Positive margin status due to incomplete removal of tumor tissue during radical prostatectomy for high-risk localized prostate cancer requires reoperation or adjuvant therapy, which increases morbidity and mortality. Adverse effects of prostate cancer treatments commonly include erectile dysfunction, urinary incontinence, and bowel dysfunction, making successful initial curative prostatectomy imperative.Current intraoperative tumor margin assessment is largely limited to frozen section analysis, which is a lengthy, labor-intensive process that is obtrusive to the clinical workflow within the operating room (OR). Therefore, a rapid method for prostate cancer margin assessment in the OR could improve outcomes for patients.Dual probe difference specimen imaging (DDSI), which uses paired antibody-based probes that are labeled with spectrally distinct fluorophores, was shown herein for prostate cancer margin assessment. The paired antibody-based probes consisted of a targeted probe to prostate-specific membrane antigen (PSMA) and an untargeted probe, which were used as a cocktail to stain resected murine tissue specimens including prostate tumor, adipose, muscle, and normal prostate. Ratiometric images  of the difference between targeted and untargeted probe uptake were calculated and evaluated for accuracy using receiver operator characteristic curve analysis with area under the curve values used to evaluate the utility of the DDSI method to detect PSMA positive prostate cancer.Targeted and untargeted probe uptake was similar between the high and low PSMA expressing tumor due to nonspecific probe uptake after topical administration. The ratiometric DDSI approach showed substantial contrast difference between the PSMA positive tumors and their respective normal tissues . Furthermore, DDSI showed substantial contrast difference between the high PSMA expressing tumors and the minimally PSMA expressing tumors due to the ratiometric correction for the nonspecific uptake patterns in resected tissues.Previous work has shown that ratiometic imaging has strong predictive value for breast cancer margin status using topical administration. Translation of the ratiometric DDSI methodology herein from breast to prostate cancers demonstrates it as a robust, ratiometric technique that provides a molecularly specific imaging modality for intraoperative margin detection. Using the validated DDSI protocol on resected prostate cancers permitted rapid and accurate assessment of PSMA status as a surrogate for prostate cancer margin status. Future studies will further evaluate the utility of this technology to quantitatively characterize prostate margin status using PSMA as a biomarker. Particularly, in the case of surgical cancer removal, where the goal is margin negative resection, staining of the resected specimen is an attractive alternative to enable molecular-specific margin assessment without the need for the contrast agent to touch the patient while leveraging the existing FGS clinical infrastructure.A variety of technologies are under development to improve intraoperative margin assessment including Raman spectroscopy using endogenous tissue contrast as well as microscopy with ultraviolet (UV) surface excitation to enable rapid slide-free histology, further highlighting this unmet clinical need.–Although conceptually simple, topical targeted probe administration to resected tissues is dominated by nonspecific uptake and thus fluorescent signal in both the tumor tissue and surrounding normal tissues, negating the bulk of biomarker targeted contrast. Notably, use of an untargeted, companion probe of equivalent size and charge can effectively map this nonspecific tissue uptake, which shows substantial intra- and intertissue variability. The use of targeted and untargeted companion probe staining has previously been investigated using antibodies labeled with spectrally distinct fluorophores and surface enhance Raman scattering (SERS) nanoparticles.,,–,––,A robust DDSI protocol with high sensitivity and specificity for differentiation between benign and malignant breast tissues22.1Alexa Fluor 647  and Cy3B  were purchased in their N-hydroxysuccinimide ester form and then solubilized in anhydrous dimethyl sulfoxide (DMSO) at 10 mM for antibody conjugation. For the targeted probe, PSMA targeted monoclonal antibody (Mab) J533 IgG was used. The mouse hybridoma Prost J533 (HB-12127) was purchased from American Type Culture Collection . The hybridoma was used to produce the J533 antibody as the IgG, K isotype, which was purified using mercaptoethylpyridine/protein A chromatography by the Monoclonal Antibody Core at Oregon Health and Science University (OHSU). For the untargeted probe, donkey anti-rabbit (DkRb) IgG  was used.2.2The human prostate carcinoma cell line LNCaP clone FGC was cultured in RPMI 1x  with 10% fetal bovine serum  and 1% penicillin–streptomycin–glutamine (Thermo Fisher Scientific). The human prostate grade IV adenocarcinoma cell line PC-3 was cultured in F-12K 1x (Gibco/Thermo Fisher Scientific) with 10% FBS and 1% penicillin–streptomycin–glutamine. All cell lines were cultured to 2.3The targeted probe J533 IgG was conjugated to AF647, and the untargeted probe DkRb IgG was conjugated to Cy3B. Each probe was prepared individually, explained briefly as follows. First, the antibodies were buffer exchanged into 1x phosphate-buffered saline (PBS) at pH 8.0. Following this, 10 mM AF647 in anhydrous DMSO was added to the J533 IgG, and 10 mM Cy3B in anhydrous DMSO was added to the DkRb IgG, resulting in a 5:1 fluorophore-to-antibody molar ratio in a total volume of 1 mL for each solution diluted in 1x PBS at pH 8.0. The solutions were shaken gently for 3 h at room temperature (RT), protected from light. The resulting solutions were then concentrated in 10 kDa molecular weight cut-off spin filters  into clean microcentrifuge tubes to remove any unconjugated fluorophore. The fluorophore-to-protein ratio for each conjugated antibody was quantified using absorbance spectroscopy . Antibody absorbance was measured at 280 nm  were quantified prior to the LNCaP and PC-3 PSMA stained samples.LNCaP and PC-3 cells were trypsinized, counted, and resuspended to 2.5LNCaP and PC-3 cells were trypsinized, counted, and resuspended to a final concentration of 2.6Separate cohorts of male athymic nude mice  weighing 19 to 21 g were used to grow LNCaP and PC-3 tumor xenografts. All animal studies were approved by the Institutional Animal Care and Use Committee at OHSU (Protocol No. TR01_IP0000202).2.7All mice were anesthetized with a 100 mg/kg ketamine  and 10 mg/kg xylazine  solution injected intraperitoneally. The toe pinch method was utilized to ensure the mice were fully anesthetized prior to tumor implantation. The lower peritoneal region of each mouse was prepared in a sterile field using povidine-iodine . For LNCaP and PC-3 xenografts, 2.8All mice were euthanized via carbon dioxide asphyxiation followed by cervical dislocation prior to tumor resection, which is compliant with the recommendations by the panel on euthanasia of the American Veterinary Medical Association. LNCaP tumor bearing mice were euthanized after 6 weeks of tumor growth or a maximum tumor size of 2.9White light and fluorescence images of tumor, adipose, muscle, and prostate tissues were collected using a previously described custom-built wide-field fluorescence imaging system consisting of a QImaging EXi Blue monochrome camera  for fluorescence detection with a removable Bayer filter and a near infrared (NIR) PhotoFluor II light source .2.10The collected targeted and untargeted fluorescence images were processed using custom written MATLAB code  to generate DDSI maps of each set of tumor, adipose, muscle, and prostate tissues described as follows. First, the median background signal from a user-selected region of interest (ROI) absent of resected tissue was subtracted from the entire image. Separate ROIs were then selected within an equal volume of each probe in solution that was imaged in a 96 well plate to calculate the mean intensity value of the targeted and untargeted staining solutions. These values were subsequently used to normalize the targeted and untargeted images permitting calculation of DDSI tissue map. Each pixel value within the ROI in both targeted and untargeted fluorescence images was divided by the average intensity value of the ROI representative of the DDSI staining solution in their respective imaging channels. Each tissue sample ROI was selected to encompass the bulk of tissue without containing any of the tumor edges so as to ensure that all pixels selected corresponded to the tissue type. User-created manual masks were used to identify tissue sample ROIs containing tumor or normal  tissues based on white light images alone to minimize selection bias that could come from processing the fluorescence images. These user-created manual masks on white light images were overlaid onto each normalized fluorescence image (targeted and untargeted), and final DDSI images were then calculated as 2.11Following DDSI, each tumor and normal tissue set were flash frozen in optical cutting temperature compound to preserve the tissue and its orientation. Frozen blocks were sectioned at 2.12All statistical analysis was performed using custom written MATLAB code. Tumor-to-normal adipose, muscle, and prostate tissue diagnostic detection ability was determined by ROC curves generated for the untargeted probe images, targeted probe images, and calculated DDSI images. The perfcurve function in MATLAB was used to calculate area under the curve (AUC) values on a pixel-by-pixel basis with individual pixel values for each tissue type used as the response variable input. A threshold variable was generated with a linearly increasing value from the minimum to maximum pixel intensity with 100 times fewer number of values than the total number of pixel values. The true positive rate (percentage of tumor pixels greater than threshold), false positive rate , ROC curves, and corresponding AUC values were then generated and plotted at each threshold value. Histogram plots for tumor-to-normal adipose, muscle, and prostate tissue were generated, on which the optimal threshold point determined via ROC curve analysis was plotted.33.1in vitro were used, where the highly PSMA expressing LNCaP cell line  and the previously published DDSI staining protocol optimized for breast cancer were used to establish the accuracy of DDSI for prostate cancer detection. LNCaP  differentiation as compared to using the targeted probe  alone in highly PSMA expressing LNCaP tumor xenografts . ROC AUCThe only intraoperative margin assessment methodology developed and in use for prostatectomy at select hospitals is FSA, which adds 20 min to the surgical time in 90% of cases on averageLimitations to the current work include the separation of tissue types into discrete entities , which would be a heterogeneous mixture in clinical samples. In addition, a limited number of prostate cancers do not overexpress PSMA and thus additional biomarker development would be required to facilitate diagnostic DDSI for margin status assessment in all prostatectomy cases. In future studies, clinical sample staining will be evaluated to assess the diagnostic accuracy of the DDSI methodology in mixed, heterogeneous samples, and additional biomarkers will be evaluated for low PSMA expressing prostate cancers. In summary, the DDSI methodology could be extended to evaluate margin status across additional cancer types, which would enhance the diagnostic and surgical capability of DDSI, improving patient outcomes.


