A systematic review of quantitative EEG findings in Fibromyalgia, Chronic Fatigue Syndrome and Long COVID.

Fibromyalgia Syndrome (FMS), Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) and Long COVID (LC) are similar multisymptom clinical syndromes but with difference in dominant symptoms in each individual. There is existing and emerging literature on possible functional alterations of the central nervous system in these conditions. This review aims to synthesise and appraise the literature on resting-state quantitative EEG (qEEG) in FMS, ME/CFS and LC, drawing on previous research on FMS and ME/CFS to help understand neuropathophysiology of the new condition LC. A systematic search of MEDLINE, Embase, CINHAL, PsycINFO and Web of Science databases for articles published between December 1994 and September 2023 was performed. Out of the initial 2510 studies identified, 17 articles were retrieved that met all the predetermined selection criteria, particularly of assessing qEEG changes in one of the three conditions compared to healthy controls. All studies scored moderate to high quality on the Newcastle-Ottawa scale. There was a general trend for decreased low-frequency EEG band activity (delta, theta, and alpha) and increased high-frequency EEG beta activity in FMS, differing to that found in ME/CFS. The limited LC studies included in this review focused mainly on cognitive impairments and showed mixed findings not consistent with patterns observed in FMS and ME/CFS. Our findings suggest different patterns of qEEG brainwave activity in FMS and ME/CFS. Further research is required to explore whether there are phenotypes within LC that have EEG signatures similar to FMS or ME/CFS. This could inform identification of reliable diagnostic markers and possible targets for neuromodulation therapies tailored to each clinical syndrome.

Sarcopenia versus clinical frailty scale in predicting the risk of postoperative mortality after emergency laparotomy: a retrospective cohort study.

OBJECTIVES: To compare predictive significance of sarcopenia and clinical frailty scale (CFS) in terms of postoperative mortality in patients undergoing emergency laparotomy METHODS: In compliance with STROCSS statement standards, a retrospective cohort study with prospective data collection approach was conducted. The study period was between January 2017 and January 2022. All adult patients with non-traumatic acute abdominal pathology who underwent emergency laparotomy in our centre were included. The primary outcome was 30-day mortality and secondary outcomes were in-hospital mortality and 90-day mortality. The predictive value of sarcopenia and CFS were compared using the receiver operating characteristic (ROC) curve analysis and multivariable binary logistic regression analysis. RESULTS: A total of 1043 eligible patients were included. The risk of 30-day mortality, in-hospital mortality, and 90-day mortality were 8%, 10%, and 11%, respectively. ROC curve analysis suggested that sarcopenia is a significantly stronger predictor of 30-day mortality (AUC: 0.87 vs. 0.70, P<0.0001), in-hospital mortality (AUC: 0.79 vs. 0.67, P=0.0011), and 90-day mortality (AUC: 0.79 vs. 0.67, P=0.0009) compared with CFS. Moreover, multivariable binary logistic regression analysis identified sarcopenia as an independent predictor of mortality [coefficient: 4.333, OR: 76.16 (95% CI 37.06-156.52), P<0.0001] but not the CFS [coefficient: 0.096, OR: 1.10 (95% CI 0.88-1.38), P=0.4047]. CONCLUSIONS: Sarcopenia is a stronger predictor of postoperative mortality compared with CFS in patients undergoing emergency laparotomy. It cancels out the predictive value of clinical frailty scale in multivariable analyses; hence among the two variables, sarcopenia deserves to be included in preoperative predictive tools.

Low versus standard central venous pressure during laparoscopic liver resection: A systematic review, meta-analysis and trial sequential analysis.

