Liver transcriptome response to hyperthermic stress in three distinct chicken lines.

BACKGROUND: High ambient temperatures cause stress in poultry, especially for broiler lines, which are genetically selected for rapid muscle growth. RNA-seq technology provides powerful insights into environmental response from a highly metabolic tissue, the liver. We investigated the effects of acute (3 h, 35 °C) and chronic (7d of 35 °C for 7 h/d) heat stress on the liver transcriptome of 3-week-old chicks of a heat-susceptible broiler line, a heat-resistant Fayoumi line, and their advanced intercross line (AIL). RESULTS: Transcriptome sequencing of 48 male chickens using Illumina HiSeq 2500 technology yielded an average of 33.9 million, 100 base-pair, single-end reads per sample. There were 8 times more differentially expressed genes (DEGs) (FDR < 0.05) in broilers (n = 627) than Fayoumis (n = 78) when comparing the acute-heat samples to the control (25 °C) samples. Contrasting genetic lines under similar heat treatments, the highest number of DEGs appeared between Fayoumi and broiler lines. Principal component analysis of gene expression and analysis of the number of DEGs suggested that the AIL had a transcriptomic response more similar to the Fayoumi than the broiler line during acute heat stress. The number of DEGs also suggested that acute heat stress had greater impact on the broiler liver transcriptome than chronic heat stress. The angiopoietin-like 4 (ANGPTL4) gene was identified as differentially expressed among all 6 contrasts. Ingenuity Pathway Analysis (IPA) created a novel network that combines the heat shock protein family with immune response genes. CONCLUSIONS: This study extends our understanding of the liver transcriptome response to different heat exposure treatments in distinct genetic chicken lines and provides information necessary for breeding birds to be more resilient to the negative impacts of heat. The data strongly suggest ANGPTL4 as a candidate gene for improvement of heat tolerance in chickens.

Pharmacologic management of comorbid post-traumatic stress disorder and addictions.

BACKGROUND AND OBJECTIVES: Post-traumatic Stress Disorder (PTSD) and substance use disorders (SUD) frequently co-occur, and their combination can increase poor health outcomes as well as mortality. METHODS: Using PUBMED and the list of references from key publications, this review article covered the epidemiology, neurobiology and pharmacotherapy of PTSD with comorbid alcohol, opiate, and cannabis use disorders. These SUD represent two with and one without FDA approved pharmacotherapies. RESULTS: SUD is two to three times more likely among individuals with lifetime PTSD, and suicide, which is made more likely by both of these disorders, appears to be additively increased by having this comorbidity of SUD and PTSD. The shared neurobiological features of these two illnesses include amygdalar hyperactivity with hippocampal, medial prefrontal and anterior cingulate cortex dysfunction. Medications for comorbid PTSD and SUD include the PTSD treatment sertraline, often used in combination with anticonvulsants, antipsychotics, and adrenergic blockers. When PTSD is comorbid with alcohol use disorder (AUD), naltrexone, acamprosate or disulfiram may be combined with PTSD treatments. Disulfiram alone may treat both PTSD and AUD. For PTSD combined with opiate use disorder methadone or buprenorphine are most commonly used with sertraline. Marijuana use has been considered by some to be a treatment for PTSD, but no FDA treatment for this addiction is approved. Pregabalin and D-cycloserine are two innovations in pharmacotherapy for PTSD and SUD. CONCLUSIONS AND SCIENTIFIC SIGNIFICANCE: Comorbid PTSD and SUD amplifies their lethality and treatment complexity. Although they share important neurobiology, these patients uncommonly respond to a single pharmacotherapy such as sertraline or disulfiram and more typically require medication combinations and consideration of the specific type of SUD.

Subunit Communication within Dimeric SF1 DNA Helicases.

