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1.  Magnesium impacts myosin V motor activity by altering key conformational changes in the mechanochemical cycle 
Biochemistry  2013;52(27):10.1021/bi4004364.
We investigated how magnesium (Mg) impacts key conformational changes during the ADP binding/release steps in myosin V and how these alterations impact the actomyosin mechanochemical cycle. The conformation of the nucleotide binding pocket was examined with our established FRET system in which myosin V labeled with FlAsH in the upper 50 kDa domain participates in energy transfer with mant labeled nucleotides. We examined the maximum actin-activated ATPase activity of MV FlAsH at a range of free Mg concentrations (0.1–9 mM) and find that the highest activity occurs at low Mg (0.1–0.3 mM), while there is a 50–60% reduction in activity at high Mg (3–9 mM). The motor activity examined with the in vitro motility assay followed a similar Mg-dependence and the trend was similar with dimeric myosin V. Transient kinetic FRET studies of mantdADP binding/release from actomyosin V FlAsH demonstrate that the transition between the weak and strong actomyosin. ADP states is coupled to movement of the upper 50 kDa domain and is dependent on Mg with the strong state stabilized by Mg. We find that the kinetics of the upper 50 kDa conformational change monitored by FRET correlates well with the ATPase and motility results over a wide range of Mg concentrations. Our results suggest the conformation of the upper 50 kDa domain is highly dynamic in the Mg free actomyosin. ADP state, which is in agreement with ADP binding being entropy driven in the absence of Mg. Overall, our results demonstrate that Mg is a key factor in coupling the nucleotide- and actin-binding regions. In addition, Mg concentrations in the physiological range can alter the structural transition that limits ADP dissociation from actomyosin V, which explains the impact of Mg on actin-activated ATPase activity and in vitro motility.
doi:10.1021/bi4004364
PMCID: PMC3855573  PMID: 23725637
Myosin; actin; FRET
2.  Curing hepatitis C with pills: a step toward global control 
Lancet  2010;376(9751):1441-1442.
doi:10.1016/S0140-6736(10)61497-3
PMCID: PMC4083467  PMID: 20951421
3.  Reproductive factors and risk of lung cancer in female textile workers in Shanghai, China 
Cancer causes & control : CCC  2013;24(7):1305-1314.
Purpose
Hormonal factors may play a role in the development of lung cancer in women. This study examined the relationship between lung cancer and reproductive factors in a large cohort of women, most of whom never smoked (97%).
Methods
A cohort of 267,400 female textile workers in Shanghai, China, enrolled in a trial of breast self-examination provided information on reproductive history, demographic factors and cigarette smoking at enrollment in 1989–91. The cohort was followed until July of 2000 for incidence of lung cancer; 824 cases were identified. Hazard ratios (HR) and 95% confidence intervals (CI) associated with selected reproductive factors were calculated using Cox proportional hazards modeling, adjusting for smoking, age, and also parity when relevant.
Results
Nulliparous women were at increased risk compared to parous women (HR= 1.33, 95% CI 1.00–1.77). Women who had gone through menopause at baseline were at increased risk compared to women of the same age who were still menstruating. Risk was higher in women with a surgical menopause (HR=1.64, 95%CI 0.96–2.79) than in those with a natural menopause (HR=1.35, 95% CI 0.84–2.18), and risk was highest in those postmenopausal women with a hysterectomy and bilateral oophorectomy at baseline (HR=1.39, 95% CI 0.96–2.00), although the risk estimates were not statistically significant.
Conclusions
These results support experimental data that demonstrate a biological role for hormones in lung carcinogenesis.
doi:10.1007/s10552-013-0208-y
PMCID: PMC3734938  PMID: 23584535
4.  A novel CT acquisition and analysis technique for breathing motion modeling 
Physics in medicine and biology  2013;58(11):L31-L36.
Purpose
To report on a novel technique for providing artifact-free quantitative 4DCT image datasets for breathing motion modeling.
Methods
Commercial clinical four-dimensional computed tomography (4DCT) methods have trouble managing irregular breathing. The resulting images contain motion-induced artifacts that can distort structures and inaccurately characterize breathing motion. We have developed a novel scanning and analysis method for motion-correlated CT that utilizes standard repeated fast helical acquisitions, a simultaneous breathing surrogate measurement, deformable image registration, and a published breathing motion model.
Results
The motion model differs from the CT-measured motion by an average of 0.72 mm, indicating the precision of the motion model. The integral of the divergence of one of the motion model parameters is predicted to be a constant 1.11 and is found in this case to be 1.09, indicating the accuracy of the motion model.
Conclusions
The proposed technique shows promise for providing motion-artifact free images at user-selected breathing phases, accurate Hounsfield units, and noise characteristics similar to non-4D CT techniques, at a patient dose similar to or less than current 4DCT techniques.
doi:10.1088/0031-9155/58/11/L31
PMCID: PMC3685480  PMID: 23640212
4DCT; Breathing Motion Modeling; radiation therapy
5.  Cytomegalovirus infection modulates the phenotype and functional profile of the T-cell immune response to mycobacterial antigens in older life☆ 
Experimental Gerontology  2014;54(100):94-100.
