Bile salt hydrolase acyltransferase activity expands bile acid diversity.

Bile acids (BAs) are steroid detergents in bile that contribute to the absorption of fats and fat-soluble vitamins while shaping the gut microbiome because of their antimicrobial properties1-4. Here we identify the enzyme responsible for a mechanism of BA metabolism by the gut microbiota involving amino acid conjugation to the acyl-site of BAs, thus producing a diverse suite of microbially conjugated bile acids (MCBAs). We show that this transformation is mediated by acyltransferase activity of bile salt hydrolase (bile salt hydrolase/transferase, BSH/T). Clostridium perfringens BSH/T rapidly performed acyl transfer when provided various amino acids and taurocholate, glycocholate or cholate, with an optimum at pH 5.3. Amino acid conjugation by C. perfringens BSH/T was diverse, including all proteinaceous amino acids except proline and aspartate. MCBA production was widespread among gut bacteria, with strain-specific amino acid use. Species with similar BSH/T amino acid sequences had similar conjugation profiles and several bsh/t alleles correlated with increased conjugation diversity. Tertiary structure mapping of BSH/T followed by mutagenesis experiments showed that active site structure affects amino acid selectivity. These MCBA products had antimicrobial properties, where greater amino acid hydrophobicity showed greater antimicrobial activity. Inhibitory concentrations of MCBAs reached those measured natively in the mammalian gut. MCBAs fed to mice entered enterohepatic circulation, in which liver and gallbladder concentrations varied depending on the conjugated amino acid. Quantifying MCBAs in human faecal samples showed that they reach concentrations equal to or greater than secondary and primary BAs and were reduced after bariatric surgery, thus supporting MCBAs as a significant component of the BA pool that can be altered by changes in gastrointestinal physiology. In conclusion, the inherent acyltransferase activity of BSH/T greatly diversifies BA chemistry, creating a set of previously underappreciated metabolites with the potential to affect the microbiome and human health.

Fibroblast-Derived Lysyl Oxidase Increases Oxidative Phosphorylation and Stemness in Cholangiocarcinoma.

BACKGROUND & AIMS: Metabolic and transcriptional programs respond to extracellular matrix-derived cues in complex environments, such as the tumor microenvironment. Here, we demonstrate how lysyl oxidase (LOX), a known factor in collagen crosslinking, contributes to the development and progression of cholangiocarcinoma (CCA). METHODS: Transcriptomes of 209 human CCA tumors, 143 surrounding tissues, and single-cell data from 30 patients were analyzed. The recombinant protein and a small molecule inhibitor of the LOX activity were used on primary patient-derived CCA cultures to establish the role of LOX in migration, proliferation, colony formation, metabolic fitness, and the LOX interactome. The oncogenic role of LOX was further investigated by RNAscope and in vivo using the AKT/NICD genetically engineered murine CCA model. RESULTS: We traced LOX expression to hepatic stellate cells and specifically hepatic stellate cell-derived inflammatory cancer-associated fibroblasts and found that cancer-associated fibroblast-driven LOX increases oxidative phosphorylation and metabolic fitness of CCA, and regulates mitochondrial function through transcription factor A, mitochondrial. Inhibiting LOX activity in vivo impedes CCA development and progression. Our work highlights that LOX alters tumor microenvironment-directed transcriptional reprogramming of CCA cells by facilitating the expression of the oxidative phosphorylation pathway and by increasing stemness and mobility. CONCLUSIONS: Increased LOX is driven by stromal inflammatory cancer-associated fibroblasts and correlates with diminished survival of patients with CCA. Modulating the LOX activity can serve as a novel tumor microenvironment-directed therapeutic strategy in bile duct pathologies.

SARS-CoV-2 Nsp8 N-terminal domain folds autonomously and binds dsRNA.

The SARS-CoV-2 Nsp8 protein is a critical component of the RNA replicase, as its N-terminal domain (NTD) anchors Nsp12, the RNA, and Nsp13. Whereas its C-terminal domain (CTD) structure is well resolved, there is an open debate regarding the conformation adopted by the NTD as it is predicted as disordered but found in a variety of complex-dependent conformations or missing from many other structures. Using NMR spectroscopy, we show that the SARS CoV-2 Nsp8 NTD features both well folded secondary structure and disordered segments. Our results suggest that while part of this domain corresponding to two long α-helices forms autonomously, the folding of other segments would require interaction with other replicase components. When isolated, the α-helix population progressively declines towards the C-termini but surprisingly binds dsRNA while preserving structural disorder.

