A Sensitive,Reproducible and Objective Immunofluorescence Analysis Method of Dystrophin in Individual Fibers in Samples from Patients with Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is characterized by the absence or reduced levels of dystrophin expression on the inner surface of the sarcolemmal membrane of muscle fibers. Clinical development of therapeutic approaches aiming to increase dystrophin levels requires sensitive and reproducible measurement of differences in dystrophin expression in muscle biopsies of treated patients with DMD. This, however, poses a technical challenge due to intra- and inter-donor variance in the occurrence of revertant fibers and low trace dystrophin expression throughout the biopsies. We have developed an immunofluorescence and semi-automated image analysis method that measures the sarcolemmal dystrophin intensity per individual fiber for the entire fiber population in a muscle biopsy. Cross-sections of muscle co-stained for dystrophin and spectrin have been imaged by confocal microscopy, and image analysis was performed using Definiens software. Dystrophin intensity has been measured in the sarcolemmal mask of spectrin for each individual muscle fiber and multiple membrane intensity parameters (mean, maximum, quantiles per fiber) were calculated. A histogram can depict the distribution of dystrophin intensities for the fiber population in the biopsy. This method was tested by measuring dystrophin in DMD, Becker muscular dystrophy, and healthy muscle samples. Analysis of duplicate or quadruplicate sections of DMD biopsies on the same or multiple days, by different operators, or using different antibodies, was shown to be objective and reproducible (inter-assay precision, CV 2–17% and intra-assay precision, CV 2–10%). Moreover, the method was sufficiently sensitive to detect consistently small differences in dystrophin between two biopsies from a patient with DMD before and after treatment with an investigational compound.     

Establishing irremediable psychiatric suffering in the context of medical assistance in dying in the Netherlands: a qualitative study

Background:Establishing irremediability of suffering is a central challenge in determining the appropriateness of medical assistance in dying (MAiD) for patients with a psychiatric disorder. We sought to evaluate how experienced psychiatrists define irremediable psychiatric suffering in the context of MAiD and what challenges they face while establishing irremediable psychiatric suffering.Methods:We conducted a qualitative study of psychiatrists in the Netherlands with experience assessing irremediable psychiatric suffering in the context of MAiD. We collected data from in-depth, semistructured interviews focused on the definition of irremediable psychiatric suffering and on the challenges in establishing irremediability. We analyzed themes using a modified grounded theory approach.Results:The study included 11 psychiatrists. Although irremediable psychiatric suffering is a prospective concept, most participants relied on retrospective dimensions to define it, such as a history of failed treatments, and expressed that uncertainty was inevitable in this process. When establishing irremediable psychiatric suffering, participants identified challenges related to diagnosis and treatment. The main diagnostic challenge identified was the frequent co-occurrence of more than 1 psychiatric diagnosis. Important challenges related to treatment included assessing the quality of past treatments, establishing when limits of treatment had been reached and managing “treatment fatigue.”Interpretation:Challenges regarding the definition, diagnosis and treatment of irremediable psychiatric suffering complicate the process of establishing it in the context of MAiD. Development of consensus clinical criteria for irremediable psychiatric suffering in this context and further research to understand “treatment fatigue” among patients with psychiatric disorders may help address these challenges. Registration: This study was preregistered under osf.io/2jrnd.

