A CTCF Code for 3D Genome Architecture.

The architectural protein CTCF plays a complex role in decoding the functional output of the genome. Guo et al. now show that the orientation of a CTCF site restricts its choice of interacting partner, thus creating a code that predicts the three-dimensional organization of the genome. We propose a DNA extrusion model to account for orientation-specific loop formation.

Evolutionarily Conserved Principles Predict 3D Chromatin Organization.

Topologically associating domains (TADs), CTCF loop domains, and A/B compartments have been identified as important structural and functional components of 3D chromatin organization, yet the relationship between these features is not well understood. Using high-resolution Hi-C and HiChIP, we show that Drosophila chromatin is organized into domains we term compartmental domains that correspond precisely with A/B compartments at high resolution. We find that transcriptional state is a major predictor of Hi-C contact maps in several eukaryotes tested, including C. elegans and A. thaliana. Architectural proteins insulate compartmental domains by reducing interaction frequencies between neighboring regions in Drosophila, but CTCF loops do not play a distinct role in this organism. In mammals, compartmental domains exist alongside CTCF loop domains to form topological domains. The results suggest that compartmental domains are responsible for domain structure in all eukaryotes, with CTCF playing an important role in domain formation in mammals.

Nascent Aß42 Fibrillization in Synaptic Endosomes Precedes Plaque Formation in a Mouse Model of Alzheimer's-like ß-Amyloidosis.

Accumulation of amyloid-ß peptide (Aß) aggregates in synapses may contribute to the profound synaptic loss characteristic of Alzheimer's disease (AD). The origin of synaptic Aß aggregates remains elusive, but loss of endosomal proteostasis may trigger their formation. In this study, we identified the synaptic compartments where Aß accumulates, and performed a longitudinal analysis of synaptosomes isolated from brains of TgCRND8 APP transgenic mice of either sex. To evaluate the specific contribution of Aß-degrading protease endothelin-converting enzyme (ECE-1) to synaptic/endosomal Aß homeostasis, we analyzed the effect of partial Ece1 KO in brain and complete ECE1 KO in SH-SY5Y cells. Global inhibition of ECE family members was used to further assess their role in preventing synaptic Aß accumulation. Results showed that, before extracellular amyloid deposition, synapses were burdened with detergent-soluble Aß monomers, oligomers, and fibrils. Levels of all soluble Aß species declined thereafter, as Aß42 turned progressively insoluble and accumulated in Aß-producing synaptic endosomal vesicles with characteristics of multivesicular bodies. Accordingly, fibrillar Aß was detected in brain exosomes. ECE-1-deficient mice had significantly increased endogenous synaptosomal Aß42 levels, and protease inhibitor experiments showed that, in TgCRND8 mice, synaptic Aß42 became nearly resistant to degradation by ECE-related proteases. Our study supports that Aß accumulating in synapses is produced locally, within endosomes, and does not require the presence of amyloid plaques. ECE-1 is a determinant factor controlling the accumulation and fibrillization of nascent Aß in endosomes and, in TgCRND8 mice, Aß overproduction causes rapid loss of Aß42 solubility that curtails ECE-mediated degradation.SIGNIFICANCE STATEMENT Deposition of aggregated Aß in extracellular plaques is a defining feature of AD. Aß aggregates also accumulate in synapses and may contribute to the profound synaptic loss and cognitive dysfunction typical of the disease. However, it is not clear whether synaptotoxic Aß is mainly derived from plaques or if it is produced and aggregated locally, within affected synaptic compartments. Filling this knowledge gap is important for the development of an effective treatment for AD, as extracellular and intrasynaptic pools of Aß may not be equally modulated by immunotherapies or other therapeutic approaches. In this manuscript, we provide evidence that Aß aggregates building up in synapses are formed locally, within synaptic endosomes, because of disruptions in nascent Aß proteostasis.

A new class of monoclonal Aß antibodies selectively targets and triggers deposition of Aß protofibrils.

