Chronic Traumatic Encephalopathy in a Routine Neuropathology Service in Australia.

Chronic traumatic encephalopathy (CTE) is a neuropathological diagnosis defined by a unique pattern of hyperphosphorylated tau (p-tau) accumulation that begins in neocortical regions of the brain. It is associated with a range of neuropsychological symptoms, but a definitive diagnosis can only be made by postmortem brain examination. In 2018, we instituted CTE screening for all autopsy brains as part of our routine departmental protocol by performing p-tau immunohistochemistry on a restricted set of 3 neocortical blocks (frontal, temporal, and parietal). This strategy allowed us to identify 4 cases of low-stage CTE from 180 consecutive autopsies. Two of the 4 cases had a documented history of brain injury; for the remaining 2 cases, there was a long history of treatment-resistant tonic/clonic epilepsy suggesting that undocumented brain injuries may have occurred. Our experience indicates that 3-block CTE screening is useful in identifying CTE in routine practice. The results of this study further support the association between prior head injuries and CTE and demonstrate that, albeit uncommon, CTE does occur in the general population. Our findings suggest that p-tau screening should be routinely pursued in brain autopsy, particularly where there is a documented or likely history of traumatic brain injury.

Molecular Analysis of pSK1 par: A Novel Plasmid Partitioning System Encoded by Staphylococcal Multiresistance Plasmids.

The segregation of prokaryotic plasmids typically requires a centromere-like site and two proteins, a centromere-binding protein (CBP) and an NTPase. By contrast, a single 245 residue Par protein mediates partition of the prototypical staphylococcal multiresistance plasmid pSK1 in the absence of an identifiable NTPase component. To gain insight into centromere binding by pSK1 Par and its segregation function we performed structural, biochemical and in vivo studies. Here we show that pSK1 Par binds a centromere consisting of seven repeat elements. We demonstrate this Par-centromere interaction also mediates Par autoregulation. To elucidate the Par centromere binding mechanism, we obtained a structure of the Par N-terminal DNA-binding domain bound to centromere DNA to 2.25 Å. The pSK1 Par structure, which harbors a winged-helix-turn-helix (wHTH), is distinct from other plasmid CBP structures but shows homology to the B. subtilis chromosome segregation protein, RacA. Biochemical studies suggest the region C-terminal to the Par wHTH forms coiled coils and mediates oligomerization. Fluorescence microscopy analyses show that pSK1 Par enhances the separation of plasmids from clusters, driving effective segregation upon cell division. Combined the data provide insight into the molecular properties of a single protein partition system.

Distribution of tau hyperphosphorylation in canine dementia resembles early Alzheimer's disease and other tauopathies.

Some aged community dogs acquire a degenerative syndrome termed Canine Cognitive Dysfunction (CCD) that resembles human dementia because of Alzheimer's Disease (AD), with comparable cognitive and behavioral deficits. Dogs also have similar neuroanatomy, share our domestic environment and develop amyloid-ß plaques, making them likely a valuable ecological model of AD. However, prior investigations have demonstrated a lack of neurofibrillary tau pathology in aged dogs, an important hallmark of AD, though elevated phosphorylated tau (p-tau) at the Serine 396 (S396) epitope has been reported in CCD. Here using enhanced immunohistochemical methods, we investigated p-tau in six CCD brains and six controls using the AT8 antibody (later stage neurofibrillary pathology), and an antibody against S396 p-tau (earlier stage tau dysfunction). For the first time, we systematically assessed the Papez circuit and regions associated with Braak staging and found that all CCD dogs displayed elevated S396 p-tau labeling throughout the circuit. The limbic thalamus was particularly implicated, with a similar labeling pattern to that reported for AD neurofibrillary pathology, especially the anterior nuclei, while the hippocampus exhibited dysfunction confined to synaptic layers and efferent pathways. The cingulate and temporal lobes displayed significantly greater tauopathy than the frontal and occipital cortices, also reflective of early Braak staging patterns in AD. Immunofluorescence confirmed that S396 was accumulating within neuronal axons, somata and oligodendrocytes. We also observed AT8 labeling in one CCD brain, near the transentorhinal cortex in layer II neurons, one of the first regions to be affected in AD. Together, these data demonstrate a concordance in regional distribution of tauopathy between CCD and AD, most evident in the limbic thalamus, an important step in further validating CCD as a translational model for human AD and understanding early AD pathogenic mechanisms.

