Dietary Marine Oils Selectively Decrease Obesogenic Diet-Derived Carbonylation in Proteins Involved in ATP Homeostasis and Glutamate Metabolism in the Rat Cerebellum.

The regular intake of diets high in saturated fat and sugars increases oxidative stress and has been linked to cognitive decline and premature brain aging. The cerebellum is highly vulnerable to oxidative stress and thus, obesogenic diets might be particularly detrimental to this tissue. However, the precise molecular mechanisms behind obesity-related brain damage are still not clear. Since protein carbonylation, a biomarker of oxidative stress, influences protein functions and is involved in metabolic control, the current investigation addressed the effect of long-term high-fat and high-sucrose diet intake on the cerebellum of Sprague-Dawley rats by deciphering the changes caused in the carbonylated proteome. The antioxidant effects of fish oil supplementation on cerebellar carbonylated proteins were also investigated. Lipid peroxidation products and carbonylated proteins were identified and quantified using immunoassays and 2D-LC-MS/MS in the cerebellum. After 21 weeks of nutritional intervention, the obesogenic diet selectively increased carbonylation of the proteins that participate in ATP homeostasis and glutamate metabolism in the cerebellum. Moreover, the data demonstrated that fish oil supplementation restrained carbonylation of the main protein targets oxidatively damaged by the obesogenic diet, and additionally protected against carbonylation of several other proteins involved in amino acid biosynthesis and neurotransmission. Therefore, dietary interventions with fish oils could help the cerebellum to be more resilient to oxidative damage. The results could shed some light on the effect of high-fat and high-sucrose diets on redox homeostasis in the cerebellum and boost the development of antioxidant-based nutritional interventions to improve cerebellum health.

Discrepancy in medications reported by elderly patients in the dental office and in their electronic medical records: A pilot study.

AIMS: This study's main objective was to analyze the discrepancy between the dental medication record (DMR) and the physician-prescribed active medications recorded in the medical medication record (MMR). METHODS: The study group consisted of 100 adults who attended the University Dental Clinic (Santiago de Compostela, Spain) requesting dental care. A dental history was created for all participants that included the DMR. The MMR were compiled from their electronic medical records. RESULTS: About 80% of the patients consumed at least one drug (94.2% of those >65 years) and 19% took more than five drugs (26.4% of those > 65 years). In total, 54% of the patients had some discrepancy between the medications recorded in the DMR and those in the MMR (48.4% for those ≤65 years and 64.7% for those >65 years). The rate of participants who omitted some drugs was higher for those >65 years. The drugs most omitted from the DMR were analgesics/opioids, antihypertensives and anxiolytics/hypnotics/sedatives. CONCLUSIONS: It is imperative to access the MMR of patients requesting dental care because a significant number of medications are not reflected in their DMR. These discrepancies may be particularly common and relevant in elderly patients, in whom multimorbidity and polypharmacy are more frequent.

Prph2 knock-in mice recapitulate human central areolar choroidal dystrophy retinal degeneration and exhibit aberrant synaptic remodeling and microglial activation.

