Lysholm and KOOS QoL Demonstrate High Responsiveness in Patients Undergoing Anterior Cruciate Ligament Reconstruction: A Systematic Review and Meta-analysis of Randomized Clinical Trials.

BACKGROUND: There have been a large number of patient-reported outcome measures (PROMs) used to assess outcomes after anterior cruciate ligament (ACL) reconstruction (ACLR). PURPOSE/HYPOTHESIS: The purpose was to determine which PROMs are being commonly used in randomized clinical trials (RCTs) to assess patients undergoing ACLR and to compare the responsiveness between them. It was hypothesized that the International Knee Documentation Committee (IKDC) score would be the most commonly used and responsive PROM among patients undergoing ACLR. STUDY DESIGN: Meta-analysis. Level of evidence, 2. METHODS: PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines were followed, and relevant studies were extracted from the PubMed/MEDLINE and Web of Science databases. The inclusion criteria were English-language RCTs reporting on PROMs after ACLR. For articles meeting our inclusion criteria for responsiveness analysis (≥2 PROMs reported, 1 year minimum follow-up, and reported pre- and postoperative PROM means and standard deviations), the responsiveness between PROMs was compared using effect size (ES) and relative efficiency (RE). RESULTS: A total of 108 articles met the inclusion criteria, comprising 9034 patients (mean age, 29.9 years; mean body mass index, 24.3; mean follow-up time, 36.1 months). There were 34 PROMs identified. The top 3 most commonly reported PROMs were the IKDC (n = 68; 63.0%), Lysholm (n = 65; 60.2%), and Tegner (n = 47; 43.5%) scores. The 2 PROMs with the highest ES were the ACL-Quality of Life (QoL) (3.37) and Knee Injury and Osteoarthritis Outcome Score (KOOS) QoL (2.07) scores. Compared with other PROMs, Lysholm and KOOS QoL scores had the greatest RE values. The Lysholm score had a greater RE than the KOOS Pain (RE, 1.17), KOOS Symptoms (RE, 1.22), KOOS Activities of Daily Living (ADL) (RE, 1.42), KOOS Sport/Recreation (RE, 1.55), KOOS QoL (RE, 1.41), and Tegner (RE, 2.89) scores. KOOS QoL had a greater RE than the IKDC (RE, 1.32), KOOS Pain (RE, 1.60), KOOS Symptoms (RE, 2.12), KOOS ADL (RE, 3.03), KOOS Sport/Recreation (RE, 1.27), and Tegner (RE, 2.06) scores. CONCLUSION: The IKDC score is the most commonly reported PROM in RCTs after ACLR; however, the Lysholm and KOOS QoL scores demonstrated the highest responsiveness in patients undergoing ACLR compared with other PROMs.

ASES and UCLA Are Responsive Patient-Reported Outcome Measures After Rotator Cuff Repair: A Systematic Review and Meta-analysis.

BACKGROUND: Multiple patient-reported outcome measures (PROMs) have been used to assess shoulder function, but it is unknown which are the most effective. PURPOSE/HYPOTHESIS: The purpose of this study was to report the multiple PROMs used after rotator cuff repair (RCR) and to compare the responsiveness between them. It was hypothesized that the Western Ontario Rotator Cuff PROM would be the most responsive and commonly used in patients undergoing RCR. STUDY DESIGN: Meta-analysis; Level of evidence, 4. METHODS: A systematic review was conducted in accordance with PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) and PRISMA-Searching extension guidelines via PubMed/MEDLINE, Embase (Elsevier), and Web of Science (Clarivate). Patient and study characteristics were extracted. From the studies that met inclusion criteria for responsiveness analysis (≥2 PROMs reported, 1-year minimum follow-up, and pre- and postoperative PROM means and standard deviations reported), 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 252 studies met inclusion criteria (32,072 patients; mean age, 59.6 years; mean body mass index, 28.7; mean follow-up time, 27.8 months). Range of motion was reported in 131 (52%) studies and imaging findings were reported in 123 (49%) studies. There were 67 PROM instruments identified; the most commonly used were the American Shoulder and Elbow Surgeon (ASES) (n = 183; 73%), Visual Analog Scale (VAS) (n = 163; 65%), and Constant (n = 118; 47%) scores. The 3 PROMs with the highest effect sizes were the University of California, Los Angeles (UCLA) (2.51), Western Ontario Rotator Cuff (2.42), and ASES (2.00) scores. Overall, UCLA and ASES scores were the most responsive PROMs. The ASES PROM was more responsive than the VAS (RE, 1.70), Constant (RE, 2.76), Simple Shoulder Test (RE, 1.67), and Single Assessment Numeric Evaluation (RE, 2.14) scores. The UCLA PROM was more responsive than the ASES (RE, 1.03), VAS (RE, 3.66), Constant (RE, 1.72), and Simple Shoulder Test (RE, 1.66) scores. CONCLUSION: ASES and UCLA scores were the most responsive PROMs after RCR. The authors recommend widespread adoption of ASES and UCLA scores for clinical and research standardization; however, the UCLA PROM requires in-person range of motion and strength testing, which is a practical limitation and barrier to long-term follow-up.

