Is the disease risk and penetrance in Leber hereditary optic neuropathy actually low?

Pedigree analysis showed that a large proportion of Leber hereditary optic neuropathy (LHON) family members who carry a mitochondrial risk variant never lose vision. Mitochondrial haplotype appears to be a major factor influencing the risk of vision loss from LHON. Mitochondrial variants, including m.14484T>C and m.11778G>A, have been added to gene arrays, and thus many patients and research participants are tested for LHON mutations. Analysis of the UK Biobank and Australian cohort studies found more than 1 in 1,000 people in the general population carry either the m.14484T>C or the m.11778G>A LHON variant. None of the subset of carriers examined had visual acuity at 20/200 or worse, suggesting a very low penetrance of LHON. Haplogroup analysis of m.14484T>C carriers showed a high rate of haplogroup U subclades, previously shown to have low penetrance in pedigrees. Penetrance calculations of the general population are lower than pedigree calculations, most likely because of modifier genetic factors. This Matters Arising Response paper addresses the Watson et al. (2022) Matters Arising paper, published concurrently in The American Journal of Human Genetics.

Recommendations for the management of critically ill patients with COVID-19 in Intensive Care Units.

The COVID-19 pandemic has led to the admission of a high number of patients to the ICU, generally due to severe respiratory failure. Since the appearance of the first cases of SARS-CoV-2 infection, at the end of 2019, in China, a huge number of treatment recommendations for this entity have been published, not always supported by sufficient scientific evidence or with methodological rigor necessary. Thanks to the efforts of different groups of researchers, we currently have the results of clinical trials, and other types of studies, of higher quality. We consider it necessary to create a document that includes recommendations that collect this evidence regarding the diagnosis and treatment of COVID-19, but also aspects that other guidelines have not considered and that we consider essential in the management of critical patients with COVID-19. For this, a drafting committee has been created, made up of members of the SEMICYUC Working Groups more directly related to different specific aspects of the management of these patients.

[Recommendations for the management of critically ill patients with COVID-19 in Intensive Care Units]. / Recomendaciones para el manejo de los pacientes críticos con COVID-19 en las Unidades de Cuidados Intensivos.

The COVID-19 pandemic has led to the admission of a high number of patients to the ICU, generally due to severe respiratory failure. Since the appearance of the first cases of SARS-CoV-2 infection, at the end of 2019, in China, a huge number of treatment recommendations for this entity have been published, not always supported by sufficient scientific evidence or with methodological rigor necessary. Thanks to the efforts of different groups of researchers, we currently have the results of clinical trials, and other types of studies, of higher quality. We consider it necessary to create a document that includes recommendations that collect this evidence regarding the diagnosis and treatment of COVID-19, but also aspects that other guidelines have not considered and that we consider essential in the management of critical patients with COVID-19. For this, a drafting committee has been created, made up of members of the SEMICYUC Working Groups more directly related to different specific aspects of the management of these patients.

MitoScape: A big-data, machine-learning platform for obtaining mitochondrial DNA from next-generation sequencing data.

The growing number of next-generation sequencing (NGS) data presents a unique opportunity to study the combined impact of mitochondrial and nuclear-encoded genetic variation in complex disease. Mitochondrial DNA variants and in particular, heteroplasmic variants, are critical for determining human disease severity. While there are approaches for obtaining mitochondrial DNA variants from NGS data, these software do not account for the unique characteristics of mitochondrial genetics and can be inaccurate even for homoplasmic variants. We introduce MitoScape, a novel, big-data, software for extracting mitochondrial DNA sequences from NGS. MitoScape adopts a novel departure from other algorithms by using machine learning to model the unique characteristics of mitochondrial genetics. We also employ a novel approach of using rho-zero (mitochondrial DNA-depleted) data to model nuclear-encoded mitochondrial sequences. We showed that MitoScape produces accurate heteroplasmy estimates using gold-standard mitochondrial DNA data. We provide a comprehensive comparison of the most common tools for obtaining mtDNA variants from NGS and showed that MitoScape had superior performance to compared tools in every statistically category we compared, including false positives and false negatives. By applying MitoScape to common disease examples, we illustrate how MitoScape facilitates important heteroplasmy-disease association discoveries by expanding upon a reported association between hypertrophic cardiomyopathy and mitochondrial haplogroup T in men (adjusted p-value = 0.003). The improved accuracy of mitochondrial DNA variants produced by MitoScape will be instrumental in diagnosing disease in the context of personalized medicine and clinical diagnostics.

