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Hydrological alteration has contributed to the global decline of stream frogs. Flows support stream frog reproduction, juvenile development, food resources, and maintain habitats for all life stages. At present, there is a lack of information regarding the specific water requirements necessary for the conservation of stream frogs. To address this gap, we developed a traits-based approach that serves as a valuable tool for grouping and prioritising water-dependent stream frog species to inform future research priorities and environmental flow design. In this study, we focussed on 53 Australian frog species and analysed eight species traits to develop water-dependent functional groups for stream frogs. We classified frogs based on their level of water dependency using an agglomerative hierarchical clustering analysis and a systematic review of water requirements and water management threats. The distinguishing traits that determined functional groups were: tadpole body type, egg clutch type, stream breeding habitat type, and documented association with flowing water and water permanence. Our study identified two distinct water-dependent groups: facultative stream spawners, capable of reproducing in both stream and non-stream habitats, and obligate stream spawners, restricted solely to stream habitats. Importantly, we highlight that the obligate stream spawners are the most sensitive group to within-channel flow alteration and should be prioritised for water management decisions in lotic environments. This study represents the first comprehensive overview of the importance of hydrology for stream frogs and identifies the critical need for additional research and validation to enhance our understanding of stream frog responses to flows regimes.

New and innovative techniques utilizing underwater cameras have the potential to track changes in turtle populations whilst being less invasive and time intensive than traditional trapping surveys. In this study, we trialled the use of Baited Remote Underwater Videos (BRUVs) in five upland streams to test if BRUVs could be used for abundance, size and sex estimates of freshwater turtles. We also compared BRUVs to cathedral traps and identified key considerations for future use of this method, such as whether the time of day influenced results. Our study successfully detected 83 turtles from 52 h of footage and identified all species known to occur in the study area. The deployment time did not significantly influence relative abundance. BRUVs also produced statistically similar catch per unit effort estimates to cathedral trapping techniques. No small turtles (i.e. juveniles) were detected, and difficulties with visibility, sex accuracy and carapace measurements are discussed for future BRUV use. Our study shows that BRUVs are a useful, time effective, non-invasive technique to collect relative abundance and species richness estimates for freshwater turtles.

Oligodendrocyte production and myelination continues lifelong in the central nervous system (CNS), and all stages of this process can be adaptively regulated by neuronal activity. While artificial exogenous stimulation of neuronal circuits greatly enhances oligodendrocyte progenitor cell (OPC) production and increases myelination during development, the extent to which physiological stimuli replicates this is unclear, particularly in the adult CNS when the rate of new myelin addition slows. Here, we used environmental enrichment (EE) to physiologically stimulate neuronal activity for 6 weeks in 9-week-old C57BL/six male and female mice and found no increase in compact myelin in the corpus callosum or somatosensory cortex. Instead, we observed a global increase in callosal axon diameter with thicker myelin sheaths, elongated paranodes and shortened nodes of Ranvier. These findings indicate that EE induced the dynamic structural remodelling of myelinated axons. Additionally, we observed a global increase in the differentiation of OPCs and pre-myelinating oligodendroglia in the corpus callosum and somatosensory cortex. Our findings of structural remodelling of myelinated axons in response to physiological neural stimuli during young adulthood provide important insights in understanding experience-dependent myelin plasticity throughout the lifespan and provide a platform to investigate axon-myelin interactions in a physiologically relevant context.

