Structure-based discovery of potent WD repeat domain 5 inhibitors that demonstrate efficacy and safety in preclinical animal models.

WD repeat domain 5 (WDR5) is a core scaffolding component of many multiprotein complexes that perform a variety of critical chromatin-centric processes in the nucleus. WDR5 is a component of the mixed lineage leukemia MLL/SET complex and localizes MYC to chromatin at tumor-critical target genes. As a part of these complexes, WDR5 plays a role in sustaining oncogenesis in a variety of human cancers that are often associated with poor prognoses. Thus, WDR5 has been recognized as an attractive therapeutic target for treating both solid and hematological tumors. Previously, small-molecule inhibitors of the WDR5-interaction (WIN) site and WDR5 degraders have demonstrated robust in vitro cellular efficacy in cancer cell lines and established the therapeutic potential of WDR5. However, these agents have not demonstrated significant in vivo efficacy at pharmacologically relevant doses by oral administration in animal disease models. We have discovered WDR5 WIN-site inhibitors that feature bicyclic heteroaryl P7 units through structure-based design and address the limitations of our previous series of small-molecule inhibitors. Importantly, our lead compounds exhibit enhanced on-target potency, excellent oral pharmacokinetic (PK) profiles, and potent dose-dependent in vivo efficacy in a mouse MV4:11 subcutaneous xenograft model by oral dosing. Furthermore, these in vivo probes show excellent tolerability under a repeated high-dose regimen in rodents to demonstrate the safety of the WDR5 WIN-site inhibition mechanism. Collectively, our results provide strong support for WDR5 WIN-site inhibitors to be utilized as potential anticancer therapeutics.

The Influence of Contextual and Theoretical Expertise on Generic and Occupation-Specific Lifting Strategy.

OBJECTIVE: To determine whether (i) low back loads and/or (ii) kinematic coordination patterns differed across theoretical expert, contextual expert and novice groups when completing both generic and occupation-specific lifts. BACKGROUND: Experience has been proposed as a factor that could reduce biomechanical exposures in lifting, but the literature reports mixed effects. The inconsistent relationship between experience and exposures may be partially attributable to the broad classification of experience and experimental lifting protocols not replicating the environment where experience was gained. METHODS: Purposive sampling was used to recruit 72 participants including theoretical experts (formal training on lifting mechanics), contextual experts (paramedics), and novices. Participants performed 10 barbell and crate (generic) lifts, as well as backboard and stretcher (occupation-specific) lifts while whole-body kinematics and ground reaction forces were collected. Peak low back compression and anteroposterior shear loads normalized to body mass, as well as kinematic coordination patterns, were calculated as dependent variables. RESULTS: No significant differences in low back loads were observed across expertise groups. However, significant differences were seen in kinematic coordination patterns across expertise groups in occupation-specific lifts, but not in generic lifts. CONCLUSION: Increasing expertise is unlikely to minimize low back loads in lifting. However, contextual expertise did influence lifting kinematics, but only when performing occupationally specific lifts. APPLICATION: Contextual expertise may help lifters adopt lifting kinematics that enhance the tolerance of their musculoskeletal system to withstand applied loads, but does not seem to reduce the applied low back loads relative to noncontextual expert groups.

Outcomes of the Arterial Switch Operation in Patients With Dextrocardia.

BACKGROUND: Patients with dextrocardia and d-transposition of the great arteries (d-TGA) present a technical challenge when performing the arterial switch operation (ASO). We sought to determine the long-term outcomes of this rare presentation. METHODS: All patients who underwent the ASO with d-TGA and dextrocardia were identified from the hospital database. RESULTS: There were four patients with dextrocardia (4 of 844, 0.5%) patients; two patients had situs solitus and two patients had situs inversus. Three patients had a ventricular septal defect. Left ventricular outflow tract obstruction was present in one patient and one patient had a hypoplastic arch with severe coarctation of aorta. Anomalous coronary anatomy occurred in two patients. There was one early mortality and the cause of death was sepsis. One patient required late reoperation for severe neo-pulmonary valve regurgitation at 23 years after the ASO. At last follow-up, all three surviving patients were in New York Heart Association Class I. CONCLUSIONS: Patients with d-TGA and dextrocardia who undergo the ASO are extremely rare and require challenging operative management.