Establishing risk of vision loss in Leber hereditary optic neuropathy.

We conducted an updated epidemiological study of Leber hereditary optic neuropathy (LHON) in Australia by using registry data to establish the risk of vision loss among different LHON mutations, sex, age at onset, and mitochondrial haplogroup. We identified 96 genetically unrelated LHON pedigrees, including 56 unpublished pedigrees, and updated 40 previously known pedigrees, comprising 620 affected individuals and 4,948 asymptomatic carriers. The minimum prevalence of vision loss due to LHON in Australia in 2020 was one in 68,403 individuals. Although our data confirm some well-established features of LHON, the overall risk of vision loss among those with a LHON mutation was lower than reported previously-17.5% for males and 5.4% for females. Our findings confirm that women, older adults, and younger children are also at risk. Furthermore, we observed a higher incidence of vision loss in children of affected mothers as well as in children of unaffected women with at least one affected brother. Finally, we confirmed our previous report showing a generational fall in prevalence of vision loss among Australian men. Higher reported rates of vision loss in males with a LHON mutation are not supported by our work and other epidemiologic studies. Accurate knowledge of risk is essential for genetic counseling of individuals with LHON mutations. This knowledge could also inform the detection and validation of potential biomarkers and has implications for clinical trials of treatments aimed at preventing vision loss in LHON because an overestimated risk may lead to an underpowered study or a false claim of efficacy.

Elevación del lactato en el postoperatorio como marcador de isquemia mesentérica aguda. Descripción de dos casos / Postoperative lactate elevation as a marker of underlying acute mesenteric ischemia. Description of two cases

El lactato sérico es un marcador inespecífico de hipoperfusión tisular. El diagnóstico diferencial de su elevación incluye la isquemia intestinal aguda. A pesar de ser controvertido para este uso, el lactato se sigue empleando, por su elevada sensibilidad y a falta de otros marcadores validados. Presentamos los casos de 2 pacientes que desarrollaron como complicación posquirúrgica una isquemia mesentérica aguda, que cursó con dolor abdominal moderado —un síntoma inespecífico en el contexto postoperatorio— y unos niveles de lactato sérico en aumento progresivo, lo que facilitó la sospecha y la posterior confirmación diagnóstica mediante una prueba de imagen. Se destaca la importancia fisiopatológica de la elevación del lactato en el contexto perioperatorio, que debe llevar a realizar un diagnóstico diferencial de sus posibles causas, incluyendo entre las mismas la isquemia mesentérica; pues aunque en el primer caso el desenlace fue negativo, la sospecha precoz permitió en el segundo, realizar un diagnóstico y tratamiento efectivos.(AU)

Serum lactate is a non-specific marker of tissue hypoperfusion. Elevated serum lactate is used in the differential diagnosis of acute intestinal ischemia. Although this practice is controversial, in the absence of other validated markers lactate is still used because of its high sensitivity. We present the cases of two patients who developed acute mesenteric ischemia as a post-surgical complication. The patients reported moderate abdominal pain —a non-specific symptom in the postoperative context— and tests showed progressively increasing serum lactate levels, which facilitated suspicion and subsequent diagnostic confirmation through an imaging test. These cases highlight the physiopathological importance of lactate elevation in the perioperative context and of performing a differential diagnosis of its possible causes, including mesenteric ischemia. Although the outcome was negative in the first case, early suspicion allowed us to make an effective diagnosis and administer appropriate treatment in the second patient.(AU)

Postoperative lactate elevation as a marker of underlying acute mesenteric ischemia. Description of two cases.

Serum lactate is a non-specific marker of tissue hypoperfusion. Elevated serum lactate is used in the differential diagnosis of acute intestinal ischemia. Although this practice is controversial, in the absence of other validated markers lactate is still used because of its high sensitivity. We present the cases of two patients who developed acute mesenteric ischemia as a post-surgical complication. The patients reported moderate abdominal pain -a non-specific symptom in the postoperative context- and tests showed progressively increasing serum lactate levels, which facilitated suspicion and subsequent diagnostic confirmation through an imaging test. These cases highlight the physiopathological importance of lactate elevation in the perioperative context and of performing a differential diagnosis of its possible causes, including mesenteric ischemia. Although the outcome was negative in the first case, early suspicion allowed us to make an effective diagnosis and administer appropriate treatment in the second patient.