To compare the outcomes of low central venous pressure (CVP) to standard CVP during laparoscopic liver resection. The study design was a systematic review following the PRISMA statement standards. The available literature was searched to identify all studies comparing low CVP with standard CVP in patients undergoing laparoscopic liver resection. The outcomes included intraoperative blood loss (primary outcome), need for blood transfusion, mean arterial pressure, operative time, Pringle time, and total complications. Random- effects modelling was applied for analyses. Type I and type II errors were assessed by trial sequential analysis (TSA). A total of 8 studies including 682 patients were included (low CVP group, 342; standard CVP group, 340). Low CVP reduced intraoperative blood loss during laparoscopic liver resection (mean difference [MD], -193.49 mL; 95% confidence interval [CI], -339.86 to -47.12; p = 0.01). However, low CVP did not have any effect on blood transfusion requirement (odds ratio [OR], 0.54; 95% CI, 0.28-1.03; p = 0.06), mean arterial pressure (MD, -1.55 mm Hg; 95% CI, -3.85-0.75; p = 0.19), Pringle time (MD, -0.99 minutes; 95% CI, -5.82-3.84; p = 0.69), operative time (MD, -16.38 minutes; 95% CI, -36.68-3.39; p = 0.11), or total complications (OR, 1.92; 95% CI, 0.97-3.80; p = 0.06). TSA suggested that the meta-analysis for the primary outcome was not subject to type I or II errors. Low CVP may reduce intraoperative blood loss during laparoscopic liver resection (moderate certainty); however, this may not translate into shorter operative time, shorter Pringle time, or less need for blood transfusion. Randomized controlled trials with larger sample sizes will provide more robust evidence.

Delivery to, and Reactivation of, the p53 Pathway in Cancer Cells Using a Grafted Cyclotide Conjugated with a Cell-Penetrating Peptide.

Peptides are promising drug modalities that can modulate protein-protein interactions, but their application is hampered by their limited ability to reach intracellular targets. Here, we improved the cytosolic delivery of a peptide blocking p53:MDM2/X interactions using a cyclotide as a stabilizing scaffold. We applied several design strategies to improve intracellular delivery and found that the conjugation of the lead cyclotide to the cyclic cell-penetrating peptide cR10 was the most effective. Conjugation allowed cell internalization at micromolar concentration and led to elevated intracellular p53 levels in A549, MCF7, and MCF10A cells, as well as inducing apoptosis in A549 cells without causing membrane disruption. The lead peptide had >35-fold improvement in inhibitory activity and increased cellular uptake compared to a previously reported cyclotide p53 activator. In summary, we demonstrated the delivery of a large polar cyclic peptide in the cytosol and confirmed its ability to modulate intracellular protein-protein interactions involved in cancer.

Design-rules for stapled peptides with in vivo activity and their application to Mdm2/X antagonists.

Although stapled α-helical peptides can address challenging targets, their advancement is impeded by poor understandings for making them cell permeable while avoiding off-target toxicities. By synthesizing >350 molecules, we present workflows for identifying stapled peptides against Mdm2(X) with in vivo activity and no off-target effects. Key insights include a clear correlation between lipophilicity and permeability, removal of positive charge to avoid off-target toxicities, judicious anionic residue placement to enhance solubility/behavior, optimization of C-terminal length/helicity to enhance potency, and optimization of staple type/number to avoid polypharmacology. Workflow application gives peptides with >292x improved cell proliferation potencies and no off-target cell proliferation effects ( > 3800x on-target index). Application of these 'design rules' to a distinct Mdm2(X) peptide series improves ( > 150x) cellular potencies and removes off-target toxicities. The outlined workflow should facilitate therapeutic impacts, especially for those targets such as Mdm2(X) that have hydrophobic interfaces and are targetable with a helical motif.

Treating recurrent metastatic breast cancer to the skin with proton therapy.

Proton therapy offers unique physical properties that make it an excellent choice for treating metastatic breast cancer, particularly when recurrent disease occurs near previously irradiated tissues. This case study demonstrates the dosimetric benefits of proton therapy in patients with metastatic breast cancer to the skin. The case consists of one patient with 5 separate areas treated with Pencil Beam Scanning (PBS) proton therapy over a period of 2 years using Monte Carlo calculation and robust optimization. The results demonstrate that proton therapy effectively spared healthy tissues while delivering the prescribed dose to the tumor. This case demonstrates the feasibility of developing effective radiation plans for skin metastases using proton therapy, highlighting its dosimetric advantages and minimal impact on nearby organs.

Stitched peptides as potential cell permeable inhibitors of oncogenic DAXX protein.

