Preclinical proof of principle for orally delivered Th17 antagonist miniproteins.

Interleukin (IL)-23 and IL-17 are well-validated therapeutic targets in autoinflammatory diseases. Antibodies targeting IL-23 and IL-17 have shown clinical efficacy but are limited by high costs, safety risks, lack of sustained efficacy, and poor patient convenience as they require parenteral administration. Here, we present designed miniproteins inhibiting IL-23R and IL-17 with antibody-like, low picomolar affinities at a fraction of the molecular size. The minibinders potently block cell signaling in vitro and are extremely stable, enabling oral administration and low-cost manufacturing. The orally administered IL-23R minibinder shows efficacy better than a clinical anti-IL-23 antibody in mouse colitis and has a favorable pharmacokinetics (PK) and biodistribution profile in rats. This work demonstrates that orally administered de novo-designed minibinders can reach a therapeutic target past the gut epithelial barrier. With high potency, gut stability, and straightforward manufacturability, de novo-designed minibinders are a promising modality for oral biologics.

De novo design of bioactive protein switches.

Allosteric regulation of protein function is widespread in biology, but is challenging for de novo protein design as it requires the explicit design of multiple states with comparable free energies. Here we explore the possibility of designing switchable protein systems de novo, through the modulation of competing inter- and intramolecular interactions. We design a static, five-helix 'cage' with a single interface that can interact either intramolecularly with a terminal 'latch' helix or intermolecularly with a peptide 'key'. Encoded on the latch are functional motifs for binding, degradation or nuclear export that function only when the key displaces the latch from the cage. We describe orthogonal cage-key systems that function in vitro, in yeast and in mammalian cells with up to 40-fold activation of function by key. The ability to design switchable protein functions that are controlled by induced conformational change is a milestone for de novo protein design, and opens up new avenues for synthetic biology and cell engineering.

Prevalence of Invasive Bacterial Infection in Hypothermic Young Infants: A Multisite Study.

OBJECTIVE: To determine the prevalence of bacteremia and meningitis (invasive bacterial infection [IBI]) in hypothermic young infants, and also to determine the prevalence of serious bacterial infections (SBI) and neonatal herpes simplex virus and to identify characteristics associated with IBI. STUDY DESIGN: We conducted a retrospective cohort study of infants ≤90 days of age who presented to 1 of 9 hospitals with historical or documented hypothermia (temperature ≤36.0°C) from September 1, 2017, to May 5, 2021. Infants were identified by billing codes or electronic medical record search of hypothermic temperatures. All charts were manually reviewed. Infants with hypothermia during birth hospitalization, and febrile infants were excluded. IBI was defined as positive blood culture and/or cerebrospinal fluid culture treated as a pathogenic organism, whereas SBI also included urinary tract infection. We used multivariable mixed-effects logistic regression to identify associations between exposure variables and IBI. RESULTS: Overall, 1098 young infants met the inclusion criteria. IBI prevalence was 2.1% (95% CI, 1.3-2.9) (bacteremia 1.8%; bacterial meningitis 0.5%). SBI prevalence was 4.4% (95% CI, 3.2-5.6), and neonatal herpes simplex virus prevalence was 1.3% (95% CI, 0.6-1.9). Significant associations were found between IBI and repeated temperature instability (OR, 4.9; 95% CI, 1.3-18.1), white blood cell count abnormalities (OR, 4.8; 95% CI, 1.8-13.1), and thrombocytopenia (OR, 5.0; 95% CI, 1.4-17.0). CONCLUSIONS: IBI prevalence in hypothermic young infants is 2.1%. Further understanding of characteristics associated with IBI can guide the development decision tools for management of hypothermic young infants.

A patient-centered approach to learning social determinants of health for first year medical students.

