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Peroxisomes are membrane-bound organelles harboring metabolic enzymes. In humans, peroxisomes are required for normal development, yet the genes regulating peroxisome function remain unclear. We performed a genome-wide CRISPRi screen to identify novel factors involved in peroxisomal homeostasis. We found that inhibition of RNF146, an E3 ligase activated by poly(ADP-ribose), reduced the import of proteins into peroxisomes. RNF146-mediated loss of peroxisome import depended on the stabilization and activity of the poly(ADP-ribose) polymerases TNKS and TNKS2, which bind the peroxisomal membrane protein PEX14. We propose that RNF146 and TNKS/2 regulate peroxisome import efficiency by PARsylation of proteins at the peroxisome membrane. Interestingly, we found that the loss of peroxisomes increased TNKS/2 and RNF146-dependent degradation of non-peroxisomal substrates, including the β-catenin destruction complex component AXIN1, which was sufficient to alter the amplitude of β-catenin transcription. Together, these observations not only suggest previously undescribed roles for RNF146 in peroxisomal regulation but also a novel role in bridging peroxisome function with Wnt/β-catenin signaling during development., (© 2024 Vu et al.)

Single-cell analysis in living humans is essential for understanding disease mechanisms, but it is impractical in non-regenerative organs, such as the eye and brain, because tissue biopsies would cause serious damage. We resolve this problem by integrating proteomics of liquid biopsies with single-cell transcriptomics from all known ocular cell types to trace the cellular origin of 5,953 proteins detected in the aqueous humor. We identified hundreds of cell-specific protein markers, including for individual retinal cell types. Surprisingly, our results reveal that retinal degeneration occurs in Parkinson's disease, and the cells driving diabetic retinopathy switch with disease stage. Finally, we developed artificial intelligence (AI) models to assess individual cellular aging and found that many eye diseases not associated with chronological age undergo accelerated molecular aging of disease-specific cell types. Our approach, which can be applied to other organ systems, has the potential to transform molecular diagnostics and prognostics while uncovering new cellular disease and aging mechanisms., Competing Interests: Declaration of interests V.B.M. has received speaker fees from Somalogic, Inc., (Published by Elsevier Inc.)

We describe a strategy to boost the efficiency of gene editing via homology-directed repair (HDR) by covalently modifying the template DNA with interstrand crosslinks. Crosslinked templates (xHDRTs) increase Cas9-mediated editing efficiencies by up to fivefold in K562, HEK293T, U2OS, iPS and primary T cells. Increased editing from xHDRTs is driven by events on the template molecule and requires ataxia telangiectasia and Rad3-related (ATR) kinase and components of the Fanconi anemia pathway., (© 2023. The Author(s).)

Ophthalmic neurodegenerative diseases encompass a wide array of molecular pathologies unified by calpain dysregulation. Calpains are calcium-dependent proteases that perpetuate cellular death and inflammation when hyperactivated. Calpain inhibition trials in other organs have faced pharmacological challenges, but the eye offers many advantages for the development and testing of targeted molecular therapeutics, including small molecules, peptides, engineered proteins, drug implants, and gene-based therapies. This review highlights structural mechanisms underlying calpain activation, distinct cellular expression patterns, and in vivo models that link calpain hyperactivity to human retinal and developmental disease. Optimizing therapeutic approaches for calpain-mediated eye diseases can help accelerate clinically feasible strategies for treating calpain dysregulation in other diseased tissues., Competing Interests: Declaration of interests The authors declare no conflict of interest., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Purpose: To identify vitreous molecular biomarkers associated with autoimmune retinopathy (AIR)., Design: Case-control study., Participants: We analyzed six eyes from four patients diagnosed with AIR and eight comparative controls diagnosed with idiopathic macular holes and epiretinal membranes., Methods: Vitreous biopsies were collected from the participants and analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) or multiplex ELISA., Outcome Measures: Protein expression changes were evaluated by 1-way ANOVA (significant p-value <0.05), hierarchical clustering, and pathway analysis to identify candidate protein biomarkers., Results: There were 16 significantly upregulated and 17 significantly downregulated proteins in the vitreous of three AIR patients compared to controls. The most significantly upregulated proteins included lysozyme C (LYSC), zinc-alpha-2-glycoprotein (ZA2G), complement factor D (CFAD), transforming growth factor-beta induced protein (BGH3), beta-crystallin B2, and alpha-crystallin A chain. The most significantly downregulated proteins included disco-interacting protein 2 homolog (DIP2C), retbindin (RTBDN), and amyloid beta precursor like protein 2 (APLP2). Pathway analysis revealed that vascular endothelial growth factor (VEGF) signaling was a top represented pathway in the vitreous of AIR patients compared to controls. In comparison to a different cohort of three AIR patients analyzed by multiplex ELISA, a commonly differentially expressed protein was neuronal cell adhesion molecule (NrCAM) with p-values of 0.027 in the LC-MS/MS dataset and 0.035 in the ELISA dataset., Conclusion: Protein biomarkers such as NrCAM in the vitreous may eventually help diagnose AIR., Competing Interests: Conflict of Interest: No conflicting relationship exists for any author.

