessment of human gastric fluid composition as a function of PPI usage 
Physiological Reports  2015;3(1):e12269.
Abstract
The standard of care for chronic gastro‐esophageal reflux disease (GERD), which affects up to 40% of the population, is the use of drugs such as proton pump inhibitors (PPIs) that block the production of stomach acid. Despite widespread use, the effects of PPIs on gastric fluid remain poorly characterized. In this study, gastric fluid was collected from patients undergoing cardiac surgery who were not (n = 40) or were (n = 25) actively taking PPIs. Various enzymatic and immunoassays as well as mass spectrometry were utilized to analyze the concentrations of bile, gastricsin, trypsin, and pepsin in the gastric fluid. Proteomic analyses by mass spectrometry suggested that degradation of trypsin at low pH might account, at least in part, for the observation that patients taking PPIs have a greater likelihood of having high concentrations of trypsin in their gastric fluid. In general, the concentrations of all analytes evaluated varied over several orders of magnitude, covering a minimum of a 2000‐fold range (gastricsin) and a maximum of a 1 × 106 –fold range (trypsin). Furthermore, the concentrations of various analytes were poorly correlated with one another in the samples. For example, trypsin and bile concentrations showed a significant (P < 0.0001) but not strong correlation (r = 0.54). Finally, direct assessment of bacterial concentrations by flow cytometry revealed that PPIs did not cause a profound increase in microbial load in the gastric fluid. These results further delineate the profound effects that PPI usage has on the physiology of the stomach.
Further delineating the profound effects that PPI usage has on the physiology of the stomach, gastric fluid was collected from patients undergoing cardiac surgery who were not (n = 40) or were (n = 25) actively taking PPIs. Proteomic analyses by mass spectrometry suggested that degradation of trypsin at low pH might account, at least in part, for the observation that patients taking PPIs have a greater likelihood of having high concentrations of trypsin in their gastric fluid. Direct assessment of bacterial concentrations by flow cytometry revealed that PPIs did not cause a profound increase in microbial load in the gastric fluid, contrary with results previously obtained using culture‐dependent methods.
doi:10.14814/phy2.12269
PMCID: PMC4387745  PMID: 25626870
Bile; gastric fluid; gastricsin; proton pump inhibitors; trypsin
8.  Long-term Mortality Associated with Oophorectomy versus Ovarian Conservation in the Nurses’ Health Study 
Obstetrics and gynecology  2013;121(4):709-716.
Objective
To report long-term mortality following oophorectomy or ovarian conservation at the time of hysterectomy in subgroups of women based on age at the time of surgery, use of estrogen therapy, presence of risk-factors for CHD and length of follow-up.
Methods
A prospective cohort study of 30,117 Nurses’ Health Study participants having a hysterectomy for benign disease Multivariable-adjusted hazard ratios [HR] for death from CHD, stroke, breast cancer, epithelial ovarian cancer, lung cancer, colorectal cancer, total cancer and all-causes were determined, comparing bilateral oophorectomy (n=16,914) with ovarian conservation (n=13,203).
Results
Over 28 years of follow-up, 16.8% of women with hysterectomy and bilateral oophorectomy died from all causes compared with 13.3% of women who had ovarian conservation (HR=1.13;95% confidence interval [CI] 1.06–1.21). Oophorectomy was associated with a lower risk of death from ovarian cancer (4v44) and prior to age 47.5 years a lower risk of death from breast cancer. However at no age was oophorectomy associated with a lower risk of other cause-specific or all-cause mortality. For women younger than 50 at the time of hysterectomy, bilateral oophorectomy was associated with significantly increased mortality in women who had never-used estrogen therapy, but not in past and current users: all-cause mortality (HR=1.41;95% CI, 1.04–1.92;Pinteraction=0.03); lung cancer mortality (HR=1.44;95% CI, 0.17–1.21;Pinteraction=0.02); and CHD mortality (HR=2.35;95% CI, 1.22–4.27;Pinteraction=0.02).
Conclusions
For women younger than 50 at the time of hysterectomy, bilateral oophorectomy was associated with significantly increased mortality in women who had never-used estrogen therapy. At no age was oophorectomy associated with increased overall survival.
doi:10.1097/AOG.0b013e3182864350
PMCID: PMC4254662  PMID: 23635669
9.  Association of age dependent liver injury and fibrosis with immune cell populations 
Background/Aim
The liver’s response to injury is fibrosis, and when chronic, cirrhosis. Age is a critical factor impacting many immune-mediated processes, potentially including the liver’s wounding response to injury.