Monomers of the Superfamily (SF) 1 helicases, E. coli Rep and UvrD, can translocate directionally along single stranded (ss) DNA, but must be activated to function as helicases. In the absence of accessory factors, helicase activity requires Rep and UvrD homo-dimerization. The ssDNA binding sites of SF1 helicases contain a conserved aromatic amino acid (Trp250 in Rep and Trp256 in UvrD) that stacks with the DNA bases. Here we show that mutation of this Trp to Ala eliminates helicase activity in both Rep and UvrD. Rep(W250A) and UvrD(W256A) can still dimerize, bind DNA, and monomers still retain ATP-dependent ssDNA translocase activity, although with ∼10-fold lower rates and lower processivities than wild type monomers. Although neither wtRep monomers nor Rep(W250A) monomers possess helicase activity by themselves, using both ensemble and single molecule methods, we show that helicase activity is achieved upon formation of a Rep(W250A)/wtRep hetero-dimer. An ATPase deficient Rep monomer is unable to activate a wtRep monomer indicating that ATPase activity is needed in both subunits of the Rep hetero-dimer. We find the same results with E. coli UvrD and its equivalent mutant (UvrD(W256A)). Importantly, Rep(W250A) is unable to activate a wtUvrD monomer and UvrD(W256A) is unable to activate a wtRep monomer indicating that specific dimer interactions are required for helicase activity. We also demonstrate subunit communication within the dimer by virtue of Trp fluorescence signals that only are present within the Rep dimer, but not the monomers. These results bear on proposed subunit switching mechanisms for dimeric helicase activity.

Transient-state kinetic analysis of transcriptional activator·DNA complexes interacting with a key coactivator.

Several lines of evidence suggest that the prototypical amphipathic transcriptional activators Gal4, Gcn4, and VP16 interact with the key coactivator Med15 (Gal11) during transcription initiation despite little sequence homology. Recent cross-linking data further reveal that at least two of the activators utilize the same binding surface within Med15 for transcriptional activation. To determine whether these three activators use a shared binding mechanism for Med15 recruitment, we characterized the thermodynamics and kinetics of Med15·activator·DNA complex formation by fluorescence titration and stopped-flow techniques. Combination of each activator·DNA complex with Med15 produced biphasic time courses. This is consistent with a minimum two-step binding mechanism composed of a bimolecular association step limited by diffusion, followed by a conformational change in the Med15·activator·DNA complex. Furthermore, the equilibrium constant for the conformational change (K(2)) correlates with the ability of an activator to stimulate transcription. VP16, the most potent of the activators, has the largest K(2) value, whereas Gcn4, the least potent, has the smallest value. This correlation is consistent with a model in which transcriptional activation is regulated at least in part by the rearrangement of the Med15·activator·DNA ternary complex. These results are the first detailed kinetic characterization of the transcriptional activation machinery and provide a framework for the future design of potent transcriptional activators.

Differential immunological response detected in mRNA expression profiles among diverse chicken lines in response to Salmonella challenge.

Salmonella enterica serovar Enteritidis is a bacterial pathogen that contributes to poultry production losses and human foodborne illness. The bacterium elicits a broad immune response involving both the innate and adaptive components of the immune system. Coordination of the immune response is largely directed by cytokines. The objective of the current study was to characterize the expression of a select set of cytokines and regulatory immune genes in three genetically diverse chicken lines after infection with S. Enteritidis. Leghorn, Fayoumi and broiler day-old chicks were orally infected with pathogenic S. Enteritidis or culture medium. At 2 and 18 h postinfection, spleens and ceca were collected and mRNA expression levels for 7 genes (GM-CSF, IL2, IL15, TGF-ß1, SOCS3, P20K, and MHC class IIß) were evaluated by real-time quantitative PCR. Genetic line had a significant effect on mRNA expression levels of IL15, TGF-ß1, SOCS3 and P20K in the spleen and on P20K and MHC class IIß in the cecum. Comparing challenged vs. unchallenged birds, the expression of SOCS3 and P20K mRNA were significantly higher in the spleen and cecum, while MHC class IIß mRNA was significantly lower in spleen. Combining the current RNA expression results with those of previously reported studies on the same samples reveals distinct RNA expression profiles among the three genetic chicken lines and the 2 tissues. This study illustrates that these diverse genetic lines have distinctively different immune response to S. Enteritidis challenge within the spleen and the cecum.

Impact of Medicaid Expansion on the Diagnosis, Treatment, and Outcomes of Stage II and III Rectal Cancer Patients.

