Microtubule lattice spacing governs cohesive envelope formation of tau family proteins.

Tau is an intrinsically disordered microtubule-associated protein (MAP) implicated in neurodegenerative disease. On microtubules, tau molecules segregate into two kinetically distinct phases, consisting of either independently diffusing molecules or interacting molecules that form cohesive 'envelopes' around microtubules. Envelopes differentially regulate lattice accessibility for other MAPs, but the mechanism of envelope formation remains unclear. Here we find that tau envelopes form cooperatively, locally altering the spacing of tubulin dimers within the microtubule lattice. Envelope formation compacted the underlying lattice, whereas lattice extension induced tau envelope disassembly. Investigating other members of the tau family, we find that MAP2 similarly forms envelopes governed by lattice spacing, whereas MAP4 cannot. Envelopes differentially biased motor protein movement, suggesting that tau family members could spatially divide the microtubule surface into functionally distinct regions. We conclude that the interdependent allostery between lattice spacing and cooperative envelope formation provides the molecular basis for spatial regulation of microtubule-based processes by tau and MAP2.

Sustainable kidney care delivery and climate change - a call to action.

The delivery of kidney care, particularly haemodialysis treatment, can result in substantial environmental impact through greenhouse emissions, natural resources depletion and waste generation. However, strategies exist to mitigate this impact and improve long term environmental sustainability for the provision of haemodialysis treatment. The nephrology community has begun taking actions to improve the environmental sustainability of dialysis, but much work remains to be done by healthcare professionals, dialysis providers and professional organisations.

Autoregulatory control of microtubule binding in doublecortin-like kinase 1.

The microtubule-associated protein, doublecortin-like kinase 1 (DCLK1), is highly expressed in a range of cancers and is a prominent therapeutic target for kinase inhibitors. The physiological roles of DCLK1 kinase activity and how it is regulated remain elusive. Here, we analyze the role of mammalian DCLK1 kinase activity in regulating microtubule binding. We found that DCLK1 autophosphorylates a residue within its C-terminal tail to restrict its kinase activity and prevent aberrant hyperphosphorylation within its microtubule-binding domain. Removal of the C-terminal tail or mutation of this residue causes an increase in phosphorylation within the doublecortin domains, which abolishes microtubule binding. Therefore, autophosphorylation at specific sites within DCLK1 has diametric effects on the molecule's association with microtubules. Our results suggest a mechanism by which DCLK1 modulates its kinase activity to tune its microtubule-binding affinity. These results provide molecular insights for future therapeutic efforts related to DCLK1's role in cancer development and progression.

A concept mobility device with multi-positional configurations and child-kind restraint for safe perioperative transfer and induction of anaesthesia in children with autistic spectrum disorder - a cross sectional study.

BACKGROUND: Transfer into the operating room, onto the operating table and mask induction of anaesthesia are major challenges faced by children with Autistic Spectrum Disorder (ASD). In a pilot study, parents observed that perioperative transfer becomes unsafe and difficult when children with ASD becomes uncooperative. METHOD: A CHILD-KIND CONCEPT mobility system comprising of multi-positioning seat configurations and restraining module was developed with inputs from multi-disciplinary healthcare professionals and parents with children with ASD. To appeal to children and motivate child-machine interaction, the seat configurations and restraining module are designed to take the form of child-friendly, non-threatening, fun and familiar items. The sitting configuration, sitting to supine transformation, the restraint modules resemble racing-car seat, reclining motion of a home massage chair, safety restraints found in airplanes and amusement rides respectively. Healthcare professionals (HCPs) involved in the perioperative patient care, parents of ASD children and children (neurotypical and ASD) experience the use of the system in a non-clinical environment and participated in a survey study. The acceptance of its functionality (HCPs, parents) for perioperative transfer and induction of anaesthesia, rating of the user experience and likes and dislikes of (parents and children) were obtained. RESULTS: Thirty-two HCPs, 30 parents and 23 children participated. Majority of parents and HCPs opined the use of the system enables improvement in the management of perioperative movement (90.0% parents, 100% HCPs), safe perioperative movement (86.7% parents, 96.9% HCPs) and promotes ease of anaesthesia induction (76.7% parents, 90.6% HCPs) for uncooperative combative ASD children. Overall, 93.8% HCPs and 86.7% parents would recommend its frequent use in their own practice and their ASD children respectively. Attractiveness and multi-functionality are attributes endorsed by parents and children. Children endorse its use for induction of anaesthesia (73.9%), dental chair (82.6%), intra-hospital transfer (95.7%). CONCLUSION: A child-kind mobility device that integrates appeal with functionality of restraint and multi-positional transformation has a potential to promote safe perioperative movement and ease of induction of anaesthesia in anxious uncooperative ASD children.