Methods
The effects of age on acute and chronic liver injury were evaluated using a carbon tetrachloride model in mice. Lymphocyte and macrophage populations were assessed by flow cytometry and immunohistochemical analysis.
Results
Acute liver injury was greater in 18-month old (old) mice than in 9-month old (middle aged) mice as judged by changes in aminotransferases. Similarly, 18-month old livers had a significantly greater fibrogenic response to injury than did 9-month old livers after chronic injury (assessed by col1α1 mRNA expression, morphometric analysis and hydroxyproline measurement). Interestingly, livers from young mice (6 weeks old) also exhibited an increase in fibrogenesis compared to 9-month old mice, albeit not to the same degree as in old mice. Consistent with a role for macrophages in fibrogenesis, the number of liver macrophages in young and 9-month old mice increased, while in chronically injured 18-month old livers, the number of macrophages was reduced, and was less than in the livers of young and 9-month old injured livers.
Conclusions
Our data indicate that the fibrogenic response to injury varies substantially with age, and moreover that macrophage recruitment and dynamics may be an important component in differential age-associated fibrotic disease.
doi:10.1111/liv.12202
PMCID: PMC4151465  PMID: 23710620
extracellular matrix; collagen; cirrhosis; smooth muscle actin; aminotransferase
10.  Using multiparametric data with missing features for learning patterns of pathology 
The paper presents a method for learning multimodal classifiers from datasets in which not all subjects have data from all modalities. Usually, subjects with a severe form of pathology are the ones failing to satisfactorily complete the study, especially when it consists of multiple imaging modalities. A classifier capable of handling subjects with unequal numbers of modalities prevents discarding any subjects, as is traditionally done, thereby broadening the scope of the classifier to more severe pathology. It also allows design of the classifier to include as much of the available information as possible and facilitates testing of subjects with missing modalities over the constructed classifier. The presented method employs an ensemble based approach where several subsets of complete data are formed and trained using individual classifiers. The output from these classifiers is fused using a weighted aggregation step giving an optimal probabilistic score for each subject. The method is applied to a spatio-temporal dataset for autism spectrum disorders (ASD)(96 patients with ASD and 42 typically developing controls) that consists of functional features from magnetoencephalography (MEG) and structural connectivity features from diffusion tensor imaging (DTI). A clear distinction between ASD and controls is obtained with an average 5-fold accuracy of 83.3% and testing accuracy of 88.4%. The fusion classifier performance is superior to the classification achieved using single modalities as well as multimodal classifier using only complete data (78.3%). The presented multimodal classifier framework is applicable to all modality combinations.
PMCID: PMC4023481  PMID: 23286164
11.  The Role of T-Type Calcium Channel Genes in Absence Seizures 
The thalamic relay neurons, reticular thalamic nucleus, and neocortical pyramidal cells form a circuit that sustains oscillatory burst firing, and is regarded as the underlying mechanism of absence seizures. T-type calcium channels play a key role in this circuit. Here, we review the role of T-type calcium channel genes in the development of absence seizures, and emphasize gain or loss of function mutations, and other variations that alter both quantity and quality of transcripts, and methylation status of isoforms of T-type calcium channel proteins might be of equal importance in understanding the pathological mechanism of absence seizures.
doi:10.3389/fneur.2014.00045
PMCID: PMC4023043  PMID: 24847307
absence epilepsy; T-type Ca2+ channels; genetics; expression; methylation
12.  Testicular artery pseudoaneurysm: a case report 
F1000Research  2014;3:2.
This is a case of an unusual cause of a testicular mass and the clinical features associated with its presentation and management.  The patient presented with testicular pain and was found to have a testicular mass on ultrasound with a central 1cm anechoic region with arterial wave-form concerning for a pseudoaneurysm. The patient underwent orchiectomy with resolution of his symptoms. This case highlights the presentation of testicular artery pseudoaneurysm and outcome following orchiectomy.
doi:10.12688/f1000research.3-2.v1
PMCID: PMC4118757  PMID: 25132959
13.  Rapamycin Prevents Seizures After Depletion of STRADA in a Rare Neurodevelopmental Disorder 
Science translational medicine  2013;5(182):182ra53.
