Native extracellular matrix probes to target patient- and tissue-specific cell-microenvironment interactions by force spectroscopy.

Atomic Force Microscopy (AFM) is successfully used for the quantitative investigation of the cellular mechanosensing of the microenvironment. To this purpose, several force spectroscopy approaches aim at measuring the adhesive forces between two living cells and also between a cell and an appropriate reproduction of the extracellular matrix (ECM), typically exploiting tips suitably functionalised with single components (e.g. collagen, fibronectin) of the ECM. However, these probes only poorly reproduce the complexity of the native cellular microenvironment and consequently of the biological interactions. We developed a novel approach to produce AFM probes that faithfully retain the structural and biochemical complexity of the ECM; this was achieved by attaching to an AFM cantilever a micrometric slice of native decellularised ECM, which was cut by laser microdissection. We demonstrate that these probes preserve the morphological, mechanical, and chemical heterogeneity of the ECM. Native ECM probes can be used in force spectroscopy experiments aimed at targeting cell-microenvironment interactions. Here, we demonstrate the feasibility of dissecting mechanotransductive cell-ECM interactions in the 10 pN range. As proof-of-principle, we tested a rat bladder ECM probe against the AY-27 rat bladder cancer cell line. On the one hand, we obtained reproducible results using different probes derived from the same ECM regions; on the other hand, we detected differences in the adhesion patterns of distinct bladder ECM regions (submucosa, detrusor, and adventitia), in line with the disparities in composition and biophysical properties of these ECM regions. Our results demonstrate that native ECM probes, produced from patient-specific regions of organs and tissues, can be used to investigate cell-microenvironment interactions and early mechanotransductive processes by force spectroscopy. This opens new possibilities in the field of personalised medicine.

Adhesion force spectroscopy with nanostructured colloidal probes reveals nanotopography-dependent early mechanotransductive interactions at the cell membrane level.

Mechanosensing, the ability of cells to perceive and interpret the microenvironmental biophysical cues (such as the nanotopography), impacts strongly cellular behaviour through mechanotransductive processes and signalling. These events are predominantly mediated by integrins, the principal cellular adhesion receptors located at the cell/extracellular matrix (ECM) interface. Because of the typical piconewton force range and nanometre length scale of mechanotransductive interactions, achieving a detailed understanding of the spatiotemporal dynamics occurring at the cell/microenvironment interface is challenging; sophisticated interdisciplinary methodologies are required. Moreover, an accurate control over the nanotopographical features of the microenvironment is essential, in order to systematically investigate and precisely assess the influence of the different nanotopographical motifs on the mechanotransductive process. In this framework, we were able to study and quantify the impact of microenvironmental nanotopography on early cellular adhesion events by means of adhesion force spectroscopy based on innovative colloidal probes mimicking the nanotopography of natural ECMs. These probes provided the opportunity to detect nanotopography-specific modulations of the molecular clutch force loading dynamics and integrin clustering at the level of single binding events, in the critical time window of nascent adhesion formation. Following this approach, we found that the nanotopographical features are responsible for an excessive force loading in single adhesion sites after 20-60 s of interaction, causing a drop in the number of adhesion sites. However, by manganese treatment we demonstrated that the availability of activated integrins is a critical regulatory factor for these nanotopography-dependent dynamics.

[COVID-19 pandemic: structured expansion of ventilation capacities using home respirators]. / COVID-19-Pandemie: strukturierte Erweiterung von Beatmungskapazitäten mithilfe von Heimrespiratoren.

Due to the current COVID-19 pandemic there is a need for a rapid increase in intensive care and ventilation capacities. Delivery times for additional intensive care respirators are currently not foreseeable. An option to increase ventilation capacities not only for COVID-19, but for all patients requiring mechanical ventilation is to use home respirators. Home respirators are turbine respirators, so they can usually be operated without high-pressure oxygen connections and can therefore also be used in areas outside the classical intensive care medical infrastructure. Due to their limited technical features, home respirators are not suitable for the treatment of severely affected patients but can be used for weaning after respiratory improvement, which means that intensive care respirators are available again more quickly. Respiratory therapists are specially trained nurses or therapists in the field of out of hospital ventilation and can independently use home ventilation respirators, for example for weaning in the intensive care unit. Thus, they relieve intensive care nursing staff in the pandemic. Due to the COVID-19 pandemic medical students from the Oldenburg University are currently being trained in operating home respirators to provide basic support in the hospital if necessary.