Aggregation and accumulation of amyloid-ß peptide (Aß) are a critical trigger for the onset of Alzheimer's disease (AD). While the plaques are the most outstanding Aß pathological feature, much of the recent research emphasis has been on soluble Aß species because of their diffusible, proinflammatory, and toxic properties. The focus on soluble aggregated Aß species has also increased the interest in antibodies that are selective for different Aß conformations. In the current study, we developed and characterized a new class of monoclonal antibodies (referred to as mAbSL) that are selective for Aß protofibrils. Cloning and sequencing of the heavy and light chain variable regions for multiple antibodies identified sequence characteristics that may impart the conformational selectivity by the antibodies. Transfection of FreeStyle 293F cells with the plasmids permitted in-house expression and purification of mAbSL antibodies along with non-conformation-selective Aß monoclonal antibodies (Aß mAbs). Several of the purified mAbSL antibodies demonstrated significant affinity and selectivity for Aß42 protofibrils compared with Aß42 monomers and Aß42 fibrils. Competition ELISA assays assessing the best overall antibody, mAbSL 113, yielded affinity constants of 7 nM for the antibody-Aß42 protofibril interaction, while the affinity for either Aß42 monomers or Aß42 fibrils was roughly 80 times higher. mAbSL 113 significantly inhibited Aß42 monomer aggregation by a unique mechanism compared with the inhibition displayed by Aß mAb 513. Aß42 protofibril dynamics were also markedly altered in the presence of mAbSL 113, whereby insoluble complex formation and protofibril deposition were stimulated by the antibody at low substoichiometric molar ratios. As the field contemplates the therapeutic effectiveness of Aß conformation-selective antibodies, the findings presented here demonstrate new information on a monoclonal antibody that selectively targets Aß protofibrils and impacts Aß dynamics.

Analysis of Hi-C data using SIP effectively identifies loops in organisms from C. elegans to mammals.

Chromatin loops are a major component of 3D nuclear organization, visually apparent as intense point-to-point interactions in Hi-C maps. Identification of these loops is a critical part of most Hi-C analyses. However, current methods often miss visually evident CTCF loops in Hi-C data sets from mammals, and they completely fail to identify high intensity loops in other organisms. We present SIP, Significant Interaction Peak caller, and SIPMeta, which are platform independent programs to identify and characterize these loops in a time- and memory-efficient manner. We show that SIP is resistant to noise and sequencing depth, and can be used to detect loops that were previously missed in human cells as well as loops in other organisms. SIPMeta corrects for a common visualization artifact by accounting for Manhattan distance to create average plots of Hi-C and HiChIP data. We then demonstrate that the use of SIP and SIPMeta can lead to biological insights by characterizing the contribution of several transcription factors to CTCF loop stability in human cells. We also annotate loops associated with the SMC component of the dosage compensation complex (DCC) in Caenorhabditis elegans and demonstrate that loop anchors represent bidirectional blocks for symmetrical loop extrusion. This is in contrast to the asymmetrical extrusion until unidirectional blockage by CTCF that is presumed to occur in mammals. Using HiChIP and multiway ligation events, we then show that DCC loops form a network of strong interactions that may contribute to X Chromosome-wide condensation in C. elegans hermaphrodites.

Psychosocial factors play a greater role in preoperative symptoms for patients with atraumatic shoulder instability: data from the MOON-Shoulder Instability group.