A rare neurodevelopmental disorder in the Old Order Mennonite population called PMSE (polyhydramnios, megalencephaly, and symptomatic epilepsy syndrome; also called Pretzel syndrome) is characterized by infantile-onset epilepsy, neurocognitive delay, craniofacial dysmorphism, and histopathological evidence of heterotopic neurons in subcortical white matter and subependymal regions. PMSE is caused by a homozygous deletion of exons 9 to 13 of the LYK5/STRADA gene, which encodes the pseudokinase STRADA, an upstream inhibitor of mammalian target of rapamycin complex 1 (mTORC1). We show that disrupted pathfinding in migrating mouse neural progenitor cells in vitro caused by STRADA depletion is prevented by mTORC1 inhibition with rapamycin or inhibition of its downstream effector p70 S6 kinase (p70S6K) with the drug PF-4708671 (p70S6Ki). We demonstrate that rapamycin can rescue aberrant cortical lamination and heterotopia associated with STRADA depletion in the mouse cerebral cortex. Constitutive mTORC1 signaling and a migration defect observed in fibroblasts from patients with PMSE were also prevented by mTORC1 inhibition. On the basis of these preclinical findings, we treated five PMSE patients with sirolimus (rapamycin) without complication and observed a reduction in seizure frequency and an improvement in receptive language. Our findings demonstrate a mechanistic link between STRADA loss and mTORC1 hyperactivity in PMSE, and suggest that mTORC1 inhibition may be a potential treatment for PMSE as well as other mTOR-associated neurodevelopmental disorders.
doi:10.1126/scitranslmed.3005271
PMCID: PMC3720125  PMID: 23616120
14.  Ovarian Conservation at the Time of Hysterectomy and Long-Term Health Outcomes in the Nurses’ Health Study 
Obstetrics and gynecology  2009;113(5):1027-1037.
Objective
To report long-term health outcomes and mortality after oophorectomy or ovarian conservation.
Methods
We conducted a prospective, observational study of 29,380 women participants of the Nurses’ Health Study who had a hysterectomy for benign disease; 16,345 (55.6%) had hysterectomy with bilateral oophorectomy and 13,035 (44.4%) had hysterectomy with ovarian conservation. We evaluated incident events or death due to coronary heart disease (CHD), stroke, breast cancer, ovarian cancer, lung cancer, colorectal cancer, total cancers, hip fracture, pulmonary embolus, and death from all causes.
Results
Over 24 years of follow-up, for women with hysterectomy and bilateral oophorectomy, compared with ovarian conservation, the multivariable hazard ratios (HR) were 1.12 (95% CI 1.03, 1.21) for total mortality, 1.17 (95% CI 1.02, 1.35) for fatal plus nonfatal CHD, and 1.14 (95% CI 0.98, 1.33) for stroke. Although the risks of breast (HR 0.75 95% CI 0.68, 0.84), ovarian (HR 0.04 95% CI 0.01, 0.09, NNT = 220), and total cancers (HR 0.92 95% CI 0.86, 0.98) decreased after oophorectomy, lung cancer incidence (HR =1.26, 95% CI 1.02, 1.56, NNH = 190) and total cancer mortality (HR=1.17, 95% CI 1.04, 1.32) increased. For never-users of estrogen therapy, bilateral oophorectomy before age 50 was associated with an increased risk of all-cause mortality, CHD, and stroke. With an approximate 35-year life span following surgery, one additional death would be expected for every 9 oophorectomies performed.
Conclusions
Compared with ovarian conservation, bilateral oophorectomy at the time of hysterectomy for benign disease is associated with a decreased risk of breast and ovarian cancer, but an increased risk of all-cause mortality, fatal and non-fatal coronary heart disease, and lung cancer. In no analysis or age-group was oophorectomy associated with increased survival.
doi:10.1097/AOG.0b013e3181a11c64
PMCID: PMC3791619  PMID: 19384117
15.  New insights into the synergism of nucleoside analogs with radiotherapy 
Nucleoside analogs have been frequently used in combination with radiotherapy in the clinical setting, as it has long been understood that inhibition of DNA repair pathways is an important means by which many nucleoside analogs synergize. Recent advances in our understanding of the structure and function of deoxycytidine kinase (dCK), a critical enzyme required for the anti-tumor activity for many nucleoside analogs, have clarified the mechanistic role this kinase plays in chemo- and radio-sensitization. A heretofore unrecognized role of dCK in the DNA damage response and cell cycle machinery has helped explain the synergistic effect of these agents with radiotherapy. Since most currently employed nucleoside analogs are primarily activated by dCK, these findings lend fresh impetus to efforts focused on profiling and modulating dCK expression and activity in tumors. In this review we will briefly review the pharmacology and biochemistry of the major nucleoside analogs in clinical use that are activated by dCK. This will be followed by discussions of recent advances in our understanding of dCK activation via post-translational modifications in response to radiation and current strategies aimed at enhancing this activity in cancer cells.