Infection with Cytomegalovirus is associated with accelerated immunosenescence. Expansions of CMV-specific T cell responses have previously been demonstrated to affect the ability of T cells to respond to other infections. Most people above 60 years of age display M. tuberculosis-specific immunity because of vaccination, exposure, or both. T-cell responses can be assessed by measuring intracellular IFN-γ in vitro after tuberculin stimulation. Here we investigated tuberculin-specific CD4 T-cell responses in independently living healthy older people in the South of England using flow-cytometry. Individuals were investigated for tuberculin and CMV-specific T-cell immunity using in vitro antigen stimulation followed by intracellular staining for IFN-γ, TNF-α, IL2, as well as degranulation and CD154 upregulation. We also examined a control group of younger individuals (20–35 years of age). There was no significant difference between older and young people in regards to tuberculin responsiveness of CD4 T-cells; however, older people seemed to show more outliers. Increased responsiveness to tuberculin was significantly correlated to CMV responsiveness but not age. In older donors, the memory phenotype of tuberculin-induced T-cells was significantly skewed towards a more terminal differentiation phenotype in CMV-infected compared to uninfected individuals and the degree of skewing correlated quantitatively with the size of the CMV-specific CD4 T-cell response. This is a fundamental advance over previous reports of changes of the tuberculin-specific CD4 T-cell response with CMV serostatus. Our results show that how the immune system responds to CMV has a fundamental impact on the phenotype and function of the immune response to mycobacterial antigens in older life.
Highlights
•We examine the CD4 T-cell response to tuberculosis antigens in older people.•The CD4 T-cell response to Cytomegalovirus is explored in parallel.•CMV infection changes the profile of the tuberculin-specific-response.•The size of the CMV T-cell response is linked to these changes in a quantitative way.•The way we respond to CMV (‘mode’) affects our T-cell immunity to other pathogens.
doi:10.1016/j.exger.2013.12.007
PMCID: PMC4003347  PMID: 24370373
Cytomegalovirus; Tuberculosis; Immunosenescence; T-cell response; Flow-cytometry
6.  Sequence artefacts in a prospective series of formalin-fixed tumours tested for mutations in hotspot regions by massively parallel sequencing 
BMC Medical Genomics  2014;7:23.
Background
Clinical specimens undergoing diagnostic molecular pathology testing are fixed in formalin due to the necessity for detailed morphological assessment. However, formalin fixation can cause major issues with molecular testing, as it causes DNA damage such as fragmentation and non-reproducible sequencing artefacts after PCR amplification. In the context of massively parallel sequencing (MPS), distinguishing true low frequency variants from sequencing artefacts remains challenging. The prevalence of formalin-induced DNA damage and its impact on molecular testing and clinical genomics remains poorly understood.
Methods
The Cancer 2015 study is a population-based cancer cohort used to assess the feasibility of mutational screening using MPS in cancer patients from Victoria, Australia. While blocks were formalin-fixed and paraffin-embedded in different anatomical pathology laboratories, they were centrally extracted for DNA utilising the same protocol, and run through the same MPS platform (Illumina TruSeq Amplicon Cancer Panel). The sequencing artefacts in the 1-10% and the 10-25% allele frequency ranges were assessed in 488 formalin-fixed tumours from the pilot phase of the Cancer 2015 cohort. All blocks were less than 2.5 years of age (mean 93 days).
Results
Consistent with the signature of DNA damage due to formalin fixation, many formalin-fixed samples displayed disproportionate levels of C>T/G>A changes in the 1-10% allele frequency range. Artefacts were less apparent in the 10-25% allele frequency range. Significantly, changes were inversely correlated with coverage indicating high levels of sequencing artefacts were associated with samples with low amounts of available amplifiable template due to fragmentation. The degree of fragmentation and sequencing artefacts differed between blocks sourced from different anatomical pathology laboratories. In a limited validation of potentially actionable low frequency mutations, a NRAS G12D mutation in a melanoma was shown to be a false positive.
Conclusions
These findings indicate that DNA damage following formalin fixation remains a major challenge in laboratories working with MPS. Methodologies that assess, minimise or remove formalin-induced DNA damaged templates as part of MPS protocols will aid in the interpretation of genomic results leading to better patient outcomes.
doi:10.1186/1755-8794-7-23
PMCID: PMC4032349  PMID: 24885028
7.  Chemical-Genetics of Rapamycin-Insensitive TORC2 in S. cerevisiae 
Cell reports  2013;5(6):1725-1736.
