Dynamics of Mouth Opening in Hydra

Hydra, a simple freshwater animal famous for its regenerative capabilities, must tear a hole through its epithelial tissue each time it opens its mouth. The feeding response of Hydra has been well-characterized physiologically and is regarded as a classical model system for environmental chemical biology. However, due to a lack of in vivo labeling and imaging tools, the biomechanics of mouth opening have remained completely unexplored. We take advantage of the availability of transgenic Hydra lines to perform the first dynamical analysis, to our knowledge, of Hydra mouth opening and test existing hypotheses regarding the underlying cellular mechanisms. Through cell position and shape tracking, we show that mouth opening is accompanied by changes in cell shape, but not cellular rearrangements as previously suggested. Treatment with a muscle relaxant impairs mouth opening, supporting the hypothesis that mouth opening is an active process driven by radial contractile processes (myonemes) in the ectoderm. Furthermore, we find that all events exhibit the same relative rate of opening. Because one individual can open consecutively to different amounts, this suggests that the degree of mouth opening is controlled through neuronal signaling. Finally, from the opening dynamics and independent measurements of the elastic properties of the tissues, we estimate the forces exerted by the myonemes to be on the order of a few nanoNewtons. Our study provides the first dynamical framework, to our knowledge, for understanding the remarkable plasticity of the Hydra mouth and illustrates that Hydra is a powerful system for quantitative biomechanical studies of cell and tissue behaviors in vivo.     

Measuring endemicity and burden of leprosy across countries and regions: A systematic review and Delphi survey

BackgroundLeprosy is a chronic infectious disease caused by Mycobacterium leprae, the annual new case detection in 2019 was 202,189 globally. Measuring endemicity levels and burden in leprosy lacks a uniform approach. As a result, the assessment of leprosy endemicity or burden are not comparable over time and across countries and regions. This can make program planning and evaluation difficult. This study aims to identify relevant metrics and methods for measuring and classifying leprosy endemicity and burden at (sub)national level.MethodsWe used a mixed-method approach combining findings from a systematic literature review and a Delphi survey. The literature search was conducted in seven databases, searching for endemicity, burden and leprosy. We reviewed the available evidence on the usage of indicators, classification levels, and scoring methods to measure and classify endemicity and burden. A two round Delphi survey was conducted to ask experts to rank and weigh indicators, classification levels, and scoring methods.ResultsThe literature review showed variation of indicators, levels, and cut-off values to measure leprosy endemicity and/or burden. The most used indicators for endemicity include new case detection rate (NCDR), new cases among children and new cases with grade 2 disability. For burden these include NCDR, MB cases, and prevalence. The classification levels ‘high’ and ‘low’ were most important. It was considered most relevant to use separate scoring methods for endemicity and burden. The scores would be derived by use of multiple indicators.ConclusionThere is great variation in the existing method for measuring endemicity and burden across countries and regions. Our findings contribute to establishing a standardized uniform approach to measure and classify leprosy endemicity and burden at (sub)national level, which would allow effective communication and planning of intervention strategies.     

Multifaceted effects of oligodendroglial exosomes on neurons: impact on neuronal firing rate,signal transduction and gene regulation

Exosomes are small membranous vesicles of endocytic origin that are released by almost every cell type. They exert versatile functions in intercellular communication important for many physiological and pathological processes. Recently, exosomes attracted interest with regard to their role in cell–cell communication in the nervous system. We have shown that exosomes released from oligodendrocytes upon stimulation with the neurotransmitter glutamate are internalized by neurons and enhance the neuronal stress tolerance. Here, we demonstrate that oligodendroglial exosomes also promote neuronal survival during oxygen–glucose deprivation, a model of cerebral ischaemia. We show the transfer from oligodendrocytes to neurons of superoxide dismutase and catalase, enzymes which are known to help cells to resist oxidative stress. Additionally, we identify various effects of oligodendroglial exosomes on neuronal physiology. Electrophysiological analysis using in vitro multi-electrode arrays revealed an increased firing rate of neurons exposed to oligodendroglial exosomes. Moreover, gene expression analysis and phosphorylation arrays uncovered differentially expressed genes and altered signal transduction pathways in neurons after exosome treatment. Our study thus provides new insight into the broad spectrum of action of oligodendroglial exosomes and their effects on neuronal physiology. The exchange of extracellular vesicles between neural cells may exhibit remarkable potential to impact brain performance.     

