Direct coupled electrical stimulation towards improved osteogenic differentiation of human mesenchymal stem/stromal cells: a comparative study of different protocols.

Electrical stimulation (ES) has been described as a promising tool for bone tissue engineering, being known to promote vital cellular processes such as cell proliferation, migration, and differentiation. Despite the high variability of applied protocol parameters, direct coupled electric fields have been successfully applied to promote osteogenic and osteoinductive processes in vitro and in vivo. Our work aims to study the viability, proliferation, and osteogenic differentiation of human bone marrow-derived mesenchymal stem/stromal cells when subjected to five different ES protocols. The protocols were specifically selected to understand the biological effects of different parts of the generated waveform for typical direct-coupled stimuli. In vitro culture studies evidenced variations in cell responses with different electric field magnitudes (numerically predicted) and exposure protocols, mainly regarding tissue mineralization (calcium contents) and osteogenic marker gene expression while maintaining high cell viability and regular morphology. Overall, our results highlight the importance of numerical guided experiments to optimize ES parameters towards improved in vitro osteogenesis protocols.

Novel approaches to motoneuron disease/ALS treatment using non-invasive brain and spinal stimulation: IFCN handbook chapter.

Non-invasive brain stimulation techniques have been exploited in motor neuron disease (MND) with multifold objectives: to support the diagnosis, to get insights in the pathophysiology of these disorders and, more recently, to slow down disease progression. In this review, we consider how neuromodulation can now be employed to treat MND, with specific attention to amyotrophic lateral sclerosis (ALS), the most common form with upper motoneuron (UMN) involvement, taking into account electrophysiological abnormalities revealed by human and animal studies that can be targeted by neuromodulation techniques. This review article encompasses repetitive transcranial magnetic stimulation methods (including low-frequency, high-frequency, and pattern stimulation paradigms), transcranial direct current stimulation as well as experimental findings with the newer approach of trans-spinal direct current stimulation. We also survey and discuss the trials that have been performed, and future perspectives.

How does the electric field induced by tDCS influence motor-related connectivity? Model-guided perspectives.

Over the last decade, transcranial direct current stimulation (tDCS) has been applied not only to modulate local cortical activation, but also to address communication between functionally-related brain areas. Stimulation protocols based on simple two-electrode placements are being replaced by multi-electrode montages to target intra- and inter-hemispheric neural networks using multichannel/high definition paradigms.Objective. This study aims to investigate the characteristics of electric field (EF) patterns originated by tDCS experiments addressing changes in functional brain connectivity.Methods. A previous selection of tDCS experimental studies aiming to modulate motor-related connectivity in health and disease was conducted. Simulations of the EF induced in the cortex were then performed for each protocol selected. The EF magnitude and orientation are determined and analysed in motor-related cortical regions for five different head models to account for inter-subject variability. Functional connectivity outcomes obtained are qualitatively analysed at the light of the simulated EF and protocol characteristics, such as electrode position, number and stimulation dosing.Main findings. The EF magnitude and orientation predicted by computational models can be related with the ability of tDCS to modulate brain functional connectivity. Regional differences in EF distributions across subjects can inform electrode placements more susceptible to inter-subject variability in terms of brain connectivity-related outcomes.Significance. Neuronal facilitation/inhibition induced by tDCS fields may indirectly influence intra and inter-hemispheric connectivity by modulating neural components of motor-related networks. Optimization of tDCS using computational models is essential for adequate dosing delivery in specific networks related to clinically relevant connectivity outcomes.

Is There a Role for Risk-Reducing Bilateral Breast Surgery in BRCA1/2 Ovarian Cancer Survivors? An Observational Study.