BACKGROUND: Previous studies have demonstrated that psychosocial factors and comorbid depression are associated with worse preoperative baseline measures, clinical outcomes, and recovery in patients undergoing shoulder surgery. It is unknown whether this potential link would differ between those with traumatic vs. atraumatic shoulder instability, as symptoms may persist longer in atraumatic instability prior to surgical intervention. The purpose of this study was to determine if psychosocial factors and/or comorbid depression more heavily influence preoperative symptoms for patients with traumatic vs. atraumatic shoulder instability. METHODS: Prospective baseline data from 1552 patients in the Multicenter Orthopaedic Outcomes Network (MOON) Shoulder Instability cohort were analyzed based on mechanism of injury while controlling for age, sex, and direction of instability. Multivariable linear regressions were performed to determine whether psychological factors (RAND 36 Mental Component Score [MCS], depression diagnosis, Personality Assessment Screener-22) were predictive of preoperative American Shoulder and Elbow Surgeons Standardized Shoulder Assessment Form (ASES) and Western Ontario Shoulder Instability Index (WOSI) scores in the atraumatic group. The same model was repeated for the traumatic instability group, and the model fit was compared between groups, with P < .05 considered statistically significant. RESULTS: Female sex and lower MCS were significantly associated with worse preoperative ASES and WOSI scores for the group with atraumatic instability (ASES R2 = 0.15, P < .001; WOSI R2 = 0.17, P < .001). The same model performed significantly worse (P < .05) for both ASES and WOSI scores in the group with traumatic instability (ASES R2 = 0.07, WOSI R2 = 0.08). CONCLUSIONS: Worse preoperative psychosocial factors were found to be more strongly associated with shoulder-related pain and function for patients with atraumatic instability. Across multiple orthopedic conditions, depression and emotional well-being have been associated with worse preoperative symptoms and inferior postoperative patient-reported outcomes. Despite the stronger and significant association in atraumatic patients, worse psychosocial factors did not have as large an impact as has been seen in other, more chronic conditions such as osteoarthritis or rotator cuff tears. In addition to medically optimizing patients prior to surgery, the current findings identify a subset of shoulder instability patients that may benefit from a behavioral health intervention either prior to surgery or early in the postoperative period to potentially improve postoperative outcomes.

The intricate biophysical puzzle of caspase-1 activation.

This review takes a closer look at the structural components of the molecules involved in the processes leading to caspase-1 activation. Interleukins 1ß and 18 (IL-1ß, IL-18) are well-known proinflammatory cytokines that are produced following cleavage of their respective precursor proteins by the cysteine protease caspase-1. Active caspase-1 is the final step of the NLRP3 inflammasome, a three-protein intracellular complex involved in inflammation and induction of pyroptosis (a proinflammatory cell-death process). NLRP3 activators facilitate assembly of the inflammasome complex and subsequent activation of caspase-1 by autoproteolysis. However, the definitive structural components of active caspase-1 are still unclear and new data add to the complexity of this process. This review outlines the historical and recent findings that provide supporting evidence for the structural aspects of caspase-1 autoproteolysis and activation.

Disentangling aggregation-prone proteins: a new method for isolating α-synuclein species: An Editorial Highlight for "A simple, versatile and robust centrifugation-based filtration protocol for the isolation and quantification of α-synuclein monomers, oligomers and fibrils: Towards improving experimental reproducibility in α-synuclein research" on page 103.

Protein aggregation plays a central role in numerous neurodegenerative diseases. The key proteins in these diseases are of significant importance, but their investigation can be challenging due to unique properties of protein misfolding and oligomerization. Alpha-synuclein protein (α-Syn) is the predominant component of Lewy Bodies in Parkinson's disease (PD) and is a member of this class of proteins. Many α-Syn studies are limited by the inability to separate various monomeric, oligomeric, and fibrillar forms of the protein from heterogeneous mixtures. This Editorial Highlight summarizes the impact of a study published in the current issue of Journal of Neurochemistry, in which Lashuel and colleagues developed a simple, rapid centrifugation- and filter-based method for separating, isolating, and quantifying different forms of α-Syn. The researchers used electron microscopy, SDS-PAGE, circular dichroism, and protein assays to carefully validate the method and quantitate α-Syn yields and loss. The publication of this new method will not only aid in future studies of α-Syn, but will likely extend to other proteins that underlie a variety of neurodegenerative diseases.

Inflammatory mechanisms in neurodegeneration.