doi:10.1186/1748-717X-8-223
PMCID: PMC3851323  PMID: 24066967
Nucleoside analogs; Radiotherapy; Deoxycytidine kinase; ATM
16.  Appendectomy and Clostridium difficile colitis: Relationships revealed by clinical observations and immunology 
Advances in understanding the interaction between the human immune system and the microbiome have led to an improved understanding of the function of the vermiform appendix as a safe-house for beneficial bacteria in the colon. These advances have been made despite long standing clinical observations that the appendectomy is a safe and effective procedure. However, more recent clinical data show that an appendectomy puts patients at increased risk for recurrent Clostridium difficile (C. difficile)-associated colitis, and probably other diseases associated with an altered microbiome. At the same time, appendectomy does not apparently put patients at risk for an initial onset of C. difficile-associated colitis. These clinical observations point toward the idea that the vermiform appendix might not effectively protect the microbiome in the face of broad spectrum antibiotics, the use of which precedes the initial onset of C. difficile-associated colitis. Further, these observations point to the idea that historically important threats to the microbiome such as infectious gastrointestinal pathogens have been supplanted by other threats, particularly the use of broad spectrum antibiotics.
doi:10.3748/wjg.v19.i34.5607
PMCID: PMC3769896  PMID: 24039352
Appendectomy; Clostridium difficile; Colitis; Diarrheal illness; Vermiform appendix
17.  In vivo antitumor activity of intratumoral fludarabine phosphate in refractory tumors expressing E. coli purine nucleoside phosphorylase 
Purpose
Systemically administered fludarabine phosphate (F-araAMP) slows growth of human tumor xenografts that express E. coli purine nucleoside phosphorylase (PNP). However, this treatment has been limited by the amount of F-araAMP that can be administered in vivo. The current study was designed to 1) determine whether efficacy of this overall strategy could be improved by intratumoral (IT) administration of F-araAMP, 2) test enhancement of the approach with external beam radiation, and 3) optimize recombinant adenovirus as a means to augment PNP delivery and bystander killing in vivo.
Methods
The effects of systemic or intratumoral F-araAMP in mice were investigated with human tumor xenografts (300 mg) in which 10% of the cells expressed E. coli PNP from a lentiviral promoter. Tumors injected with an adenoviral vector expressing E. coli PNP (Ad/PNP; 2 × 1011 viral particles, 2×/day × 3 days) and the impact of radiotherapy on tumors treated by this approach were also studied. Radiolabeled F-araAMP was used to monitor prodrug activation in vivo.
Results
Intratumoral administration of F-araAMP in human tumor xenografts expressing E. coli PNP resulted in complete regressions and/or prolonged tumor inhibition. External beam radiation significantly augmented this effect. Injection of large human tumor xenografts (human glioma, non-small cell lung cancer, or malignant prostate tumors) with Ad/PNP followed by intratumoral F-araAMP resulted in excellent antitumor activity superior to that observed following systemic administration of prodrug.
Conclusions
Activation of F-araAMP by E. coli PNP results in destruction of large tumor xenografts in vivo, augments radiotherapy, and promotes robust bystander killing. Our results indicate that intratumoral injection of F-araAMP leads to ablation of tumors in vivo with minimal toxicity.
doi:10.1007/s00280-012-1908-9
PMCID: PMC3423194  PMID: 22760227
tumor sensitization; low growth fraction malignancy; fludarabine; viral gene transfer; E. coli PNP
18.  Spontaneous bacterial cell lysis and biofilm formation in the colon of the Cape Dune mole-rat and the laboratory rabbit 
A wide range of techniques, including highthroughput DNA sequencing methods, have been applied to the evaluation of the normal intestinal flora. However, the inability to grow many of those species in culture imposes substantial constraints on the techniques used to evaluate this important community. The presence of biofilms in the normal gut adds further complexity to the issue. In this study, a flow cytometric analysis was used to separate intact bacterial cells, cell debris, and other particulate matter based on bacteria-specific staining and particle size. In addition, an analysis of biofilm formation using fluorescent light microscopy was conducted. Using these approaches, the ratio of bacterial cell debris to intact bacterial cells as a measure of spontaneous lysis of bacterial cells in the gut of the Cape dune mole-rat (Bathyergus suillus) and the laboratory rabbit (Oryctolagus cuniculus) was examined, and the degree of biofilm formation was semi-quantitatively assessed. The results suggest that the degree of spontaneous cell lysis was greater in the appendix than in the cecum in both the mole-rat and the rabbit. Further, the results point toward extensive epithelial-associated biofilm formation in the proximal mole-rat and rabbit large bowel, although the biofilms may be less structured than those found in laboratory rodents and in humans.