BACKGROUND: Insurance status has been associated with disparities in stage at cancer diagnosis. We examined how Medicaid expansion (ME) impacted diagnoses, surgical treatment, use of neoadjuvant therapies (NCRT), and outcomes for Stage II and III rectal cancer. STUDY DESIGN: We used 2010-2017 American College of Surgeons National Cancer Database (NCDB) to identify patients ages 18-65, with Medicaid as primary form of payment, and were diagnosed with Stage II or III rectal cancer. Patients were stratified based on Census bureau division's ME adoption rates of High, Medium, Low. Overall trends were examined, and patient characteristics and outcomes were compared before and after ME date of 1/1/2014. RESULTS: Over 8 years of NCDB data examined, there was an increasing trend of Stage II and III rectal cancer diagnoses, surgical resection, and use of NCRT for Medicaid patients. We observed an increase in age, proportion of White Medicaid patients in Low ME divisions, and proportion of fourth income quartile patients in High ME divisions. Univariate analysis showed decreased use of open surgery for all 3 categories after ME, but adjusted odds ratios (aOR) were not significant based on multivariate analysis. NCRT utilization increased after ME for all 3 ME adoption categories and aOR significantly increased for Low and High ME divisions. ME significantly decreased 90-day mortality. CONCLUSIONS: Medicaid expansion had important impacts on increasing Stage II and III rectal cancer diagnoses, use of NCRT, and decreased 90-day mortality for patients with Medicaid. Our study supports increasing health insurance coverage to improve Medicaid patient outcomes in rectal cancer care.

Discovery of BLU-945, a Reversible, Potent, and Wild-Type-Sparing Next-Generation EGFR Mutant Inhibitor for Treatment-Resistant Non-Small-Cell Lung Cancer.

While epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have changed the treatment landscape for EGFR mutant (L858R and ex19del)-driven non-small-cell lung cancer (NSCLC), most patients will eventually develop resistance to TKIs. In the case of first- and second-generation TKIs, up to 60% of patients will develop an EGFR T790M mutation, while third-generation irreversible TKIs, like osimertinib, lead to C797S as the primary on-target resistance mutation. The development of reversible inhibitors of these resistance mutants is often hampered by poor selectivity against wild-type EGFR, resulting in potentially dose-limiting toxicities and a sub-optimal profile for use in combinations. BLU-945 (compound 30) is a potent, reversible, wild-type-sparing inhibitor of EGFR+/T790M and EGFR+/T790M/C797S resistance mutants that maintains activity against the sensitizing mutations, especially L858R. Pre-clinical efficacy and safety studies supported progression of BLU-945 into clinical studies, and it is currently in phase 1/2 clinical trials for treatment-resistant EGFR-driven NSCLC.

Conformational change in the Bacillus subtilis RNase P holoenzyme--pre-tRNA complex enhances substrate affinity and limits cleavage rate.

Ribonuclease P (RNase P) is a ribonucleoprotein complex that catalyzes the 5' maturation of precursor tRNAs. To investigate the mechanism of substrate recognition in this enzyme, we characterize the thermodynamics and kinetics of Bacillus subtilis pre-tRNA(Asp) binding to B. subtilis RNase P holoenzyme using fluorescence techniques. Time courses for fluorescein-labeled pre-tRNA binding to RNase P are biphasic in the presence of both Ca(II) and Mg(II), requiring a minimal two-step association mechanism. In the first step, the apparent bimolecular rate constant for pre-tRNA associating with RNase P has a value that is near the diffusion limit and is independent of the length of the pre-tRNA leader. Following formation of the initial enzyme-substrate complex, a unimolecular step enhances the overall affinity of pre-tRNA by eight- to 300-fold as the length of the leader sequence increases from 2 to 5 nucleotides. This increase in affinity is due to a decrease in the reverse rate constant for the conformational change that correlates with the formation of an optimal leader-protein interaction in the RNase P holoenzyme-pre-tRNA complex. Furthermore, the forward rate constant for the conformational change becomes rate limiting for cleavage under single-turnover conditions at high pH, explaining the origin of the observed apparent pK(a) in the RNase P-catalyzed cleavage reaction. These data suggest that a conformational change in the RNase P*pre-tRNA complex is coupled to the interactions between the 5' leader and P protein and aligns essential functional groups at the cleavage active site to enhance efficient cleavage of pre-tRNA.

Pre-tRNA turnover catalyzed by the yeast nuclear RNase P holoenzyme is limited by product release.

