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Seizure threshold 2 (SZT2) is a component of the KICSTOR complex which, under catabolic conditions, functions as a negative regulator in the amino acid-sensing branch of mTORC1. Mutations in this gene cause a severe neurodevelopmental and epileptic encephalopathy whose main symptoms include epilepsy, intellectual disability, and macrocephaly. As SZT2 remains one of the least characterized regulators of mTORC1, in this work we performed a systematic interactome analysis under catabolic and anabolic conditions. Besides numerous mTORC1 and AMPK signaling components, we identified clusters of proteins related to autophagy, ciliogenesis regulation, neurogenesis, and neurodegenerative processes. Moreover, analysis of SZT2 ablated cells revealed increased mTORC1 signaling activation that could be reversed by Rapamycin or Torin treatments. Strikingly, SZT2 KO cells also exhibited higher levels of autophagic components, independent of the physiological conditions tested. These results are consistent with our interactome data, in which we detected an enriched pool of selective autophagy receptors/regulators. Moreover, preliminary analyses indicated that SZT2 alters ciliogenesis. Overall, the data presented form the basis to comprehensively investigate the physiological functions of SZT2 that could explain major molecular events in the pathophysiology of developmental and epileptic encephalopathy in patients with SZT2 mutations.

Pathogen discovery contributes to our knowledge of bat-borne viruses and is linked to the heightened interest globally in bats as recognised reservoirs of zoonotic agents. The transmission of lyssaviruses from bats-to-humans, domestic animals, or other wildlife species is uncommon, but interest in these pathogens remains due to their ability to cause an acute, progressive, invariably fatal encephalitis in humans. Consequently, the detection and characterisation of bat lyssaviruses continues to expand our knowledge of their phylogroup definition, viral diversity, host species association, geographical distribution, evolution, mechanisms for perpetuation, and the potential routes of transmission. Although the opportunity for lyssavirus cross-species transmission seems rare, adaptation in a new host and the possibility of onward transmission to humans requires continued investigation. Considering the limited efficacy of available rabies biologicals it is important to further our understanding of protective immunity to minimize the threat from these pathogens to public health. Hence, in addition to increased surveillance, the development of a niche pan-lyssavirus vaccine or therapeutic biologics for post-exposure prophylaxis for use against genetically divergent lyssaviruses should be an international priority as these emerging lyssaviruses remain a concern for global public health.

Lyssaviruses are the causative agents for rabies, a zoonotic and fatal disease. Bats are the ancestral reservoir host for lyssaviruses, and at least three different lyssaviruses have been found in bats from Germany. Across Europe, novel lyssaviruses were identified in bats recently and occasional spillover infections in other mammals and human cases highlight their public health relevance. Here, we report the results from an enhanced passive bat rabies surveillance that encompasses samples without human contact that would not be tested under routine conditions. To this end, 1236 bat brain samples obtained between 2018 and 2020 were screened for lyssaviruses via several RT-qPCR assays. European bat lyssavirus type 1 (EBLV-1) was dominant, with 15 positives exclusively found in serotine bats (Eptesicus serotinus) from northern Germany. Additionally, when an archived set of bat samples that had tested negative for rabies by the FAT were screened in the process of assay validation, four samples tested EBLV-1 positive, including two detected in Pipistrellus pipistrellus. Subsequent phylogenetic analysis of 17 full genomes assigned all except one of these viruses to the A1 cluster of the EBLV-1a sub-lineage. Furthermore, we report here another Bokeloh bat lyssavirus (BBLV) infection in a Natterer’s bat (Myotis nattereri) found in Lower Saxony, the tenth reported case of this novel bat lyssavirus.