Architectonic principles of polyproline II helix bundle protein domains.

Glycine rich polyproline II helix assemblies are an emerging class of natural domains found in several proteins with different functions and diverse origins. The distinct properties of these domains relative to those composed of α-helices and ß-sheets could make glycine-rich polyproline II helix assemblies a useful building block for protein design. Whereas the high population of polyproline II conformers in disordered state ensembles could facilitate glycine-rich polyproline II helix folding, the architectonic bases of these structures are not well known. Here, we compare and analyze their structures to uncover common features. These protein domains are found to be highly tolerant of distinct flanking sequences. This speaks to the robustness of this fold and strongly suggests that glycine rich polyproline II assemblies could be grafted with other protein domains to engineer new structures and functions. These domains are also well packed with few or no cavities. Moreover, a significant trend towards antiparallel helix configuration is observed in all these domains and could provide stabilizing interactions among macrodipoles. Finally, extensive networks of Cα-H···OC hydrogen bonds are detected in these domains. Despite their diverse evolutionary origins and activities, glycine-rich polyproline II helix assemblies share architectonic features which could help design novel proteins.

Phe-Gly motifs drive fibrillization of TDP-43's prion-like domain condensates.

Transactive response DNA-binding Protein of 43 kDa (TDP-43) assembles various aggregate forms, including biomolecular condensates or functional and pathological amyloids, with roles in disparate scenarios (e.g., muscle regeneration versus neurodegeneration). The link between condensates and fibrils remains unclear, just as the factors controlling conformational transitions within these aggregate species: Salt- or RNA-induced droplets may evolve into fibrils or remain in the droplet form, suggesting distinct end point species of different aggregation pathways. Using microscopy and NMR methods, we unexpectedly observed in vitro droplet formation in the absence of salts or RNAs and provided visual evidence for fibrillization at the droplet surface/solvent interface but not the droplet interior. Our NMR analyses unambiguously uncovered a distinct amyloid conformation in which Phe-Gly motifs are key elements of the reconstituted fibril form, suggesting a pivotal role for these residues in creating the fibril core. This contrasts the minor participation of Phe-Gly motifs in initiation of the droplet form. Our results point to an intrinsic (i.e., non-induced) aggregation pathway that may exist over a broad range of conditions and illustrate structural features that distinguishes between aggregate forms.

Nose-Ocomial Pulmonary Edema: A Rare Complication of Oxymetazoline.

A 72-year-old female with epiphora presented for outpatient punctoplasty with probing and lacrimal stent placement. Oxymetazoline was administered intranasally and the case was completed in standard fashion. Postoperatively, the patient desaturated with a workup revealing elevated cardiac enzymes, pulmonary congestion, and sinus bradycardia. However, the final cardiac testing was noncontributory, suggesting flash pulmonary edema secondary to intranasal oxymetazoline. This case highlights a rare presentation of pulmonary compromise secondary to oxymetazoline, emphasizing the importance of intraoperative and postoperative vigilance in simple outpatient procedures.

Longitudinal microbial and molecular dynamics in the cystic fibrosis lung after Elexacaftor-Tezacaftor-Ivacaftor therapy.

BACKGROUND: Cystic fibrosis (CF) is a genetic disorder causing poor mucociliary clearance in the airways and subsequent respiratory infection. The recently approved triple therapy Elexacaftor-Tezacaftor-Ivacaftor (ETI) has significantly improved lung function and decreased airway infection in persons with CF (pwCF). This improvement has been shown to occur rapidly, within the first few weeks of treatment. The effects of longer term ETI therapy on lung infection dynamics, however, remain mostly unknown. RESULTS: Here, we applied 16S rRNA gene amplicon sequencing, untargeted metabolomics, and neutral models to high-resolution, longitudinally collected sputum samples from pwCF on ETI therapy (162 samples, 7 patients) and compared to similarly collected data set from pwCF not taking ETI (630 samples, 9 patients). Because ETI reduces sputum production, samples were collected in freezers provided in the subject's homes at least 3 months after first taking ETI, with those on ETI collecting a sample approximately weekly. The lung function (%ppFEV1) of those in our longitudinal cohort significantly improved after ETI (6.91, SD = 7.74), indicating our study cohort was responsive to ETI. The daily variation of alpha- and beta-diversity of both the microbiome and metabolome was higher for those on ETI, reflecting a more dynamic microbial community and chemical environment during treatment. Four of the seven subjects on ETI were persistently infected with Pseudomonas or Burkholderia in their sputum throughout the sampling period while the total bacterial load significantly decreased with time (R = - 0.42, p = 0.01) in only one subject. The microbiome and metabolome dynamics on ETI were personalized, where some subjects had a progressive change with time on therapy, whereas others had no association with time on treatment. To further classify the augmented variance of the CF microbiome under therapy, we fit the microbiome data to a Hubbell neutral dynamics model in a patient-stratified manner and found that the subjects on ETI had better fit to a neutral model. CONCLUSION: This study shows that the longitudinal microbiology and chemistry in airway secretions from subjects on ETI has become more dynamic and neutral and that after the initial improvement in lung function, many are still persistently infected with CF pathogens.