Can we enable individuals to reach further down without rounding their backs before beginning a lift? Examining the influence of starting foot and trunk position on reach depth.

There is disagreement regarding the efficacy of 'safe' lifting recommendations for reducing low back disorder risk. These recommendations commonly focus on minimising lumbar spine flexion, which limits the range of allowable starting lift positions for that person. This study evaluated whether starting postural adaptations could allow a person to reach down further without rounding their lumbar spine before beginning a lift. Reach displacement was measured as participants performed a series of maximal reach tasks under different combinations of stance width, foot orientation and trunk inclination, with their lumbar spine motion restricted. There were no interactions between any of the three postural adaptations or any effect of stance width or trunk inclination. Seventy-nine percent of participants achieved their greatest reach displacement with their feet externally rotated, which contributed to a 4 cm greater reach displacement compared to a neutral foot orientation (p < 0.001).Practitioner summary: This study examined whether aspects of initial posture could influence the ability to adhere to 'safe' lifting recommendations across a range of lift heights. As a component of lifting (re)training interventions, practitioners should consider starting lift posture adaptations (e.g. manipulating foot external rotation) to improve capacity to adhere to recommendations.

Quantifying how functional and structural personal factors influence biomechanical exposures in paramedic lifting tasks.

It is unknown how structural (sex, stature, body mass) and functional (strength, flexibility) personal factors influence lifting strategy in paramedic work. We explored whether variance in peak low back forces and kinematic coordination patterns could be explained by structural and functional personal factors in paramedic lifting tasks. Seventy-two participants performed backboard and stretcher lifts. Peak low back forces normalised to body mass, as well as kinematic coordination patterns, were calculated as dependent variables. Being female, stronger, shorter, having higher body mass, and/or having greater lower body range of motion (ROM) were all independently associated with lower normalised low back forces across backboard and stretcher lifting. Females and stronger individuals seemed to define a movement objective to consistently minimise compressive forces, while individuals with greater hip ROM consistently minimised anteroposterior shear forces. The efficacy of improving strength and hip ROM to reduce low back forces in paramedic lifting should be investigated.Practitioner summary: Females, stronger individuals, and individuals with greater hip range of motion consistently exhibited lower normalised low back forces in paramedic lifting. Improving strength and hip range of motion via training is a potential proactive ergonomics approach to reduce peak low back forces in paramedic lifting tasks.

Activation of the innate signaling molecule MAVS by bunyavirus infection upregulates the adaptor protein SARM1, leading to neuronal death.

La Crosse virus (LACV), a zoonotic Bunyavirus, is a major cause of pediatric viral encephalitis in the United States. A hallmark of neurological diseases caused by LACV and other encephalitic viruses is the induction of neuronal cell death. Innate immune responses have been implicated in neuronal damage, but no mechanism has been elucidated. By using in vitro studies in primary neurons and in vivo studies in mice, we have shown that LACV infection induced the RNA helicase, RIG-I, and mitochondrial antiviral signaling protein (MAVS) signaling pathway, resulting in upregulation of the sterile alpha and TIR-containing motif 1 (SARM1), an adaptor molecule that we found to be directly involved in neuronal damage. SARM1-mediated cell death was associated with induced oxidative stress response and mitochondrial damage. These studies provide an innate-immune signaling mechanism for virus-induced neuronal death and reveal potential targets for development of therapeutics to treat encephalitic viral infections.

Functional Movement Screen Task Scores and Joint Range-of-motion: A Construct Validity Study.

