Neuropathological Assessment as an Endpoint in Clinical Trial Design.

Different neurodegenerative conditions can have complex, overlapping clinical presentations that make accurate diagnosis during life very challenging. For this reason, confirmation of the clinical diagnosis still requires postmortem verification. This is particularly relevant for clinical trials of novel therapeutics where it is important to ascertain what disease- and/or pathology-modifying effects the therapeutics have had. Furthermore, it is important to confirm that patients in the trial had the correct clinical diagnosis as this will have a major bearing on the interpretation of trial results. Here we present a simple protocol for pathological assessment of neurodegenerative changes.

Strain-dependent elastography of cancer cells reveals heterogeneity and stiffening due to attachment.

Because cells vary in thickness and in biomechanical properties, the use of a constant force trigger during atomic force microscopy (AFM) stiffness mapping produces a varied nominal strain that can obfuscate the comparison of local material properties. In this study, we measured the biomechanical spatial heterogeneity of ovarian and breast cancer cells by using an indentation-dependent pointwise Hertzian method. Force curves and surface topography were used together to determine cell stiffness as a function of nominal strain. By recording stiffness at a particular strain, it may be possible to improve comparison of the material properties of cells and produce higher contrast representations of cell mechanical properties. Defining a linear region of elasticity that corresponds to a modest nominal strain, we were able to clearly distinguish the mechanics of the perinuclear region of cells. We observed that, relative to the lamelopodial stiffness, the perinuclear region was softer for metastatic cancer cells than their nonmetastatic counterparts. Moreover, contrast in the strain-dependent elastography in comparison to conventional force mapping with Hertzian model analysis revealed a significant stiffening phenomenon in the thin lamellipodial region in which the modulus scales inversely and exponentially with cell thickness. The observed exponential stiffening is not affected by relaxation of cytoskeletal tension, but finite element modeling indicates it is affected by substrate adhesion. The novel cell mapping technique explores cancer cell mechanical nonlinearity that results from regional heterogeneity, which could help explain how metastatic cancer cells can show soft phenotypes while simultaneously increasing force generation and invasiveness.

Acute urinary retention in a 27-year-old male secondary to benign prostatic hyperplasia treated with Holmium Enucleation of the Prostate (HOLEP).

INTRODUCTION: Benign prostatic hyperplasia (BPH) is common in the ageing male. Clinical manifestations like retention impact on a patient's quality of life. Alterations in androgen activity at the androgen receptor complex level in the prostate contribute to prostatic hyperplasia with the highest incidence occurring in males in their 70's. There remains a paucity of cases in young males who develop acute urinary retention secondary to BPH. We present a case of a 27-year-old male who developed acute urinary retention secondary to BPH who required a Holmium Laser Enucleation of his Prostate (HOLEP). CASE DESCRIPTION: A 27 year old man was admitted in acute urinary retention. BPH was diagnosed via way of radiological imaging and histological assessment. After pre-operative sperm banking and suprapubic catheterisation, the patient underwent a HOLEP. He had biochemically confirmed hypogonadotrophic hypogonadism which was at odds with his muscular, physical appearance. Total testosterone levels had fluctuated following admission suggesting an exogenous substance was interfering with the hypothalamic-pituitary-gonadal axis but he denied exogenous steroid use. RESULT: The patient successfully passed his voiding trial on the second post-operative day and remained catheter free. Post-operative uroflowmetry and sexual function remain unknown as patient disengaged with follow up. CONCLUSION: HOLEP prostatectomy is a safe and effective way of managing BPH in younger patients following sperm banking and assessment by endocrinology.

Physiologic Fidelity as a Domain in Assessing Mixed Reality Trauma Simulation.

