Corrigendum: Measuring Locomotor Activity and Behavioral Aspects of Rodents Living in the Home-Cage.

[This corrects the article DOI: 10.3389/fnbeh.2022.877323.].

Measuring Locomotor Activity and Behavioral Aspects of Rodents Living in the Home-Cage.

Automatization and technological advances have led to a larger number of methods and systems to monitor and measure locomotor activity and more specific behavior of a wide variety of animal species in various environmental conditions in laboratory settings. In rodents, the majority of these systems require the animals to be temporarily taken away from their home-cage into separate observation cage environments which requires manual handling and consequently evokes distress for the animal and may alter behavioral responses. An automated high-throughput approach can overcome this problem. Therefore, this review describes existing automated methods and technologies which enable the measurement of locomotor activity and behavioral aspects of rodents in their most meaningful and stress-free laboratory environment: the home-cage. In line with the Directive 2010/63/EU and the 3R principles (replacement, reduction, refinement), this review furthermore assesses their suitability and potential for group-housed conditions as a refinement strategy, highlighting their current technological and practical limitations. It covers electrical capacitance technology and radio-frequency identification (RFID), which focus mainly on voluntary locomotor activity in both single and multiple rodents, respectively. Infrared beams and force plates expand the detection beyond locomotor activity toward basic behavioral traits but discover their full potential in individually housed rodents only. Despite the great premises of these approaches in terms of behavioral pattern recognition, more sophisticated methods, such as (RFID-assisted) video tracking technology need to be applied to enable the automated analysis of advanced behavioral aspects of individual animals in social housing conditions.

Sumoylation of Notch1 represses its target gene expression during cell stress.

The Notch signaling pathway is a key regulator of stem cells during development, and its deregulated activity is linked to developmental defects and cancer. Transcriptional activation of Notch target genes requires cleavage of the Notch receptor in response to ligand binding, production of the Notch intracellular domain (NICD1), NICD1 migration into the nucleus, and assembly of a transcriptional complex. Post-translational modifications of Notch regulate its trafficking, turnover, and transcriptional activity. Here, we show that NICD1 is modified by small ubiquitin-like modifier (SUMO) in a stress-inducible manner. Sumoylation occurs in the nucleus where NICD1 is sumoylated in the RBPJ-associated molecule (RAM) domain. Although stress and sumoylation enhance nuclear localization of NICD1, its transcriptional activity is attenuated. Molecular modeling indicates that sumoylation can occur within the DNA-bound ternary transcriptional complex, consisting of NICD1, the transcription factor Suppressor of Hairless (CSL), and the co-activator Mastermind-like (MAML) without its disruption. Mechanistically, sumoylation of NICD1 facilitates the recruitment of histone deacetylase 4 (HDAC4) to the Notch transcriptional complex to suppress Notch target gene expression. Stress-induced sumoylation decreases the NICD1-mediated induction of Notch target genes, which was abrogated by expressing a sumoylation-defected mutant in cells and in the developing central nervous system of the chick in vivo. Our findings of the stress-inducible sumoylation of NICD1 reveal a novel context-dependent regulatory mechanism of Notch target gene expression.

Keratins regulate colonic epithelial cell differentiation through the Notch1 signalling pathway.