Bystander chronic infection negatively impacts development of CD8(+) T cell memory.

Epidemiological evidence suggests that chronic infections impair immune responses to unrelated pathogens and vaccines. The underlying mechanisms, however, are unclear and distinguishing effects on priming versus development of immunological memory has been challenging. We investigated whether bystander chronic infections impact differentiation of memory CD8(+) T cells, the hallmark of protective immunity against intracellular pathogens. Chronic bystander infections impaired development of memory CD8(+) T cells in several mouse models and humans. These effects were independent of initial priming and were associated with chronic inflammatory signatures. Chronic inflammation negatively impacted the number of bystander CD8(+) T cells and their memory development. Distinct underlying mechanisms of altered survival and differentiation were revealed with the latter regulated by the transcription factors T-bet and Blimp-1. Thus, exposure to prolonged bystander inflammation impairs the effector to memory transition. These data have relevance for immunity and vaccination during persisting infections and chronic inflammation.

A randomized phase 2 study of sapanisertib in combination with paclitaxel versus paclitaxel alone in women with advanced, recurrent, or persistent endometrial cancer.

OBJECTIVE: This phase 2 study investigated sapanisertib (selective dual inhibitor of mTORC1/2) alone, or in combination with paclitaxel or TAK-117 (a selective small molecule inhibitor of PI3K), versus paclitaxel alone in advanced, recurrent, or persistent endometrial cancer. METHODS: Patients with histologic diagnosis of endometrial cancer (1-2 prior regimens) were randomized to 28-day cycles on four treatment arms: 1) weekly paclitaxel 80 mg/m2 (days 1, 8, and 15); 2) weekly paclitaxel 80 mg/m2 + oral sapanisertib 4 mg on days 2-4, 9-11, 16-18, and 23-25; 3) weekly sapanisertib 30 mg, or 4) sapanisertib 4 mg + TAK-117 200 mg on days 1-3, 8-10, 15-17, and 22-24. RESULTS: Of 241 patients randomized, 234 received treatment (paclitaxel, n = 87 [3 ongoing]; paclitaxel+sapanisertib, n = 86 [3 ongoing]; sapanisertib, n = 41; sapanisertib+TAK-117, n = 20). The sapanisertib and sapanisertib+TAK-117 arms were closed to enrollment after futility analyses. After a median follow-up of 14.4 (paclitaxel) versus 17.2 (paclitaxel+sapanisertib) months, median progression-free survival (PFS; primary endpoint) was 3.7 versus 5.6 months (hazard ratio [HR] 0.82; 95% confidence interval [CI] 0.58-1.15; p = 0.139); in patients with endometrioid histology (n = 116), median PFS was 3.3 versus 5.7 months (HR 0.66; 95% CI 0.43-1.03). Grade ≥ 3 treatment-emergent adverse event rates were 54.0% with paclitaxel versus 89.5% paclitaxel+sapanisertib. CONCLUSIONS: Our findings support inclusion of chemotherapy combinations with investigational agents for advanced or metastatic disease. The primary endpoint was not met and toxicity was manageable. TRIAL REGISTRATION: ClinicalTrials.gov number, NCT02725268.

Quality of life and symptoms among patients with relapsed/refractory AL amyloidosis treated with ixazomib-dexamethasone versus physician's choice.