Current approaches for identifying synergistic targets use cell culture models with combinations of clinically available drugs to see if the combined effect of the combination is better than predicted by their individual efficacy. New techniques are needed to systematically and rationally identify targets and pathways that have a high potential as synergistic targets. In this study, we create a tool to screen and identify molecular targets that may synergize with new inhibitors of TOR (Target of Rapamycin), a conserved protein that is a major integrator of cell proliferation signals in the nutrient-signaling pathway. While clinical results from TORC1 inhibition using rapamycin analogs (that only inhibit TORC1) have been disappointing, trials using inhibitors that also target TORC2 have been promising. To understand the molecular basis for this increased therapeutic efficacy and to discover secondary targets that may have potential in targeted combination therapy, we engineered TOR2 in S. cerevisiae to accept an orthogonal inhibitor in order to create the first chemical tool to selectively inhibit TORC2. We used this tool to create a Chemical Epistasis Mini-Array Profile, or ChE-MAP, by measuring interactions between the chemically inhibited TOR2 kinase and a diverse library of deletion mutants. The ChE-MAP identified known TOR components and distinguished between TORC1 (assessed using rapamycin) and TORC2 dependent functions. Results showed a novel TORC2-specific interaction with the pentose phosphate pathway (PPP). We used global metabolic profiling to show that that TORC2 inhibition led to decreases in metabolites specific to the PPP and confirmed that TOR2 was regulating this process using metabolic flux analysis. Regulation of the PPP is a previously unappreciated role for TORC2 that may suggest a role for the complex in balancing the high energy demand required for ribosome biogenesis.
doi:10.1016/j.celrep.2013.11.040
PMCID: PMC4007695  PMID: 24360963
8.  Workshop report on the 2nd Joint ENCCA/EuroSARC European bone sarcoma network meeting: integration of clinical trials with tumour biology 
This is the report of the 2nd Joint ENCCA/EuroSARC European Bone Sarcoma Network Meeting held in Leiden, The Netherlands, on 26-27 September 2013, bringing together preclinical and clinical investigators on bone sarcoma. The purpose of this workshop was to present the achievements of biological research and clinical trials in bone sarcomas and to stimulate crosstalk.
doi:10.1186/2045-3329-4-4
PMCID: PMC4014081
Osteosarcoma; Ewing sarcoma; Bone tumours; Translational research; ENCCA; EuroSARC
9.  Use of Laser Capture Microdissection to Map Hepatitis C Virus–Positive Hepatocytes in Human Liver 
Gastroenterology  2013;145(6):1404-13.e1-10.
BACKGROUND & AIMS
Hepatitis C virus (HCV) predominantly infects hepatocytes, but many hepatocytes are not infected; studies have shown that HCV antigens cluster within the liver. We investigated spatial distribution and determinants of HCV replication in human liver samples.
METHODS
We analyzed liver samples from 4 patients with chronic HCV infection (genotype 1, Metavir scores 0–1) to estimate the proportion of infected hepatocytes and the amount of HCV viral RNA (vRNA) per cell. Single-cell laser capture microdissection was used to capture more than 1000 hepatocytes in grids, to preserve geometric relationships. HCV vRNA and interferon-induced transmembrane protein 3 (IFITM3) messenger RNA (the transcript of an interferon-stimulated gene) were measured in the same hepatocytes by quantitative polymerase chain reaction and assembled in maps to identify areas of high and low HCV replication.
RESULTS
Patients’ serum levels of HCV RNA ranged from 6.87 to 7.40 log10 IU/mL; the proportion of HCV-infected hepatocytes per person ranged from 21% to 45%, and the level of vRNA ranged from 1 to 50 IU/hepatocyte. Infection was not random; we identified clustering of HCV-positive hepatocytes using infected-neighbor analysis (P < .0005) and distance to the kth nearest neighbor compared with random distributions, obtained by bootstrap simulations (P < .02). Hepatocytes that expressed IFITM3 did not appear to cluster and were largely HCV negative.
CONCLUSIONS
We used single-cell laser capture and high-resolution analysis to show that in human liver HCV infects hepatocytes in nonrandom clusters, whereas expression of antiviral molecules is scattered among hepatocytes. These findings show that quantitative single-cell RNA measurements can be used to estimate the abundance of HCV vRNA per infected human hepatocyte and are consistent with cell–cell propagation of infection in the absence of clustered IFITM3.
doi:10.1053/j.gastro.2013.08.034
PMCID: PMC4005338  PMID: 23973767
ISG; Intrahepatic Infection; Virology; scLCM
10.  Microsecond Molecular Dynamics Simulations of Mg2+- and K+- Bound E1 Intermediate States of the Calcium Pump 
PLoS ONE  2014;9(4):e95979.
We have performed microsecond molecular dynamics (MD) simulations to characterize the structural dynamics of cation-bound E1 intermediate states of the calcium pump (sarcoendoplasmic reticulum Ca2+-ATPase, SERCA) in atomic detail, including a lipid bilayer with aqueous solution on both sides. X-ray crystallography with 40 mM Mg2+ in the absence of Ca2+ has shown that SERCA adopts an E1 structure with transmembrane Ca2+-binding sites I and II exposed to the cytosol, stabilized by a single Mg2+ bound to a hybrid binding site I′. This Mg2+-bound E1 intermediate state, designated E1•Mg2+, is proposed to constitute a functional SERCA intermediate that catalyzes the transition from E2 to E1•2Ca2+ by facilitating H+/Ca2+ exchange. To test this hypothesis, we performed two independent MD simulations based on the E1•Mg2+ crystal structure, starting in the presence or absence of initially-bound Mg2+. Both simulations were performed for 1 µs in a solution containing 100 mM K+ and 5 mM Mg2+ in the absence of Ca2+, mimicking muscle cytosol during relaxation. In the presence of initially-bound Mg2+, SERCA site I′ maintained Mg2+ binding during the entire MD trajectory, and the cytosolic headpiece maintained a semi-open structure. In the absence of initially-bound Mg2+, two K+ ions rapidly bound to sites I and I′ and stayed loosely bound during most of the simulation, while the cytosolic headpiece shifted gradually to a more open structure. Thus MD simulations predict that both E1•Mg2+ and E•2K+ intermediate states of SERCA are populated in solution in the absence of Ca2+, with the more open 2K+-bound state being more abundant at physiological ion concentrations. We propose that the E1•2K+ state acts as a functional intermediate that facilitates the E2 to E1•2Ca2+ transition through two mechanisms: by pre-organizing transport sites for Ca2+ binding, and by partially opening the cytosolic headpiece prior to Ca2+ activation of nucleotide binding.
