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.

Dual control of Kinesin-1 recruitment to microtubules by Ensconsin in Drosophila neuroblasts and oocytes.

Drosophila Ensconsin (also known as MAP7) controls spindle length, centrosome separation in brain neuroblasts (NBs) and asymmetric transport in oocytes. The control of spindle length by Ensconsin is Kinesin-1 independent but centrosome separation and oocyte transport require targeting of Kinesin-1 to microtubules by Ensconsin. However, the molecular mechanism used for this targeting remains unclear. Ensconsin contains a microtubule (MT)-binding domain (MBD) and a Kinesin-binding domain (KBD). Rescue experiments show that only full-length Ensconsin restores the spindle length phenotype. KBD expression rescues ensc centrosome separation defects in NBs, but not the fast oocyte streaming and the localization of Staufen and Gurken. Interestingly, the KBD can stimulate Kinesin-1 targeting to MTs in vivo and in vitro We propose that a KBD and Kinesin-1 complex is a minimal activation module that increases Kinesin-1 affinity for MTs. Addition of the MBD present in full-length Ensconsin allows this process to occur directly on the MT and triggers higher Kinesin-1 targeting. This dual regulation by Ensconsin is essential for optimal Kinesin-1 targeting to MTs in oocytes, but not in NBs, illustrating the importance of adapting Kinesin-1 recruitment to different biological contexts.

EFFECTS OF TRICAINE METHANESULFONATE IN A MANAGED COLLECTION OF MOON JELLYFISH (AURELIA AURITA).

The moon jellyfish (Aurelia aurita) is a scyphozoan frequently maintained in public and private aquaria. Little research has been conducted to investigate the effects of various drugs, such as anesthetics, in this species. Tricaine methanesulfonate (MS-222), a common immersion anesthetic for fish and amphibians, was evaluated in a managed population of moon jellyfish. Twenty-four clinically healthy jellyfish were assigned into three groups of eight for trials of 0.3 g/L MS-222 (low concentration [LC]), 0.6 g/L MS-222 (high concentration [HC]), and a saltwater control. The goal was to evaluate the effects of MS-222 administration on moon jellyfish movement and response to stimuli. Movement and response to stimuli were measured via rocking and probe stimulus tests and observations of bell contraction quality and body tone. These tests were performed at baseline and throughout both drug exposure and recovery periods. A threshold drug effect was defined based on systematic scoring criteria. Additionally, elastomer tags were administered to four of eight animals in each MS-222 group to evaluate response to tag placement after drug exposure. Threshold drug effect was achieved in six of eight individuals in the LC group and eight of eight individuals in the HC group. The LC group had median threshold and recovery times of 12.2 and 10.1 min, respectively, while the HC group had median threshold and recovery times of 4.0 and 19.9 min, respectively. The HC group had significantly faster time to threshold drug effect (P < 0.001) and longer recovery times (P= 0.005) than the LC group. In both the LC and HC tagged group, three of four jellyfish had no reaction to tag placement. All animals recovered uneventfully, and there were no mortalities. MS-222 at 0.3 and 0.6 g/L decreased movement and response to stimuli in moon jellyfish.

Functional Analysis of Calcium-Sensing Receptor Variants Identified in Families Provisionally Diagnosed with Familial Hypocalciuric Hypercalcaemia.