BACKGROUND: Risk-reducing surgeries are an option for cancer risk management in BRCA1/2 individuals. However, while adnexectomy is commonly recommended in breast cancer (BC) survivors, risk-reducing bilateral breast surgery (RRBBS) is controversial in ovarian cancer (OC) survivors due to relapse rates and mortality. METHODS: We conducted a retrospective analysis of BRCA1/2-OC survivors, with OC as first cancer diagnosis. RESULTS: Median age at OC diagnosis for the 69 BRCA1/2-OC survivors was 54 years. Median overall survival was 8 years, being significantly higher for BRCA2 patients than for BRCA1 patients (p = 0.011). Nine patients (13.2%) developed BC at a median age of 61 years. The mean overall BC-free survival was 15.5 years (median not reached). Eight patients (11.8%) underwent bilateral mastectomy (5 simultaneous with BC treatment; 3 RRBBS) at a median age of 56.5 years. The median time from OC to bilateral mastectomy/RRBBS was 5.5 years. CONCLUSIONS: This study adds evidence regarding a lower BC risk after BRCA1/2-OC and higher survival for BRCA2-OC patients. A comprehensive analysis of the competing risks of OC mortality and recurrence against the risk of BC should be individually addressed. Surgical BC risk management may be considered for longer BRCA1/2-OC disease-free survivors. Ultimately, these decisions should always be tailored to patients' characteristics and preferences.

Extending AAV Packaging Cargo through Dual Co-Transduction: Efficient Protein Trans-Splicing at Low Vector Doses.

Adeno-associated viral (AAV) vectors represent one of the leading platforms for gene delivery. Nevertheless, their small packaging capacity restricts their use for diseases requiring large-gene delivery. To overcome this, dual-AAV vector systems that rely on protein trans-splicing were developed, with the split-intein Npu DnaE among the most-used. However, the reconstitution efficiency of Npu DnaE is still insufficient, requiring higher vector doses. In this work, two split-inteins, Cfa and Gp41-1, with reportedly superior trans-splicing were evaluated in comparison with Npu DnaE by transient transfections and dual-AAV in vitro co-transductions. Both Cfa and Gp41-1 split-inteins enabled reconstitution rates that were over two-fold higher than Npu DnaE and 100% of protein reconstitution. The impact of different vector preparation qualities in split-intein performances was also evaluated in co-transduction assays. Higher-quality preparations increased split-inteins' performances by three-fold when compared to low-quality preparations (60-75% vs. 20-30% full particles, respectively). Low-quality vector preparations were observed to limit split-gene reconstitutions by inhibiting co-transduction. We show that combining superior split-inteins with higher-quality vector preparations allowed vector doses to be decreased while maintaining high trans-splicing rates. These results show the potential of more-efficient protein-trans-splicing strategies in dual-AAV vector co-transduction, allowing the extension of its use to the delivery of larger therapeutic genes.

Mammalian Stable Cell Platforms for Recombinant Adeno-associated Virus (rAAV) Production: Development Strategies and their Impact on Viral Productivity.

Adeno-associated viruses (AAV) are widely used as a recombinant vectors in gene therapy. AAVs are non-pathogenic. They present reduced cytotoxicity and can transduce both dividing and non-dividing cells. The existence of different serotypes provides flexibility for targeting different tissues and organs. Its therapeutic success was already shown by the approval of three products by the European and American regulatory agencies. To satisfy the high dosage, safety, and reproducibility required in each clinical trial, production platforms based on stable mammalian cell lines have been proposed as the best strategy. However, the methodologies employed must be adapted to each cell line, which often results in distinct productivities. In this article, we review the published and commercially available mammalian stable cell lines, discussing the key factors that impact viral production yields, such as integration sites and copy numbers.

ANKRD11 pathogenic variants and 16q24.3 microdeletions share an altered DNA methylation signature in patients with KBG syndrome.

Pathogenic variants in ANKRD11 or microdeletions at 16q24.3 are the cause of KBG syndrome (KBGS), a neurodevelopmental syndrome characterized by intellectual disability, dental and skeletal anomalies, and characteristic facies. The ANKRD11 gene encodes the ankyrin repeat-containing protein 11A transcriptional regulator, which is expressed in the brain and implicated in neural development. Syndromic conditions caused by pathogenic variants in epigenetic regulatory genes show unique patterns of DNA methylation (DNAm) in peripheral blood, termed DNAm signatures. Given ANKRD11's role in chromatin modification, we tested whether pathogenic ANKRD11 variants underlying KBGS are associated with a DNAm signature. We profiled whole-blood DNAm in 21 individuals with ANKRD11 variants, 2 individuals with microdeletions at 16q24.3 and 28 typically developing individuals, using Illumina's Infinium EPIC array. We identified 95 differentially methylated CpG sites that distinguished individuals with KBGS and pathogenic variants in ANKRD11 (n = 14) from typically developing controls (n = 28). This DNAm signature was then validated in an independent cohort of seven individuals with KBGS and pathogenic ANKRD11 variants. We generated a machine learning model from the KBGS DNAm signature and classified the DNAm profiles of four individuals with variants of uncertain significance (VUS) in ANKRD11. We identified an intermediate classification score for an inherited missense variant transmitted from a clinically unaffected mother to her affected child. In conclusion, we show that the DNAm profiles of two individuals with 16q24.3 microdeletions were indistinguishable from the DNAm profiles of individuals with pathogenic variants in ANKRD11, and we demonstrate the diagnostic utility of the new KBGS signature by classifying the DNAm profiles of individuals with VUS in ANKRD11.