doi:10.1371/journal.pone.0095979
PMCID: PMC3997511  PMID: 24760008
11.  Dairy sheep production research at the University of Wisconsin-Madison, USA – a review 
Commercial milking of sheep is a new agricultural industry in the United States starting approximately 30 yr ago. The industry is still small, but it is growing. The majority of the sheep milk is used in the production of specialty cheeses. The United States is the major importer of sheep milk cheeses with 50 to 60% of annual world exports coming to the United States during the past 20 yr. Therefore, there is considerable growth potential for the industry in the United States. The only dairy sheep research flock in North America is located at the Spooner Agricultural Research Station of the University of Wisconsin-Madison. The research program started in 1993 and has been multifaceted; dealing with several areas important to commercial dairy sheep farmers. The East Friesian and Lacaune dairy breeds were compared and introduced to the industry through the research program. Both dairy breeds produced significantly more milk than traditional meat-wool breeds found in the U.S., but the two breeds differed in their production traits. East Friesian-cross ewes produced more lambs and slightly more milk than Lacaune-cross ewes whereas Lacaune-cross ewes produced milk with a higher percentage of fat and protein than East Friesian-cross ewes. Lactation physiology studies have shown that ewes with active corpora lutea have increased milk yields, oxytocin release during milking is required to obtain normal fat percentages in the milk, large udder cisterns of dairy ewes can allow for increased milking intervals, and short daylengths during late pregnancy results in increased milk yield. In the nutrition area, legume-grass pastures and forages with a higher percentage of legume will result in increased milk production. Grazing ewes respond to additional supplementation with increased milk yield, but it is important to match the supplement to the quality of the grazing. Ewes on high quality legume-grass pastures that are high in rumen degradable protein respond with increased milk production to supplements high in energy and/or high in rumen undegraded protein.
doi:10.1186/2049-1891-5-22
PMCID: PMC4004524  PMID: 24739956
Dairy sheep; East Friesian; Grazing; Lacaune; Lactation physiology; Nitrogen efficiency; RDP; RUP; Supplementation
12.  Mephedrone Does not Damage Dopamine Nerve Endings of the Striatum but Enhances the Neurotoxicity of Methamphetamine, Amphetamine and MDMA 
Journal of neurochemistry  2013;125(1):102-110.
Mephedrone (4-methylmethcathinone) is a β-ketoamphetamine stimulant drug of abuse with close structural and mechanistic similarities to methamphetamine. One of the most powerful actions associated with mephedrone is the ability to stimulate dopamine (DA) release and block its reuptake through its interaction with the dopamine transporter (DAT). Although mephedrone does not cause toxicity to DA nerve endings, its ability to serve as a DAT blocker could provide protection against methamphetamine-induced neurotoxicity like other DAT inhibitors. To test this possibility, mice were treated with mephedrone (10, 20 or 40 mg/kg) prior to each injection of a neurotoxic regimen of methamphetamine (4 injections of 2.5 or 5.0 mg/kg at 2 hr intervals). The integrity of DA nerve endings of the striatum was assessed through measures of DA, DAT and tyrosine hydroxylase levels. The moderate to severe DA toxicity associated with the different doses of methamphetamine was not prevented by any dose of mephedrone but was, in fact, significantly enhanced. The hyperthermia caused by combined treatment with mephedrone and methamphetamine was the same as seen after either drug alone. Mephedrone also enhanced the neurotoxic effects of amphetamine and MDMA on DA nerve endings. In contrast, nomifensine protected against methamphetamine-induced neurotoxicity. Because mephedrone increases methamphetamine neurotoxicity, the present results suggest that it interacts with the DAT in a manner unlike that of other typical DAT inhibitors. The relatively innocuous effects of mephedrone alone on DA nerve endings mask a potentially dangerous interaction with drugs that are often co-abused with it, leading to heightened neurotoxicity.
doi:10.1111/jnc.12114
PMCID: PMC3604033  PMID: 23205838
mephedrone; methamphetamine; MDMA; amphetamine; dopamine; neurotoxicity
13.  Genome-wide Association Study Identifies Two Susceptibility Loci for Osteosarcoma 
Nature genetics  2013;45(7):799-803.
