A Medium-Throughput Single Cell CRISPR-Cas9 Assay to Assess Gene Essentiality.

BACKGROUND: Target selection for oncology is a crucial step in the successful development of therapeutics. Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 editing of specific loci offers an alternative method to RNA interference and small molecule inhibitors for determining whether a cell line is dependent on a specific gene product for proliferation or survival. In our initial studies using CRISPR-Cas9 to verify the dependence on EZH2 activity for proliferation of a SMARCB1/SNF5/INI1 mutant malignant rhabdoid tumor (MRT) cell line, we noted that the initial reduction in proliferation was lost over time. We hypothesized that in the few cells that retain proliferative capacity, at least one allele of EZH2 remains functional. To verify this, we developed an assay to analyze 10s-100s of clonal cell populations for target gene disruption using restriction digest and fluorescent fragment length analyses. RESULTS: Our results clearly show that in cell lines in which EZH2 is essential for proliferation, at least one potentially functional allele of EZH2 is retained in the clones that survive. CONCLUSION: This assay clearly indicates whether or not a specific gene is essential for survival and/or proliferation in a given cell line. Such data can aid the development of more robust therapeutics by increasing confidence in target selection.

Tree species partition N uptake by soil depth in boreal forests.

It is recognized that the coexistence of herbaceous species in N-depleted habitats can be facilitated by N partitioning; however, the existence of such a phenomenon for trees has not yet been demonstrated. Here, we show from both foliage and soil 15N natural abundance values and from a 12-year in situ 15N addition experiment, that black spruce (Picea mariana) and jack pine (Pinus banksiana), two widespread species of the Canadian boreal forest, take up N at different depths. While black spruce takes up N from the organic soil, jack pine acquires it deeper within the highly N-depleted mineral soil. Systematic difference in foliar 15N natural abundance between the two species across seven sites distributed throughout the eastern Canadian boreal forest shows that N spatial partitioning is a widespread phenomenon. Distinct relationships between delta15N and N concentration in leaves of both species further emphasize their difference in N acquisition strategies. This result suggests that such complementary mechanisms of N acquisition could facilitate tree species coexistence in such N-depleted habitats and could contribute to the positive biodiversity-productivity relationship recently revealed for the eastern Canadian boreal forest, where jack pine is present. It also has implications for forest management and provides new insights to interpret boreal forest regeneration following natural or anthropogenic perturbations.

TreeMos: a high-throughput phylogenomic approach to find and visualize phylogenetic mosaicism.

SUMMARY: TreeMos is a novel high-throughput graphical analysis application that allows the user to search for phylogenetic mosaicism among one or more DNA or protein sequence multiple alignments and additional unaligned sequences. TreeMos uses a sliding window and local alignment algorithm to identify the nearest neighbour of each sequence segment, and visualizes instances of sequence segments whose nearest neighbour is anomalous to that identified using the global alignment. Data sets can include whole genome sequences allowing phylogenomic analyses in which mosaicism may be attributed to recombination between any two points in the genome. TreeMos can be run from the command line, or within a web browser allowing the relationships between taxa to be explored by drill-through. AVAILABILITY: http://www2.warwick.ac.uk/fac/sci/whri/research/archaeobotany.

Nuclear import of Cdk/cyclin complexes: identification of distinct mechanisms for import of Cdk2/cyclin E and Cdc2/cyclin B1.

Reversible phosphorylation of nuclear proteins is required for both DNA replication and entry into mitosis. Consequently, most cyclin-dependent kinase (Cdk)/cyclin complexes are localized to the nucleus when active. Although our understanding of nuclear transport processes has been greatly enhanced by the recent identification of nuclear targeting sequences and soluble nuclear import factors with which they interact, the mechanisms used to target Cdk/cyclin complexes to the nucleus remain obscure; this is in part because these proteins lack obvious nuclear localization sequences. To elucidate the molecular mechanisms responsible for Cdk/cyclin transport, we examined nuclear import of fluorescent Cdk2/cyclin E and Cdc2/cyclin B1 complexes in digitonin-permeabilized mammalian cells and also examined potential physical interactions between these Cdks, cyclins, and soluble import factors. We found that the nuclear import machinery recognizes these Cdk/cyclin complexes through direct interactions with the cyclin component. Surprisingly, cyclins E and B1 are imported into nuclei via distinct mechanisms. Cyclin E behaves like a classical basic nuclear localization sequence-containing protein, binding to the alpha adaptor subunit of the importin-alpha/beta heterodimer. In contrast, cyclin B1 is imported via a direct interaction with a site in the NH2 terminus of importin-beta that is distinct from that used to bind importin-alpha.

