Radiosensitization of noradrenaline transporter-expressing tumour cells by proteasome inhibitors and the role of reactive oxygen species

Background The radiopharmaceutical 131I-metaiodobenzylguanidine (131I-MIBG) is used for the targeted radiotherapy of noradrenaline transporter (NAT)-expressing neuroblastoma. Enhancement of 131I-MIBG's efficacy is achieved by combination with the topoisomerase I inhibitor topotecan - currently being evaluated clinically. Proteasome activity affords resistance of tumour cells to radiation and topoisomerase inhibitors. Therefore, the proteasome inhibitor bortezomib was evaluated with respect to its cytotoxic potency as a single agent and in combination with 131I-MIBG and topotecan. Since elevated levels of reactive oxygen species (ROS) are induced by bortezomib, the role of ROS in tumour cell kill was determined following treatment with bortezomib or the alternative proteasome inhibitor, MG132.<p></p> Methods Clonogenic assay and growth of tumour xenografts were used to investigate the effects of proteasome inhibitors alone or in combination with radiation treatment. Synergistic interactions in vitro were evaluated by combination index analysis. The dependency of proteasome inhibitor-induced clonogenic kill on ROS generation was assessed using antioxidants.<p></p> Results Bortezomib, in the dose range 1 to 30 nM, decreased clonogenic survival of both SK-N-BE(2c) and UVW/NAT cells, and this was prevented by antioxidants. It also acted as a sensitizer in vitro when administered with X-radiation, with 131I-MIBG, or with 131I-MIBG and topotecan. Moreover, bortezomib enhanced the delay of the growth of human tumour xenografts in athymic mice when administered in combination with 131I-MIBG and topotecan. MG132 and bortezomib had similar radiosensitizing potency, but only bortezomib-induced cytotoxicity was ROS-dependent.<p></p> Conclusions Proteasome inhibition shows promise for the treatment of neuroblastoma in combination with 131I-MIBG and topotecan. Since the cytotoxicity of MG132, unlike that of bortezomib, was not ROS-dependent, the latter proteasome inhibitor may have a favourable toxicity profile in normal tissues.<p></p&gt

The role of copper in disulfiram-induced toxicity and radiosensitisation of cancer cells.

Abstract Disulfiram has been used for several decades in the treatment of alcoholism. It now shows promise as an anti-cancer drug and radiosensitizer. Proposed mechanisms of action include the induction of oxidative stress and inhibition of proteasome activity. Our purpose was to determine the potential of disulfiram to enhance the anti-tumor efficacy of external beam -irradiation and 131I-metaiodobenzylguanidine (131I-MIBG), a radiopharmaceutical used for the therapy of neuroendocrine tumors. Methods: The role of copper in disulfiram-induced toxicity was investigated by clonogenic assay after treatment of human SK-N-BE(2c) neuroblastoma and UVW/NAT glioma cells. Synergistic interaction between disulfiram and radiotherapy was evaluated by combination index analysis. Tumor growth delay was determined in vitro using multicellular tumor spheroids and in vivo using human tumor xenografts in athymic mice. Results: Escalating disulfiram dosage caused a biphasic reduction in the surviving fraction of clonogens. Clonogenic cell kill after treatment with disulfiram concentrations less than 4 M was copper-dependent, whereas cytotoxicity at concentrations greater than 10 M was caused by oxidative stress. The cytotoxic effect of disulfiram was maximal when administered with equimolar copper. Likewise, disulfiram’s radiosensitization of tumor cells was copper-dependent. Furthermore, disulfiram treatment enhanced the toxicity of 131I-MIBG to spheroids and xenografts expressing the noradrenaline transporter. Conclusions: The results demonstrate that (i) the cytotoxicity of disulfiram was copper-dependent; (ii) molar excess of disulfiram relative to copper resulted in attenuation of disulfiram-mediated cytotoxicity; (iii) copper was required for the radiosensitizing activity of disulfiram and (iv) copper-complexed disulfiram enhanced the efficacy not only of external beam radiation but also of targeted radionuclide therapy in the form of 131I-MIBG. Therefore disulfiram may have anti-cancer potential in combination with radiotherapy