This review discusses the profound connection between microglia, neuroinflammation, and Alzheimer's disease (AD). Theories have been postulated, tested, and modified over several decades. The findings have further bolstered the belief that microglia-mediated inflammation is both a product and contributor to AD pathology and progression. Distinct microglia phenotypes and their function, microglial recognition and response to protein aggregates in AD, and the overall role of microglia in AD are areas that have received considerable research attention and yielded significant results. The following article provides a historical perspective of microglia, a detailed discussion of multiple microglia phenotypes including dark microglia, and a review of a number of areas where microglia intersect with AD and other pathological neurological processes. The overall breadth of important discoveries achieved in these areas significantly strengthens the hypothesis that neuroinflammation plays a key role in AD. Future determination of the exact mechanisms by which microglia respond to, and attempt to mitigate, protein aggregation in AD may lead to new therapeutic strategies.

An integrated -omics analysis of the epigenetic landscape of gene expression in human blood cells.

BACKGROUND: Gene expression can be influenced by DNA methylation 1) distally, at regulatory elements such as enhancers, as well as 2) proximally, at promoters. Our current understanding of the influence of distal DNA methylation changes on gene expression patterns is incomplete. Here, we characterize genome-wide methylation and expression patterns for ~ 13 k genes to explore how DNA methylation interacts with gene expression, throughout the genome. RESULTS: We used a linear mixed model framework to assess the correlation of DNA methylation at ~ 400 k CpGs with gene expression changes at ~ 13 k transcripts in two independent datasets from human blood cells. Among CpGs at which methylation significantly associates with transcription (eCpGs), > 50% are distal (> 50 kb) or trans (different chromosome) to the correlated gene. Many eCpG-transcript pairs are consistent between studies and ~ 90% of neighboring eCpGs associate with the same gene, within studies. We find that enhancers (P < 5e-18) and microRNA genes (P = 9e-3) are overrepresented among trans eCpGs, and insulators and long intergenic non-coding RNAs are enriched among cis and distal eCpGs. Intragenic-eCpG-transcript correlations are negative in 60-70% of occurrences and are enriched for annotated gene promoters and enhancers (P < 0.002), highlighting the importance of intragenic regulation. Gene Ontology analysis indicates that trans eCpGs are enriched for transcription factor genes and chromatin modifiers, suggesting that some trans eCpGs represent the influence of gene networks and higher-order transcriptional control. CONCLUSIONS: This work sheds new light on the interplay between epigenetic changes and gene expression, and provides useful data for mining biologically-relevant results from epigenome-wide association studies.

APP Regulates Microglial Phenotype in a Mouse Model of Alzheimer's Disease.

UNLABELLED: Prior work suggests that amyloid precursor protein (APP) can function as a proinflammatory receptor on immune cells, such as monocytes and microglia. Therefore, we hypothesized that APP serves this function in microglia during Alzheimer's disease. Although fibrillar amyloid ß (Aß)-stimulated cytokine secretion from both wild-type and APP knock-out (mAPP(-/-)) microglial cultures, oligomeric Aß was unable to stimulate increased secretion from mAPP(-/-) cells. This was consistent with an ability of oligomeric Aß to bind APP. Similarly, intracerebroventricular infusions of oligomeric Aß produced less microgliosis in mAPP(-/-) mice compared with wild-type mice. The mAPP(-/-) mice crossed to an APP/PS1 transgenic mouse line demonstrated reduced microgliosis and cytokine levels and improved memory compared with wild-type mice despite robust fibrillar Aß plaque deposition. These data define a novel function for microglial APP in regulating their ability to acquire a proinflammatory phenotype during disease. SIGNIFICANCE STATEMENT: A hallmark of Alzheimer's disease (AD) brains is the accumulation of amyloid ß (Aß) peptide within plaques robustly invested with reactive microglia. This supports the notion that Aß stimulation of microglial activation is one source of brain inflammatory changes during disease. Aß is a cleavage product of the ubiquitously expressed amyloid precursor protein (APP) and is able to self-associate into a wide variety of differently sized and structurally distinct multimers. In this study, we demonstrate both in vitro and in vivo that nonfibrillar, oligomeric forms of Aß are able to interact with the parent APP protein to stimulate microglial activation. This provides a mechanism by which metabolism of APP results in possible autocrine or paracrine Aß production to drive the microgliosis associated with AD brains.