Coupled afterslip and transient mantle flow after the 2011 Tohoku earthquake.

Modeling of postseismic deformation following great earthquakes has revealed the viscous structure of the mantle and the frictional properties of the fault interface. However, for giant megathrust events, viscoelastic flow and afterslip mechanically interplay with each other during the postseismic period. We explore the role of afterslip and viscoelastic relaxation and their interaction in the aftermath of the 2011 M w (moment magnitude) 9.0 Tohoku earthquake based on a detailed model analysis of the postseismic deformation with laterally varying, experimentally constrained, rock rheology. Mechanical coupling between viscoelastic relaxation and afterslip notably modifies both the afterslip distribution and surface deformation. Thus, we highlight the importance of addressing mechanical coupling for long-term studies of postseismic relaxation, especially in the context of the geodynamics of the Japan trench across the seismic cycle.

Heat capacity and latent heat measurements of CoMnSi using a microcalorimeter.

A new method of utilizing a commercial silicon nitride membrane calorimeter to measure the latent heat at a first order phase transition is presented. The method is a direct measurement of the thermoelectric voltage jump induced by the latent heat, in a thermally isolated system ideally suited for single crystal and small microgram samples. We show that when combined with the ac calorimetry technique previously developed, the resultant thermal measurement capabilities are extremely powerful. We demonstrate the applicability of the combined method with measurements on a 100 microm size fragment of CoMnSi exhibiting a sizable magnetocaloric effect near room temperature, and obtain good agreement with previously reported values on bulk samples.

Expression of the mouse c-abl type IV proto-oncogene product in the insect cell baculovirus system.

The cellular gene c-abl is the normal homologue of the transforming gene (v-abl) within the genome of the Abelson leukaemia virus. The cDNA sequence coding for the cellular form of the murine abl gene (c-abl type IV) has been inserted into the baculovirus transfer vector, pAc36C, so that the c-abl gene is under the control of the polyhedrin promoter of Autographa californica nuclear polyhedrosis virus (AcNPV). Spodoptera frugiperda cells infected with the recombinant transfer vector in the presence of wild type AcNPV DNA yielded recombinant, polyhedrin negative virus that expressed moderate levels of the c-Abl protein (representing approx. 0.5-1% of the stained cellular proteins as determined by densitometric scanning). The insect derived c-Abl protein was compared to the P210-BCR/ABL protein from K562 cells, a cell line derived from a patient with chronic myelogenous leukaemia. Antibodies raised against synthetic peptides based on c-abl encoded peptides react with the insect derived c-Abl. In addition, the baculovirus derived c-Abl protein has a tyrosine kinase activity as demonstrated by phosphorylation of a synthetic polypeptide and also by autophosphorylation. Phosphoamino acid analysis of immunoprecipitated, autophosphorylated baculovirus derived c-Abl protein indicates that the majority of label incorporated is on the tyrosine residues. Immunofluorescence microscopy has been used to show that the majority of the c-Abl protein expressed in cells infected with recombinant virus is located in the nuclear and plasma membranes.

Crystallization of a type II dehydroquinase from Mycobacterium tuberculosis.

A type II 3-dehydroquinase from Mycobacterium tuberculosis has been crystallized in the presence of 6% polyethyleneglycol 6000. Data from these crystals have been collected to a resolution of 2.2 A on a rotating anode X-ray source. The space group has been determined as F23 with unit cell dimensions of a = b = c = 127.8 A. There is one molecule in the asymmetric unit.

Crystallization of a type I 3-dehydroquinase from Salmonella typhi.

Crystals have been grown of a type I 3-dehydroquinase from both Escherichia coli and Salmonella typhi. However, only those from S. typhi diffract to a resolution of 2.3 A on a conventional X-ray source and are suitable for structure determination. The space group has been determined as P2(1)2(1)2 with unit cell dimensions a = 48.01 A, b = 114.29 A, c = 42.87 A. There is one subunit in the asymmetric unit.

