Generation of human induced pluripotent stem cell lines (LUMCi051-A,B and LUMCi052-A,B,C) of two patients with Spinocerebellar ataxia type 7.

Spinocerebellar Ataxia Type 7 (SCA7) is an autosomal dominantly inherited disorder, primarily characterized by cerebellar ataxia and visual loss. SCA7 is caused by a CAG repeat expansion in exon 3 of the ATXN7 gene. We generated human induced pluripotent stem cells (hiPSCs) from peripheral blood-derived erythroblasts from two SCA7 patients (LUMCi051-A,B and LUMCi052-A,B,C) using integration-free episomal vectors. All hiPSC clones express pluripotency factors, show a normal karyotype, and can differentiate into the three germ layers. These lines can be used for in vitro disease modeling and therapy testing.

Methods behind oncolytic virus-based DC vaccines in cancer: Toward a multiphase combined treatment strategy for Glioblastoma (GBM) patients.

Glioblastoma (GBM) remains an orphan cancer disease with poor outcome. Novel treatment strategies are needed. Immunotherapy has several modes of action. The addition of active specific immunotherapy with dendritic cell vaccines resulted in improved overall survival of patients. Integration of DC vaccination within the first-line combined treatment became a challenge, and immunogenic cell death immunotherapy during chemotherapy was introduced. We used a retrospective analysis using real world data to evaluate the complex combined treatment, which included individualized multimodal immunotherapy during and after standard of care, and which required adaptations during treatment, and found a further improvement of overall survival. We also discuss the use of real world data as evidence. Novel strategies to move the field of individualized multimodal immunotherapy forward for GBM patients are reviewed.

Representation of Patients With Chronic Kidney Disease in Clinical Trials of Cardiovascular Disease Medications: A Systematic Review.

Importance: Patients with chronic kidney disease (CKD) are at high risk for cardiovascular disease, but their systematic underrepresentation in cardiovascular randomized clinical trials (RCTs) limits the generation of appropriate evidence to guide cardiovascular risk management (CVRM). Objective: To evaluate the underrepresentation of patients with CKD in cardiovascular RCTs, and to highlight evidence gaps in CVRM medications in this population. Evidence Review: A systematic search was conducted in ClinicalTrials.gov from February 2000 through October 2021 for RCTs with full-text publications. If no full-text publications were found in ClinicalTrials.gov, MEDLINE, Embase, and Google Scholar were also searched. Eligible RCTs were those evaluating the effectiveness of antiplatelets, anticoagulants, blood pressure-lowering drugs, glucose-lowering drugs, or cholesterol-lowering drugs in adults with cardiovascular disease or cardiovascular risk factors. Trials with a sample size of fewer than 100 patients were excluded. Findings: In total, 1194 RCTs involving 2 207 677 participants (mean [SD] age, 63 [6] years; 1 343 970 males [64%]) were included. Since 2000, the percentage of cardiovascular RCTs excluding patients with CKD has increased from 66% to 79% (74% overall [884 RCTs]). In 864 RCTs (72%), more patients were excluded than anticipated on safety grounds (63% [306] of trials required no dose adjustment, and 79% [561] required dose adjustment). In total, 158 RCTs (13%) reported results for patients with CKD separately (eg, in subgroup analyses). Significant evidence gaps exist in most CVRM interventions for patients with CKD, particularly for those with CKD stages 4 to 5. Twenty-three RCTs (2%) reported results for patients with an estimated glomerular filtration rate less than 30 mL/min/1.73 m2, 15 RCTs (1%) reported for patients receiving dialysis, and 1 RCT (0.1%) reported for recipients of kidney transplant. Conclusions and Relevance: Results of this systematic review suggest that representation of patients with CKD in cardiovascular RCTs has not improved in the past 2 decades and that these RCTs excluded more patients with CKD than expected on safety grounds. Lack of reporting or underreporting of results for this patient population is associated with evidence gaps in the effectiveness of most CVRM medications in patients with all stages of CKD, particularly CKD stages 4 to 5.

Both clinical trial register and electronic bibliographic database searches were needed to identify randomized clinical trials for systematic reviews: an evaluation study.