INTRODUCTION: To address health disparities, future physicians must understand the role of social determinants of health (SDH). Teaching SDH can be challenging. We created an authentic SDH curriculum using four real myocardial infarction (MI) patients. METHODS: During the three academic years from 2019-2020 to 2021-2022, 579 first year medical students participated in the four day curriculum. Day 1: students interviewed and learned about their patient's MI. Day 2: students met in small groups and shared their patient's history. At session end, students were familiar with four patient stories. Day 3: students explored their patient's neighborhood and then interviewed their patient again, focusing on SDH. Day 4: students gave formal patient presentations that highlighted SDH. Group discussion followed and reinforced the role of SDH. Students wrote reflections on SDH that were read and graded. End of course evaluations were reviewed. RESULTS: Five hundred and seventy-nine students completed the curriculum. Course directors graded SDH reflections on a six-point rubric for the years of 2020-2021 and 2021-2022. Ninety percent and 96% of the SDH reflections during the respective years contained 5-6/6 of the rubric components. Ninety-six percent to 98% of students 'agreed' or 'strongly agreed' that the curriculum was effective for their learning. DISCUSSION: For educators in need of an SDH curriculum that is both engaging and effective, we have found this activity to be feasible, low cost, and highly impactful for first year medical students.[Box: see text].

High-throughput characterization of protein-protein interactions by reprogramming yeast mating.

High-throughput methods for screening protein-protein interactions enable the rapid characterization of engineered binding proteins and interaction networks. While existing approaches are powerful, none allow quantitative library-on-library characterization of protein interactions in a modifiable extracellular environment. Here, we show that sexual agglutination of Saccharomyces cerevisiae can be reprogrammed to link interaction strength with mating efficiency using synthetic agglutination (SynAg). Validation of SynAg with 89 previously characterized interactions shows a log-linear relationship between mating efficiency and protein binding strength for interactions with Kds ranging from below 500 pM to above 300 µM. Using induced chromosomal translocation to pair barcodes representing binding proteins, thousands of distinct interactions can be screened in a single pot. We demonstrate the ability to characterize protein interaction networks in a modifiable environment by introducing a soluble peptide that selectively disrupts a subset of interactions in a representative network by up to 800-fold. SynAg enables the high-throughput, quantitative characterization of protein-protein interaction networks in a fully defined extracellular environment at a library-on-library scale.

Drip System for Admissions to Resident Teams: Impact on Workload and Education.

OBJECTIVES: Assigning patients to a call team every fourth day (bolus system) caused the maldistribution of patients among resident teams and required additional faculty effort for overflow patient care. We changed to a continuous daily rotation (drip system) and examined the effect on clinical workload among resident teams, resident education, and faculty utilization. METHODS: This is a retrospective study based on the daily records of 7 am team census, the attending physician schedules for a pediatric hospital medicine service with 5 teams, and the measures of resident education, including noon conference attendance, scores on in-service examinations, and duty hour violations. Data from the bolus system (May 2014-June 2015) were compared with the drip system (May 2016-June 2017). RESULTS: Data from 348 bolus days and 338 drip days were analyzed. There was a decrease in interteam variation from 6.2 to 3.9 patients (P < 0.001). There were fewer days with the following: large interteam variation (143 to 25, P < 0.001), days with resident teams at or above capacity (26 to 11, P = 0.01), resident teams below a minimum 7 am census (133 to 18, P < 0.001), and days when additional faculty were pulled for clinical care (61 to 9, P < 0.001). Resident noon conference attendance was unchanged and there was no adverse effect on examination scores or duty hour violations. CONCLUSIONS: Changing from a bolus to a drip model for admissions to inpatient teams resulted in a more even distribution of the workload and a more efficient use of physician resources without negatively affecting resident education.

Larotrectinib (LOXO-101).

One of the most challenging issues in oncology research and treatment is identifying oncogenic drivers within an individual patient's tumor which can be directly targeted by a clinically available therapeutic drug. In this context, gene fusions as one important example of genetic aberrations leading to carcinogenesis follow the widely accepted concept that cell growth and proliferation are driven by the accomplished fusion (usually involving former proto-oncogenes) and may therefore be successfully inhibited by substances directed against the fusion. This concept has already been established with oncogenic gene fusions like BCR-ABL in chronic myelogenous leukemia (CML) or anaplastic lymphoma kinase (ALK) in lung cancer, including special tyrosine kinase inhibitors (TKIs) which are able to block the activation of the depending downstream proliferation pathways and, consequently, tumor growth. During the last decade, the NTRK1, 2, and 3 genes, encoding the TRKA, B, and C proteins, have attracted increasing attention as another significant and targetable gene fusion in a variety of cancers. Several TRK inhibitors have been developed, and one of them, Larotrectinib (formerly known as LOXO-101), represents an orally available, selective inhibitor of the TRK receptor family that has already shown substantial clinical benefit in both pediatric and adult patients harboring an NTRK gene fusion over the last few years.