Ribonuclease P (RNase P) is a ribonucleoprotein that catalyzes the 5' maturation of precursor transfer RNA in the presence of magnesium ions. The bacterial RNase P holoenzyme consists of one catalytically active RNA component and a single essential but catalytically inactive protein. In contrast, yeast nuclear RNase P is more complex with one RNA subunit and nine protein subunits. We have devised an affinity purification protocol to gently and rapidly purify intact yeast nuclear RNase P holoenzyme for transient kinetic studies. In pre-steady-state kinetic studies under saturating substrate concentrations, we observed an initial burst of tRNA formation followed by a slower, linear, steady-state turnover, with the burst amplitude equal to the concentration of the holoenzyme used in the reaction. These data indicate that the rate-limiting step in turnover occurs after pre-tRNA cleavage, such as mature tRNA release. Additionally, the steady-state rate constants demonstrate a large dependence on temperature that results in nonlinear Arrhenius plots, suggesting that a kinetically important conformational change occurs during catalysis. Finally, deletion of the 3' trailer in pre-tRNA has little or no effect on the steady-state kinetic rate constants. These data suggest that, despite marked differences in subunit composition, the minimal kinetic mechanism for cleavage of pre-tRNA catalyzed by yeast nuclear RNase P holoenzyme is similar to that of the bacterial RNase P holoenzyme.

Transcriptome Response of Liver and Muscle in Heat-Stressed Laying Hens.

Exposure to high ambient temperature has detrimental effects on poultry welfare and production. Although changes in gene expression due to heat exposure have been well described for broiler chickens, knowledge of the effects of heat on laying hens is still relatively limited. In this study, we profiled the transcriptome for pectoralis major muscle (n = 24) and liver (n = 24), during a 4-week cyclic heating experiment performed on layers in the early phase of egg production. Both heat-control and time-based contrasts were analyzed to determine differentially expressed genes (DEGs). Heat exposure induced different changes in gene expression for the two tissues, and we also observed changes in gene expression over time in the control animals suggesting that metabolic changes occurred during the transition from onset of lay to peak egg production. A total of 73 DEGs in liver were shared between the 3 h heat-control contrast, and the 4-week versus 3 h time contrast in the control group, suggesting a core set of genes that is responsible for maintenance of metabolic homeostasis regardless of the physiologic stressor (heat or commencing egg production). The identified DEGs improve our understanding of the layer's response to stressors and may serve as targets for genetic selection in the future to improve resilience.

Pharmacologic Management of Comorbid Post-Traumatic Stress Disorder and Addictions.

(Reprinted with permission From The American Journal on Addictions, 24: 705-712, 2015).

Evaluation of thrombolysis using tissue plasminogen activator in lower extremity deep venous thrombosis with concomitant femoral-popliteal venous segment involvement.

OBJECTIVE: Current guidelines recommend thrombolytic therapy for iliofemoral deep venous thrombosis (DVT). Anticoagulation is the standard treatment for femoral-popliteal and tibial-level DVT. The objective of this study was to evaluate the efficacy of catheter-directed thrombolysis (CDT) using tissue plasminogen activator vs standard anticoagulation alone in patients with lower extremity DVT involving the femoral-popliteal segment. METHODS: A retrospective review was performed of patients referred to the vascular surgery service with lower extremity DVT from 2006 to 2015. Patients who had DVT involving the femoral-popliteal segment were identified, including some patients who had concomitant involvement of iliofemoral and tibial veins. Patients with pure iliofemoral and tibial vein DVT were excluded from this analysis. Review of medical records, follow-up ultrasound studies, hypercoagulable panel, and venography were performed. Comparison of outcomes between patients who received thrombolytic therapy using tissue plasminogen activator and patients who received standard anticoagulation alone was performed. The primary outcomes measured were restoration of patency of the femoral-popliteal segment at 3 months, incidence of post-thrombotic syndrome (PTS), and valvular dysfunction. Secondary outcomes were incidence of bleeding, in-hospital mortality, and pulmonary embolism. RESULTS: The study cohort was composed of 191 patients (CDT, n = 89; anticoagulation alone, n = 102) who met inclusion criteria. Most patients with thrombus involving the femoral-popliteal segment also had proximal venous segment involvement, with 93% of the patient cohort having proximal iliofemoral DVT. Patients who did not receive CDT were older (mean age of 64 years vs 51 years; P < .001) and had more associated comorbidities, such as diabetes, immobility, and cancer. A significant number of patients who received CDT had a positive family history for DVT (21.3% vs 8.8%; P = .023), and it was more likely to be their first episode of DVT (73.0% vs 55.9%; P = .016). Patients who received CDT were more likely to have restoration of patency (74.7% vs 11.1%; P < .001) and lower incidence of PTS (21.3% vs 73.4%; P < .001) and valvular dysfunction (23.0% vs 66.7%; P < .001) compared with patients who were treated with anticoagulation alone. Incidence of bleeding was significantly more for patients treated with anticoagulation alone (14.7% vs 5.6%; P = .018) compared with patients who received CDT. On multivariate analysis, age was the predominant risk factor for bleeding. There was no significant difference in mortality and pulmonary embolism. CONCLUSIONS: In patients with acute proximal DVT and concomitant femoral-popliteal venous segment involvement, CDT resulted in superior patency at 3 months and less PTS and valvular reflux. This was achieved without increase in bleeding complications compared with anticoagulation alone. Age was the major factor predictive of bleeding in either group. The results of this study may not be applicable to patients with pure femoral-popliteal venous segment DVT because only 3% of patients had this finding.