N-desmethyldiazepam: a new metabolite of chlordiazepoxide in man.

Following administration of chlordiazepoxide HCl to man, N-desmethyldiazepam, a known metabolite of diazepam (Valium), was identified in plasma. The metabolite was identified on the basis of its thin-layer chromatographic (TLC) mobility, electron-capture gas-chromatographic (EC-GC) retention time, and mass spectrum relative to authentic N-desmethyldiazepam. Plasma levels of N-desmethyldiazepam in subjects receiving both single and chronic doses of chlordiazepoxide were determined by an EC-GC method with a limit of sensitivity of 10 ng/ml using 2-ml samples and by a radioimmunoassay procedure which had a limit of sensitivity of 20 ng/ml using a 0.1-ml sample. Both assay methods gave good agreement for the levels of N-desmethyldiazepam. In subjects receiving a single 30-mg oral or intravenous dose of chlordiazepoxide, measurable levels of N-desmethyldiazepam in plasma (10 to 60 ng/ml) were obtained 24 to 72 hr after administration. In 5 subjects receiving 10 mg of chlordiazepoxide three times a day, steady-state levels of N-desmethyldiazepam in plasma were reached after about 1 wk of administration. The mean maximum and minimum steady-state levels of N-desmethyldiazepam were 260 and 220 ng/ml of plasma, respectively. Similar steady-state levels were observed on treatment with 30 mg of chlordiazepoxide over 24 hr.

Evolution of transcription-regulating proteins by enzyme recruitment: molecular models for nitrogen metabolite repression and ethanol utilisation in eukaryotes.

Studies on the quinic acid utilisation gene (qut) cluster in Aspergillus nidulans showed that the genes encoding transcriptional activator and repressor proteins evolved by co-opting duplicated copies of genes encoding metabolic enzymes. In order to test the hypothesis that this was a general route for the genesis of regulatory proteins, the origins of the major control protein mediating nitrogen metabolite repression (an example of inter-pathway regulation) and ethanol utilisation (an example of intra-pathway regulation) in filamentous fungi were sought. The regulatory proteins mediating nitrogen metabolite repression were deduced to have originated in a duplication of genes encoding the anthranilate synthase complex which is active in the shikimate pathway. The major protein regulating ethanol utilisation was deduced to have its origin in the fusion of duplicated genes encoding the aldehyde and alcohol dehydrogenases (ALDA and ALCA). These data strongly support the view that transcriptional regulatory proteins evolve by the recruitment of functional domains provided by metabolic enzymes.

Genesis of eukaryotic transcriptional activator and repressor proteins by splitting a multidomain anabolic enzyme.

The genes necessary for the correctly regulated catabolism of quinate in Aspergillus nidulans and Neurospora crassa are controlled at the level of transcription by a DNA-binding activator protein and a repressor protein that directly interact with one another. The repressor protein is homologous throughout its length with the three C-terminal domains of a pentafunctional enzyme catalysing five consecutive steps in the related anabolic shikimate pathway. We now report that the activator protein is homologous to the two N-terminal domains of the same pentafunctional enzyme and that this proposed structural similarity suggests a molecular mechanism by which the repressor recognises the activator protein. We believe that this is the first report of the genesis of a pair of interacting eukaryotic regulatory proteins by the splitting of a multidomain anabolic enzyme. The recruitment of preformed enzymatically active domains to a regulatory role may represent a general mechanism for the evolution of pathway-specific regulator proteins in dispensable pathways.

Potassium affects actigraph-identified sleep.