Olaparib.

Olaparib (Lynparza [AstraZeneca, Cambridge, UK], formerly referred to as AZD2281 or KU0059436) is an oral poly(ADP-ribose) polymerase (PARP) inhibitor. It is rationally designed to act as a competitive inhibitor of NAD+ at the catalytic site of PARP1 and PARP2, both members of the PARP family of enzymes that are central to the repair of DNA single-strand breaks (SSBs) mediated via the base excision repair (BER) pathway. Inhibition of the BER pathway by olaparib leads to the accumulation of unrepaired SSBs, which leads to the formation of deleterious double-strand breaks (DSBs). In cells with an intact homologous recombination (HR) pathway, these DSBs can be repaired effectively. However, in tumors with homologous recombination repair deficiencies, olaparib causes synthetic lethality through the combination of two molecular events that are otherwise nonlethal when occurring in isolation. Olaparib is already approved for the treatment of patients with recurrent ovarian cancer and a BRCA mutation, and it has been shown to provide clinically meaningful benefits among such patients. It has also shown promising activity in patients with metastatic breast or prostate cancer and a germline BRCA mutation. Besides its usage as a single agent, olaparib can also act either as a chemo- and/or radiosensitizer, due to its ability to potentiate the cytotoxic effects of these therapeutic agents. However, a clear patient benefit for the latter application has not been demonstrated yet.

Unraveling graft-versus-host disease and graft-versus-leukemia responses using TCR Vß spectratype analysis in a murine bone marrow transplantation model.

The optimum use of allogeneic blood and marrow transplantation (BMT) as a curative therapy for hematological malignancies lies in the successful separation of mature donor T cells that are host reactive and induce graft-versus-host disease (GVHD) from those that are tumor reactive and mediate graft-versus-leukemia (GVL) effects. To study whether this separation was possible in an MHC-matched murine BMT model (B10.BR→CBA) with a CBA-derived myeloid leukemia line, MMC6, we used TCR Vß CDR3-size spectratype analysis to first show that the Vß13 family was highly skewed in the B10.BR anti-MMC6 CD8(+) T cell response but not in the alloresponse against recipient cells alone. Transplantation of CD8(+)Vß13(+) T cells at the dose equivalent of their constituency in 1 × 10(7) CD8(+) T cells, a dose that had been shown to mediate lethal GVHD in recipient mice, induced a slight GVL response with no concomitant GVHD. Increasing doses of CD8(+)Vß13(+) T cells led to more significant GVL responses but also increased GVHD symptoms and associated mortality. Subsequent spectratype analysis of GVHD target tissues revealed involvement of gut-infiltrating CD8(+)Vß13(+) T cells accounting for the observed in vivo effects. When BMT recipients were given MMC6-presensitized CD8(+)Vß13(+) T cells, they displayed a significant GVL response with minimal GVHD. Spectratype analysis of tumor-presensitized, gut-infiltrating CD8(+)Vß13(+) T cells showed preferential usage of tumor-reactive CDR3-size lengths, and these cells expressed increased effector memory phenotype (CD44(+)CD62L(-/lo)). Thus, Vß spectratyping can identify T cells involved in antihost and antitumor reactivity and tumor presensitization can aid in the separation of GVHD and GVL responses.

Rapid Cycle Deliberate Practice Training for Simulated Cardiopulmonary Resuscitation in Resident Education.