Patient's perception: shorter and more severe prodromal phase in GBA-associated PD.

BACKGROUND: Prevalence and time of occurrence of prodromal symptoms of Parkinson's disease (PD) in relation to the onset of classical motor manifestation varies between patients. Possible modifying factors might be different genetic architectures predisposing to varying burden of manifestations. OBJECTIVES: To characterize the prodromal phase in PD patients with heterozygous mutations in the GBA gene compared to PD patients without GBA mutation. METHODS: In a retrospective design, 151 participants [47 PD patients carrying a GBA mutation (PDGBA ), 52 idiopathic PD patients (PDidiopathic ), 52 healthy elderly (CON)] underwent a validated structured interview designed to assess prevalence and time of occurrence of prodromal symptoms. RESULTS: PDGBA showed a higher prevalence of prodromal symptoms and almost simultaneous occurrence of non-motor and early motor symptoms shortly before PD diagnosis whereas PDidiopathic reported a longer prodromal phase starting with non-motor symptoms. CONCLUSION: The short and severe prodromal phase in PDGBA might call for shorter assessment intervals in yet premanifest GBA mutation carriers.

Effects of fat supplementation to diets high in nonforage fiber on production responses of midlactation dairy cows.

The effects of dietary nonforage fiber sources on production responses of lactating dairy cattle have been well described, but interactions with other components of the diet have been less thoroughly explored. We investigated the effects of adding 2 commonly fed fat sources to a ration featuring high levels of nonforage fiber supplied by a corn milling by-product. Midlactation Holstein cows were blocked by parity, stratified by days in milk, and randomly assigned to 1 of 6 pens (12 cows/pen). Pens were randomly assigned to treatment sequences in a 3 × 3 Latin square design, where the treatments consisted of prilled saturated fat (SAT; Energy Booster 100, Milk Specialties Co., Dundee, IL), calcium salts of long-chain fatty acids (UNS; Megalac, Church and Dwight Co. Inc., Princeton, NJ), or no added dietary fat (control), with fat sources included to provide 1.2% added fat (dry matter basis). Treatment periods were 21 d; milk and feed samples were collected and milk yield and feed intake were recorded for the last 4 d of each period. Results were analyzed with mixed models with pen as the experimental unit, and orthogonal contrasts were employed to evaluate the overall effect of added fat and the effect of fat source. Dry matter intake and milk yield tended to increase with added fat. Protein content decreased with fat supplementation, to a greater degree for UNS than for SAT, but protein yield was not affected. Fat content, fat yield, and energy-corrected milk yield were not affected by treatment. Conversion of feed to milk tended to increase for UNS compared with SAT. Fat supplementation to diets high in nonforage fiber had effects that were similar to those reported for more traditional lactation diets, except for the dry matter intake response.

Inflammatory profile discriminates clinical subtypes in LRRK2-associated Parkinson's disease.

BACKGROUND AND PURPOSE: The presentation of Parkinson's disease patients with mutations in the LRRK2 gene (PDLRRK2 ) is highly variable, suggesting a strong influence of modifying factors. In this context, inflammation is a potential candidate inducing clinical subtypes. METHODS: An extensive battery of peripheral inflammatory markers was measured in human serum in a multicentre cohort of 142 PDLRRK2 patients from the MJFF LRRK2 Consortium, stratified by three different subtypes as recently proposed for idiopathic Parkinson's disease: diffuse/malignant, intermediate and mainly pure motor. RESULTS: Patients classified as diffuse/malignant presented with the highest levels of the pro-inflammatory proteins interleukin 8 (IL-8), monocyte chemotactic protein 1 (MCP-1) and macrophage inflammatory protein 1-ß (MIP-1-ß) paralleled by high levels of the neurotrophic protein brain-derived neurotrophic factor (BDNF). It was also possible to distinguish the clinical subtypes based on their inflammatory profile by using discriminant and area under the receiver operating characteristic curve analysis. CONCLUSIONS: Inflammation seems to be associated with the presence of a specific clinical subtype in PDLRRK2 that is characterized by a broad and more severely affected spectrum of motor and non-motor symptoms. The pro-inflammatory metabolites IL-8, MCP-1 and MIP-1-ß as well as BDNF are interesting candidates to be included in biomarker panels that aim to differentiate subtypes in PDLRRK2 and predict progression.