The p75 neurotrophin receptor (p75NTR ) is required for maintaining peripheral sensory neuron survival and function; however, the underlying cellular mechanism remains unclear. The general view is that expression of p75NTR by the neuron itself is required for maintaining sensory neuron survival and myelination in the peripheral nervous system (PNS). Adopting a neuronal-specific conditional knockout strategy, we demonstrate the partial depletion of p75NTR in neurons exerts little influence upon maintaining sensory neuron survival and peripheral nerve myelination in health and after demyelinating neuropathy. Our data show that the density and total number of dorsal root ganglion (DRG) neurons in 2-month-old mice is not affected following the deletion of p75NTR in large-diameter myelinating neurons, as assessed by stereology. Adopting experimental autoimmune neuritis induced in adult male mice, an animal model of demyelinating peripheral neuropathy, we identify that deleting p75NTR in myelinating neurons exerts no influence upon the disease progression, the total number of DRG neurons, and the extent of myelin damage in the sciatic nerve, indicating that the expression of neuronal p75NTR is not essential for maintaining peripheral neuron survival and myelination after a demyelinating insult in vivo. Together, results of this study suggest that the survival and myelination of peripheral sensory neurons is independent of p75NTR expressed by a subtype of neurons in vivo. Thus, our findings provide new insights into the mechanism underpinning p75NTR -mediated neuronal survival in the PNS.

Schwann cell reprogramming and differentiation are crucial prerequisites for neuronal regeneration and re-myelination to occur following injury to peripheral nerves. The neurotrophin receptor p75NTR has been identified as a positive modulator for Schwann cell myelination during development and implicated in promoting nerve regeneration after injury. However, most studies base this conclusion on results obtained from complete p75NTR knockout mouse models and cannot dissect the specific role of p75NTR expressed by Schwann cells. In this present study, a conditional knockout model selectively deleting p75NTR expression in Schwann cells was generated, where p75NTR expression is replaced with that of an mCherry reporter. Silencing of Schwann cell p75NTR expression was confirmed in the sciatic nerve in vivo and in vitro, without altering axonal expression of p75NTR. No difference in sciatic nerve myelination during development or following sciatic nerve crush injury was observed, as determined by quantification of both myelinated and unmyelinated nerve fiber densities, myelinated axonal diameter and myelin thickness. However, the absence of Schwann cell p75NTR reduced motor nerve conduction velocity after crush injury. Our data indicate that the absence of Schwann cell p75NTR expression in vivo is not critical for axonal regrowth or remyelination following sciatic nerve crush injury, but does play a key role in functional recovery. Overall, this represents the first step in redefining the role of p75NTR in the peripheral nervous system, suggesting that the Schwann cell-axon unit functions as a syncytium, with the previous published involvement of p75NTR in remyelination most likely depending on axonal/neuronal p75NTR and/or mutual glial-axonal interactions.

The neurotrophin, brain-derived neurotrophic factor (BDNF) promotes central nervous system (CNS) myelination during development and after injury. This is achieved via activation of oligodendrocyte-expressed tropomyosin-related kinase (Trk) B receptors. However, while administration of BDNF has shown beneficial effects, BDNF itself has a poor pharmacokinetic profile. Here, we compare two TrkB-targeted BDNF-mimetics, the structural-mimetic, tricyclic dimeric peptide-6 (TDP6) and the non-peptide small molecule TrkB agonist LM22A-4 in a cuprizone model of central demyelination in female mice. Both mimetics promoted remyelination, increasing myelin sheath thickness and oligodendrocyte densities after 1-week recovery. Importantly, LM22A-4 exerts these effects in an oligodendroglial TrkB-dependent manner. However, analysis of TrkB signaling by LM22A-4 suggests rather than direct activation of TrkB, LM22A-4 exerts its effects via indirect transactivation of Trk receptors. Overall, these studies support the therapeutic strategy to selectively targeting TrkB activation to promote remyelination in the brain.