A Combinatorial MAP Code Dictates Polarized Microtubule Transport.

Many eukaryotic cells distribute their intracellular components asymmetrically through regulated active transport driven by molecular motors along microtubule tracks. While intrinsic and extrinsic regulation of motor activity exists, what governs the overall distribution of activated motor-cargo complexes within cells remains unclear. Here, we utilize in vitro reconstitution of purified motor proteins and non-enzymatic microtubule-associated proteins (MAPs) to demonstrate that MAPs exhibit distinct influences on the motility of the three main classes of transport motors: kinesin-1, kinesin-3, and cytoplasmic dynein. Further, we dissect how combinations of MAPs affect motors and unveil MAP9 as a positive modulator of kinesin-3 motility. From these data, we propose a general "MAP code" that has the capacity to strongly bias directed movement along microtubules and helps elucidate the intricate intracellular sorting observed in highly polarized cells such as neurons.

Microtubules gate tau condensation to spatially regulate microtubule functions.

Tau is an abundant microtubule-associated protein in neurons. Tau aggregation into insoluble fibrils is a hallmark of Alzheimer's disease and other types of dementia1, yet the physiological state of tau molecules within cells remains unclear. Using single-molecule imaging, we directly observe that the microtubule lattice regulates reversible tau self-association, leading to localized, dynamic condensation of tau molecules on the microtubule surface. Tau condensates form selectively permissible barriers, spatially regulating the activity of microtubule-severing enzymes and the movement of molecular motors through their boundaries. We propose that reversible self-association of tau molecules, gated by the microtubule lattice, is an important mechanism of the biological functions of tau, and that oligomerization of tau is a common property shared between the physiological and disease-associated forms of the molecule.

Salvage of exhausted neck access using a novel inside-out device in dialysis-dependent patients.

Chronic central venous occlusion represents a challenge for upper body central dialysis access for both surgical and endovascular interventions. The Surfacer (Surfacer Inside-Out Access Catheter System) is a novel technique that offers a device-based solution over various physician-modified techniques in salvaging exhausted right-sided neck veins for catheter placement. We report a retrospective series of four cases in which this system is used to gain upper body venous access from a single center in Singapore. This report seeks to integrate the understanding of the technical aspects of a new inside-out device and its utility in recanalization of occluded right-sided neck veins as it relates to Asian patients.

A mouthful - airway matters in intraoperative neuromonitoring in auditory brainstem implant surgery for the pediatric patient: a case series.

BACKGROUND: The Auditory brainstem implant (ABI) is a new surgical option for hearing impaired children. Intraoperative neurophysiology monitoring includes brainstem mapping of cranial nerve (CN) IX, X, XI, XII and their motor nuclei, and corticobulbar tract motor-evoked potential. These require laryngeal electrodes and intra-oral pins, posing a challenge to airway management especially in the pediatric airway, where specialized electromyogram (EMG) tracheal tubes are not available. Challenges include determining the optimum position on the endotracheal tube (ETT) in which to place laryngeal electrode, and the increase in external diameter of ETT contributed by the wrapping the electrode around the shaft of ETT; this may necessitate downsizing of the tracheal tube. An appropriate size ETT minimizes displacement, which in turn can affect electrode contact with the vocal cords. Finally, a small thus crowded pediatric airway makes for difficult visualization during placement of intraoral neuromonitoring electrodes. The use of a videolaryngoscope helps determine optimum electrode placement. CASE PRESENTATION: We describe intraoperative neurophysiology monitoring and airway management for the first two ABI procedures in Singapore, conducted for children with congenitally absent cochlear nerves. CONCLUSION: Neurophysiology cranial nerve IX, X, XII monitoring in the ABI procedure requires intraoral placement of electrodes. Care should be exercised during placement and removal. Vagus nerve monitoring in children requires attention to tube preparation, and consideration should be given to avoidance of airway topicalization.