OBJECTIVES: To determine whether clinical trial register (CTR) searches can accurately identify a greater number of completed randomized clinical trials (RCTs) than electronic bibliographic database (EBD) searches for systematic reviews of interventions, and to quantify the number of eligible ongoing trials. STUDY DESIGN AND SETTING: We performed an evaluation study and based our search for RCTs on the eligibility criteria of a systematic review that focused on the underrepresentation of people with chronic kidney disease in cardiovascular RCTs. We conducted a combined search of ClinicalTrials.gov and the WHO International Clinical Trials Registry Platform through the Cochrane Central Register of Controlled Trials to identify eligible RCTs registered up to June 1, 2023. We searched Cochrane Central Register of Controlled Trials, EMBASE, and MEDLINE for publications of eligible RCTs published up to June 5, 2023. Finally, we compared the search results to determine the extent to which the two sources identified the same RCTs. RESULTS: We included 92 completed RCTs. Of these, 81 had results available. Sixty-six completed RCTs with available results were identified by both sources (81% agreement [95% CI: 71-88]). We identified seven completed RCTs with results exclusively by CTR search (9% [95% CI: 4-17]) and eight exclusively by EBD search (10% [95% CI: 5-18]). Eleven RCTs were completed but lacked results (four identified by both sources (36% [95% CI: 15-65]), one exclusively by EBD search (9% [95% CI: 1-38]), and six exclusively by CTR search (55% [95% CI: 28-79])). Also, we identified 42 eligible ongoing RCTs: 16 by both sources (38% [95% CI: 25-53]) and 26 exclusively by CTR search (62% [95% CI: 47-75]). Lastly, we identified four RCTs of unknown status by both sources. CONCLUSION: CTR searches identify a greater number of completed RCTs than EBD searches. Both searches missed some included RCTs. Based on our case study, researchers (eg, information specialists, systematic reviewers) aiming to identify all available RCTs should continue to search both sources. Once the barriers to performing CTR searches alone are targeted, CTR searches may be a suitable alternative.

SMCHD1 has separable roles in chromatin architecture and gene silencing that could be targeted in disease.

The interplay between 3D chromatin architecture and gene silencing is incompletely understood. Here, we report a novel point mutation in the non-canonical SMC protein SMCHD1 that enhances its silencing capacity at endogenous developmental targets. Moreover, it also results in enhanced silencing at the facioscapulohumeral muscular dystrophy associated macrosatellite-array, D4Z4, resulting in enhanced repression of DUX4 encoded by this repeat. Heightened SMCHD1 silencing perturbs developmental Hox gene activation, causing a homeotic transformation in mice. Paradoxically, the mutant SMCHD1 appears to enhance insulation against other epigenetic regulators, including PRC2 and CTCF, while depleting long range chromatin interactions akin to what is observed in the absence of SMCHD1. These data suggest that SMCHD1's role in long range chromatin interactions is not directly linked to gene silencing or insulating the chromatin, refining the model for how the different levels of SMCHD1-mediated chromatin regulation interact to bring about gene silencing in normal development and disease.

The Application of Evidence-Based Medicine in Individualized Medicine.

The fundamental aim of healthcare is to improve overall health of the population by providing state-of-the-art healthcare for individuals at an affordable cost. The foundation for this system is largely referred to as "evidence-based medicine". Too often, evidence-based medicine is based solely on so-called "best research evidence", collected through randomized controlled trials while disregarding clinical expertise and patient expectations. As healthcare gravitates towards personalized and individualized medicine, such external clinical (research) evidence can inform, but never replace, individual clinical expertise. This applies in particular to orphan diseases, for which clinical trials are methodologically particularly problematic, and evidence derived from them is often questionable. Evidence-based medicine constitutes a complex process to allow doctors and patients to select the best possible solutions for each individual based on rapidly developing new therapeutic directions. This requires a revisit of the foundations of evidence-based medicine. A proposition as to how to manage evidence-based data in individualized immune-oncology is presented here.

Exploring the Impact of Tensions in Stakeholder Norms on Designing for Value Change: The Case of Biosafety in Industrial Biotechnology.

Synthetic biologists design and engineer organisms for a better and more sustainable future. While the manifold prospects are encouraging, concerns about the uncertain risks of genome editing affect public opinion as well as local regulations. As a consequence, biosafety and associated concepts, such as the Safe-by-design framework and genetic safeguard technologies, have gained notoriety and occupy a central position in the conversation about genetically modified organisms. Yet, as regulatory interest and academic research in genetic safeguard technologies advance, the implementation in industrial biotechnology, a sector that is already employing engineered microorganisms, lags behind. The main goal of this work is to explore the utilization of genetic safeguard technologies for designing biosafety in industrial biotechnology. Based on our results, we posit that biosafety is a case of a changing value, by means of further specification of how to realize biosafety. Our investigation is inspired by the Value Sensitive Design framework, to investigate scientific and technological choices in their appropriate social context. Our findings discuss stakeholder norms for biosafety, reasonings about genetic safeguards, and how these impact the practice of designing for biosafety. We show that tensions between stakeholders occur at the level of norms, and that prior stakeholder alignment is crucial for value specification to happen in practice. Finally, we elaborate in different reasonings about genetic safeguards for biosafety and conclude that, in absence of a common multi-stakeholder effort, the differences in informal biosafety norms and the disparity in biosafety thinking could end up leading to design requirements for compliance instead of for safety.