INTRODUCTION: Mixed reality has been used in trauma and emergency medicine simulation for more than a decade. As mixed reality potential in trauma simulation continues to expand, so too does the need to validate it as a surrogate for real-life emergency scenarios. Validation of these simulations can occur by measuring fidelity, or the degree to which a computing system can reproduce real-world experiences. After performing a literature review, we determined that most fidelity assessments of trauma and emergency simulations focus on how the user subjectively experiences the simulation. Although subjective user assessment is an important component of determining fidelity, we pose an introductory three-part framework that may assess mixed reality trauma simulation more adequately. MATERIALS AND METHODS: A literature review was conducted using Google Scholar, PubMed, and the Uniformed Services University PowerER search database. Relevant articles were assessed to identify how studies measured fidelity in trauma simulation. We then designed the three-part framework to aid researchers in assessing the fidelity of mixed reality trauma simulations. RESULTS: The domains we determined to best assess mixed reality emergency simulation are as follows:1. Continue assessing fidelity via subjective user assessments. This allows the researcher to know how real the simulation looked and felt to the user based on their individual report.2. Determine whether the trauma simulation changes the medical decision-making capacity of the user. If the user's decision-making capacity changes with a stress-inducing trauma simulation versus a non-stress-inducing simulation, then the stress-inducing trauma environment would be approaching greater fidelity.3. Study the domain of our newly proposed concept: physiologic fidelity. We define physiologic fidelity as the degree to which the simulation elicits a measurable, autonomic response independent of observed emotion or perceived affect. Recreating objective autonomic arousal may be the best way to ensure a trauma simulation reaches fidelity. CONCLUSION: We propose a methodology to assess mixed reality trauma simulation fidelity. Once fidelity is more fully known to the researcher and the simulation user, adjustments can be made to approach reality more closely. Improved simulators may enrich the preparedness of both junior and senior learners for real-life emergencies. We believe assessing the three domains using the Wide Area Virtual Experience at the Val G. Hemming simulation center in Bethesda, MD, will validate mixed reality-trauma simulators as invaluable surrogates for real-life emergency scenarios and ultimately contribute to improved clinical outcomes for clinicians and their patients.

Prominent astrocytic alpha-synuclein pathology with unique post-translational modification signatures unveiled across Lewy body disorders.

Alpha-synuclein (aSyn) is a pre-synaptic monomeric protein that can form aggregates in neurons in Parkinson's disease (PD), Parkinson's disease with dementia (PDD) and dementia with Lewy bodies (DLB), and in oligodendrocytes in multiple system atrophy (MSA). Although aSyn in astrocytes has previously been described in PD, PDD and DLB, the biochemical properties and topographical distribution of astrocytic aSyn have not been studied in detail. Here, we present a systematic investigation of aSyn astrocytic pathology using an expanded antibody toolset covering the entire sequence and key post-translational modifications (PTMs) of aSyn in Lewy body disorders (LBDs) and in MSA. Astrocytic aSyn was detected in the limbic cortical regions of LBDs but were absent in main pathological regions of MSA. The astrocytic aSyn was revealed only with antibodies against the mid N-terminal and non-amyloid component (NAC) regions covering aSyn residues 34-99. The astroglial accumulations were negative to canonical aSyn aggregation markers, including p62, ubiquitin and aSyn pS129, but positive for phosphorylated and nitrated forms of aSyn at Tyrosine 39 (Y39), and not resistant to proteinase K. Our findings suggest that astrocytic aSyn accumulations represent a major part of aSyn pathology in LBDs and possess a distinct sequence and PTM signature that is characterized by both N- and C-terminal truncations and modifications at Y39. This is the first description that aSyn accumulations are made solely from N- and C-terminally cleaved aSyn species and the first report demonstrating that astrocytic aSyn is a mixture of Y39 phosphorylated and nitrated species. These observations underscore the importance of systematic characterization of aSyn accumulations in different cell types to capture the aSyn pathological diversity in the brain. Our findings combined with further studies on the role of astrocytic pathology in the progression of LBDs can pave the way towards identifying novel disease mechanisms and therapeutic targets.

Modeling Studies of Gravity Wave Dynamics in Highly Structured Environments: Reflection, Trapping, Instability, Momentum Transport, Secondary Gravity Waves, and Induced Flow Responses.