Changes in Near-Infrared Spectroscopy After Congenital Cyanotic Heart Surgery.

BACKGROUND: Since oxygen saturation from pulse oximetry (SpO2) and partial pressure of arterial oxygen (PaO2) are observed to improve immediately after surgical correction of cyanotic congenital heart disease (CHD), we postulate that cerebral (CrO2) and somatic (SrO2) oximetry also improves immediately post-correction. We aim to prospectively examine CrO2 and SrO2, before, during, and after surgical correction as well as on hospital discharge in children with cyanotic CHD to determine if and when these variables increase. METHODS: This is a prospective observational trial. Eligibility criteria included children below 18 years of age with cyanotic CHD who required any cardiac surgical procedure. CrO2 and SrO2 measurements were summarized at six time-points for comparison: (1) pre-cardiopulmonary bypass (CPB); (2) during CPB; (3) post-CPB; (4) Day 1 in the pediatric intensive care unit (PICU); (5) Day 2 PICU; and (6) discharge. Categorical and continuous variables are presented as counts (percentages) and median (interquartile range), respectively. RESULTS: Twenty-one patients were analyzed. 15 (71.4%) and 6 (28.6%) patients underwent corrective and palliative surgeries, respectively. In the corrective surgery group, SpO2 increased immediately post-CPB compared to pre-CPB [99 (98, 100) vs. 86% (79, 90); p < 0.001] and remained in the normal range through to hospital discharge. Post-CPB CrO2 did not change from pre-CPB [72.8 (58.8, 79.0) vs. 72.1% (63.0, 78.3); p = 0.761] and even decreased on hospital discharge [60.5 (53.6, 62.9) vs. 72.1% (63.0, 78.3); p = 0.005]. Post-CPB SrO2 increased compared to pre-CPB [87.3 (77.2, 89.5) vs. 72.7% (65.6, 77.3); p = 0.001] but progressively decreased during PICU stay to a value lower than baseline at hospital discharge [66.9 (57.3, 76.9) vs. 72.7% (65.6, 77.3); p = 0.048]. CONCLUSION: CrO2 and SrO2 did not increase after corrective surgery of cyanotic CHD even up to hospital discharge. Future larger studies are required to validate these findings. (This study is registered with ClinicalTrials.gov ID: NCT02417259.).

Competition between microtubule-associated proteins directs motor transport.

Within cells, motor and non-motor microtubule-associated proteins (MAPs) simultaneously converge on the microtubule. How the binding activities of non-motor MAPs are coordinated and how they contribute to the balance and distribution of motor transport is unknown. Here, we examine the relationship between MAP7 and tau owing to their antagonistic roles in vivo. We find that MAP7 and tau compete for binding to microtubules, and determine a mechanism by which MAP7 displaces tau from the lattice. MAP7 promotes kinesin-based transport in vivo and strongly recruits kinesin-1 to the microtubule in vitro, providing evidence for direct enhancement of motor motility by a MAP. Both MAP7 and tau strongly inhibit kinesin-3 and have no effect on cytoplasmic dynein, demonstrating that MAPs differentially control distinct classes of motors. Overall, these results reveal a general principle for how MAP competition dictates access to the microtubule to determine the correct distribution and balance of motor activity.

Pigmented Porokeratosis. A Further Variant?

Porokeratosis is a clonal disorder of keratinization characterized by the presence of the cornoid lamella. A number of variants of porokeratosis have been described, based on the clinical features and histologic features of the lesions. The authors present a case of porokeratosis with prominent melanocytic hyperplasia, which was biopsied to clinically exclude melanoma. The authors retrospectively studied cases of porokeratosis to look for the presence of melanocytic hyperplasia. Melanocytic hyperplasia was identified in 8 of 31 cases (25.8%). All of the cases except the index case were clinically nonpigmented but arose in solar damaged skin. This case represents a distinct variant of porokeratosis, and the authors propose the designation pigmented porokeratosis. Melanocytic hyperplasia is a benign condition, and it is important that this is not histologically confused with melanoma in situ, particularly in a context of clinically pigmented lesion. Increased recognition of pigmented porokeratosis is essential to avoid an erroneous diagnosis of melanoma in situ.

