The Application of CRISPR/Cas Technology to Efficiently Model Complex Cancer Genomes in Stem Cells

CRISPR/Cas gene editing technologies have emerged as powerful tools in the study of oncogenic transformation. The system's specificity, versatility, and ease of implementation allow researchers to identify important molecular markers and pathways which grant cancers stem cell like properties. This technology has already been applied to researching specific cancers, but has seen restricted therapeutic applications due to inherent ethical and technical limitations. Active development and adaptation of the CRISPR/Cas system has produced new methods to take advantage of both non‐homologous end joining and homologous recombination repair mechanisms in attempts to remedy these limitations and improve the versatility of gene edits that can be created. Nonetheless, until issues with specificity and in vivo efficiency are resolved, utilization of CRISPR/Cas systems would be best employed in the modeling and study of various cancer genes. While it may have potential therapeutic applications to targeted cancer therapies in the future, presently CRISPR/Cas is a remarkable technique that can be utilized for easy and efficient gene editing when it comes to cancer research. J. Cell. Biochem. 119: 134–140, 2018. © 2017 Wiley Periodicals, Inc.     

Cellular Localization, Expression, and Structure of the Nuclear Dot Protein 52

Nuclear dots containing PML and Sp100 proteins (NDs) play a role in the development of acute promyelocytic leukemia, are modified after infection with various viruses, and are autoimmunogenic in patients with primary biliary cirrhosis (PBC). PML and Sp100 gene expression is strongly enhanced by interferons (IFN). Based on immunostaining with a monoclonal antibody (mAb C8A2), a third protein, nuclear dot protein 52 (NDP52), was recently localized in NDs. Here we analyzed the cellular localization, expression, and structure of NDP52 in more detail. Our NDP52-specific sera revealed mainly cytoplasmic staining but no ND pattern, neither in untreated nor in IFN-treated cells. Cells transfected with NDP52 expression vectors showed exclusively cytoplasmic staining. In subcellular fractionation experiments, NDP52 was found in cytoplasmic and nuclear fractions. Unlike as described for Sp100 and PML, NDP52 mRNA and protein levels were only marginally enhanced by IFN γ and not enhanced at all by IFN β. NDP52 homodimerization but no heterodimerization with Sp100 or PML could be demonstrated. None of the 93 PBC sera tested contained autoantibodies against NDP52. Finally, mAb C8A2 reacted not only with NDP52 but also with a conformation-dependent epitope on the Sp100 protein. These data imply that NDP52 forms homodimers but no heterodimers with Sp100 and PML, lacks autoantigenicity in PBC, localizes mainly in the cytoplasm, and is associated with the nucleus, but not with NDs. Finally, unlike Sp100 and PML, NDP52 expression is neither markedly enhanced nor localization detectably altered by type I and II IFNs.     

Selective mortality of a coral reef damselfish: role of predator–competitor synergisms

Phenotypic variability within cohorts of juvenile organisms can serve as the basis for selective mortality. Previous studies have demonstrated the important role that predators play in this process but not the impact of competitors on selective predation. We use a combination of lab and field studies to evaluate the effect of the presence of adult competitor damselfish (Dascyllus aruanus and Pomacentrus moluccensis; family Pomacentridae) on the selective mortality of newly-arrived (settled) lemon damselfish (P. moluccensis) by resident predator fishes (Pseudochromis fuscus Pseudochromidae and Cheilodipterus quinquelineatus Apogonidae). Lab trials consisted of mesocosm experiments in which the behaviour, mortality, and physiological condition (measured as lipid content) of surviving P. moluccensis settlers from each of three treatments: (1) predators absent, (2) predators present, and (3) predators and competitors present, were compared. The field study involved stocking newly settled P. moluccensis on natural bommies (patch reefs) which had either been subject to a partial removal of resident fish (predators and competitors) or left alone. Results indicated there was very strong condition-based selective mortality in both the lab and field trials. In both cases there was a strong positive relationship between mortality and the lipid content of surviving fish; implying low-condition fish were selectively removed. The mesocosm trials indicated that the strength of mortality as well as condition selectivity was higher when competitors were present than when they were absent. Behavioural observations in the mesocosm study suggest that attention by juvenile P. moluccensis to the movements and occasional chases of the competitors (especially D. aruanus) reduced their vigilance to the predators. These results suggest the important and interactive roles which condition of newly settled reef fish and interspecific competition can play in the outcomes of post-settlement predation. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Ultraviolet light sensitivity of carotene-and cytochrome-c-accumulating strains of the smut fungusUstilago violacea

White, pink, orange, and yellow strains ofUstilago violacea containing high and low levels of cytochrome c and various carotenes were exposed to ultraviolet light. The survival curves for all strains were of exponential decay form, but the carotene-accumulating strains were generally more resistant to UV than those strains with no carotenes at all. The UV exposure time leading to 90% loss in viability, LD₉₀, was quantitatively related to the carotene content in the form of a power function, where LD₉₀ increased as the fifth root of the total carotene content per cell. We also determined that the ratio of total carotenes to total cytochrome c per cell was quantitatively related to the rate of viability loss during UV exposure.     