Osteosarcoma is the most common primary bone malignancy of adolescents and young adults. In order to better understand the genetic etiology of osteosarcoma, we performed a multi-stage genome-wide association study (GWAS) consisting of 941 cases and 3,291 cancer-free adult controls of European ancestry. Two loci achieved genome-wide significance: rs1906953 at 6p21.3, in the glutamate receptor metabotropic 4 [GRM4] gene (P = 8.1 ×10-9), and rs7591996 and rs10208273 in a gene desert on 2p25.2 (P = 1.0 ×10-8 and 2.9 ×10-7). These two susceptibility loci warrant further exploration to uncover the biological mechanisms underlying susceptibility to osteosarcoma.
doi:10.1038/ng.2645
PMCID: PMC3910497  PMID: 23727862
14.  Measuring biexponential transverse relaxation of the ASL signal at 9.4 T to estimate arterial oxygen saturation and the time of exchange of labeled blood water into cortical brain tissue 
The transverse decay of the arterial spin labeling (ASL) signal was measured at four inflow times in the rat brain cortex at 9.4 T. Biexponential T2 decay was observed that appears to derive from different T2 values associated with labeled water in the intravasculature (IV) and extravascular (EV) compartments. A two compartment biexponential model was used to assess the relative contribution of the IV and EV compartments to the ASL signal, without assuming a value for T2 of labeled blood water in the vessels. This novel methodology was applied to estimate the exchange time of blood water into EV tissue space and the oxygen saturation of blood on the arterial side of the vasculature. The mean exchange time of labeled blood water was estimated to be 370±40 ms. The oxygen saturation of the arterial side of the vasculature was significantly less than 100% (∼85%), which may have implications for quantitative functional magnetic resonance imaging studies where the arterial oxygen saturation is frequently assumed to be 100%.
doi:10.1038/jcbfm.2012.156
PMCID: PMC3564190  PMID: 23168531
arterial spin labeling; ASL; cerebral hemodynamics; exchange; functional MRI (fMRI); MRI
15.  Identification of small molecules for human hepatocyte expansion and iPS differentiation 
Nature chemical biology  2013;9(8):514-520.
Cell-based therapies hold the potential to alleviate the growing burden of liver diseases. Such therapies require human hepatocytes, which, within the stromal context of the liver, are capable of many rounds of replication. However, this ability is lost ex vivo and human hepatocyte sourcing has been limiting many fields of research for decades. Here, we developed a high-throughput screening platform for primary human hepatocytes to identify small molecules in two different classes that can be used to generate renewable sources of functional human hepatocytes. One class induced functional proliferation of primary human hepatocytes in vitro. The second class enhanced hepatocyte functions and promoted differentiation of iPS-derived hepatocytes, toward a phenotype more mature than what was previously obtainable. The identification of these small molecules can help to address a major challenge impacting many facets of liver research and may lead to the development of novel therapeutics for liver diseases.
doi:10.1038/nchembio.1270
PMCID: PMC3720805  PMID: 23728495
16.  Bacterial community dynamics and activity in relation to dissolved organic matter availability during sea-ice formation in a mesocosm experiment 
MicrobiologyOpen  2014;3(1):139-156.
The structure of sea-ice bacterial communities is frequently different from that in seawater. Bacterial entrainment in sea ice has been studied with traditional microbiological, bacterial abundance, and bacterial production methods. However, the dynamics of the changes in bacterial communities during the transition from open water to frozen sea ice is largely unknown. Given previous evidence that the nutritional status of the parent water may affect bacterial communities during ice formation, bacterial succession was studied in under ice water and sea ice in two series of mesocosms: the first containing seawater from the North Sea and the second containing seawater enriched with algal-derived dissolved organic matter (DOM). The composition and dynamics of bacterial communities were investigated with terminal restriction fragment length polymorphism (T-RFLP), and cloning alongside bacterial production (thymidine and leucine uptake) and abundance measurements (measured by flow cytometry). Enriched and active sea-ice bacterial communities developed in ice formed in both unenriched and DOM-enriched seawater (0–6 days). γ-Proteobacteria dominated in the DOM-enriched samples, indicative of their capability for opportunistic growth in sea ice. The bacterial communities in the unenriched waters and ice consisted of the classes Flavobacteria, α-and γ-Proteobacteria, which are frequently found in natural sea ice in polar regions. Furthermore, the results indicate that seawater bacterial communities are able to adapt rapidly to sudden environmental changes when facing considerable physicochemical stress such as the changes in temperature, salinity, nutrient status, and organic matter supply during ice formation.
doi:10.1002/mbo3.157
PMCID: PMC3937737  PMID: 24443388
16S rDNA; bacteria; DOM; flow cytometry; mesocosm; sea ice; T-RFLP
17.  A microscale human liver platform that supports the hepatic stages of Plasmodium falciparum and vivax 
Cell host & microbe  2013;14(1):104-115.