Patient-reported outcomes in AL amyloidosis have not been well-studied. We analyzed health-related quality of life (HRQOL) and AL amyloidosis symptoms data from the phase 3 TOURMALINE-AL1 trial (NCT01659658) (ixazomib-dexamethasone, n = 85; physician's choice of chemotherapy [PC], n = 83). HRQOL and symptom burden were measured with the SF-36v2, Functional Assessment of Cancer Therapy/Gynecologic Oncology Group Neurotoxicity subscale (FACT/GOG-Ntx), and an amyloidosis symptom questionnaire (ASQ). Score changes during treatment were analyzed descriptively and using repeated-measures linear mixed models; analyses were not adjusted for multiplicity. Least-squares (LS) mean changes from baseline were significantly higher (better HRQOL) for ixazomib-dexamethasone at several cycles for SF-36v2 Role Physical and Vitality subscales (p < .05); no subscales demonstrated significant differences favoring PC. For FACT/GOG-Ntx, small but significant differences in LS mean changes favored ixazomib-dexamethasone over PC at multiple cycles for seven items and both summary scores; significant differences favored PC for one item (trouble hearing) at multiple cycles. ASQ total score trended downward (lower burden) in both arms; significant LS mean differences favored ixazomib-dexamethasone over PC at some cycles (p < .05). Patients with relapsed/refractory AL amyloidosis treated with ixazomib-dexamethasone experienced HRQOL and symptoms that were similar to or trended better than patients treated with PC despite longer duration of therapy.

Sequential regression and simulation: a method for estimating causal effects from heterogeneous clinical trials without a common control group.

BACKGROUND: The advent of clinical trial data sharing platforms has created opportunities for making new discoveries and answering important questions using already collected data. However, existing methods for meta-analyzing these data require the presence of shared control groups across studies, significantly limiting the number of questions that can be confidently addressed. We sought to develop a method for meta-analyzing potentially heterogeneous clinical trials even in the absence of a common control group. METHODS: This work was conducted within the context of a broader effort to study comparative efficacy in Crohn's disease. Following a search of clnicaltrials.gov we obtained access to the individual participant data from nine trials of FDA-approved treatments in Crohn's Disease (N = 3392). We developed a method involving sequences of regression and simulation to separately model the placebo- and drug-attributable effects, and to simulate head-to-head trials against an appropriately normalized background. We validated this method by comparing the outcome of a simulated trial comparing the efficacies of adalimumab and ustekinumab against the recently published results of SEAVUE, an actual head-to-head trial of these drugs. This study was pre-registered on PROSPERO (#157,827) prior to the completion of SEAVUE. RESULTS: Using our method of sequential regression and simulation, we compared the week eight outcomes of two virtual cohorts subject to the same patient selection criteria as SEAVUE and treated with adalimumab or ustekinumab. Our primary analysis replicated the corresponding published results from SEAVUE (p = 0.9). This finding proved stable under multiple sensitivity analyses. CONCLUSIONS: This new method may help reduce the bias of individual participant data meta-analyses, expand the scope of what can be learned from these already-collected data, and reduce the costs of obtaining high-quality evidence to guide patient care.

Expanded Conformations of Monomeric Tau Initiate Its Amyloidogenesis.

Understanding early amyloidogenesis is key to rationally develop therapeutic strategies. Tau protein forms well-characterized pathological deposits but its aggregation mechanism is still poorly understood. Using single-molecule force spectroscopy based on a mechanical protection strategy, we studied the conformational landscape of the monomeric tau repeat domain (tau-RD244-368 ). We found two sets of conformational states, whose frequency is influenced by mutations and the chemical context. While pathological mutations Δ280K and P301L and a pro-amyloidogenic milieu favored expanded conformations and destabilized local structures, an anti-amyloidogenic environment promoted a compact ensemble, including a conformer whose topology might mask two amyloidogenic segments. Our results reveal that to initiate aggregation, monomeric tau-RD244-368 decreases its polymorphism adopting expanded conformations. This could account for the distinct structures found in vitro and across tauopathies.

Insight into polyproline II helical bundle stability in an antifreeze protein denatured state.