Some patients prescribed opioid analgesic (OA) medications for pain experience serious side effects, including dependence, sedation, and overdose. As most patients are at low risk for OA-related harms, risk reduction interventions requiring multiple counseling sessions are impractical on a large scale.This study evaluates whether an intervention based on reinforcement learning (RL), a field of artificial intelligence, learned through experience to personalize interactions with patients with pain discharged from the emergency department (ED) and decreased self-reported OA misuse behaviors while conserving counselors’ time.We used data representing 2439 weekly interactions between a digital health intervention (“Prescription Opioid Wellness and Engagement Research in the ED” [PowerED]) and 228 patients with pain discharged from 2 EDs who reported recent opioid misuse. During each patient’s 12 weeks of intervention, PowerED used RL to select from 3 treatment options: a brief motivational message delivered via an interactive voice response (IVR) call, a longer motivational IVR call, or a live call from a counselor. The algorithm selected session types for each patient each week, with the goal of minimizing OA risk, defined in terms of a dynamic score reflecting patient reports during IVR monitoring calls. When a live counseling call was predicted to have a similar impact on future risk as an IVR message, the algorithm favored IVR to conserve counselor time. We used logit models to estimate changes in the relative frequency of each session type as PowerED gained experience. Poisson regression was used to examine the changes in self-reported OA risk scores over calendar time, controlling for the ordinal session number (1st to 12th).P=.006) and extended IVR sessions (P<.001). Live counseling sessions were selected 33.5% of the time in the first 5 weeks of interactions (95% CI 27.4%-39.7%) but only for 16.4% of sessions (95% CI 12.7%-20%) after 125 weeks. Controlling for each patient’s changes during the course of treatment, this adaptation of treatment-type allocation led to progressively greater improvements in self-reported OA risk scores (P<.001) over calendar time, as measured by the number of weeks since enrollment began. Improvement in risk behaviors over time was especially pronounced among patients with the highest risk at baseline (P=.02).Participants on average were 40 (SD 12.7) years of age; 66.7% (152/228) were women and 51.3% (117/228) were unemployed. Most participants  reported chronic pain, and 46.2% (104/225) had moderate to severe depressive symptoms. As PowerED gained experience through interactions over a period of 142 weeks, it delivered fewer live counseling sessions than brief IVR sessions (The RL-supported program learned which treatment modalities worked best to improve self-reported OA risk behaviors while conserving counselors’ time. RL-supported interventions represent a scalable solution for patients with pain receiving OA prescriptions.Clinicaltrials.gov NCT02990377; https://classic.clinicaltrials.gov/ct2/show/NCT02990377 The related public health crises of chronic pain and opioid analgesic (OA) misuse continue to challenge the United States ,2. AlthoMany patients with chronic pain and risks of OA misuse can at least theoretically be identified during emergency department (ED) encounters. However, although risk reduction interventions based on motivational interviewing and cognitive behavioral therapies can be helpful -10, the Digital communication with patients via cell phone apps, text messaging, or interactive voice response (IVR) calls can facilitate OA risk monitoring and behavior change support for patients discharged from EDs -14. IdenP=.01) and pain intensity (P=.03).In a previous randomized trial of 278 patients with musculoskeletal pain, we evaluated the effectiveness of reinforcement learning (RL), a type of artificial intelligence (AI), as a strategy for increasing the efficiency of cognitive behavioral therapy for chronic pain by automatically adjusting the type and duration of counseling sessions based on patient feedback regarding their status . Compareguesses among a limited set of potential treatment options or action choices and evaluate the impact of those decisions based on some dynamic measure of feedback (the reward in RL). Over time, RL systems systematically incorporate experience with previous action choices as well as information about the user to make progressively more educated guesses that optimize the reward function. Unlike more deterministic digital interventions that use tree-structured protocols to decide what actions to take , RL systems use these repeated cycles of action choices and feedback in a dynamic, probabilistic manner to determine which actions optimize the reward score. In our previous trial of RL support for pain-related cognitive behavioral therapy  scores ,21).The methods and findings of this study are presented here in accordance with the CONSORT -AI Extension Guidelines . All parThe study was approved by the University of Michigan Human Subjects Committee (#HUM00105229) before any patient contact. The protocol is registered in ClinicalTrials.gov (#NCT02990377).Diagnostic and Statistical Manual for Mental Disorders Version 5 (DSM-V) moderate or severe opioid use disorder, were experiencing opioid withdrawal symptoms at the time of screening, were unable to communicate in English, were at acute risk for self-harm, were incarcerated, were classified as level 1 trauma , were pregnant, were under treatment for sexual assault, or had a family member or significant other enrolled in the study. Patients were approached for recruitment in person in the ED or were recruited via telephone following discharge because of COVID-19 restrictions. Participants were not compensated for completing IVR calls or telephone counseling sessions. Participants received US $20 to US $40 for completing surveys at baseline and follow-up time points and received US $5 for providing saliva samples at various time points .Potential participants were identified between November 8, 2018, and May 8, 2021, based on medical record evidence of visits to EDs affiliated with 2 large health systems. Both EDs are level I trauma centers located in the same county in Southeast Michigan, which has a median annual household income of US $75,730. Demographically, 70% of county residents are White non-Hispanic, 12% are Black or African American, 5% are Hispanic, and 13% are Asian or multiracial. In total, 11% of the county residents use Medicaid as their primary form of health insurance. Eligible patients were adults aged 18 to 70 years, presenting at the ED primarily for a pain-related complaint, and who reported any OA misuse in the previous 3 months. Misuse was defined as any score >0 on the modified COMM questionnaire . PatientAfter completing the baseline assessments, the patients were randomized with equal probability to the PowerED intervention or enhanced usual care. These analyses focused exclusively on patients randomized to the PowerED group.PowerED patients participated in 12 weeks of behavioral intervention addressing OA risk and pain management. Counseling and psychoeducation were based on motivational enhancement and cognitive behavioral principles and focused on improving patients’ OA safety profile, non–OA-related strategies for coping with pain , improving overall health , and effective communication with clinicians. Before initiating the randomized trial, the PowerED intervention content was pilot-tested and found to have positive effects on opioid misuse behaviors after discharge from the ED . In addiPowerED patients received brief (<5 minute) IVR calls up to 5 times per week, in which they responded to queries recorded in a human, female voice using their touchtone keypad. During the calls, the patients reported their use of OA prescription medications, levels of pain, and related risks. During IVR assessments, patients’ pain severity over the previous 24 hours was assessed using the pain Numerical Rating Scale . PatientEach week for each patient, PowerED selected from three options:A brief (<5-minute) IVR call including motivational messages focused on safe OA use and healthy strategies for pain management: During these calls, patients heard messages selected from 6 categories determined based on their most recent reports about pain severity  and OA use (yes vs no).An extended (5- to 10-minute) IVR call focused on OA-related risks and behavior change strategies: In these calls, patients received rotating questions focused on strategies for improving their pain and overall health, their beliefs regarding opioid use, and the importance of pain management. Using the motivational enhancement principle of supporting patients’ decisional autonomy .,21.20,21The patients’ baseline characteristics were described for the overall sample and separately within each of the 3 subgroups defined by baseline COMM scores. We calculated the total number and percentage of attempted IVR assessments that were completed within the baseline COMM score subgroups.We hypothesized that the relative frequency of decisions resulting in the selection of each of the 3 session types would evolve as PowerED gained experience with the population and learned which options were most successful in optimizing patients’ weekly OA risk scores while conserving counselor time. To test this hypothesis, we fitted a multinomial logit model predicting the 3-level outcome of selected session type , controlling for the potential correlation of weekly decisions within patients. Predictors of interest included the ordinal week of enrollment  and the session number for that