Identification of variants in the calcium-sensing receptor (CASR) gene is an important means of distinguishing between familial hypocalciuric hypercalcaemia (FHH) and primary hyperparathyroidism. However, identification and bioinformatics analysis of genetic variants alone is now considered insufficient as definitive proof; additional functional assessment is required to diagnose FHH with certainty. We identified two novel variants, D433Y and C739Y, and one previously reported variant G509R in the CASR of four kindreds provisionally diagnosed with FHH and aimed to functionally characterise these variants to confirm the diagnosis. Variant receptors were cloned as FLAG-tagged constructs into the mammalian expression vector, pcDNA3.1. Wild type and variant receptor constructs were expressed in HEK293 cells and their expression assessed by Western blot analysis and their functionality analysed using an IP-One assay which measures myo-inositol 1-phosphate accumulation following CaSR activation. Western blot analysis showed that the D433Y receptor had diminished mature glycosylated receptor compared with wild type CaSR whereas the G509R receptor had a complete lack of mature receptor. The C739Y receptor was consistently overexpressed. Functional assessment showed the D433Y receptor to be mildly inactivating at physiological calcium concentrations whereas the G509R receptor was inactive at all calcium concentrations. By contrast, the C739Y variant was activating compared to wild type receptor which is inconsistent with it causing FHH. We conclude that functional assessment of CaSR variants using the IP-One assay was useful in the investigation of suspected FHH probands, confirming the D433Y and G509R variants as likely pathogenic/pathogenic, but dismissing the C739Y variant as causing FHH.

'Wargaming with the chiefs': A novel pre-mortem exercise to enhance communication and well-being among 'top gun' chief residents.

Pilots who complete the 'TOPGUN' program return to their squadrons as elite leaders and instructors. Physicians from all specialties who are selected to become Chief residents can also be viewed as 'Top Guns', as they are the 'cream of the crop'; having been chosen to be the leaders and role-models for all the other residents in their programs. Yet, unlike Top Gun pilots, Chief residents are arguably only minimally prepared for this new role. Wargaming involves generating every possible bad outcome and brainstorming contingencies for these possible outcomes. We developed an exercise for the incoming Chiefs (ICs) and outgoing Chiefs (OCs) in four specialties: Family Medicine, Internal Medicine, Neurosurgery, and Neurology. Following this exercise, 100% of the Chiefs in all four programs indicated that this activity was beneficial and the majority agreed that wargaming improved communication and their own feelings of well-being. Based on our trainees' feedback, it appears that wargaming is a simple, fun, and highly-interactive exercise which increases perceived control among ICs and allows OCs the chance to reflect and share their knowledge and experience with the new Chiefs. This activity also seems to enhance communication and feelings of well-being among both incoming and outgoing Chief residents.

Regeneration of Drosophila sensory neuron axons and dendrites is regulated by the Akt pathway involving Pten and microRNA bantam.

Both cell-intrinsic and extrinsic pathways govern axon regeneration, but only a limited number of factors have been identified and it is not clear to what extent axon regeneration is evolutionarily conserved. Whether dendrites also regenerate is unknown. Here we report that, like the axons of mammalian sensory neurons, the axons of certain Drosophila dendritic arborization (da) neurons are capable of substantial regeneration in the periphery but not in the CNS, and activating the Akt pathway enhances axon regeneration in the CNS. Moreover, those da neurons capable of axon regeneration also display dendrite regeneration, which is cell type-specific, developmentally regulated, and associated with microtubule polarity reversal. Dendrite regeneration is restrained via inhibition of the Akt pathway in da neurons by the epithelial cell-derived microRNA bantam but is facilitated by cell-autonomous activation of the Akt pathway. Our study begins to reveal mechanisms for dendrite regeneration, which depends on both extrinsic and intrinsic factors, including the PTEN-Akt pathway that is also important for axon regeneration. We thus established an important new model system--the fly da neuron regeneration model that resembles the mammalian injury model--with which to study and gain novel insights into the regeneration machinery.

ReMAPping the microtubule landscape: How phosphorylation dictates the activities of microtubule-associated proteins.

Classical microtubule-associated proteins (MAPs) were originally identified based on their co-purification with microtubules assembled from mammalian brain lysate. They have since been found to perform a range of functions involved in regulating the dynamics of the microtubule cytoskeleton. Most of these MAPs play integral roles in microtubule organization during neuronal development, microtubule remodeling during neuronal activity, and microtubule stabilization during neuronal maintenance. As a result, mutations in MAPs contribute to neurodevelopmental disorders, psychiatric conditions, and neurodegenerative diseases. MAPs are post-translationally regulated by phosphorylation depending on developmental time point and cellular context. Phosphorylation can affect the microtubule affinity, cellular localization, or overall function of a particular MAP and can thus have profound implications for neuronal health. Here we review MAP1, MAP2, MAP4, MAP6, MAP7, MAP9, tau, and DCX, and how each is regulated by phosphorylation in neuronal physiology and disease. Developmental Dynamics 247:138-155, 2018. © 2017 Wiley Periodicals, Inc.