Changes in public order after the opening of a medically supervised safer injecting facility for illicit injection drug users

Background

North America''s first medically supervised safer injecting facility for illicit injection drug users was opened in Vancouver on Sept. 22, 2003. Although similar facilities exist in a number of European cities and in Sydney, Australia, no standardized evaluations of their impact have been presented in the scientific literature.

Methods

Using a standardized prospective data collection protocol, we measured injection-related public order problems during the 6 weeks before and the 12 weeks after the opening of the safer injecting facility in Vancouver. We measured changes in the number of drug users injecting in public, publicly discarded syringes and injection-related litter. We used Poisson log-linear regression models to evaluate changes in these public order indicators while considering potential confounding variables such as police presence and rainfall.

Results

In stratified linear regression models, the 12-week period after the facility''s opening was independently associated with reductions in the number of drug users injecting in public (p < 0.001), publicly discarded syringes (p < 0.001) and injection-related litter (p < 0.001). The predicted mean daily number of drug users injecting in public was 4.3 (95% confidence interval [CI] 3.5–5.4) during the period before the facility''s opening and 2.4 (95% CI 1.9–3.0) after the opening; the corresponding predicted mean daily numbers of publicly discarded syringes were 11.5 (95% CI 10.0–13.2) and 5.4 (95% CI 4.7–6.2). Externally compiled statistics from the city of Vancouver on the number of syringes discarded in outdoor safe disposal boxes were consistent with our findings.

Interpretation

The opening of the safer injecting facility was independently associated with improvements in several measures of public order, including reduced public injection drug use and public syringe disposal.Many cities are experiencing epidemics of bloodborne diseases as a result of illicit injection drug use,^1,2,3 and drug overdoses have become a leading cause of death in many urban areas.^4,5,6 Public drug use also plagues many inner city neighbourhoods, and the unsafe disposal of syringes in these settings is a major community concern.^{7,8,9,10,11,12,13}In over 2 dozen European cities and, more recently, in Sydney, Australia, medically supervised safer injecting facilities, where injection drug users (IDUs) can inject previously obtained illicit drugs under the supervision of medical staff, have been established in an effort to reduce the community and public health impacts of illicit drug use.¹⁴ Inside these facilities IDUs are typically provided with sterile injecting equipment, emergency care in the event of overdose, as well as primary care services and referral to addiction treatment.^13,15 Although anecdotal reports have suggested that such sites may improve public order,¹² reduce the number of deaths from overdose¹⁶ and improve access to care,¹⁷ no standardized evaluations of their impact are available in the scientific literature.¹⁸On Sept. 22, 2003, health officials in Vancouver opened a government-sanctioned safer injecting facility as pilot project. The facility, the first in North America, is centrally located in Vancouver''s Downtown Eastside, which is the most impoverished urban neighbourhood in Canada and home to well-documented overdose and HIV epidemics among the estimated 5000 IDUs who reside there.^19,20 Federal approval for the 3-year project was granted on the condition that the health and social impacts of the facility be rigorously evaluated. Although evaluation of the facility''s impact on certain outcomes (e.g., HIV incidence) is ongoing and will take several years, it is now possible to examine the impacts of the site on public order. Therefore, we conducted this study to test the hypothesis that changes in improperly discarded syringes and public drug use would be observed after the opening of the safer injecting facility.     

Adherence to Chemoprophylaxis and Plasmodium falciparum Anti-Circumsporozoite Seroconversion in a Prospective Cohort Study of Dutch Short-Term Travelers

Background

We conducted a prospective study in a cohort of short-term travelers assessing the incidence rate of anti-circumsporozoite seroconversion, adherence to chemoprophylaxis, symptoms of malaria during travel, and malaria treatment abroad.

Methods

Adults were recruited from the travel clinic of the Public Health Service Amsterdam. They kept a structured daily travel diary and donated blood samples before and after travel. Blood samples were serologically tested for the presence of Plasmodium falciparum anti-circumsporozoite antibodies.