Little is known about the construct validity of the Functional Movement Screen (FMS). We aimed to assess associations between FMS task scores and measures of maximum joint range-of-motion (ROM) among university varsity student-athletes from 4 sports (volleyball, basketball, ice hockey, and soccer). Athletes performed FMS tasks and had their maximum ankle, hip and shoulder ROM measured. Multivariable linear regression was used to estimate associations between FMS task scores and ROM measurements. 101 university student-athletes were recruited (52 W/49 M; mean age 20.4±1.9 years). In general, athletes with higher FMS task scores had greater ROM compared to those with lower task scores. For example, athletes who scored 2 on the FMS squat task had 4° (95% CI, 1° to 7°) more uni-articular ankle dorsiflexion ROM compared with those who scored 1, while those who scored 3 on the FMS squat task had 10° (4° to 17°) more uni-articular ankle dorsiflexion ROM compared with those who scored 1. Large variation in ROM measurements was observed. In sum, substantial overlap in joint ROM between groups of athletes with different FMS task scores weakens the construct validity of the FMS as an indicator of specific joint ROM.

Mechanical Energy Expenditure at Lumbar Spine and Lower Extremity Joints During the Single-Leg Squat Is Affected by the Nonstance Foot Position.

ABSTRACT: Hirsch, SM, Chapman, CJ, Frost, DM, and Beach, TAC. Mechanical energy expenditure at lumbar spine and lower extremity joints during the single-leg squat is affected by the nonstance foot position. J Strength Cond Res 36(9): 2417-2426, 2022-Previous research has shown that discrete kinematic and kinetic quantities during bodyweight single-leg squat (SLS) movements are affected by elevated foot positioning and sex of the performer, but generalizations are limited by the high-dimensional data structure reported. Using a 3D inverse dynamical linked-segment model, we quantified mechanical energy expenditure (MEE) at each joint in the kinetic chain, the total MEE (sum of MEE across aforesaid joints), and the relative contribution of each joint to total MEE during SLSs performed with elevated foot positioned beside stance leg (SLS-Side), and in-front of (SLS-Front) and behind (SLS-Back) the body. Total MEE differed between SLS variations ( p = 0.002), with the least amount observed in the SLS-Back (effect size [ES] = 0.066-0.069). Approximately 50% of total MEE was contributed by the knee joint in each SLS variation, whereas MEE at the ankle, hip, and lumbar spine (in absolute and relative terms) varied complexly as a function of the elevated foot position. Total MEE ( p = 0.0192, ES = 0.852) and the absolute MEE at the knee and spine was greater in men across the SLS variations performed ( p = 0.025-0.036, ES = 0.715-0.766), but only the lumbar spine contribution to total MEE was larger in men across all SLS variations ( p = 0.045, ES = 0.607). Otherwise, there were no other sex-specific responses observed. Biomechanically, SLS movements are generally "knee-dominant," but changing elevated foot position effectively redistributes MEE among other joints in the linkage. Consistent with the previous conclusions reached based on discrete kinematic and kinetic data, not all SLSs are equal.

Comparison of 5 Normalization Methods for Knee Joint Moments in the Single-Leg Squat.

Ratio scaling is the most common magnitude normalization approach for net joint moment (NJM) data. Generally, researchers compute a ratio between NJM and (some combination of) physical body characteristics (eg, mass, height, limb length, etc). However, 3 assumptions must be verified when normalizing NJM data this way. First, the regression line between NJM and the characteristic(s) used passes through the origin. Second, normalizing NJM eliminates its correlation with the characteristic(s). Third, the statistical interpretations following normalization are consistent with adjusted linear models. The study purpose was to assess these assumptions using data collected from 16 males and 16 females who performed a single-leg squat. Standard inverse dynamics analyses were conducted, and ratios were computed between the mediolateral and anteroposterior components of the knee NJM and participant mass, height, leg length, mass × height, and mass × leg length. Normalizing NJM-mediolateral by mass × height and mass × leg length satisfied all 3 assumptions. Normalizing NJM-anteroposterior by height and leg length satisfied all 3 assumptions. Therefore, if normalization of the knee NJM is deemed necessary to address a given research question, it can neither be assumed that using (any combination of) participant mass, height, or leg length as the denominator is appropriate nor consistent across joint axes.