Background: A point mutation in sickle cell disease (SCD) alters one amino acid in the β-globin subunit of hemoglobin, with resultant anemia and multiorgan damage that typically shortens lifespan by decades. Because SCD is caused by a single mutation, and hematopoietic stem cells (HSCs) can be harvested, manipulated, and returned to an individual, it is an attractive target for gene correction., Results: An optimized Cas9 ribonucleoprotein (RNP) with an ssDNA oligonucleotide donor together generated correction of at least one β-globin allele in more than 30% of long-term engrafting human HSCs. After adopting a high-fidelity Cas9 variant, efficient correction with minimal off-target events also was observed. In vivo erythroid differentiation markedly enriches for corrected β-globin alleles, indicating that erythroblasts carrying one or more corrected alleles have a survival advantage., Significance: These findings indicate that the sickle mutation can be corrected in autologous HSCs with an optimized protocol suitable for clinical translation., Competing Interests: The authors have no conflicts of interest. MCW is medical director of All Cells, Inc., from which some of the human CD34+ cells for the studies were purchased. Matthew S.McNeill, Garrett R. Rettig, Yongming Sun, Yu Wang, and Mark A. Behlke are employees of IDT, Inc. Mark A. De-Witt is a consultant for Synthego Inc. and CellFE Inc. Synthego Inc. did not contribute to this work. MCW is medical director of All Cells, Inc., from which some of the human CD34+ cells for the studies were purchased. Matthew S.McNeill, Garrett R. Rettig, Yongming Sun, Yu Wang, and Mark A. Behlke are employees of IDT, Inc. Mark A. De-Witt is a consultant for Synthego Inc. and CellFE Inc. Synthego Inc. did not contribute to this work. GRR is an employee of Integrated DNA Technologies and Danaher Corp and shareholder of Danaher Corp A single patent application was filed from this project: USPAT APPLICATION NUMBER: 17047025 - Methods for Treating Sickle Cell Disease. Products and tools supplied by IDT are for research use only and not intended for diagnostic or therapeutic purposes. Purchaser and/or user is solely responsible for all decisions regarding the use of these products and any associated regulatory or legal obligations., (© 2022.)

Naturally occurring point mutations in the HBG promoter switch hemoglobin synthesis from defective adult beta-globin to fetal gamma-globin in sickle cell patients with hereditary persistence of fetal hemoglobin (HPFH) and ameliorate the clinical severity. Inspired by this natural phenomenon, we tiled the highly homologous HBG proximal promoters using adenine and cytosine base editors that avoid the generation of large deletions and identified novel regulatory regions including a cluster at the -123 region. Base editing at -123 and -124 bp of HBG promoter induced fetal hemoglobin (HbF) to a higher level than disruption of well-known BCL11A binding site in erythroblasts derived from human CD34+ hematopoietic stem and progenitor cells (HSPC). We further demonstrated in vitro that the introduction of -123T > C and -124T > C HPFH-like mutations drives gamma-globin expression by creating a de novo binding site for KLF1. Overall, our findings shed light on so far unknown regulatory elements within the HBG promoter and identified additional targets for therapeutic upregulation of fetal hemoglobin., Competing Interests: NR, BW, SW, HB, AG, GM, JV, KP, BB, ND, VR, NS, AP, YN, RK, MN, PB, ST, SM, SV, AS, MD, MC, JC, KM No competing interests declared, (© 2022, Ravi et al.)