The distribution of human Lyme borreliosis (LB) is assumed random in Germany, indicating that the human pathogenic species of the Borrelia burgdorferi sensu lato complex (Bb) are similarly distributed as part of the tick microbiome. The aim of this study was to differentiate if the presence of Bb occurs with a defined tick microbiome composition. Furthermore, the effect of location on tick microbiome composition was addressed for two German locations. Therefore, nucleic acid extracts from 82 Borrelia-positive and 118 Borrelia-negative Ixodes ricinus ticks sampled from human hosts in both districts were selected. Nucleic acid extracts were used for human pathogenic Bb species diagnostics based on qPCR and multilocus sequence typing (MLST) and bacterial 16S rRNA gene amplicon sequencing followed by network analyses. As a result, the presence of Bb shifted the sequence read abundances of Candidatus Midichloria, Rickettsia, Pseudomonas, Staphylococcus, and Candidatus Neoehrlichia and their topological roles in the tick microbiome. Moreover, the location was less important in the tick microbiome composition but shifted significantly sequence read abundances of Pseudomonas and Wolbachia as well as the topological role of microbial members. Since the presence of human pathogenic Bb species with other tick-associated pathogens varies regionally, we suggest that a bacterial 16S rRNA gene-based microbiome survey should be implemented in the routine diagnostics for both tick and host if human pathogenic species of Bb were detected. This diagnostic extension will help to optimize therapeutic approaches against Bb infection and co-occurring pathogens.

Subcellular localization of RNAs and proteins is important for polarized cells such as neurons. Here the authors differentiate mouse embryonic stem cells into neurons, and analyze the local transcriptome, proteome, and translated transcriptome in their cell bodies and neurites, providing a unique resource for future studies on neuronal polarity.

Abstract Background As rabies still represents a major public threat with tens of thousands of deaths per year, particularly in developing countries, adequate surveillance based on rapid and reliable rabies diagnosis for both humans and animals is essential. Rabies diagnosis relies on highly sensitive and specific laboratory tests for detection of viral antigens. Among those tests, at present the immunofluorescence antibody test is the “gold standard test” for rabies diagnosis, followed by virus isolation in either mice or cell culture. Because of the advantages of molecular assays in terms of sensitivity and applicability their approval as confirmatory diagnostic test by international organizations (OIE, WHO) is envisaged. Therefore, the objective was to develop and validate novel molecular assays and RNA extraction methods for rabies that reduce the turnaround time but remain highly sensitive and specific. Methods Here, novel assays, i.e. HighSpeed RT-qPCR and isothermal recombinase polymerase amplification (RPA) were designed and tested. Furthermore, three magnetic bead-based rapid extraction methods for manual or automated extraction were validated and combined with the new downstream assays. Results While the conventional column based RNA extraction method showed the highest intra-run variations, all magnetic bead-based rapid extraction methods delivered nearly comparable sensitivity and efficiency of RNA recovery. All newly developed molecular tests were able to detect different rabies virus strains in a markedly reduced timeframe in comparison to the standard diagnostic assays. The observed detection limit for the HighSpeed RT-qPCR was 10 genome copies per reaction, and 1000 genome copies per reaction for the RPA assay. Conclusion Magnetic bead-based rapid RNA extraction methods are highly sensitive and show a high level of reproducibility and therefore, are particularly suitable for molecular diagnostic assays including rabies. In addition, with a detection limit of 10 genome copies per reaction, the HighSpeed RT-qPCR is suitable for rapid ante mortem rabies diagnosis in humans as well as confirmatory test in integrated bite management and subsequent post-exposure prophylaxis.

Silk fibres currently used in biotechnology are chemically reconstituted silk fibroins (RSF), which are more stable than native silk fibroin (NSF) but possess different biophysical properties. Here, the authors use microfluidic droplets to encapsulate and store NSF, preserving their native structure.

A connection between centrosomes and chromosomes is a key feature of mitotic spindles. Here the authors generate 3D reconstructions of whole mitotic spindles in earlyC. elegansembryos and show that chromosomes are anchored by the entire spindle network and that connections through kinetochore microtubules are few and likely very transient.