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.

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.

International Knee Documentation Committee (IKDC) Is the Most Responsive Patient Reported Outcome Measure After Meniscal Surgery.

Purpose: To report the variability in outcome measures after meniscal surgery and to compare responsiveness between patient-reported outcome measures (PROMs). Methods: A systematic search of the PubMed/MEDLINE and Web of Science databases was performed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analysis guidelines. A total of 257 studies met inclusion criteria. Patient and study attributes were extracted, including pre- and postoperative means for PROMs. Of the studies that met inclusion criteria for responsiveness analysis (2+ PROMs reported, 1-year minimum follow-up; n = 172), we compared the responsiveness between PROM instruments using effect size and relative efficiency (RE) if a PROM could be compared with another in at least 10 articles. Results: In total, 18,612 patients (18,690 menisci, mean age = 38.6 years, mean body mass index = 26.3) were included in this study. Radiographic measures were reported in 167 (65.0%) studies, range of motion was reported in 53 (20.6%) studies, and 35 different PROM instruments were identified. The mean number of PROMs in each article was 3.6 and 83.8% reported 2 or more PROMs. The most used PROMs were Lysholm (74.5%) and IKDC (51.0%). IKDC was found to be more responsive than other PROMs, which include Lysholm (RE = 1.03), Tegner (RE = 3.90), and Knee Injury and Osteoarthritis Outcome Score (KOOS) Activities of Daily Living (ADL) (RE = 1.12). KOOS Quality of Life (QoL) was also more responsive than other PROMs, such as IKDC (RE = 1.45) and KOOS ADL (RE = 1.48). Lysholm was more responsive compared with KOOS QoL (RE = 1.14), KOOS ADL (RE = 1.96), and Tegner (RE = 3.53). Conclusions: Our study found that IKDC, KOOS QoL, and Lysholm were the most responsive PROMs. However, because of the previously reported risks of either floor effects (KOOS QoL) or ceiling effects (Lysholm), the IKDC may offer a more complete psychometric profile when quantifying outcomes after meniscus procedures. Clinical Relevance: To improve clinical outcomes, surgical decision-making, and research methodology, it is important to determine which PROMs are the most responsive after meniscal surgery.

Chromatin alternates between A and B compartments at kilobase scale for subgenic organization.

Nuclear compartments are prominent features of 3D chromatin organization, but sequencing depth limitations have impeded investigation at ultra fine-scale. CTCF loops are generally studied at a finer scale, but the impact of looping on proximal interactions remains enigmatic. Here, we critically examine nuclear compartments and CTCF loop-proximal interactions using a combination of in situ Hi-C at unparalleled depth, algorithm development, and biophysical modeling. Producing a large Hi-C map with 33 billion contacts in conjunction with an algorithm for performing principal component analysis on sparse, super massive matrices (POSSUMM), we resolve compartments to 500 bp. Our results demonstrate that essentially all active promoters and distal enhancers localize in the A compartment, even when flanking sequences do not. Furthermore, we find that the TSS and TTS of paused genes are often segregated into separate compartments. We then identify diffuse interactions that radiate from CTCF loop anchors, which correlate with strong enhancer-promoter interactions and proximal transcription. We also find that these diffuse interactions depend on CTCF's RNA binding domains. In this work, we demonstrate features of fine-scale chromatin organization consistent with a revised model in which compartments are more precise than commonly thought while CTCF loops are more protracted.

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.

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.

Development of ß-sheet structure in Aß aggregation intermediates diminishes exposed hydrophobic surface area and enhances proinflammatory activity.