GBA-associated parkinsonism and dementia: beyond α-synucleinopathies?

BACKGROUND AND PURPOSE: To date the role of GBA mutations beyond α-synucleinopathies in the parkinsonism-dementia spectrum is still unclear. The aim of the study was to screen for GBA mutations in progressive supranuclear palsy (PSP), corticobasal syndrome (CBS), primary progressive aphasia (PPA) and the behavioural variant of frontotemporal dementia (bvFTD). METHODS: In all, 303 patients with a clinical diagnosis of PSP (n = 157), CBS (n = 39), PPA (n = 35) and bvFTD (n = 72) and 587 neurologically healthy controls were screened for the most common GBA mutations. RESULTS: GBA mutations were detected in one healthy control and four patients with a clinical diagnosis of PSP (n = 1), probable CBS (n = 2) and PPA (n = 1, with concomitant C9orf72 expansion). Overall the prevalence of GBA mutations was low in non-α-synucleinopathies but significantly higher in the CBS subgroup compared to controls. CONCLUSION: Although numbers are small, our findings indicate that the clinical phenotype of GBA-associated neurodegenerative disease is more heterogeneous than previously assumed, including phenotypes not usually associated with underlying α-synucleinopathies. This may be of relevance, once causal therapeutic strategies for GBA-associated neurodegenerative disease are developed.

[Assistive Services in the Workplace of People with Hearing Impairment in the State of North Rhine-Westphalia]. / Inanspruchnahme begleitender Hilfen im Arbeitsleben von hörgeschädigten Arbeitnehmerinnen und Arbeitnehmern in Nordrhein-Westfalen.

AIM OF THE STUDY: Assistive services in the workplace are an important aspect of the participation of people with hearing impairment in working life. This article presents the results of the GINKO study and an survey conducted by the University of Cologne on behalf of the MAIS in order to provide a comprehensive examination of the employment situation of hearing impaired people in North Rhine-Westphalia. The GINKO study examines the impact of laws on the integration of hard-of-hearing and deaf people as well as people who have become deaf as adults, focusing on communication and organizations; this project was funded by the German Federal Ministry for Labour and Social Affairs (BMAS). METHOD: In the GINKO study, conducted in cooperation with the German Association of the Hard of Hearing and the German Association of the Deaf, a standardised questionnaire with questions about the workplace was administered to employed people with hearing impairments. The questionnaire was administered on paper and was also available online accompanied by sign language videos. The University of Cologne study in North Rhine-Westphalia examined the service situation of hard-of-hearing, deaf and deaf-blind people through face-to-face interviews and government statistics. RESULTS: The results of the nationwide GINKO study show that hearing-impaired people in North Rhine-Westphalia draw on assistive services in employment more often than hearing-impaired people in the rest of Germany. The study found statistically significant differences in the categories of "maintenance and development of professional knowledge and skills" and "psychosocial support in conflict situations resulting from disability". CONCLUSION: One reason for the more positive evaluations of the participants in North Rhine-Westphalia as compared to other regions in Germany could be the particular network of support services in that state. However, the overall positive results from North Rhine-Westphalia should not obscure the fact that a majority of participants in many areas of North Rhine-Westphalia reported much less positive evaluations. They reported that they did not (yet) have an accessible workplace and that assistive services are not available to all hearing impaired workers.

[Knowledge of laws by students with hearing impairment: results from the GINKO-project]. / Gesetzeskenntnis bei Studierenden mit Hör-behinde-rung: Ergebnisse aus dem Projekt GINKO.