Molybdenum disulphide (MoS2) thin films have received increasing interest as device-active layers in low-dimensional electronics and also as novel catalysts in electrochemical processes such as the hydrogen evolution reaction (HER) in electrochemical water splitting. For both types of applications, industrially scalable fabrication methods with good control over the MoS2 film properties are crucial. Here, we investigate scalable physical vapour deposition (PVD) of MoS2 films by magnetron sputtering. MoS2 films with thicknesses from ≈10 to ≈1000 nm were deposited on SiO2/Si and reticulated vitreous carbon (RVC) substrates. Samples deposited at room temperature (RT) and at 400 °C were compared. The deposited MoS2 was characterized by macro- and microscopic X-ray, electron beam and light scattering, scanning and spectroscopic methods as well as electrical device characterization. We find that room-temperature-deposited MoS2 films are amorphous, of smooth surface morphology and easily degraded upon moderate laser-induced annealing in ambient conditions. In contrast, films deposited at 400 °C are nano-crystalline, show a nano-grained surface morphology and are comparatively stable against laser-induced degradation. Interestingly, results from electrical transport measurements indicate an unexpected metallic-like conduction character of the studied PVD MoS2 films, independent of deposition temperature. Possible reasons for these unusual electrical properties of our PVD MoS2 thin films are discussed. A potential application for such conductive nanostructured MoS2 films could be as catalytically active electrodes in (photo-)electrocatalysis and initial electrochemical measurements suggest directions for future work on our PVD MoS2 films.

Infectious diseases are often transmitted through local interactions. Yet, both surveillance and control measures are implemented within administrative units. Capturing local transmission processes and spatial coupling between regions from aggregate level data is therefore a technical challenge that can shed light on both theoretical questions and practical decisions. Fox rabies has been eliminated from much of Europe through oral rabies vaccination (ORV) programmes. The European Union (EU) co-finances ORV to maintain rabies freedom in EU member and border states via a cordon sanitaire. Models to capture local transmission dynamics and spatial coupling have immediate application to the planning of these ORV campaigns and to other parts of the world considering oral vaccination. We fitted a hierarchical Bayesian state-space model to data on three decades of fox rabies cases and ORV campaigns from Eastern Germany. Specifically, we find that (i) combining regional spatial coupling and heterogeneous local transmission allows us to capture regional rabies dynamics; (ii) incursions from other regions account for less than 1% of cases, but allow for re-emergence of disease; (iii) herd immunity achieved through bi-annual vaccination campaigns is short-lived due to population turnover. Together, these findings highlight the need for regular and sustained vaccination efforts and our modelling approach can be used to provide strategic guidance for ORV delivery. Moreover, we show that biological understanding can be gained from inference from partially observed data on wildlife disease.

Rabies is a fatal zoonotic disease that causes a heavy burden on societies. Namibia, a country in southern Africa, is aiming at controlling the disease in its main reservoir, the domestic dog. To facilitate the implementation comprehensive information on the ecology and epidemiology of the disease and surveillance is of utmost importance. The study presented assesses the baseline data for both human and animal rabies surveillance in Namibia in recent times and establishes correlations with ecological and socio-economic data in order to provide an up-to-date picture on the epidemiology of rabies in Namibia. For instance, it was important to identify the main drivers in the epidemiology, and whether the control strategy by mass vaccination of dogs is undermined by cycles of rabies in wildlife. Rabies in humans was reported mainly from the Northern Communal Areas (NCAs), with a total of 113 cases from 2011 to 2017, representing an incidence of between 1.0 and 2.4 annual human rabies deaths per 100,000 inhabitants. Kavango, the region with the highest human rabies incidence was also the region with the lowest animal rabies surveillance intensity. Generally, the vast majority (77%) of dog samples originated from communal farm land, followed by urban areas (17%), while only a small fraction (3%) was submitted from freehold farm areas. In contrast, kudu and eland submissions were almost exclusively from freehold farmland (76%) and urban areas (19%), whereas the submission of cattle samples was evenly distributed among freehold farms (46%) and communal farm land (46%). The likelihood of sample submission decreased exponentially with distance to one of the two laboratories. Overall, 67% (N = 1,907) of all samples submitted tested rabies-positive, with the highest positivity rate observed in kudus (89%) and jackals (87%). The transmission cycle of rabies in dogs appears restricted to the northern communal areas of Namibia, whilst rabies in wildlife species is predominately reported from farmland in central Namibia, mostly affecting kudu (Tragelaphus strepsiceros) and livestock with a likely reservoir in wildlife canids such as jackals or bat-eared foxes. The analysis confirms the presence of two independent transmission cycles in Namibia with little geographic overlap, thus allowing for a sustainable control of rabies in dogs in the NCAs.