Deciphering systemic lupus erythematosus-associated serum biomarkers reflecting apoptosis and disease activity.

Systemic lupus erythematosus (SLE) is a severe chronic inflammatory autoimmune connective tissue disease. Despite major efforts, SLE remains a poorly understood disease with unpredictable course, unknown etiology and complex pathogenesis. Apoptosis combined with deficiency in clearing apoptotic cells is an important etiopathogenic event in SLE, which could contribute to the increased load of potential autoantigen(s); however, the lack of disease-specific protein signatures deciphering SLE and the underlying biological processes is striking and represents a key limitation. In this retrospective pilot study, we explored the immune system as a specific sensor for disease, in order to advance our understanding of SLE. To this end, we determined multiplexed serum protein expression profiles of crude SLE serum samples, using antibody microarrays. The aim was to identify differential immunoprofiles, or snapshots of the immune response modulated by the disease, reflecting apoptosis, a key process in the etiology of SLE and disease activity. The results showed that multiplexed panels of SLE-associated serum biomarkers could be decoded, in particular reflecting disease activity, but potentially the apoptosis process as well. While the former biomarkers could display a potential future use for prognosis, the latter biomarkers might help shed further light on the apoptosis process taking place in SLE.

Microtubule-associated protein MAP7 promotes tubulin posttranslational modifications and cargo transport to enable osmotic adaptation.

Cells remodel their cytoskeletal networks to adapt to their environment. Here, we analyze the mechanisms utilized by the cell to tailor its microtubule landscape in response to changes in osmolarity that alter macromolecular crowding. By integrating live-cell imaging, ex vivo enzymatic assays, and in vitro reconstitution, we probe the impact of cytoplasmic density on microtubule-associated proteins (MAPs) and tubulin posttranslational modifications (PTMs). We find that human epithelial cells respond to fluctuations in cytoplasmic density by modulating microtubule acetylation, detyrosination, or MAP7 association without differentially affecting polyglutamylation, tyrosination, or MAP4 association. These MAP-PTM combinations alter intracellular cargo transport, enabling the cell to respond to osmotic challenges. We further dissect the molecular mechanisms governing tubulin PTM specification and find that MAP7 promotes acetylation and inhibits detyrosination. Our data identify MAP7 in modulating the tubulin code, resulting in microtubule cytoskeleton remodeling and alteration of intracellular transport as an integrated mechanism of cellular adaptation.

Knowledge Gaps Regarding Indigenous Health in Occupational Therapy: A National Survey.

Background. There is a need for the occupational therapy profession to respond to the Truth and Reconciliation Commission of Canada Calls to Action and work towards supporting the health and well-being of Indigenous Peoples. Purpose. (1) To determine the knowledge gaps of occupational therapists about Indigenous health and (2) to create recommendations to address identified gaps and inform responses from the profession. Method. A national needs survey was created and distributed to occupational therapists across Canada to determine the knowledge of occupational therapists about Indigenous health. Survey results were analyzed using thematic analysis and descriptive statistics. Findings. Data collected from 364 survey responses informed six distinct themes representing knowledge gaps of occupational therapists related to Indigenous health as follows: lack of foundational knowledge, power relations, lifelong learner, need for appropriate tools/approaches, respectful collaboration, and environmental influences. Implications. The project offers insight into the role of the occupational therapy profession in the process of reconciliation. Insights are focused on decolonizing occupational therapy practice, building trusting relationships with Indigenous Peoples, and the provision of appropriate training for occupational therapists to engage in culturally safer practices.

Global oceanic oxygenation controlled by the Southern Ocean through the last deglaciation.