Developing a high-throughput approach to quantify the extent of myelin integrity in preclinical models of demyelinating diseases will enhance our capacity to identify novel therapies for myelin repair. In light of the technical limitations of electron microscopy and immunohistochemical analyses of myelination, we have utilized a novel imaging technique, spectral confocal reflectance (SCoRe) microscopy. SCoRe takes advantage of the optically reflective properties of compact myelin, allowing the integrity of compact myelin to be quantified over the course of the cuprizone-induced model of central demyelination. We applied SCoRe imaging on fixed frozen brain sections. SCoRe analysis of control mice identified an increase in corpus callosum myelination during the period of cuprizone administration and recovery, suggesting that the normal developmental processes of myelination are ongoing at this time. Importantly, analysis of mice subjected to cuprizone identified a significant reduction in compact myelin in both rostral and caudal corpus callosum compared to age-matched control mice. SCoRe microscopy also allowed the visualization and quantification of the amount of myelin debris in demyelinating lesions. Combining SCoRe imaging with immunohistochemistry, we quantified the amount of myelin debris within IBA-1+ microglia and found that 11% of myelin debris colocalized in microglia irrespective of the callosal regions, with the vast majority of debris outside of microglia. In summary, we have demonstrated that SCoRe microscopy is an effective and powerful tool to perform both quantitative and qualitative analyses of compact myelin integrity in health or after injury in vivo, demonstrating its future application in high-throughput assessments and screening of the therapeutic efficacy of myelin repair therapies in preclinical animal models of demyelinating diseases.

Blocking inhibitory factors within CNS demyelinating lesions is regarded as a promising strategy to promote remyelination. Bone morphogenetic protein 4 (BMP4) is an inhibitory factor present in demyelinating lesions. Noggin, an endogenous antagonist to BMP, has previously been shown to increase the number of oligodendrocytes and promote remyelination in vivo. However, it remains unclear how BMP4 signaling inhibits remyelination. Here we investigated the downstream signaling pathway that mediates the inhibitory effect that BMP4 exerts upon remyelination through pharmacological and transgenic approaches. Using the cuprizone mouse model of central demyelination, we demonstrate that selectively blocking BMP4 signaling via the pharmacological inhibitor LDN-193189 significantly promotes oligodendroglial differentiation and the extent of remyelination in vivo This was accompanied by the downregulation of transcriptional targets that suppress oligodendrocyte differentiation. Further, selective deletion of BMP receptor type IA (BMPRIA) within primary mouse oligodendrocyte progenitor cells (OPCs) significantly enhanced their differentiation and subsequent myelination in vitro Together, the results of this study identify that BMP4 signals via BMPRIA within OPCs to inhibit oligodendroglial differentiation and their capacity to myelinate axons, and suggest that blocking the BMP4/BMPRIA pathway in OPCs is a promising strategy to promote CNS remyelination.

The pool of quality control proteins (QC) that maintains protein-folding homeostasis (proteostasis) is dynamic but can become depleted in human disease. A challenge has been in quantitatively defining the depth of the QC pool. With a new biosensor, flow cytometry-based methods and mathematical modeling we measure the QC capacity to act as holdases and suppress biosensor aggregation. The biosensor system comprises a series of barnase kernels with differing folding stability that engage primarily with HSP70 and HSP90 family proteins. Conditions of proteostasis stimulation and stress alter QC holdase activity and aggregation rates. The method reveals the HSP70 chaperone cycle to be rate limited by HSP70 holdase activity under normal conditions, but this is overcome by increasing levels of the BAG1 nucleotide exchange factor to HSPA1A or activation of the heat shock gene cluster by HSF1 overexpression. This scheme opens new paths for biosensors of disease and proteostasis systems.

When proteostasis becomes unbalanced, unfolded proteins can accumulate and aggregate. Here we report that the dye, tetraphenylethene maleimide (TPE-MI) can be used to measure cellular unfolded protein load. TPE-MI fluorescence is activated upon labelling free cysteine thiols, normally buried in the core of globular proteins that are exposed upon unfolding. Crucially TPE-MI does not become fluorescent when conjugated to soluble glutathione. We find that TPE-MI fluorescence is enhanced upon reaction with cellular proteomes under conditions promoting accumulation of unfolded proteins. TPE-MI reactivity can be used to track which proteins expose more cysteine residues under stress through proteomic analysis. We show that TPE-MI can report imbalances in proteostasis in induced pluripotent stem cell models of Huntington disease, as well as cells transfected with mutant Huntington exon 1 before the formation of visible aggregates. TPE-MI also detects protein damage following dihydroartemisinin treatment of the malaria parasites Plasmodium falciparum. TPE-MI therefore holds promise as a tool to probe proteostasis mechanisms in disease.Proteostasis is maintained through a number of molecular mechanisms, some of which function to protect the folded state of proteins. Here the authors demonstrate the use of TPE-MI in a fluorigenic dye assay for the quantitation of unfolded proteins that can be used to assess proteostasis on a cellular or proteome scale.