Medical assistance in dying (MAiD), also known as physician-assisted death, has been legalized in an increasing number of jurisdictions around the world.¹ In 2023, Canada will join a small group of countries that allow MAiD for people with mental illness, more commonly referred to as persons with a psychiatric disorder. ² In the Netherlands, MAiD for irremediable psychiatric suffering has been approved by jurisprudence since the 1990s and it has been regulated by law since 2002. The last decade has seen a marked increase in MAiD for irremediable psychiatric suffering; in 2020, MAiD was performed 88 times for psychiatric suffering (1.3% of all MAiD cases), compared with just 2 instances in 2010.³ The number of requests by patients with a psychiatric disorder is much higher, but 90% of requests do not end in MAiD. Sometimes, they are retracted by patients, but most are denied by psychiatrists.⁴The main legal requirements for MAiD in the Netherlands are that the patient must be able to make a competent request, that the patient’s suffering must be unbearable and irremediable, and that the patient and physician agree that there are no other reasonable treatment options. The process for MAiD requires an assessment by an independent physician and, in the case of psychiatric suffering, a third assessment by an independent psychiatrist, preferably one with specific expertise regarding the patient’s disorder.⁵ The Canadian legal requirements under the amended Bill C-7, which will come into effect in 2023, will be largely similar to the Dutch requirements.⁶ However, Canadian legislation, which does not yet permit MAiD for mental illness, has 1 important difference: patients with decision-making capacity can qualify for MAiD if they refuse treatments that they do not find acceptable. It is as yet unknown whether this approach to refusal of treatment will also be applied to MAiD where mental illness is the sole underlying condition.Although there are concerns about decision-making capacity, the central dilemma of MAiD for patients with a psychiatric disorder appears to revolve around applying the concept of irremediability to psychiatric disorders. The 2018 guideline by the Dutch Psychiatry Association defines irremediable psychiatric suffering in the context of MAiD as follows: “irremediability means that there is no longer any prospect of alleviating, mitigating, enduring or removing suffering. There is no longer a reasonable treatment perspective.”⁷ Elsewhere, the guideline states that reasonable treatment perspective means that “there is a prospect of improvement with adequate treatment, within a foreseeable period, and with a reasonable ratio between the expected results and the burden of the treatment for the patient.”⁷A recent scoping review identified a multitude of conceptual articles addressing irremediability in the context of psychiatric MAiD but few empirical studies.¹ This suggests that the assessment of irremediable psychiatric suffering is particularly difficult relative to suffering arising from other types of conditions.Surveys estimate that 46% of psychiatrists in the Netherlands have received an explicit MAiD request at least once in their career, and 4% actually assisted in the death of a patient with a psychiatric disorder.⁸ The experiences of psychiatrists who have handled MAiD requests can be seen as an important source of knowledge about the challenges of establishing irremediable psychiatric suffering in practice. The aim of this study was to learn how experienced psychiatrists define psychiatric suffering as irremediable in the context of a MAiD request and what challenges they face while establishing irremediable psychiatric suffering.     

Analyses of factors influencing the birth weight and neonatal growth rate of Cheetah (Acinonyx jubatus) cubs

Birth weight and the neonatal growth rate are reliable indicators of neonatal survival prospects. Data on weight at birth and consecutive weights until 40 days of age were recorded for cheetah cubs in 16 litters. Growth was found to be linear during the first 40 days of life. Weight data were used to evaluate the influence of several factors on birth weight and neonatal growth. The factors used in these analyses were sex, litter identity, litter size, average litter size over the first 40 days, birth weight, parents, gestation length, parity of the dam, and inbreeding. For birth weight and neonatal growth, litter identity was the major explanatory factor (81.8 and 85.3%). For birth weight, a significant influence of gestation length was found (p < 0.05), whereas inbreeding coefficient tended to decrease the birth weight (p = 0.09). Together, gestation length and inbreeding coefficient account for 57.5% of the between‐litter variation for birth weight. Factors with significant influences on neonatal growth are gestation length and parity (p < 0.05). The average litter size over the first 40 days tended to influence neonatal growth (p = 0.07). These three variables together account for 99.9% of the between‐litter variation for neonatal growth during the first 40 days of life. A comparison of neonatal growth between mother‐raised and hand‐raised cubs revealed a lower growth rate in hand‐raised cubs (45 vs. 27 g/day). Zoo Biol 18:129–139, 1999. © 1999 Wiley‐Liss, Inc.     

Worker reproductive parasitism in naturally orphaned colonies of the Asian red dwarf honey bee, Apis florea

The truce between honey bee (Apis spp.) workers over reproduction is broken in the absence of their queen. Queenright workers generally abstain from personal reproduction, raising only the queen’s offspring. Queenless workers activate their ovaries, produce eggs, and reduce the rate at which they destroy worker-laid eggs, so that some eggs are reared to maturity. Reduced policing of worker-laid eggs renders queenless nests vulnerable to worker reproductive parasitism (WRP), and may result in the colony raising eggs of unrelated (non-natal) workers that parasitize it. Queenless colonies of A. florea are heavily parasitized with the eggs of non-natal workers. However, queenless colonies often abscond upon disturbance and build a small comb in which to rear their own male offspring. We investigated three naturally occurring orphaned colonies to determine if they are also parasitized. We show that WRP is present in orphaned colonies, and non-natal workers have significantly higher rates of ovary activation than natal workers. In contrast to experimentally manipulated colonies, in our samples, natal and non-natal workers had statistically equal reproductive success, but this may have been due to the small number of non-natals present.     