SUMMARY
The Plasmodium liver stage is an attractive target for the development of anti-malarial drugs and vaccines, as it provides an opportunity to interrupt the life cycle of the parasite at a critical early stage. However, targeting the liver stage has been difficult. Undoubtedly, a major barrier has been the lack of robust, reliable and reproducible in vitro liver stage cultures. Here, we establish the liver stages for both Plasmodium falciparum and Plasmodium vivax in a microscale human liver platform composed of cryopreserved, micropatterned human primary hepatocytes surrounded by supportive stromal cells. Using this system, we have successfully recapitulated the full liver stage of P. falciparum including the release of infected merozoites and infection of overlaid erythrocytes, and also the establishment of small forms in late liver stages of P. vivax. Finally, we validate the potential of this platform as a tool for medium-throughput anti-malarial drug screening and vaccine development.
doi:10.1016/j.chom.2013.06.005
PMCID: PMC3780791  PMID: 23870318
18.  RB1-mediated cell-autonomous and host-dependent oncosuppressor mechanisms in radiation-induced osteosarcoma 
Oncoimmunology  2014;3(1):e27569.
The mechanisms by which retinoblastoma 1 (RB1) mediates oncosuppressive functions are still being elucidated. We found that radiation-induced senescence in the bone depends on RB1 and is associated with the secretion of multiple bioactive factors, including interleukin-6 (IL-6), as well as with the infiltration of natural killer T (NKT) cells. Importantly, the inhibition of RB1, IL-6 or NKT cells predisposed mice to radiation-induced osteosarcomas, unveiling a cancer cell-extrinsic mechanisms that underlie the oncosuppressive activity of RB1.
doi:10.4161/onci.27569
PMCID: PMC4006854  PMID: 24800165
interleukin-6; natural killer T cells; radiation; retinoblastoma 1; senescence; senescence-associated secretory phenotype (SASP)
19.  Anterior Glenohumeral Laxity and Stiffness After a Shoulder-Strengthening Program in Collegiate Cheerleaders 
Journal of Athletic Training  2013;48(1):25-30.
Context
Approximately 62% of all cheerleaders sustain some type of orthopaedic injury during their cheerleading careers. Furthermore, the occurrence of such injuries has led to inquiry regarding optimal prevention techniques. One possible cause of these injuries may be related to inadequate conditioning in cheerleaders.
Objective
To determine whether a strength and conditioning program produces quantifiable improvements in anterior glenohumeral (GH) laxity and stiffness.
Design
Descriptive laboratory study.
Setting
University laboratory.
Patients or Other Participants
A sample of 41 collegiate cheerleaders (24 experimental and 17 control participants) volunteered. No participants had a recent history (in the past 6 months) of upper extremity injury or any history of upper extremity surgery.
Intervention(s)
The experimental group completed a 6-week strength and conditioning program between the pretest and posttest measurements; the control group did not perform any strength training between tests.
Main Outcome Measure(s)
We measured anterior GH laxity and stiffness with an instrumented arthrometer. We conducted a group × time analysis of variance with repeated measures on time (P < .05) to determine differences between groups.
Results
A significant interaction was demonstrated, with the control group having more anterior GH laxity at the posttest session than the strengthening group (P = .03, partial η2 = 0.11). However, no main effect for time (P = .92) or group (P = .97) was observed. In another significant interaction, the control group had less anterior GH stiffness at the posttest session than the strengthening group (P = .03, partial η2 = 0.12). Main effects for time (P = .02) and group (P = .004) were also significant.
Conclusions
Cheerleaders who participate in a shoulder-strengthening program developed less anterior GH laxity and more stiffness than cheerleaders in the control group.
doi:10.4085/1062-6050-47.6.03
PMCID: PMC3554029  PMID: 23672322
instability; conditioning; injury prevention
20.  High-Throughput FRET Assay Yields Allosteric SERCA Activators 
Using fluorescence resonance energy transfer (FRET), we performed a high-throughput screen (HTS) in a reconstituted membrane system, seeking compounds that reverse inhibition of sarco-/endoplasmic reticulum Ca-ATPase (SERCA) by its endogenous regulator, phospholamban (PLB). Such compounds have long been sought to correct aberrant Ca2+ regulation in heart failure. Donor-SERCA was reconstituted in phospholipid membranes with or without acceptor-PLB, and FRET was measured in a steady-state fluorescence microplate reader. A 20,000-compound library was tested in duplicate. Compounds that decreased FRET by more than three standard deviations were considered hits. From 43 primary hits (0.2%), 31 (72%) were found to be false positives upon more thorough testing. The remaining 12 hits were tested in assays of Ca-ATPase activity, and six of these activated SERCA significantly, by as much as 60%, and several also enhanced cardiomyocyte contractility. These compounds directly activated SERCA from heart and other tissues. These results validate our FRET approach and set the stage for medicinal chemistry and pre-clinical testing. We were concerned about the high rate of false positives, resulting from the low precision of steady-state fluorescence. Preliminary studies with a novel fluorescence lifetime plate reader show 20-fold higher precision. This instrument can dramatically increase the quality of future HT.
doi:10.1177/1087057112456878
PMCID: PMC3721969  PMID: 22923787
calcium pump; calcium transport; phospholamban; reconstituted membrane; fluorescence lifetime
21.  Diabetic nephropathy: Is it time yet for routine kidney biopsy? 
World Journal of Diabetes  2013;4(6):245-255.