Awake airway endoscopy in mucopolysaccharidosis: a case report.

Mucopolysaccharidosis (MPS) are a group of rare genetic inherited diseases with a progressive course due to the accumulation of glycosaminoglycans resulting in anatomic abnormalities and organ dysfunction, including the respiratory, cardiovascular, skeletal, and neurological systems that can increase the risk of anesthesia complications. Clinical manifestations are variable, multisystemic, and include severe morphological changes. The anesthetic management of these patients is complex, particularly airway management, which can be planned to include a fiberoptic airway investigation prior to surgery. We present two cases of patients with MPS type VI and VII who underwent fiberoptic airway mapping under conscious sedation, with no complications. Since MPS is a rare but challenging disease concerning the airway management, we propose a safe and effective anesthetic technique that could be used for fiberoptic bronchoscopy and allow fiberoptic-assisted tracheal intubation at the time of surgery.

Transcriptome analysis of Sf9 insect cells during production of recombinant Adeno-associated virus.

The insect cell-baculovirus expression vector system (IC-BEVS) has emerged as an alternative time- and cost-efficient production platform for recombinant Adeno-associated virus (AAV) for gene therapy. However, a better understanding of the underlying biological mechanisms of IC-BEVS is fundamental to further optimize this expression system toward increased product titer and quality. Here, gene expression of Sf9 insect cells producing recombinant AAV through a dual baculovirus expression system, with low multiplicity of infection (MOI), was profiled by RNA-seq. An 8-fold increase in reads mapping to either baculovirus or AAV transgene sequences was observed between 24 and 48 h post-infection (hpi), confirming a take-over of the host cell transcriptome by the baculovirus. A total of 336 and 4784 genes were identified as differentially expressed at 24 hpi (vs non-infected cells) and at 48 hpi (vs. infected cells at 24 hpi), respectively, including dronc, birc5/iap5, and prp1. Functional annotation found biological processes such as cell cycle, cell growth, protein folding, and cellular amino acid metabolic processes enriched along infection. This work uncovers transcriptional changes in Sf9 in response to baculovirus infection, which provide new insights into cell and/or metabolic engineering targets that can be leveraged for rational bioprocess engineering of IC-BEVS for AAV production.

A randomised trial of anti-GM-CSF otilimab in severe COVID-19 pneumonia (OSCAR).

BACKGROUND: Granulocyte-macrophage colony-stimulating factor (GM-CSF) and dysregulated myeloid cell responses are implicated in the pathophysiology and severity of COVID-19. METHODS: In this randomised, sequential, multicentre, placebo-controlled, double-blind study, adults aged 18-79 years (Part 1) or ≥70 years (Part 2) with severe COVID-19, respiratory failure and systemic inflammation (elevated C-reactive protein/ferritin) received a single intravenous infusion of otilimab 90 mg (human anti-GM-CSF monoclonal antibody) plus standard care (NCT04376684). The primary outcome was the proportion of patients alive and free of respiratory failure at Day 28. RESULTS: In Part 1 (n=806 randomised 1:1 otilimab:placebo), 71% of otilimab-treated patients were alive and free of respiratory failure at Day 28 versus 67% who received placebo; the model-adjusted difference of 5.3% was not statistically significant (95% CI -0.8-11.4%, p=0.09). A nominally significant model-adjusted difference of 19.1% (95% CI 5.2-33.1%, p=0.009) was observed in the predefined 70-79 years subgroup, but this was not confirmed in Part 2 (n=350 randomised) where the model-adjusted difference was 0.9% (95% CI -9.3-11.2%, p=0.86). Compared with placebo, otilimab resulted in lower serum concentrations of key inflammatory markers, including the putative pharmacodynamic biomarker CC chemokine ligand 17, indicative of GM-CSF pathway blockade. Adverse events were comparable between groups and consistent with severe COVID-19. CONCLUSIONS: There was no significant difference in the proportion of patients alive and free of respiratory failure at Day 28. However, despite the lack of clinical benefit, a reduction in inflammatory markers was observed with otilimab, in addition to an acceptable safety profile.