Systemic delivery of a DUX4-targeting antisense oligonucleotide to treat facioscapulohumeral muscular dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is one of the most prevalent skeletal muscle dystrophies. Skeletal muscle pathology in individuals with FSHD is caused by inappropriate expression of the transcription factor DUX4, which activates different myotoxic pathways. At the moment there is no molecular therapy that can delay or prevent skeletal muscle wasting in FSHD. In this study, a systemically delivered antisense oligonucleotide (ASO) targeting the DUX4 transcript was tested in vivo in ACTA1-MCM;FLExDUX4 mice that express DUX4 in skeletal muscles. We show that the DUX4 ASO was well tolerated and repressed the DUX4 transcript, DUX4 protein, and mouse DUX4 target gene expression in skeletal muscles. In addition, the DUX4 ASO alleviated the severity of skeletal muscle pathology and partially prevented the dysregulation of inflammatory and extracellular matrix genes. DUX4 ASO-treated ACTA1-MCM;FLExDUX4 mice performed better on a treadmill; however, the hanging grid and four-limb grip strength tests were not improved compared to control ASO-treated ACTA1-MCM;FLExDUX4 mice. This study shows that systemic delivery of ASOs targeting DUX4 is a promising therapeutic strategy for FSHD and strategies that further improve the ASO efficacy in skeletal muscle are warranted.

From Innovation to Application: Bridging the Valley of Death in Industrial Biotechnology.

Few biotechnology innovations make it through the Valley of Death to markets. Based on our experience with academia, technology transfer offices, and industry, we provide insights into differences in operating levels, how to best traverse the Valley of Death, and ways to foster more innovation towards market implementation.

A metabolic and physiological design study of Pseudomonas putida KT2440 capable of anaerobic respiration.

BACKGROUND: Pseudomonas putida KT2440 is a metabolically versatile, HV1-certified, genetically accessible, and thus interesting microbial chassis for biotechnological applications. However, its obligate aerobic nature hampers production of oxygen sensitive products and drives up costs in large scale fermentation. The inability to perform anaerobic fermentation has been attributed to insufficient ATP production and an inability to produce pyrimidines under these conditions. Addressing these bottlenecks enabled growth under micro-oxic conditions but does not lead to growth or survival under anoxic conditions. RESULTS: Here, a data-driven approach was used to develop a rational design for a P. putida KT2440 derivative strain capable of anaerobic respiration. To come to the design, data derived from a genome comparison of 1628 Pseudomonas strains was combined with genome-scale metabolic modelling simulations and a transcriptome dataset of 47 samples representing 14 environmental conditions from the facultative anaerobe Pseudomonas aeruginosa. CONCLUSIONS: The results indicate that the implementation of anaerobic respiration in P. putida KT2440 would require at least 49 additional genes of known function, at least 8 genes encoding proteins of unknown function, and 3 externally added vitamins.

Dnmt3b regulates DUX4 expression in a tissue-dependent manner in transgenic D4Z4 mice.

BACKGROUND: Facioscapulohumeral muscular dystrophy (FSHD) is a skeletal muscle disorder that is caused by derepression of the transcription factor DUX4 in skeletal muscle cells. Apart from SMCHD1, DNMT3B was recently identified as a disease gene and disease modifier in FSHD. However, the exact role of DNMT3B at the D4Z4 repeat array remains unknown. METHODS: To determine the role of Dnmt3b on DUX4 repression, hemizygous mice with a FSHD-sized D4Z4 repeat array (D4Z4-2.5 mice) were cross-bred with mice carrying an in-frame exon skipping mutation in Dnmt3b (Dnmt3bMommeD14 mice). Additionally, siRNA knockdowns of Dnmt3b were performed in mouse embryonic stem cells (mESCs) derived from the D4Z4-2.5 mouse model. RESULTS: In mESCs derived from D4Z4-2.5 mice, Dnmt3b was enriched at the D4Z4 repeat array and DUX4 transcript levels were upregulated after a knockdown of Dnmt3b. In D4Z4-2.5/Dnmt3bMommeD14 mice, Dnmt3b protein levels were reduced; however, DUX4 RNA levels in skeletal muscles were not enhanced and no pathology was observed. Interestingly, D4Z4-2.5/Dnmt3bMommeD14 mice showed a loss of DNA methylation at the D4Z4 repeat array and significantly higher DUX4 transcript levels in secondary lymphoid organs. As these lymphoid organs seem to be more sensitive to epigenetic modifiers of the D4Z4 repeat array, different immune cell populations were quantified in the spleen and inguinal lymph nodes of D4Z4-2.5 mice crossed with Dnmt3bMommeD14 mice or Smchd1MommeD1 mice. Only in D4Z4-2.5/Smchd1MommeD1 mice the immune cell populations were disturbed. CONCLUSIONS: Our data demonstrates that loss of Dnmt3b results in derepression of DUX4 in lymphoid tissues and mESCs but not in myogenic cells of D4Z4-2.5/Dnmt3bMommeD14 mice. In addition, the Smchd1MommeD1 variant seems to have a more potent role in DUX4 derepression. Our studies suggest that the immune system is particularly but differentially sensitive to D4Z4 chromatin modifiers which may provide a molecular basis for the yet underexplored immune involvement in FSHD.