patient . We used Stata’s  postestimation commands to calculate the probability distribution across session types over enrollment weeks and present those findings graphically.calendar time should be independent of within-patient temporal changes and represent a reasonable estimate of improvements over time in the intervention’s ability to target session types. As noted earlier, scores representing the patient’s OA risk behaviors in a given week had a possible range of 1 to 7, and 80.89% (1316/1627) of completed call-weeks had the best possible score of 1. Poisson regression was used to accommodate this skewed outcome distribution with a limited possible range. Regression models treated patients as random effects and other predictors as fixed effects. As we were particularly interested in PowerED’s performance within patient subgroups defined by baseline levels of OA risk, we first fitted a base model including only patients’ enrollment week and session number as predictors. We then fitted a second model including these predictors as well as dummy variables representing the main effects for baseline COMM scores of 2 to 5 and ≥6 (vs a score of 1) and interaction terms between those indicators and enrollment week. Using Stata’s postestimation command, we plotted the changes in predicted reward scores over enrollment weeks separately for the 3 levels of baseline COMM scores.We hypothesized that IVR-reported OA risk scores would improve as PowerED interacted with the population and made increasingly effective decisions. To test this hypothesis, we fitted 2 nested multilevel regression models with the week’s OA risk score as the outcome and independent variables representing enrollment week, session number, and baseline OA risk severity (3-level COMM score). The session number was included to control for within-patient changes in risk scores resulting from regression to the mean or intervention effectiveness. By controlling for session number, coefficients associated with enrollment week or P values for both the COMM by race and COMM by education differences were <.03). Participants with higher baseline COMM scores were also more likely to have moderate or severe symptoms of depression (P=.002).A total of 228 patients were randomized to receive the PowerED intervention  over 130Participants collectively received 11,544 days of IVR assessment calls and responded approximately half of the time . Response rates were similar across the groups, as defined by the baseline COMM scores . The RL P=.006) and extended IVR sessions (P<.001).PowerED’s decisions evolved as it gained experience through interactions with patients . AnalyseP=.02) and extended IVR sessions . For example, after 120 weeks of interactions with the sample, PowerED chose live counseling 21.2% of the time for patients’ first session (95% CI 16.8%-25.6%) but only 13.8% of the time if the patient was in their final, week-12 session (95% CI 9.5%-18.1%).The coefficients presented in P≤.004). Modeling also indicated that significant improvements in weekly reward scores over calendar time were experienced specifically among patients with baseline COMM scores of ≥6 indicating the highest baseline OA risk (model 2 in P=.02). Using coefficients from model 2 in Multilevel models predicting reward scores reflecting patients’ OA risk behaviors indicated that scores improved as PowerED gained more experience through patient interactions. The model predicting scores based on both enrollment week and ordinal session number indicated that both of these 2 factors were predictive of improvements in OA risk . Moreover, improvements in patients’ risk scores over time occurred during some of the most challenging months of the COVID-19 pandemic—the time when opioid deaths were increasing and access to care for many patients was restricted . ServiceThese findings are important because despite studies demonstrating the effectiveness of brief behavioral interventions for improving pain and decreasing OA risk ,10,33, tAs our goal was to reach the broadest possible population, this study included patients with relatively low baseline COMM scores. Having a lower threshold for inclusion in OA risk reduction could have substantial public health benefits, but monitoring and behavior change support for such a population using only live counseling sessions is not a scalable option, given the millions of eligible patients. Therefore, programs such as PowerED that seek to improve patients’ risk profile while minimizing human resource use are especially vital. It is particularly encouraging that analyses demonstrated that PowerED was able to improve patients’ self-reported risk behaviors as it gained experience interacting with patients who had COMM scores of ≥6. As shown in In a previous study, an RL-supported program of pain cognitive behavioral therapy achieved outcomes that were noninferior to a standard therapist-delivered program . DespiteThe PowerED intervention used patients’ IVR feedback to make decisions regarding the most effective mode of treatment while conserving counselor time. However, a limitation of this study is that response rates to IVR assessment calls were relatively low compared with the 88% response rate in a study using a similar feedback loop to adapt treatment among US Department of Veterans Affairs patients with chronic back pain . Even whThis study and our previous study, in which we used a similar AI approach to deliver cognitive behavioral therapy to veterans with chronic musculoskeletal pain , suggestDespite these opportunities, the development and adoption of AI-supported behavioral interventions must proceed with caution and a deep understanding of the ways in which such algorithms can perpetuate or even exacerbate disparities in care. Limited diversity in AI training samples may lead to biased decisions in more heterogeneous populations ,39. PoteIn conclusion, this study demonstrates that an OA risk reduction intervention supported by RL can learn to target clinician resources through interactions with patients, with the goal of maximizing population benefits. Such programs may be particularly scalable and effective for the large population of patients with pain at risk of OA-related harms who lack access to standard behavioral health services. Given that RL systems such as this one improve through experience with patients, large-scale studies in diverse patient populations are recommended to maximize intervention effectiveness and make OA risk reduction programs available for the millions of Americans at risk for OA-related harms.


This on-demandlight-based conformational control directly alters helicase activityas demonstrated by both bulk phase experiments and single-moleculeoptical tweezers analysis of one of the engineered helicases. The“opto-helicase” may be useful in future applicationsthat require spatiotemporal control of DNA hybridization states.Engineering a protein variant with a desired role relieson deepknowledge of the relationship between a protein's native structureand function. Using our structural understanding of a regulatory subdomainfound in a family of DNA helicases, we engineered novel helicasesfor which the subdomain orientation is designed to switch betweenunwinding-inactive and -active conformations upon  While weknow how to design and apply DNA nanostructures and how the DNA duplexis unwound to initiate biological processes such as transcription,replication, and repair, our ability to control the hybridizationstate of the DNA duplex at high spatiotemporal resolution is stilllimited. The ability to use nucleic-acid processing enzymes in a controlledmanner to manipulate DNA structures at predetermined locations andtime points would thus be highly valuable.DNA is an instruction manual, efficientlyencoding informationneeded for gene expression and protein synthesis. Additionally, itsstructural characteristics as a long, flexible, polymeric moleculethat is programmable, able to form secondary structures and to bindproteins and other nucleic acids, lend themselves well to many technologicaland biological applications. For example, custom DNA origami shapescan be synthesized based on the complementarity of nucleobases,6 To carry out their functions, especially those requiring directionalmotion, helicases consume chemical energy from nucleoside triphosphates,most commonly adenosine triphosphate (ATP), and turn it into mechanicalenergy allowing them to move along strands of a nucleic acid. Thismovement allows helicases to displace nucleic-acid-bound proteins,7 resolve DNA secondary structures,8 and dissolve the double helix.7 However, their ability to do so is context-dependent, requiringfine-tuned adaptation to specific conformations, oligomerization states,or stimulation by other external factors.9Helicases are a broadclass of enzymes that are responsible forprocessing nucleic acids in all domains of life and play importantroles in DNA replication, recombination, and repair, among other vitalnucleic acid metabolism processes.12 These helicaseshave been shown to unwind DNA efficiently as dimers,13 although monomer unwinding can be promoted through destabilizingthe duplex by force,14 interactions withprotein partners,15 or conformational controlof a regulatory subdomain, 2B.18 This structural subdomain canadopt multiple orientations,19 the extremes of which correspond to conformations known as “open”and “closed”  DNA, accompaniedby an increase in the speed of translocation on single-stranded (ss)DNA.17 Single-molecule experiments have furthershown that a single UvrD molecule adopts the “closed”conformation during duplex unwinding.21 The transition from the “closed” to “open”conformation correlates