Keratins (K) are intermediate filament proteins important in stress protection and mechanical support of epithelial tissues. K8, K18 and K19 are the main colonic keratins, and K8-knockout (K8-/-) mice display a keratin dose-dependent hyperproliferation of colonic crypts and a colitis-phenotype. However, the impact of the loss of K8 on intestinal cell differentiation has so far been unknown. Here we show that K8 regulates Notch1 signalling activity and differentiation in the epithelium of the large intestine. Proximity ligation and immunoprecipitation assays demonstrate that K8 and Notch1 co-localize and interact in cell cultures, and in vivo in the colonic epithelial cells. K8 with its heteropolymeric partner K18 enhance Notch1 protein levels and activity in a dose dependent manner. The levels of the full-length Notch1 receptor (FLN), the Notch1 intracellular domain (NICD) and expression of Notch1 downstream target genes are reduced in the absence of K8, and the K8-dependent loss of Notch1 activity can be rescued with re-expression of K8/K18 in K8-knockout CRISPR/Cas9 Caco-2 cells protein levels. In vivo, K8 deletion with subsequent Notch1 downregulation leads to a shift in differentiation towards a goblet cell and enteroendocrine phenotype from an enterocyte cell fate. Furthermore, the K8-/- colonic hyperproliferation results from an increased number of transit amplifying progenitor cells in these mice. K8/K18 thus interact with Notch1 and regulate Notch1 signalling activity during differentiation of the colonic epithelium.

Regulation of Treg-associated CD39 in multiple sclerosis and effects of corticotherapy during relapse.

BACKGROUND: Accumulating data highlight proinflammatory processes leading to MS relapses. Whether anti-inflammatory mechanisms are concomitantly activated is unclear. The ectonucleotidase CD39 has been described as a novel T regulatory cell (Treg) marker. The purpose of this study was to explore whether regulatory mechanisms are activated during MS relapses and reinforced by intravenous methylprednisolone (ivMP). METHODS: Blood samples were collected from stable and relapsing MS patients and healthy controls. We used FOXP3 methylation-specific qPCR and CD4(+)CD25(high)FOXP3(+) analysis to quantify Tregs. Cytokine mRNA expression levels were measured in peripheral blood mononuclear cells (PBMCs) and in CD4(+) T cells. CD39 expression was determined by flow cytometry in monocytes, NK, T and B cells. CD39 enzymatic activity was assessed by ATP luminometry. RESULTS: The proportion of Tregs was similar in relapsing MS patients and healthy controls. CD39 mRNA level was higher in PBMCs of relapsing MS patients than in controls. The proportion of CD39-expressing Tregs was higher in MS patients. IvMP decreased the overall proportion of Tregs while it increased CD39 mRNA levels, the proportions of CD39-expressing Tregs and monocytes as well as CD39 ectonucleotidase activity. CONCLUSIONS: Our data suggest that immunoregulatory mechanisms are ongoing in MS patients, particularly during relapses, and strengthened by ivMP.

Fingolimod increases CD39-expressing regulatory T cells in multiple sclerosis patients.

BACKGROUND: Multiple sclerosis (MS) likely results from an imbalance between regulatory and inflammatory immune processes. CD39 is an ectoenzyme that cleaves ATP to AMP and has been suggested as a novel regulatory T cells (Treg) marker. As ATP has numerous proinflammatory effects, its degradation by CD39 has anti-inflammatory influence. The purpose of this study was to explore regulatory and inflammatory mechanisms activated in fingolimod treated MS patients. METHODS AND FINDINGS: Peripheral blood mononuclear cells (PBMCs) were isolated from relapsing-remitting MS patients before starting fingolimod and three months after therapy start. mRNA expression was assessed in ex vivo PBMCs. The proportions of CD8, B cells, CD4 and CD39-expressing cells were analysed by flow cytometry. Treg proportion was quantified by flow cytometry and methylation-specific qPCR. Fingolimod treatment increased mRNA levels of CD39, AHR and CYP1B1 but decreased mRNA expression of IL-17, IL-22 and FOXP3 mRNA in PBMCs. B cells, CD4+ cells and Treg proportions were significantly reduced by this treatment, but remaining CD4+ T cells were enriched in FOXP3+ cells and in CD39-expressing Tregs. CONCLUSIONS: In addition to the decrease in circulating CD4+ T cells and CD19+ B cells, our findings highlight additional immunoregulatory mechanisms induced by fingolimod.

Effects of pool isolation on trophic ecology of fishes in a highland stream.