Results

Overall, the incidence rate (IR) of anti-circumsporozoite seroconversion was 0.8 per 100 person-months. Of 945 travelers, 620 (66%) visited high-endemic areas and were advised about both chemoprophylaxis and preventive measures against mosquito bites. Most subjects (520/620 = 84%) took at least 75% of recommended prophylaxis during travel. Travel to Africa, use of mefloquine, travel duration of 14–29 days in endemic areas, and concurrent use of DEET (N,N-diethyl-meta-toluamide) were associated with good adherence practices. Four travelers without fever seroconverted, becoming anti-circumsporozoite antibody-positive. All four had been adherent to chemoprophylaxis; two visited Africa, one Suriname, one India. Ten subjects with fever were tested for malaria while abroad and of these, three received treatment. All three were adherent to chemoprophylaxis and tested negative for anti-circumsporozoite antibodies.

Conclusion

Travel to Africa, using mefloquine, travel duration of 14–29 days in endemic areas, and use of DEET were associated with good adherence to chemoprophylaxis. The combination of chemoprophylaxis and other preventive measures were sufficient to protect seroconverting travelers from clinical malaria. Travelers who were treated for malaria abroad did not seroconvert.     

PIC-1/SUMO-1-Modified PML-Retinoic Acid Receptor α Mediates Arsenic Trioxide-Induced Apoptosis in Acute Promyelocytic Leukemia

Fusion proteins involving the retinoic acid receptor alpha (RARalpha) and PML or PLZF nuclear protein are the genetic markers of acute promyelocytic leukemia (APL). APLs with PML-RARalpha or PLZF-RARalpha fusion protein differ only in their response to retinoic acid (RA) treatment: the t(15;17) (PML-RARalpha-positive) APL blasts are sensitive to RA in vitro, and patients enter disease remission after RA treatment, while those with t(11;17) (PLZF-RARalpha-positive) APLs do not. Recently it has been shown that complete remission can be achieved upon treatment with arsenic trioxide (As2O3) in PML-RARalpha-positive APL, even when the patient has relapsed and the disease is RA resistant. This appears to be due to apoptosis induced by As2O3 in the APL blasts by poorly defined mechanisms. Here we report that (i) As2O3 induces apoptosis only in cells expressing the PML-RARalpha, not the PLZF-RARalpha, fusion protein; (ii) PML-RARalpha is partially modified by covalent linkage with a PIC-1/SUMO-1-like protein prior to As2O3 treatment, whereas PLZF-RARalpha is not; (iii) As2O3 treatment induces a change in the modification pattern of PML-RARalpha toward highly modified forms; (iv) redistribution of PML nuclear bodies (PML-NBs) upon As2O3 treatment is accompanied by recruitment of PIC-1/SUMO-1 into PML-NBs, probably due to hypermodification of both PML and PML-RARalpha; (v) As2O3-induced apoptosis is independent of the DNA binding activity located in the RARalpha portion of the PML-RARalpha fusion protein; and (vi) the apoptotic process is bcl-2 and caspase 3 independent and is blocked only partially by a global caspase inhibitor. Taken together, these data provide novel insights into the mechanisms involved in As2O3-induced apoptosis in APL and predict that treatment of t(11;17) (PLZF-RARalpha-positive) APLs with As2O3 will not be successful.     

Novel benzimidazole-based MCH R1 antagonists

The identification of an MCH R1 antagonist screening hit led to the optimization of a class of benzimidazole-based MCH R1 antagonists. Structure-activity relationships and efforts to optimize pharmacokinetic properties are detailed along with the demonstration of the effectiveness of an MCH R1 antagonist in an animal model of obesity.     

Distribution of Phytophthora cinnamomi at different spatial scales: When can a negative result be considered positively?

Abstract In October 1999, patches of dead and dying trees were identified in rainforest vegetation throughout the Tully Falls area in north Queensland, Australia. Previous incidents of patch death in the region had been attributed to Phytophthora cinnamomi. The distribution of P. cinnamomi was assessed by testing for its presence in seven sites displaying signs of dieback and seven sites that appeared healthy. Each site was a circular quadrat, 20‐m radius (total area = 1256.6 m²). Within each quadrat, two perpendicular line transects were constructed. A single soil sample (250 g) was taken at the centre point and at 1‐m intervals along each transect. All soil samples were tested for the presence of P. cinnamomi using a combination of lupin baiting, subsequent culturing and microscopic identification. Of the 1134 samples, 783 recorded positive responses. The mean number of positive responses was not significantly greater in patch death sites than in control sites, suggesting that at this scale of resolution the distribution of P. cinnamomi was uniform. However, at spatial scales of 1‐m intervals across transects the distribution of P. cinnamomi was random.