with a “strand-switching” behaviorwhere the helicase loads on the opposite strand of the helix and movesin the opposite direction to unwinding, allowing the DNA to rezip.22 Additional single-molecule experiments have revealed that permanentsite-specific chemical crosslinking of Rep into the “closed”conformation (Rep-X) creates a “superhelicase” thatis capable of highly processive unwinding of long stretches (>6kb)of duplex DNA as a monomer.16 AlthoughRep-X is an SF1 variant that is the most successful at uninterruptedunwinding of long stretches of DNA, it requires a free 3′-tailto load onto the DNA, and its direction of motion (the ability toswitch-strands) cannot be controlled. Crosslinked superhelicases basedon Rep and PcrA have been used for mimicking cotranscriptional RNAfolding,24 genome imaging via local denaturing,26 remodeling of small catalytic DNA hairpins,27 and isothermal DNA amplification.28 Theconformation of the 2B domain of Rep is clearly important for itsenzymatic function and controlling this conformation has led to thedevelopment of several molecular tools. The ability to control the2B domain in a precise and reversible manner would enable furtherstudy of the association between Rep structure and function and maybe applied to novel technologies.Of the large structural superfamily 1 (SF1) of DNA helicases, thethree most extensively studied proteins are Rep, UvrD, and PcrA, whichexhibit high sequence and structural homology.“closed” 1A, that trans to cis states upon exposure to UV (365 nm) light (S1A–D).Here, we created light-activatable variants ofRep helicase, RepLAs,where we aimed to control in real time the 2B subdomain orientation1A with am) light 1B. This trans and cis isomer lengths of azobenzene . Our selectionof residues was assisted by Chimera32 whichallowed us to replace each single amino acid in both the open andclosed conformations of Rep in the crystallographic structure10 (Protein Data Bank entry 1UAA) with cysteines.Replacements were made using the cysteine rotamer that most closelyapproximated the conformation of the native residue. We calculatedthe accessible surface area for each mutated residue33 as well as the distance between the estimated gamma atom coordinatesfor every mutated pair of cysteines in each conformation . We selected the residue pairs thatfit the criteria for the distance constraints of cis and trans azobenzene and maximized the solventaccessible surface area of the least-accessible member of each “open”/”“closed”pair considering both conformations that could be present during thechemical crosslinking . Manualinspection of each candidate cysteine pair minimized the likelihoodthat the selected residues had a functional role in ATP or DNA binding.35To design light-activatable versions ofRep helicase RepLA, 1C, we muobenzene 1B–D. We 34 in a pET28a vector. This cysteine-free template was then used toinsert the desired cysteines selected for crosslinking in two roundsof single-cysteine insertion using site-directed mutagenesis usingmutagenic oligonucleotide primers and the Q5 polymerase chain reaction(PCR) Kit (NEB). Cloning was performed with DH5α Escherichia coli cells. Cloned and Sanger-sequencing-confirmedplasmids were then moved to BL21 E. coli cells where proteins were expressed with IPTG induction and purifiedwith a Ni-NTA column as done previously34 and confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) for purity. Proteins were stored in Buffer B: 50 mM TrisHCl(pH 7.6 at room temperature), 1 mM ethylenediaminetetraacetic acid(EDTA), 600 mM NaCl, and appropriate amounts of glycerol (25–50%)when storing at −80 °C.To make the differentversions of RepLA, we cloned a cysteine-free mutant of Rep helicasecontaining a 6xHis tag as described previouslyFigure S1B) dissolved in dimethylformamide (DMF). Crosslinker solution wasadded to eluted protein solution for a final ratio of 4.4:1 crosslinker/proteinsuch that the final solution contained <2% DMF. The reaction wasrotated at room temperature in the dark for 1.5 h, spun down to removeprecipitated crosslinkers, and dialyzed into Buffer B at 4 °Covernight. Crosslinking was confirmed by a denaturing SDS-PAGE gel(S2) that displays a characteristic band retardationof the protein for the crosslinked fraction due to chemical crosslinkingbetween residues that are sufficiently separated in the primary proteinsequence. A secondary confirmation was performed for all proteinsusing UV–vis spectroscopy where a characteristic absorbancepeak at 365 nm (A365) is expected for the incorporated azobenzenemoiety in its trans state azobenzene  or bismaleimioethane  and determining the time point at which the A365 reachedthe calculated midpoint between the A365 of the 10 tail to allow for helicase loading.All protein samples were pretreated with 455 nm light (M455L4-C1 ThorLabs)in thin-walled PCR tubes for 1 min prior to addition to the reactionmixture to ensure a consistent trans isomerizationstate. UV-treated samples were irradiated for 30s with 365 nm light(uvBeast 365 nm mini flashlight) in thin-walled PCR tubes prior toaddition to the reaction buffer. Reactions were initiated by addingthe substrate DNA alongside adenosine triphosphate (ATP) to solutionscontaining the protein. All experiments were performed at room temperaturein the dark. The final reaction contained 20 nM protein, 50 nM substrateDNA, 1 mM ATP, 10 mM MgCl2, 100 mM TrisHCl (pH 8.0), 10%glycerol, and 15 mM NaCl. Reactions were quenched after 10 min bythe addition of an equal volume of twofold concentrated quenchingbuffer containing 20 mM EDTA, 0.5% SDS, and 250 nM of an unlabeledsingle-stranded 18 nt reporter DNA in the final-quenched volume. Finalfluorescence was measured 10 min after quenching with a Qubit 3.0fluorometer using 480 nm excitation and emission read at 510–580nm. Fluorescence was reported in relative fluorescence units (RFUs)using the default values of the Qubit instrument. All values depictedhere were normalized to a control condition containing all reactioncomponents except ATP , which,as expected, showed the highest fluorescence for each experiment toallow for better comparison across conditions. For assays performedunder reducing conditions, reaction buffer was prepared as above withthe addition of 100 mM dithiothreitol (DTT). Proteins were exposedto light as described above, added to the appropriate amount of reactionbuffer, and then incubated for 10 min at room temperature in the dark.The protein and reaction buffer were then combined with appropriateamounts of DNA reporter and ATP, and the assay was quenched and fluorescencewas measured as described above. DNA constructs are included in Table S1.Helicase activity assaysperformed in bulk utilized an 18 bp dsDNA duplex consisting of an18 nt DNA strand labeled with a fluorescein at the 5′ end anda dabcyl quencher on the 3′ end and hybridized to a 28 nt strandcontaining a (dT)21 Briefly, the construct consists of an 89 bphairpin stem capped by a (dT)4 loop  and contains at its 3′ end a 10dT single-stranded DNA region that serves as a Rep helicase loadingsite  and collimated the light beam priorto introducing UV into the path of the optical trap via a beam-splittingcube . We focused the lightat the back focal plane of the front objective to ensure epi-illuminationof the specimen plane . TheUV power was kept low within a range of ∼1–10 mW toavoid possible photodamage to the sample. We controlled the UV lightthrough a computer interface. When noted, we tracked the timing ofUV illumination (Supporting Information text). We acquired all optical trap data at 100 Hz.We modified a dual-trapoptical tweezers instrument37 consisting of Nescofilm mask  patterned withthree flow channels and adhered to two microscope coverslips . The coverslips were passivated withpolyethylene glycol to reduce protein adsorption. The outer channelswere connected to the central channel via glass capillaries  embedded in the Nescofilm. The top and bottomchannels contained DNA-coated streptavidin- and anti-digoxigenin antibodybeads, respectively, that flowed into the central channel where theycould be captured by the optical traps. The central channel containedtwo adjacent laminar flow streams composed of different buffers thatdid not mix significantly. One stream contained ATP (250 μM)but not Rep helicase, while the other contained Rep helicase (25 nM)but not ATP.Measurements werecarried out in a microfluidic sample chamber2, and 2% glycerol. The media contained an oxygen-scavenging systemconsisting of 1.2% glucose, 0.29 mg/mL pyranose oxidase from Coriolus sp. ,and 0.15 mg/mL catalase from Apergillus niger  to increase the lifetime ofthe DNA tethers and reduce photodamage to the sample.38 Unless otherwise noted, we used 25 nM Rep and 250 μMATP for all measurements.We performedour experiments in the dark (unless otherwise noted), at 22 °C,in 35 mM Tris–HCl (pH 8.0), 20 mM NaCl, 5 mM MgClFigure S8). First, a single-DNA hairpinwas tethered between two optically trapped beads in the ATP stream.The tether and beads were then translated into the protein stream,allowing a single Rep molecule to bind to the 10 nt loading site butnot to unwind DNA due to the absence of ATP. Finally, the complexwas returned to the ATP stream where unwinding could initiate. Anactive feedback loop maintained a constant tension of 11 pN throughout.In measurements with continuous UV illumination (S8A), the UV light source was turned on during all the steps describedabove, i.e., when loading Rep onto the DNA in the protein stream andwhen unwinding in the ATP stream. For experiments performed underdiscontinuous UV illumination , UV illumination was only initiated once the DNA with boundprotein had entered the ATP