Development and validation of an acid/alkaline digestion method for efficient microplastic extraction from wastewater treatment plant effluents: Sulfuric acid concentration and contact time do matter.

Accurate analysis of microplastic particles (MPs) in environmental samples requires removal of interferences during sample preparation. Wastewater samples are interference-rich and thus particularly challenging, with concentrated sulfuric acid currently deemed impractical as a reagent. Therefore, this study aimed to establish a straightforward, effective, and safe method employing concentrated sulfuric acid and potassium hydroxide to eliminate interferents from effluent samples obtained from wastewater treatment plants (WWTPs). We found that 80 % sulfuric acid at room temperature with a brief contact time of 5 min was viable through a qualitative spot test involving 37 plastics categorized into three types (I, II, and III) based on their polymer structure's oxygen position. A quantitative assessment revealed that treatments involving H2SO4 and KOH (20 %, 24 h, 48 °C), either separately or in combination, had no discernible physical impact on the overall plastics, except for a subtle one for Type III plastics (e.g., nylon and PMMA) known to be labile under harsh pH conditions. This acid/alkaline digestion (AAD) method, incorporating such conditions for H2SO4 and KOH treatments, yielded a high mass removal efficacy (97.8 ± 2.4 %, n = 13) for eliminating natural particle interferents for primary, secondary, and tertiary effluent samples. Furthermore, the AAD method allowed for the determination of MPs in effluents with high surrogate particle recoveries (e.g., 95.1 % for larger than 500 µm size fraction). This method is readily adaptable to create appropriate protocols for different types of environmental matrices.

Signature-driven repurposing of Midostaurin for combination with MEK1/2 and KRASG12C inhibitors in lung cancer.

Drug combinations are key to circumvent resistance mechanisms compromising response to single anti-cancer targeted therapies. The implementation of combinatorial approaches involving MEK1/2 or KRASG12C inhibitors in the context of KRAS-mutated lung cancers focuses fundamentally on targeting KRAS proximal activators or effectors. However, the antitumor effect is highly determined by compensatory mechanisms arising in defined cell types or tumor subgroups. A potential strategy to find drug combinations targeting a larger fraction of KRAS-mutated lung cancers may capitalize on the common, distal gene expression output elicited by oncogenic KRAS. By integrating a signature-driven drug repurposing approach with a pairwise pharmacological screen, here we show synergistic drug combinations consisting of multi-tyrosine kinase PKC inhibitors together with MEK1/2 or KRASG12C inhibitors. Such combinations elicit a cytotoxic response in both in vitro and in vivo models, which in part involves inhibition of the PKC inhibitor target AURKB. Proteome profiling links dysregulation of MYC expression to the effect of both PKC inhibitor-based drug combinations. Furthermore, MYC overexpression appears as a resistance mechanism to MEK1/2 and KRASG12C inhibitors. Our study provides a rational framework for selecting drugs entering combinatorial strategies and unveils MEK1/2- and KRASG12C-based therapies for lung cancer.

Kras oncogene ablation prevents resistance in advanced lung adenocarcinomas.

KRASG12C inhibitors have revolutionized the clinical management of patients with KRASG12C-mutant lung adenocarcinoma. However, patient exposure to these inhibitors leads to the rapid onset of resistance. In this study, we have used genetically engineered mice to compare the therapeutic efficacy and the emergence of tumor resistance between genetic ablation of mutant Kras expression and pharmacological inhibition of oncogenic KRAS activity. Whereas Kras ablation induces massive tumor regression and prevents the appearance of resistant cells in vivo, treatment of KrasG12C/Trp53-driven lung adenocarcinomas with sotorasib, a selective KRASG12C inhibitor, caused a limited antitumor response similar to that observed in the clinic, including the rapid onset of resistance. Unlike in human tumors, we did not observe mutations in components of the RAS-signaling pathways. Instead, sotorasib-resistant tumors displayed amplification of the mutant Kras allele and activation of xenobiotic metabolism pathways, suggesting that reduction of the on-target activity of KRASG12C inhibitors is the main mechanism responsible for the onset of resistance. In sum, our results suggest that resistance to KRAS inhibitors could be prevented by achieving a more robust inhibition of KRAS signaling mimicking the results obtained upon Kras ablation.

KRAS4A induces metastatic lung adenocarcinomas in vivo in the absence of the KRAS4B isoform.