Genome editing often takes the form of either error-prone sequence disruption by non-homologous end joining (NHEJ) or sequence replacement by homology-directed repair (HDR). Although NHEJ is generally effective, HDR is often difficult in primary cells. Here, we use a combination of immunophenotyping, next-generation sequencing, and single-cell RNA sequencing to investigate and reprogram genome editing outcomes in subpopulations of adult hematopoietic stem and progenitor cells. We find that although quiescent stem-enriched cells mostly use NHEJ, non-quiescent cells with the same immunophenotype use both NHEJ and HDR. Inducing quiescence before editing results in a loss of HDR in all cell subtypes. We develop a strategy of controlled cycling and quiescence that yields a 6-fold increase in the HDR/NHEJ ratio in quiescent stem cells ex vivo and in vivo. Our results highlight the tension between editing and cellular physiology and suggest strategies to manipulate quiescent cells for research and therapeutic genome editing., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Of the side effects of prostaglandin analogues (PGAs), uveitis and cystoid macular oedema (CME) have significant potential for vision loss based on postmarket reports. Caution has been advised due to concerns of macular oedema and uveitis. In this report, we researched and summarised the original data suggesting these effects and determined their incidence., Methods: Preferred Reporting Items for Systematic review and Meta-Analyses guidelines were followed. Studies evaluating topical PGAs in patients with ocular hypertension or open angle glaucoma were included. MEDLINE, PubMed, EMBASE, CINAHL, Web of Science, Cochrane Library, LILACS and ClinicalTrials.gov were searched between 1946 and 2019. Experimental studies, animal studies and randomised studies with other intraocular pressure-lowering eye drops were excluded., Results: 214 studies (28 232 patients) met the inclusion criteria. Using prospective data, the incidence of uveitis and CME among PGA users were 62/28 232 (0.22%) and 25/28 232 (0.09%), respectively. A higher frequency of both uveitis and CME were found among latanoprost users compared with bimatoprost. There were 21 case studies reporting CME including 48 eyes in 43 patients. 47 of 48 eyes (97.9%) had previous incisional ocular surgery. 8 eyes were re-challenged, of which 7 (87.5%) recurred. 7 case studies reported uveitis in 15 eyes of 10 patients. 7 of 15 eyes (46.7%) were either pseudophakic or aphakic. 6 eyes were re-challenged, and all 6 (100%) recurred., Conclusions: Cases of uveitis or CME revealed a confounding effect of ocular surgery, aphakia or subluxed intraocular lens. PGAs may be used in non-surgical patients without concern of causing CME or uveitis. The incidences of PGA-associated CME and uveitis are rare with limited prospective studies on the cause-effect relationship., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Cas9 is a prokaryotic RNA-guided DNA endonuclease that binds substrates tightly in vitro but turns over rapidly when used to manipulate genomes in eukaryotic cells. Little is known about the factors responsible for dislodging Cas9 or how they influence genome engineering. Unbiased detection through proximity labeling of transient protein interactions in cell-free Xenopus laevis egg extract identified the dimeric histone chaperone facilitates chromatin transcription (FACT) as an interactor of substrate-bound Cas9. FACT is both necessary and sufficient to displace dCas9, and FACT immunodepletion converts Cas9's activity from multi-turnover to single turnover. In human cells, FACT depletion extends dCas9 residence times, delays genome editing, and alters the balance between indel formation and homology-directed repair. FACT knockdown also increases epigenetic marking by dCas9-based transcriptional effectors with a concomitant enhancement of transcriptional modulation. FACT thus shapes the intrinsic cellular response to Cas9-based genome manipulation most likely by determining Cas9 residence times., Competing Interests: Declaration of Interests J.E.C. is a co-founder of Spotlight Therapeutics., (Copyright © 2020 Elsevier Inc. All rights reserved.)

CRISPR-Cas genome editing induces targeted DNA damage but can also affect off-target sites. Current off-target discovery methods work using purified DNA or specific cellular models but are incapable of direct detection in vivo. We developed DISCOVER-Seq (discovery of in situ Cas off-targets and verification by sequencing), a universally applicable approach for unbiased off-target identification that leverages the recruitment of DNA repair factors in cells and organisms. Tracking the precise recruitment of MRE11 uncovers the molecular nature of Cas activity in cells with single-base resolution. DISCOVER-Seq works with multiple guide RNA formats and types of Cas enzymes, allowing characterization of new editing tools. Off-targets can be identified in cell lines and patient-derived induced pluripotent stem cells and during adenoviral editing of mice, paving the way for in situ off-target discovery within individual patient genotypes during therapeutic genome editing., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

In the search for molecular machinery for custom biosynthesis of valuable compounds, the modular type I polyketide synthases (PKSs) offer great potential. In this study, we investigate the flexibility of BorM5, the iterative fifth module of the borrelidin synthase, with a panel of non-native priming substrates in vitro. BorM5 differentially extends various aliphatic and substituted substrates. Depending on substrate size and substitution BorM5 can exceed the three iterations it natively performs. To probe the effect of methyl branching on chain length regulation, we engineered a BorM5 variant capable of incorporating methylmalonyl- and malonyl-CoA into its intermediates. Intermediate methylation did not affect overall chain length, indicating that the enzyme does not to count methyl branches to specify the number of iterations. In addition to providing regulatory insight about BorM5, we produced dozens of novel methylated intermediates that might be used for production of various hydrocarbons or pharmaceuticals. These findings enable rational engineering and recombination of BorM5 and inform the study of other iterative modules.