channel. The details of each protocolare described in the Supporting Information Text.All experimentswere done under single-turnover conditions, where a single helicasewas allowed to bind and unwind the DNA hairpin before dissociating, were verified by fitting theforce-extension behavior of a single molecule of the DNA hairpin constructobtained from mechanical unfolding and refolding by the optical traps,as described previously.21 The measurementof RepLA2 activation time is described in Supporting Information text.The amount of DNA unwound (in units ofbase pairs) at each time point was calculated by dividing the changein tether extension (in nm) by the extension of the two nucleotidesof ssDNA released per unwound base pair at the measured force. Thelatter was determined from the extensible worm-like chain (XWLC) modelfor ssDNA using the following parameters:39 . We excluded by visual inspection residues likely toplay an important role in ATP or DNA binding. We picked three residuepairs that satisfied our criteria (We set out to design crosslinkedvariants of Rep that under the ormation 1C. We naS2) andby UV–vis spectroscopy to ensure the incorporation of the azobenzenelinker . The gel-band intensities .Following the cloningand purification of all three of the RepLAproteins, a crosslinking reaction was performed with the azobenzenecrosslinker, and the proteins were subsequently analyzed by SDS-PAGEto determine the extent of crosslinking .To confirm the incorporation of the azobenzenecrosslinker, weused the characteristic absorbance of t 365 nm 2C which t 365 nm 2C or unctrans to cis upon UV (365 nm) irradiation and returnsto the trans state either spontaneously or upon blue(455 nm) light irradiation.40 To characterizethe isomerization of the incorporated azobenzene, we next performeda temporal study of isomerization after UV irradiation in one of theproteins, RepLA2, which displayed the largest light dependence ina bulk unwinding assay. The UV–vis spectra were measured beforeUV (365 nm) irradiation to establish the profile for the trans state of azobenzene, and then the sample was irradiated for 2 minwith UV light to convert azobenzene into the cis state.The UV–vis spectra were then measured immediately after UVirradiation as well as at various time points over several hours,while the protein was held in the dark at room temperature, allowingazobenzene to relax to the trans state. Upon exposureto UV, the absorbance at 365 nm decreased, indicating conversion tothe cis state . Weadditionally tested the isomerization kinetics of RepLA2 with consistentexposure to ambient room light after UV exposure . Samples that were exposed to ambient light exhibitedmore rapid isomerization to the trans state whichis consistent with greater exposure to blue wavelengths of light thanultraviolet wavelengths of light from ambient white light. Subsequentexperiments were, therefore, performed in the dark and can be assumedto occur in the cis state when UV-treated due toits long-lasting nature.Azobenzeneundergoes photoisomerization from is state 2D. The tof hours 2E, in ag10 overhang for helicase loading (Table S1). The strand complementary to the loading strand was labeledwith a fluorescein on the 5′-end and a fluorescence quencheron the 3′-end. When RepLA unwinds this substrate and displacesthe labeled strand, the released single strand will coil because ofits high flexibility,43 and the fluorophorewill be quenched  and at increasing concentrationsof helicase .To characterize the unwinding activity of the RepLA helicases inbulk solution, we developed a fluorescence-based DNA unwinding assay.The unwinding substrate contained 18 bp dsDNA with a 3′ (dT)quenched 3A. We pr13 As expected, at the protein concentrations tested,uncrosslinked RepLA2 and RepLA3 do not unwind any significant amountof duplex , possibly because the greater distance betweenthe crosslinked cysteines in these constructs minimizes disulfideformation in solution.We posited that the RepLA proteins would exhibitnegligible unwindingwhen they are left in their native, uncrosslinked state, as the short10 nt overhang on the dsDNA reporter would not accommodate dimersof Rep, which are necessary for unwinding activity in its native state.f duplex 3B. Inter2 or LA3 1D, consi2 or LA3 2B, as di2 or LA3 3C.. The As a positive control, we tested whetherRepLAs would exhibit enhancedunwinding when crosslinked with the same ethane crosslinker utilizedfor Rep-X. All three helicase constructs, when crosslinked with theethane crosslinker, exhibit the expected increase in unwinding capacityas measured by the decrease in fluorescence intensity relative tothe sample without helicase. The drop in fluorescence intensity inthe two-carbon crosslinked proteins compared to that of the uncrosslinkedhelicases demonstrates that the ethane crosslinker is indeed capableof enhancing the unwinding activity 3B. Variatrans state occupancy prior to the start of the experiment.Helicases were activated separately through 30 s illumination witha 365 nm light before being added to the reaction mix to ensure thatUV light acted on the proteins alone. Reactions were initiated withthe addition of substrate DNA and ATP. All three azobenzene-containingRepLA proteins exhibit a significant enhancement of their unwindingwhen pre-activated with UV light . Placing the tethered hairpin into the protein channel allowedRepLA2 to bind to the 10 nt ssDNA site, which can accommodate onlya monomer of the protein.13 Unwinding wasthen initiated by moving the tethered hairpin with bound RepLA2 intothe ATP channel. Experiments were performed with and without UV exposure(SI text). Light activation was carried out in situ with epi-illuminationof the sample chamber with 365 nm light from an LED . The UV light was continuously on during RepLA2loading in the protein channel and during unwinding in the ATP channel. The UV power at the specimenplane was kept at 1–10 mW.Next, we employeda DNA hairpin unwinding assay using optical tweezerst = 0. Entry into the ATP channel was determinedby recording when the tethered construct crossed the mid-point betweenthe protein and ATP channels. While the locations were constant, andthe capillaries used to introduce beads and the tip of the parafilmwere used as fiducial markers to initiate the positions, some variabilityin flow and position is inevitable. Traces in which unwinding beginsbefore or after t = 0 most likely represent replicatesin which the sample encountered ATP before or after its recorded entryinto the ATP channel, which is due to inherent variability from experimentto experiment.At a constant force, the DNAextension increased over time as the loaded helicase unwound eachbase pair releasing two nucleotides of ssDNA. 45 behavior which is more consistent with uncrosslinked Rep. In contrast,the hairpin was often unwound completely under light-on conditions.Here, RepLA2 was first illuminated by 465 nm light and then loadedonto the hairpin in the protein channel in the dark (Supporting Information text). The tethered hairpin was nextmoved into the ATP channel, and UV light was turned on only aftera delay of several seconds (ranging between ∼1 and 17 s) . To ensure that no RepLA2 was exposed to 365 nm light while inthe protein channel, the microfluidic chamber was flushed out (with40 μL of buffer) after every illumination cycle (Supporting Information text). In addition, alltubes and syringes containing protein were covered to prevent unwantedlight exposure. We observed two types of traces: a fraction whereunwinding initiated upon exposure to ATP but before UV illumination and a fraction whereunwinding started only after UV exposure . We attribute the first trace type to the populationof RepLA2 that are not crosslinked. Consistent with our interpretation,the average processivity and velocity for this fraction of helicasesthat can unwind in the absence of light  are consistent with the unwinding dynamicsof wtRep .To differentiate the unwinding activity of uncrosslinked RepLA2that remains in the protein solution, we performed additional opticaltweezer experiments with discontinuous UV illumination where light induces the open conformation that allows strand switchingto reverse its movement on DNA.21Control of the 2B subdomain of Rep helicaseusing the light-responsiveazobenzene moiety will allow more precise study of the relationshipbetween helicase conformation and enzymatic activity. Additionally,spatiotemporal control of a DNA-modifying enzyme may ultimately allowfor precise in situ modulation of small DNA structures and, eventually,larger DNA origami-based systems. Helicases have been previously usedto control the hybridization state of small DNA constructs to speedup DNA reaction networkscis-state lifetime48 and the wavelength at which the molecule isomerizes,moving away from the UV range of the light spectrum.49 Further structural modulations of the crosslinker could also expandthe distances between which the residue pairs need to fluctuate, ifa similar domain motion is to be modified in another family of helicases.While we used an azobenzene moiety to synthesize RepLA that isresponsive in the UV range, alternate photoswitchable crosslinkersmay also be possible. Other azobenzene derivatives have been shownto change both the 16 which limits its modularityfor some technological applications. We suspect that because the crosslinkingsites are much closer to the hinge for the 2B subdomain rotation inRepLAs, single-stranded DNA may gain access to the DNA-binding siteswithout hindrance from the crosslinker.Using single-molecule optical tweezers analysis, we showed thatRepLA2 can unwind a hairpin DNA substrate. Interestingly, RepLA2 canbind to the loading site