Background: Simulation-based medical education has been used in medical training for decades. Rapid cycle deliberate practice (RCDP) is a novel simulation strategy that uses iterative practice and feedback to achieve skill mastery. To date, there has been minimal evaluation of RCDP vs standard immersive simulation (IS) for the teaching of cardiopulmonary resuscitation to graduate medical education (GME) learners. Our primary objective was to compare the time to performance of Advanced Cardiac Life Support (ACLS) actions between trainees who completed RCDP vs IS. Methods: This study was a prospective, randomized, controlled curriculum evaluation. A total of 55 postgraduate year-1 internal medicine and emergency medicine residents participated in the study. Residents were randomized to instruction by RCDP (28) or IS (27). Stress and ability were self-assessed before and after training using an anonymous survey that incorporated five-point Likert-type questions. We measured and compared times to initiate critical ACLS actions between the two groups during a subsequent IS. Results: Prior learner experience between RCDP and IS groups was similar. Times to completion of the first pulse check, chest compression initiation, backboard placement, pad placement, initial rhythm analysis, first defibrillation, epinephrine administration, and antiarrhythmic administration were similar between RCDP and IS groups. However, RCDP groups took less time to complete the pulse check between compression cycles (6.2 vs 14.2 seconds, P = 0.01). Following training, learners in the RCDP and IS groups scored their ability to lead and their levels of anticipated stress similarly (3.43 vs 3.30, (P = 0.77), 2.43 vs. 2.41, P = 0.98, respectively). However, RCDP groups rated their ability to participate in resuscitation more highly (4.50 vs 3.96, P = 0.01). The RCDP groups also reported their realized stress of participating in the event as lower than that of the IS groups (2.36 vs 2.85, P = 0.01). Conclusion: Rapid cycle deliberate practice learners demonstrated a shorter pulse check duration, reported lower stress levels associated with their experience, and rated their ability to participate in ACLS care more highly than their IS-trained peers. Our results support further investigation of RCDP in other simulation settings.

Time to Positive Blood and Cerebrospinal Fluid Cultures in Hypothermic Young Infants.

BACKGROUND AND OBJECTIVE: Hypothermia in young infants may be secondary to an invasive bacterial infection. No studies have explored culture time-to-positivity (TTP) in hypothermic infants. Our objective was to compare TTP of blood and cerebrospinal fluid (CSF) cultures between pathogenic and contaminant bacteria in hypothermic infants ≤90 days of age. METHODS: Secondary analysis of a retrospective cohort of 9 children's hospitals. Infants ≤90 days of age presenting to the emergency department or inpatient setting with hypothermia from September 1, 2017, to May 5, 2021, with positive blood or CSF cultures were included. Differences in continuous variables between pathogenic and contaminant organism groups were tested using a 2-sample t test and 95% confidence intervals for the mean differences reported. RESULTS: Seventy-seven infants met inclusion criteria. Seventy-one blood cultures were positive, with 20 (28.2%) treated as pathogenic organisms. Five (50%) of 10 positive CSF cultures were treated as pathogenic. The median (interquartile range [IQR]) TTP for pathogenic blood cultures was 16.8 (IQR 12.7-19.2) hours compared with 26.11 (IQR 20.5-48.1) hours for contaminant organisms (P < .001). The median TTP for pathogenic organisms on CSF cultures was 34.3 (IQR 2.0-53.7) hours, compared with 58.1 (IQR 52-72) hours for contaminant CSF organisms (P < .186). CONCLUSIONS: Our study is the first to compare the TTP of blood and CSF cultures between pathogenic and contaminant bacteria in hypothermic infants. All pathogenic bacteria in the blood grew within 36 hours. No difference in TTP of CSF cultures between pathogenic and contaminant bacteria was detected.

Performance of Febrile Infant Decision Tools on Hypothermic Infants Evaluated for Infection.

BACKGROUND: Given the lack of evidence-based guidelines for hypothermic infants, providers may be inclined to use febrile infant decision-making tools to guide management decisions. Our objective was to assess the diagnostic performance of febrile infant decision tools for identifying hypothermic infants at low risk of bacterial infection. METHODS: We conducted a secondary analysis of a retrospective cohort study of hypothermic (≤36.0 C) infants ≤90 days of age presenting to the emergency department or inpatient unit among 9 participating sites between September 1, 2016 and May 5, 2021. Well-appearing infants evaluated for bacterial infections via laboratory testing were included. Infants with complex chronic conditions or premature birth were excluded. Performance characteristics for detecting serious bacterial infection (SBI; urinary tract infection, bacteremia, bacterial meningitis) and invasive bacterial infection (IBI; bacteremia, bacterial meningitis) were calculated for each tool. RESULTS: Overall, 314 infants met the general inclusion criteria, including 14 cases of SBI (4.5%) and 7 cases of IBI (2.2%). The median age was 5 days, and 68.1% of the infants (214/314) underwent a full sepsis evaluation. The Philadelphia, Boston, IBI Score, and American Academy of Pediatrics Clinical Practice Guideline did not misclassify any SBI or IBI as low risk; however, they had low specificity and positive predictive value. Rochester and Pediatric Emergency Care Applied Research Network tools misclassified infants with bacterial infections. CONCLUSIONS: Several febrile infant decision tools were highly sensitive, minimizing missed SBIs and IBIs in hypothermic infants. However, the low specificity of these decision tools may lead to unnecessary testing, antimicrobial exposure, and hospitalization.