Central areolar choroidal dystrophy is an inherited disorder characterized by progressive choriocapillaris atrophy and retinal degeneration and is usually associated with mutations in the PRPH2 gene. We aimed to generate and characterize a mouse model with the p.Arg195Leu mutation previously described in patients. Heterozygous (Prph2WT/KI) and homozygous (Prph2KI/KI) mice were generated using the CRISPR/Cas9 system to introduce the p.Arg195Leu mutation. Retinal function was assessed by electroretinography and optomotor tests at 1, 3, 6, 9, 12, and 20 months of age. The structural integrity of the retinas was evaluated at the same ages using optical coherence tomography. Immunofluorescence and transmission electron microscopy images of the retina were also analyzed. Genetic sequencing confirmed that both Prph2WT/KI and Prph2KI/KI mice presented the p.Arg195Leu mutation. A progressive loss of retinal function was found in both mutant groups, with significantly reduced visual acuity from 3 months of age in Prph2KI/KI mice and from 6 months of age in Prph2WT/KI mice. Decreased amplitudes in the electroretinography responses were observed from 1 month of age in Prph2KI/KI mice and from 6 months of age in Prph2WT/KI mice. Morphological analysis of the retinas correlated with functional findings, showing a progressive decrease in retinal thickness of mutant mice, with earlier and more severe changes in the homozygous mutant mice. We corroborated the alteration of the outer segment structure, and we found changes in the synaptic connectivity in the outer plexiform layer as well as gliosis and signs of microglial activation. The new Prph2WT/KI and Prph2KI/KI murine models show a pattern of retinal degeneration similar to that described in human patients with central areolar choroidal dystrophy and appear to be good models to study the mechanisms involved in the onset and progression of the disease, as well as to test the efficacy of new therapeutic strategies.

Fish oil supplementation counteracts the effect of high-fat and high-sucrose diets on the carbonylated proteome in the rat cerebral cortex.

High daily intake of saturated fats and refined carbohydrates, which often leads to obesity and overweight, has been associated with cognitive impairment, premature brain aging and the aggravation of neurodegenerative diseases. Although the molecular pathology of obesity-related brain damage is not fully understood, the increased levels of oxidative stress induced by the diet seem to be definitively involved. Being protein carbonylation determinant for protein activity and function and a main consequence of oxidative stress, this study aims to investigate the effect of the long-term high-fat and sucrose diet intake on carbonylated proteome of the cerebral cortex of Sprague-Dawley rats. To achieve this goal, the study identified and quantified the carbonylated proteins and lipid peroxidation products in the cortex, and correlated them with biometrical, biochemical and other redox status parameters. Results demonstrated that the obesogenic diet selectively increased oxidative damage of specific proteins that participate in fundamental pathways for brain function, i.e. energy production, glucose metabolism and neurotransmission. This study also evaluated the antioxidant properties of fish oil to counteract diet-induced brain oxidative damage. Fish oil supplementation demonstrated a stronger capacity to modulate carbonylated proteome in the brain cortex. Data indicated that fish oils did not just decrease carbonylation of proteins affected by the obesogenic diet, but also decreased the oxidative damage of other proteins participating in the same metabolic functions, reinforcing the beneficial effect of the supplement on those pathways. The results could help contribute to the development of successful nutritional-based interventions to prevent cognitive decline and promote brain health.

Low-Toxicity Solvents for the Extraction of Valuable Lipid Compounds from Octopus (Octopus vulgaris) Waste.

This study focused on the recovery of valuable lipid compounds from octopus (Octopus vulgaris) by-products. Extraction conditions of total lipids (TLs), phospholipids (PLs), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) were optimized by employing a Simplex-Lattice design; for it, different relative concentrations of three low-toxicity solvents (ethanol, acetone, and ethyl acetate) were considered. The optimization process was also addressed in reference to fatty acid (FA) ratios (total polyunsaturated FAs/total saturated FAs and total ω3 FAs/total ω6 FAs). The variance analysis of multiple regression data demonstrated that the quadratic model was significant (p < 0.05) for TL, PL, and DHA values and the ω3/ω6 ratio. As a result, the following optimized values were obtained: 113.8 g·kg-1 dry by-products (TLs), 217.3 g·kg-1 lipids (PLs), 22.55 g·100 g-1 total FAs (DHA), and 3.70 (ω3/ω6 ratio). According to the model developed, optimized values were shown to correspond to the following relative solvent concentrations (ethanol/acetone/ethyl acetate): 0.46/0.00/0.54, 0.93/0.07/0.00, 0.83/0.17/0.00, and 0.64/0.00/0.36, respectively. Comparison to yields obtained by the conventional chloroform/methanol method was carried out. A novel strategy based on the employment of low-toxicity solvents is proposed for the extraction of valuable lipid constituents from octopus waste. A different solvent ratio would be necessary according to the lipid compound concerned.