Age influences susceptibility of brain capillary endothelial cells to La Crosse virus infection and cell death.

BACKGROUND: A key factor in the development of viral encephalitis is a virus crossing the blood-brain barrier (BBB). We have previously shown that age-related susceptibility of mice to the La Crosse virus (LACV), the leading cause of pediatric arbovirus encephalitis in the USA, was associated with the ability of the virus to cross the BBB. LACV infection in weanling mice (aged around 3 weeks) results in vascular leakage in the olfactory bulb/tract (OB/OT) region of the brain, which is not observed in adult mice aged > 6-8 weeks. Thus, we studied age-specific differences in the response of brain capillary endothelial cells (BCECs) to LACV infection. METHODS: To examine mechanisms of LACV-induced BBB breakdown and infection of the CNS, we analyzed BCECs directly isolated from weanling and adult mice as well as established a model where these cells were infected in vitro and cultured for a short period to determine susceptibility to virus infection and cell death. Additionally, we utilized correlative light electron microscopy (CLEM) to examine whether changes in cell morphology and function were also observed in BCECs in vivo. RESULTS: BCECs from weanling, but not adult mice, had detectable infection after several days in culture when taken ex vivo from infected mice suggesting that these cells could be infected in vitro. Further analysis of BCECs from uninfected mice, infected in vitro, showed that weanling BCECs were more susceptible to virus infection than adult BCECs, with higher levels of infected cells, released virus as well as cytopathic effects (CPE) and cell death. Although direct LACV infection is not detected in the weanling BCECs, CLEM analysis of brain tissue from weanling mice indicated that LACV infection induced significant cerebrovascular damage which allowed virus-sized particles to enter the brain parenchyma. CONCLUSIONS: These findings indicate that BCECs isolated from adult and weanling mice have differential viral load, infectivity, and susceptibility to LACV. These age-related differences in susceptibility may strongly influence LACV-induced BBB leakage and neurovascular damage allowing virus invasion of the CNS and the development of neurological disease.

Relationship between operative duration and perioperative outcomes after radical cystectomy.

INTRODUCTION Prolonged operative times have been associated with an increased risk in complications in other major abdominal surgeries. This study tests the hypothesis that longer operative times will be associated with an increased risk in perioperative complications after radical cystectomy (RC). MATERIALS AND METHODS: Adult patients who underwent RC from January 1, 2012, through December 31, 2016, were identified from the National Surgical Quality Improvement Program (NSQIP) database. A natural log transformation was used to determine cutoff points for operative times at 33rd, 67th, and 90th percentiles: 272, 371, and 479 minutes, respectively. Cohorts were A (≤ 272 min), B (273-371 min), C (372-479 min), and D (> 479 min). Multivariable logistic regression analysis was performed to identify associations between operative time and perioperative complications. RESULTS: Among 5,610 patients, the distribution across cohorts was A, 1,993 patients; B, 1,818; C, 1,171; and D, 628. Cohort D had a higher incidence of pulmonary embolism (PE), deep vein thrombosis (DVT), urinary tract infection (UTI), sepsis, 30-day readmission, and blood transfusion rate and had a longer median hospital length of stay. Multivariable analysis showed that operative time (per 60 min) was associated with increased risk of DVT (OR 1.10, p = .04), PE (OR 1.15, p = .01), UTI (OR 1.08, p = .004), readmission (OR 1.04, p = .03), and blood transfusion (OR 1.23, p < .001). CONCLUSIONS: Longer operative times during RC are associated with a higher rate of perioperative complications. These findings may be confounded by disease stage, surgeon experience, variations in perioperative management protocols, or a combination of the above.

