Genomic profiling of solid tumors harboring BRD4-NUT and response to immune checkpoint inhibitors

BackgroundThe translocation t(15:19) produces the oncogenic BRD4-NUT fusion which is pathognomonic for NUT carcinoma (NC), which is a rare, but extremely aggressive solid tumor. Comprehensive genomic profiling (CGP) by hybrid-capture based next generation sequencing of 186+ genes of a cohort of advanced cancer cases with a variety of initial diagnoses harboring BRD4-NUT may shed further insight into the biology of these tumors and possible options for targeted treatment.Case presentationThirty-one solid tumor cases harboring a BRD4-NUT translocation are described, with only 16% initially diagnosed as NC and the remainder carrying other diagnoses, most commonly NSCLC—NOS (22%) and lung squamous cell carcinoma (NSCLC-SCC) (16%). The cohort was all microsatellite stable and harbored a low Tumor Mutational Burden (TMB, mean 1.7 mut/mb, range 0–4). In two index cases, patients treated with immune checkpoint inhibitors (ICPI) had unexpected partial or better responses of varying duration. Notably, four cases – including the two index cases - were negative for PD-L1 expression. Neo-antigen prediction for BRD4-NUT and then affinity modeling of the peptide-MHC (pMHC) complex for an assessable index case predicted very high affinity binding, both on a ranked (99.9%) and absolute (33 nM) basis.ConclusionsCGP identifies BRD4-NUT fusions in advanced solid tumors which carry a broad range of initial diagnoses and which should be re-diagnosed as NC per guidelines. A hypothesized mechanism underlying responses to ICPI in the low TMB, PD-L1 negative index cases is the predicted high affinity of the BRD4-NUT fusion peptide to MHC complexes. Further study of pMHC affinity and response to immune checkpoint inhibitors in patients with NC harboring BRD4-NUT is needed to validate this therapeutic hypothesis.     

Antioxidant properties of aged garlic extract: an in vitro study incorporating human low density lipoprotein

Oxidation of low-density lipoprotein (LDL) has been recognized as playing an important role in the development and progression of atherosclerotic heart disease. Human LDL was isolated and challenged with a range of oxidants either in the presence or absence of AGE or its diethyl ether extract. Oxidative modification of the LDL fraction using CuSO(4), 5-lipoxygenase and xanthine/xanthine oxidase was monitored by both the appearance of thiobarbituric-acid substances (TBA-RS) and an increase in electrophoretic mobility.This study indicates that AGE is an effective antioxidant as it scavenged superoxide ions and reduced lipid peroxide formation in cell free assays. Superoxide production was completely inhibited in the presence of a 10% (v/v) aqueous preparation of AGE and reduced by 34% in the presence of a 10% (v/v) diethyl ether extract of AGE. The presence of 10% (v/v) diethyl ether extract of AGE significantly reduced Cu(2+) and 15-lipoxygenase-mediated lipid peroxidation of isolated LDL by 81% and 37%, respectively. In addition, it was found that AGE also had the capacity to chelate copper ions. In contrast, the diethyl ether extract of AGE displayed no copper binding capacity, but demonstrated distinct antioxidant properties. These results support the view that AGE inhibits the in vitro oxidation of isolated LDL by scavenging superoxide and inhibiting the formation of lipid peroxides. AGE was also shown to reduce LDL oxidation by the chelation of Cu(2+). Thus, AGE may have a role to play in preventing the development and progression of atherosclerotic disease.     

Recapitulation of Tumor Heterogeneity and Molecular Signatures in a 3D Brain Cancer Model with Decreased Sensitivity to Histone Deacetylase Inhibition

Introduction

Physiologically relevant pre-clinical ex vivo models recapitulating CNS tumor micro-environmental complexity will aid development of biologically-targeted agents. We present comprehensive characterization of tumor aggregates generated using the 3D Rotary Cell Culture System (RCCS).

Methods

CNS cancer cell lines were grown in conventional 2D cultures and the RCCS and comparison with a cohort of 53 pediatric high grade gliomas conducted by genome wide gene expression and microRNA arrays, coupled with immunohistochemistry, ex vivo magnetic resonance spectroscopy and drug sensitivity evaluation using the histone deacetylase inhibitor, Vorinostat.

Results

Macroscopic RCCS aggregates recapitulated the heterogeneous morphology of brain tumors with a distinct proliferating rim, necrotic core and oxygen tension gradient. Gene expression and microRNA analyses revealed significant differences with 3D expression intermediate to 2D cultures and primary brain tumors. Metabolic profiling revealed differential profiles, with an increase in tumor specific metabolites in 3D. To evaluate the potential of the RCCS as a drug testing tool, we determined the efficacy of Vorinostat against aggregates of U87 and KNS42 glioblastoma cells. Both lines demonstrated markedly reduced sensitivity when assaying in 3D culture conditions compared to classical 2D drug screen approaches.

Conclusions

Our comprehensive characterization demonstrates that 3D RCCS culture of high grade brain tumor cells has profound effects on the genetic, epigenetic and metabolic profiles of cultured cells, with these cells residing as an intermediate phenotype between that of 2D cultures and primary tumors. There is a discrepancy between 2D culture and tumor molecular profiles, and RCCS partially re-capitulates tissue specific features, allowing drug testing in a more relevant ex vivo system.     