Stages and Conformations of the Tau Repeat Domain during Aggregation and Its Effect on Neuronal Toxicity

Several neurodegenerative diseases are characterized by the aggregation and posttranslational modifications of Tau protein. Its “repeat domain” (TauRD) is mainly responsible for the aggregation properties, and oligomeric forms are thought to dominate the toxic effects of Tau. Here we investigated the conformational transitions of this domain during oligomerization and aggregation in different states of β-propensity and pseudo-phosphorylation, using several complementary imaging and spectroscopic methods. Although the repeat domain generally aggregates more readily than full-length Tau, its aggregation was greatly slowed down by phosphorylation or pseudo-phosphorylation at the KXGS motifs, concomitant with an extended phase of oligomerization. Analogous effects were observed with pro-aggregant variants of TauRD. Oligomers became most evident in the case of the pro-aggregant mutant TauRDΔK280, as monitored by atomic force microscopy, and the fluorescence lifetime of Alexa-labeled Tau (time-correlated single photon counting (TCSPC)), consistent with its pronounced toxicity in mouse models. In cell models or primary neurons, neither oligomers nor fibrils of TauRD or TauRDΔK280 had a toxic effect, as seen by assays with lactate dehydrogenase and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, respectively. However, oligomers of pro-aggregant TauRDΔK280 specifically caused a loss of spine density in differentiated neurons, indicating a locally restricted impairment of function.     

A Disease-causing Mutation Illuminates the Protein Membrane Topology of the Kidney-expressed Prohibitin Homology (PHB) Domain Protein Podocin

Mutations in the NPHS2 gene are a major cause of steroid-resistant nephrotic syndrome, a severe human kidney disorder. The NPHS2 gene product podocin is a key component of the slit diaphragm cell junction at the kidney filtration barrier and part of a multiprotein-lipid supercomplex. A similar complex with the podocin ortholog MEC-2 is required for touch sensation in Caenorhabditis elegans. Although podocin and MEC-2 are membrane-associated proteins with a predicted hairpin-like structure and amino and carboxyl termini facing the cytoplasm, this membrane topology has not been convincingly confirmed. One particular mutation that causes kidney disease in humans (podocin^P118L) has also been identified in C. elegans in genetic screens for touch insensitivity (MEC-2^P134S). Here we show that both mutant proteins, in contrast to the wild-type variants, are N-glycosylated because of the fact that the mutant C termini project extracellularly. Podocin^P118L and MEC-2^P134S did not fractionate in detergent-resistant membrane domains. Moreover, mutant podocin failed to activate the ion channel TRPC6, which is part of the multiprotein-lipid supercomplex, indicative of the fact that cholesterol recruitment to the ion channels, an intrinsic function of both proteins, requires C termini facing the cytoplasmic leaflet of the plasma membrane. Taken together, this study demonstrates that the carboxyl terminus of podocin/MEC-2 has to be placed at the inner leaflet of the plasma membrane to mediate cholesterol binding and contribute to ion channel activity, a prerequisite for mechanosensation and the integrity of the kidney filtration barrier.     

Plastin 1 Binds to Keratin and Is Required for Terminal Web Assembly in the Intestinal Epithelium

Plastin 1 (I-plastin, fimbrin) along with villin and espin is a prominent actin-bundling protein of the intestinal brush border microvilli. We demonstrate here that plastin 1 accumulates in the terminal web and interacts with keratin 19, possibly contributing to anchoring the rootlets to the keratin network. This prompted us to investigate the importance of plastin 1 in brush border assembly. Although in vivo neither villin nor espin is required for brush border structure, plastin 1-deficient mice have conspicuous ultrastructural alterations: microvilli are shorter and constricted at their base, and, strikingly, their core actin bundles lack true rootlets. The composition of the microvilli themselves is apparently normal, whereas that of the terminal web is profoundly altered. Although the plastin 1 knockout mice do not show any overt gross phenotype and present a normal intestinal microanatomy, the alterations result in increased fragility of the epithelium. This is seen as an increased sensitivity of the brush border to biochemical manipulations, decreased transepithelial resistance, and increased sensitivity to dextran sodium sulfate-induced colitis. Plastin 1 thus emerges as an important regulator of brush border morphology and stability through a novel role in the organization of the terminal web, possibly by connecting actin filaments to the underlying intermediate filament network.     