Legislation for people with disabilities has also changed due to other changes in the law, especially due to the recent ratification of the UN Convention on the Rights of Persons with Disabilities. These laws, in particular the UN Convention on the Rights of Persons with Disabilities, with its inclusion of the right to equitable and universal access to education for people with disabilities and their implementation, are of central importance for students who are impaired. As part of the GINKO (the legislative effect laws have on the professional integration of those who are hard of hearing, people who have gone deaf and those who are deaf through communication and organization; promotion: BMAS) project, the following questions were also brought up for discussion and were investigated: to what extent hearing-impaired students are aware of legislation that benefits them, whether these laws will be implemented, and what factors have an impact on this legal knowledge or its implementation. Overall, 4,825 handicapped individuals with hearing impairments - including n=166 students - took part in the survey. The results of the evaluation of the group of hearing-impaired students indicate that many of them are not informed about laws important to them. It was also found that the knowledge of a law cannot be equated with its implementation. This survey also resulted in a resolve for the future, to demand information about legal options be reinforced, and to adjust this information to fit the needs of specific target groups, e.g. this information could be disseminated through sign language films. On the other hand, these results also apply to higher education, for these institutions to create learning conditions where existing regulatory design options for students with disabilities are implemented, thereby affording students an equal opportunity to participate in higher education.

Honey bee promoter sequences for targeted gene expression.

The honey bee, Apis mellifera, displays a rich behavioural repertoire, social organization and caste differentiation, and has an interesting mode of sex determination, but we still know little about its underlying genetic programs. We lack stable transgenic tools in honey bees that would allow genetic control of gene activity in stable transgenic lines. As an initial step towards a transgenic method, we identified promoter sequences in the honey bee that can drive constitutive, tissue-specific and cold shock-induced gene expression. We identified the promoter sequences of Am-actin5c, elp2l, Am-hsp83 and Am-hsp70 and showed that, except for the elp2l sequence, the identified sequences were able to drive reporter gene expression in Sf21 cells. We further demonstrated through electroporation experiments that the putative neuron-specific elp2l promoter sequence can direct gene expression in the honey bee brain. The identification of these promoter sequences is an important initial step in studying the function of genes with transgenic experiments in the honey bee, an organism with a rich set of interesting phenotypes.

GBA-associated PD presents with nonmotor characteristics.

OBJECTIVE: To evaluate whether there exists distinct characteristics in glucocerebrosidase (GBA)-associated Parkinson disease (PD) with regard to motor and nonmotor symptoms as well as imaging characteristics assessed by transcranial sonography (TCS). METHODS: Twenty patients with PD with heterozygous GBA mutations (N370S, L444P) (GBA-PD) in comparison to 20 patients with sporadic PD negative for GBA mutations (sPD) were included. We assessed motor impairment with the Unified Parkinson's Disease Rating Scale-III. Nonmotor symptoms were evaluated using the Montreal Cognitive Assessment, Neuropsychiatric Inventory, revised form of the Beck Depression Inventory, Parkinson Disease Sleep Scale, Sniffin' Sticks, and Unified Multiple System Atrophy Rating Scale items 9-12. TCS imaging was used to detect morphologic characteristics. RESULTS: Patients with GBA-PD more often had a variety of nonmotor symptoms, namely dementia, neuropsychiatric disturbances, and autonomic dysfunction, and had more severe cases, than patients with sPD. They also demonstrated a higher prevalence of a reduced echogenicity of the brainstem raphe assessed by TCS. CONCLUSIONS: Especially nonmotor symptoms seem to be very common in GBA-PD. Further studies are needed to validate these observations in order to better understand the pathogenesis of GBA-PD and develop specific therapeutic concepts.

A polymorphism in the gene encoding AdipoR1 affects olfactory recognition.

Polymorphisms in the gene encoding adiponectin receptor 1 (AdipoR1) are associated with insulin resistance, fatty liver, increased risk for type 2 diabetes and cardiovascular disease. AdipoR1 is expressed in the central nervous system and in the olfactory mucosa of mice and humans. We therefore hypothesized that a common polymorphism in AdipoR1 might alter olfactory function. We investigated a group of 222 healthy subjects (male: n = 147, female: n = 75) for olfactory recognition, and genotyped them for the polymorphism rs6666089 in the human AdipoR1 gene. This polymorphism has been previously shown to be associated with insulin resistance. Olfactory recognition was tested using standardized sniffing sticks, and parameters of glucose metabolism and serum adiponectin levels were assessed. We found a significant olfactory impairment in carriers of the AdipoR1 polymorphism rs6666089 (olfactory recognition: GG: 89.4 ± 1.2%, GA: 86.9 ± 1.4%, AA: 77.2 ± 4.8%, additive model, P = 0.0004, adjusted for age). Adiponectin levels had no impact on olfactory recognition. Fasting plasma glucose, fasting plasma insulin, body mass index and HbA1c did not differ between the genotype groups. In conclusion, the presence of a genetic variation in AdipoR1 is associated with decreased olfactory recognition in healthy subjects. Adiponectin signalling may have an important role in olfactory function and regulation of appetite.