Diabetic nephropathy (DN) is one of the most important long-term complications of diabetes. Patients with diabetes and chronic kidney disease have an increased risk of all-cause mortality, cardiovascular mortality, and kidney failure. The clinical diagnosis of DN depends on the detection of microalbuminuria. This usually occurs after the first five years from the onset of diabetes, and predictors of DN development and progression are being studied but are not yet implemented into clinical practice. Diagnostic tests are useful tools to recognize onset, progression and response to therapeutic interventions. Microalbuminuria is an indicator of DN, and it is considered the only noninvasive marker of early onset. However, up to now there is no diagnostic tool that can predict which patients will develop DN before any damage is present. Pathological renal injury is hard to predict only with clinical and laboratory findings. An accurate estimate of damage in DN can only be achieved by the histological analysis of tissue samples. At the present time, renal biopsy is indicated on patients with diabetes under the suspicion of the presence of nephropathies other than DN. Results from renal biopsies in patients with diabetes had made possible the classification of renal biopsies in three major groups associated with different prognostic features: diabetic nephropathy, non-diabetic renal disease (NDRD), and a superimposed non-diabetic condition on underlying diabetic nephropathy. In patients with type 2 diabetes with a higher degree of suspicion for NDRD, it is granted the need of a renal biopsy. It is important to identify and differentiate these pathologies at an early stage in order to prevent progression and potential complications. Therefore, a more extensive use of biopsy is advisable.
doi:10.4239/wjd.v4.i6.245
PMCID: PMC3874483  PMID: 24379914
Diabetic nephropathy; Kidney biopsy; Non-diabetic renal disease
22.  Intra-patient dose escalation in Ewing’s sarcoma treated with vincristine, doxorubicin, cyclophosphamide alternating with ifosfamide and etoposide: a retrospective review 
Background
Data suggests that males experience less toxicity and poorer survival than females treated for Ewing’s sarcoma. We instituted an intra-patient dose escalation (DE) policy with Vincristine/Doxorubicin/Cyclophosphamide (VDC) alternating with Ifosfamide/Etoposide (IE) based on hematological nadirs and report its feasibility and safety.
Methods
A retrospective review of adherence to DE guidelines and toxicities was conducted for patients who received DE with VDC/IE over 3 years at a single cancer center. Absolute neutrophil counts (ANC) was collected on days 8, 12 and 15 for cycles 1–6. DE of 10%/cycle was applied if ANC > 1.5×109/L and platelet > 100×109/L on all blood results. The primary endpoint was the proportion of patients who received appropriate DE. The secondary endpoint was to assess morbidity, changes in hematologic nadirs between gender and age and a comparison with a prior cohort of ESFT patients who did not receive DE. Gender comparisons were assessed via independent 2-sample t-tests assuming unequal variances. Within cycle changes in hematologic nadirs were assessed using repeated measures ANOVA. Relapse free survival and overall survival (OS) curves were estimated using the Kaplan-Meier method.
Results
23 patients were identified (mean age: 27; range 17–54). 91 decisions for DE were made (1 decision excluded because of progressive disease) with 90% concordance with guidelines. No adverse outcomes occurred as a result of the inappropriate escalation. Grade 3/4 febrile neutropenia (FN) during VDC and IE was 26.1% (6/23 patients) and 17.4% respectively with no difference for those who were DE. Males were less neutropenic after C1 and C3 of VDC compared to females (P-value C1 = 0.003; C3 = 0.005). VDC was associated with greater neutropenia on day 8 whereas IE had greater neutropenia on day 12 (P-value <0.001). During VDC, a non statistical difference in neutropenia was seen for individuals aged 15–25 (n = 13) compared with older individuals (P-value = 0.09). OS comparison for those with localized disease with a prior cohort who were not DE showed similar outcomes (P-value = 0.37).
Conclusions
DE is deliverable without increased adverse outcomes. Males have less myelosuppression during VDC, and should be especially considered for DE.
doi:10.1186/2045-3329-3-15
PMCID: PMC3866566  PMID: 24321600
Ewing’s sarcoma; Dose escalation; Toxicity; Neutropenia; Chemotherapy
23.  The oncogenic properties of EWS/WT1 of desmoplastic small round cell tumors are unmasked by loss of p53 in murine embryonic fibroblasts 
BMC Cancer  2013;13:585.
Background
Desmoplastic small round cell tumor (DSRCT) is characterized by the presence of a fusion protein EWS/WT1, arising from the t (11;22) (p13;q12) translocation. Here we examine the oncogenic properties of two splice variants of EWS/WT1, EWS/WT1-KTS and EWS/WT1 + KTS.
Methods
We over-expressed both EWS/WT1 variants in murine embryonic fibroblasts (MEFs) of wild-type, p53+/- and p53-/- backgrounds and measured effects on cell-proliferation, anchorage-independent growth, clonogenicity after serum withdrawal, and sensitivity to cytotoxic drugs and gamma irradiation in comparison to control cells. We examined gene expression profiles in cells expressing EWS/WT1. Finally we validated our key findings in a small series of DSRCT.