Cross-skilling training to support medical redeployment in the COVID-19 pandemic.

During the response to the COVID-19 pandemic, doctors will be redeployed into roles with which they are unfamiliar. Adequate training must be provided to reacquaint doctors with medical ward practice, supporting psychological wellbeing and patient safety. Here we describe a cross-skilling programme in North Bristol NHS Trust designed to address colleague anxiety and support wellbeing during redeployment.

Clustering of activated microglia occurs before the formation of dystrophic neurites in the evolution of Aß plaques in Alzheimer's disease.

Alzheimer's disease (AD) is a late-onset disease that has proved difficult to model. Microglia are implicated in AD, but reports vary on precisely when and how in the sequence of pathological changes they become involved. Here, post-mortem human tissue from two differentially affected regions of the AD brain and from non-demented individuals with a high load of AD-type pathology (high pathology controls) was used to model the disease time course in order to determine how microglial activation relates temporally to the deposition of hallmark amyloid-ß (Aß) and hyperphosphorylated microtubule associated protein tau pathology. Immunofluorescence against the pan-microglial marker, ionised calcium-binding adapter molecule 1 (IBA1), Aß and tau, was performed in the primary motor cortex (PMC), a region relatively spared of AD pathological changes, and compared to the severely affected inferior temporal cortex (ITC) in the same cases. Unlike the ITC, the PMC in the AD cases was spared of any degenerative changes in cortical thickness and the density of Betz cells and total neurons. The clustering of activated microglia was greatest in the PMC of AD cases and high pathology controls compared to the ITC. This suggests microglial activation is most prominent in the early phases of AD pathophysiology. Nascent tau inclusions were found in neuritic plaques in the PMC but were more numerous in the ITC of the same case. This shows that tau positive neuritic plaques begin early in AD which is likely of pathogenic importance, however major tau deposition follows the accumulation of Aß and clustering of activated microglia. Importantly, findings presented here demonstrate that different states of microglial activation, corresponding to regional accumulations of Aß and tau, are present simultaneously in the same individual; an important factor for consideration if targeting these cells for therapeutic intervention.

The relationship between the morphological subtypes of microglia and Alzheimer's disease neuropathology.

Microglial associations with both the major Alzheimer's disease (AD) pathognomonic entities, ß-amyloid-positive plaques and tau-positive neurofibrillary tangles, have been noted in previous investigations of both human tissue and mouse models. However, the precise nature of their role in the pathogenesis of AD is debated; the major working hypothesis is that pro-inflammatory activities of activated microglia contribute to disease progression. In contrast, others have proposed that microglial dystrophy with a loss of physiological and neuroprotective activities promotes neurodegeneration. This immunohistochemical study sought to gain clarity in this area by quantifying the morphological subtypes of microglia in the mildly-affected primary visual cortex (PVC), the moderately affected superior frontal cortex (SFC) and the severely affected inferior temporal cortex (ITC) of 8 AD cases and 15 age and gender-matched, non-demented controls with ranging AD-type pathology. AD cases had increased ß-amyloid and tau levels compared to controls in all regions. Neuronal loss was observed in the SFC and ITC, and was associated with atrophy in the latter. A major feature of the ITC in AD was a decrease in ramified (healthy) microglia with image analysis confirming reductions in arborized area and skeletal complexity. Activated microglia were not associated with AD but were increased in non-demented controls with greater AD-type pathology. Microglial clusters were occasionally associated with ß-amyloid- and tau-positive plaques but represented less than 2% of the total microglial population. Dystrophic microglia were not associated with AD, but were inversely correlated with brain pH suggesting that agonal events were responsible for this morphological subtype. Overall these novel findings suggest that there is an early microglial reaction to AD-type pathology but a loss of healthy microglia is the prominent feature in severely affected regions of the AD brain.