The conformational epitope for a new Aß42 protofibril-selective antibody partially overlaps with the peptide N-terminal region.

Aggregation and accumulation of amyloid-ß peptide (Aß) is a key component of Alzheimer's disease (AD). While monomeric Aß appears to be benign, oligomers adopt a biologically detrimental structure. These soluble structures can be detected in AD brain tissue by antibodies that demonstrate selectivity for aggregated Aß. Protofibrils are a subset of soluble oligomeric Aß species and are described as small (< 100 nm) curvilinear assemblies enriched in ß-sheet structure. Our own in vitro studies demonstrate that microglial cells are much more sensitive to soluble Aß42 protofibrils compared to Aß42 monomer or insoluble Aß42 fibrils. Protofibrils interact with microglia, trigger Toll-like receptor signaling, elicit cytokine transcription and expression, and are rapidly taken up by the cells. Because of the importance of this Aß species, we sought to develop an antibody that selectively recognizes protofibrils over other Aß species. Immunization of rabbits with isolated Aß42 protofibrils generated a high-titer anti serum with a strong affinity for Aß42 protofibrils. The antiserum, termed AbSL, was selective for Aß42 protofibrils over Aß42 monomers and Aß42 fibrils. AbSL did not react with amyloid precursor protein and recognized distinct pathological features in AD transgenic mouse brain slices. Competition studies with an Aß antibody that targets residues 1-16 indicated that the conformational epitope for AbSL involved the N-terminal region of protofibrils in some manner. The newly developed antibody may have potential diagnostic and therapeutic uses in AD tissue and patients, and targeting of protofibrils in AD may have beneficial effects. Read the Editorial Highlight for this article on page 621. Cover Image for this issue: doi. 10.1111/jnc.13827.

Integrated tRNA, transcript, and protein profiles in response to steroid hormone signaling.

The accurate and efficient transfer of genetic information into amino acid sequences is carried out through codon-anticodon interactions between mRNA and tRNA, respectively. In this way, tRNAs function at the interface between gene expression and protein synthesis. Whether tRNA levels are dynamically regulated and to what degree tRNA abundance influences the cellular proteome remains largely unexplored. Here we profile tRNA, transcript and protein levels in Drosophila Kc167 cells, a plasmatocyte cell line that, upon treatment with 20-hydroxyecdysone, differentiates into macrophages. We find that high abundance tRNAs associate with codons that are overrepresented in the Kc167 cell proteome, whereas tRNAs that are in low supply associate with codons that are underrepresented. Ecdysone-induced differentiation of Kc167 cells leads to changes in mRNA codon usage in a manner consistent with the developmental progression of the cell. At both early and late time points, ecdysone treatment concomitantly increases the abundance of tRNAThr(CGU), which decodes a differentiation-associated codon that becomes enriched in the macrophage proteome. These results together suggest that tRNA levels may provide a meaningful regulatory mechanism for defining the cellular proteomic landscape.

Chemotherapy impedes in vitro microcirculation and promotes migration of leukemic cells with impact on metastasis.

