APOB CRISPR-Cas9 Engineering in Hypobetalipoproteinemia: A Promising Tool for Functional Studies of Novel Variants

Hypobetalipoproteinemia is characterized by LDL-cholesterol and apolipoprotein B (apoB) plasma levels below the fifth percentile for age and sex. Familial hypobetalipoproteinemia (FHBL) is mostly caused by premature termination codons in the APOB gene, a condition associated with fatty liver and steatohepatitis. Nevertheless, many families with a FHBL phenotype carry APOB missense variants of uncertain significance (VUS). We here aimed to develop a proof-of-principle experiment to assess the pathogenicity of VUS using the genome editing of human liver cells. We identified a novel heterozygous APOB-VUS (p.Leu351Arg), in a FHBL family. We generated APOB knock-out (KO) and APOB-p.Leu351Arg knock-in Huh7 cells using CRISPR-Cas9 technology and studied the APOB expression, synthesis and secretion by digital droplet PCR and ELISA quantification. The APOB expression was decreased by 70% in the heterozygous APOB-KO cells and almost abolished in the homozygous-KO cells, with a consistent decrease in apoB production and secretion. The APOB-p.Leu351Arg homozygous cells presented with a 40% decreased APOB expression and undetectable apoB levels in cellular extracts and supernatant. Thus, the p.Leu351Arg affected the apoB secretion, which led us to classify this new variant as likely pathogenic and to set up a hepatic follow-up in this family. Therefore, the functional assessment of APOB-missense variants, using gene-editing technologies, will lead to improvements in the molecular diagnosis of FHBL and the personalized follow-up of these patients.     

Micro-RNA and Proteomic Profiles of Plasma-Derived Exosomes from Irradiated Mice Reveal Molecular Changes Preventing Apoptosis in Neonatal Cerebellum

Cell communication via exosomes is capable of influencing cell fate in stress situations such as exposure to ionizing radiation. In vitro and in vivo studies have shown that exosomes might play a role in out-of-target radiation effects by carrying molecular signaling mediators of radiation damage, as well as opposite protective functions resulting in resistance to radiotherapy. However, a global understanding of exosomes and their radiation-induced regulation, especially within the context of an intact mammalian organism, has been lacking. In this in vivo study, we demonstrate that, compared to sham-irradiated (SI) mice, a distinct pattern of proteins and miRNAs is found packaged into circulating plasma exosomes after whole-body and partial-body irradiation (WBI and PBI) with 2 Gy X-rays. A high number of deregulated proteins (59% of WBI and 67% of PBI) was found in the exosomes of irradiated mice. In total, 57 and 13 miRNAs were deregulated in WBI and PBI groups, respectively, suggesting that the miRNA cargo is influenced by the tissue volume exposed to radiation. In addition, five miRNAs (miR-99b-3p, miR-200a-3p, miR-200a, miR-182-5p, miR-182) were commonly overexpressed in the exosomes from the WBI and PBI groups. In this study, particular emphasis was also given to the determination of the in vivo effect of exosome transfer by intracranial injection in the highly radiosensitive neonatal cerebellum at postnatal day 3. In accordance with a major overall anti-apoptotic function of the commonly deregulated miRNAs, here, we report that exosomes from the plasma of irradiated mice, especially in the case of WBI, prevent radiation-induced apoptosis, thus holding promise for exosome-based future therapeutic applications against radiation injury.     

Preclinical Evaluation of CAR T Cell Function: In Vitro and In Vivo Models

Immunotherapy using chimeric antigen receptor (CAR) T cells is a rapidly emerging modality that engineers T cells to redirect tumor-specific cytotoxicity. CAR T cells have been well characterized for their efficacy against B cell malignancies, and rigorously studied in other types of tumors. Preclinical evaluation of CAR T cell function, including direct tumor killing, cytokine production, and memory responses, is crucial to the development and optimization of CAR T cell therapies. Such comprehensive examinations are usually performed in different types of models. Model establishment should focus on key challenges in the clinical setting and the capability to generate reliable data to indicate CAR T cell therapeutic potency in the clinic. Further, modeling the interaction between CAR T cells and tumor microenvironment provides additional insight for the future endeavors to enhance efficacy, especially against solid tumors. This review will summarize both in vitro and in vivo models for CAR T cell functional evaluation, including how they have evolved with the needs of CAR T cell research, the information they can provide for preclinical assessment of CAR T cell products, and recent technology advances to test CAR T cells in more clinically relevant models.     

Oxadiazole Derivatives as Dual Orexin Receptor Antagonists: Synthesis,Structure–Activity Relationships,and Sleep-Promoting Properties in Rats

The orexin system plays an important role in the regulation of wakefulness. Suvorexant, a dual orexin receptor antagonist (DORA) is approved for the treatment of primary insomnia. Herein, we outline our optimization efforts toward a novel DORA. We started our investigation with rac-[3-(5-chloro-benzooxazol-2-ylamino)piperidin-1-yl]-(5-methyl-2-[1,2,3]triazol-2-ylphenyl)methanone ( 3 ), a structural hybrid of suvorexant and a piperidine-containing DORA. During the optimization, we resolved liabilities such as chemical instability, CYP3A4 inhibition, and low brain penetration potential. Furthermore, structural modification of the piperidine scaffold was essential to improve potency at the orexin 2 receptor. This work led to the identification of (5-methoxy-4-methyl-2-[1,2,3]triazol-2-ylphenyl)-{(S)-2-[5-(2-trifluoromethoxyphenyl)-[1,2,4]oxadiazol-3-yl]pyrrolidin-1-yl}methanone ( 51 ), a potent, brain-penetrating DORA with in vivo efficacy similar to that of suvorexant in rats.     