Variation in Care of Well-Appearing Hypothermic Young Infants: A Multisite Study.

BACKGROUND AND OBJECTIVES: Numerous decision tools have emerged to guide management of febrile infants, but limited data exist to guide the care of young infants presenting with hypothermia. We evaluated the variation in care for well-appearing hypothermic young infants in the hospital and/or emergency department setting between participating sites. METHODS: This is a retrospective cohort study of well-appearing infants ≤90 days old across 9 academic medical centers from September 1, 2016 to May 5, 2021. Infants were identified via billing codes for hypothermia or an initial temperature ≤36.0°C with manual chart review performed. Primary outcomes included assessment of variation in diagnostic evaluation, disposition, empirical antimicrobial therapy, and length of stay. RESULTS: Of 14 278 infants originally identified, 739 met inclusion criteria. Significant interhospital variation occurred across all primary outcomes. Across sites, a full serious bacterial illness evaluation was done in 12% to 76% of hypothermic infants. Empirical antibiotics were administered 20% to 87% of the time. Performance of herpes simplex viral testing ranged from 7% to 84%, and acyclovir was empirically started 8% to 82% of the time. Hospital admission rates ranged from 45% to 100% of patients. CONCLUSIONS: Considerable variation across multiple aspects of care exists for well-appearing young infants presenting with hypothermia. An improved understanding of hypothermic young infants and their risk of infection can lead to the development of clinical decision tools to guide appropriate evaluation and management.

Computational Design of Structured and Functional Peptide Macrocycles.

Structure-based computational design methods have been developed to create proteins in silico with diverse shapes and sizes that accurately fold in vitro, from 7-residue macrocycles to megadalton-scale self-assembling nanomaterials. Precise control over protein shape has further enabled design and optimization of functional therapeutic proteins, including agonists, antagonists, enzymes, and vaccines. Computational design of functional peptides of smaller size presents a persistent challenge, with few successful examples to date. Herein we describe validated general methods for computational design of peptides using the Rosetta molecular modeling suite and discuss outstanding challenges and future directions.

Delayed Diagnosis of Pediatric Sternoclavicular Joint Infections and Clavicular Osteomyelitis During the COVID-19 Pandemic: A Report of 3 Cases.

Sternoclavicular joint infections and osteomyelitis of the clavicle are extremely rare infections, especially in the pediatric population. Early signs of these infections are nonspecific and can be mistaken for common upper respiratory infections such as COVID-19 and influenza. Rapid diagnosis and treatment are critical for preventing potentially fatal complications such as mediastinitis. We present three cases of sternoclavicular joint infections in the past year during the COVID-19 pandemic. All three patients had delayed diagnoses likely secondary to COVID-19 workup. Each patient underwent surgical irrigation and débridement. Two of three patients required multiple surgeries and prolonged antibiotic courses. Placement of antibiotic-impregnated calcium sulfate beads into the surgical site cleared the infection in all cases where they were used. All three patients made a full recovery; however, the severity of their situations should not be overlooked. Children presenting to the hospital with chest pain, fever, and shortness of breath should not simply be discharged based on a negative COVID-19 test or other viral assays. A higher index of suspicion for bacterial infections such as clavicular osteomyelitis is important. Close attention must be placed on the physical examination to locate potential areas of concentrated pain, erythema, or swelling to prompt advanced imaging if necessary.

The immunological impact of genetic drift in the B10.BR congenic inbred mouse strain.