Associations and community health workers: analysis and time trends over ten years of training-action / Asociaciones y agentes de salud de base comunitaria: análisis y tendencias temporales durante diez años de formación-acción

Objective: To analyse the profile of the persons and associations that participated in the course, quantify peer education activities and analyse their evolution. Method: A quantitative study using an analysis of the course records from 2009 to 2018 was designed for this purpose inside mihsalud program designed to promote health amongst persons in vulnerable situations in the city of Valencia (Spain). It offers a yearly training-action course of community health workers (CHW) that is attended by persons who have been proposed by associations. The associations were defined according to their population (immigrant, local or intercultural) and the CHWs according to gender, country of birth, year of course, association and continuity after training. Means and confidence intervals were calculated at 95% and a bivariate analysis was conducted in order to compare the activities that took place in 2009 to 2013 with those of 2014 to 2018. The time trends were analysed by applying linear regression models that included the different years studied as the dependent variable. Results: 201 CHW of 31 nationalities were trained, 81.6% (95% confidence interval [95% CI]: 75.5-86.7] were women. Eighty-two associations participated, 51.2% (95% CI: 39.9-62.4] worked with culturally diverse populations. Participation by associations (p=.017) and CHWs (p=.377) increased in a statistically significant manner over the years. After the course, 35.3% (95% CI: 28.7-42.4] of the CHWs continued to collaborate voluntarily in the associations. (AU)

Objetivo: Analizar el perfil de las personas y asociaciones que participaron en los cursos, cuantificar las actividades de educación entre iguales y analizar su evolución. Método: Se diseñó un estudio cuantitativo utilizando un análisis de los registros del curso de 2009 a 2018, dentro del programa mihsalud diseñado para promover la salud entre las personas en situación de vulnerabilidad en la ciudad de Valencia (España). Anualmente se ofrece un curso de formación-acción para agentes de salud de base comunitaria (ASBC), al que asisten personas propuestas por asociaciones. Las asociaciones se definieron según su población (inmigrantes, locales o interculturales) y los ASBC según el género, el país de nacimiento, el año de curso, la asociación y la continuidad después de la capacitación. Las medias y los intervalos de confianza se calcularon al 95% y se realizó un análisis bivariado para comparar las actividades que tuvieron lugar en 2009 a 2013 con las de 2014 a 2018. Las tendencias temporales se analizaron aplicando modelos de regresión lineal que incluyeron el estudio de diferentes años como la variable dependiente. Resultados: 201 ASBC de 31 nacionalidades fueron entrenados, 81.6% (intervalo de confianza del 95% [IC 95%]: 75,5 a 86,7) eran mujeres. Participaron 82 asociaciones, el 51,2% (IC 95%: 39,9 a 62,4) trabajaron con poblaciones culturalmente diversas. La participación de asociaciones (p = 0,017) y ASBC (p = 0,377) aumentó de manera estadísticamente significativa a lo largo de los años. Después del curso, el 35,3% (IC 95%: 28,7 a 42,4] de los ASBC continuaron colaborando voluntariamente en las asociaciones.(AU)

Calidad de vida en el trastorno por déficit de atención con hiperactividad: percepción de padres e hijos / Quality of life in attention deficit hyperactivity disorder: perception of parents and children

