Generation of two patient specific GABRD variants and their isogenic controls for modeling epilepsy.

Developmental and epileptic encephalopathies (DEEs) are early-onset conditions that cause intractable seizures and developmental delays. Missense variants in Gamma-aminobutyric acid type A receptor (GABAAR) subunits commonly cause DEEs. Ahring et al. (2022) showed a variant in the gene that encodes the delta subunit (GABRD) is strongly associated with the gain-of-function of extrasynaptic GABAAR. Here, we report the generation of two patient-specific human induced pluripotent stem cells (hiPSC) lines with (i) a de novo variant and (ii) a maternal variant, both for the pathogenic GABRD c.872 C>T, (p.T291I). The variants in the generated cell line were corrected using the CRISPR-Cas9 gene editing technique (respective isogenic control lines).

Hsp90 inhibition sensitizes DLBCL cells to cisplatin.

PURPOSE: Platinum-containing therapy is standard treatment for relapsed Diffuse Large B-Cell Lymphoma (DLBCL). However, the efficacy of treatment is limited by drug resistance leading to relapse. Cisplatin resistance has been linked to impairments of the DNA damage response, and several DNA repair proteins have been identified as clients of the molecular chaperone Hsp90. Here, we investigated the combinatory treatment of cisplatin and the Hsp90 inhibitor, 17AAG, in DLBCL cells to evaluate if inhibition of Hsp90 could sensitize DLBCL cells to cisplatin treatment. METHODS: Cell viability was assessed for cisplatin and 17AAG as monotherapies and for 25 different combinations in 7 DLBCL cell lines, where the Bliss Independence Model and the Combination Index were applied to assess their interaction. Induction of apoptosis and DNA damage response were evaluated by measuring Annexin V and γH2AX levels after 48 h of exposure. RESULTS: 17AAG synergized with cisplatin in DLBCL cells as detected in both interaction assessment models, resulting in a lower viability after 48 h for the combination-treated cells compared to both vehicle and single drug-treated cells. The combination also induced a stronger apoptotic response and an increase in DNA damage in 17AAG, cisplatin- and combination-treated cells compared to vehicle-treated cells, with the effect of the combination generally being higher than compared to both single drugs. CONCLUSION: This study demonstrates that 17AAG sensitizes DLBCL cells to cisplatin treatment. This effect is correlated with increased apoptotic and DNA damage response, potentially mediated by downregulation of Hsp90 clients in DNA repair pathways. Thus, cisplatin resistance could plausibly be overcome by combining the treatment with an Hsp90 inhibiting drug.

Downregulating carnitine palmitoyl transferase 1 affects disease progression in the SOD1 G93A mouse model of ALS.

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease characterized by death of motor neurons. The etiology and pathogenesis remains elusive despite decades of intensive research. Herein, we report that dysregulated metabolism plays a central role in the SOD1 G93A mouse model mimicking ALS. Specifically, we report that the activity of carnitine palmitoyl transferase 1 (CPT1) lipid metabolism is associated with disease progression. Downregulation of CPT1 activity by pharmacological and genetic methods results in amelioration of disease symptoms, inflammation, oxidative stress and mitochondrial function, whereas upregulation by high-fat diet or corticosterone results in a more aggressive disease progression. Finally, we show that downregulating CPT1 shifts the gut microbiota communities towards a protective phenotype in SOD1 G93A mice. These findings reveal that metabolism, and specifically CPT1 lipid metabolism plays a central role in the SOD1 G93A mouse model and shows that CPT1 might be a therapeutic target in ALS.

Opportunistic screening for atrial fibrillation in a real-life setting in general practice in Denmark-The Atrial Fibrillation Found On Routine Detection (AFFORD) non-interventional study.

Atrial fibrillation (AF) is a chronic disease with an incidence increasing steeply by age and affecting more than 11 million patients in Europe and the United States. Diagnosing AF is essential for the prevention of stroke by oral anticoagulation. Opportunistic screening for AF in patients ≥65 years of age is recommended by the European and Danish Societies of Cardiology. The study aim was to examine the detection rate of AF in consecutively screened patients in the primary care setting in Denmark. In an open, non-interventional, cluster, multicenter, cross-sectional, observational study patients ≥65 years of age entering consecutively into general practice clinics were invited to nurse-assisted opportunistic screening for AF. The General Practice (GP) clinics participating were randomized to patient inclusion in three age groups: 65-74, 75-84, and ≥85 years respectively. All patients underwent pulse palpation followed by 12-led electrocardiogram in case of irregular pulse. Two cardiologists validated all electrocardiogram examinations. Forty-nine general practice clinics recruited in total 970 patients split into three age groups; 480 patients (65-74 years), 372 (75-84 years), and 118 patients ≥85 years of age. Co-morbidities increased by age with hypertension being most frequent. Eighty-seven patients (9%) were detected with an irregular pulse, representing 4.4%, 10.5% and 22.9%, respectively in the three age groups. Assessment of electrocardiograms by the GP showed suspicion of AF in 13 patients with final verification of electrocardiograms by cardiologists revealing 10 AF-patients. The highest detection rate of AF was found in the ≥85 age group (3.39%) followed by the 65-74 age group (0.83%) and the 75-84 age group (0.54%). Opportunistic screening of AF in primary care is feasible and do result in the detection of new AF-patients. Close collaboration with cardiologists is advisable to avoid false positive screening results.

Evaluation of objective measures of smoking status--a prospective clinical study in a group of head and neck cancer patients treated with radiotherapy.

The present study was undertaken to evaluate objective measures of the smoking status of head and neck cancer patients during the course of radiotherapy. This was done by conducting a weekly structured interview, and measurement of carbon monoxide in expired air and of serum concentration of cotinine, the major metabolite of nicotine. These methods were tested prospectively in a series of 20 patients with head and neck cancer treated with radiotherapy. The results showed significant differences in the levels of end-expired carbon monoxide as well as serum cotinine among the different self-reported smoking groups. Combining the two objective measures and the interview data, the study revealed that up to 50% of self-reported non-smokers were in fact smoking actively. Measurement of end-expired carbon monoxide levels was found to be a precise indicator of smoking in the hours preceding measurement. Serum cotinine was a valuable measure of true smoking status. Assuming that this assay reflects the true smoking status, sensitivity, specificity and positive predictive value of self-reporting in this patient population was 79%, 80%, and 92%, respectively. In research aiming to investigate possible relations between smoking and radiotherapy, it is recommended that patients' smoking status be evaluated objectively as a supplement to self-reporting, at least in the head and neck cancer patients.