Integrated host and viral transcriptome analyses reveal pathology and inflammatory response mechanisms to ALV-J injection in SPF chickens.

Avian leukosis virus (ALV) is detrimental to poultry health and causes substantial economic losses from mortality and decreased performance. Because tumorigenesis is a complex mechanism, the regulatory architecture of the immune system is likely to include the added dimensions of modulation by miRNAs and long-noncoding RNA (lncRNA). To characterize the response to ALV challenge, we developed a novel methodology that combines four datasets: mRNA expression and the associated regulatory factors of miRNA and lncRNA, and ALV gene expression. Specific Pathogen-Free (SPF) layer chickens were infected with ALV-J or maintained as non-injected controls. Spleen samples were collected at 40 days post injection (dpi), and sequenced. There were 864 genes, 7 miRNAs and 17 lncRNAs differentially expressed between infected and non-infected birds. The combined analysis of the 4 RNA expression datasets revealed that ALV infection is detected by pattern-recognition receptors (TLR9 and TLR3) leading to a type-I IFN mediated innate immune response that is modulated by IRF7 and IRF1. Co-expression network analysis of mRNA with miRNA, lncRNA and virus genes identified key elements within the complex networks utilized during ALV response. The integration of information from the host transcriptomic, epigenetic and virus response also has the potential to provide deeper insights into other host-pathogen interactions.

Heat Stress and Lipopolysaccharide Stimulation of Chicken Macrophage-Like Cell Line Activates Expression of Distinct Sets of Genes.

Acute heat stress requires immediate adjustment of the stressed individual to sudden changes of ambient temperatures. Chickens are particularly sensitive to heat stress due to development of insufficient physiological mechanisms to mitigate its effects. One of the symptoms of heat stress is endotoxemia that results from release of the lipopolysaccharide (LPS) from the guts. Heat-related cytotoxicity is mitigated by the innate immune system, which is comprised mostly of phagocytic cells such as monocytes and macrophages. The objective of this study was to analyze the molecular responses of the chicken macrophage-like HD11 cell line to combined heat stress and lipopolysaccharide treatment in vitro. The cells were heat-stressed and then allowed a temperature-recovery period, during which the gene expression was investigated. LPS was added to the cells to mimic the heat-stress-related endotoxemia. Semi high-throughput gene expression analysis was used to study a gene panel comprised of heat shock proteins, stress-related genes, signaling molecules and immune response genes. HD11 cell line responded to heat stress with increased mRNA abundance of the HSP25, HSPA2 and HSPH1 chaperones as well as DNAJA4 and DNAJB6 co-chaperones. The anti-apoptotic gene BAG3 was also highly up-regulated, providing evidence that the cells expressed pro-survival processes. The immune response of the HD11 cell line to LPS in the heat stress environment (up-regulation of CCL4, CCL5, IL1B, IL8 and iNOS) was higher than in thermoneutral conditions. However, the peak in the transcriptional regulation of the immune genes was after two hours of temperature-recovery. Therefore, we propose the potential influence of the extracellular heat shock proteins not only in mitigating effects of abiotic stress but also in triggering the higher level of the immune responses. Finally, use of correlation networks for the data analysis aided in discovering subtle differences in the gene expression (i.e. the role of the CASP3 and CASP9 genes).