Although most cancer drugs target the proliferation of cancer cells, it is metastasis, the complex process by which cancer cells spread from the primary tumor to other tissues and organs of the body where they form new tumors, that leads to over 90% of all cancer deaths. Thus, there is an urgent need for anti-metastasis therapy. Surprisingly, emerging evidence suggests that certain anti-cancer drugs such as paclitaxel and doxorubicin can actually promote metastasis, but the mechanism(s) behind their pro-metastatic effects are still unclear. Here, we use a microfluidic microcirculation mimetic (MMM) platform which mimics the capillary constrictions of the pulmonary and peripheral microcirculation, to determine if in-vivo-like mechanical stimuli can evoke different responses from cells subjected to various cancer drugs. In particular, we show that leukemic cancer cells treated with doxorubicin and daunorubicin, commonly used anti-cancer drugs, have over 100% longer transit times through the device, compared to untreated leukemic cells. Such delays in the microcirculation are known to promote extravasation of cells, a key step in the metastatic cascade. Furthermore, we report a significant (p < 0.01) increase in the chemotactic migration of the doxorubicin treated leukemic cells. Both enhanced retention in the microcirculation and enhanced migration following chemotherapy, are pro-metastatic effects which can serve as new targets for anti-metastatic drugs.

Aß40 has a subtle effect on Aß42 protofibril formation, but to a lesser degree than Aß42 concentration, in Aß42/Aß40 mixtures.

Recent findings suggest that the senile plaques in Alzheimer's disease may contain soluble amyloid-ß peptide (Aß) fibril precursors along with insoluble fibrils. These soluble Aß species, including oligomers and protofibrils, have been well-studied in vitro and are formed via non-covalent self-assembly of Aß monomers. While both 40- and 42-residue forms of Aß are observed in the human body, the majority of the Aß aggregation work has been conducted on Aß42 or Aß40 separately, with relatively few investigations of mixtures. In order to study the effect of different combinations of Aß40 and Aß42 on protofibril formation, mixtures of either dry solid peptide, or purified Aß40 and Aß42 monomer solutions were mixed together and protofibril/monomer distributions were quantified. Increases in the Aß42/Aß40 ratio increased protofibril formation but the presence of Aß40 in the mixed Aß solutions had a significant negative impact on protofibril formation compared to equivalent solutions of pure Aß42. Protofibril size was less affected, but ß-sheet structure increased with protofibrils formed from higher Aß42/Aß40 ratio solutions. Direct measurement of Aß42/Aß40 ratios by C-terminal-selective ELISA found very little Aß40 incorporated into protofibrils. The cumulative data emphasizes the critical importance of Aß42, yet establishes Aß40 as a regulator of Aß42 aggregation.

Biophysical comparison of soluble amyloid-ß(1-42) protofibrils, oligomers, and protofilaments.

Some of the pathological hallmarks of the Alzheimer's disease brain are senile plaques composed of insoluble amyloid-ß protein (Aß) fibrils. However, much of the recent emphasis in research has been on soluble Aß aggregates in response to a growing body of evidence that shows that these species may be more neurotoxic than fibrils. Within this subset of soluble aggregated Aß are protofibrils and oligomers. Although each species has been widely investigated separately, few studies have directly compared and contrasted their physical properties. In this work, we examined well-recognized preparations of Aß(1-42) oligomers and protofibrils with multiangle (MALS) and dynamic (DLS) light scattering in line with, or following, size-exclusion chromatography (SEC). Multiple SEC-MALS analyses of protofibrils revealed molecular weight (Mw) gradients ranging from 200 to 2600 kDa. Oligomeric Aß species are generally considered to be a smaller and more nascent than protofibrils. However, oligomer Mw values ranged from 225 to 3000 kDa, larger than that for protofibrils. Root-mean-square radius (Rg) values correlated with the Mw trends with protofibril Rg values ranging from 16 to 35 nm, while oligomers produced one population at 40-43 nm with a more disperse population from 22 to 39 nm. Hydrodynamic radius (RH) measurements by DLS and thioflavin T fluorescence measurements indicated that protofibrils and oligomers had commonalities, yet electron microscopy revealed morphological differences between the two. SEC-purified Aß(1-42) monomer at lower concentrations was slower to nucleate but formed protofibrils (1500 kDa) or soluble protofilaments (3000 kDa) depending on the buffer type. The findings from these studies shed new light on the similarities and differences between distinct soluble aggregated Aß species.