On FO2 quantifier alternation over words

We show that each level of the quantifier alternation hierarchy within FO^2[<] -- the 2-variable fragment of the first order logic of order on words -- is a variety of languages. We then use the notion of condensed rankers, a refinement of the rankers defined by Weis and Immerman, to produce a decidable hierarchy of varieties which is interwoven with the quantifier alternation hierarchy -- and conjecturally equal to it. It follows that the latter hierarchy is decidable within one unit: given a formula alpha in FO^2[<], one can effectively compute an integer m such that alpha is equivalent to a formula with at most m+1 alternating blocks of quantifiers, but not to a formula with only m-1 blocks. This is a much more precise result than what is known about the quantifier alternation hierarchy within FO[<], where no decidability result is known beyond the very first levels

DiffCoEx: a simple and sensitive method to find differentially coexpressed gene modules

Background: Large microarray datasets have enabled gene regulation to be studied through coexpression analysis. While numerous methods have been developed for identifying differentially expressed genes between two conditions, the field of differential coexpression analysis is still relatively new. More specifically, there is so far no sensitive and untargeted method to identify gene modules (also known as gene sets or clusters) that are differentially coexpressed between two conditions. Here, sensitive and untargeted means that the method should be able to construct de novo modules by grouping genes based on shared, but subtle, differential correlation patterns. Results: We present DiffCoEx, a novel method for identifying correlation pattern changes, which builds on the commonly used Weighted Gene Coexpression Network Analysis (WGCNA) framework for coexpression analysis. We demonstrate its usefulness by identifying biologically relevant, differentially coexpressed modules in a rat cancer dataset. Conclusions: DiffCoEx is a simple and sensitive method to identify gene coexpression differences between multiple conditions

The Word Problem for Omega-Terms over the Trotter-Weil Hierarchy

For two given $\omega$-terms $\alpha$ and $\beta$, the word problem for $\omega$-terms over a variety $\boldsymbol{\mathrm{V}}$ asks whether $\alpha=\beta$ in all monoids in $\boldsymbol{\mathrm{V}}$. We show that the word problem for $\omega$-terms over each level of the Trotter-Weil Hierarchy is decidable. More precisely, for every fixed variety in the Trotter-Weil Hierarchy, our approach yields an algorithm in nondeterministic logarithmic space (NL). In addition, we provide deterministic polynomial time algorithms which are more efficient than straightforward translations of the NL-algorithms. As an application of our results, we show that separability by the so-called corners of the Trotter-Weil Hierarchy is witnessed by $\omega$-terms (this property is also known as $\omega$-reducibility). In particular, the separation problem for the corners of the Trotter-Weil Hierarchy is decidable

Childhood Allergy and the NeOnatal Environment (CANOE) Research Protocol and Recruitment Redesign during the COVID-19 Pandemic

Rationale: Recruitment for research studies is a challenging endeavor that has been further complicated by the COVID-19 pandemic. While clinical research was temporarily halted due to the pandemic, it was hypothesized that study and recruitment restructuring would enable brisk enrollment when research resumed. Methods: A new NIH/ECHO-supported multi-center birth cohort, “Childhood Allergy and the NeOnatal Environment” (CANOE) was launched in January 2019 across four sites to determine how pre-, peri-, and post-natal factors influence development of recurrent wheezing and atopic dermatitis. Study recruitment was halted for nine months due to the COVID-19 pandemic, during which recruitment and study procedures were redesigned. Results: Recruitment strategies were modified to limit in-person contact, shifting toward alternative HIPAA-compliant methods like clinician referrals, institutional social media, and telemedicine consenting. Protocol changes included reducing frequency of in-person visits, leveraging clinical care visits to collect bio-samples, expanded self-collection of samples at home, and posting study materials online. Recruitment rates range from 3-12 families per month per site. In-clinic recruitment with modifications for social distancing has been successful across all sites. Other successful strategies have included targeted social media posts, mailed letters, and email. Rates of consent have been similar across recruitment strategies and the implementation of multiple recruitment strategies has yielded the highest rates of ongoing consent and enrollment of mother-infant dyads. Conclusions: Study procedures that prioritize health and safety measures such as social distancing, study participant convenience, and diversification of recruitment strategies enable continued birth cohort recruitment and data collection while adhering to public health restrictions during the pandemic