Ocean dissolved oxygen (DO) can provide insights on how the marine carbon cycle affects global climate change. However, the net global DO change and the controlling mechanisms remain uncertain through the last deglaciation. Here, we present a globally integrated DO reconstruction using thallium isotopes, corroborating lower global DO during the Last Glacial Maximum [19 to 23 thousand years before the present (ka B.P.)] relative to the Holocene. During the deglaciation, we reveal reoxygenation in the Heinrich Stadial 1 (~14.7 to 18 ka B.P.) and the Younger Dryas (11.7 to 12.9 ka B.P.), with deoxygenation during the Bølling-Allerød (12.9 to 14.7 ka B.P.). The deglacial DO changes were decoupled from North Atlantic Deep Water formation rates and imply that Southern Ocean ventilation controlled ocean oxygen. The coherence between global DO and atmospheric CO2 on millennial timescales highlights the Southern Ocean's role in deglacial atmospheric CO2 rise.

Patients' perceptions on outcomes after mechanical thrombectomy in acute ischemic stroke.

BACKGROUND: The modified Rankin Scale (mRS) is a clinician-reported scale that measures the degree of disability in patients who suffered a stroke. Patients' perception of a meaningful recovery from severe stroke, expected value of a stroke intervention, and the effect of disparities are largely unknown. METHODS: We conducted a survey of patients, their family members, and accompanying visitors to understand their personal preferences and expectations for acute strokes potentially eligible for acute endovascular intervention using a hypothetical scenario of a severe stroke in a standardized questionnaire. RESULTS: Of 164 survey respondents, 65 (39.6%) were the patient involved, 93 (56.7%) were a family member, and six (3.7%) were accompanied visitors (friends, other). Minimally acceptable disability after a stroke intervention was considered as mRS 2 by 42 respondents (25.6%), as mRS 3 by 79 (48.2%), and as mRS 4 by 43 (26.2%) respondents. Race was associated with different views on this question (p < 0.001; Hispanic and Black patients being more likely to accept disability than Caucasian and Asian patients), while sex (p = 0.333) and age (p = 0.560) were not. Sixty-three respondents (38.4%) viewed minimally acceptable probability of improvement with an intervention as over 50%, 57 (34.8%) as 10-50%, and 44 (26.8%) as less than 10%. CONCLUSIONS: A wide range of acceptable outcomes were reported regardless of gender or age. However, race was associated with different acceptable outcome. This is an important finding to demonstrate because of the persistent racial and ethnic disparities in the utilization of endovascular therapy for acute stroke in the United States.

Development and application of in vivo molecular traps reveals that dynein light chain occupancy differentially affects dynein-mediated processes.

The ability to rapidly and specifically regulate protein activity combined with in vivo functional assays and/or imaging can provide unique insight into underlying molecular processes. Here we describe the application of chemically induced dimerization of FKBP to create nearly instantaneous high-affinity bivalent ligands capable of sequestering cellular targets from their endogenous partners. We demonstrate the specificity and efficacy of these inducible, dimeric "traps" for the dynein light chains LC8 (Dynll1) and TcTex1 (Dynlt1). Both light chains can simultaneously bind at adjacent sites of dynein intermediate chain at the base of the dynein motor complex, yet their specific function with respect to the dynein motor or other interacting proteins has been difficult to dissect. Using these traps in cultured mammalian cells, we observed that induction of dimerization of either the LC8 or TcTex1 trap rapidly disrupted early endosomal and lysosomal organization. Dimerization of either trap also disrupted Golgi organization, but at a substantially slower rate. Using either trap, the time course for disruption of each organelle was similar, suggesting a common regulatory mechanism. However, despite the essential role of dynein in cell division, neither trap had a discernable effect on mitotic progression. Taken together, these studies suggest that LC occupancy of the dynein motor complex directly affects some, but not all, dynein-mediated processes. Although the described traps offer a method for rapid inhibition of dynein function, the design principle can be extended to other molecular complexes for in vivo studies.

Macromolecular Crowding Tailors the Microtubule Cytoskeleton Through Tubulin Modifications and Microtubule-Associated Proteins.

