Detecting material state changes in the nucleolus by label-free digital holographic microscopy.

Ribosome biogenesis is initiated in the nucleolus, a multiphase biomolecular condensate formed by liquid-liquid phase separation. The nucleolus is a powerful disease biomarker and stress biosensor whose morphology reflects function. Here we have used digital holographic microscopy (DHM), a label-free quantitative phase contrast microscopy technique, to detect nucleoli in adherent and suspension human cells. We trained convolutional neural networks to detect and quantify nucleoli automatically on DHM images. Holograms containing cell optical thickness information allowed us to define a novel index which we used to distinguish nucleoli whose material state had been modulated optogenetically by blue-light-induced protein aggregation. Nucleoli whose function had been impacted by drug treatment or depletion of ribosomal proteins could also be distinguished. We explored the potential of the technology to detect other natural and pathological condensates, such as those formed upon overexpression of a mutant form of huntingtin, ataxin-3, or TDP-43, and also other cell assemblies (lipid droplets). We conclude that DHM is a powerful tool for quantitatively characterizing nucleoli and other cell assemblies, including their material state, without any staining.

Somatic copy number gains of α-synuclein (SNCA) in Parkinson's disease and multiple system atrophy brains.

The α-synuclein protein, encoded by SNCA, has a key role in the pathogenesis of Parkinson's disease and other synucleinopathies. Although usually sporadic, Parkinson's disease can result from inherited copy number variants in SNCA and other genes. We have hypothesized a role of somatic SNCA mutations, leading to mosaicism, in sporadic synucleinopathies. The evidence for mosaicism in healthy and diseased brain is increasing rapidly, with somatic copy number gains of APP reported in Alzheimer's brain. Here we demonstrate somatic SNCA copy number gains in synucleinopathies (Parkinson's disease and multiple system atrophy), focusing on substantia nigra. We selected sporadic cases with relatively young onset or short disease duration, and first excluded high level copy number variant mosaicism by DNA analysis using digital PCR for SNCA, and/or customized array comparative genomic hybridization. To detect low level SNCA copy number variant mosaicism, we used fluorescent in situ hybridization with oligonucleotide custom-designed probes for SNCA, validated on brain and fibroblasts with known copy number variants. We determined SNCA copy number in nigral dopaminergic neurons and other cells in frozen nigra sections from 40 cases with Parkinson's disease and five with multiple system atrophy, and 25 controls, in a blinded fashion. Parkinson's disease cases were significantly more likely than controls to have any SNCA gains in dopaminergic neurons (P = 0.0036), and overall (P = 0.0052). The average proportion of dopaminergic neurons with gains in each nigra was significantly higher in Parkinson's disease than controls (0.78% versus 0.45%; P = 0.017). There was a negative correlation between the proportion of dopaminergic neurons with gains and onset age in Parkinson's disease (P = 0.013), but not with disease duration, or age of death in cases or controls. Cases with tremor at onset were less likely to have gains (P = 0.035). All multiple system atrophy cases had gains, and the highest levels in dopaminergic neurons were in two of these cases (2.76%, 2.48%). We performed selective validation with different probes after dye swapping. All three control probes used showed minimal or no gains (≤0.1% in dopaminergic neurons). We also found occasional SNCA gains in frontal neurons of cases with Parkinson's disease, and the putamen of one multiple system atrophy case. We present evidence of somatic SNCA gains in brain, more commonly in nigral dopaminergic neurons of Parkinson's disease than controls, negatively correlated with onset age, and possibly commonest in some multiple system atrophy cases. Somatic SNCA gains may be a risk factor for sporadic synucleinopathies, or a result of the disease process.10.1093/brain/awy157_video1awy157media15813519976001.

DNA isolation protocol effects on nuclear DNA analysis by microarrays, droplet digital PCR, and whole genome sequencing, and on mitochondrial DNA copy number estimation.

Potential bias introduced during DNA isolation is inadequately explored, although it could have significant impact on downstream analysis. To investigate this in human brain, we isolated DNA from cerebellum and frontal cortex using spin columns under different conditions, and salting-out. We first analysed DNA using array CGH, which revealed a striking wave pattern suggesting primarily GC-rich cerebellar losses, even against matched frontal cortex DNA, with a similar pattern on a SNP array. The aCGH changes varied with the isolation protocol. Droplet digital PCR of two genes also showed protocol-dependent losses. Whole genome sequencing showed GC-dependent variation in coverage with spin column isolation from cerebellum. We also extracted and sequenced DNA from substantia nigra using salting-out and phenol / chloroform. The mtDNA copy number, assessed by reads mapping to the mitochondrial genome, was higher in substantia nigra when using phenol / chloroform. We thus provide evidence for significant method-dependent bias in DNA isolation from human brain, as reported in rat tissues. This may contribute to array "waves", and could affect copy number determination, particularly if mosaicism is being sought, and sequencing coverage. Variations in isolation protocol may also affect apparent mtDNA abundance.