A qualitative study of the perceived effects of blue lights in washrooms on people who use injection drugs.

BACKGROUND: Blue lights are sometimes placed in public washrooms to discourage injection drug use. Their effectiveness has been questioned and concerns raised that they are harmful but formal research on the issue is limited to a single study. We gathered perceptions of people who use injection drugs on the effects of blue lights with the aim of informing harm reduction practice. METHODS: We interviewed 18 people in two Canadian cities who currently or previously used injection drugs to better understand their perceptions of the rationale for and consequences of blue lights in public washrooms. RESULTS: Participants described a preference for private places to use injection drugs, but explained that the need for an immediate solution would often override other considerations. While public washrooms were in many cases not preferred, their accessibility and relative privacy appear to make them reasonable compromises in situations involving urgent injecting. Participants understood the aim of blue lights to be to deter drug use. The majority had attempted to inject in a blue-lit washroom. While there was general agreement that blue lights do make injecting more difficult, a small number of participants were entirely undeterred by them, and half would use a blue-lit washroom if they needed somewhere to inject urgently. Participants perceived that, by making veins less visible, blue lights make injecting more dangerous. By dispersing public injection drug use to places where it is more visible, they also make it more stigmatizing. Despite recognizing these harms, more than half of the participants were not opposed to the continued use of blue lights. CONCLUSIONS: Blue lights are unlikely to deter injection drugs use in public washrooms, and may increase drug use-related harms. Despite recognizing these negative effects, people who use injection drugs may be reluctant to advocate against their use. We attempt to reconcile this apparent contradiction by interpreting blue lights as a form of symbolic violence and suggest a parallel with other emancipatory movements for inspiration in advocating against this and other oppressive interventions.

Humor as a coping strategy for adult-child caregivers of individuals with Alzheimer's disease.

Guided by a phenomenological theoretical perspective, 6 young adult-child caregivers of individuals with Alzheimer's disease were interviewed. The purpose of this study was to gain a better understanding of the experiences of young adult-child caregivers, with the key focus being the use of humor as a coping strategy in this caregiving circumstance. Qualitative analysis revealed a unique young adult-child caregiver experience in relation to humor including an acknowledgment of 3 key determinants that must be considered if humor is to be effective as a coping strategy, the identification of 4 factors that influence humor use, and finally a description of the perceived benefits of humor. Moreover, these research findings highlight the potential for the use of humor to be incorporated as a coping strategy for caregivers in the hope of preventing caregiver burnout and optimizing patient care.

The ESAT-6/CFP-10 secretion system of Mycobacterium marinum modulates phagosome maturation.

Virulence of Mycobacterium tuberculosis and related pathogenic mycobacteria requires the secretion of early secretory antigenic 6 kDa (ESAT-6) and culture filtrate protein 10 (CFP-10), two small proteins that lack traditional signal sequences and are exported through an alternative secretion pathway encoded primarily by the RD1 genetic locus. Mutations affecting the synthesis or secretion of ESAT-6 or CFP-10 attenuate the virulence of M. tuberculosis in murine models of infection. However, the specific functions of these proteins and of their secretion system are currently unclear. In this study, we isolated a mutant of Mycobacterium marinum defective in the secretion of ESAT-6 and CFP-10. The mutation was localized within MM5446, which is orthologous to Rv3871 of M. tuberculosis H37Rv and encodes an ATPase that is a component of the ESAT-6/CFP-10 secretion system. The mutant bacteria were unable to replicate within J774 macrophages although their growth in 7H9 medium was equivalent to the parental strain. Phagosome maturation and acidification were analysed in infected macrophages by confocal and electron microscopy using the late endosome/lysosome marker LAMP-1, along with various fluid-phase markers such as rhodamine-dextran and ferritin and the acidotropic dye LysoTracker Red. These studies demonstrated that while the wild-type parental strain of M. marinum primarily resides in a poorly acidified, non-lysosomal compartment, a significantly higher percentage of the MM5446 mutant organisms are in acidified compartments. These results suggest that the ESAT-6/CFP-10 secretion system plays a role in preventing phagolysosomal fusion, a novel function that accounts for the ability of bacteria to survive inside host cells. This finding provides a mechanism by which the ESAT-6/CFP-10 secretion system potentiates the virulence of pathogenic mycobacteria.