The use of polyproline II (PPII) helices in protein design is currently hindered by limitations in our understanding of their conformational stability and folding. Recent studies of the snow flea antifreeze protein (sfAFP), a useful model system composed of six PPII helices, suggested that a low denatured state entropy contributes to folding thermodynamics. Here, circular dichroism spectroscopy revealed minor populations of PPII like conformers at low temperature. To get atomic level information on the conformational ensemble and entropy of the reduced, denatured state of sfAFP, we have analyzed its chemical shifts and {1H}-15N relaxation parameters by NMR spectroscopy at four experimental conditions. No significant populations of stable secondary structure were detected. The stiffening of certain N-terminal residues at neutral versus acidic pH and shifted pKa values leads us to suggest that favorable charge-charge interactions could bias the conformational ensemble to favor the formation the C1-C28 disulfide bond during nascent folding, although no evidence for preferred contacts between these positions was detected by paramagnetic relaxation enhancement under denaturing conditions. Despite a high content of flexible glycine residues, the mobility of the sfAFP denatured ensemble is similar for denatured α/ß proteins both on fast ps/ns as well as slower µs/ms timescales. These results are in line with a conformational entropy in the denatured ensemble resembling that of typical proteins and suggest that new structures based on PPII helical bundles should be amenable to protein design.

Baat Gene Knockout Alters Post-Natal Development, the Gut Microbiome, and Reveals Unusual Bile Acids in Mice.

Bile acids (BAs) are steroid detergents in bile that contribute to fat absorption, cell signaling, and microbiome interactions. The final step in their synthesis is amino acid conjugation with either glycine or taurine in the liver by the enzyme bile acid-CoA:amino acid N-acyltransferase (BAAT). Here, we describe the microbial, chemical, and physiological consequences of Baat gene knockout. Baat-/- mice were underweight after weaning but quickly exhibited catch-up growth. At three weeks of age, KO animals had increased phospholipid excretion and decreased subcutaneous fat pad mass, liver mass, glycogen staining in hepatocytes, and hepatic vitamin A stores, but these were less marked in adulthood. Additionally, KO mice had an altered microbiome in early life. Their BA pool was highly enriched in cholic acid but not completely devoid of conjugated BAs. KO animals had 27-fold lower taurine-conjugated BAs than wild type in their liver but similar concentrations of glycine-conjugated BAs and higher microbially conjugated BAs. Furthermore, the BA pool in Baat-/- was enriched in a variety of unusual BAs that were putatively sourced from cysteamine conjugation with subsequent oxidation and methylation of the sulfur group mimicking taurine. Antibiotic treatment of KO mice indicated the microbiome was not the likely source of the unusual conjugations, instead, the unique BAs in KO animals were likely derived from the peroxisomal acyltransferases Acnat1 and Acnat2, which are duplications of Baat in the mouse genome that are inactivated in humans. This study demonstrates that BA conjugation is important for early life development of mice.

Phase 1 study to evaluate the effects of rifampin on pharmacokinetics of pevonedistat, a NEDD8-activating enzyme inhibitor in patients with advanced solid tumors.

Pevonedistat (TAK-924/MLN4924) is an investigational small molecule inhibitor of the NEDD8-activating enzyme that has demonstrated clinical activity across solid tumors and hematological malignancies. Here we report the results of a phase 1 study evaluating the effect of rifampin, a strong CYP3A inducer, on the pharmacokinetics (PK) of pevonedistat in patients with advanced solid tumors (NCT03486314). Patients received a single 50 mg/m2 pevonedistat dose via a 1-h infusion on Days 1 (in the absence of rifampin) and 10 (in the presence of rifampin), and daily oral dosing of rifampin 600 mg on Days 3-11. Twenty patients were enrolled and were evaluable for PK and safety. Following a single dose of pevonedistat at 50 mg/m2, the mean terminal half-life of pevonedistat was 5.7 and 7.4 h in the presence and in the absence of rifampin, respectively. The geometric mean AUC0-inf of pevonedistat in the presence of rifampin was 79% of that without rifampin (90% CI: 69.2%-90.2%). The geometric mean Cmax of pevonedistat in the presence of rifampin was similar to that in the absence of rifampin (96.2%; 90% CI: 79.2%-117%). Coadministration of pevonedistat with rifampin, a strong metabolic enzyme inducer, did not result in clinically meaningful decreases in systemic exposures of pevonedistat. The study results support the recommendation that no pevonedistat dose adjustment is needed for patients receiving concomitant CYP3A inducers. CLINICALTRIALS.GOV IDENTIFIER: NCT03486314.

The protease-inhibitor SerpinB3 as a critical modulator of the stem-like subset in human cholangiocarcinoma.