Plated Cambrian bilaterians reveal the earliest stages of echinoderm evolution

Echinoderms are unique in being pentaradiate, having diverged from the ancestral bilaterian body plan more radically than any other animal phylum. This transformation arises during ontogeny, as echinoderm larvae are initially bilateral, then pass through an asymmetric phase, before giving rise to the pentaradiate adult. Many fossil echinoderms are radial and a few are asymmetric, but until now none have been described that show the original bilaterian stage in echinoderm evolution. Here we report new fossils from the early middle Cambrian of southern Europe that are the first echinoderms with a fully bilaterian body plan as adults. Morphologically they are intermediate between two of the most basal classes, the Ctenocystoidea and Cincta. This provides a root for all echinoderms and confirms that the earliest members were deposit feeders not suspension feeders.     

Changing domesticity of Aedes aegypti in northern peninsular Malaysia: reproductive consequences and potential epidemiological implications

Background

The domestic dengue vector Aedes aegypti mosquitoes breed in indoor containers. However, in northern peninsular Malaysia, they show equal preference for breeding in both indoor and outdoor habitats. To evaluate the epidemiological implications of this peridomestic adaptation, we examined whether Ae. aegypti exhibits decreased survival, gonotrophic activity, and fecundity due to lack of host availability and the changing breeding behavior.

Methodology/Principal Findings

This yearlong field surveillance identified Ae. aegypti breeding in outdoor containers on an enormous scale. Through a sequence of experiments incorporating outdoors and indoors adapting as well as adapted populations, we observed that indoors provided better environment for the survival of Ae. aegypti and the observed death patterns could be explained on the basis of a difference in body size. The duration of gonotrophic period was much shorter in large-bodied females. Fecundity tended to be greater in indoor acclimated females. We also found increased tendency to multiple feeding in outdoors adapted females, which were smaller in size compared to their outdoors breeding counterparts.

Conclusion/Significance

The data presented here suggest that acclimatization of Ae. aegypti to the outdoor environment may not decrease its lifespan or gonotrophic activity but rather increase breeding opportunities (increased number of discarded containers outdoors), the rate of larval development, but small body sizes at emergence. Size is likely to be correlated with disease transmission. In general, small size in Aedes females will favor increased blood-feeding frequency resulting in higher population sizes and disease occurrence.     

Auxin and ethylene: collaborators or competitors?

The individual roles of auxin and ethylene in controlling the growth and development of young seedlings have been well studied. In recent years, these two hormones have been shown to act synergistically to control specific growth and developmental processes, such as root elongation and root hair formation, as well as antagonistically in other processes, such as lateral root formation and hypocotyl elongation. This review examines the growth and developmental processes that are regulated by crosstalk between these two hormones and explores the mechanistic basis for the regulation of these processes. The emerging trend from these experiments is that ethylene modulates auxin synthesis, transport, and signaling with unique targets and responses in a range of tissues to fine-tune seedling growth and development.     

Cloning and characterization of two new thermostable and alkalitolerant α-amylases from the <Emphasis Type="Italic">Anoxybacillus</Emphasis> species that produce high levels of maltose

Two genes that encode α-amylases from two Anoxybacillus species were cloned and expressed in Escherichia coli. The genes are 1,518 bp long and encode 506 amino acids. Both sequences are 98% similar but are distinct from other well-known α-amylases. Both of the recombinant enzymes, ASKA and ADTA, were purified using an α-CD–Sepharose column. They exhibited an optimum activity at 60°C and pH 8. Both amylases were stable at pH 6–10. At 60°C in the absence of Ca²⁺, negligible reduction in activity for up to 48 h was observed. The activity half-life at 65°C was 48 and 3 h for ASKA and ADTA, respectively. In the presence of Ca²⁺ ions, both amylases were highly stable for at least 48 h and had less than a 10% decrease in activity at 70°C. Both enzymes exhibited similar end-product profiles, and the predominant yield was maltose (69%) from starch hydrolysis. To the best of our knowledge, most α-amylases that produce high levels of maltose are active at an acidic to neutral pH. This is the first report of two thermostable, alkalitolerant recombinant α-amylases from Anoxybacillus that produce high levels of maltose and have an atypical protein sequence compared with known α-amylases.     

Comparison of the estimation capabilities of response surface methodology and artificial neural network for the optimization of recombinant lipase production by E. coli BL21

Response surface methodology (RSM) and artificial neural network (ANN) were used to optimize the effect of four independent variables, viz. glucose, sodium chloride (NaCl), temperature and induction time, on lipase production by a recombinant Escherichia coli BL21. The optimization and prediction capabilities of RSM and ANN were then compared. RSM predicted the dependent variable with a good coefficient of correlation determination (R ²) and adjusted R ² values for the model. Although the R ² value showed a good fit, absolute average deviation (AAD) and root mean square error (RMSE) values did not support the accuracy of the model and this was due to the inferiority in predicting the values towards the edges of the design points. On the other hand, ANN-predicted values were closer to the observed values with better R ², adjusted R ², AAD and RMSE values and this was due to the capability of predicting the values throughout the selected range of the design points. Similar to RSM, ANN could also be used to rank the effect of variables. However, ANN could not predict the interactive effect between the variables as performed by RSM. The optimum levels for glucose, NaCl, temperature and induction time predicted by RSM are 32 g/L, 5 g/L, 32°C and 2.12 h, and those by ANN are 25 g/L, 3 g/L, 30°C and 2 h, respectively. The ANN-predicted optimal levels gave higher lipase activity (55.8 IU/mL) as compared to RSM-predicted levels (50.2 IU/mL) and the predicted lipase activity was also closer to the observed data at these levels, suggesting that ANN is a better optimization method than RSM for lipase production by the recombinant strain.