Enhancement of Lipid Stability of Frozen Fish by Octopus-Waste Glazing.

The antioxidant properties of the liquor resulting from commercial octopus cooking were analysed for this study. Two different concentrations of octopus-cooking liquor (OCL) were tested as glazing systems during the frozen storage period (-18 °C for up to 6 months) of whole Atlantic horse mackerel (Trachurus trachurus). Compared to water-control glazing samples, an inhibitory effect (p < 0.05) on lipid oxidation development (the formation of thiobarbituric acid reactive substances and fluorescent compounds) was detected in frozen fish treated with the most concentrated OCL-glazing system. Additionally, a preservative effect (p < 0.05) on polyunsaturated fatty acids (measurement of polyene index) was also proved. However, no effect (p > 0.05) on the free fatty acid content and on the ω3/ω6 ratio was detected with the presence of the OCL in the glazing system. An increased lipid quality in frozen horse mackerel was established by including the OCL solution in the glazing system. According to previous research, the observed preserving properties were explained on the basis of the presence of antioxidant compounds in the cooking liquor. A novel and valuable combination of glazing processing and the employment of a marine waste substrate is proposed to enhance the lipid stability of frozen fish.

Integration of Food-based Learning With Science in the Preschool Classroom: Implementation Gaps and Opportunities.

OBJECTIVE: To explore Head Start teachers' use and integration of food-based learning (FBL) with science learning in the Head Start classroom. DESIGN: Phenomenological approach using in-depth semistructured telephone interviews. SETTING: North Carolina Head Start preschools. PARTICIPANTS: Thirty-five Head Start lead and assistant teachers. PHENOMENON OF INTEREST: All interviews were transcribed verbatim. Authors coded interview data for emergent themes. ANALYSIS: Eleven primary themes were identified during analysis and inductively organized using the Systems Thinking Iceberg Model. RESULTS: Teachers described most frequently using FBL during mealtimes. Teachers stated they felt successful when children were engaged and willing to try a new food. However, they struggled to connect food to science concepts. Teachers reported several motivators (eg, improving health) and barriers (eg, food waste) to integrating FBL. Teachers prioritized preparing children for kindergarten, but most teachers did not see how FBL could help them achieve this goal. CONCLUSIONS AND IMPLICATIONS: Head Start teacher professional development programs could impact all 4 levels of the Systems Thinking Model to improve teachers' perceptions, underlying structures, and mental models regarding integrative FBL. Additional research is warranted to investigate the adoption, implementation, and potential impact of FBL on academic outcomes.

Combined Intake of Fish Oil and D-Fagomine Prevents High-Fat High-Sucrose Diet-Induced Prediabetes by Modulating Lipotoxicity and Protein Carbonylation in the Kidney.

Obesity has been recognized as a major risk factor for chronic kidney disease, insulin resistance being an early common metabolic feature in patients suffering from this syndrome. This study aims to investigate the mechanism underlying the induction of kidney dysfunction and the concomitant onset of insulin resistance by long-term high-fat and sucrose diet feeding in Sprague Dawley rats. To achieve this goal, our study analyzed renal carbonylated protein patterns, ectopic lipid accumulation and fatty acid profiles and correlated them with biometrical and biochemical measurements and other body redox status parameters. Rats fed the obesogenic diet developed a prediabetic state and incipient kidney dysfunction manifested in increased plasma urea concentration and superior levels of renal fat deposition and protein carbonylation. An obesogenic diet increased renal fat by preferentially promoting the accumulation of saturated fat, arachidonic, and docosahexaenoic fatty acids while decreasing oleic acid. Renal lipotoxicity was accompanied by selectively higher carbonylation of proteins involved in the blood pH regulation, i.e., bicarbonate reclamation and synthesis, amino acid, and glucose metabolisms, directly related to the onset of insulin resistance. This study also tested the combination of antioxidant properties of fish oil with the anti-diabetic properties of buckwheat D-Fagomine to counteract diet-induced renal alterations. Results demonstrated that bioactive compounds combined attenuated lipotoxicity, induced more favorable lipid profiles and counteracted the excessive carbonylation of proteins associated with pH regulation in the kidneys, resulting in an inhibition of the progression of the prediabetes state and kidney disease.