Roles of Lsr2 in colony morphology and biofilm formation of Mycobacterium smegmatis.

The lipid-rich cell wall is a defining feature of Mycobacterium species. Individual cell wall components affect diverse mycobacterial phenotypes including colony morphology, biofilm formation, antibiotic resistance, and virulence. In this study, we describe a transposon insertion mutant of Mycobacterium smegmatis mc2 155 that exhibits altered colony morphology and defects in biofilm formation. The mutation was localized to the lsr2 gene. First identified as an immunodominant T-cell antigen of Mycobacterium leprae, lsr2 orthologs have been identified in all sequenced mycobacterial genomes, and homologs are found in many actinomycetes. Although its precise function remains unknown, localization experiments indicate that Lsr2 is a cytosolic protein, and cross-linking experiments demonstrate that it exists as a dimer. Characterization of cell wall lipid components reveals that the M. smegmatis lsr2 mutant lacks two previously unidentified apolar lipids. Characterization by mass spectrometry and thin-layer chromatography indicate that these two apolar lipids are novel mycolate-containing compounds, called mycolyl-diacylglycerols (MDAGs), in which a mycolic acid (alpha- or alpha'-mycolate) molecule is esterified to a glycerol. Upon complementation with an intact lsr2 gene, the mutant reverts to the parental phenotypes and MDAG production is restored. This study demonstrates that due to its impact on the biosynthesis of the hydrophobic MDAGs, Lsr2 plays an important role in the colony morphology and biofilm formation of M. smegmatis.

Cutting edge: microbial products elicit formation of dendritic cell aggresome-like induced structures in macrophages.

In response to a maturation stimulus, dendritic cells undergo the formation of ubiquitinated protein aggregates known as dendritic cell aggresome-like induced structures (DALIS). DALIS are thought to act as Ag storage structures, allowing for the prioritized degradation of proteins during infection. In this study, we demonstrate that murine macrophages can also form ubiquitinated protein aggregates that are indistinguishable from DALIS. These were formed in a dose- and time-dependent manner, and in response to a variety of microbial products. Surprisingly, the proteasome did not accumulate on these ubiquitinated protein structures, further underlining the difference between DALIS and aggresomes. Our studies suggest that DALIS formation is important for the function of Ag-presenting immune cells during infection.

PimF, a mannosyltransferase of mycobacteria, is involved in the biosynthesis of phosphatidylinositol mannosides and lipoarabinomannan.

Phosphatidylinositol mannosides (PIMs) and their related molecules lipomannan (LM) and lipoarabinomannan (LAM) are important components of the mycobacterial cell wall. These molecules mediate host-pathogen interactions and exhibit immunomodulatory activities. The biosynthesis of these lipoglycans is not fully understood. In this study, we have identified a mycobacterial gene (Rv1500) that is involved in the synthesis of PIMs. We have named this gene pimF. Transposon mutagenesis of pimF of Mycobacterium marinum resulted in multiple phenotypes, including altered colony morphology, disappearance of tetracyl-PIM(7), and accumulation of tetraacyl-PIM(5). The syntheses of LAM and LM were also affected. In addition, the pimF mutant exhibited a defect during infection of cultured macrophage cells. Although the mutant was able to replicate and persist within macrophages, the initial cell entry step was inefficient. Transformation of the M. marinum mutant with the pimF homolog of Mycobacterium tuberculosis complemented all of the above mentioned phenotypes. These results provide evidence that PimF is a mannosyltransferase. However, sequence analysis indicates that PimF is distinct from mannosyltransferases involved in the early steps of PIM synthesis. PimF catalyzes the formation of high molecular weight PIMs, which are precursors for the synthesis of LAM and LM. As such, this work marks the first analysis of a mannosyltransferase involved in the later stages of PIM synthesis.