The objective of this study was to examine the effects of pool isolation on fish diet and to answer three questions: Were food resources different or reduced in isolated compared with connected pools? Were fishes more selective and did they increase resource partitioning in isolated pools? Would individuals exhibit increased gastrointestinal tract (GIT) length in isolation to aid nutrient absorption? Benthic macroinvertebrate density and richness were significantly lower in isolated pools compared with connected pools; fishes became more selective and partitioned resources more in isolated pools and all three species showed an increase in GIT length in isolated pools compared with connected pools. With a changing climate predicted to increase intermittency within headwater streams, the results show that these fishes have the adaptive ability to respond and continue to survive.

Infectious neuropathies.

PURPOSE OF REVIEW: Infectious neuropathies are heterogeneous neuropathies with multiple causes. They still represent an important world health burden and some of them have no current available therapy. RECENT FINDINGS: Leprosy incidence has decreased by 50% during the last years, but leprosy-related neuropathies still cause severe disability. The pure neuritic leprosy is a diagnostic challenge that may require nerve biopsy or nerve aspiration cytology. The treatment itself may lead to a 'reversal reaction', which further causes injuries to the nerve. HCV-related neuropathies may be related or not to the presence of cryoglobulins. The absence of vasculitis, the most frequent form is a peripheral sensory neuropathy involving small nerve fibers, and more accurately diagnosed by pain-related evoked potentials. HIV-related neuropathy has become the major neurological complication of HIV infection. Both HIV-induced neuropathy and antiretroviral toxic neuropathy are clinically indistinguishable. The existence of an isolated chronic polyneuropathy due to Borrelia burgdorferi remains highly controversial. Lastly, an active infectious ganglioneuritis caused by varicella zoster virus, producing shingles, is the most frequent infectious neuropathy in the world and may cause various neurological complications. Zoster sine herpete remains frequently undiagnosed. SUMMARY: Recent data have improved our knowledge and diagnostic tools of infectious neuropathies. Treatment of the injured nerves is not yet available, and prevention and rapid diagnosis remain the main priorities for the clinician.

The glove-like structure of the conserved membrane protein TatC provides insight into signal sequence recognition in twin-arginine translocation.

In bacteria, two signal-sequence-dependent secretion pathways translocate proteins across the cytoplasmic membrane. Although the mechanism of the ubiquitous general secretory pathway is becoming well understood, that of the twin-arginine translocation pathway, responsible for translocation of folded proteins across the bilayer, is more mysterious. TatC, the largest and most conserved of three integral membrane components, provides the initial binding site of the signal sequence prior to pore assembly. Here, we present two crystal structures of TatC from the thermophilic bacteria Aquifex aeolicus at 4.0 Å and 6.8 Å resolution. The membrane architecture of TatC includes a glove-shaped structure with a lipid-exposed pocket predicted by molecular dynamics to distort the membrane. Correlating the biochemical literature to these results suggests that the signal sequence binds in this pocket, leading to structural changes that facilitate higher order assemblies.

Spatiotemporal dispersion and wave envelopes with relativistic and pseudorelativistic characteristics.

A generic nonparaxial model for pulse envelopes is presented. Classic Schrödinger-type descriptions of wave propagation have their origins in slowly-varying envelopes combined with a Galilean boost to the local time frame. By abandoning these two simplifications, a picture of pulse evolution emerges in which frame-of-reference considerations and space-time transformations take center stage. A wide range of effects, analogous to those in special relativity, then follows for both linear and nonlinear systems. Explicit demonstration is presented through exact bright and dark soliton pulse solutions.

The complex process of GETting tail-anchored membrane proteins to the ER.

Biosynthesis of membrane proteins requires that hydrophobic transmembrane (TM) regions be shielded from the cytoplasm while being directed to the correct membrane. Tail-anchored (TA) membrane proteins, characterized by a single C-terminal TM, pose an additional level of complexity because they must be post-translationally targeted. In eukaryotes, the GET pathway shuttles TA-proteins to the endoplasmic reticulum. The key proteins required in yeast (Sgt2 and Get1-5) have been under extensive structural and biochemical investigation during recent years. The central protein Get3 utilizes nucleotide linked conformational changes to facilitate substrate loading and targeting. Here we analyze this complex process from a structural perspective, as understood in yeast, and further postulate on similar pathways in other domains of life.