Amyloid-ß(1-42) protofibrils stimulate a quantum of secreted IL-1ß despite significant intracellular IL-1ß accumulation in microglia.

Neuroinflammation is a characteristic feature of the Alzheimer's disease (AD) brain. Significant inflammatory markers such as activated microglia and cytokines can be found surrounding the extracellular senile plaques predominantly composed of amyloid-ß protein (Aß). Several innate immune pathways, including Toll-like receptors (TLRs) and the NLRP3 inflammasome, have been implicated in AD inflammation. Aß plays a primary role in activating these pathways which likely contributes to the progressive neurodegeneration in AD. In order to better understand the complexities of this interaction we investigated the inflammatory response of primary microglia to Aß(1-42) protofibrils. Aß(1-42) protofibrils triggered a time- and MyD88-dependent process that produced tumor necrosis factor alpha (TNFα) and interleukin-1ß (IL-1ß) mRNA, and intracellular pro and mature forms of IL-1ß protein. The accumulation of both IL-1ß forms indicated that Aß(1-42) protofibrils were able to prime and activate the NLRP3 inflammasome. Surprisingly, Aß-induced accumulation of intracellular mature IL-1ß did not translate into greater IL-1ß secretion. Instead, we found that Aß elicited a quantized burst of secreted IL-1ß and this process occurred even prior to Aß priming of the microglia suggesting a basal level of either pro or mature IL-1ß in the cultured primary microglia. The IL-1ß secretion burst was rapid but not sustained, yet could be re-evoked with additional Aß stimulation. The findings from this study demonstrated multiple sites of IL-1ß regulation by Aß(1-42) protofibrils including TLR/MyD88-mediated priming, NLRP3 inflammasome activation, and modulation of the IL-1ß secretory process. These results underscore the wide-ranging effects of Aß on the innate immune response.

Stability of early-stage amyloid-ß(1-42) aggregation species.

Accumulation of aggregated amyloid-ß protein (Aß) is an important feature of Alzheimer's disease. There is significant interest in understanding the initial steps of Aß aggregation due to the recent focus on soluble Aß oligomers. In vitro studies of Aß aggregation have been aided by the use of conformation-specific antibodies which recognize shape rather than sequence. One of these, OC antiserum, recognizes certain elements of fibrillar Aß across a broad range of sizes. We have observed the presence of these fibrillar elements at very early stages of Aß incubation. Using a dot blot assay, OC-reactivity was found in size exclusion chromatography (SEC)-purified Aß(1-42) monomer fractions immediately after isolation (early-stage). The OC-reactivity was not initially observed in the same fractions for Aß(1-40) or the aggregation-restricted Aß(1-42) L34P but was detected within 1-2weeks of incubation. Stability studies demonstrated that early-stage OC-positive Aß(1-42) aggregates were resistant to 4M urea or guanidine hydrochloride but sensitive to 1% sodium dodecyl sulfate (SDS). Interestingly, the sensitivity to SDS diminished over time upon incubation of the SEC-purified Aß(1-42) solution at 4°C. Within 6-8days the OC-positive Aß42 aggregates were resistant to SDS denaturation. The progression to, and development of, SDS resistance for Aß(1-42) occurred prior to thioflavin T fluorescence. In contrast, Aß(1-40) aggregates formed after 6days of incubation were sensitive to both urea and SDS. These findings reveal information on some of the earliest events in Aß aggregation and suggest that it may be possible to target early-stage aggregates before they develop significant stability.

CD47 does not mediate amyloid-ß(1-42) protofibril-stimulated microglial cytokine release.