Diagnosis and Management of Anorectal Disorders in the Primary Care Setting.

Anorectal disorders are very common among a wide population of patients. Because patients may be embarrassed about the anatomic location of their symptoms, they may present to care late in the course of their illness. Care should be taken to validate patient concerns and normalize fears. This article discusses the diagnoses and management of common anorectal disorders among patients presenting to a primary care physician.

Microglia show altered morphology and reduced arborization in human brain during aging and Alzheimer's disease.

Changes in microglia function are involved in Alzheimer's disease (AD) for which ageing is the major risk factor. We evaluated microglial cell process morphologies and their gray matter coverage (arborized area) during ageing and in the presence and absence of AD pathology in autopsied human neocortex. Microglial cell processes were reduced in length, showed less branching and reduced arborized area with aging (case range 52-98 years). This occurred during normal ageing and without microglia dystrophy or changes in cell density. There was a larger reduction in process length and arborized area in AD compared to aged-matched control microglia. In AD cases, on average, 49%-64% of microglia had discontinuous and/or punctate Iba1 labeled processes instead of continuous Iba1 distribution. Up to 16% of aged-matched control microglia displayed discontinuous or punctate features. There was no change in the density of microglial cell bodies in gray matter during ageing or AD. This demonstrates that human microglia show progressive cell process retraction without cell loss during ageing. Additional changes in microglia occur with AD including Iba1 protein puncta and discontinuity. We suggest that reduced microglial arborized area may be an aging-related correlate of AD in humans. These variations in microglial cells during ageing and in AD could reflect changes in neural-glial interactions which are emerging as key to mechanisms involved in ageing and neurodegenerative disease.

STED imaging of tau filaments in Alzheimer's disease cortical grey matter.

Alzheimer's disease (AD) involves the propagation of filaments of tau protein throughout the cerebral cortex. Imaging tau filaments and oligomers in human brain at high resolution would help contribute insight into the mechanism and progression of tauopathic diseases. STED microscopy is a nano-scale imaging technique and we aimed to test the abilities of this method for resolving tau structures within human brain. Using autopsied 50µm AD brain sections, we demonstrate that STED microscopy can resolve immunolabelled tau filaments at 77nm resolution. Ribbon-like tau filaments imaged by STED appeared smooth along their axis with limited axial undulations. STED also resolved 70-80nm wide tau puncta. Of the fluorophores tested, STAR635p was optimal for STED imaging in this tissue. This was in part due to brain tissue autofluorescence within the lower wavelength ranges (488-590nm). Further, the stability and minimal photobleaching of STAR635p allowed STED z-stacks of neurons packed with tau filaments (neurofibrillary tangles) to be collated. There was no loss of x-y image resolution of individual tau filaments through the 20µm z-stack. This demonstrates that STED can contribute to nano-scale analysis and characterisation of pathologies within banked human autopsied brain tissue. Resolving tau structures at this level of resolution provides promising avenues for understanding mechanisms of pathology propagation in the different tauopathies as well as illuminating what contributes to disease heterogeneity.

Dynamic Filament Formation by a Divergent Bacterial Actin-Like ParM Protein.

Actin-like proteins (Alps) are a diverse family of proteins whose genes are abundant in the chromosomes and mobile genetic elements of many bacteria. The low-copy-number staphylococcal multiresistance plasmid pSK41 encodes ParM, an Alp involved in efficient plasmid partitioning. pSK41 ParM has previously been shown to form filaments in vitro that are structurally dissimilar to those formed by other bacterial Alps. The mechanistic implications of these differences are not known. In order to gain insights into the properties and behavior of the pSK41 ParM Alp in vivo, we reconstituted the parMRC system in the ectopic rod-shaped host, E. coli, which is larger and more genetically amenable than the native host, Staphylococcus aureus. Fluorescence microscopy showed a functional fusion protein, ParM-YFP, formed straight filaments in vivo when expressed in isolation. Strikingly, however, in the presence of ParR and parC, ParM-YFP adopted a dramatically different structure, instead forming axial curved filaments. Time-lapse imaging and selective photobleaching experiments revealed that, in the presence of all components of the parMRC system, ParM-YFP filaments were dynamic in nature. Finally, molecular dissection of the parMRC operon revealed that all components of the system are essential for the generation of dynamic filaments.