Safety, tolerability, and pharmacokinetics of escalating high doses of ivermectin in healthy adult subjects.

Safety and pharmacokinetics (PK) of the antiparasitic drug ivermectin, administered in higher and/or more frequent doses than currently approved for human use, were evaluated in a double-blind, placebo-controlled, dose escalation study. Subjects (n = 68) were assigned to one of four panels (3:1, ivermectin/placebo): 30 or 60 mg (three times a week) or 90 or 120 mg (single dose). The 30 mg panel (range: 34 7-594 microg/kg) also received a single dose with food after a 1-week washout. Safety assessments addressed both known ivermectin CNS effects and general toxicity. The primary safety endpoint was mydriasis, accurately quantitated by pupillometry. Ivermectin was generally well tolerated, with no indication of associated CNS toxicity for doses up to 10 times the highest FDA-approved dose of 200 microg/kg. All dose regimens had a mydriatic effect similar to placebo. Adverse experiences were similar between ivermectin and placebo and did not increase with dose. Following single doses of 30 to 120 mg, AUC and Cmax were generally dose proportional, with t(max) approximately 4 hours and t1/2 approximately 18 hours. The geometric mean AUC of 30 mg ivermectin was 2.6 times higher when administered with food. Geometric mean AUC ratios (day 7/day 1) were 1.24 and 1.40 for the 30 and 60 mg doses, respectively, indicating that the accumulation of ivermectin given every fourth day is minimal. This study demonstrated that ivermectin is generally well tolerated at these higher doses and more frequent regimens.

Modified high-performance liquid chromatographic method for the determination of ertapenem in human urine: enhanced selectivity and automation.

A column-switching, reversed-phase high-performance liquid chromatographic (HPLC) assay for a new structurally unique carbapenem antibiotic, ertapenem, in urine has been improved for selectivity and automated using a Packard MultiPROBE II EX pipetting station. The method uses column-switching for on-line extraction of the urine sample. The extraction column, analytical column, mobile phase, and timing of the column-switching valve have been changed to enhance selectivity for the analyte over endogenous background material. Sample transfer and dilution prior to direct-injection into the HPLC system have been accomplished using a Packard MultiPROBE II EX robotic liquid handling system.

Assay methodology for the quantitation of unbound ertapenem, a new carbapenem antibiotic, in human plasma.

Ertapenem is a new once-a-day antibiotic with excellent coverage of common community gram negative and gram positive aerobes and anaerobes. It demonstrates nonlinear protein binding in human plasma (about 94% bound). An assay for unbound drug was developed to study the pharmacokinetics of unbound ertapenem in plasma. Unbound drug is separated from plasma samples (1.0 ml) by ultrafiltration using a Centrifree((R)) centrifugal filter device. Ertapenem (vulnerable to hydrolysis of the beta-lactam moiety) is stabilized in the filtrate by adding an equal volume of 0.1 M MES buffer, pH 6.5 and then is analyzed by reversed-phase high-performance liquid chromatography (HPLC) with ultraviolet (UV) absorbance detection (300 nm). Non-specific binding to the Centrifree((R)) device is <3%. A suitable internal standard is not available. The assay is specific and linear over the concentration range of 0.25 to 100 microgram/ml in plasma filtrate. The lower limit of quantitation (LLOQ) is 0.25 microgram/ml. Intra-day precision is C.V.<10% and accuracy ranges from 97 to 101% of nominal concentration. Inter-day precision and accuracy were determined using quality control samples (QCs) prepared in plasma ultrafiltrate at 0.5, 12 and 80 microgram/ml and stored at -70 degrees C with stabilizer. Inter-day assay accuracy and precision ranged from 100 to 111% of nominal concentration and 1.8 to 5.3% C.V. (n=40), respectively. The assay has been used to analyze plasma samples from subjects receiving 500 and 2000 mg i.v. doses of ertapenem (30 min infusion).

Determination of simvastatin-derived HMG-CoA reductase inhibitors in biomatrices using an automated enzyme inhibition assay with radioactivity detection.