INTRODUCCIÓN: la calidad de vida relacionada con la salud (CVRS) es una dimensión relevante en la evaluación y consideración de los efectos de un tratamiento en el trastorno por déficit de atención con hiperactividad (TDAH). El objetivo del estudio es analizar las diferencias entre la percepción de padres e hijos en la CVRS en casos TDAH tratados farmacológicamente (TDAH-T), casos no tratados (TDAH-N) y controles. MATERIAL Y MÉTODOS: muestra de 228 participantes entre 8 y 14 años (114 controles, 57 TDAH-T y 57 TDAH-N). Muestreo consecutivo de TDAH según DSM-IV (ADHD RS-IV) y muestreo aleatorio de controles emparejados por sexo y edad. Evaluación de CVRS mediante las diez dimensiones del KIDSCREEN-52 versión padres y versión hijos. RESULTADOS: en los controles existen diferencias significativas entre padres e hijos en tres de las diez dimensiones del KIDSCREEN-52 (autonomía, autopercepción y recursos económicos), en cuatro dimensiones en TDAH-T (bienestar psicológico, autopercepción, entorno escolar y recursos económicos) y en seis dimensiones en TDAH-N (bienestar psicológico y físico, estado de ánimo, autopercepción, entorno escolar y recursos económicos). En todas las dimensiones donde existen diferencias significativas los hijos perciben mejor CVRS que la atribuida por los padres, excepto en la dimensión económica que sucede a la inversa. No existen diferencias significativas entre padres e hijos en controles, TDAH-N o TDAH-T en las dimensiones de aceptación social, relación con padres y amigos. CONCLUSIONES: es necesario que en la evaluación que precede a cualquier intervención clínica se deban tener en cuenta las perspectivas de padres e hijos sobre la CVRS

INTRODUCTION: health-related quality of life (HRQL) is a relevant dimension in the evaluation and consideration of the effects of a treatment in Attention Deficit Hyperactivity Disorder (ADHD). The objective of the study is to analyze the differences on the perception between parents and children in the HRQL in ADHD cases treated pharmacologically (ADHD-T), untreated cases (ADHD-N) and controls. MATERIAL AND METHODS: sample of 228 participants between 8 and 14 years old (114 controls, 57 ADHD-T and 57 ADHD-N). Consecutive sampling of ADHD according to DSM-IV (ADHD Rating Scales IV) and random sampling of controls matched by sex and age. HRQL assessment using the ten dimensions of the KIDSCREEN-52 parent version and child version. RESULTS: there are significant differences between parents/children in three out of ten dimensions of KIDSCREEN-52 (autonomy, self-perception, and financial resources), in four ADHD-T dimensions (psychological well-being, self-perception, school environment, and financial resources), and in six ADHD-N dimensions (psychological and physical well-being, mood, self-perception, school environment and financial resources) in controls. Children perceive HRQL better than parents in all dimensions with significant differences, except for economic dimension (the opposite). There are no significant differences between parents/children in controls, ADHD-N or ADHD-T in the dimensions of social acceptance, relationship with parents and friends. CONCLUSIONS: it is necessary to take into account the perspectives of parents and children regarding HRQL in the evaluation preceding any clinical intervention

Associations and community health workers: analysis and time trends over ten years of training-action.

OBJECTIVE: To analyse the profile of the persons and associations that participated in the course, quantify peer education activities and analyse their evolution. METHOD: A quantitative study using an analysis of the course records from 2009 to 2018 was designed for this purpose inside mihsalud program designed to promote health amongst persons in vulnerable situations in the city of Valencia (Spain). It offers a yearly training-action course of community health workers (CHW) that is attended by persons who have been proposed by associations. The associations were defined according to their population (immigrant, local or intercultural) and the CHWs according to gender, country of birth, year of course, association and continuity after training. Means and confidence intervals were calculated at 95% and a bivariate analysis was conducted in order to compare the activities that took place in 2009 to 2013 with those of 2014 to 2018. The time trends were analysed by applying linear regression models that included the different years studied as the dependent variable. RESULTS: 201 CHW of 31 nationalities were trained, 81.6% (95% confidence interval [95% CI]: 75.5-86.7] were women. Eighty-two associations participated, 51.2% (95% CI: 39.9-62.4] worked with culturally diverse populations. Participation by associations (p=.017) and CHWs (p=.377) increased in a statistically significant manner over the years. After the course, 35.3% (95% CI: 28.7-42.4] of the CHWs continued to collaborate voluntarily in the associations. CONCLUSIONS: The results of the CHW training-action course improve over time given that a significant increase in participation by associations and women can be seen, along with a greater number of activities completed during the training. One effect of this is that CHWs are contracted or carry out voluntary activities in the associations.

Critically ill COVID-19 patients attended by anesthesiologists in northwestern Spain: a multicenter prospective observational study. / Pacientes críticos COVID-19 atendidos por anestesiólogos en el Noroeste de España: estudio multicéntrico, prospectivo, observacional.