DAXX (Death Domain Associated Protein 6) is frequently upregulated in various common cancers, and its suppression has been linked to reduced tumor progression. Consequently, DAXX has gained significant interest as a therapeutic target in such cancers. DAXX is known to function in several critical biological pathways including chromatin remodelling, transcription regulation, and DNA repair. Leveraging structural information, we have designed and developed a novel set of stapled/stitched peptides that specifically target a surface on the N-terminal helical bundle domain of DAXX. This surface serves as the anchor point for binding to multiple interaction partners, such as Rassf1C, p53, Mdm2, and ATRX, as well as for the auto-regulation of the DAXX N-terminal SUMO interaction motif (SIM). Our experiments demonstrate that these peptides effectively bind to and inhibit DAXX with a higher affinity than the known interaction partners. Furthermore, these peptides release the auto-inhibited SIM, enabling it to interact with SUMO-1. Importantly, we have developed stitched peptides that can enter cells, maintaining their intracellular concentrations at nanomolar levels even after 24 hours, without causing any membrane perturbation. Collectively, our findings suggest that these stitched peptides not only serve as valuable tools for probing the molecular interactions of DAXX but also hold potential as precursors to the development of therapeutic interventions.

Subtotal pyloric obstruction by atypical hypertrophic pyloric muscle in a newborn.

This is a case of a neonate with suspected duodenal atresia on prenatal imaging. However, distal bowel gas was identified postnatally on regular X-rays with a possible pyloric obstructing mass visualised on ultasound. No contrast was visualised passing through the stomach on fluoroscopic studies. Operative evaluation revealed an atypical asymmetric hypertrophic pylorus with exophytic lesions of ectopic glandular tissue. Longitudinal open pyloromyotomy was performed which relieved the gastric obstruction resulting in symptomatic relief without any anatomy altering procedure required.

A high-throughput microfluidic mechanoporation platform to enable intracellular delivery of cyclic peptides in cell-based assays.

Cyclic peptides are poised to target historically difficult to drug intracellular protein-protein interactions, however, their general cell impermeability poses a challenge for characterizing function. Recent advances in microfluidics have enabled permeabilization of the cytoplasmic membrane by physical cell deformation (i.e., mechanoporation), resulting in intracellular delivery of impermeable macromolecules in vector- and electrophoretic-free approaches. However, the number of payloads (e.g., peptides) and/or concentrations delivered via microfluidic mechanoporation is limited by having to pre-mix cells and payloads, a manually intensive process. In this work, we show that cells are momentarily permeable (t 1/2 = 1.1-2.8 min) after microfluidic vortex shedding (µVS) and that lower molecular weight macromolecules can be cytosolically delivered upon immediate exposure after cells are processed/permeabilized. To increase the ability to screen peptides, we built a system, dispensing-microfluidic vortex shedding (DµVS), that integrates a µVS chip with inline microplate-based dispensing. To do so, we synced an electronic pressure regulator, flow sensor, on/off dispense valve, and an x-y motion platform in a software-driven feedback loop. Using this system, we were able to deliver low microliter-scale volumes of transiently mechanoporated cells to hundreds of wells on microtiter plates in just several minutes (e.g., 96-well plate filled in <2.5 min). We validated the delivery of an impermeable peptide directed at MDM2, a negative regulator of the tumor suppressor p53, using a click chemistry- and NanoBRET-based cell permeability assay in 96-well format, with robust delivery across the full plate. Furthermore, we demonstrated that DµVS could be used to identify functional, low micromolar, cellular activity of otherwise cell-inactive MDM2-binding peptides using a p53 reporter cell assay in 96- and 384-well format. Overall, DµVS can be combined with downstream cell assays to investigate intracellular target engagement in a high-throughput manner, both for improving structure-activity relationship efforts and for early proof-of-biology of non-optimized peptide (or potentially other macromolecular) tools.

Thiol-triggered deconstruction of bifunctional silyl ether terpolymers via an SNAr-triggered cascade.

While Si-containing polymers can often be deconstructed using chemical triggers such as fluoride, acids, and bases, they are resistant to cleavage by mild reagents such as biological nucleophiles, thus limiting their end-of-life options and potential environmental degradability. Here, using ring-opening metathesis polymerization, we synthesize terpolymers of (1) a "functional" monomer (e.g., a polyethylene glycol macromonomer or dicyclopentadiene); (2) a monomer containing an electrophilic pentafluorophenyl (PFP) substituent; and (3) a cleavable monomer based on a bifunctional silyl ether . Exposing these polymers to thiols under basic conditions triggers a cascade of nucleophilic aromatic substitution (SNAr) at the PFP groups, which liberates fluoride ions, followed by cleavage of the backbone Si-O bonds, inducing polymer backbone deconstruction. This method is shown to be effective for deconstruction of polyethylene glycol (PEG) based graft terpolymers in organic or aqueous conditions as well as polydicyclopentadiene (pDCPD) thermosets, significantly expanding upon the versatility of bifunctional silyl ether based functional polymers.