The prospects of targeting DUX4 in facioscapulohumeral muscular dystrophy.

PURPOSE OF REVIEW: Facioscapulohumeral muscular dystrophy (FSHD) is a neuromuscular disorder, which is caused by incomplete repression of the transcription factor double homeobox 4 (DUX4) in skeletal muscle. To date, there is no DUX4-targeting treatment to prevent or delay disease progression. In the present review, we summarize developments in therapeutic strategies with the focus on inhibiting DUX4 and DUX4 target gene expression. RECENT FINDINGS: Different studies show that DUX4 and its target genes can be repressed with genetic therapies using diverse strategies. Additionally, different small compounds can reduce DUX4 and its target genes in vitro and in vivo. SUMMARY: Most studies that show DUX4 repression by genetic therapies have only been tested in vitro. More efforts should be made to test them in vivo for clinical translation. Several compounds have been shown to prevent DUX4 and target gene expression in vitro and in vivo. However, their efficiency and specificity has not yet been shown. With emerging clinical trials, the clinical benefit from DUX4 repression in FSHD will likely soon become apparent.

In silico-guided engineering of Pseudomonas putida towards growth under micro-oxic conditions.

BACKGROUND: Pseudomonas putida is a metabolically versatile, genetically accessible, and stress-robust species with outstanding potential to be used as a workhorse for industrial applications. While industry recognises the importance of robustness under micro-oxic conditions for a stable production process, the obligate aerobic nature of P. putida, attributed to its inability to produce sufficient ATP and maintain its redox balance without molecular oxygen, severely limits its use for biotechnology applications. RESULTS: Here, a combination of genome-scale metabolic modelling and comparative genomics is used to pinpoint essential [Formula: see text]-dependent processes. These explain the inability of the strain to grow under anoxic conditions: a deficient ATP generation and an inability to synthesize essential metabolites. Based on this, several P. putida recombinant strains were constructed harbouring acetate kinase from Escherichia coli for ATP production, and a class I dihydroorotate dehydrogenase and a class III anaerobic ribonucleotide triphosphate reductase from Lactobacillus lactis for the synthesis of essential metabolites. Initial computational designs were fine-tuned by means of adaptive laboratory evolution. CONCLUSIONS: We demonstrated the value of combining in silico approaches, experimental validation and adaptive laboratory evolution for microbial design by making the strictly aerobic Pseudomonas putida able to grow under micro-oxic conditions.

Pseudomonas putida KT2440 is HV1 certified, not GRAS.

Pseudomonas putida is rapidly becoming a workhorse for industrial production due to its metabolic versatility, genetic accessibility and stress-resistance properties. The P. putida strain KT2440 is often described as Generally Regarded as Safe, or GRAS, indicating the strain is safe to use as food additive. This description is incorrect. P. putida KT2440 is classified by the FDA as HV1 certified, indicating it is safe to use in a P1 or ML1 environment.

Displacement of popliteal sciatic nerve catheters after major foot and ankle surgery: a randomized controlled double-blinded magnetic resonance imaging study.