A paralogue of the phosphomutase-like gene family in Candida glabrata, CgPmu2, gained broad-range phosphatase activity due to a small number of clustered substitutions.

Inorganic phosphate is required for a range of cellular processes, such as DNA/RNA synthesis and intracellular signalling. The phosphate starvation-inducible phosphatase activity of Candida glabrata is encoded by the gene CgPMU2 (C. glabrata phosphomutase-like protein). CgPMU2 is part of a three-gene family (∼75% identical) created through gene duplication in the C. glabrata clade; only CgPmu2 is a PHO-regulated broad range acid phosphatase. We identified amino acids that confer broad range phosphatase activity on CgPmu2 by creating fusions of sections of CgPMU2 with CgPMU1, a paralogue with little broad range phosphatase activity. We used site-directed mutagenesis on various fusions to sequentially convert CgPmu1 to CgPmu2. Based on molecular modelling of the Pmu proteins on to a histidine phosphatase crystal structure, clusters of amino acids were found in two distinct regions that were able to confer phosphatase activity. Substitutions in these two regions together conferred broad phosphatase activity on CgPmu1. Interestingly, one change is a histidine adjacent to the active site histidine of CgPmu2 and it exhibits a novel ability to partially replace the conserved active site histidine in CgPmu2. Additionally, a second amino acid change was able to confer nt phosphatase activity to CgPmu1, suggesting single amino acid changes neofunctionalize CgPmu2.

Cofilin rods and aggregates concur with tau pathology and the development of Alzheimer's disease.

BACKGROUND: Imaging of human brain as well as cellular and animal models has highlighted a role for the actin cytoskeleton in the development of cell pathology in Alzheimer's disease (AD). Rods and aggregates of the actin-associated protein cofilin are abundant in grey matter of postmortem AD brain and rods are found inside neurites in animal and cell models of AD. OBJECTIVE: We sought further understanding of the significance of cofilin rods/aggregates to the disease process: Do rods/aggregates correlate with AD progression and the development of hallmark neurofibrillary tangles and neuropil threads? Are cofilin rods/aggregates found in the same neurites as hyperphosphorylated tau? METHODS: The specificity of rods/aggregates to AD compared with general aging and their spatial relationship to tau protein was examined in postmortem human hippocampus, inferior temporal cortex, and anterior cingulate cortex. RESULTS: The presence of cofilin rods/aggregates correlated with the extent of tau pathology independent of patient age. Densities of rods/aggregates were fourfold greater in AD compared with aged-matched control brains and rods/aggregates were significantly larger in AD brain. We did not find evidence for our hypothesis that intracellular cofilin rods are localized to tau-positive neuropil threads. Instead, data suggest the involvement of microglia in the clearance of cofilin rods/aggregates and/or in their synthesis in and around amyloid plaques and surrounding neuropil. CONCLUSION: Cofilin rods and aggregates signify events initiated early in the pathological cascade. Further definition of the mechanisms leading to their formation in the human brain will provide insights into the cellular causes of AD.

Novel acid phosphatase in Candida glabrata suggests selective pressure and niche specialization in the phosphate signal transduction pathway.