doi:10.1007/s00253-011-3207-5
PMCID: PMC3726217  PMID: 21538116
Biofilm; Cecal appendix; Flow cytometry; Mole-rat; Rabbit; Spontaneous lysis
19.  Lymphocyte phenotypes in wild-caught rats suggest potential mechanisms underlying increased immune sensitivity in post-industrial environments 
Cellular & molecular immunology  2012;9(2):163-174.
The immune systems of wild rats and of laboratory rats can been utilized as models of the human immune system in pre-industrial and post-industrial societies, respectively. In this study, lymphocyte phenotypes in wild rats were broadly characterized, and the results were compared to those obtained by us and by others using cells derived from various strains of laboratory rats. Although not expected, the production of regulatory T cells was not apparently different in wild rats compared to laboratory rats. On the other hand, differences in expression of markers involved in complement regulation, adhesion, signaling and maturation suggest increased complement regulation and decreased sensitivity in wild-caught rats compared to laboratory rats, and point toward complex differences between the maturation of T cells. The results potentially lend insight into the pathogenesis of post-industrial epidemics of allergy and autoimmune disease.
doi:10.1038/cmi.2011.61
PMCID: PMC3719982  PMID: 22327212
allergy; autoimmunity; biome; hygiene; immune regulation
20.  Genomics of sablefish (Anoplopoma fimbria): expressed genes, mitochondrial phylogeny, linkage map and identification of a putative sex gene 
BMC Genomics  2013;14:452.
Background
The sablefish (order: Scorpaeniformes) is an economically important species in commercial fisheries of the North Pacific and an emerging species in aquaculture. Aside from a handful of sequences in NCBI and a few published microsatellite markers, little is known about the genetics of this species. The development of genetic tools, including polymorphic markers and a linkage map will allow for the successful development of future broodstock and mapping of phenotypes of interest. The significant sexual dimorphism between females and males makes a genetic test for early identification of sex desirable.
Results
A full mitochondrial genome is presented and the resulting phylogenetic analysis verifies the placement of the sablefish within the Scorpaeniformes. Nearly 35,000 assembled transcript sequences are used to identify genes and obtain polymorphic SNP and microsatellite markers. 360 transcribed polymorphic loci from two sablefish families produce a map of 24 linkage groups. The sex phenotype maps to sablefish LG14 of the male map. We show significant conserved synteny and conservation of gene-order between the threespine stickleback Gasterosteus aculeatus and sablefish. An additional 1843 polymorphic SNP markers are identified through next-generation sequencing techniques. Sex-specific markers and sequence insertions are identified immediately upstream of the gene gonadal-soma derived factor (gsdf), the master sex determinant locus in the medaka species Oryzias luzonensis.
Conclusions
The first genomic resources for sablefish provide a foundation for further studies. Over 35,000 transcripts are presented, and the genetic map represents, as far as we can determine, the first linkage map for a member of the Scorpaeniformes. The observed level of conserved synteny and comparative mapping will allow the use of the stickleback genome in future genetic studies on sablefish and other related fish, particularly as a guide to whole-genome assembly. The identification of sex-specific insertions immediately upstream of a known master sex determinant implicates gsdf as an excellent candidate for the master sex determinant for sablefish.
doi:10.1186/1471-2164-14-452
PMCID: PMC3708741  PMID: 23829495
Sablefish; Black cod; Microsatellite; SNP; Linkage map; Conserved synteny; Threespine stickleback; Sex-specific sequences; Gonadal soma-derived factor
21.  Circulating angiogenic modulatory factors predict survival and functional class in pulmonary arterial hypertension 
Pulmonary Circulation  2013;3(2):369-380.