Effect of nitrogen source and concentration to produce proteins in mass cultures of the microalgae <i>Chaetoceros muelleri</i>

Proteins are one of the major metabolites in biomass from microalgae that constitute the diet of marine organisms grown in aquaculture, and are essential for their growth. The quantity of this component is influenced by nutrients, temperature and light intensity, among others. We examined the growth, biomass production and protein of Chaetoceros muelleri with two sources of nitrogen (nitrate and urea) at three concentrations, using the medium f/2 (0.88 mol/L) (nitrates) as control. The treatments were the medium 2f (3.53 mol/L) and 4f (7.05 mol/L) with NO_3^-, and the medium f/2 (0.88 mol/L), 2f (3.53mol/L) and 4f (7.05 mol/L) with urea. In general, the productive parameters were greater using urea than nitrate in the media. Higher cell concentrations (2.83 x 10⁶ cell/mL), average and cumulative growth rates (1.50 div/day and 6.01 divisions), dry weight (0.0044 g/L), and proportion of proteins (23.74%) were found when urea was used as the N source. However, most of the bands on the electrophoretic profile were present in the mediums with NO_3^- (~6.5 to 90 kDa).     

The GDS-8; a short, client- and user-friendly shortened version of the Geriatric Depression Scale for nursing homes

The objective of this study was to construct a patient- and user-friendly shortened version of the Geriatric Depression Scale (GDS) that is especially suitable for nursing home patients. The study was carried out on two different data bases including 23 Dutch nursing homes. Data on the GDS (n=410), the Mini Mental State Examination (n=410) and a diagnostic interview (SCAN; n=333), were collected by trained clinicians. Firstly, the items of the GDS-15 were judged on their clinical applicability by three clinical experts. Subsequently, seven items that were identified as unsuitable were removed using the GDS-data of the Assess-project (n=77), and internal consistency was calculated. Secondly, with respect to criterion validity (sensitivity, specificity, area under ROC and positive and negative predictive values), the newly constructed 8-item version of the GDS was validated in the AGED data set (n=333), using DSM-IV diagnosis for depression as measured by the SCAN as 'gold standard'. In the AGED dataset, the GDS-8 was internally consistent (alpha=.80) and high sensitivity rates of 96.3% for major depression and 83.0% for minor depression were found, with a specificity rate of 71.7% at a cut-off point of 2/3. The GDS-8 has good psychometric properties. Given that the GDS-8 is less burdening for the patient, more comfortable to use and less time consuming, it may be a more feasible screening test for the frail nursing home population.     

HIBCH mutations can cause Leigh-like disease with combined deficiency of multiple mitochondrial respiratory chain enzymes and pyruvate dehydrogenase

Background

Deficiency of 3-hydroxy-isobutyryl-CoA hydrolase (HIBCH) caused by HIBCH mutations is a rare cerebral organic aciduria caused by disturbance of valine catabolism. Multiple mitochondrial respiratory chain (RC) enzyme deficiencies can arise from a number of mechanisms, including defective maintenance or expression of mitochondrial DNA. Impaired biosynthesis of iron-sulphur clusters and lipoic acid can lead to pyruvate dehydrogenase complex (PDHc) deficiency in addition to multiple RC deficiencies, known as the multiple mitochondrial dysfunctions syndrome.

Methods

Two brothers born to distantly related Pakistani parents presenting in early infancy with a progressive neurodegenerative disorder, associated with basal ganglia changes on brain magnetic resonance imaging, were investigated for suspected Leigh-like mitochondrial disease. The index case had deficiencies of multiple RC enzymes and PDHc in skeletal muscle and fibroblasts respectively, but these were normal in his younger brother. The observation of persistently elevated hydroxy-C4-carnitine levels in the younger brother led to suspicion of HIBCH deficiency, which was investigated by biochemical assay in cultured skin fibroblasts and molecular genetic analysis.