without a free 3′end, although incorporationof azobenzene may create a small topological ring. The topologicalring in crosslinked Rep-X prevents it from unwinding without a free3′-end,Although we observeda strong UV-induced enhancement in unwindingprocessivity and speed from RepLA2 in our single-molecule experiments,we still observed significant DNA unwinding activities without UVillumination. The processivity, speed, and pattern of unwinding inindividual RepLA2 (no UV) traces closely match what we observe forwtRep under monomeric conditions, indicating that this backgroundunwinding may arise from uncrosslinked RepLA2 unwinding as a monomer.trans state of azobenzenein RepLA2 is not entirely incompatible with the closed conformationof Rep. There may be a conformational heterogeneity where a subpopulationof RepLA2 with trans azobenzene exists in the closedconformation, responsible for some of the unwinding activities observed.While we note that there may be some intermolecular crosslinking thatoccurs during the maleimide reaction, the small percentage of Repmultimers are unlikely to play a large role in unwinding DNA in solution.We note that performing bulk unwinding experiments under reducingconditions partially abrogates background unwinding activity, especiallyfor RepLA1 implying that the formation of disulfide bonds may affecta subset of RepLA designs and require further optimization of proteinstorage and reaction conditions. However, we also note that the effectof disulfide reduction on RepLA2 and RepLA3 is negligible, and singlemolecule unwinding traces of RepLA2 do not show the same pattern ofactivity as Rep-X, which we may expect if a disulfide bridge formedbetween the two cysteine residues. Another possibility to explainbackground unwinding is that the


Objective. Spinal cord injury (SCI) leads to debilitating sensorimotor deficits that greatly limit quality of life. This work aims to develop a mechanistic understanding of how to best promote functional recovery following SCI. Electrical spinal stimulation is one promising approach that is effective in both animal models and humans with SCI. Optogenetic stimulation is an alternative method of stimulating the spinal cord that allows for cell-type-specific stimulation. The present work investigates the effects of preferentially stimulating neurons within the spinal cord and not glial cells, termed ‘neuron-specific’ optogenetic spinal stimulation. We examined forelimb recovery, axonal growth, and vasculature after optogenetic or sham stimulation in rats with cervical SCI. Approach. Adult female rats received a moderate cervical hemicontusion followed by the injection of a neuron-specific optogenetic viral vector ipsilateral and caudal to the lesion site. Animals then began rehabilitation on the skilled forelimb reaching task. At four weeks post-injury, rats received a micro-light emitting diode (µLED) implant to optogenetically stimulate the caudal spinal cord. Stimulation began at six weeks post-injury and occurred in conjunction with activities to promote use of the forelimbs. Following six weeks of stimulation, rats were perfused, and tissue stained for GAP-43, laminin, Nissl bodies and myelin. Location of viral transduction and transduced cell types were also assessed. Main Results. Our results demonstrate that neuron-specific optogenetic spinal stimulation significantly enhances recovery of skilled forelimb reaching. We also found significantly more GAP-43 and laminin labeling in the optogenetically stimulated groups indicating stimulation promotes axonal growth and angiogenesis. Significance. These findings indicate that optogenetic stimulation is a robust neuromodulator that could enable future therapies and investigations into the role of specific cell types, pathways, and neuronal populations in supporting recovery after SCI. Therapeutic electrical stimulation applied to the spinal cord via epidural  with the goal of guiding new circuitry towards accurate downstream targets that support enhanced FRT performance  completed the long-term treatment experiments. Rats were divided across two groups: optogenetic stimulation (N = 6) and unstimulated sham (N = 5). Each rat began the study by acclimating to the three behavioral tasks after which they received a cervical hemicontusion (C4) and injection of an optogenetic virus ipsilateral and caudal to the lesion site  implant over C6 to deliver optogenetic light stimulation  at ipsilateral spinal segment C6 based on our prior work  and xylazine (10 mg kg−1). Supplemental dosages of ketamine were administered throughout the procedure as needed to maintain a surgical plane of anesthesia. Rats were placed on a liquid heating pad and shaved along the upper back and received an intradermal injection of lidocaine (1 mg kg−1) and bupivacaine (1 mg kg−1) at the site of incision, as well as a subcutaneous injection of enrofloxacin (5 mg kg−1). A subcutaneous injection of sterile saline (5 ml) was also provided to maintain hydration, and the eyes were coated with ophthalmic ointment to prevent dryness. The surgical field was cleaned with betadine and alcohol. An incision was made in the skin and through muscle layers covering vertebral segments C2–T2. A hemi-laminectomy was made over spinal segment C4 and a hemicontusion was produced using the Infinite Horizon Impactor .All rats received a C4 hemicontusion and intraspinal injections of the optogenetic vector μl deposits of AAV2-hSyn-ChR2-YFP  were then injected using a Hamilton syringe  with a glass-pulled pipette tip attached to a model 5000 microinjection unit . The targeted region of injection was just midline to the dorsal root entry zone and at a depth of 0.5 mm for both injection sites. A dwell time of 10 min was maintained for all injections.Rats received AAV spinal injections immediately after the hemicontusion procedure. This process began with a partial laminectomy ipsilateral to the injury at the caudal aspect of spinal segment C6. Rats were positioned in a spinal stereotaxic frame . Two, 1.5 −1) for pain management. This continued twice daily for the following two days. Weight and hydration were monitored daily for several days post-operatively.Following the injections, gelfoam was placed over the exposed spinal cord at C4 and C6. The muscle and skin layers were sutured closed; the rats were then placed on a heating pad and provided intraperitoneal saline injections and a reapplication of ophthalmic ointment. Once awake, the rats received a subcutaneous injection of buprenorphine (0.05 mg kgForelimb rehabilitation—dailySkilled FRTThe skilled FRT is used to assess forepaw movement after SCI. Rats were placed in a clear arena with a slit leading to a platform 1 cm away from the arena containing a sugar pellet. Rats then (1) reached through the slit with their affected forelimb to (2) grab the pellet, (3) bring it into the arena, and (4) to their mouth to eat. The successful completion of this entire process is considered a ‘success’. If any of these steps could not be completed, animals ‘failed’ the attempt and could not eat the pellet. This task was performed 15 min per day, five days a week for the entirety of the study, acting both as rehabilitation and the primary outcome. Reaching success was scored during each of these rehabilitation sessions. The ‘handedness’ of each rat was determined prior to injury by identifying the favored forepaw and perfroming the injury to target the dominant limb.et alWeekly videos of the FRT task were then used to determine the successful reaching attempts by researchers blinded to treatment groups. In cases where the performance on the filmed footage was a standard deviation above or below the weekly training average, the score from the day prior to filming was used. Video footage was also used to complete 12-point analysis .Two to four weeks after receiving a spinal injury and viral injections, rats underwent a second surgical procedure to implant a −1)/xylazine (10 mg kg−1) mixture. Supplemental dosages of ketamine were provided throughout the procedure to maintain a surgical plane of anesthesia. The upper back and top of head were shaved and cleaned with betadine and alcohol. Intradermal injections of lidocaine (1 mg kg−1) and bupivacaine (1 mg kg−1) were applied near the incision site and a subcutaneous injection of sterile saline (5 ml) was made for hydration. Ophthalmic ointment was applied to the eyes to prevent dryness and the rat was positioned onto a liquid heating pad. An incision was made over C2–T2 through the skin and muscle layers. A small amount of vertebral lamina was removed over caudal C6, the site of stimulation. A small hole was then drilled into ipsilesional and contralesional C5 using a Dremel tool with a .0210′′/.533 mm drill bit (Gyros Precision Tools). A 5/32 inch #000 flat fillister head screw (JI Morris) was inserted into the contralesional hole at C5 and a second screw passed through the hole of the μLED implant and into the bone.To begin the procedure, rats were anesthetized with a ketamine  was applied to fill any space between the implant and top of the exposed spinal cord to minimize scar tissue accumulation. Next, UV-curable FusioTM liquid dentin  was applied over the top of the implant and contralesional lamina and then cured using an LED cure lamp .The LED portion of the implant was then positioned over the exposed spinal cord at ipsilesional C6 where the hemi-laminectomy and μLED to the head-mounted pedestal was routed under the skin. The pedestal was then affixed to the top of the skull using a combination of microscrews , C & B Metabond Quick Adhesive System , and Ortho Jet Liquid Acrylic and Jet Denture Repair Powder . The muscle layers and