Three decades of research, both in vitro and in vivo, have demonstrated the conformational heterogeneity that is displayed by the amyloid ß peptide (Aß) in Alzheimer's disease (AD). Understanding the distinct properties between Aß conformations and how conformation may impact cellular activity remain open questions, yet still continue to provide new insights into protein misfolding and aggregation. In particular, there is interest in the group of soluble oligomeric prefibrillar Aß species comprising lower molecular weight oligomers up to larger protofibrils. In the current study, a number of strategies were utilized to separate Aß protofibrils and oligomers and show that the smaller Aß oligomers have a much different conformation than Aß protofibrils. The differences were consistent for both Aß40 and Aß42. Protofibrils bound thioflavin T to a greater extent than oligomers, and were highly enriched in ß-sheet secondary structure. Aß oligomers possessed a more open structure with significant solvent exposure of hydrophobic domains as determined by tryptophan fluorescence and bis-ANS binding, respectively. The protofibril-selective antibody AbSL readily discerned conformational differences between protofibrils and oligomers. The more developed structure for Aß protofibrils ultimately proved critical for provoking the release of tumor necrosis factor α from microglial cells. The findings demonstrated a dependency on ß-sheet structure for soluble Aß aggregates to cause a microglial inflammatory response. The Aß aggregation process yields many conformationally-varied species with different levels of ß-structure and exposed hydrophobicity. The conformation elements likely determine biological activity and pathogenicity.

Development of a Simple and Effective Lipid-A Antagonist Based on Computational Prediction.

Sepsis is a serious medical condition characterized by bacterial infection and a subsequent massive systemic inflammatory response. In an effort to identify compounds that block lipopolysaccharide (LPS)-induced inflammation reported herein is the development of simple Lipid-A analogues that lack a disaccharide core yet still possess potent antagonistic activity against LPS. The structure of the new lead compound was developed based on predictive computational experiments. LPS antagonism by the lead compound was not straightforward, and a biphasic effect was observed suggesting a possibility of more than one binding site. An IC50 value of 13 nM for the new compound was determined for the possible high affinity site. The combination of computational, synthetic, and biological studies revealed new structural determinants of these simplified analogues. It is expected that the acquired information will aid future design of LPS targeting glycopharmaceuticals.

Development and characterization of phasor-based analysis for FLIM to evaluate the metabolic and epigenetic impact of HER2 inhibition on squamous cell carcinoma cultures.

SIGNIFICANCE: Deranged metabolism and dysregulated growth factor signaling are closely associated with abnormal levels of proliferation, a recognized hallmark in tumorigenesis. Fluorescence lifetime imaging microscopy (FLIM) of endogenous nicotinamide adenine dinucleotide (NADH), a key metabolic coenzyme, offers a non-invasive, diagnostic indicator of disease progression, and treatment response. The model-independent phasor analysis approach leverages FLIM to rapidly evaluate cancer metabolism in response to targeted therapy. AIM: We combined lifetime and phasor FLIM analysis to evaluate the influence of human epidermal growth factor receptor 2 (HER2) inhibition, a prevalent cancer biomarker, on both nuclear and cytoplasmic NAD(P)H of two squamous cell carcinoma (SCC) cultures. While better established, the standard lifetime analysis approach is relatively slow and potentially subject to intrinsic fitting errors and model assumptions. Phasor FLIM analysis offers a rapid, model-independent alternative, but the sensitivity of the bound NAD(P)H fraction to growth factor signaling must also be firmly established. APPROACH: Two SCC cultures with low- and high-HER2 expression, were imaged using multiphoton-excited NAD(P)H FLIM, with and without treatment of the HER2 inhibitor AG825. Cells were challenged with mitochondrial inhibition and uncoupling to investigate AG825's impact on the overall metabolic capacity. Phasor FLIM and lifetime fitting analyses were compared within nuclear and cytoplasmic compartments to investigate epigenetic and metabolic impacts of HER2 inhibition. RESULTS: NAD(P)H fluorescence lifetime and bound fraction consistently decreased following HER2 inhibition in both cell lines. High-HER2 SCC74B cells displayed a more significant response than low-HER2 SCC74A in both techniques. HER2 inhibition induced greater changes in nuclear than cytoplasmic compartments, leading to an increase in NAD(P)H intensity and concentration. CONCLUSIONS: The use of both, complementary FLIM analysis techniques together with quantitative fluorescence intensity revealed consistent, quantitative changes in NAD(P)H metabolism associated with inhibition of growth factor signaling in SCC cell lines. HER2 inhibition promoted increased reliance on oxidative phosphorylation in both cell lines.

Principles of 3D compartmentalization of the human genome.