Results

Specific spectrophotometric enzyme assay revealed HIBCH activity to be below detectable limits in cultured skin fibroblasts from both brothers. Direct Sanger sequence analysis demonstrated a novel homozygous pathogenic missense mutation c.950G <A; p.Gly317Glu in the HIBCH gene, which segregated with infantile-onset neurodegeneration within the family.

Conclusions

HIBCH deficiency, a disorder of valine catabolism, is a novel cause of the multiple mitochondrial dysfunctions syndrome, and should be considered in the differential diagnosis of patients presenting with multiple RC deficiencies and/or pyruvate dehydrogenase deficiency.

     

IL7R gene expression network associates with human healthy ageing

Background

The level of expression of the interleukin 7 receptor (IL7R) gene in blood has recently been found to be associated with familial longevity and healthy ageing. IL7R is crucial for T cell development and important for immune competence. To further investigate the IL7R pathway in ageing, we identified the closest interacting genes to construct an IL7R gene network that consisted of IL7R and six interacting genes: IL2RG, IL7, TSLP, CRLF2, JAK1 and JAK3. This network was explored for association with chronological age, familial longevity and immune-related diseases (type 2 diabetes, chronic obstructive pulmonary disease and rheumatoid arthritis) in 87 nonagenarians, 337 of their middle-aged offspring and 321 middle-aged controls from the Leiden Longevity Study (LLS).

Results

We observed that expression levels within the IL7R gene network were significantly different between the nonagenarians and middle-aged controls (P?=?4.6 × 10^?4), being driven by significantly lower levels of expression in the elderly of IL7, IL2RG and IL7R. After adjustment for multiple testing and white blood cell composition and in comparison with similarly aged controls, middle-aged offspring of nonagenarian siblings exhibit a lower expression level of IL7R only (P?=?0.006). Higher IL7R gene expression in the combined group of middle-aged offspring and controls is associated with a higher prevalence of immune-related disease (P?=?0.001). On the one hand, our results indicate that lower IL7R expression levels, as exhibited by the members of long-lived families that can be considered as ‘healthy agers’, are beneficial in middle age. This is augmented by the observation that higher IL7R gene expression associates with immune-related disease. On the other hand, IL7R gene expression in blood is lower in older individuals, indicating that low IL7R gene expression might associate with reduced health. Interestingly, this contradictory result is supported by the observation that a higher IL7R gene expression level is associated with better prospective survival, both in the nonagenarians (Hazard ratio (HR)?=?0.63, P?=?0.037) and the middle-aged individuals (HR?=?0.33, P?=?1.9 × 10^–4).

Conclusions

Overall, we conclude that the IL7R network reflected by gene expression levels in blood may be involved in the rate of ageing and health status of elderly individuals.

     

Reproductive interference between honeybee species in artificial sympatry

Reproductive isolation between closely related species is often incomplete. The Western honeybee, Apis mellifera, and the Eastern hive bee, Apis cerana, have been allopatric for millions of years, but are nonetheless similar in morphology and behaviour. During the last century, the two species were brought into contact anthropogenically, providing potential opportunities for interspecific matings. Hybrids between A. mellifera and A. cerana are inviable, so natural interspecific matings are of concern because they may reduce the viability of A. cerana and A. mellifera populations – two of the world's most important pollinators. We examined the mating behaviour of A. mellifera and A. cerana queens and drones from Caoba Basin, China and Cairns, Australia. Drone mating flight times overlap in both areas. Analysis of the spermathecal contents of queens with species‐specific genetic markers indicated that in Caoba Basin, 14% of A. mellifera queens mated with at least one A. cerana male, but we detected no A. cerana queens that had mated with A. mellifera males. Similarly, in Cairns, no A. cerana queens carried A. mellifera sperm, but one‐third of A. mellifera queens had mated with at least one A. cerana male. No hybrid embryos were detected in eggs laid by interspecifically mated A. mellifera queens in either location. However, A. mellifera queens artificially inseminated with A. cerana sperm produced inviable hybrid eggs or unfertilized drones. This suggests that reproductive interference will impact the viability of honeybee populations wherever A. cerana and A. mellifera are in contact.