The MHC-matched, minor histocompatibility Ag (miHA)-mismatched B10.BR-->CBA strain combination has been used to elucidate the immunobiology of graft-vs-host disease (GVHD) following allogeneic bone marrow transplantation. Studies conducted in the 1980s had established that B10.BR CD8+ T cells were capable of mediating GVHD in the absence of CD4+ T cells, and that CD4+ T cells were unable to induce lethal disease. In more recent studies with this GVHD model, we detected etiological discrepancies with the previously published results, which suggested that genetic drift might have occurred within the B10.BR strain. In particular, there was increased allorecognition of CBA miHA by B10.BR CD4+ T cells, as determined by both TCR Vbeta spectratype analysis and the induction of lethal GVHD in CBA recipients. Additionally, alloreactivity was observed between the genetically drifted mice (B10.BR/Jdrif) and mice rederived from frozen embryos of the original strain (B10.BR/Jrep) using Vbeta spectratype analysis and IFN-gamma ELISPOT assays, suggesting that new miHA differences had arisen between the mice. Furthermore, T cell-depleted B10.BR/Jdrif bone marrow cells were unable to provide long-term survival following either allogeneic or syngeneic bone marrow transplantation. Gene expression analysis revealed several genes involved in hematopoiesis that were overexpressed in the lineage-negative fraction of B10.BR/Jdrif bone marrow, as compared with B10.BR/Jrep mice. Taken together, these results suggest that genetic drift in the B10.BR strain has significantly impacted the immune alloreactive response in the GVHD model by causing altered expression of miHA and diminished capacity for survival following transplantation into lethally irradiated recipients.

Overlap between in vitro donor antihost and in vivo posttransplantation TCR Vbeta use: a new paradigm for designer allogeneic blood and marrow transplantation.

Following allogeneic blood and marrow transplantation (BMT), mature donor T cells can enhance engraftment, counteract opportunistic infections, and mount graft-versus-tumor (GVT) responses, but at the risk of developing graft-versus-host disease (GVHD). With the aim of separating the beneficial effects of donor T cells from GVHD, one approach would be to selectively deplete subsets of alloreactive T cells in the hematopoietic cell inoculum. In this regard, TCR Vbeta repertoire analysis by CDR3-size spectratyping can be a powerful tool for the characterization of alloreactive T-cell responses. We investigated the potential of this spectratype approach by comparing the donor T-cell alloresponses generated in vitro against patient peripheral blood lymphocytes (PBLs) with those detected in vivo posttransplantation. The results indicated that for most Vbeta families that exhibited alloreactive CDR3-size skewing, there was a robust overlap between the in vitro antipatient and in vivo spectratype histograms. Thus, in vitro spectratype analysis may be useful for determining the alloreactive T-cell response involved in GVHD development and, thereby, could serve to guide select Vbeta family depletion for designer transplants to improve outcomes.

Prognostic Value of Functional Capacity in Different Exercise Protocols.

Background Functional capacity is associated with mortality, although the prognostic value of achieved estimated metabolic equivalents (METs) across various exercise protocols is not established. We sought to determine whether achieved METs had different prognostic implications according to the protocol employed. Methods and Results From 1991 to 2015, we identified 120 705 consecutive patients from a stress testing registry who underwent the following 7 different standardized exercise protocols: Bruce, modified Bruce, Cornell 0%, Cornell 5%, Cornell 10%, Naughton, and modified Naughton. The primary outcome was all-cause mortality. There were 74 953 Bruce, 8368 modified Bruce, 2648 Cornell 0%, 9972 Cornell 5%, 20 425 Cornell 10%, 1226 Naughton, and 3113 modified Naughton protocols. During a mean follow-up of 8.7 years, a total of 8426 deaths (6.9%) occurred. When compared with the Bruce protocol, after multivariable adjustment for clinical risk factors, medications, and functional capacity, test protocol was independently associated with mortality (modified Naughton [hazard ratio (HR), 2.51; 95% CI, 2.26-2.8], Naughton [HR, 1.79; 95% CI, 1.57-2.04], Cornell 0% [HR, 1.79; 95% CI, 1.59-2.01], modified Bruce [HR, 1.62; 95% CI, 1.48-1.76], Cornell 5% [HR, 1.61; 95% CI, 1.47-1.75], and Cornell 10% [HR, 1.32; 95% CI, 1.22-1.42]). Across all protocols, higher estimated METs were associated with lower mortality, although the equivalent METs achieved were associated with a worse prognosis in less-demanding protocols. Conclusions Higher estimated METs are reliably associated with lower mortality in all exercise protocols, although the prognostic value is not transferable across different tests. Consequently, the prognostic value of METs achieved during a stress test should be considered protocol dependent.