Resident perspectives on the value of interdisciplinary conference calls for geriatric patients.

BACKGROUND: There are limited competency-based educational curricula for transitions of care education (TOC) for internal medicine (IM) residency programs. The University of Colorado implemented a virtual interdisciplinary conference call, TEAM (Transitions Expectation and Management), between providers on the inpatient Acute Care of the Elder (ACE) unit and the outpatient Seniors Clinic at the University of Colorado Hospital. Residents rotating on the ACE unit participated in weekly conferences discussing Seniors Clinic patients recently discharged, or currently hospitalized, to address clinical concerns pertaining to TOC. Our goals were to understand resident perceptions of the educational value of these conferences, and to determine if these experiences changed attitudes or practice related to care transitions. METHODS: We performed an Institutional Review Board-approved qualitative study of IM housestaff who rotated on the ACE unit during 2018-2019. Semi-structured interviews were conducted to understand perceptions of the value of TEAM calls for residents' own practice and the impact on patient care. Data was analyzed inductively, guided by thematic analysis. RESULTS: Of the 32 IM residents and interns who rotated on ACE and were invited to participate, 11 agreed to an interview. Three key themes emerged from interviews that highlighted residents' experiences identifying and navigating some of their educational 'blind spots:' 1) Awareness of patient social complexities, 2) Bridging gaps in communication across healthcare settings, 3) Recognizing the value of other disciplines during transitions. CONCLUSIONS: This study highlights learner perspectives of the benefit of interdisciplinary conference calls between inpatient and outpatient providers to enhance transitions of care, which provide meaningful feedback and serve as a vehicle for residents to recognize the impact of their care decisions in the broader spectrum of patients' experience during hospital discharge. Educators can maximize the value of these experiences by promoting reflective debriefs with residents and bringing to light previously unrecognized knowledge gaps around hospital discharge.

Regulating Movement Frequency and Speed: Implications for Lumbar Spine Load Management Strategies Demonstrated Using an In Vitro Porcine Model.

The relationship between internal loading dose and low-back injury risk during lifting is well known. However, the implications of movement parameters that influence joint loading rates-movement frequency and speed-on time-dependent spine loading responses remain less documented. This study quantified the effect of loading rate and frequency on the tolerated cumulative loading dose and its relation to joint lifespan. Thirty-two porcine spinal units were exposed to biofidelic compression loading paradigms that differed by joint compression rate (4.2 and 8.3 kN/s) and frequency (30 and 60 cycles per minute). Cyclic compression testing was applied until failure was detected or 10,800 continuous cycles were tolerated. Instantaneous weighting factors were calculated to evaluate the cumulative load and Kaplan-Meier survival probability functions were examined following nonlinear dose normalization of the cyclic lifespan. Significant reductions in cumulative compression were tolerated when spinal units were compressed at 8.3 kN/s (P < .001, 67%) and when loaded at 30 cycles per minute (P = .008, 45%). There was a positive moderate relationship between cumulative load tolerance and normalized cyclic lifespan (R2 = .52), which was supported by joint survivorship functions. The frequency and speed of movement execution should be evaluated in parallel to loading dose for the management of low-back training exposures.

Cutting Edge: CCR2 Is Not Required for Ly6Chi Monocyte Egress from the Bone Marrow but Is Necessary for Migration within the Brain in La Crosse Virus Encephalitis.

Inflammatory monocyte (iMO) recruitment to the brain is a hallmark of many neurologic diseases. Prior to entering the brain, iMOs must egress into the blood from the bone marrow through a mechanism, which for known encephalitic viruses, is CCR2 dependent. In this article, we show that during La Crosse Virus-induced encephalitis, egress of iMOs was surprisingly independent of CCR2, with similar percentages of iMOs in the blood and brain of heterozygous and CCR2-/- mice following infection. Interestingly, CCR2 was required for iMO trafficking from perivascular areas to sites of virus infection within the brain. Thus, CCR2 was not essential for iMO trafficking to the blood or the brain but was essential for trafficking within the brain parenchyma. Analysis of other orthobunyaviruses showed that Jamestown Canyon virus also induced CCR2-independent iMO egress to the blood. These studies demonstrate that the CCR2 requirement for iMO egress to the blood is not universal for all viruses.