The diagnosis of pulmonary arterial hypertension (PAH) is frequently delayed. We hypothesized that circulating angiogenic modulatory protein levels might correspond with vascular remodeling activity and serve as sensitive biomarkers of PAH. Levels of soluble endoglin (sEng), soluble vascular endothelial growth factor receptor-1 (sVEGFR1), N-terminal brain natriuretic peptide (NT-proBNP), C-reactive protein (CRP), and other biomarkers were measured in peripheral blood from 97 PAH patients, 16 first-degree relatives of idiopathic or heritable pulmonary arterial hypertension (HPAH) patients, and 56 controls, and correlated with disease, functional class, hemodynamic parameters, exercise capacity, and transplant-free survival. Endoglin expression was analyzed in lung tissues of six individuals with idiopathic or HPAH and four individuals without PAH. Levels of sEng, sVEGFR1, CRP, and NT-proBNP were elevated in Group I PAH of diverse etiologies, with sEng performing better than NT-proBNP in detecting PAH (receiver operator characteristic-area-under-the curve [ROC-AUC] of 0.82 ± 0.03 vs. 0.71 ± 0.05, P = 0.016). While sEng, sVEGFR1, and NT-proBNP correlated with New York Heart Association (NYHA) class, sEng levels were more sensitive than NT-proBNP in detecting NYHA Class I-II disease (ROC-AUC of 0.88 ± 0.05 vs. 0.67 ± 0.08, P = 0.028). sEng, sVEGFR1, CRP, and NT-proBNP predicted transplant-free survival by univariate Cox regression. After adjusting for NT-proBNP levels, each of the other three markers predicted transplant-free survival. In multivariate analysis, sEng and CRP were independent predictors of survival. Endoglin expression was markedly enhanced in the microvascular endothelium and endovascular lesions of PAH versus control lung tissues. Circulating angiogenic proteins sEng and sVEGFR1 are sensitive markers of prognosis and function in Group I PAH, including mildly symptomatic disease, and may provide unique noninvasive data reflecting underlying remodeling activity.
doi:10.4103/2045-8932.110445
PMCID: PMC3757832  PMID: 24015338
angiogenesis; biomarkers; endoglin; pulmonary arterial hypertension; VEGF receptor-1
22.  Identification of Rv0535 as methylthioadenosine phosphorylase from Mycobacterium tuberculosis 
5′-methylthioadenosine (MTA) is a natural purine that is metabolized by methylthioadenosine phosphorylase (MTAP, E.C 2.4.2.28) in Eukarya and Archaea but generally not in bacteria. In this work, Rv0535, which has been annotated as a probable MTAP in M. tuberculosis, was expressed in and purified from E. coli BL21 (DE3). The purified protein displayed properties of a phosphorylase and MTA was the preferred substrate. Adenosine and S-adenosyl-L-homocysteine were poor substrates and no activity was detected with 5′-methylthioinosine, the other natural purines or the natural pyrimidines. Kinetic analysis of M. tuberculosis MTAP showed that the Km value for MTA was 9.1 μM. Rv0535 was estimated as a 30 kDa protein on a denaturing SDS-PAGE gel, which agreed with the molecular mass predicted by its gene sequence. Using gel filtration chromatography, the native molecular mass of the enzyme was determined to be 60 ± 4 kDa, and thus indicates that M. tuberculosis MTAP is a dimer. Differences in active site between mycobacterial and human MTAPs were identified by homology modeling based on the crystal of the human enzyme. A complete structure activity relationship analysis could identify differences in substrate specificity between the two enzymes to aid in the development of purine-based, anti-tuberculosis drugs.
doi:10.1016/j.tube.2011.11.010
PMCID: PMC3288397  PMID: 22225784
5′-methylthioadenosine phosphorylase; Rv0535; purine metabolism; Mycobacterium tuberculosis
23.  Evolutionary biology and anthropology suggest biome reconstitution as a necessary approach toward dealing with immune disorders 
Industrialized society currently faces a wide range of non-infectious, immune-related pandemics. These pandemics include a variety of autoimmune, inflammatory and allergic diseases that are often associated with common environmental triggers and with genetic predisposition, but that do not occur in developing societies. In this review, we briefly present the idea that these pandemics are due to a limited number of evolutionary mismatches, the most damaging being ‘biome depletion’. This particular mismatch involves the loss of species from the ecosystem of the human body, the human biome, many of which have traditionally been classified as parasites, although some may actually be commensal or even mutualistic. This view, evolved from the ‘hygiene hypothesis’, encompasses a broad ecological and evolutionary perspective that considers host-symbiont relations as plastic, changing through ecological space and evolutionary time. Fortunately, this perspective provides a blueprint, termed ‘biome reconstitution’, for disease treatment and especially for disease prevention. Biome reconstitution includes the controlled and population-wide reintroduction (i.e. domestication) of selected species that have been all but eradicated from the human biome in industrialized society and holds great promise for the elimination of pandemics of allergic, inflammatory and autoimmune diseases.