Induction of cytotoxic effects and changes in DNA methylation-related gene expression in a human fibroblast cell line by the metal-organic framework [H2NMe2]3 [Tb(III)(2,6 pyridinedicarboxylate)3] (Tb-MOF).

Lanthanide metal-organic frameworks (lanthanide MOFs) may be utilized for a variety of environmental and human health applications due to their luminescent properties and high thermal and water stability. However, the cytotoxic and epigenetic effects produced in human cells are not known. Therefore, we evaluated the cytotoxic effects, internalization, and changes in the mRNA abundance of DNA methylation and demethylation enzymes by exposing human fibroblast cells to a metal-organic framework [H2NMe2]3 [Tb(III)(2,6 pyridinedicarboxylate)3] (Tb-MOF). For this purpose, the cells were exposed to six concentrations (0.05 to 1.6 mg/mL) of Tb-MOF for 48 h. Field emission electron microscopy coupled to linear energy dispersive spectroscopy (FESEM‒EDS) and confocal microscopy analysis were performed. The cytotoxicity was determined with crystal violet and MTT assays. The results demonstrated the internalization of Tb-MOF at concentrations as low as 0.05 mg/mL, as well as concentration-dependent toxicity. Additionally, we detected significant changes in the gene expression levels of DNA methyltransferases and demethylases due to the presence of Tb-MOF, suggesting that Tb-MOF could generate epigenetic changes even at low concentrations. The results of our study may establish a foundation for future research attempting to develop and apply secure nanomaterials (e.g., MOFs) to minimize damage to the environment and human health.

Fiber-like Action of d-Fagomine on the Gut Microbiota and Body Weight of Healthy Rats.

The goal of this work is to explore if the changes induced by d-fagomine in the gut microbiota are compatible with its effect on body weight and inflammation markers in rats. Methods: Sprague Dawley rats were fed a standard diet supplemented with d-fagomine (or not, for comparison) for 6 months. The variables measured were body weight, plasma mediators of inflammation (hydroxyeicosatetraenoic acids, leukotriene B4, and IL-6), and the concentration of acetic acid in feces and plasma. The composition and diversities of microbiota in cecal content and feces were estimated using 16S rRNA metabarcoding and high-throughput sequencing. We found that after just 6 weeks of intake d-fagomine significantly reduced body weight gain, increased the plasma acetate concentration, and reduced the plasma concentration of the pro-inflammatory biomarkers' leukotriene B4, interleukin 6 and 12 hydroxyeicosatetraenoic acids. These changes were associated with a significantly increased prevalence of Bacteroides and Prevotella feces and increased Bacteroides, Prevotella, Clostridium, and Dysgonomonas while reducing Anaerofilum, Blautia, and Oribacterium in cecal content. In conclusion, d-fagomine induced changes in the composition and diversity of gut microbiota similar to those elicited by dietary fiber and compatible with its anti-inflammatory and body-weight-reducing effects.

Antioxidant Effect of Octopus Byproducts in Canned Horse Mackerel (Trachurus trachurus) Previously Subjected to Different Frozen Storage Times.