Neuroinflammation triggered by accumulation of amyloid-ß protein (Aß) is a significant component of the Alzheimer's disease (AD) brain. Senile plaques composed of Aß attract and activate microglia cells resulting in cytokine secretion and a proinflammatory environment. The mechanism by which Aß activates microglia is complex and involves numerous cellular components. One receptor potentially involved in Aß recognition and the ensuing microglia proinflammatory response is CD47. Since there is significant interest in soluble aggregated Aß species, we sought to determine if CD47 plays a key role in microglia cytokine release stimulated by soluble Aß(1-42) protofibrils. Pretreatment of primary murine microglia with the CD47 antagonist peptide 4N1K significantly and potently inhibited both tumor necrosis factor-α (TNFα) and interleukin-1ß (IL-1ß) secretion stimulated by Aß(1-42) protofibrils. 4N1K displayed toxicity to the microglia but only at concentrations much higher than the observed inhibition. Surprisingly, 4N1K also potently inhibited TNFα secretion triggered by lipopolysaccharide which is not known to signal through CD47. Treatment of the microglia with a neutralizing anti-CD47 antibody failed to block the Aß protofibril response even though comparable samples were completely inhibited by 4N1K. Finally, Aß(1-42) protofibrils stimulated similar levels of secreted TNFα production in both wild-type and CD47(-/-) microglia and 4N1K still potently inhibited the Aß protofibril response even in the CD47(-/-) microglia. The overall findings demonstrated that the microglial proinflammatory response to Aß(1-42) protofibril is not dependent on CD47 and that 4N1K exhibits CD47-independent inhibitory activity.

Responsiveness of Patient-Reported Outcome Measures After Large Knee Articular Cartilage Transplantation: A Systematic Review and Meta-analysis.

BACKGROUND: Cartilage transplantation is commonly used to treat large (>4 cm2) articular cartilage defects of the knee. The 2 most common transplants are osteochondral allograft transplantation and autologous chondrocyte implantation. Several patient-reported outcome measures (PROMs) have been used to determine the efficacy of treatment, but it is unknown which measures are the most effective. PURPOSE: To report the multiple PROMs used after large knee articular cartilage transplantation surgery and to compare the responsiveness between them. STUDY DESIGN: Meta-analysis; Level of evidence, 4. METHODS: Following Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, a systematic search of the PubMed/MEDLINE and Web of Science databases was performed. A total of 181 articles met inclusion criteria. Patient and study characteristics were extracted, including pre- and postoperative means for PROMs. From the articles that met inclusion criteria for responsiveness analysis (2+ PROMs reported, 1-year minimum follow-up, reported pre- and postoperative means and standard deviations; n = 131), the authors compared the responsiveness between PROM instruments using effect size and relative efficiency (RE) if a PROM could be compared with another in ≥10 articles. RESULTS: A total of 10,015 patients (10,093 knees; mean age, 34.8 years; mean body mass index, 26.1) were included in this study. The mean follow-up time was 58.3 months (range, 1.5-247.2 months), imaging findings were reported in 80 articles (44.2%), patient satisfaction was reported in 39 articles (21.5%), and range of motion was reported in 10 articles (5.5%). There were 58 unique PROM instruments identified, with the most used being the International Knee Documentation Committee (IKDC) score (n = 118; 65.2%), followed by Knee injury and Osteoarthritis Outcome Score (KOOS) Pain (n = 58; 32.0%), KOOS Sport and Recreation (n = 58; 32.0%), KOOS Quality of Life (n = 57; 31.5%), KOOS Activities of Daily Living (n = 57; 31.5%), and KOOS Symptoms (n = 57; 31.5%). Overall, IKDC was found to have the greatest effect size (1.68) and the best responsiveness of the other PROMs, which include KOOS Pain (RE, 1.38), KOOS Symptoms (RE, 3.06), KOOS Activities of Daily Living (RE, 1.65), KOOS Sport and Recreation (RE, 1.44), Lysholm (RE, 1.76), and Tegner (RE, 1.56). CONCLUSION: The IKDC is the most responsive PROM after large knee articular cartilage transplantation surgery. The IKDC score is recommended for assessing outcomes after cartilage transplantation surgery.