Background: Vitamin D has a steroid- and an anabolic-resembling chemical structure. Vitamin D is essential for many processes in the human body after hydroxylation. Aims of the Study: To investigate the impact of 25-hydroxy-vitamin D plasma concentrations on the blood parameters number of erythrocytes, hematocrit, mean corpuscular hemoglobin and mean corpuscular volume. Methods: Serial assessments were done in 290 patients with multiple sclerosis and repeated after a mean interval of 245 days. A recommendation for vitamin D supplementation was given in case of a concentration lower than 20 ng/mL combined with a prescription of a formulation containing vitamin D but not vitamin K. Results: There was a fall of vitamin D in 119 subjects and a rise in 164, while no change appeared in 7 participants. When vitamin D values went down between both assessments moments, the computed increase of mean corpuscular haemoglobin was significantly lower compared with the rise of mean corpuscular haemoglobin associated with a vitamin D elevation. When vitamin D declined, the computed fall of mean corpuscular volume fall was significantly lower compared with the decrease of mean corpuscular volume, when vitamin D rose. Positive correlations were found between differences of vitamin D and mean corpuscular haemoglobin, respectively mean corpuscular volume. Inverse relations appeared between disparities of vitamin D and erythrocytes, respectively haematocrit. Conclusions: The elevation of vitamin D plasma levels provides enhanced preconditions for a better tissue oxygenation on a cellular level.

Animal activity is an indicator for its welfare and manual observation is time and cost intensive. To this end, automatic detection and monitoring of live captive animals is of major importance for assessing animal activity, and, thereby, allowing for early recognition of changes indicative for diseases and animal welfare issues. We demonstrate that machine learning methods can provide a gap-less monitoring of red foxes in an experimental lab-setting, including a classification into activity patterns. Therefore, bounding boxes are used to measure fox movements, and, thus, the activity level of the animals. We use computer vision, being a non-invasive method for the automatic monitoring of foxes. More specifically, we train the existing algorithm ‘you only look once’ version 4 (YOLOv4) to detect foxes, and the trained classifier is applied to video data of an experiment involving foxes. As we show, computer evaluation outperforms other evaluation methods. Application of automatic detection of foxes can be used for detecting different movement patterns. These, in turn, can be used for animal behavioral analysis and, thus, animal welfare monitoring. Once established for a specific animal species, such systems could be used for animal monitoring in real-time under experimental conditions, or other areas of animal husbandry.

Tufting enteropathy (TE) is caused by recessive EPCAM mutations, and is characterized by intractable diarrhea of congenital onset and disorganization of enterocytes. TE generally requires parenteral nutrition (PN) during childhood or intestinal bowel transplantation. We report three unrelated families with six children with TE. We highlight the high rate of disease-related mortality. We observe adequate weight gain with PN, but low to normal and stunted body length, supporting the recent notion that a short stature might be intrinsic to TE. The diagnosis of TE in the index patients from each family was delayed for months to years, even when clinical data, duodenal biopsies, or exome sequencing data were obtained early on. We identified three novel pathogenic EPCAM variants: a deletion of exon 1 that removes the ATG initiation codon, a missense variant c.326A > G (p.Gln109Arg), and nonsense mutation c.429G > A (p.Trp143*) in a compound heterozygous state with the Mediterranean splice site variant c.556-14A > G (Tyr186Phefs*6). Homozygosity for p.Gln109Arg was associated with absent EPCAM staining, and compound heterozygosity for p.Trp143*/Tyr186Phefs*6 was associated with reduced EPCAM staining in duodenal biopsies; such observations might contribute to a genotype–phenotype correlation in larger cohorts of TE patients. This study extends the clinical and molecular spectrum of TE.