Influence of Membrane Intrusion on Permeate‐Sided Pressure Drop During High‐Pressure Reverse Osmosis

By replacing thermal concentration processes, high‐pressure reverse osmosis has the potential to contribute to cost and energy savings regarding concepts for industrial water reuse. To provide a better understanding of the spiral‐wound element behavior during high‐pressure operation, this study focusses on the investigation of their performance by scrutinizing the crucial effect of the permeate‐sided pressure drop induced by membrane‐spacer interactions. The experiments show a considerable influence of membrane intrusion on the element performance with a strong dependence on the feed pressure.     

A triple spin-labeling strategy coupled with DEER analysis to detect DNA modifications and enzymatic repair

In a spin: Spin-labeled oligonucleotides produced by click chemistry can be studied by EPR, by using a DEER sequence. This was used to test a complex triple-labeling strategy with damaged DNA. Extensive and accurate analysis of DNA structure and enzymatic repair processes were performed after digestion by EndoIV. Modified DNA structures and DNA-protein interactions can now be readily studied.     

Influence of pigment on biodeterioration of acrylic paint films in Southern Brazil

Biodeterioration of paint films leads to loss of durability and increased repainting costs. The influence of pigments on the biodeterioration of architectural paint films in the city of Florianópolis, Brazil, was evaluated using ten differently colored acrylic paint films exposed to the environment for 34 months. Fouling (biofilm formation) on the surfaces was assessed macroscopically, using British Standard BS 3900/1989 G6, and microscopically. After 20 months, major colonizers were bacteria and fungi, with some cyanobacteria and few algae; north-facing suede and peach and south-facing ice colors showed 100% cover at this time. The least affected color at all times was blue, with a maximum of 30% cover on south-facing panels after 34 months. North-facing panels were generally more fouled than South-facing. Blue, red, and ceramic colors always performed best. Resistance to fouling may have been due to copper in blue and acidity from sulfur oxides in ceramic pigments. Pigments may prolong paint film life and reduce the need for biocides.     

Experimental Validation of a Fundamental Model for PCR Efficiency

Recently a theoretical analysis of PCR efficiency has been published by Booth et al. (2010). The PCR yield is the product of three efficiencies: (i) the annealing efficiency is the fraction of templates that form binary complexes with primers during annealing, (ii) the polymerase binding efficiency is the fraction of binary complexes that bind to polymerase to form ternary complexes and (iii) the elongation efficiency is the fraction of ternary complexes that extend fully. Yield is controlled by the smallest of the three efficiencies and control could shift from one type of efficiency to another over the course of a PCR experiment. Experiments have been designed that are specifically controlled by each one of the efficiencies and the results are consistent with the mathematical model. The experimental data has also been used to quantify six key parameters of the theoretical model. An important application of the fully characterized model is to calculate initial template concentration from real-time PCR data. Given the PCR protocol, the midpoint cycle number (where the template concentration is half that of the final concentration) can be theoretically determined and graphed for a variety of initial DNA concentrations. Real-time results can be used to calculate the midpoint cycle number and consequently the initial DNA concentration, using this graph. The application becomes particularly simple if a conservative PCR protocol is followed where only the annealing efficiency is controlling.     

Cyp1 Inhibition Prevents Doxorubicin-Induced Cardiomyopathy in a Zebrafish Heart-Failure Model

Doxorubicin is a highly effective chemotherapy agent used to treat many common malignancies. However, its use is limited by cardiotoxicity, and cumulative doses exponentially increase the risk of heart failure. To identify novel heart failure treatment targets, a zebrafish model of doxorubicin-induced cardiomyopathy was previously established for small-molecule screening. Using this model, several small molecules that prevent doxorubicin-induced cardiotoxicity both in zebrafish and in mouse models have previously been identified. In this study, exploration of doxorubicin cardiotoxicity is expanded by screening 2271 small molecules from a proprietary, target-annotated tool compound collection. It is found that 120 small molecules can prevent doxorubicin-induced cardiotoxicity, including 7 highly effective compounds. Of these, all seven exhibited inhibitory activity towards cytochrome P450 family 1 (CYP1). These results are consistent with previous findings, in which visnagin, a CYP1 inhibitor, also prevents doxorubicin-induced cardiotoxicity. Importantly, genetic mutation of cyp1a protected zebrafish against doxorubicin-induced cardiotoxicity phenotypes. Together, these results provide strong evidence that CYP1 is an important contributor to doxorubicin-induced cardiotoxicity and highlight the CYP1 pathway as a candidate therapeutic target for clinical cardioprotection.