High cell density cultivation and recombinant protein production with <Emphasis Type="Italic">Escherichia coli</Emphasis> in a rocking-motion-type bioreactor

Background

Single-use rocking-motion-type bag bioreactors provide advantages compared to standard stirred tank bioreactors by decreased contamination risks, reduction of cleaning and sterilization time, lower investment costs, and simple and cheaper validation. Currently, they are widely used for cell cultures although their use for small and medium scale production of recombinant proteins with microbial hosts might be very attractive. However, the utilization of rocking- or wave-induced motion-type bioreactors for fast growing aerobic microbes is limited because of their lower oxygen mass transfer rate. A conventional approach to reduce the oxygen demand of a culture is the fed-batch technology. New developments, such as the BIOSTAT^® CultiBag RM system pave the way for applying advanced fed-batch control strategies also in rocking-motion-type bioreactors. Alternatively, internal substrate delivery systems such as EnBase^® Flo provide an opportunity for adopting simple to use fed-batch-type strategies to shaken cultures. Here, we investigate the possibilities which both strategies offer in view of high cell density cultivation of E. coli and recombinant protein production.

Results

Cultivation of E. coli in the BIOSTAT^® CultiBag RM system in a conventional batch mode without control yielded an optical density (OD₆₀₀) of 3 to 4 which is comparable to shake flasks. The culture runs into oxygen limitation. In a glucose limited fed-batch culture with an exponential feed and oxygen pulsing, the culture grew fully aerobically to an OD₆₀₀ of 60 (20 g L^-1 cell dry weight). By the use of an internal controlled glucose delivery system, EnBase^® Flo, OD₆₀₀ of 30 (10 g L^-1 cell dry weight) is obtained without the demand of computer controlled external nutrient supply. EnBase^® Flo also worked well in the CultiBag RM system with a recombinant E. coli RB791 strain expressing a heterologous alcohol dehydrogenase (ADH) to very high levels, indicating that the enzyme based feed supply strategy functions well for recombinant protein production also in a rocking-motion-type bioreactor.

Conclusions

Rocking-motion-type bioreactors may provide an interesting alternative to standard cultivation in bioreactors for cultivation of bacteria and recombinant protein production. The BIOSTAT^® Cultibag RM system with the single-use sensors and advanced control system paves the way for the fed-batch technology also to rocking-motion-type bioreactors. It is possible to reach cell densities which are far above shake flasks and typical for stirred tank reactors with the improved oxygen transfer rate. For more simple applications the EnBase^® Flo method offers an easy and robust solution for rocking-motion-systems which do not have such advanced control possibilities.

     

The core of tau-paired helical filaments studied by scanning transmission electron microscopy and limited proteolysis

In Alzheimer's disease and frontotemporal dementias the microtubule-associated protein tau forms intracellular paired helical filaments (PHFs). The filaments formed in vivo consist mainly of full-length molecules of the six different isoforms present in adult brain. The substructure of the PHF core is still elusive. Here we applied scanning transmission electron microscopy (STEM) and limited proteolysis to probe the mass distribution of PHFs and their surface exposure. Tau filaments assembled from the three repeat domain have a mass per length (MPL) of approximately 60 kDa/nm and filaments from full-length tau (htau40DeltaK280 mutant) have approximately 160 kDa/nm, compared with approximately 130 kDa/nm for PHFs from Alzheimer's brain. Polyanionic cofactors such as heparin accelerate assembly but are not incorporated into PHFs. Limited proteolysis combined with N-terminal sequencing and mass spectrometry of fragments reveals a protease-sensitive N-terminal half and semiresistant PHF core starting in the first repeat and reaching to the C-terminus of tau. Continued proteolysis leads to a fragment starting at the end of the first repeat and ending in the fourth repeat. PHFs from tau isoforms with four repeats revealed an additional cleavage site within the middle of the second repeat. Probing the PHFs with antibodies detecting epitopes either over longer stretches in the C-terminal half of tau or in the fourth repeat revealed that they grow in a polar manner. These data describe the physical parameters of the PHFs and enabled us to build a model of the molecular arrangement within the filamentous structures.