doi:10.1093/emph/eot008
PMCID: PMC3868394  PMID: 24481190
allergy; autoimmunity; inflammation; helminths; microbiome; mutualism; autism
24.  Synthesis and Evaluation of the Substrate Activity of C-6 Substituted Purine Ribosides with E. coli Purine Nucleoside Phosphorylase: Palladium Mediated Cross-Coupling of Organozinc Halides with 6-Chloropurine Nucleosides [1] 
A series of C-6 alkyl, cycloalkyl, and aryl-9-(β-d-ribofuranosyl)purines were synthesized and their substrate activities with Escherichia coli purine nucleoside phosphorylase (E. coli PNP) were evaluated. (Ph3P)4Pd-mediated cross-coupling reactions of 6-chloro-9-(2,3,5-tri-O-acetyl-β-d-ribofuranosyl)-purine (6) with primary alkyl (Me, Et, n-Pr, n-Bu, isoBu) zinc halides followed by treatment with NH3/MeOH gave the corresponding 6-alkyl-9-(β-d-ribofuranosyl) purine derivatives 7–11, respectively, in good yields. Reactions of 6 with cycloalkyl(propyl, butyl, pentyl)zinc halides and aryl (phenyl, 2-thienyl)zinc halides gave under similar conditions the corresponding 6-cyclopropyl, cyclobutyl, cyclopentyl, phenyl, and thienyl -9-(β-d-ribofuranosyl)purine derivatives 12–16, respectively in high yields. E. coli PNP showed a high tolerance to the steric and hydrophobic environment at the 6-position of the synthesized purine ribonucleosides. Significant cytotoxic activity was observed for 8, 12, 15, and 16. Evaluation of 12 and 16 against human tumor xenografts in mice did not demonstrate any selective antitumor activity. In addition, 6-methyl-9-(β-d-arabinofuranosyl)purine (18) was prepared and evaluated.
doi:10.1016/j.ejmech.2011.10.039
PMCID: PMC3259247  PMID: 22112758
purine nucleoside phosphorylase; organozinc halides; cross-coupling reactions; 6-alkyl; cycloalkyl/aryl/heterocyclylpurine ribonucleosides
25.  The Crystal Structure of Streptococcus pyogenes Uridine Phosphorylase Reveals a Distinct Subfamily of Nucleoside Phosphorylases†‡ 
Biochemistry  2011;50(30):6549-6558.
Uridine phosphorylase (UP), a key enzyme in the pyrimidine salvage pathway, catalyzes the reversible phosphorolysis of uridine or 2′-deoxyuridine to uracil and ribose 1-phosphate or 2′-deoxyribose 1-phosphate. This enzyme belongs to the nucleoside phosphorylase I superfamily whose members show diverse specificity for nucleoside substrates. Phylogenetic analysis shows Streptococcus pyogenes uridine phosphorylase (SpUP) is found in a distinct branch of the pyrimidine subfamily of nucleoside phosphorylases. To further characterize SpUP, we determined the crystal structure in complex with the products, ribose 1-phosphate and uracil, at 1.8 Å resolution. Like Escherichia coli UP (EcUP), the biological unit of SpUP is a hexamer with an α/β monomeric fold. A novel feature of the active site is the presence of His169, which structurally aligns with Arg168 of the EcUP structure. A second active site residue, Lys162, is not present in previously determined UP structures and interacts with O2 of uracil. Biochemical studies of wild type SpUP showed that substrate specificity is similar to that of EcUP, while EcUP is about sevenfold more efficient than SpUP. Biochemical studies on active site mutant SpUP showed that mutations of His169 reduced activity, while mutation of Lys162 abolished all activity, suggesting that negative charge in the transition state resides mostly on uracil O2. This is in contrast to EcUP for which transition state stabilization occurs mostly at O4.
doi:10.1021/bi200707z
PMCID: PMC3144492  PMID: 21707079

Results 1-25 (66)