Cells remodel their cytoskeletal networks to adapt to their environment. Here, we analyze the mechanisms utilized by the cell to tailor its microtubule landscape in response to changes in osmolarity that alter macromolecular crowding. By integrating live cell imaging, ex vivo enzymatic assays, and in vitro reconstitution, we probe the impact of acute perturbations in cytoplasmic density on microtubule-associated proteins (MAPs) and tubulin posttranslational modifications (PTMs), unraveling the molecular underpinnings of cellular adaptation via the microtubule cytoskeleton. We find that cells respond to fluctuations in cytoplasmic density by modulating microtubule acetylation, detyrosination, or MAP7 association, without differentially affecting polyglutamylation, tyrosination, or MAP4 association. These MAP-PTM combinations alter intracellular cargo transport, enabling the cell to respond to osmotic challenges. We further dissect the molecular mechanisms governing tubulin PTM specification, and find that MAP7 promotes acetylation by biasing the conformation of the microtubule lattice, and directly inhibits detyrosination. Acetylation and detyrosination can therefore be decoupled and utilized for distinct cellular purposes. Our data reveal that the MAP code dictates the tubulin code, resulting in remodeling of the microtubule cytoskeleton and alteration of intracellular transport as an integrated mechanism of cellular adaptation.

Addressing Natural Killer Cell Dysfunction and Plasticity in Cell-Based Cancer Therapeutics.

Natural killer (NK) cells are cytotoxic group 1 innate lymphoid cells (ILC), known for their role as killers of stressed, cancerous, and virally infected cells. Beyond this cytotoxic function, NK cell subsets can influence broader immune responses through cytokine production and have been linked to central roles in non-immune processes, such as the regulation of vascular remodeling in pregnancy and cancer. Attempts to exploit the anti-tumor functions of NK cells have driven the development of various NK cell-based therapies, which have shown promise in both pre-clinical disease models and early clinical trials. However, certain elements of the tumor microenvironment, such as elevated transforming growth factor (TGF)-ß, hypoxia, and indoalemine-2,3-dioxygenase (IDO), are known to suppress NK cell function, potentially limiting the longevity and activity of these approaches. Recent studies have also identified these factors as contributors to NK cell plasticity, defined by the conversion of classical cytotoxic NK cells into poorly cytotoxic, tissue-resident, or ILC1-like phenotypes. This review summarizes the current approaches for NK cell-based cancer therapies and examines the challenges presented by tumor-linked NK cell suppression and plasticity. Ongoing efforts to overcome these challenges are discussed, along with the potential utility of NK cell therapies to applications outside cancer.

Knowledge Gaps Regarding Indigenous Health in Occupational Therapy: A Delphi Process.

Background. The occupational therapy profession needs to respond to the calls to action from the Truth and Reconciliation Commission (TRC) to engage in the process of reconciliation with Indigenous populations. Purpose. To inform development of a survey intended to determine the knowledge gaps of occupational therapists in relation to Indigenous health. Method. A Delphi process engaging 18 occupational therapists with membership in an Indigenous health network was used to prioritize and refine potential themes identified via literature review. Findings. Results of three consensus rounds and Dunn-Bonferroni post-hoc testing demonstrated three statistically distinct hierarchical tiers of 10 priority themes to inform survey development. Implications. The consensus prioritized themes from the literature to underpin further research on occupational therapists' knowledge in relation to Indigenous health and can provide a learning scaffold for occupational therapists to support a continued response to the TRC calls to action.

Constitutive ALS3 expression in Candida albicans enhances adhesion and biofilm formation of efg1, but not cph1 mutant strains.

Adhesion to living and non-living surfaces is an important virulence trait of the fungal pathogen Candida albicans. Biofilm formation in this organism depends on the expression of a number of cell surface proteins including the hypha-specific protein Als3p. Loss of ALS3 impairs biofilm formation and decreases cell-cell adhesion. We wanted to test whether constitutively expressing ALS3 could compensate for defects in adhesion and biofilm formation observed in mutant strains that lack key transcriptional regulators of biofilm formation Efg1p and Cph1p. We found that ALS3 improved adhesion and biofilm formation in the efg1Δ and efg1Δ cph1Δ mutant strains, but had less effect on the cph1Δ strain.