Awake airway endoscopy in mucopolysaccharidosis: a case report

Abstract Mucopolysaccharidosis (MPS) are a group of rare genetic inherited diseases with a progressive course due to the accumulation of glycosaminoglycans resulting in anatomic abnormalities and organ dysfunction, including the respiratory, cardiovascular, skeletal, and neurological systems that can increase the risk of anesthesia complications. Clinical manifestations are variable, multisystemic, and include severe morphological changes. The anesthetic management of these patients is complex, particularly airway management, which can be planned to include a fiberoptic airway investigation prior to surgery. We present two cases of patients with MPS type VI and VII who underwent fiberoptic airway mapping under conscious sedation, with no complications. Since MPS is a rare but challenging disease concerning the airway management, we propose a safe and effective anesthetic technique that could be used for fiberoptic bronchoscopy and allow fiberoptic-assisted tracheal intubation at the time of surgery.

JANUS: an open-source 3D printable perfusion bioreactor and numerical model-based design strategy for tissue engineering.

Bioreactors have been employed in tissue engineering to sustain longer and larger cell cultures, managing nutrient transfer and waste removal. Multiple designs have been developed, integrating sensor and stimulation technologies to improve cellular responses, such as proliferation and differentiation. The variability in bioreactor design, stimulation protocols, and cell culture conditions hampered comparison and replicability, possibly hiding biological evidence. This work proposes an open-source 3D printable design for a perfusion bioreactor and a numerical model-driven protocol development strategy for improved cell culture control. This bioreactor can simultaneously deliver capacitive-coupled electric field and fluid-induced shear stress stimulation, both stimulation systems were validated experimentally and in agreement with numerical predictions. A preliminary in vitro validation confirmed the suitability of the developed bioreactor to sustain viable cell cultures. The outputs from this strategy, physical and virtual, are openly available and can be used to improve comparison, replicability, and control in tissue engineering applications.

Posterior Reversible Encephalopathy Syndrome in a Pediatric Intensive Care Unit: A Case Series.

Posterior reversible encephalopathy syndrome (PRES) is a reversible clinical-radiographic abnormality. It is characterized by headache, altered consciousness, seizures, and visual disruption, in addition to characteristic white matter edema lesions in the parieto-occipital areas of the brain. Early detection and treatment are crucial to prevent irreversible damage. This paper presents the cases of three patients with PRES with concurrent diagnoses of glomerulonephritis, Guillain-Barré syndrome, and sickle cell disease. All patients experienced systemic hypertension, seizures, and altered consciousness. All patients were admitted to intensive care for decreased level of awareness or status epilepticus requiring invasive mechanical ventilation. Anticonvulsants and antihypertensive therapy were essential. No chronic complications were recorded.

The Prevalence of Potentially Inappropriate Prescribing in Two Family Health Units in Portugal.

OBJECTIVE: Polypharmacy and potentially inappropriate prescribing (PIP) are growing concerns in the ageing population. They carry the risk of increasing adverse effects, medical interactions, and difficulties managing the medication. Few studies in Portugal evaluate the prevalence of polypharmacy and PIP in primary care. No previous studies in the primary care setting in Portugal have been conducted using the European Union (EU)(7)-PIM (potentially inappropriate medication) list tool. In this study, we aimed to estimate the prevalence of polypharmacy and PIP in two family health units in Portugal.  Methods: To answer this question, we enrolled a sample of 361 elderly patients from two family health units in a descriptive observational transversal study. We randomly selected patients, consulted their prescription records in the previous 12 months, and applied the EU(7)-PIM list tool, validated for the Portuguese population. The data was then analyzed using descriptive and inferential statistics and the Statistical Package for the Social Sciences (IBM SPSS Statistics for Windows, IBM Corp., Version 24.0, Armonk, NY). RESULTS: Our results showed a prevalence of 79.8% of polypharmacy in the elderly population and 73.4% of PIP. These values are higher than predicted in the literature, but different screening tools have been used among papers. The mean number of prescribed drugs per patient was nine in one unit and seven in the other, and the mode was eleven per patient. The most identified PIP-associated drugs were proton pump inhibitors in 46.4% of the patients in one unit and 43.7% in the other. We also found a statistically significant higher prevalence of PIP and polypharmacy in females and patients over 75 years. CONCLUSION: From a prevalence perspective, we found higher-than-expected prevalences of PIP and polypharmacy in our population. Contributing factors might be a higher ageing index in the Portuguese population, modern practices using combination therapy, and the use of a screening tool that does not take into account the personal clinical history of patients. Further limitations involve only including patients with follow-up in the units studied. Even so, it suggests both PIP and polypharmacy as concerns to address, and we will strive to educate both health teams on PIP, polypharmacy, and deprescribing. We also emphasize the need to widen the study to other family health units.