A compressible numerical model is applied for three-dimensional (3-D) gravity wave (GW) packets undergoing momentum deposition, self-acceleration (SA), breaking, and secondary GW (SGW) generation in the presence of highly-structured environments enabling thermal and/or Doppler ducts, such as a mesospheric inversion layer (MIL), tidal wind (TW), or combination of MIL and TW. Simulations reveal that ducts can strongly modulate GW dynamics. Responses modeled here include reflection, trapping, suppressed transmission, strong local instabilities, reduced SGW generations, higher altitude SGW responses, and induced large-scale flows. Instabilities that arise in ducts experience strong dissipation after they emerge, while trapped smaller-amplitude and smaller-scale GWs can survive in ducts to much later times. Additionally, GW breaking and its associated dynamics enhance the local wind along the GW propagation direction in the ducts, and yield layering in the wind field. However, these dynamics do not yield significant heat transport in the ducts. The failure of GW breaking to induce stratified layers in the temperature field suggests that such heat transport might not be as strong as previously assumed or inferred from observations and theoretical assessments. The present numerical simulations confirm previous finding that MIL generation may not be caused by the breaking of a transient high-frequency GW packet alone.

Pathologic mechanobiological interactions between red blood cells and endothelial cells directly induce vasculopathy in iron deficiency anemia.

The correlation between cardiovascular disease and iron deficiency anemia (IDA) is well documented but poorly understood. Using a multi-disciplinary approach, we explore the hypothesis that the biophysical alterations of red blood cells (RBCs) in IDA, such as variable degrees of microcytosis and decreased deformability may directly induce endothelial dysfunction via mechanobiological mechanisms. Using a combination of atomic force microscopy and microfluidics, we observed that subpopulations of IDA RBCs (idRBCs) are significantly stiffer and smaller than both healthy RBCs and the remaining idRBC population. Furthermore, computational simulations demonstrated that the smaller and stiffer idRBC subpopulations marginate toward the vessel wall causing aberrant shear stresses. This leads to increased vascular inflammation as confirmed with perfusion of idRBCs into our "endothelialized" microfluidic systems. Overall, our multifaceted approach demonstrates that the altered biophysical properties of idRBCs directly lead to vasculopathy, suggesting that the IDA and cardiovascular disease association extends beyond correlation and into causation.

Possible Contribution of Altered Cholinergic Activity in the Visual Cortex in Visual Hallucinations in Parkinson's Disease.

OBJECTIVE: Up to one-third of patients with Parkinson's disease (PD) experience visual hallucinations (VHs). Lewy bodies are sparse in the visual cortices and seem unlikely to explain the hallucinations. Some neuroimaging studies have found that perfusion is reduced in the occipital lobe in individuals with VHs. Recent work has suggested that decreased cholinergic input may directly lead to the decreased perfusion. The investigators hypothesized that individuals with PD and VHs would have biochemical evidence of reduced microvascular perfusion and reduced cholinergic activity in areas of the brain that process visual images. METHODS: Tissue from Brodmann's area (BA) 18 and BA 19 was obtained from a well-characterized cohort matched for age, gender, and postmortem interval in 69 individuals (PD without VHs, N=11; PD without dementia plus VHs N=10, N=10; PD with dementia plus VHs, N=16; and control subjects, N=32). Von Willebrand factor, vascular endothelial growth factor A, and myelin-associated glycoprotein:proteolipid protein-1 (MAG:PLP1) ratio-a measure of tissue oxygenation relative to metabolic demand, acetylcholinesterase (AChE), butyrylcholinesterase (BChE), choline acetyltransferase, and α-synuclein-were quantified by enzyme-linked immunosorbent assay. The primary outcome was the MAG:PLP1 ratio. RESULTS: There was no biochemical evidence of chronic hypoperfusion in PD, although microvessel density was decreased in ventral BA 18 and BA 19. There was no between-group difference in BChE in either dorsal BA 18 or BA 19. AChE concentration was reduced in individuals with PD compared with control subjects in dorsal and ventral BA 18 and dorsal BA 19, and it was increased in ventral BA 19. These changes were most marked in the PD plus VHs group. CONCLUSIONS: These results suggest that changes in cholinergic activity rather than chronic hypoperfusion may underlie VHs in PD.

The diagonal band of Broca in health and disease.