The effects on lipid damage in canned horse mackerel (Trachurus trachurus) of a prior frozen storage (−18 °C) period and the presence of an octopus (Octopus vulgaris) cooking juice (OCJ) in the packing medium were investigated. An increase of the frozen storage time favoured an increase (p < 0.05) of free fatty acid (FFA), peroxide, and thiobarbituric acid reactive substance contents and a decrease (p < 0.05) of the phospholipid (PL) value and polyene index. Furthermore, an increased presence of OCJ in the packing medium led to an inhibitory effect (p < 0.05) on fluorescent compound formation as well as to a retention (p < 0.05) of the PL and FFA compounds. Colour determination showed a substantial increase (p < 0.05) of L* and b* values in canned fish with previous frozen storage time. Nevertheless, this increase was partly reduced (p < 0.05) by the OCJ presence in the packing medium. It is concluded that previous holding time has led to an increased lipid oxidation development and loss of beneficial lipid constituents (i.e., PLs and polyunsaturated fatty acids). Remarkably, the presence in the packing medium of preservative compounds (i.e., antioxidants) included in waste juice obtained from octopus processing provided an effective tool for lipid preservation and quality enhancement in canned fish.

Recurrent NOMO1 Gene Deletion Is a Potential Clinical Marker in Early-Onset Colorectal Cancer and Is Involved in the Regulation of Cell Migration.

The incidence of early-onset colorectal cancer (EOCRC; age younger than 50 years) has been progressively increasing over the last decades globally, with causes unexplained. A distinct molecular feature of EOCRC is that compared with cases of late-onset colorectal cancer, in EOCRC cases, there is a higher incidence of Nodal Modulator 1 (NOMO1) somatic deletions. However, the mechanisms of NOMO1 in early-onset colorectal carcinogenesis are currently unknown. In this study, we show that in 30% of EOCRCs with heterozygous deletion of NOMO1, there were pathogenic mutations in this gene, suggesting that NOMO1 can be inactivated by deletion or mutation in EOCRC. To study the role of NOMO1 in EOCRC, CRISPR/cas9 technology was employed to generate NOMO1 knockout HCT-116 (EOCRC) and HS-5 (bone marrow) cell lines. NOMO1 loss in these cell lines did not perturb Nodal pathway signaling nor cell proliferation. Expression microarrays, RNA sequencing, and protein expression analysis by LC-IMS/MS showed that NOMO1 inactivation deregulates other signaling pathways independent of the Nodal pathway, such as epithelial-mesenchymal transition and cell migration. Significantly, NOMO1 loss increased the migration capacity of CRC cells. Additionally, a gut-specific conditional NOMO1 KO mouse model revealed no subsequent tumor development in mice. Overall, these findings suggest that NOMO1 could play a secondary role in early-onset colorectal carcinogenesis because its loss increases the migration capacity of CRC cells. Therefore, further study is warranted to explore other signalling pathways deregulated by NOMO1 loss that may play a significant role in the pathogenesis of the disease.

First Outbreak of NDM-1-Producing Klebsiella pneumoniae ST11 in a Portuguese Hospital Centre during the COVID-19 Pandemic.

New Delhi metallo-ß-lactamase (NDM) carbapenemase has been considered a global threat due to its worldwide widespread in recent years. In Portugal, a very low number of infections with NDM-producing Enterobacterales has been reported. A total of 52 strains from 40 patients and 1 environmental sample isolated during COVID-19 pandemic were included in this study. Wholegenome sequencing (WGS) was performed on 20 carbapenemase-producing strains, including 17 NDM-1-producing Klebsiella pneumoniae ST11-KL105 lineage strains, one NDM-1-producing Escherichia coli ST58 strain and one KPC-3-producing K. pneumoniae ST147 strain, recovered from a total of 19 patients. Of interest, also one NDM-1-producing K. pneumoniae ST11-KL105 was collected from the hospital environment. Genome-wide phylogenetic analysis revealed an ongoing dissemination of NDM-1-producing K. pneumoniae ST11 strains (n = 18) with the same genetic features seen across multiple wards. Furthermore, the ST58 E. coli strain, collected from a patient rectal swab that was also colonised with a K. pneumoniae strain, also showed the IncFIA plasmid replicon and the blaNDM-1 gene (preceded by IS30 and followed by genes bleMBL, trpF, dsbC, cutA, groES and groEL). The blaNDM-1 is part of Tn125-like identical to those reported in Poland, Italy and India. The blaKPC-3 K. pneumoniae ST147-KL64 strain has the genetic environment Tn4401d isoform. In conclusion, herein we report the molecular epidemiology, resistome, virulome and mobilome of the first NDM-1 carbapenemase outbreak caused by K. pneumoniae ST11-KL105 lineage during the COVID-19 pandemic in Portugal. Moreover, the outbreak strains characterised included seventeen different patients (infected and colonised) and one environmental sample which also emphasises the role of commensal and hospital environment strains in the dissemination of the outbreak.