Chromatin is organized in the nucleus via CTCF loops and compartmental domains. Here, we compare different cell types to identify distinct paradigms of compartmental domain formation in human tissues. We identify and quantify compartmental forces correlated with histone modifications characteristic of transcriptional activity and previously underappreciated roles for distinct compartmental domains correlated with the presence of H3K27me3 and H3K9me3, respectively. We present a computer simulation model capable of predicting compartmental organization based on the biochemical characteristics of independent chromatin features. Using this model, we show that the underlying forces responsible for compartmental domain formation in human cells are conserved and that the diverse compartmentalization patterns seen across cell types are due to differences in chromatin features. We extend these findings to Drosophila to suggest that the same principles are at work beyond humans. These results offer mechanistic insights into the fundamental forces driving the 3D organization of the genome.

Expression of NLRP3 inflammasome proteins in ExpiCHO-S mammalian cells reveals oligomerization properties that are highly sensitive to solution conditions.

The NLRP3 inflammasome is a key intracellular component of the innate immune response. It is a three-protein complex essential for the production of mature interleukin 1-ß. The complex, which is comprised of three proteins, NLRP3, ASC, and pro-caspase-1, has been implicated in the physiological response to pathogenic elements of cardiovascular disease and Alzheimer's disease. Investigations into the properties of the three proteins can be aided by larger-scale recombinant expression to produce adequate amounts. In the current study, a variety of NLRP3 inflammasome proteins were expressed in the ExpiCHO-S mammalian cell system with a particular focus on ASC. ASC fusion proteins with glutathione-S transferase, maltose-binding protein, and SUMO increased solubility and aided in determining the stability and oligomerization propensity of individual ASC domains and full-length ASC. ASC oligomerization was highly sensitive to protein concentration, ionic strength, and mutation. These observations provided strategic ways to enhance protein purification and characterize ASC oligomerization. The ExpiCHO-S expression system consistently produced high-yield recombinant NLRP3 inflammasome proteins which led to a further understanding of ASC oligomerization.

Inhibition of matrix metalloproteinase-9 secretion by dimethyl sulfoxide and cyclic adenosine monophosphate in human monocytes.

BACKGROUND: Matrix metalloproteinases (MMPs), including MMP-9, are an integral part of the immune response and are upregulated in response to a variety of stimuli. New details continue to emerge concerning the mechanistic and regulatory pathways that mediate MMP-9 secretion. There is significant evidence for regulation of inflammation by dimethyl sulfoxide (DMSO) and 3',5'-cyclic adenosine monophosphate (cAMP), thus investigation of how these two molecules may regulate both MMP-9 and tumor necrosis factor α (TNFα) secretion by human monocytes was of high interest. The hypothesis tested in this study was that DMSO and cAMP regulate MMP-9 and TNFα secretion by distinct mechanisms. AIM: To investigate the regulation of lipopolysaccharide (LPS)-stimulated MMP-9 and tumor necrosis factor α secretion in THP-1 human monocytes by dimethyl sulfoxide and cAMP. METHODS: The paper describes a basic research study using THP-1 human monocyte cells. All experiments were conducted at the University of Missouri-St. Louis in the Department of Chemistry and Biochemistry. Human monocyte cells were grown, cultured, and prepared for experiments in the University of Missouri-St. Louis Cell Culture Facility as per accepted guidelines. Cells were treated with LPS for selected exposure times and the conditioned medium was collected for analysis of MMP-9 and TNFα production. Inhibitors including DMSO, cAMP regulators, and anti-TNFα antibody were added to the cells prior to LPS treatment. MMP-9 secretion was analyzed by gel electrophoresis/western blot and quantitated by ImageJ software. TNFα secretion was analyzed by enzyme-linked immuno sorbent assay. All data is presented as the average and standard error for at least 3 trials. Statistical analysis was done using a two-tailed paired Student t-test. P values less than 0.05 were considered significant and designated as such in the Figures. LPS and cAMP regulators were from Sigma-Aldrich, MMP-9 standard and antibody and TNFα antibodies were from R&D Systems, and amyloid-ß peptide was from rPeptide. RESULTS: In our investigation of MMP-9 secretion from THP-1 human monocytes, we made the following findings. Inclusion of DMSO in the cell treatment inhibited LPS-induced MMP-9, but not TNFα, secretion. Inclusion of DMSO in the cell treatment at different concentrations inhibited LPS-induced MMP-9 secretion in a dose-dependent fashion. A cell-permeable cAMP analog, dibutyryl cAMP, inhibited both LPS-induced MMP-9 and TNFα secretion. Pretreatment of the cells with the adenylyl cyclase activator forskolin inhibited LPS-induced MMP-9 and TNFα secretion. Pretreatment of the cells with the general cAMP phosphodiesterase inhibitor IBMX reduced LPS-induced MMP-9 and TNFα in a dose-dependent fashion. Pre-treatment of monocytes with an anti-TNFα antibody blocked LPS-induced MMP-9 and TNFα secretion. Amyloid-ß peptide induced MMP-9 secretion, which occurred much later than TNFα secretion. The latter two findings strongly suggested an upstream role for TNFα in mediating LPS-stimulate MMP-9 secretion. CONCLUSION: The cumulative data indicated that MMP-9 secretion was a distinct process from TNFα secretion and occurred downstream. First, DMSO inhibited MMP-9, but not TNFα, suggesting that the MMP-9 secretion process was selectively altered. Second, cAMP inhibited both MMP-9 and TNFα with a similar potency, but at different monocyte cell exposure time points. The pattern of cAMP inhibition for these two molecules suggested that MMP-9 secretion lies downstream of TNFα and that TNFα may a key component of the pathway leading to MMP-9 secretion. This temporal relationship fit a model whereby early TNFα secretion directly led to later MMP-9 secretion. Lastly, antibody-blocking of TNFα diminished MMP-9 secretion, suggesting a direct link between TNFα secretion and MMP-9 secretion.