The diagonal band of Broca (DBB) contains the second largest cholinergic cell group in the human brain, known as the nucleus of the vertical limb of the DBB (nvlDBB). It has major projections to the hippocampus, but it is often underinvestigated, partly due to its ill-defined anatomical boundaries and hence the difficulty of reliable sampling. In this chapter, we have reviewed the historical literature to reestablish the anatomy of the nvlDBB, distinguishing it from neighboring basal forebrain cholinergic nuclei. Although varying degrees of neuronal loss in the nvlDBB have been reported in a range of neurological disorders, and in the aged brain, the significant nvlDBB cholinergic neuronal loss reported in Lewy body dementias is of particular interest. Retrograde tracer study in rodents has demonstrated reciprocal connections between the DBB and the hippocampal CA2 subfield, an area particularly susceptible to Lewy pathologies. Previous functional studies have demonstrated that the nvlDBB is particularly involved in memory retrieval, a cognitive domain severely affected in Lewy body disorders. Based on these observations, we propose an anatomical and functional connection between the cholinergic component of the nvlDBB (Ch2) and the hippocampal CA2.

Nanofiber-Based Delivery of Bioactive Lipids Promotes Pro-regenerative Inflammation and Enhances Muscle Fiber Growth After Volumetric Muscle Loss.

Volumetric muscle loss (VML) injuries after extremity trauma results in an important clinical challenge often associated with impaired healing, significant fibrosis, and long-term pain and functional deficits. While acute muscle injuries typically display a remarkable capacity for regeneration, critically sized VML defects present a dysregulated immune microenvironment which overwhelms innate repair mechanisms leading to chronic inflammation and pro-fibrotic signaling. In this series of studies, we developed an immunomodulatory biomaterial therapy to locally modulate the sphingosine-1-phosphate (S1P) signaling axis and resolve the persistent pro-inflammatory injury niche plaguing a critically sized VML defect. Multiparameter pseudo-temporal 2D projections of single cell cytometry data revealed subtle distinctions in the altered dynamics of specific immune subpopulations infiltrating the defect that were critical to muscle regeneration. We show that S1P receptor modulation via nanofiber delivery of Fingolimod (FTY720) was characterized by increased numbers of pro-regenerative immune subsets and coincided with an enriched pool of muscle stem cells (MuSCs) within the injured tissue. This FTY720-induced priming of the local injury milieu resulted in increased myofiber diameter and alignment across the defect space followed by enhanced revascularization and reinnervation of the injured muscle. These findings indicate that localized modulation of S1P receptor signaling via nanofiber scaffolds, which resemble the native extracellular matrix ablated upon injury, provides great potential as an immunotherapy for bolstering endogenous mechanisms of regeneration following VML injury.

Modulating local S1P receptor signaling as a regenerative immunotherapy after volumetric muscle loss injury.

Regeneration of skeletal muscle after volumetric injury is thought to be impaired by a dysregulated immune microenvironment that hinders endogenous repair mechanisms. Such defects result in fatty infiltration, tissue scarring, chronic inflammation, and debilitating functional deficits. Here, we evaluated the key cellular processes driving dysregulation in the injury niche through localized modulation of sphingosine-1-phosphate (S1P) receptor signaling. We employ dimensionality reduction and pseudotime analysis on single cell cytometry data to reveal heterogeneous immune cell subsets infiltrating preclinical muscle defects due to S1P receptor inhibition. We show that global knockout of S1P receptor 3 (S1PR3) is marked by an increase of muscle stem cells within injured tissue, a reduction in classically activated relative to alternatively activated macrophages, and increased bridging of regenerating myofibers across the defect. We found that local S1PR3 antagonism via nanofiber delivery of VPC01091 replicated key features of pseudotime immune cell recruitment dynamics and enhanced regeneration characteristic of global S1PR3 knockout. Our results indicate that local S1P receptor modulation may provide an effective immunotherapy for promoting a proreparative environment leading to improved regeneration following muscle injury.

Decrease of Clone Diversity in IgM Repertoires of HBV Chronically Infected Individuals With High Level of Viral Replication.