skin were sutured closed. Ophthalmic ointment was reapplied, and buprenorphine was administered subcutaneously twice daily for the following two days. Enrofloxacin (25 mg kg−1) was added to water bottles for 7 d to provide additional protection against infections.The catheter connecting the Therapeutic optogenetic stimulationet alp = 1.00). Any sign of infection occurring below the implant for rats in either group was cause for exclusion from the study.Rats in the optogenetic stimulation group received stimulation beginning six weeks post-injury. The stimulation parameters were based on those determined to be thermally-safe in our previous work  and were transcardially perfused using 0.9% sodium chloride, followed by 10% formalin. The cervical spinal cord was dissected and placed in 10% formalin overnight for 24 h at 4 °C. Spinal cord tissue was cryopreserved in 30% sucrose for 24 h at 4 °C. Just prior to freezing, the cervical spinal cord was divided into three blocks: ‘rostral’ , ‘lesion’ (the lesion site including 1 mm of spared tissue above and below), and ‘caudal’ . Each block of tissue was embedded in O.C.T mounting compound  and then placed in a mixture of dry ice and acetone to rapidly freeze. Tissue was sectioned into 40 μm-thick cross sections using a cryostat.Assessment of AAV vector transductionTo assess the location of viral transduction in the stimulated rats, tissue sections from cervical segment C6 were mounted onto Superfrost plus slides  and covered with a coverslip using polyvinyl alcohol (PVA) mounting media . Sections were then imaged on a Zeiss Axio Zoom microscope  to confirm the presence of fluorescence of the reporter gene, YFP, indicating AAV transduction.Quantification of spared tissue after injuryet alSections from lesion site at C4 were mounted onto subbed Superfrost plus slides  and stained with cresyl violet and myelin dyes as described in our previous work  mixed with 1% normal donkey serum  and triton X-100  for 3 × 10 min on a shaker table at room temperature. Tissue was then incubated in a blocking solution of 5% DS-PBS-T for 1 h at room temperature. Sections were transferred to an overnight incubation in diluted primary antibody at 4 °C on a shaker table. Specific antibodies and dilutions include: GAP-43 , Laminin , VGLUT1 , VGLUT2 , GAD65 , and GAD67 . The following day, sections were washed with 1% DS-PBS-T for 3 × 10 min. Then, sections were incubated first in a biotinylated secondary antibody  for 1 h on a shaker table at room temperature, followed by streptavidin-680 . Sections were then washed 3 × 10 min with PBS, mounted onto Superfrost plus slides (Fisher Scientific) and coverslipped with PVA mountant (Sigma-Aldrich). Sections were viewed on a Zeiss Axio Zoom microscope .GAP-43 quantitative analysis of axons: The number of GAP-43-labeled fibers was determined using FIJI open-source software  ipsilateral dorsal, (2) contralateral dorsal, (3) ipsilateral lateral, (4) contralateral lateral, (5) ipsilateral ventral, (6) contralateral ventral, (7) ipsilateral dorsal gray matter, (8) contralateral dorsal gray matter, (9) ipsilateral ventral gray matter, and (10) contralateral ventral gray matter. Values were averaged across the four sections and then across the respective group.Analysis in the white matter: GAP-43-labeled fibers are easily visualized in the white matter. In these regions, the brightness/contrast was toggled within FIJI  using the exposure range indicator to normalize exposure across each section. Images were captured at a magnification of 0.50 pixels μm−1. Vessel counts were determined within the same ten regions of interest using FIJI software  and optogenetically stimulated (N = 6) groups, non-parametric tests were used to assess statistical significance. The Mann–Whitney U-test was used for mean comparisons on the FRT , IBB , GAP-43 and laminin (30 tests were performed for each antibody). The Wilcoxon rank-sum was used for paired-wise tests to compare GAP-43 and laminin across the rostral, lesion, and caudal regions of the cord within groups . To identify outliers, the interquartile range was determined for each data set and any data points falling 1.5 × above or below this range were considered outliers .Prior to the start of stimulation at six weeks post-injury, the group average of successful reaches on the FRT at five weeks post-injury was 22% ± 0.09% and 20% ± 0.07% for the unstimulated and optogenetically stimulated groups, respectively (figure p = 0.009). Following four weeks of optogenetic stimulation, animals receiving optogenetic stimulation had recovered a total of 38% ± 0.09% above pre-stimulation function, significantly more than the recovery observed in the unstimulated group . The optogenetically stimulated group continued to demonstrate significantly greater success (47% ± 0.09% above pre-stimulation function) compared to the unstimulated group for the final two weeks of stimulation . At this final time point, an outlier was removed from the optogenetic stimulation group as they were 1.5 × below the interquartile range. Inclusion of the outlier adjusts the p-value to 0.116 at this final time point only.Within two weeks of stimulation (seven weeks post-injury), animals receiving optogenetic stimulation recovered significantly more forelimb function (29% ± 0.05% above pre-stimulation function) compared to the unstimulated group . This was determined from IBB video analysis.Changes to specific limb characteristics during the FRT within each group, such as digit movement or aim, did not suggest any specific aspects of limb movement were responsible for the improved reaching success in the stimulated group (figure S2). Instead, each rat seemed to utilize different movement strategies to successfully retrieve pellets. Similarly, there were no significant group differences on the IBB or LUAT tasks (figures S3–S5). The optogenetic group, however, trended toward an increase in volar contact compared to pre-stimulation at nine weeks post-injury on the IBB that was not seen in the unstimulated group , at the level of the lesion (C4/5), and caudal to the lesion (C6/7) at the site of stimulation  rostral to the lesion, (2) centered on the lesion, and (3) caudal to the lesion. In the unstimulated group, the number of GAP-43-labeled fibers was similar between the rostral versus lesion segments, and rostral versus caudal segments. However, a significant decrease in GAP-43 fibers was noted when comparing lesion segments to caudal segments  at spinal segment C6. Most of the stimulated rats had viral transduction throughout the hemicord gray matter (figures Each of the rats that received optogenetic stimulation were assessed for overall viral vector transduction (4.This is the first study to describe the functional effects of optogenetic stimulation after cervical SCI. Our overall findings suggest optogenetic stimulation promotes robust functional recovery that is affected by lesion magnitude and supported in part by new axonal growth and angiogenesis. Several key aspects of these findings are discussed below.Effects of optogenetic stimulation on forelimbfunctionbioluminescent optogenetic stimulation after SCI in the lumbar cord (Petersen et alet alet alThe current study shows significantly enhanced recovery of forelimb reaching in rats that received optogenetic stimulation. While this is the first study to investigate the therapeutic effects of long-term optogenetic stimulation in the cervical spinal cord, a parallel study looked at the effects of Stimulation effects on unpaired taskset alet alThe present study found significantly enhanced recovery on the forelimb reaching task, but not the IBB and LUAT tests. This may be due to pairing stimulation with only the FRT, leading to the formation of new circuitry tailored to FRT performance via Hebbian plasticity in which ‘neurons that fire together wire together’ Hebb . SpecifiLocation of induced axonal growthet alet alet alet alet alet alOptogenetic stimulation increases GAP-43 expression, similar to other long-term optogenetic studies following stroke (Cheng et alet alet alet alet alBoth unstimulated and stimulated rats share a similar amount of axonal growth within the gray matter, where-as stimulated rats had significantly more GAP-43 in the white matter. This finding highlights the importance of plasticity within the white matter for promoting functional recovery. Propriospinal pathways have been shown in several studies to be particularly plastic after SCI and likely represent a significant portion of new growth induced by optogenetic stimulation (Bareyre et alet alet alet alet alet alin-vitro enhances neurite outgrowth in dorsal root ganglia cells (Park et alet alet alThe neuronal activation induced by optogenetic stimulation is likely the driving force behind the enhanced axonal growth reported here. Neuronal activation produces numerous neurophysiological effects that ultimately support increased axonal growth (Mondello Effect of stimulation on vasculatureet alet alet alet alet alet alet alStudies have shown that neuro-vasculature and endogenous neural activity are tightly coupled such that excitatory neural activity increases blood flow (Cox et alet alet alet alet alet alet alet alIn addition to the well-known benefits of increased neuro-vasculature, such as supplying tissue with oxygen and nutrients, a tight relationship between neuro-vasculature and improved functional outcomes has been reported (Glaser It is important to note that since we did not measure vascular volume, our results may reveal only part of the neuro-vasculature changes. Nonetheless, these findings suggest that functional improvements seen after optogenetic stimulation may be partly attributed to increased gray matter vasculature, which could translate into healthier neuronal populations that are more supportive of new circuitry.Neuron-specific stimulation is a powerfulneuromodulator within the spinal cordet alet alet alet alet alet alet alet al2+ signaling (Cornell-Bell et alet alet alet alet alet alet alet alOur findings suggest that stimulation of neurons in the absence of direct glial stimulation can promote significant functional recovery after SCI. This is notable as the majority of past therapeutic stimulation studies utilized electrical stimulation, which activates both neurons and glial cells (Roitbak and Fanardjian 5.et alet alet alet alet alet alet alet alet alThe current study demonstrates that optogenetic stimulation has robust neuromodulatory effects after injury, and shows therapeutic promise for treating SCI. Similar findings have been reported in animal models for other neurological diseases including stroke (Shah