TASK channels belong to the two-pore-domain potassium (K2P) channels subfamily. These channels modulate cellular excitability, input resistance, and response to synaptic stimulation. TASK-channel inhibition led to membrane depolarization. TASK-3 is expressed in different cancer cell types and neurons. Thus, the discovery of novel TASK-3 inhibitors makes these bioactive compounds very appealing to explore new cancer and neurological therapies. TASK-3 channel blockers are very limited to date, and only a few heterofused compounds have been reported in the literature. In this article, we combined a pharmacophore hypothesis with molecular docking to address for the first time the rational design, synthesis, and evaluation of 5-(indol-2-yl)pyrazolo[3,4-b]pyridines as a novel family of human TASK-3 channel blockers. Representative compounds of the synthesized library were assessed against TASK-3 using Fluorometric imaging plate reader—Membrane Potential assay (FMP). Inhibitory properties were validated using two-electrode voltage-clamp (TEVC) methods. We identified one active hit compound (MM-3b) with our systematic pipeline, exhibiting an IC50 ≈ 30 μM. Molecular docking models suggest that compound MM-3b binds to TASK-3 at the bottom of the selectivity filter in the central cavity, similar to other described TASK-3 blockers such as A1899 and PK-THPP. Our in silico and experimental studies provide a new tool to predict and design novel TASK-3 channel blockers.

European bat lyssavirus type 1 (EBLV-1) is the causative agent for almost all reported rabies cases found in European bats. In recent years, increasing numbers of available EBLV-1 full genomes and their phylogenetic analyses helped to further elucidate the distribution and genetic characteristics of EBLV-1 and its two subtypes, namely EBLV-1a and EBLV-1b. Nonetheless, the absence of full-genome sequences from regions with known detections of EBLV-1 still limit the understanding of the phylogeographic relations between viruses from different European regions. In this study, a set of 21 archived Danish EBLV-1 samples from the years 1985 to 2009 was processed for the acquisition of full-genome sequences using a high-throughput sequencing approach. Subsequent phylogenetic analysis encompassing all available EBLV-1 full genomes from databases revealed the Danish sequences belong to the EBLV-1a subtype and further highlighted the distinct, close phylogenetic relationship of Danish, Dutch and German isolates in this region. In addition, the formation of five putative groups nearly exclusively formed by Danish isolates and the overall increased resolution of the EBLV-1a branch indicate a higher genetic diversity and spatial segregation for this sublineage than was previously known. These results emphasize the importance of phylogenetic analyses of full-genome sequences of lyssaviruses for genetic geography.

Symptomatic treatments are available for Parkinson’s disease and Alzheimer’s disease. An unmet need is cure or disease modification. This review discusses possible reasons for negative clinical study outcomes on disease modification following promising positive findings from experimental research. It scrutinizes current research paradigms for disease modification with antibodies against pathological protein enrichment, such as α-synuclein, amyloid or tau, based on post mortem findings. Instead a more uniform regenerative and reparative therapeutic approach for chronic neurodegenerative disease entities is proposed with stimulation of an endogenously existing repair system, which acts independent of specific disease mechanisms. The repulsive guidance molecule A pathway is involved in the regulation of peripheral and central neuronal restoration. Therapeutic antagonism of repulsive guidance molecule A reverses neurodegeneration according to experimental outcomes in numerous disease models in rodents and monkeys. Antibodies against repulsive guidance molecule A exist. First clinical studies in neurological conditions with an acute onset are under way. Future clinical trials with these antibodies should initially focus on well characterized uniform cohorts of patients. The efficiency of repulsive guidance molecule A antagonism and associated stimulation of neurogenesis should be demonstrated with objective assessment tools to counteract dilution of therapeutic effects by subjectivity and heterogeneity of chronic disease entities. Such a research concept will hopefully enhance clinical test strategies and improve the future therapeutic armamentarium for chronic neurodegeneration.

Here, Dirk Lefeber and colleagues identify functional mutations in ATP6AP1 encoding Ac45. The authors show that Ac45 is the functional ortholog of yeast V-ATPase assembly factor Voa1 and provide evidence for tissue-specific Ac45 processing, associated with the clinical phenotype of immunodeficiency, hepatopathy, and neurocognitive abnormalities.