The present study examines the effects of potassium supplementation on sleep quality and phase, as indirectly inferred from wrist actigraphy and sleep logs, in normal young males on a low-potassium diet. A randomized, double-blind, placebo-controlled, counterbalanced crossover design compared 1 wk of oral potassium chloride supplements (96 meq/day) to 1 wk of identical placebo capsules. Outcome measures were taken from sleep logs and wrist actigraphy. Sleep was indirectly inferred from wrist-actigraph data using a computer algorithm. Potassium supplementation significantly delayed sleep-log-identified Bedtime (p less than 0.001). Potassium reduced Sleeping Interval for both sleep-log (p less than 0.01) and wrist-actigraph (p less than 0.1) data. Potassium significantly increased actigraphic Sleep Efficiency (p less than 0.05) due to a reduction in actigraphic Wake after Sleep Onset (WASO) (p less than 0.05). No effect of potassium on actigraphic sleep phase was observed. Side effects were minimal and not significantly different between treatment conditions. The results may indicate an improvement in sleep consolidation with potassium supplementation. Further studies using standard polysomnography are required to define potassium's effects on human sleep.

Simultaneous quantitation of the 5'-triphosphate metabolites of zidovudine, lamivudine, and stavudine in peripheral mononuclear blood cells of HIV infected patients by high-performance liquid chromatography tandem mass spectrometry.

A high-performance liquid chromatography (HPLC) method utilizing triple quadrupole mass spectrometry (MS) detection was developed and validated for the simultaneous measurement of the intracellular nucleoside 5'-triphosphate anabolites of zidovudine (ZDV-TP), lamivudine (3TC-TP), and stavudine (d4T-TP). These compounds were extracted from patient peripheral blood mononuclear cells (PBMCs) which are the sites of HIV replication and drug action. Ion-exchange solid phase extraction (SPE) followed by enzymatic digestion with alkaline phosphatase was utilized to yield the measurable nucleoside forms of the nucleotides. Reversed phase C-18 SPE with addition of a nucleoside internal standard, 3'-azido-2',3'-dideoxyuridine (AzdU) allowed for the indirect measurement of the original 5'-triphosphate concentration by HPLC/MS/MS. Quantitation was performed from calibration curves generated from authentic 5'-triphosphate standards spiked in PBMCs from healthy volunteers. Analytical range for the three 5'-triphosphates was equivalent to 50-45,000 pg. Mean interassay accuracies for 3TC-TP, d4T-TP, and ZDV-TP (n > 90) were 99.4%, 100.1%, and 108.0%, respectively. Mean interassay precisions (%C.V.) for 3TC-TP, d4T-TP, and ZDV-TP (n > 90) were 8.8%, 10.4%, and 8.2%, respectively. Recovery of the extraction method was 79.2%, 83.1%, and 98.3% for 3TC-TP, d4T-TP, and ZDV-TP, respectively. This method can be utilized to measure the intracellular 5'-triphosphate levels in HIV infected patients receiving antiretroviral therapy containing the nucleoside reverse transcriptase inhibitors 3TC, d4T, or ZDV.

Arteriovenous malformations of the gastrointestinal tract.

During an eight-year period, 17 patients ranging in age from 7 months to 81 years were found to have arteriovenous malformations (AVMs) of the gastrointestinal tract complicated by major hemorrhage. After review of these cases, a clinical classification of AVMs was developed, based on angiographic characteristics, localization, age of the patient, and family history. Type 1 AVMs were solitary, localized lesions within the right side of the colon. They occurred in seven patients 55 years of age or older. None were palpable or visible at operation. Type 2 AVMs occurred in seven patients. They were larger and occasionally visible, most commonly in the small intestine, and probably of congenital origin. Symptoms all began before 50 years of age. Type 3 AVMs were punctate angiomas causing gastrointestinal hemorrhage. They occurred in three patients with the classical findings of hereditary hemorrhagic telangiectasia. The diagnosis of most AVMs can be determined preoperatively only by selective angiographic studies.

Effect of flurazepam on common clinical laboratory tests.

Twenty-two clinical laboratory tests performed on blood samples from 16 normal subjects following one week of either flurazepam or placebo administered in a double-blind method showed no apparent chemical interference by flurazepam on any of the testing procedures.

Phase I dose escalation pharmacokinetics of O-(chloroacetylcarbamoyl) fumagillol (TNP-470) and its metabolites in AIDS patients with Kaposi's sarcoma.