Tail-anchor targeting by a Get3 tetramer: the structure of an archaeal homologue.

Efficient delivery of membrane proteins is a critical cellular process. The recently elucidated GET (Guided Entry of TA proteins) pathway is responsible for the targeted delivery of tail-anchored (TA) membrane proteins to the endoplasmic reticulum. The central player is the ATPase Get3, which in its free form exists as a dimer. Biochemical evidence suggests a role for a tetramer of Get3. Here, we present the first crystal structure of an archaeal Get3 homologue that exists as a tetramer and is capable of TA protein binding. The tetramer generates a hydrophobic chamber that we propose binds the TA protein. We use small-angle X-ray scattering to provide the first structural information of a fungal Get3/TA protein complex showing that the overall molecular envelope is consistent with the archaeal tetramer structure. Moreover, we show that this fungal tetramer complex is capable of TA insertion. This allows us to suggest a model where a tetramer of Get3 sequesters a TA protein during targeting to the membrane.

Automatic miniaturized fluorometric flow system for chemical and toxicological control of glibenclamide.

In this work, and for the first time, it was developed an automatic and fast screening miniaturized flow system for the toxicological control of glibenclamide in beverages, with application in forensic laboratory investigations, and also, for the chemical control of commercially available pharmaceutical formulations. The automatic system exploited the multipumping flow (MPFS) concept and allowed the implementation of a new glibenclamide determination method based on the fluorometric monitoring of the drug in acidic medium (λ(ex)=301 nm; λ(em)=404 nm), in the presence of an anionic surfactant (SDS), promoting an organized micellar medium to enhance the fluorometric measurements. The developed approach assured good recoveries in the analysis of five spiked alcoholic beverages. Additionally, a good agreement was verified when comparing the results obtained in the determination of glibenclamide in five commercial pharmaceutical formulations by the proposed method and by the pharmacopoeia reference procedure.

Structural characterization of the Get4/Get5 complex and its interaction with Get3.

The recently elucidated Get proteins are responsible for the targeted delivery of the majority of tail-anchored (TA) proteins to the endoplasmic reticulum. Get4 and Get5 have been identified in the early steps of the pathway mediating TA substrate delivery to the cytoplasmic targeting factor Get3. Here we report a crystal structure of Get4 and an N-terminal fragment of Get5 from Saccharomyces cerevisae. We show Get4 and Get5 (Get4/5) form an intimate complex that exists as a dimer (two copies of Get4/5) mediated by the C-terminus of Get5. We further demonstrate that Get3 specifically binds to a conserved surface on Get4 in a nucleotide dependent manner. This work provides further evidence for a model in which Get4/5 operates upstream of Get3 and mediates the specific delivery of a TA substrate.

Model for eukaryotic tail-anchored protein binding based on the structure of Get3.

The Get3 ATPase directs the delivery of tail-anchored (TA) proteins to the endoplasmic reticulum (ER). TA-proteins are characterized by having a single transmembrane helix (TM) at their extreme C terminus and include many essential proteins, such as SNAREs, apoptosis factors, and protein translocation components. These proteins cannot follow the SRP-dependent co-translational pathway that typifies most integral membrane proteins; instead, post-translationally, these proteins are recognized and bound by Get3 then delivered to the ER in the ATP dependent Get pathway. To elucidate a molecular mechanism for TA protein binding by Get3 we have determined three crystal structures in apo and ADP forms from Saccharomyces cerevisae (ScGet3-apo) and Aspergillus fumigatus (AfGet3-apo and AfGet3-ADP). Using structural information, we generated mutants to confirm important interfaces and essential residues. These results point to a model of how Get3 couples ATP hydrolysis to the binding and release of TA-proteins.