PREHOSPITAL PLASMA IS NONINFERIOR TO WHOLE BLOOD FOR RESTORATION OF CEREBRAL OXYGENATION IN A RHESUS MACAQUE MODEL OF TRAUMATIC SHOCK AND HEMORRHAGE.

ABSTRACT: Introduction: Traumatic shock and hemorrhage (TSH) is a leading cause of preventable death in military and civilian populations. Using a TSH model, we compared plasma with whole blood (WB) as prehospital interventions, evaluating restoration of cerebral tissue oxygen saturation (CrSO 2 ), systemic hemodynamics, colloid osmotic pressure (COP) and arterial lactate, hypothesizing plasma would function in a noninferior capacity to WB, despite dilution of hemoglobin (Hgb). Methods: Ten anesthetized male rhesus macaques underwent TSH before randomization to receive a bolus of O(-) WB or AB(+) plasma at T0. At T60, injury repair and shed blood (SB) to maintain MAP > 65 mm Hg began, simulating hospital arrival. Hematologic data and vital signs were analyzed via t test and two-way repeated measures ANOVA, data presented as mean ± SD, significance = P < 0.05. Results: There were no significant group differences for shock time, SB volume, or hospital SB. At T0, MAP and CrSO 2 significantly declined from baseline, though not between groups, normalizing to baseline by T10. Colloid osmotic pressure declined significantly in each group from baseline at T0 but restored by T30, despite significant differences in Hgb (WB 11.7 ± 1.5 vs. plasma 6.2 ± 0.8 g/dL). Peak lactate at T30 was significantly higher than baseline in both groups (WB 6.6 ± 4.9 vs. plasma 5.7 ± 1.6 mmol/L) declining equivalently by T60. Conclusions: Plasma restored hemodynamic support and CrSO 2 , in a capacity not inferior to WB, despite absence of additional Hgb supplementation. This was substantiated via return of physiologic COP levels, restoring oxygen delivery to microcirculation, demonstrating the complexity of restoring oxygenation from TSH beyond simply increasing oxygen carrying capacity.

Survey of colleges and schools of pharmacy to determine restrictions for teaching, research, and advocacy related to contraception.

INTRODUCTION: The pharmacist's role in reproductive health is evolving. Since 46 states allow providers to refuse to provide reproductive health services, it is important to consider whether learning is impacted by institution restrictions on contraception teaching, advocacy, and research. METHODS: An electronic survey was emailed to deans of all pharmacy schools on the American Association of Colleges of Pharmacy Institutional Membership list with a request to share with faculty teaching women's health content within their curriculum. The survey collected information about contraception teaching, research, and advocacy. RESULTS: Of 145 schools contacted, 39 (27%) provided complete responses. Of these, 22 (56%) were public, not religiously-affiliated, seven (18%) were private, not religiously-affiliated, six (15%) were private, currently religiously-affiliated, and four were (10%) private, historically religiously-affiliated. All respondents taught hormonal contraception in the required curriculum and 15 (39%) taught miscarriage management/abortifacients. None reported restrictions on contraception teaching or research. One respondent cited an advocacy restriction for contraception methods due to violation of the school's beliefs, and another cited an advocacy restriction for miscarriage management/abortifacients. Respondents noted students expressed ethical questions/concerns about refusing to dispense contraception (59%), dispensing certain contraceptives (54%), dispensing to minors (46%), and dispensing all contraceptives (21%). Additionally, respondents reported pharmacists/faculty expressed ethical questions/concerns about refusing to dispense contraception (31%), dispensing to minors (21%), dispensing certain contraceptives (15%), and all contraceptives (13%). CONCLUSIONS: Overall, respondents reported no restrictions in contraception teaching and scholarship and minimal advocacy restrictions. Faculty should consider ethical questions/concerns from students, faculty, and pharmacists when teaching this material.