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.

Ultrasmall Mixed Eu-Gd Oxide Nanoparticles for Multimodal Fluorescence and Magnetic Resonance Imaging of Passive Accumulation and Retention in TBI.

Traumatic brain injury (TBI) is a leading cause of death and disability worldwide. TBI can have a long-term impact on the quality of life for survivors of all ages. However, there remains no approved treatment that improves outcomes following TBI, which is partially due to poor delivery of therapies into the brain. Therefore, there is a significant unmet need to develop more effective delivery strategies that increase the accumulation and retention of potentially efficacious treatments in the injured brain. Recent work has revealed that nanoparticles (NPs) may offer a promising approach for site-specific delivery; however, a detailed understanding of the specific NP properties that promote brain accumulation and retention are still being developed. Multimodal imaging plays a vital role in the understanding of physicochemical properties that initiate the uptake and accumulation of NPs in the brain at both high spatial (e.g., fluorescence imaging) and temporal (e.g., magnetic resonance imaging, MRI) frequency. However, many NP systems that are currently used in TBI only provide contrast in a single imaging modality limiting the imaging data that can be obtained, and those that offer multimodal imaging capabilities have complicated multistep synthesis methods. Therefore, the goal of this work was to develop an ultrasmall NP with simple fabrication capable of multimodal imaging. Here, we describe the development, characterization, accumulation, and retention of poly(ethylene glycol) (PEG)-coated europium-gadolinium (Eu-Gd) mixed magnetic NPs (MNPs) in a controlled cortical impact mouse model of TBI. We find that these NPs having an ultrasmall core size of 2 nm and a small hydrodynamic size of 13.5 nm can be detected in both fluorescence and MR imaging modalities and rapidly accumulate and are retained in injured brain parenchyma. These NPs should allow for further testing of NP physicochemical properties that promote accumulation and retention in TBI and other disease models.

Author Correction: Human and mouse single-nucleus transcriptomics reveal TREM2-dependent and TREM2-independent cellular responses in Alzheimer's disease.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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.

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.

Human and mouse single-nucleus transcriptomics reveal TREM2-dependent and TREM2-independent cellular responses in Alzheimer's disease.

Glia have been implicated in Alzheimer's disease (AD) pathogenesis. Variants of the microglia receptor triggering receptor expressed on myeloid cells 2 (TREM2) increase AD risk, and activation of disease-associated microglia (DAM) is dependent on TREM2 in mouse models of AD. We surveyed gene-expression changes associated with AD pathology and TREM2 in 5XFAD mice and in human AD by single-nucleus RNA sequencing. We confirmed the presence of Trem2-dependent DAM and identified a previously undiscovered Serpina3n+C4b+ reactive oligodendrocyte population in mice. Interestingly, remarkably different glial phenotypes were evident in human AD. Microglia signature was reminiscent of IRF8-driven reactive microglia in peripheral-nerve injury. Oligodendrocyte signatures suggested impaired axonal myelination and metabolic adaptation to neuronal degeneration. Astrocyte profiles indicated weakened metabolic coordination with neurons. Notably, the reactive phenotype of microglia was less evident in TREM2-R47H and TREM2-R62H carriers than in non-carriers, demonstrating a TREM2 requirement in both mouse and human AD, despite the marked species-specific differences.