Sudden ST-Segment Changes in a Critically Ill Patient.

A patient in his mid-60s underwent bilateral orthotopic lung transplant and is later hospitalized for sepsis from pneumonia complicated by acute transplant rejection, without any cardiac symptoms. He develops severe acute respiratory distress syndrome requiring intubation and paralysis. Electrocardiography shows sudden ST-segment changes. What would you do next?

Specialties with Few Underrepresented Applicants Lack Diversity Information on Residency Websites.

INTRODUCTION: With the advent of virtual interviews and the increasing accessibility of internet resources, students increasingly rely on program websites for residency application decisions. In this cross-sectional study, we evaluated the presence of diversity or inclusion information in the least diverse US specialties' residency program websites, including dermatology, orthopedic surgery, otolaryngology, plastic surgery, and urology residency programs. METHODS: Two authors independently reviewed each Accreditation Council for Graduate Medical Education-accredited non-military US residency program website and ranked the websites' diversity and inclusion information using six pre-determined criteria based on previous studies in the literature. RESULTS: This study reveals that more than half of residency programs of each specialty met zero of the diversity and inclusion information criteria. CONCLUSIONS: Residency program websites in the least diverse specialties are lacking important information for prospective applicants that may help signal programs' commitment to inclusivity and attract a diverse candidate pool.

Cardiovascular events more than 6 months after pregnancy in patients with congenital heart disease.

OBJECTIVES: Patients with congenital heart disease (CHD) are increasingly pursuing pregnancy, highlighting the need for data on late cardiovascular events (more than 6 months after delivery). We aimed to determine the incidence of late cardiovascular events in postpartum patients with CHD and evaluate the accuracy of the existing risk scores in predicting these events. STUDY DESIGN: We identified patients with CHD who delivered between 2008 and 2020 at a tertiary centre and had follow-up data for greater than 6 months post partum. Late cardiovascular events were defined as heart failure, arrhythmia, thromboembolic events, endocarditis, urgent cardiovascular interventions or death. Survival analysis and Cox proportional model were used to estimate the incidence of late cardiovascular events and determine the hazard ratio of factors associated with these events. RESULTS: Of 117 patients, 19% had 36 late cardiovascular events over a median follow-up of 3.8 years. Annual incidence of any late cardiovascular event was 5.7%. Hazards of late cardiovascular events were significantly higher among those with higher Cardiac Disease in Pregnancy Study (CARPREG) II and Zwangerschap bij Aangeboren HARtAfwijking-Pregnancy in Women With Congenital Heart Disease (ZAHARA) risk scores and among patients with prepregnancy New York Heart Association class≥II. C-statistic to predict the late cardiovascular events was highest for ZAHARA (0.7823), followed by CARPREG II (0.6902) and prepregnancy New York Heart Association class≥ II (0.6677). CONCLUSIONS: Currently available risk tools designed for prognostication during the peripartum period can also be used to determine risks of late maternal cardiovascular events among those with CHD. These findings provide important new information for counselling and risk modification.

Identification of nurse shark VNAR single-domain antibodies targeting the spike S2 subunit of SARS-CoV-2.

SARS-CoV-2 is the etiological agent of the COVID-19 pandemic. Antibody-based therapeutics targeting the spike protein, specifically the S1 subunit or the receptor binding domain (RBD) of SARS-CoV-2, have gained attention due to their clinical efficacy in treating patients diagnosed with COVID-19. An alternative to conventional antibody therapeutics is the use of shark new antigen variable receptor domain (VNAR ) antibodies. VNAR s are small (<15 kDa) and can reach deep into the pockets or grooves of the target antigen. Here, we have isolated 53 VNAR s that bind to the S2 subunit by phage panning from a naïve nurse shark VNAR phage display library constructed in our laboratory. Among those binders, S2A9 showed the best neutralization activity against the original pseudotyped SARS-CoV-2 virus. Several binders, including S2A9, showed cross-reactivity against S2 subunits from other ß coronaviruses. Furthermore, S2A9 showed neutralization activity against all variants of concern (VOCs) from alpha to omicron (including BA1, BA2, BA4, and BA5) in both pseudovirus and live virus neutralization assays. Our findings suggest that S2A9 could be a promising lead molecule for the development of broadly neutralizing antibodies against SARS-CoV-2 and emerging variants. The nurse shark VNAR phage library offers a novel platform that can be used to rapidly isolate single-domain antibodies against emerging viral pathogens.

Site-Specifically Conjugated Single-Domain Antibody Successfully Identifies Glypican-3-Expressing Liver Cancer by Immuno-PET.