Results
Neither isoform of EWS/WT1 was sufficient to transform wild-type MEFs however the oncogenic potential of both was unmasked by p53 loss. Expression of EWS/WT1 in MEFs lacking at least one allele of p53 enhanced cell-proliferation, clonogenic survival and anchorage-independent growth. EWS/WT1 expression in wild-type MEFs conferred resistance to cell-cycle arrest after irradiation and daunorubicin induced apoptosis. We show DSRCT commonly have nuclear localization of p53, and copy-number amplification of MDM2/MDMX. Expression of either isoform of EWS/WT1 induced characteristic mRNA expression profiles. Gene-set enrichment analysis demonstrated enrichment of WNT pathway signatures in MEFs expressing EWS/WT1 + KTS. Wnt-activation was validated in cell lines with over-expression of EWS/WT1 and in DSRCT.
Conclusion
In conclusion, we show both isoforms of EWS/WT1 have oncogenic potential in MEFs with loss of p53. In addition we provide the first link between EWS/WT1 and Wnt-pathway signaling. These data provide novel insights into the function of the EWS/WT1 fusion protein which characterize DSRCT.
doi:10.1186/1471-2407-13-585
PMCID: PMC4029184  PMID: 24321497
24.  Structural dynamics of muscle protein phosphorylation 
We have used site-directed spectroscopic probes to detect structural changes, motions, and interactions due to phosphorylation of proteins involved in the regulation of muscle contraction and relaxation. Protein crystal structures provide static snapshots that provide clues to the conformations that are sampled dynamically by proteins in the cellular environment. Our site-directed spectroscopic experiments, combined with computational simulations, extend these studies into functional assemblies in solution, and reveal details of protein regions that are too dynamic or disordered for crystallographic approaches. Here, we discuss phosphorylation-mediated structural transitions in the smooth muscle myosin regulatory light chain (RLC), the striated muscle accessory protein myosin binding protein-C (MyBP-C), and the cardiac membrane Ca2+ pump modulator phospholamban (PLB). In each of these systems, phosphorylation near the N terminus of the regulatory protein relieves an inhibitory interaction between the phosphoprotein and its regulatory target. Several additional unifying themes emerge from our studies: (a) The effect of phosphorylation is not to change the affinity of the phosphoprotein for its regulated binding partner, but to change the structure of the bound complex without dissociation. (b) Phosphorylation induces transitions between order and dynamic disorder. (c) Structural states are only loosely coupled to phosphorylation; i.e., complete phosphorylation induces dramatic functional effects with only a partial shift in the equilibrium between ordered and disordered structural states. These studies, which offer atomic-resolution insight into the structural and functional dynamics of these phosphoproteins, were inspired in part by the ground-breaking work in this field by Michael and Kate Barany.
doi:10.1007/s10974-012-9317-6
PMCID: PMC3652605  PMID: 22930331
Muscle Regulation; Spectroscopy; Structural Dynamics; Phospholamban; Regulatory Light Chain; Myosin Binding Protein-C
25.  Post-Weaning Diet Affects Faecal Microbial Composition but Not Selected Adipose Gene Expression in the Cat (Felis catus) 
PLoS ONE  2013;8(11):e80992.
The effects of pre- (i.e., gestation and during lactation) and post-weaning diet on the composition of faecal bacterial communities and adipose expression of key genes in the glucose and insulin pathways were investigated in the cat. Queens were maintained on a moderate protein:fat:carbohydrate kibbled (“Diet A”; 35:20:28% DM; n  =  4) or high protein:fat:carbohydrate canned (“Diet B”; 45:37:2% DM; n = 3) diet throughout pregnancy and lactation. Offspring were weaned onto these diets in a nested design (n  =  5 per treatment). Faecal samples were collected at wk 8 and 17 of age. DNA was isolated from faeces and bacterial 16S rRNA gene amplicons were analysed by pyrosequencing. RNA was extracted from blood (wk 18) and adipose tissue and ovarian/testicular tissues (wk 24) and gene expression levels determined using RT-qPCR. Differences (P<0.05) in composition of faecal bacteria were observed between pregnant queens fed Diet A or B. However, pre-weaning diet had little effect on faecal bacterial composition in weaned kittens. In contrast, post-weaning diet altered bacterial population profiles in the kittens. Increased (P<0.05) abundance of Firmicutes (77% vs 52% of total reads) and Actinobacteria (0.8% vs 0.2% of total reads), and decreased (P<0.05) abundance of Fusobacteria (1.6% vs 18.4% of total reads) were observed for kittens fed the Diet A compared to those fed Diet B post-weaning. Feeding Diet B pre-weaning increased (P<0.05) the expression levels of INRS, LEPT, PAI-1 and tended to increase GLUT1, while the expression levels of IRS-1 in blood increased in kittens fed Diet A pre-weaning. Post-weaning diet had no effect on expression levels of target genes. Correlations between the expression levels of genes involved in glucose and insulin pathways and faecal Bacteriodetes and Firmicutes phyla were identified. The reasons for why post-weaning diet affects microbial populations and not gene expression levels are of interest.
doi:10.1371/journal.pone.0080992
PMCID: PMC3842929  PMID: 24312255

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