Lumbar trans-spinal direct current stimulation: A modeling-experimental approach to dorsal root ganglia stimulation.

Introduction: The excitability of spinal motor neurons (MN) can be altered through subthreshold currents, such as transcutaneous spinal direct-current stimulation (tsDCS). Current evidence shows that tsDCS can interfere with ascending somatosensory pathways and lower motor neurons' (LMN) excitability, which points to its therapeutic potential for repairing altered spinal responses. We aim to define the best tsDCS montage for maximizing the electric field (E-field) in the lumbar spinal cord (L-SC) by computer modeling; and to apply this montage to measure the effect on LMN excitability and somatosensory evoked potentials (SSEP). Methods: A human volume conductor model was obtained from an available database. The E-field distribution was calculated considering three different electrode settings aiming at maximizing the field at L-SC and right dorsal root ganglia (DRG). The best electrode setting was then selected and applied in a blind crossover pseudo-randomized study including 14 subjects. tsDCS was delivered for 15 min (cathodal vs. sham) over L2 vertebra level (4 mA, 144 mC/cm2), and its effect on F-waves, H-reflex (including homosynaptic depression, HD) and SSEPs was investigated in the lower limbs. Results: All simulated montages showed higher current density and E-field magnitudes between the electrodes (>0.15 V/m), with a major longitudinal component and with rostral-caudal direction. The induced E-field involved the sensory ganglia and was maximum in the right T8-left L2 montage, which was the one selected for the experimental protocol. We disclosed a statistically significant increase of the H-reflex amplitude at 0.1 Hz, after cathodal tsDCS (c-tsDCS) on both sides. No other significant change was observed. Discussion: Our results can suggest the c-tsDCS applied to the L-SC and DRG can modulate synaptic efficiency increasing lower motor neurons response to Ia fibers excitation. The possible implications of our findings for treating clinical conditions will be addressed in future studies.

Validation study of the Functional Assessment of Cancer Therapy-Cognitive Function-Version 3 for the Portuguese population.

BACKGROUND: Cancer-related cognitive impairment is a common and potentially debilitating symptom experienced by patients with non-central nervous system (CNS) cancers, with negative impact on their quality of life. The Functional Assessment of Cancer Therapy-Cognitive Function-Version 3 (FACT-Cog-v3) is the most extensively used instrument specifically developed to evaluate cognitive complaints in adult cancer patients. Nevertheless, this self-report measure is not yet validated for the Portuguese population. Therefore, the purpose of this study was to evaluate the psychometric properties of the FACT-Cog-v3 among patients with non-CNS cancers in Portugal. METHODS: The validation study was conducted based on a convenience sample of 281 patients with non-CNS cancers, aged between 18 and 65 years, recruited online. A confirmatory factor analysis (CFA) was used to test the factor structure of the Portuguese FACT-Cog-v3 version; internal consistency analysis was also conducted. The European Organization for Research and Treatment of Cancer Quality of Life Questionnaire Core-30 (EORTC QLQ-C30-version 3) and the Hospital Anxiety and Depression Scale (HADS) were also used to test the concurrent, convergent, and discriminant validity of the scale. RESULTS: CFA supported a four-factor model with good fix indexes and internal consistencies: perceived cognitive impairments (α = 0.97), comments from others (α = 0.92), perceived cognitive abilities (α = 0.93), and impact on quality of life (α = 0.92). Concurrent, convergent, and discriminant validities were confirmed. Moderate and strong correlations were found between the FACT-Cog-v3 subscales and the QLQ-C30 cognitive functioning subscale. Good convergent validity, with moderate correlations, was found between the FACT-Cog-v3 subscales and the HADS-A, HADS-D, and QLQ-C30 fatigue, sleep disturbance, and global health status subscales. Acceptable discriminant validity, with weak and moderate correlations, was demonstrated between the FACT-Cog-v3 subscales and the QLQ-C30 pain and nausea/vomiting subscales. CONCLUSIONS: The Portuguese FACT-Cog-v3 version can be considered a reliable and valid measure to assess cognitive concerns of patients with non-CNS cancers, with relevance for research and clinical practice.