ZyFISH: A Simple, Rapid and Reliable Zygosity Assay for Transgenic Mice

Microinjection of DNA constructs into fertilized mouse oocytes typically results in random transgene integration at a single genomic locus. The resulting transgenic founders can be used to establish hemizygous transgenic mouse lines. However, practical and experimental reasons often require that such lines be bred to homozygosity. Transgene zygosity can be determined by progeny testing assays which are expensive and time-consuming, by quantitative Southern blotting which is labor-intensive, or by quantitative PCR (qPCR) which requires transgene-specific design. Here, we describe a zygosity assessment procedure based on fluorescent in situ hybridization (zyFISH). The zyFISH protocol entails the detection of transgenic loci by FISH and the concomitant assignment of homozygosity using a concise and unbiased scoring system. The method requires small volumes of blood, is scalable to at least 40 determinations per assay, and produces results entirely consistent with the progeny testing assay. This combination of reliability, simplicity and cost-effectiveness makes zyFISH a method of choice for transgenic mouse zygosity determinations

Examining virtual research recruitment and participant diversity in a multi-center birth cohort, Childhood Allergy and the NeOnatal Environment (CANOE)

Rationale: Recruitment for a NIH/ECHO-supported multi-center birth cohort, “Childhood Allergy and the NeOnatal Environment” (CANOE) stopped due to the COVID-19 pandemic. Redesign of study procedures emphasized virtual and socially distanced activities. We hypothesized that “virtual” recruitment methods (social media, websites, email) would surpass “traditional” methods (in-clinic, telephone, flyers/print materials) and increase enrollment of families from diverse backgrounds and communities. Methods: Pregnant women (n=439, target 500) were recruited from four academic medical centers in Detroit MI, Madison WI, Nashville TN, and St. Louis MO. We collected demographic and social information by questionnaires and examined race, ethnicity, age, parity, and employment status in relation to recruitment method using chi-square tests. Results: In-clinic and telephone recruitment comprised 55% of enrollment, followed by print materials (17%), and social media and email (15%). The cohort includes families self-identifying as Caucasian/White (63%), African American/Black (27%), Hispanic/Latino (3.3%), Asian (3.5%), and mixed races (1.2%). This reflects site demographics for White and Black patients, while other populations are not as well recruited into this cohort. Recruitment method success did not vary by race, ethnicity, maternal age, or employment status (p=ns for each comparison). Most (63%) multigravida mothers (9.1% of participants) were recruited in clinic, while primigravida participants were recruited more evenly via all methods. Conclusions: “Virtual” recruitment methods comprised a smaller proportion of cohort enrollment than hypothesized and study recruitment method did not vary by race/ethnicity; however, consideration of combined, varied, and novel recruitment methods may add to the development of best practices for more representative research study recruitment

Genetical genomics: spotlight on QTL hotspots

No abstract available

Ice Nucleation Activity and Aeolian Dispersal Success in Airborne and Aquatic Microalgae

Microalgae are common members of the atmospheric microbial assemblages. Diverse airborne microorganisms are known to produce ice nucleation active (INA) compounds, which catalyze cloud and rain formation, and thus alter cloud properties and their own deposition patterns. While the role of INA bacteria and fungi in atmospheric processes receives considerable attention, the numerical abundance and the capacity for ice nucleation in atmospheric microalgae are understudied. We isolated 81 strains of airborne microalgae from snow samples and determined their taxonomy by sequencing their ITS markers, 18S rRNA genes or 23S rRNA genes. We studied ice nucleation activity of airborne isolates, using droplet freezing assays, and their ability to withstand freezing. For comparison, we investigated 32 strains of microalgae from a culture collection, which were isolated from polar and temperate aqueous habitats. We show that ∼17% of airborne isolates, which belonged to taxa Trebouxiphyceae, Chlorophyceae and Stramenopiles, were INA. A large fraction of INA strains (over 40%) had ice nucleation activity at temperatures ≥-6°C. We found that 50% of aquatic microalgae were INA, but the majority were active at temperatures &lt;-12°C. Most INA compounds produced by microalgae were proteinaceous and associated with the cells. While there were no deleterious effects of freezing on the viability of airborne microalgae, some of the aquatic strains were killed by freezing. In addition, the effect of desiccation was investigated for the aquatic strains and was found to constitute a limiting factor for their atmospheric dispersal. In conclusion, airborne microalgae possess adaptations to atmospheric dispersal, in contrast to microalgae isolated from aquatic habitats. We found that widespread taxa of both airborne and aquatic microalgae were INA at warm, sub-zero temperatures (&gt;-15°C) and may thus participate in cloud and precipitation formation