Epidemiological Characterization and the Impact of Healthcare-Associated Pneumonia in Patients Admitted in a Northern Portuguese Hospital.

Pneumonia is one of the main causes of hospitalization and mortality. It's the fourth leading cause of death worldwide. Healthcare-associated infections are the most frequent complication of healthcare and affect hundreds of millions of patients around the world, although the actual number of patients affected is unknown due to the difficulty of reliable data. The main goal of this manuscript is to describe the epidemiological characteristics of patients admitted with pneumonia and the impact of healthcare-associated pneumonia (HCAP) in those patients. It is a quantitative descriptive study with retrospective analysis of the clinical processes of 2436 individuals for 1 year (2018) with the diagnosis of pneumonia. The individuals with ≤5 years old represented 10.4% (n = 253) and ≥65 were 72.6% (n = 1769). 369 cases resulted in death, which gives a sample lethality rate of 15.2%. The severity and mortality index were not sensitive to the death event. We found 30.2% (n = 735) individuals with HCAP and 0.41% (n = 59) with ventilator-associated pneumonia (VAP). In only 59 individuals (2.4%) the agent causing pneumonia was isolated. The high fatality rate obtained shows that pneumonia is a major cause of death in vulnerable populations. Moreover, HCAP is one of the main causes of hospital admissions from pneumonia and death and the most pneumonias are treated empirically. Knowledge of the epidemiology characterization of pneumonia, especially associated with healthcare, is essential to increase the skills of health professionals for the prevention and efficient treatment of pneumonia.

Psychometric Comparison of the English and Spanish Western-Hemisphere Versions of the Modified Checklist for Autism in Toddlers-Revised.

OBJECTIVES: Parent-report screening tools for autism spectrum disorder (ASD) are widely used to promote early identification of children with or at risk for ASD. Most screening tools have been developed in English in the United States or United Kingdom; thus, translated versions are needed for use with culturally and linguistically diverse populations. Traditional translation methods include a forward translation, back translation, and review. However, when used in new cultural and linguistic contexts, this "forward-back" approach may have limitations, including differing psychometric properties compared with original instruments. This study presents a psychometric analysis of the forward-back translation methodology of an ASD screening tool. METHODS: A retrospective chart review design was used to examine Modified Checklist for Autism in Toddlers-Revised (M-CHAT-R; Robins et al.) records from 2974 toddlers. Data were compared between caregivers who completed the original English M-CHAT-R and caregivers who completed its forward-back "Spanish-Western Hemisphere" translation to compare select psychometric properties of the 2 instruments. RESULTS: Significant differences were observed between the 2 versions, including a higher overall risk score, higher initial screen-positive rate, and increased likelihood of leaving items blank among Spanish-speaking respondents. CONCLUSION: Traditional translation methods seemed to affect select psychometric properties between translations of the M-CHAT-R. A more rigorous cultural adaptation approach may be necessary to maintain equivalence with the original instrument. Until new rigorous translations are available, it is recommended that language-specific screening tools continue to be used, along with recommended follow-up interviews, to avoid exacerbating existing health disparities.

Hafnia alvei Pneumonia: A Rare Cause of Infection in a Patient with COVID-19.