In mammals, the KRAS locus encodes two protein isoforms, KRAS4A and KRAS4B, which differ only in their C terminus via alternative splicing of distinct fourth exons. Previous studies have shown that whereas KRAS expression is essential for mouse development, the KRAS4A isoform is expendable. Here, we have generated a mouse strain that carries a terminator codon in exon 4B that leads to the expression of an unstable KRAS4B154 truncated polypeptide, hence resulting in a bona fide Kras4B-null allele. In contrast, this terminator codon leaves expression of the KRAS4A isoform unaffected. Mice selectively lacking KRAS4B expression developed to term but died perinatally because of hypertrabeculation of the ventricular wall, a defect reminiscent of that observed in embryos lacking the Kras locus. Mouse embryonic fibroblasts (MEFs) obtained from Kras4B-/- embryos proliferated less than did wild-type MEFs, because of limited expression of KRAS4A, a defect that can be compensated for by ectopic expression of this isoform. Introduction of the same terminator codon into a KrasFSFG12V allele allowed expression of an endogenous KRAS4AG12V oncogenic isoform in the absence of KRAS4B. Exposure of Kras+/FSF4AG12V4B- mice to Adeno-FLPo particles induced lung tumors with complete penetrance, albeit with increased latencies as compared with control Kras+/FSFG12V animals. Moreover, a significant percentage of these mice developed proximal metastasis, a feature seldom observed in mice expressing both mutant isoforms. These results illustrate that expression of the KRAS4AG12V mutant isoform is sufficient to induce lung tumors, thus suggesting that selective targeting of the KRAS4BG12V oncoprotein may not have significant therapeutic consequences.

RASless MEFs as a Tool to Study RAS-Dependent and RAS-Independent Functions.

RAS proteins are key players in multiple cellular processes. To study the role of RAS proteins individually or in combination, we have developed MEFs that can be rendered RASless, i.e., devoid of all endogenous RAS isoforms. These cells have significantly contributed to our understanding of the requirements for RAS functions in cell proliferation as well as their implications in diverse cellular processes. Here, we describe methods using RASless MEFs to study RAS-dependent cellular activities with special emphasis on proliferation. We provide the details to identify inducers of RAS-independent proliferation in colony assays. We recommend following these stringent guidelines to avoid false-positive results. Moreover, this protocol can be adapted to generate RASless MEFs ectopically expressing RAS variants to interrogate their function in the absence of endogenous RAS isoforms or to perform experiments in the absence of RAS. Finally, we also describe protocols to generate and use RASless MEFs for cell cycle analyses using the FUCCI cell cycle indicator.

Desertomycin G, a New Antibiotic with Activity against Mycobacterium tuberculosis and Human Breast Tumor Cell Lines Produced by Streptomyces althioticus MSM3, Isolated from the Cantabrian Sea Intertidal Macroalgae Ulva sp.

The isolation and structural elucidation of a structurally new desertomycin, designated as desertomycin G (1), with strong antibiotic activity against several clinically relevant antibiotic resistant pathogens are described herein. This new natural product was obtained from cultures of the marine actinomycete Streptomyces althioticus MSM3, isolated from samples of the intertidal seaweed Ulva sp. collected in the Cantabrian Sea (Northeast Atlantic Ocean). Particularly interesting is its strong antibiotic activity against Mycobacterium tuberculosis clinical isolates, resistant to antibiotics in clinical use. To the best of our knowledge, this is the first report on a member of the desertomycin family displaying such activity. Additionally, desertomycin G shows strong antibiotic activities against other relevant Gram-positive clinical pathogens such as Corynebacterium urealyticum, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes, Enterococcus faecium, Enterococcus faecalis, and Clostridium perfringens. Desertomycin G also displays moderate antibiotic activity against relevant Gram-negative clinical pathogens such as Bacteroides fragilis, Haemophilus influenzae and Neisseria meningitidis. In addition, the compound affects viability of tumor cell lines, such as human breast adenocarcinoma (MCF-7) and colon carcinoma (DLD-1), but not normal mammary fibroblasts.

Inactivation of Capicua in adult mice causes T-cell lymphoblastic lymphoma.

CIC (also known as Capicua) is a transcriptional repressor negatively regulated by RAS/MAPK signaling. Whereas the functions of Cic have been well characterized in Drosophila, little is known about its role in mammals. CIC is inactivated in a variety of human tumors and has been implicated recently in the promotion of lung metastases. Here, we describe a mouse model in which we inactivated Cic by selectively disabling its DNA-binding activity, a mutation that causes derepression of its target genes. Germline Cic inactivation causes perinatal lethality due to lung differentiation defects. However, its systemic inactivation in adult mice induces T-cell acute lymphoblastic lymphoma (T-ALL), a tumor type known to carry CIC mutations, albeit with low incidence. Cic inactivation in mice induces T-ALL by a mechanism involving derepression of its well-known target, Etv4 Importantly, human T-ALL also relies on ETV4 expression for maintaining its oncogenic phenotype. Moreover, Cic inactivation renders T-ALL insensitive to MEK inhibitors in both mouse and human cell lines. Finally, we show that Ras-induced mouse T-ALL as well as human T-ALL carrying mutations in the RAS/MAPK pathway display a genetic signature indicative of Cic inactivation. These observations illustrate that CIC inactivation plays a key role in this human malignancy.