Protein aggregation is a hallmark of the major neurodegenerative diseases including Alzheimer's, Parkinson's, Huntington's and motor neuron and is a symptom of a breakdown in the management of proteome foldedness. Indeed, it is remarkable that under normal conditions cells can keep their proteome in a highly crowded and confined space without uncontrollable aggregation. Proteins pose a particular challenge relative to other classes of biomolecules because upon synthesis they must typically follow a complex folding pathway to reach their functional conformation (native state). Non-native conformations, including the unfolded nascent chain, are highly prone to aberrant interactions, leading to aggregation. Here we review recent advances in knowledge of proteostasis, approaches to monitor proteostasis and the impact that protein aggregation has on biology. We also include discussion of the outstanding challenges. © 2017 IUBMB Life, 69(2):49-54, 2017.

Axonal damage and demyelination are major determinants of disability in patients with peripheral demyelinating neuropathies. The neurotrophin family of growth factors are essential for the normal development and myelination of the peripheral nervous system (PNS), and as such are potential therapeutic candidates for ameliorating axonal and myelin damage. In particular, BDNF promotes peripheral nerve myelination via p75 neurotrophin receptor (p75NTR) receptors. Here, we investigated the therapeutic efficacy of a small structural mimetic of the region of BDNF that binds to p75NTR (cyclo-dPAKKR) in experimental autoimmune neuritis (EAN), an established animal model of peripheral demyelinating neuropathy. Examination of rodents induced with EAN revealed that p75NTR is abundantly expressed in affected peripheral nerves. We found that systemic administration of cyclo-dPAKKR ameliorates EAN disease severity and accelerates recovery. Animals treated with cyclo-dPAKKR displayed significantly better motor performance compared to control animals. Histological assessment revealed that cyclo-dPAKKR administration limits the extent of inflammatory demyelination and axonal damage, and protects against the disruption of nodal architecture in affected peripheral nerves. In contrast, a structural control peptide of cyclo-dPAKKR exerted no influence. Moreover, all the beneficial effects of cyclo-dPAKKR in EAN are abrogated in p75NTR heterozygous mice, strongly suggesting a p75NTR-dependent effect. Taken together, our data demonstrate that cyclo-dPAKKR ameliorates functional and pathological defects of EAN in a p75NTR-dependant manner, suggesting that p75NTR is a therapeutic target to consider for future treatment of peripheral demyelinating diseases and targeting of p75NTR is a strategy worthy of further investigation.

Undulatory motion of the body is the dominant mode of locomotion in fishes, and numerous studies of body kinematics and muscle activity patterns have provided insights into the mechanics of swimming. However, it has not been possible to investigate how key parameters such as the extent of bilateral muscle activation affect propulsive performance due to the inability to manipulate muscle activation in live, freely swimming fishes. In this article we extend previous work on passive flexible mechanical models of undulatory propulsion by using actively controlled pneumatic actuators attached to a flexible foil to gain insight into undulatory locomotion and mechanisms for body stiffness control. Two soft actuators were attached on each side of a flexible panel with stiffness comparable to that of a fish body. To study how bilateral contraction can be used to modify axial body stiffness during swimming, we ran a parameter sweep of actuator contraction phasing and frequency. Thrust production by the soft pneumatic actuators was tested at cyclic undulation frequencies ranging from 0.3 to 1.2 Hz in a recirculating flow tank at flow speeds up to 28 cm/s. Overall, this system generated more thrust at higher tail beat frequencies, with a plateau in thrust above 0.8 Hz. Self-propelled speed was found to be 0.8 foil lengths per second or ∼13 cm/s when actuated at 0.55 Hz. This active pneumatic model is capable of producing substantial trailing edge amplitudes with a maximum excursion equivalent to 1.4 foil lengths, and of generating considerable thrust. Altering the extent of bilateral co-contraction in a range from -22% to 17% of the cycle period showed that thrust was maximized with some amount of simultaneous left-right actuation of ∼3% to 6% of the cycle period. When the system is exposed to water flow, thrust was substantially reduced for conditions of greatest antagonistic overlap in left-right actuation, and also for the largest latencies introduced. This experimental plat