A robust, automated enzyme inhibition assay method was developed and validated for the determination of HMG-CoA reductase inhibitory activities in plasma and urine samples following simvastatin (SV) administration. The assay was performed on Tecan Genesis 150 and 200 systems equipped with 8-probe and 96-well plates. Plasma samples containing HMG-CoA reductase inhibitors were treated with acetonitrile for protein precipitation before being incubated with HMG-CoA reductase, [14C]-HMG-CoA, and NADPH for a fixed length of time at a fixed temperature. The product, [14C]-mevalonic acid, was lactonized and separated from excess substrate via a small ion exchange resin column, and radioactivity was counted on a scintillation counter. HMG-CoA reductase inhibitors were measured before and after base hydrolysis. The two values obtained for each sample are referred to as 'active' and 'total' HMG-CoA reductase inhibitor concentrations. Simvastatin acid (SVA), the beta-hydroxy acid of SV, was used as a standard to generate a calibration curve of HMG-CoA reductase activity versus SVA concentration (ng/ml). Three calibration ranges, 0.4-20, 2-50, and 50, 100 ng/ml, in human and animal plasma and urine were validated. The assay precision was less than 8.5%, CV in plasma and less than 10.4% in urine. The assay accuracy was 93.6-103.0 and 98.1-103.9% for the 0.4 20 and 2-50 ng/ml calibration ranges, respectively, in human plasma, and was 97.3-105.1, 94.4- 105.2, and 90.2-95.7%, for calibration range 5-100 ng/ml in rat plasma, dog plasma and human urine, respectively.

Large chromosome deletions, duplications, and gene conversion events accumulate with age in normal human colon crypts.

Little is known about the types and numbers of mutations that may accumulate in normal human cells with age. Such information would require obtaining enough DNA from a single cell to accurately carry out reliable analysis despite extensive amplification; and complete genomic coverage under these circumstances is difficult. We have compared colon crypts, which are putatively clonal and contain ~2000 cells each, to determine how much somatic genetic variation occurs in vivo (without ex vivo cell culturing). Using high-density SNP microarrays, we find that chromosome deletions, duplications, and gene conversions were significantly more frequent in colons from the older individuals. These changes affected lengths ranging from 73 kb to 46 Mb. Although detection requires progeny of a single mutant stem cell to reach niche dominance over neighboring stem cells, none of the deletions appear likely to confer a selective advantage. Mutations can become fixed randomly during stem cell evolution through neutral drift in normal human crypts. The fact that chromosomal changes are detected in individual crypts with increasing age suggests that either such changes accumulate with age or single stem cell dominance increases with age, and the former is more likely. This progressive genome-wide divergence of human somatic cells with age has implications for aging and disease in multicellular organisms.

Heterogeneity and randomness of DNA methylation patterns in human embryonic stem cells.

DNA methylation has been proposed to be important in many biological processes and is the subject of intense study. Traditional bisulfite genomic sequencing allows detailed high-resolution methylation pattern analysis of each molecule with haplotype information across a few hundred bases at each locus, but lacks the capacity to gather voluminous data. Although recent technological developments are aimed at assessing DNA methylation patterns in a high-throughput manner across the genome, the haplotype information cannot be accurately assembled when the sequencing reads are short or when each hybridization target only includes one or two cytosine-phosphate-guanine (CpG) sites. Whether a distinct and nonrandom DNA methylation pattern is present at a given locus is difficult to discern without the haplotype information, and the DNA methylation patterns are much less apparent because the data are often obtained only as methylation frequencies at each CpG site with some of these methods. It would facilitate the interpretation of data obtained from high-throughput bisulfite sequencing if the loci with nonrandom DNA methylation patterns could be distinguished from those that are randomly methylated. In this study, we carried out traditional genomic bisulfite sequencing using the normal diploid human embryonic stem (hES) cell lines, and utilized Hamming distance analysis to evaluate the existence of a distinct and nonrandom DNA methylation pattern at each locus studied. Our findings suggest that Hamming distance is a simple, quick, and useful tool to identify loci with nonrandom DNA methylation patterns and may be utilized to discern links between biological changes and DNA methylation patterns in the high-throughput bisulfite sequencing data sets.