BACKGROUND AND OBJECTIVES: There are limited information on outcome, complications and treatments of critically ill COVID-19 patients requiring admission to an intensive care unit (ICU). The aim of this study is to describe the clinical ICU course, treatments used, complications and outcomes, of critically ill COVID-19 patients admitted in seven ICU in Galicia region during the 2020 March-April pandemic peak. METHODS: Between March 21 and April 19, 2020, we evaluated critically ill COVID-19 patients admitted to the ICU of Anesthesia of seven hospitals in Galicia, northwestern Spain. Outcome, complications, and treatments were monitored until May 6, 2020, the final date of follow-up. RESULTS: A total of 97 critically ill COVID-19 patients were included. During ICU stay, mechanical ventilation became necessary in 80 (82.5%) patients, and tracheostomy in 22 (22.7%) patients. Prone position was used frequently in both intubated (67.5%) and awake (27.8%) patients. Medications consisted of antivirals agents (92.7%), corticosteroids (93.8%), tocilizumab (57.7%), and intermediate or high doses of anticoagulants (83.5%). The most frequent complications were ICU-acquired infection (52.6%), thrombosis events (16.5%), and reintubation (9.3%). After a median follow-up of 42 (34-45) days, 15 patients (15.5%) deceased, 73 patients (75.2%) had been discharged from ICU, and nine patients (9.3%) were still in the ICU. CONCLUSIONS: A high proportion of our critically ill COVID-19 patients required mechanical ventilation, prone positioning, antiviral medication, corticosteroids, and anticoagulants. ICU complications were frequent, mainly infections and thrombotic events. We had a relatively low mortality of 15,5%.

Postoperative lactate elevation as a marker of underlying acute mesenteric ischemia. Description of two cases. / Elevación del lactato en el postoperatorio como marcador de isquemia mesentérica aguda. Descripción de dos casos.

Serum lactate is a non-specific marker of tissue hypoperfusion. Elevated serum lactate is used in the differential diagnosis of acute intestinal ischemia. Although this practice is controversial, in the absence of other validated markers lactate is still used because of its high sensitivity. We present the cases of two patients who developed acute mesenteric ischemia as a post-surgical complication. The patients reported moderate abdominal pain -a non-specific symptom in the postoperative context- and tests showed progressively increasing serum lactate levels, which facilitated suspicion and subsequent diagnostic confirmation through an imaging test. These cases highlight the physiopathological importance of lactate elevation in the perioperative context and of performing a differential diagnosis of its possible causes, including mesenteric ischemia. Although the outcome was negative in the first case, early suspicion allowed us to make an effective diagnosis and administer appropriate treatment in the second patient.

Nuclear response to divergent mitochondrial DNA genotypes modulates the interferon immune response.

Mitochondrial OXPHOS generates most of the energy required for cellular function. OXPHOS biogenesis requires the coordinated expression of the nuclear and mitochondrial genomes. This represents a unique challenge that highlights the importance of nuclear-mitochondrial genetic communication to cellular function. Here we investigated the transcriptomic and functional consequences of nuclear-mitochondrial genetic divergence in vitro and in vivo. We utilized xenomitochondrial cybrid cell lines containing nuclear DNA from the common laboratory mouse Mus musculus domesticus and mitochondrial DNA (mtDNA) from Mus musculus domesticus, or exogenous mtDNA from progressively divergent mouse species Mus spretus, Mus terricolor, Mus caroli and Mus pahari. These cybrids model a wide range of nuclear-mitochondrial genetic divergence that cannot be achieved with other research models. Furthermore, we used a xenomitochondrial mouse model generated in our laboratory that harbors wild-type, C57BL/6J Mus musculus domesticus nuclear DNA and homoplasmic mtDNA from Mus terricolor. RNA sequencing analysis of xenomitochondrial cybrids revealed an activation of interferon signaling pathways even in the absence of OXPHOS dysfunction or immune challenge. In contrast, xenomitochondrial mice displayed lower baseline interferon gene expression and an impairment in the interferon-dependent innate immune response upon immune challenge with herpes simplex virus, which resulted in decreased viral control. Our work demonstrates that nuclear-mitochondrial genetic divergence caused by the introduction of exogenous mtDNA can modulate the interferon immune response both in vitro and in vivo, even when OXPHOS function is not compromised. This work may lead to future insights into the role of mitochondrial genetic variation and the immune function in humans, as patients affected by mitochondrial disease are known to be more susceptible to immune challenges.