High-throughput antibody sequencing allows in-depth insights into human antibody repertoires. To investigate the characteristics of antibody repertoires in patients with chronic HBV infection, we performed Illumina sequencing and IMGT/HighV-QUEST analysis of B lymphocytes from healthy adults and the HBV carriers with high or low level of viral replication. The comparative study revealed high levels of similarity between the IgM and IgG repertoires of the HBV carriers and the healthy adults, including the somatic mutations in V regions, the average CDR3 length, and the occurrence of junctional modifications. Nevertheless, the diversity of the unique clones decreased and some clusters of unique clones expanded in the IgM repertoire of chronic HBV carriers (CHB) compared with healthy adults (HH) and inactive HBV carriers (IHB). Such difference in clone diversity and expansion was not observed in the IgG repertoires of the three populations. More shared antibody clones were found between the IgM repertoires of IHB and HH than that found between CHB and HH (7079 clones vs. 2304 clones). Besides, the biased used IGHD genes were IGHD2-2 and IGHD3-3 in CHB library but were IGHD3-10 and IGHD3-22 in IHB and HH library. In contrast, for IgG repertories, the preferred used VDJ genes were similar in all the three populations. These results indicated that low level of serum HBV might not induce significant changes in BCR repertoires, and high level of HBV replication could have more impacts on IgM repertories than IgG repertoires. Taken together, our findings provide a better understanding of the antibody repertoires of HBV chronically infected individuals.

Automated buffer preparation using quaternary valve in fast performance liquid chromatography for protein purification from a cell membrane.

There is a great need for high-throughput protein purification to produce protein molecules for research and therapeutics. Although there have been significant advancements made in automated multi-step chromatography and preparative in-process design-of-experiment (DOE) capabilities in commercial fast performance liquid chromatography (FPLC) instruments, almost all commercial FPLCs rely on a binary buffer mixing system, which hinders automated buffer preparation. Nevertheless, current-generation FPLCs are equipped with a quaternary mixer designed for limited in-line buffer preparation and preparative pH scouting DOE experiments. We decided to leverage the quaternary mixing capability by extending and re-programming AkTA Avant's quaternary valve into an automated in-process buffer preparation system to simplify automated purification requiring complex washing steps. We accomplished this by using two extra inlet valves, a sample valve, and versatile valve to split inputs of the quaternary valve into software-selectable stock solutions of pH buffers, salts, eluents, and additives. We also devised a new flow scheme to perform automated two-step chromatography using only one versatile valve. This was accomplished by using only stock parts and software to facilitate reproduction. To demonstrate the versatility and capability of the system, we purified a transmembrane protein that requires a detergent to stay soluble and needs an in-column, high-salt washing step to achieve high purity.

Hippocampal CA2 Lewy pathology is associated with cholinergic degeneration in Parkinson's disease with cognitive decline.

Although the precise neuropathological substrates of cognitive decline in Parkinson's disease (PD) remain elusive, it has long been regarded that pathology in the CA2 hippocampal subfield is characteristic of Lewy body dementias, including dementia in PD (PDD). Early non-human primate tracer studies demonstrated connections from the nucleus of the vertical limb of the diagonal band of Broca (nvlDBB, Ch2) to the hippocampus. However, the relationship between Lewy pathology of the CA2 subfield and cholinergic fibres has not been explored. Therefore, in this study, we investigated the burden of pathology in the CA2 subsector of PD cases with varying degrees of cognitive impairment and correlated this with the extent of septohippocampal cholinergic deficit. Hippocampal sections from 67 PD, 34 PD with mild cognitive impairment and 96 PDD cases were immunostained for tau and alpha-synuclein, and the respective pathology burden was assessed semi-quantitatively. In a subset of cases, the degree of CA2 cholinergic depletion was quantified using confocal microscopy and correlated with cholinergic neuronal loss in Ch2. We found that only cases with dementia have a significantly greater Lewy pathology, whereas cholinergic fibre depletion was evident in cases with mild cognitive impairment and this was significantly correlated with loss of cholinergic neurons in Ch2. In addition, multiple antigen immunofluorescence demonstrated colocalisation between cholinergic fibres and alpha-synuclein but not tau pathology. Such specific Lewy pathology targeting the cholinergic system within the CA2 subfield may contribute to the unique memory retrieval deficit seen in patients with Lewy body disorders, as distinct from the memory storage deficit seen in Alzheimer's disease.