© 2017 Elsevier B.V. Diffuse agricultural runoff into rivers can result in contamination with herbicides for prolonged periods of time. Chronic exposure to herbicides has the potential to alter toxic impacts in primary producers such as benthic diatoms. Determining how individual diatom taxa respond to herbicide exposure over varied exposure durations is essential for assessing herbicide impacts. This study investigated the responses of various benthic diatom taxa and effects at the community level over 12 days of atrazine exposure. Diatom communities were collected from two sites with differing exposure histories; a relatively unpolluted site (Alligator Creek) and an agricultural stream (Barratta Creek) known to be polluted by atrazine and other herbicides. Diatom community composition and the proportion of healthy cells per taxon were assessed at 0, 2, 3, 6, 9 and 12 days of atrazine exposure. Pollution history altered the response of the diatom community to atrazine exposure. In the Alligator Creek diatom community there was a shift in composition towards more tolerant taxa and the loss of sensitive taxa in atrazine exposed treatments. The sensitive taxon (Gomphonema truncatum) was consistently affected by atrazine toxicity. Conversely, the polluted Barratta Creek diatom community was not strongly affected by atrazine exposure. Our study shows that during chronic atrazine exposure some taxa demonstrated the ability to recover despite initial toxicity response. Recovery could be an important trait for understanding the ecological effect of herbicide exposure on diatom species in nature and in applied circumstances such as biomonitoring indices.

c-Src kinase activity is regulated by phosphorylation of Y527 and Y416. Y527 phosphorylation stabilizes a closed conformation, which suppresses kinase activity towards substrates, whereas phosphorylation at Y416 promotes an elevated kinase activity by stabilizing the activation loop in a manner permissive for substrate binding. Here we investigated the correlation of Y416 phosphorylation with c-Src activity when c-Src was locked into the open and closed conformations (by mutations Y527F and Q528E, P529E, G530I respectively). Consistent with prior findings, we found Y416 to be more greatly phosphorylated when c-Src was in an open, active conformation. However, we also observed an appreciable amount of Y416 was phosphorylated when c-Src was in a closed, repressed conformation under conditions by which c-Src was unable to phosphorylate substrate STAT3. The phosphorylation of Y416 in the closed conformation arose by autophosphorylation, since abolishing kinase activity by mutating the ATP binding site (K295M) prevented phosphorylation. Basal Y416 phosphorylation correlated positively with cellular levels of c-Src suggesting autophosphorylation depended on self-association. Using sedimentation velocity analysis on cell lysate with fluorescence detection optics, we confirmed that c-Src forms monomers and dimers, with the open conformation also forming a minor population of larger mass complexes. Collectively, our studies suggest a model by which dimerization of c-Src primes c-Src via Y416 phosphorylation to enable rapid potentiation of activity when Src adopts an open conformation. Once in the open conformation, c-Src can amplify the response by recruiting and phosphorylating substrates such as STAT3 and increasing the extent of autophosphorylation.

Carboni's regular completion doctrine is extended to a KZ-doctrine on a 2-category whose objects are all categories and whose arrows are functors which preserve kernel arrows. The algebras for the extended doctrine are categories with regular factorizations in which regular epimorphisms are closed under composition. (C) 2002 Elsevier Science B.V. All rights reserved.