Single-ventricle palliation in children with atrioventricular septal defect and transposition of the great arteries: 45 years of experience.

BACKGROUND: The association of atrioventricular septal defect and transposition of the great arteries is very rare. As a rule, these patients have unbalanced ventricles. However, there have been no studies describing the results of single-ventricle palliation in these children. METHODS: All children who underwent surgery with a diagnosis of atrioventricular septal defect and transposition of the great arteries were included in the study. Data were obtained from medical records. RESULTS: A total of 38 patients with atrioventricular septal defect and transposition of the great arteries underwent single-ventricle palliation at the study institution between 1971 and 2016. The mean follow-up was 12.4 years (median: 14.6 years, range 2-43.3 years). Most children had unbalanced atrioventricular septal defect (94.7%, 36/38). Survival was 67.6% (95% confidence interval [CI]: 50.0-80.2%) at 10 years and 57.8% (95% CI: 38.0-73.4%) at 20 years. By 10 years, 58.6% (95% CI: 40.8-72.7%) had progressed to Fontan completion, while 32.5% (95% CI: 18.2-47.6%) had died. In patients achieving Fontan completion, 20-year event-free survival was 73.3% (95% CI: 34.8-91.3%), while 5.0% (95% CI: 0.4-20.5%) had undergone cardiac transplantation and 21.7% (95% CI: 3.2-50.8%) had undergone takedown of the Fontan circulation. Freedom from atrioventricular valve surgery was 57.0% (95% CI: 37.2-72.7%) at 10 and 20 years. CONCLUSIONS: The association of atrioventricular septal defect and transposition of the great arteries is very rare, and most of these children have unbalanced ventricles. Single-ventricle palliation results in 25-year overall survival of 50%. However, in patients, who had Fontan completion, survival was 75% at 25 years after Fontan operation.

Pregnancy Outcomes in Women After the Arterial Switch Operation.

BACKGROUND: Pregnancy outcomes after the arterial switch operation (ASO) are rare. We sought to determine outcomes of ASO survivors who underwent pregnancy. METHODS: Female patients who had an ASO and underwent pregnancy were identified from the congenital heart disease pregnancy clinic at The Royal Melbourne Hospital. All follow-up data were collected retrospectively by medical record review. RESULTS: Eleven (11) women were identified as having undergone medical care during pregnancy, from the adult congenital database, at The Royal Melbourne Hospital. There were 17 successful pregnancies, and nine women have been followed post pregnancy. Of the 17 successful deliveries, eight were delivered by Caesarean section, seven were vaginal deliveries and two were instrumented vaginal deliveries. Of the eight Caesarean sections, five were emergency and three were elective. The indications for emergency Caesarean section were obstructed labour (n = 2), abnormal cardiotocography (n = 1), obstructed labour and abnormal cardiotocography (n = 1) and congestive cardiac failure (n = 1). There was one neonatal complication (respiratory distress requiring intubation) in a child born at 31 weeks. There were maternal obstetric complications in 10 patients. There were two maternal cardiac complications during pregnancy (heart failure and rapid atrial fibrillation/flutter). There was no change in left ventricular function post-pregnancy. There was progression of severity of neo-aortic valve regurgitation in two patients post pregnancy (trivial to mild and moderate-severe to severe respectively). CONCLUSION: Pregnancy post ASO appears to be safe in the majority of women. Maternal cardiac complications are uncommon in patients without residual significant haemodynamic lesions, although maternal obstetric complications may be common.

Neuronal maturation reduces the type I IFN response to orthobunyavirus infection and leads to increased apoptosis of human neurons.