How the Number and Distance of Electrodes Change the Induced Electric Field in the Cortex during Multichannel tDCS.

Multichannel transcranial direct current stimulation (tDCS) is a promising approach to target neuromodulation of neural networks by making use of variable number of electrodes and distances to facilitate/inhibit specific connectivity patterns. Optimization of the electric field (EF) spatial distribution through computational models can provide a more accurate definition of the stimulation settings that are more effective. In this study, we investigate the effect of increasing the number of cathodes around a central anode placed over the target. We demonstrate that anode-cathode distance has the largest influence in the EF and using more than 3 cathodes did not result in considerable changes in the EF magnitude and direction. This could be relevant for simultaneous tDCS-electroencephalography (EEG) applications, by saving electrode positions for EEG acquisition. Clinical Relevance- This study demonstrates that distance between electrodes is more relevant than electrode number in determining the electric field distribution, and that a highly-focused stimulation can be equally effective with fewer electrodes.

Cognitive functioning and work-related outcomes of non-central nervous system cancer survivors: protocol for a systematic review with meta-analysis.

INTRODUCTION: In recent years, growing attention has been given to the study of the impact of cancer-related cognitive impairment (CRCI) in working non-central nervous system (CNS) cancer survivors. Available literature has shown that working cancer survivors identify cognitive problems at work as very problematic and worrisome. Some reviews have discussed the association between CRCI and work-related outcomes; however, none to date have investigated this association through comprehensive systematic review with meta-analysis. Hence, this work will comprehensively summarise existing evidence from quantitative studies assessing the relationship between CRCI and work-related outcomes of adult non-CNS cancer survivors at working age. METHODS AND ANALYSIS: The systematic review procedures and its report will follow the Preferred Reporting Items for Systematic Reviews and Meta-Analyses statement. Electronic searches in the databases Web of Science, Scopus, PubMed, ProQuest, PsycINFO and CINAHL, complemented by a manual search of other relevant articles, will be performed from 2000 onwards to identify relevant publications. Two independent reviewers will assess studies for inclusion and extract data from each article using a standardised form. Studies eligible for inclusion must be quantitative, contain adult non-CNS cancer survivors with CRCI, and a measure of cognitive functioning and work-related outcomes. To assess risk of bias, the Joanna Briggs Institute Critical Appraisal Tool Studies checklists will be independently used by the two researchers. Synthesis of the included articles will be conducted using a narrative method and through meta-analysis. Meta-analysis will be reported via correlation for the association between CRCI and work-related outcomes. The cumulative evidence will be assessed using the Grading of Recommendations Assessment, Development and Evaluation system. ETHICS AND DISSEMINATION: Ethics approval is not required since individual patient data will not be collected. The findings will be published in a peer-review indexed journal, presented at scientific meetings and included in a chapter of a Doctoral thesis. PROSPERO REGISTRATION NUMBER: CRD42020165458.

How to correctly estimate the electric field in capacitively coupled systems for tissue engineering: a comparative study.

Capacitively Coupled (CCoupled) electric fields are used to stimulate cell cultures in Tissue Engineering. Knowing the electric field (E-Field) magnitude in the culture medium is fundamental to establish a relationship between stimulus strength and cellular effects. We analysed eight CCoupled studies and sought to corroborate the reported estimates of the E-Field in the culture medium. First, we reviewed the basic physics underlying CCoupled stimulation and delineated three approaches to estimate the E-field. Using these approaches, we found that the reported values were overestimated in five studies, four of which were based on incorrect assumptions. In all studies, insufficient information was provided to reproduce the setup exactly. Creating electrical models of the experimental setup should improve the accuracy of the E-field estimates and enhance reproducibility. For this purpose, we developed a free open-source tool, the E-field Calculator for CCoupled systems, which is available for download from an internet hosting platform.