BACKGROUND AND AIMS: Cholangiocarcinoma (CCA) is a form of primary liver cancer with limited therapeutic options. Recently, cancer stem cells (CSCs) have been proposed as a driving force of tumour initiation and dissemination, thus representing a crucial therapeutic target. The protease inhibitor SerpinB3 (SB3) has been identified in several malignancies including hepatocellular carcinoma. SB3 has been involved in the early events of hepatocarcinogenesis and is highly expressed in hepatic progenitor cells and in a mouse model of liver progenitor cell activation. However, only limited information on the possible role of SB3 in CCA stem-like compartment is available. METHODS: Enrichment of CCA stem-like subset was performed by sphere culture (SPH) in CCA cell lines (CCLP1, HUCCT1, MTCHC01 and SG231). Quantitative RT-PCR and Western blotting were used to detect SB3 in both SPH and parental monolayer (MON) cells. Acquired CSC-like features were analysed using an endogenous and a paracrine in vitro model, with transfection of SB3 gene or addition of recombinant SB3 to cell medium respectively. SB3 tumorigenic role was explored in an in vivo mouse model of CCA by subcutaneous injection of SB3-transfected MON (MONSB3+ ) cells in immune-deficient NOD-SCID/IL2Rgnull  (NSG) mice. SB3 expression in human CCA sections was investigated by immunohistochemistry. Overall survival (OS) and time to recurrence (TTR) analyses were carried out from a transcriptome database of 104 CCA patients. RESULTS: SB3, barely detected in parental MON cells, was overexpressed in the same CCA cells grown as 3D SPH. Notably, MONSB3+ showed significant overexpression of genes associated with stemness (CD24, CD44, CD133), pluripotency (c-MYC, NOTCH1, STAT3, YAP, NANOG, BMI1, KLF4, OCT4, SOX2), epithelial mesenchymal transition (ß-catenin, SLUG) and extracellular matrix remodelling (MMP1, MMP7, MMP9, ADAM9, ADAM10, ADAM17, ITGB3). SB3-overexpressing cells showed superior spherogenic capacity and invasion ability compared to control. Importantly, MONSB3+ exhibited activation of MAP kinases (ERK1/2, p38, JNK) as well as phosphorylation of NFκB (p65) in addition to up-regulation of the proto-oncogene ß-catenin. All these effects were reversed after transient silencing of SB3. According to the in vitro finding, MONSB3+ cells retained high tumorigenic potential in NSG mice. SB3 overexpression was observed in human CCA tissues and analysis of OS as well as TTR indicated a worse prognosis in SB3+ CCA patients. CONCLUSION: These findings indicate a SB3 role in mediating malignant phenotype of CCA and identify a new therapeutic target.

Rosmarinus officinalis extract-loaded emulgel prevents UVB irradiation damage to the skin.

UVB-irradiation increases the risk of various skin disorders, therefore leading to inflammation and oxidative stress. In this sense, antioxidant-rich herbs such as Rosmarinus officinalis may be useful in minimizing the damage promoted by reactive oxygen species. In this work, we report the efficacy of a R. officinalis hydroethanolic extract (ROe)-loaded emulgel in preventing UVB-related skin damage. Total phenols were determined using Folin-Ciocalteu assay, and the main phytocomponents in the extract were identified by UHPLC-HRMS. Moreover, in vitro sun protection factor (SPF) value of ROe was also assessed, and we investigated the in vivo protective effect of an emulgel containing ROe against UVB-induced damage in an animal model. The ROe exhibited commercially viable SPF activity (7.56 ± 0.16) and remarkable polyphenolic content (24.15 ± 0.11 mg (Eq.GA)/g). HPLC-MS and UHPLC-HRMS results showcased that the main compounds in ROe were: rosmarinic acid, carnosic acid and carnosol. The evaluation of the in vitro antioxidant activity demonstrated a dose-dependent effect of ROe against several radicals and the capacity to reduce iron. Therefore, we demonstrated that topical application of the formulation containing ROe inhibited edema formation, myeloperoxidase activity, GSH depletion and maintained ferric reducing (FRAP) and ABTS scavenging abilities of the skin after UVB exposure.

Divergent CPEB prion-like domains reveal different assembly mechanisms for a generic amyloid-like fold.