BACKGROUND: La Crosse virus (LACV) is the leading cause of pediatric arboviral encephalitis in the USA. LACV encephalitis can result in learning and memory deficits, which may be due to infection and apoptosis of neurons in the brain. Despite neurons being the primary cell infected in the brain by LACV, little is known about neuronal responses to infection. METHODS: Human cerebral organoids (COs), which contain a spectrum of developing neurons, were used to examine neuronal responses to LACV. Plaque assay and quantitative reverse transcription (qRT) PCR were used to determine the susceptibility of COs to LACV infection. Immunohistochemistry, flow cytometry, and single-cell transcriptomics were used to determine specific neuronal subpopulation responses to the virus. RESULTS: Overall, LACV readily infected COs causing reduced cell viability and increased apoptosis. However, it was determined that neurons at different stages of development had distinct responses to LACV. Both neural progenitors and committed neurons were infected with LACV, however, committed neurons underwent apoptosis at a higher rate. Transcriptomic analysis showed that committed neurons expressed fewer interferon (IFN)-stimulated genes (ISGs) and genes involved IFN signaling in response to infection compared to neural progenitors. Furthermore, induction of interferon signaling in LACV-infected COs by application of recombinant IFN enhanced cell viability. CONCLUSIONS: These findings indicate that neuronal maturation increases the susceptibility of neurons to LACV-induced apoptosis. This susceptibility is likely due, at least in part, to mature neurons being less responsive to virus-induced IFN as evidenced by their poor ISG response to LACV. Furthermore, exogenous administration of recombinant IFN to LACV COs rescued cellular viability suggesting that increased IFN signaling is overall protective in this complex neural tissue. Together these findings indicate that induction of IFN signaling in developing neurons is an important deciding factor in virus-induced cell death.

CRISPR/Cas9-targeted enrichment and long-read sequencing of the Fuchs endothelial corneal dystrophy-associated TCF4 triplet repeat.

PURPOSE: To demonstrate the utility of an amplification-free long-read sequencing method to characterize the Fuchs endothelial corneal dystrophy (FECD)-associated intronic TCF4 triplet repeat (CTG18.1). METHODS: We applied an amplification-free method, utilizing the CRISPR/Cas9 system, in combination with PacBio single-molecule real-time (SMRT) long-read sequencing, to study CTG18.1. FECD patient samples displaying a diverse range of CTG18.1 allele lengths and zygosity status (n = 11) were analyzed. A robust data analysis pipeline was developed to effectively filter, align, and interrogate CTG18.1-specific reads. All results were compared with conventional polymerase chain reaction (PCR)-based fragment analysis. RESULTS: CRISPR-guided SMRT sequencing of CTG18.1 provided accurate genotyping information for all samples and phasing was possible for 18/22 alleles sequenced. Repeat length instability was observed for all expanded (≥50 repeats) phased CTG18.1 alleles analyzed. Furthermore, higher levels of repeat instability were associated with increased CTG18.1 allele length (mode length ≥91 repeats) indicating that expanded alleles behave dynamically. CONCLUSION: CRISPR-guided SMRT sequencing of CTG18.1 has revealed novel insights into CTG18.1 length instability. Furthermore, this study provides a framework to improve the molecular diagnostic accuracy for CTG18.1-mediated FECD, which we anticipate will become increasingly important as gene-directed therapies are developed for this common age-related and sight threatening disease.

Fragment-based screening of programmed death ligand 1 (PD-L1).

The PD-1 immune checkpoint pathway is a highly validated target for cancer immunotherapy. Despite the potential advantages of small molecule inhibitors over antibodies, the discovery of small molecule checkpoint inhibitors has lagged behind. To discover small molecule inhibitors of the PD-1 pathway, we have utilized a fragment-based approach. Small molecules were identified that bind to PD-L1 and crystal structures of these compounds bound to PD-L1 were obtained.