Herein, we describe a case report of a critically ill patient, a 48-year-old man without comorbidities admitted to the hospital with a serious type 1 (hypoxemic) respiratory insufficiency and confirmed diagnosis of COVID-19. After 5 days with invasive mechanical ventilation, the patient developed a bacterial co-infection, namely a pneumonia by Hafnia alvei, requiring the last line of respiratory support: extracorporeal membrane oxygenation (ECMO). Subsequently, his clinical situation gradually stabilized, until he was discharged from the hospital on day 61, being accompanied in ambulatory consultation by the physical medicine and pulmonology department during the post-COVID-19 recovery. H. alvei is a Gram-negative bacterium that is rarely isolated from human specimens and is rarely considered to be pathogenic. However, COVID-19 disease can cause substantial organ dysfunction and can be associated with bacterial secondary infections which can favor the emergence of rare infectious diseases by uncommon microorganisms.

Effects of a Fish Oil Rich in Docosahexaenoic Acid on Cardiometabolic Risk Factors and Oxidative Stress in Healthy Rats.

Omega-3 polyunsaturated fatty acids are associated with a lower risk of cardiometabolic diseases. However, docosahexaenoic acid (DHA) is easily oxidized, leading to cellular damage. The present study examined the effects of an increased concentration of DHA in fish oil (80% of total fatty acids) on cardiometabolic risk factors and oxidative stress compared to coconut oil, soybean oil, and fish oil containing eicosapentaenoic acid (EPA) and DHA in a balanced ratio. Forty healthy male Sprague-Dawley rats were supplemented with corresponding oil for 10 weeks. Supplementation with the fish oil containing 80% DHA decreased plasma fat, plasma total cholesterol and muscle fat compared to the coconut oil and the soybean oil. Increasing concentrations of DHA induced incorporation of DHA and EPA in cell membranes and tissues along with a decrease in ω-6 arachidonic acid. The increase in DHA promoted lipid peroxidation, protein carbonylation and antioxidant response. Taken together, the increased concentration of DHA in fish oil reduced fat accumulation compared to the coconut oil and the soybean oil. This benefit was accompanied by high lipid peroxidation and subsequent protein carbonylation in plasma and in liver. In our healthy framework, the slightly higher carbonylation found after receiving fish oil containing 80% DHA might be a protecting mechanism, which fit with the general improvement of antioxidant defense observed in those rats.

CRISPR-Cas9 Technology as a Tool to Target Gene Drivers in Cancer: Proof of Concept and New Opportunities to Treat Chronic Myeloid Leukemia.

Chronic myeloid leukemia (CML) is a hematopoietic malignancy produced by a unique oncogenic event involving the constitutively active tyrosine-kinase (TK) BCR/ABL1. TK inhibitors (TKI) changed its prognosis and natural history. Unfortunately, ABL1 remains unaffected by TKIs. Leukemic stem cells (LSCs) remain, and resistant mutations arise during treatment. To address this problem, we have designed a therapeutic CRISPR-Cas9 deletion system targeting BCR/ABL1. The system was efficiently electroporated to cell lines, LSCs from a CML murine model, and LSCs from CML patients at diagnosis, generating a specific ABL1 null mutation at high efficiency and allowing the edited leukemic cells to be detected and tracked. The CRISPR-Cas9 deletion system triggered cell proliferation arrest and apoptosis in murine and human CML cell lines. Patient and murine-derived xenografts with CRISPR-edited LSCs in NOD SCID gamma niches revealed that normal multipotency and repopulation ability of CRISPR edited LSCs were fully restored. Normal hematopoiesis was restored, avoiding myeloid bias. To the best of our knowledge, we show for the first time how a CRISPR-Cas9 deletion system efficiently interrupts BCR/ABL1 oncogene in primary LSCs to bestow a therapeutic benefit. This study is a proof of concept for genome editing in all those diseases, like CML, sustained by a single oncogenic event, opening up new therapeutic opportunities.