Methylation of Ribosomal RNA: A Mitochondrial Perspective.

Ribosomal RNA (rRNA) from all organisms undergoes post-transcriptional modifications that increase the diversity of its composition and activity. In mitochondria, specialized mitochondrial ribosomes (mitoribosomes) are responsible for the synthesis of 13 oxidative phosphorylation proteins encoded by the mitochondrial genome. Mitoribosomal RNA is also modified, with 10 modifications thus far identified and all corresponding modifying enzymes described. This form of epigenetic regulation of mitochondrial gene expression affects mitoribosome biogenesis and function. Here, we provide an overview on rRNA methylation and highlight critical work that is beginning to elucidate its role in mitochondrial gene expression. Given the similarities between bacterial and mitochondrial ribosomes, we focus on studies involving Escherichia coli and human models. Furthermore, we highlight the use of state-of-the-art technologies, such as cryoEM in the study of rRNA methylation and its biological relevance. Understanding the mechanisms and functional relevance of this process represents an exciting frontier in the RNA biology and mitochondrial fields.

OXPHOS bioenergetic compensation does not explain disease penetrance in Leber hereditary optic neuropathy.

Leber hereditary optic neuropathy (LHON) is one of the most common primary mitochondrial diseases. It is caused by point mutations in mitochondrial DNA (mtDNA) genes and in some cases, it can result in irreversible vision loss, primarily in young men. It is currently unknown why LHON mutations affect only some carriers and whether bioenergetic compensation enables unaffected carriers to overcome mitochondrial impairment and preserve cellular function. Here, we conducted bioenergetic metabolic assays and RNA sequencing to address this question using male-only, age-matched, m.11778G > A lymphoblasts and primary fibroblasts from both unaffected carriers and affected individuals. Our work indicates that OXPHOS bioenergetic compensation in LHON peripheral cells does not explain disease phenotype. We show that complex I impairment is similar in cells from unaffected carrier and affected patients, despite a transcriptional downregulation of metabolic pathways including glycolysis in affected cells relative to carriers detected by RNA sequencing. Although we did not detect OXPHOS bioenergetic compensation in carrier cells under basal conditions, our results indicate that cells from affected patients suffer a growth impairment under metabolic challenge compared to carrier cells, which were unaffected by metabolic challenge. If recapitulated in retinal ganglion cells, decreased susceptibility to metabolic challenge in unaffected carriers may help preserve metabolic homeostasis in the face of the mitochondrial complex I bioenergetic defect.

Pioglitazone and Deoxyribonucleoside Combination Treatment Increases Mitochondrial Respiratory Capacity in m.3243A>G MELAS Cybrid Cells.

The lack of effective treatments for mitochondrial disease has seen the development of new approaches, including those that aim to stimulate mitochondrial biogenesis to boost ATP generation above a critical disease threshold. Here, we examine the effects of the peroxisome proliferator-activated receptor γ (PPARγ) activator pioglitazone (PioG), in combination with deoxyribonucleosides (dNs), on mitochondrial biogenesis in cybrid cells containing >90% of the m.3243A>G mutation associated with mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS). PioG + dNs combination treatment increased mtDNA copy number and mitochondrial mass in both control (CON) and m.3243A>G (MUT) cybrids, with no adverse effects on cell proliferation. PioG + dNs also increased mtDNA-encoded transcripts in CON cybrids, but had the opposite effect in MUT cybrids, reducing the already elevated transcript levels. Steady-state levels of mature oxidative phosphorylation (OXPHOS) protein complexes were increased by PioG + dNs treatment in CON cybrids, but were unchanged in MUT cybrids. However, treatment was able to significantly increase maximal mitochondrial oxygen consumption rates and cell respiratory control ratios in both CON and MUT cybrids. Overall, these findings highlight the ability of PioG + dNs to improve mitochondrial respiratory function in cybrid cells containing the m.3243A>G MELAS mutation, as well as their potential for development into novel therapies to treat mitochondrial disease.

Correction to: Amyloid Precursor Protein Mediates Neuronal Protection from Rotenone Toxicity.

The original version of this article unfortunately contained a mistake in the author name. The family name of Dr. Vanessa A. Johannsen should be written as "Johanssen."