BACKGROUND: Popliteal sciatic nerve catheters (PSNCs) are associated with a high frequency of displacement. We aimed to estimate the frequency of catheter displacement after 48 h with magnetic resonance imaging (MRI) in patients with PSNCs after major foot and ankle surgery randomized to catheter insertion either with a short-axis in-plane (SAX-IP) approach perpendicular to the nerve or with a short-axis out-of-plane (SAX-OOP) approach parallel to the nerve. METHODS: Forty patients were randomly allocated to SAX-IP or SAX-OOP PSNC. Ropivacaine 0.75% 20 ml was injected via the catheter followed by ropivacaine 0.2% 10 ml h(-)1 infusion. Correct primary catheter placement was ensured after initial injection of local anaesthetic via the catheter. Forty-eight hours after insertion, MRI was performed after injection of saline with added contrast (Dotarem) via the catheter. The primary outcome was catheter displacement estimated as the frequency of spread of contrast exclusively outside the paraneurium. RESULTS: All patients had correct primary catheter placement. The frequency of displacement 48 h after insertion of the PSNC was 40% when inserted perpendicular to the nerve vs 10% parallel to the nerve (difference was 30 percentage points, 95% CI: 3-53 percentage points). The relative risk of displacement was four times larger (95% CI: 0.8-10, P<0.028) in the SAX-IP vs the SAX-OOP group. The morphine consumption was 150% greater in the SAX-IP compared with the SAX-OOP group. CONCLUSION: Popliteal sciatic nerve catheters for major foot and ankle surgery inserted with ultrasound guidance parallel to the sciatic nerve have a significantly lower frequency of displacement compared with those inserted perpendicular to the nerve. CLINICAL TRIAL REGISTRATION: NCT02200016.

Prevalence of endometriosis in epithelial ovarian cancer. Analysis of the associated clinical features and study on molecular mechanisms involved in the possible causality.

PURPOSE OF INVESTIGATION: To determine the prevalence of endometriosis in patients with epithelial ovarian cancer and explore the differences between women with endometrioid and clear-cell histologic subtypes with and without associated endometriosis. MATERIALS AND METHODS: The medical charts of 496 patients with epithelial ovarian cancer at the Hospital Virgin de la Arrixaca (Murcia, Spain) between 1971 and 2010 were reviewed. RESULTS: Endometriosis was present in 27 (5.4%) of the 496 cases (p < 0001), and was associated with the endometrioid histotype in 13/45 cases (29%) and with the clear cell histotype in 7/22 (32%). The prevalence of an association with endometriosis according to histologic type was 28.8% (13/45) for endometrioid carcinoma and 31.8% (7/22) for clear-cell carcinoma. CONCLUSION: Both endometrioid and clear-cell ovarians tumours are associated with pelvic endometriosis. Patients with endometiosis associated ovarian cancer differ from non-endometiosis associated ovarian cancer in their clinical characteristics.

Reproductive outcome after uterine-sparing surgery for endometrial stromal sarcoma.

► This is the second report of pregnancy following endometrial stromal sarcoma (ESS). ► The role of adnexectomy is controversial in stage I ESS. ► Adnexectomy does not appear to affect survival in stage I ESS.

Assessment of cellular and plasma oxidative stress in SAHS patients before and after continuous positive airway pressure treatment.

BACKGROUND: Several indirect techniques have been used for measuring oxidative stress in sleep apnoea-hypopnoea syndrome (SAHS) patients. The purpose of this study was to find out if both, cellular or plasma oxidative stress evaluations, are good estimators to assess oxidative stress in SAHS patients before and after one month's CPAP treatment. METHODS: The study included 28 SAHS patients requiring CPAP treatment and 15 healthy control subjects. Plasma and serum oxidative stress biomarkers (lipid peroxidation, total antioxidant capacity, and the activities of glutathione peroxidase, glutathione reductase, glutathione s-transferase, catalase and superoxide dismutase) were measured using commercial kits. Cellular oxidative stress biomarkers (mitochondrial membrane potential, intracellular glutathione, superoxide anion, and hydrogen peroxide) were analysed by flow cytometry. The Wilcoxon test for paired samples was used to compare oxidative stress and clinical parameters in patients before and after treatment with CPAP. Relationships in oxidative stress markers between controls and patients were analyzed using the Mann-Whitney U test. The Spearman correlation coefficient was calculated to estimate the linear correlations between variables. RESULTS: Oxidative stress was notably decreased after CPAP. Before CPAP, SAHS severity positively correlated with hydrogen peroxide levels, while negative correlations were observed between SAHS severity and plasma TAC in patients. Also, plasma glutathione peroxidase activity negatively correlated with cellular superoxide anion, while plasma superoxide dismutase activity positively correlated with intracellular glutathione. After CPAP, plasma TAC and glutathione peroxidase activity negatively correlated with cellular hydrogen peroxide and superoxide anion. CONCLUSIONS: In conclusion, this study seems to confirm that plasma and cellular assessment reflect, in the same way, the oxidative stress status of the studied patients. Furthermore, plasma total antioxidant capacity as well as cellular hydrogen peroxide levels can be good markers for assessing oxidative stress in SAHS patients.