BACKGROUND: Amyloids are ordered, insoluble protein aggregates, characterized by a cross-ß sheet quaternary structure in which molecules in a ß-strand conformation are stacked along the filament axis via intermolecular interactions. While amyloids are typically associated with pathological conditions, functional amyloids have also been identified and are present in a wide variety of organisms ranging from bacteria to humans. The cytoplasmic polyadenylation element-binding (CPEB) prion-like protein is an mRNA-binding translation regulator, whose neuronal isoforms undergo activity-dependent aggregation, a process that has emerged as a plausible biochemical substrate for memory maintenance. CPEB aggregation is driven by prion-like domains (PLD) that are divergent in sequence across species, and it remains unknown whether such divergent PLDs follow a similar aggregating assembly pathway. Here, we describe the amyloid-like features of the neuronal Aplysia CPEB (ApCPEB) PLD and compare them to those of the Drosophila ortholog, Orb2 PLD. RESULTS: Using in vitro single-molecule and bulk biophysical methods, we find transient oligomers and mature amyloid-like filaments that suggest similarities in the late stages of the assembly pathway for both ApCPEB and Orb2 PLDs. However, while prior to aggregation the Orb2 PLD monomer remains mainly as a random coil in solution, ApCPEB PLD adopts a diversity of conformations comprising α-helical structures that evolve to coiled-coil species, indicating structural differences at the beginning of their amyloid assembly pathways. CONCLUSION: Our results indicate that divergent PLDs of CPEB proteins from different species retain the ability to form a generic amyloid-like fold through different assembly mechanisms.

Structural transitions in Orb2 prion-like domain relevant for functional aggregation in memory consolidation.

The recent structural elucidation of ex vivo Drosophila Orb2 fibrils revealed a novel amyloid formed by interdigitated Gln and His residue side chains belonging to the prion-like domain. However, atomic-level details on the conformational transitions associated with memory consolidation remain unknown. Here, we have characterized the nascent conformation and dynamics of the prion-like domain (PLD) of Orb2A using a nonconventional liquid-state NMR spectroscopy strategy based on 13C detection to afford an essentially complete set of 13Cα, 13Cß, 1Hα, and backbone 13CO and 15N assignments. At pH 4, where His residues are protonated, the PLD is disordered and flexible, except for a partially populated α-helix spanning residues 55-60, and binds RNA oligos, but not divalent cations. At pH 7, in contrast, His residues are predominantly neutral, and the Q/H segments adopt minor populations of helical structure, show decreased mobility and start to self-associate. At pH 7, the His residues do not bind RNA or Ca2+, but do bind Zn2+, which promotes further association. These findings represent a remarkable case of structural plasticity, based on which an updated model for Orb2A functional amyloidogenesis is suggested.

Single-cell transcriptomic profiling of satellite glial cells in stellate ganglia reveals developmental and functional axial dynamics.

Stellate ganglion neurons, important mediators of cardiopulmonary neurotransmission, are surrounded by satellite glial cells (SGCs), which are essential for the function, maintenance, and development of neurons. However, it remains unknown whether SGCs in adult sympathetic ganglia exhibit any functional diversity, and what role this plays in modulating neurotransmission. We performed single-cell RNA sequencing of mouse stellate ganglia (n = 8 animals), focusing on SGCs (n = 11,595 cells). SGCs were identified by high expression of glial-specific transcripts, S100b and Fabp7. Microglia and Schwann cells were identified by expression of C1qa/C1qb/C1qc and Ncmap/Drp2, respectively, and excluded from further analysis. Dimensionality reduction and clustering of SGCs revealed six distinct transcriptomic subtypes, one of which was characterized the expression of pro-inflammatory markers and excluded from further analyses. The transcriptomic profiles and corresponding biochemical pathways of the remaining subtypes were analyzed and compared with published astrocytic transcriptomes. This revealed gradual shifts of developmental and functional pathways across the subtypes, originating from an immature and pluripotent subpopulation into two mature populations of SGCs, characterized by upregulated functional pathways such as cholesterol metabolism. As SGCs aged, these functional pathways were downregulated while genes and pathways associated with cellular stress responses were upregulated. These findings were confirmed and furthered by an unbiased pseudo-time analysis, which revealed two distinct trajectories involving the five subtypes that were studied. These findings demonstrate that SGCs in mouse stellate ganglia exhibit transcriptomic heterogeneity along maturation or differentiation axes. These subpopulations and their unique biochemical properties suggest dynamic physiological adaptations that modulate neuronal function.