Long-term outcomes of SBRT for PSMA PET detected oligometastatic prostate cancer.

BACKGROUND: Oligometastatic disease in prostate cancer (PCa) is a challenging clinical scenario encountered more frequently with the widespread adoption of PSMA-PET. SBRT aims to defer androgen deprivation and may deliver sustained biochemical failure (BF) free survival in selected patients. Little long-term data is currently available regarding the effectiveness of this approach. METHODS: A retrospective single institution study of PSMA-PET directed SBRT without initial ADT for oligo-metachronous PCa. Median dose/fractionation was 24 Gy in 2# to bones and 30 Gy in 3# to lymph nodes. The primary endpoint was time to BF (PSA + 0.2 ug/L above nadir). Secondary endpoints included time to ADT for relapse (i.e. palliative ADT), BF defined as PSA nadir + 2 ug/L, toxicity, patterns of failure and survival. Patients were excluded if they received ADT with their SBRT, had short disease-free interval, or > 3 metastases on PSMA-PET. RESULTS: 103 patients treated from November-2014 to December-2019 were analysed from our prospective database. Median follow-up was 5 years. 64 patients were treated for nodal only disease, 35 bone only and 4 mixed. 15% were free of any BF at 5 years with median time to BF of 1.1 years. 32% (33/103) of patients had further curative-intent radiation treatment following their first BF after SBRT, including subsequent SBRT. Eight patients underwent potentially curative treatment for their second or third relapse. Allowing for salvage treatment, 29/103 (28%) were biochemically disease free at last follow up. At 5 years, 39% of patients had never received any ADT and 55% had not started ADT for relapse with a median time to ADT for relapse of 5.5 years. There were 2 grade 3 toxicities (rib fracture and lymphoedema), and no local failures. CONCLUSION: PSMA-PET guided SBRT for oligo-metachronous PCa recurrence in appropriately triaged patients results in excellent local control, low toxicity and over 50% ADT free at 5 years.

New era for myelofibrosis treatment with novel agents beyond Janus kinase-inhibitor monotherapy: Focus on clinical development of BCL-XL /BCL-2 inhibition with navitoclax.

Myelofibrosis is a heterogeneous myeloproliferative neoplasm characterized by chronic inflammation, progressive bone marrow failure, and hepatosplenic extramedullary hematopoiesis. Treatments like Janus kinase inhibitor monotherapy (e.g., ruxolitinib) provide significant spleen and symptom relief but demonstrate limited ability to lead to a durable disease modification. There is an urgent unmet medical need for treatments with a novel mechanism of action that can modify the underlying pathophysiology and affect the disease course of myelofibrosis. This review highlights the role of B-cell lymphoma (BCL) protein BCL-extra large (BCL-XL ) in disease pathogenesis and the potential role that navitoclax, a BCL-extra large/BCL-2 inhibitor, may have in myelofibrosis treatment.

Novel CRISPR-Associated Gene-Editing Systems Discovered in Metagenomic Samples Enable Efficient and Specific Genome Engineering.

Development of medicines using gene editing has been hampered by enzymological and immunological impediments. We described previously the discovery and characterization of improved, novel gene-editing systems from metagenomic data. In this study, we substantially advance this work with three such gene-editing systems, demonstrating their utility for cell therapy development. All three systems are capable of reproducible, high-frequency gene editing in primary immune cells. In human T cells, disruption of the T cell receptor (TCR) alpha-chain was induced in >95% of cells, both paralogs of the TCR beta-chain in >90% of cells, and >90% knockout of ß2-microglobulin, TIGIT, FAS, and PDCD1. Simultaneous double knockout of TRAC and TRBC was obtained at a frequency equal to that of the single edits. Gene editing with our systems had minimal effect on T cell viability. Furthermore, we integrate a chimeric antigen receptor (CAR) construct into TRAC (up to ∼60% of T cells), and demonstrate CAR expression and cytotoxicity. We next applied our novel gene-editing tools to natural killer (NK) cells, B cells, hematopoietic stem cells, and induced pluripotent stem cells, generating similarly efficient cell-engineering outcomes including the creation of active CAR-NK cells. Interrogation of our gene-editing systems' specificity reveals a profile comparable with or better than Cas9. Finally, our nucleases lack preexisting humoral and T cell-based immunity, consistent with their sourcing from nonhuman pathogens. In all, we show these new gene-editing systems have the activity, specificity, and translatability necessary for use in cell therapy development.