Differential chemokine expression in chronic GVHD of the conjunctiva.

In chronic GVHD after BMT, the conjunctiva represents a target organ. GVHD can lead to severe inflammation and dry-eye syndrome (sicca syndrome). The molecular mechanisms are largely unknown. We examined the expression of chemokines in the conjunctiva in cases of chronic GVHD. In this study, we included 10 patients with chronic GVHD and 10 healthy controls. Clinical data were collected and tear film analysis and conjunctival cytology were carried out. Conjunctival biopsies were taken from all participants. Gene expression profiles of chemokines and their corresponding receptors were evaluated by means of quantitative real-time PCR. Chemokine protein expression was analysed by immunohistochemical analyses. Expressions of the Th1-associated chemokines, chemokine (C-X-C motif) ligand (CXCL) 9 (Mig), CXCL10 (IP-10), and their receptor chemokine (C-X-C motif) receptor 3 (CXCR3) were significantly increased in GVHD patients. Immunohistochemical analysis confirmed marked expression of the inflammatory CXCR3 ligands. A total of six patients had a moderate or severe sicca syndrome. Impression cytology revealed a mild keratinisation, moderate keratinisation or severe squamous metaplasia in three patients, respectively. Chronic GVHD of the conjunctiva is characterised by the expression of Th1-associated chemokines. Taken together, our results confirm that the conjunctiva is a target organ in this T cell-mediated process and add to molecular understanding of conjunctival GVHD.

Multicenter analysis of glucocerebrosidase mutations in Parkinson's disease.

BACKGROUND: Recent studies indicate an increased frequency of mutations in the gene encoding glucocerebrosidase (GBA), a deficiency of which causes Gaucher's disease, among patients with Parkinson's disease. We aimed to ascertain the frequency of GBA mutations in an ethnically diverse group of patients with Parkinson's disease. METHODS: Sixteen centers participated in our international, collaborative study: five from the Americas, six from Europe, two from Israel, and three from Asia. Each center genotyped a standard DNA panel to permit comparison of the genotyping results across centers. Genotypes and phenotypic data from a total of 5691 patients with Parkinson's disease (780 Ashkenazi Jews) and 4898 controls (387 Ashkenazi Jews) were analyzed, with multivariate logistic-regression models and the Mantel-Haenszel procedure used to estimate odds ratios across centers. RESULTS: All 16 centers could detect two GBA mutations, L444P and N370S. Among Ashkenazi Jewish subjects, either mutation was found in 15% of patients and 3% of controls, and among non-Ashkenazi Jewish subjects, either mutation was found in 3% of patients and less than 1% of controls. GBA was fully sequenced for 1883 non-Ashkenazi Jewish patients, and mutations were identified in 7%, showing that limited mutation screening can miss half the mutant alleles. The odds ratio for any GBA mutation in patients versus controls was 5.43 across centers. As compared with patients who did not carry a GBA mutation, those with a GBA mutation presented earlier with the disease, were more likely to have affected relatives, and were more likely to have atypical clinical manifestations. CONCLUSIONS: Data collected from 16 centers demonstrate that there is a strong association between GBA mutations and Parkinson's disease.

Screening for LRRK2 R1441 mutations in a cohort of PSP patients from Germany.

BACKGROUND AND PURPOSE: Mutations in the leucine-rich repeat kinase gene (LRRK2) have been shown to be the most common genetic cause of both familial and sporadic Parkinson's disease. Patients harboring LRRK2 mutations develop late onset PD that in most cases cannot be clinically distinguished from idiopathic PD. Furthermore, LRRK2 mutations have been reported to result in a broad spectrum of neuropathological alterations including progressive supranuclear palsy (PSP)-like Tau pathology. METHODS: We screened a cohort of 88 clinically confirmed PSP patients for mutations in exon 31. RESULTS: We did not find any of the known mutations or any new variants. CONCLUSIONS: Thus, there is no evidence that mutations in exon 31 of LRRK2 are a major risk factor for PSP. Our study, however, cannot rule out that other genetic variations in LRRK2 may be associated with PSP.