The pharmacokinetics of TNP-470 and its major metabolites were investigated in AIDS patients enrolled in a phase I dose escalation trial for the treatment of Kaposi's sarcoma. The patients received TNP-470 by 1-h intravenous infusion in dose cohorts of 10, 20, 30, 40, 50 and 70 mg/m2. The parent drug and metabolites, MII and MIV, were measured by high-performance liquid chromatography/mass spectrometry (HPLC/MS) in plasma samples collected during and out to 168 h after the beginning of the infusion. Both metabolites were detected in all patients' plasma, while the parent drug was undetectable at time-points as early as 5 min after the end of infusion for some patients. A large interpatient variability of pharmacokinetic parameters among the dosing cohorts was observed for TNP-470, with a mean (+/- SD) plasma elimination half-life (t1/2) of 0.06 +/- 0.04 h, plasma clearance (CL) of 1487 +/- 1216 l/h and an area under the concentration versus time curve (AUC) of 49.9 +/- 35.8 ng/ml x h. Time to maximum plasma concentration (Tmax) typically occurred before the end of the infusion. The predominant plasma metabolite was MII with a t1/2 of 1.21 +/- 0.43 h, AUC of 1226 +/- 2303 l/h and a Tmax occurring between 5 and 15 min after infusion. The reported active metabolite MIV had a t1/2 of 0.24 +/- 0.13 h, AUC of 24.9 +/- 32.6 ng/ml x h and a Tmax occurring between the midpoint of the infusion and 15 min after infusion. The parent drug was undetectable by HPLC/MS/MS in urine samples collected and pooled between 0-6 and 6-24 h from the beginning of drug administration. Metabolite MIV was present in the 0-6-h urine pool of two patients enrolled in the highest dosing cohorts, equivalent to 0.4% of the administered dose. Metabolite MII was present in all 0-6-h samples analyzed and represented 1.12 +/- 0.9% of the administered dose. Renal clearance (CLR) for MII was 140 +/- 70 ml/h.

The prevalence of serological markers to hepatitis A and hepatitis B in drug abuse personnel.

A seroepidemiologic survey was conducted in 25 clinical and 31 nonclinical personnel engaged in substance abuse research and treatment. The antibody to hepatitis A virus (anti-HAV) was identified in 16 percent of the clinical employees and 39 percent of the nonclinical personnel, for a total prevalence of 29 percent. The prevalence of anti-HAV was age related, and the overall frequency was not greater than expected attack rates. Hepatitis B virus (HBV) markers were present in 16 percent of the clinical personnel tested but in only 3 percent of nonclinical personnel, for a total prevalence of 9 percent among the two groups. The nonclinical risk did not exceed the background prevalence of the disease, but the risk for clinical employees was three to five times greater. This risk appeared to be associated with exposure to blood and other potentially infectious body fluids; however, in no case could an employee with HBV markers recall a percutaneous injury, and covert means of transmission could not be excluded. Only one case (20 percent) of HBV infection manifested clinical symptomatology. Even though proper environmental safety measures may decrease the incidence of HBV infection in at-risk groups, some health care workers and researchers in the field of substance abuse may be at sufficiently increased risk of HBV infection to warrant immunization with hepatitis B vaccine.

Depression and dysthymia.

The advances made in the 1980s and 1990s have yielded many advances in the diagnosis and treatment of depression and dysthymia. Skill of the clinician is important in sorting out the diagnosis, taking care to consider the various medical conditions that can cause depression or disguise themselves as depression. Depressive disorders are highly treatable conditions. Clinicians must overcome the stigma associated with these disorders to alleviate the pain and suffering of those afflicted. The advances in treatment have been enormous and continue to grow. The keys to these treatments lie in continuing to acquire the knowledge to unlock all of the causes of depression. An appendix follows listing medications commonly used in the treatment of depression or for other conditions in patients under treatment for depression.

Diazepam blocks fear-potentiated startle in humans.

The effects of an anxiolytic drug (diazepam) on emotional responses to aversive stimuli were investigated using physiological measures, including the startle probe reflex. Participants were 54 university students assigned to either a placebo group or a 10 mg or 15 mg diazepam group in a double-blind design. Blink responses to intermittent noise probes were recorded during viewing of neutral and unpleasant slides. Consistent with prior animal work, diazepam blocked startle potentiation during aversive stimulus processing without decreasing the overall magnitude of startle responses. These findings suggest that a common defensive state mediates startle reflex potentiation in animals and humans and that this index of fear can be used to assess the emotional effects of different drugs.