RNAi-mediated rheostat for dynamic control of AAV-delivered transgenes.

Adeno-associated virus (AAV)-based gene therapy could be facilitated by the development of molecular switches to control the magnitude and timing of expression of therapeutic transgenes. RNA interference (RNAi)-based approaches hold unique potential as a clinically proven modality to pharmacologically regulate AAV gene dosage in a sequence-specific manner. We present a generalizable RNAi-based rheostat wherein hepatocyte-directed AAV transgene expression is silenced using the clinically validated modality of chemically modified small interfering RNA (siRNA) conjugates or vectorized co-expression of short hairpin RNA (shRNA). For transgene induction, we employ REVERSIR technology, a synthetic high-affinity oligonucleotide complementary to the siRNA or shRNA guide strand to reverse RNAi activity and rapidly recover transgene expression. For potential clinical development, we report potent and specific siRNA sequences that may allow selective regulation of transgenes while minimizing unintended off-target effects. Our results establish a conceptual framework for RNAi-based regulatory switches with potential for infrequent dosing in clinical settings to dynamically modulate expression of virally-delivered gene therapies.

Predicting patient-reported outcomes following lumbar spine surgery: development and external validation of multivariable prediction models.

BACKGROUND: This study aimed to develop and externally validate prediction models of spinal surgery outcomes based on a retrospective review of a prospective clinical database, uniquely comparing multivariate regression and random forest (machine learning) approaches, and identifying the most important predictors. METHODS: Outcomes were change in back and leg pain intensity and Core Outcome Measures Index (COMI) from baseline to the last available postoperative follow-up (3-24 months), defined as minimal clinically important change (MCID) and continuous change score. Eligible patients underwent lumbar spine surgery for degenerative pathology between 2011 and 2021. Data were split by surgery date into development (N = 2691) and validation (N = 1616) sets for temporal external validation. Multivariate logistic and linear regression, and random forest classification and regression models, were fit to the development data and validated on the external data. RESULTS: All models demonstrated good calibration in the validation data. Discrimination ability (area under the curve) for MCID ranged from 0.63 (COMI) to 0.72 (back pain) in regression, and from 0.62 (COMI) to 0.68 (back pain) in random forests. The explained variation in continuous change scores spanned 16%-28% in linear, and 15%-25% in random forests regression. The most important predictors included age, baseline scores on the respective outcome measures, type of degenerative pathology, previous spinal surgeries, smoking status, morbidity, and duration of hospital stay. CONCLUSIONS: The developed models appear robust and generalisable across different outcomes and modelling approaches but produced only borderline acceptable discrimination ability, suggesting the need to assess further prognostic factors. External validation showed no advantage of the random forest approach.

"Candidatus Nealsonbacteria" Are Likely Biomass Recycling Ectosymbionts of Methanogenic Archaea in a Stable Benzene-Degrading Enrichment Culture.

The Candidate Phyla Radiation (CPR), also referred to as superphylum Patescibacteria, is a very large group of bacteria with no pure culture representatives discovered by 16S rRNA sequencing or genome-resolved metagenomic analyses of environmental samples. Within the CPR, candidate phylum Parcubacteria, previously referred to as OD1, is prevalent in anoxic sediments and groundwater. Previously, we had identified a specific member of the Parcubacteria (referred to as DGGOD1a) as an important member of a methanogenic benzene-degrading consortium. Phylogenetic analyses herein place DGGOD1a within the clade "Candidatus Nealsonbacteria." Because of its persistence over many years, we hypothesized that "Ca. Nealsonbacteria" DGGOD1a must play an important role in sustaining anaerobic benzene metabolism in the consortium. To try to identify its growth substrate, we amended the culture with a variety of defined compounds (pyruvate, acetate, hydrogen, DNA, and phospholipid), as well as crude culture lysate and three subfractions thereof. We observed the greatest (10-fold) increase in the absolute abundance of "Ca. Nealsonbacteria" DGGOD1a only when the consortium was amended with crude cell lysate. These results implicate "Ca. Nealsonbacteria" in biomass recycling. Fluorescence in situ hybridization and cryogenic transmission electron microscope images revealed that "Ca. Nealsonbacteria" DGGOD1a cells were attached to larger archaeal Methanothrix cells. This apparent epibiont lifestyle was supported by metabolic predictions from a manually curated complete genome. This is one of the first examples of bacterial-archaeal episymbiosis and may be a feature of other "Ca. Nealsonbacteria" found in anoxic environments. IMPORTANCE An anaerobic microbial enrichment culture was used to study members of candidate phyla that are difficult to grow in the lab. We were able to visualize tiny "Candidatus Nealsonbacteria" cells attached to a large Methanothrix cell, revealing a novel episymbiosis.