Activation of the maternal caregiving system by childhood fever--a qualitative study of the experiences made by mothers with a German or a Turkish background in the care of their children.

BACKGROUND: Childhood fever represents a frequent cause to consult a primary care physician. "Fever phobia" describes a fearful and irrational view of fever shared by many parents with different cultural backgrounds. The study aims to explain the experiences of mothers of children having a fever and to analyze the role of the mothers' cultural background with regard to their experiences by comparing the accounts of mothers with a German with those from a Turkish background. Disease and context specific knowledge about the influence of culture can be important for effective counselling. METHODS: We applied a qualitative approach using in-depth interviews with 11 mothers with a Turkish and 9 with a German background living in Germany. The interviews were conducted at the participants' homes from May to October 2008. Data was audio-recorded and transcribed verbatim. Grounded Theory was used as a framing methodology including open, axial and selective coding. Analysis was performed in a group with members of different professional and cultural backgrounds. RESULTS: Mothers experienced their child's fever not merely as elevated temperature but as a potentially dangerous event. A deeply rooted urge to protect the child from harm was central to all participants' experience. The caregiving system model offers a good theoretical foundation to explain the findings as it incorporates the unique relational quality of care giving mothers to their children. The cultural background represents an important context variable influencing the explanatory models and strategies of dealing with fever. The identified culturally influenced concepts sometimes match and sometimes conflict with medical knowledge. CONCLUSION: By applying the caregiving system model which is a part of attachment theory (Bowlby) maternal actions can be understood as an understandable attempt to protect the child from harm. The mothers' decisions what to do when a child has a fever can be culturally influenced. This may lead either to a frequent use of services or to an underestimation of the child's state of health. The mothers' caring role and emotional state should be acknowledged; her concerns, explanatory models and strategies should be elicited and taken seriously in order to maintain a trustful relationship, provide effective counselling and thereby insuring optimal care for the children.

Fearful or functional--a cross-sectional survey of the concepts of childhood fever among German and Turkish mothers in Germany.

BACKGROUND: Fever is one of the most common presenting complaints in paediatrics and general practice. In the majority of cases nothing harmful is diagnosed. However, the subjective meaning of fever often varies between doctors and parents. Knowledge of the parents' concept of fever may help tailor counselling to their needs.In this study we determine 1) the influence of socio-economic status and cultural background on two concepts of fever which we labelled "functional" and "fearful", each representing typical experiences of mothers, and 2) the actions taken by the mothers related to these concepts. METHODS: A standardized interview study was conducted among German and Turkish mothers in Germany in 2009. The questionnaire consisted of 36 questions and 205 items. Interviews were conducted in 16 private practices of paediatricians and 2 paediatric emergency departments in an urban region of Germany. The two fever concepts were represented in 6 statements that could be rated with a six-point Likert scale. The association of the socio-economic status and the cultural background with one of the fever concepts was determined by a multiple logistic regression. RESULTS: A total of 338 mothers (49% with a Turkish background) completed the interview (response rate 92%). The average age of mothers with a German background was higher (34.1 years vs. 32.0 years, p=0.0001). Mothers with a Turkish background were more likely to relate to the concept "fearful" [adjusted Odds Ratio (AOR) 1.99; confidence interval (CI) 1.16-3.44]. Mothers with a middle or high socio-economic status were more likely to respond to the concept "functional" [middle: AOR, 0.53; CI, 0.30-0.92; high: AOR, 0.44; CI, 0.21-0.95].Mothers adhering to the concept "fearful" more often gave acetaminophen before the recommended interval of 6 hours (46.8% vs. 31.3%, p=0.005) and visited out-of-hours services more frequently in the preceding 9 months than the other group (0.7 vs. 0.4, p=0.001). CONCLUSIONS: A Turkish migrant background and a low socio-economic status are associated with the fever concept "fearful". Mothers with these attributes seem to require specific and reassuring counselling as they use antipyretic drugs extensively and out-of-hours services frequently.