Venous thromboembolic (VTE) risk assessment remains an important issue in hospitalised, acutely-ill medical patients, and several VTE risk assessment models (RAM) have been proposed. The purpose of this large retrospective cohort study was to externally validate the IMPROVE RAM using a large database of three acute care hospitals. We studied 41,486 hospitalisations (28,744 unique patients) with 1,240 VTE hospitalisations (1,135 unique patients) in the VTE cohort and 40,246 VTE-free hospitalisations (27,609 unique patients) in the control cohort. After chart review, 139 unique VTE patients were identified and 278 randomly-selected matched patients in the control cohort. Seven independent VTE risk factors as part of the RAM in the derivation cohort were identified. In the validation cohort, the incidence of VTE was 0.20%; 95% confidence interval (CI) 0.18-0.22, 1.04%; 95%CI 0.88-1.25, and 4.15%; 95%CI 2.79-8.12 in the low, moderate, and high VTE risk groups, respectively, which compared to rates of 0.45%, 1.3%, and 4.74% in the three risk categories of the derivation cohort. For the derivation and validation cohorts, the total percentage of patients in low, moderate and high VTE risk occurred in 68.6% vs 63.3%, 24.8% vs 31.1%, and 6.5% vs 5.5%, respectively. Overall, the area under the receiver-operator characteristics curve for the validation cohort was 0.7731. In conclusion, the IMPROVE RAM can accurately identify medical patients at low, moderate, and high VTE risk. This will tailor future thromboprophylactic strategies in this population as well as identify particularly high VTE risk patients in whom multimodal or more intensive prophylaxis may be beneficial.

The main objective of the study was to investigate the ability of frequency-domain photothermal radiometry (PTR) and modulated luminescence (LUM) to detect secondary caries lesions on the walls of restorations (wall lesions). Changes in experimental PTR-LUM signals due to sequential demineralization on entire vertical walls of sectioned tooth samples were investigated. In addition, transverse micro-radiography (TMR) analysis (used as a gold standard) was conducted to measure the degree of demineralization that occurred in each sample. Statistical correlation between TMR results and PTR-LUM signals was determined using Pearson's correlation coefficient. LUM signals were found to be dominated by the scattered component of the incident laser beam. The more clinically relevant cases of localized demineralization and remineralization on vertical walls were also investigated to examine whether PTR-LUM signals are sensitive to demineralization and remineralization of much smaller areas. The overall results demonstrated that PTR-LUM is sensitive to progressive demineralization and remineralization on vertical walls of sectioned tooth samples.

The accepted mechanism of toxicity of many organophosphorous and carbamate insecticides is inhibition of acetylcholinesterase activity. In mammals, part of the toxicity assessment usually includes monitoring blood and/or brain acetylcholinesterase inhibition. Other tissues, however, contain cholinesterase activity (i.e. acetyl- and butyryl-cholinesterase), and the inhibition of that activity may be informative for a full appraisal of the toxicity profile. The present group of studies first optimized the variables for extraction and solubilization of cholinesterase activity from various rat tissues and then refined an existing automated method, in order to differentially assess acetyl and butyrylcholinesterase activity in those tissues. All these studies were conducted using tissues from untreated, Long-Evans, adult rats. The first studies determined the effect of Triton X-100 or salt (NaCl) on the extraction and solubilization of cholinesterase activity from retina, brain, striated muscle, diaphragm, and heart: phosphate buffer plus detergent (1% Triton X-100) yielded the highest activity for most tissues. For striated muscle, however, slightly more activity was extracted if the phosphate buffer contained both 1% Triton X-100 and 0.5 M NaCl. It was also noted that the degree of homogenization of some tissues (e.g. striated muscle) must be increased for maximal solubilization of all cholinesterase activity. Subsequent studies developed a method for assessing the level of acetylcholinesterase, butyrylcholinesterase and total cholinesterase activity in these tissues using an automated analyzer. In conclusion, automated assay of acetylcholinesterase activity in cholinergically innervated tissues in the rat (other than brain) is achievable and relatively convenient.