Engineering kinetics of TLR7/8 agonist release from bottlebrush prodrugs enables tumor-focused immune stimulation.

Imidazoquinolines (IMDs), such as resiquimod (R848), are of great interest as potential cancer immunotherapies because of their ability to activate Toll-like receptor 7 (TLR7) and/or TLR8 on innate immune cells. Nevertheless, intravenous administration of IMDs causes severe immune-related toxicities, and attempts to improve their tissue-selective exposure while minimizing acute systemic inflammation have proven difficult. Here, using a library of R848 "bottlebrush prodrugs" (BPDs) that differ only by their R848 release kinetics, we explore how the timing of R848 exposure affects immune stimulation in vitro and in vivo. These studies led to the discovery of R848-BPDs that exhibit optimal activation kinetics to achieve potent stimulation of myeloid cells in tumors and substantial reductions in tumor growth following systemic administration in mouse syngeneic tumor models without any observable systemic toxicity. These results suggest that release kinetics can be tuned at the molecular level to provide safe yet effective systemically administered immunostimulant prodrugs for next-generation cancer immunotherapies.

Modification of a Quadrupole/Orbitrap/Linear Quadrupole Ion Trap Tribrid Mass Spectrometer for Diagnostic Gas-Phase Ion-Molecule Reactions.

Tandem mass spectrometry based on diagnostic gas-phase ion-molecule reactions represents a robust method for functional group identification in unknown compounds. To date, most of these reactions have been studied using unit-resolution instruments, such as linear quadrupole ion traps and triple quadrupoles, which cannot be used to obtain elemental composition information for the species of interest. In this study, a high-resolution mass spectrometer, a quadrupole/orbitrap/linear quadrupole ion trap tribrid, was modified by installing a portable reagent inlet system to obtain high-resolution data for ion-molecule reactions. Examination of a previously published test system, the reaction between protonated 1,1'-sulfonyldiimizadole with 2-methoxypropene, demonstrated the ability to perform ion-molecule reactions on the modified tribrid mass spectrometer. High-resolution data were obtained for ion-molecule reactions of three isobaric ions (protonated glycylalanine, protonated glutamine, and protonated lysine) with diethylmethoxyborane. On the basis of these data, the isobaric ions can be differentiated based on both their measured accurate mass as well as the different product ions they generated upon the ion-molecule reactions. In a different experiment, analyte ions were subjected to collision-induced dissociation (CID), and the structures of the resulting fragment ions were examined via diagnostic ion-molecule reactions. This experiment allows for the functional group interrogation of fragment ions and can be used to improve the understanding of the structures of fragment ions generated in the gas phase.

Development of a selection assay for small guide RNAs that drive efficient site-directed RNA editing.

A major challenge confronting the clinical application of site-directed RNA editing (SDRE) is the design of small guide RNAs (gRNAs) that can drive efficient editing. Although many gRNA designs have effectively recruited endogenous Adenosine Deaminases that Act on RNA (ADARs), most of them exceed the size of currently FDA-approved antisense oligos. We developed an unbiased in vitro selection assay to identify short gRNAs that promote superior RNA editing of a premature termination codon. The selection assay relies on hairpin substrates in which the target sequence is linked to partially randomized gRNAs in the same molecule, so that gRNA sequences that promote editing can be identified by sequencing. These RNA substrates were incubated in vitro with ADAR2 and the edited products were selected using amplification refractory mutation system PCR and used to regenerate the substrates for a new round of selection. After nine repetitions, hairpins which drove superior editing were identified. When gRNAs of these hairpins were delivered in trans, eight of the top ten short gRNAs drove superior editing both in vitro and in cellula. These results show that efficient small gRNAs can be selected using our approach, an important advancement for the clinical application of SDRE.