Author Correction: Next generation histology methods for three-dimensional imaging of fresh and archival human brain tissues.

In the original version of this Article, the concentration of boric acid buffer for the SDS clearing solution was given incorrectly as '1 M sodium borate' and should have read '0.2 M boric acid'. Also, the composition of PBST incorrectly read '1% Triton X-100 (vol/vol) and 0.1% sodium azide (wt/vol)' and should have read '0.1% Triton X-100 (vol/vol) and 0.01% sodium azide (wt/vol)'. Further, the pH of the OPTIClear solution was not stated, and should have read 'with a pH between 7 to 8 adjusted with hydrochloric acid'. These errors have been corrected in both the PDF and HTML versions of the Article.

Low dark current III-V on silicon photodiodes by heteroepitaxy.

Top-illuminated PIN and modified uni-traveling carrier (MUTC) photodiodes based on InGaAs/InAlAs/InP were epitaxially grown on Si templates. Photodiodes with 30-µm diameter have dark currents as low as 10 nA at 3 V corresponding to a dark current density of only 0.8 mA/cm2. The responsivity, 3-dB bandwidth, output power and third-order output intercept point (OIP3) were 0.79 A/W, 9 GHz, 2.6 dBm and 15 dBm, respectively.

Next generation histology methods for three-dimensional imaging of fresh and archival human brain tissues.

Modern clearing techniques for the three-dimensional (3D) visualisation of neural tissue microstructure have been very effective when used on rodent brain but very few studies have utilised them on human brain material, mainly due to the inherent difficulties in processing post-mortem tissue. Here we develop a tissue clearing solution, OPTIClear, optimised for fresh and archival human brain tissue, including formalin-fixed paraffin-embedded material. In light of practical challenges with immunostaining in tissue clearing, we adapt the use of cresyl violet for visualisation of neurons in cleared tissue, with the potential for 3D quantification in regions of interest. Furthermore, we use lipophilic tracers for tracing of neuronal processes in post-mortem tissue, enabling the study of the morphology of human dendritic spines in 3D. The development of these different strategies for human tissue clearing has wide applicability and, we hope, will provide a baseline for further technique development.

Neuropathological Assessment as an Endpoint in Clinical Trial Design.

Different neurodegenerative conditions can have complex, overlapping clinical presentations that make accurate diagnosis during life very challenging. For this reason, confirmation of the clinical diagnosis still requires postmortem verification. This is particularly relevant for clinical trials of novel therapeutics where it is important to ascertain what disease and/or pathology modifying effects the therapeutics have had. Furthermore, it is important to confirm that patients in the trial actually had the correct clinical diagnosis as this will have a major bearing on the interpretation of trial results. Here we present a simple protocol for pathological assessment of neurodegenerative changes.

Free-of-Acrylamide SDS-based Tissue Clearing (FASTClear) for three dimensional visualization of myocardial tissue.

Several pathologic conditions of the heart lead to cardiac structural remodelling. Given the high density and the opaque nature of the myocardium, deep three dimensional (3D) imaging is difficult to achieve and structural analysis of pathological myocardial structure is often limited to two dimensional images and of thin myocardial sections. Efficient methods to obtain optical clearing of the tissue for 3D visualisation are therefore needed. Here we describe a rapid, simple and versatile Free-of-Acrylamide SDS-based Tissue Clearing (FASTClear) protocol specifically designed for cardiac tissue. With this method 3D information regarding collagen content, collagen localization and distribution could be easily obtained across a whole 300 µm-thick myocardial slice. FASTClear does not induce structural or microstructural distortion and it can be combined with immunostaining to identify the micro- and macrovascular networks. In summary, we have obtained decolorized myocardial tissue suitable for high resolution 3D imaging, with implications for the study of complex cardiac tissue structure and its changes during pathology.