ATP11A, a gene recently associated with autosomal dominant hearing loss and auditory neuropathy type 2. In a rare opportunity, we genome-sequenced a member of the original family to determine whether the DFNA33 locus may also be assigned to ATP11A. We identified a deep intronic variant in ATP11A that showed evidence of functionally normal splicing. Furthermore, we re-assessed haplotypes from the originally published DFNA33 family and identified two double recombination events and one triple recombination event in the pedigree, a highly unlikely occurrence, especially at this scale. This brief research report also serves as a call to the community to revisit families who have previously been involved in gene mapping studies, provide closure, and resolve these historical loci.Genetic heterogeneity makes it difficult to identify the causal genes for hearing loss. Studies from previous decades have mapped numerous genetic loci, providing critical supporting evidence for gene discovery studies. Despite widespread sequencing accessibility, many historically mapped loci remain without a causal gene. The DFNA33 locus was mapped in 2009 and coincidentally contains  Meiotic recombination  underlies genomic diversity and maintains fidelity during chromosome segregation. Although each chromosome generally presents at least one crossover , the recBy definition, linkage is an occurrence in which adjacent genes tend to be inherited together . Linkagehttps://hereditaryhearingloss.org/) has diligently documented mapped loci and regularly updates content with new genes as they are discovered. Despite the advent of widely accessible sequencing technologies, many of these families have been lost to follow up for a variety of reasons. As a result, it is a reasonable assumption that many of these loci will remain without a causal gene assigned. Furthermore, in spite of the success of mapping deafness loci and cost-effective genome sequencing technologies, the identification of causal genes is not always straightforward, even in families that are large and, therefore, informative by providing more information about meiotic recombination events. This is additionally reflected in the large number of families who have participated in historical linkage studies but remain without a causal gene identified. At least 20 autosomal dominant (DFNA) loci have been mapped (Hereditary Hearing Loss Homepage) that remain without a causal gene assigned. The majority of them were mapped before the widespread use of high-throughput sequencing. There are also several examples of independent investigators discovering the same gene under a pre-assigned DFN locus. For example, the DFNA20 and DFNA26 loci merged as DFNA20/26 when two teams discovered ACTG1 as a gene responsible for autosomal dominant hearing loss for these asynchronously mapped loci    and audi(DFNA84) , which cThrough extensive re-recruitment efforts, an affected individual from the originally published DFNA33 family  was re-aThe affected individual underwent a complete ear, nose, and throat examination that included binocular ear microscopy and external ear inspection. Pure-tone audiometry was performed according to current standards to determine hearing thresholds at routinely measured frequencies . Both air and bone conduction thresholds were measured. Tympanometry was performed to measure tympanic membrane compliance and middle ear pressure.https://github.com/imgag/megSAP) developed at the Institute of Medical Genetics and Applied Genomics, University Hospital Tübingen, Germany, and includes an in-house genome database called the NGS Database (NGSD), which consists data on more than 3,000 individuals to ascertain sequencing artifacts and variant frequencies. Visualization was performed using the Integrative Genomics Viewer.Whole genome sequencing was performed on genomic DNA extracted from peripheral blood using standard protocols, as previously described . The seqBioinformatics filters were used to support the analysis of variants, following autosomal dominant and recessive modes of inheritance, as described previously . This inVariant classification applied the American College of Medical Genetics and Genomics (ACMG) guidelines that are adapted for hereditary hearing loss  and refeATP11A exons 8 to 10 was PCR-amplified from the genomic DNA of the patient and a healthy control using gene-specific primers with a XhoI restriction site (ATP11A Ex8-10 forward: 5′-aattctcgagAAAATCACCGAAGCCATGAG-3′) and a BamHI restriction site (ATP11A Ex8-10 reverse: 5′-attggatccATGGTGAGAGGAGCTGTTGG-3′). The 5,014-bp amplicon was ligated into a multiple cloning site between native exons A and B in a linearized pSPL3 exon-trapping vector. The vector was transformed into DH5α competent cells  and plated overnight. The wild-type and mutant-containing vector sequences were confirmed by Sanger sequencing and transfected into HEK 293T cells using the FuGENE Transfection Reagent (Promega). Total RNA was prepared from 24-h post-transfected cells using an miRNeasy Mini Kit (QIAGEN), and reverse transcription was carried out using a High-Capacity RNA-to-cDNA Kit (Applied Biosystems). Amplified fragments were visualized on 1.5% agarose gel and, subsequently, Sanger sequenced.A region spanning The affected individual, who was not in the original DFNA33 report, is a direct descendent of a hearing-impaired father who had four affected siblings, three of whom were previously described  Figure . The indin silico pathogenicity prediction scores, no predicted effect on splicing, or an obvious clinical mismatch.Whole-genome sequencing resulted in an average 51.7 × coverage of the genome. An analysis of sequence variants and copy number variations in 289 known hearing loss-associated genes  excludedATP11A may be the gene for the DFNA33 locus and the growing clinical and functional evidence asserting ATP11A is an essential protein for a normal functioning auditory system, our analysis then focused on the previously mapped DFNA33 locus coordinates , as well as ATP11A (ATP11A intron 8 (chr13:113421269C>G (GRCh37), ENST00000375630:c.725 + 737C>T) was predicted via in silico tools to activate exonic splice enhancers. This variant was tested with an in vitro splicing assay using established protocols (ATP11A variant (ATP11A as being the gene for the DFNA33 locus and acknowledge that we have been extremely fortunate to re-recruit an individual from the DFNA33 family.Following the initial hypothesis that s ATP11A , and uncrotocols  and yielrotocols . Taken t variant , we presUpon close inspection, we noted that the haplotypes that were used to define the DFNA33 boundaries of the disease interval in the original Bönsch et al. paper show several unlikely events (Although a conventional understanding of recombination rates approximates about one crossover per homologous pair of chromosomes, it varies dramatically on a number of different scales and shows non-uniform distribution. However, mapping recombination patterns have uncovered chromosomal regions enriched for hotspots that are separated from regions that do not appear to recombine. The deCODE genetic map created chromosomal genetic maps that also included sex-averaged recombination rates . AnalysiClosely adjacent double or triple recombination events involving phasing errors are suggestive of genotyping errors . AlthougWith increased access to advanced sequencing technologies, the continuous follow-up of undiagnosed families with historical linkage is essential for the discernment of correct DFN-locus assignment. Our study highlights the necessity and potential to refine understanding of the number of total unresolved loci through a simple review of historical loci for such unlikely events. A problematic bottleneck will be re-contacting and re-establishing contact with the original families as maintaining connections to families over decades can be challenging. Considering the size of this unresolved family, although unlikely, we cannot exclude multi-locus heterogeneity or the presence of phenocopies. Furthermore, it was unfortunate that we could not recruit the entire family for repeat linkage analysis and genome sequencing of additional affected individuals to increase the chances of uncovering the causal genetic variant. The most convincing case for excluding DFNA33 altogether would be through the identification of a causal variant, either within the DFNA33 locus or elsewhere in the genome, which is a limiting factor of our work. Using a short-read genome-sequencing approach, we cannot exclude epigenetic modifications or short-inverted duplications. Nonetheless, re-assessment of haplotypes combined with new genome sequencing data can provide some value in better understanding unsolved DFNA loci.