Evolution through natural selection suggests unnecessary genes are lost. We observed that the yeast Candida glabrata lost the gene encoding a phosphate-repressible acid phosphatase (PHO5) present in many yeasts including Saccharomyces cerevisiae. However, C. glabrata still had phosphate starvation-inducible phosphatase activity. Screening a C. glabrata genomic library, we identified CgPMU2, a member of a three-gene family that contains a phosphomutase-like domain. This small-scale gene duplication event could allow for sub- or neofunctionalization. On the basis of phylogenetic and biochemical characterizations, CgPMU2 has neofunctionalized to become a broad range, phosphate starvation-regulated acid phosphatase, which functionally replaces PHO5 in this pathogenic yeast. We determined that CgPmu2, unlike ScPho5, is not able to hydrolyze phytic acid (inositol hexakisphosphate). Phytic acid is present in fruits and seeds where S. cerevisiae grows, but is not abundant in mammalian tissues where C. glabrata grows. We demonstrated that C. glabrata is limited from an environment where phytic acid is the only source of phosphate. Our work suggests that during evolutionary time, the selection for the ancestral PHO5 was lost and that C. glabrata neofunctionalized a weak phosphatase to replace PHO5. Convergent evolution of a phosphate starvation-inducible acid phosphatase in C. glabrata relative to most yeast species provides an example of how small changes in signal transduction pathways can mediate genetic isolation and uncovers a potential speciation gene.

The functional outcome of the fractured clavicle.

BACKGROUND: The severity of symptoms, rate and completeness of recovery after closed treatment of the fractured clavicle has not been fully explored. METHODS: The severity and duration of pain, analgesic requirements, ability to perform daily activities, return to work, driving and sport along with appearance of the shoulder were recorded in 56 patients between 1 and 2 years after fracture. Radiographs were assessed by Robinson's classification [8]. RESULTS: Twenty-two out of 56 patients described functional restriction 1-2 years after injury. The majority of patients ceased to experience pain (34/56) and returned to work (19/46) within 3 months. Thirty-eight out of 56 patients noted cosmetic deformity, the severity of which was associated with worse functional outcome (p = 0.002). The degree of fracture displacement was associated with cosmetic deformity (p = 0.02) and functional outcome (p = 0.015). CONCLUSION: The majority of patients perceive a cosmetic deformity and 22/56 impaired function after clavicular fracture.

Spitzenkörper, vacuoles, ring-like structures, and mitochondria of Phanerochaete velutina hyphal tips visualized with carboxy-DFFDA, CMAC and DiOC6(3).

Growth and organelle morphology in the wood rotting basidiomycete fungus Phanerochaete velutina were examined in Petri dishes, on agar-coated slides, and in submerged cultures, using DIC, fluorescence and four-dimensional (4-D; x,y,z,t) confocal microscopy, with several fluorescent probes. Phanerochaete is ideal for this work because of its fast growth, robustness, and use in a wide range of other studies. The probe carboxy-DFFDA, widely used for labelling vacuoles, has no effect either on hyphal tip extension or colony growth at the concentrations usually applied in labelling experiments. Carboxy-DFFDA labels the vacuoles and these form a tubular reticulum in hyphal tip cells. The probe also labels extremely small vesicles (punctate fluorescence) in the apex of tip cells, the Spitzenkörper, and short tubules that undergo sequences of characteristic movements and transformations to produce various morphologies, including ring-like structures. Their location and behaviour suggest that they are a distinct group of structures, possibly a subset of vacuoles, but as yet to be fully identified. Regular incursions of tubules extending from these structures and from the vacuolar reticulum into the apical dome indicate the potential for delivery of material to the apex via tubules as well as vesicles. Such structures are potential candidates for delivering chitin synthases to the apex. Spitzenkörper behaviour has been followed as hyphal tips with linear growth encounter obstacle hyphae and, as the hydrolysis product of carboxy-DFFDA only accumulates in membrane-enclosed compartments, it can be inferred that the labelled structures represent the Spitzenkörper vesicle cloud. Mitochondria also form a reticular continuum of branched tubules in growing hyphal tips, and dual localisation with DiOC6(3) and CMAC allows this to be distinguished from the vacuolar reticulum. Like vacuolar tubules, mitochondrial tubules also span the septa, indicating that they may also be a conduit for intercellular transport.