Primary liver cancer is the third leading cause of cancer-related deaths, and its incidence and mortality are increasing worldwide. Hepatocellular carcinoma (HCC) accounts for 80% of primary liver cancer cases. Glypican-3 (GPC3) is a heparan sulfate proteoglycan that histopathologically defines HCC and represents an attractive tumor-selective marker for radiopharmaceutical imaging and therapy for this disease. Single-domain antibodies are a promising scaffold for imaging because of their favorable pharmacokinetic properties, good tumor penetration, and renal clearance. Although conventional lysine-directed bioconjugation can be used to yield conjugates for radiolabeling full-length antibodies, this stochastic approach risks negatively affecting target binding of the smaller single-domain antibodies. To address this challenge, site-specific approaches have been explored. Here, we used conventional and sortase-based site-specific conjugation methods to engineer GPC3-specific human single-domain antibody (HN3) PET probes. Methods: Bifunctional deferoxamine (DFO) isothiocyanate was used to synthesize native HN3 (nHN3)-DFO. Site-specifically modified HN3 (ssHN3)-DFO was engineered using sortase-mediated conjugation of triglycine-DFO chelator and HN3 containing an LPETG C-terminal tag. Both conjugates were radiolabeled with 89Zr, and their binding affinity in vitro and target engagement of GPC3-positive (GPC3+) tumors in vivo were determined. Results: Both 89Zr-ssHN3 and 89Zr-nHN3 displayed nanomolar affinity for GPC3 in vitro. Biodistribution and PET/CT image analysis in mice bearing isogenic A431 and A431-GPC3+ xenografts, as well as in HepG2 liver cancer xenografts, showed that both conjugates specifically identify GPC3+ tumors. 89Zr-ssHN3 exhibited more favorable biodistribution and pharmacokinetic properties, including higher tumor uptake and lower liver accumulation. Comparative PET/CT studies on mice imaged with both 18F-FDG and 89Zr-ssHN3 showed more consistent tumor accumulation for the single-domain antibody conjugate, further establishing its potential for PET imaging. Conclusion: 89Zr-ssHN3 showed clear advantages in tumor uptake and tumor-to-liver signal ratio over the conventionally modified 89Zr-nHN3 in xenograft models. Our results establish the potential of HN3-based single-domain antibody probes for GPC3-directed PET imaging of liver cancers.

Humanization of the Shark VNAR Single Domain Antibody Using CDR Grafting.

The variable domain of the new antigen receptor (VNAR ) of shark single domain antibodies is evolutionarily distant from the variable regions (VH ) of mammalian immunoglobulins, yet it still has complementarity-determining regions (CDRs) that are involved in antigen recognition, therefore making it possible to humanize by grafting these CDRs to the framework of human VH homologs. Here, we show the VNAR CDR based on an analysis of currently available VNAR -antigen structure complexes in the global Protein Data Bank archive of 3D structure data, and describe the detailed protocol to humanize VNAR by CDR grafting, using B6 (an anti-Pseudomonas exotoxin VNAR ), the most common type (Type II) of shark VNAR s, as an example. Ongoing efforts will further optimize the protocol for moving shark VNAR s to the clinic for treating cancer and other human diseases. Published 2023. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol: Humanize shark VNAR sequence by CDR grafting Support Protocol 1: VNAR structure prediction and comparison Support Protocol 2: Measure binding kinetics of humanized VNAR using bio-layer interferometry (BLI).

Million spot binding array platform for exploring and optimizing multiple simultaneous detection events.

Large-scale, high-throughput specificity assays to characterize binding properties within a competitive and complex environment of potential binder-target pairs remain challenging and cost prohibitive. Barcode cycle sequencing (BCS) is a molecular binding assay for proteins, peptides, and other small molecules that is built on a next-generation sequencing (NGS) chip. BCS uses a binder library and targets labeled with unique DNA barcodes. Upon binding, binder barcodes are ligated to target barcodes and sequenced to identify encoded binding events. For complete details on the use and execution of this protocol, please refer to Hong et al. (2022).

Target-switch SELEX: Screening with alternating targets to generate aptamers to conserved terminal dipeptides.

Systematic evolution of ligands by exponential enrichment (SELEX) encompasses a wide variety of high-throughput screening techniques for producing nucleic acid binders to molecular targets through directed evolution. We describe here the design and selection steps for discovery of DNA aptamers with specificity for the two consecutive N-terminal amino acids (AAs) of a small peptide (8-10 amino acids). This bead-based method may be adapted for applications requiring binders which recognize a specific portion of the desired target. For complete details on the use and execution of this protocol, please refer to Hong et al. (2022).

Immunotherapy for hepatobiliary cancers: Emerging targets and translational advances.

Over the past several decades, primary liver cancer (PLC), mostly hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (iCCA), has become the focus of rising concern mainly due to the increasing rates of incidence and high global mortality. Immunotherapy, as an emerging treatment approach, represents an effective and promising option against PLC. However, the selection of immunotherapeutic targets while considering tumor heterogeneity and immunosuppressive tumor microenvironment is a major challenge. The purpose of this review is to summarize and present the emerging immunotherapeutic targets for HCC and iCCA and to evaluate their translation advances in currently ongoing clinical trials. To better provide a framework for the liver cancer target selection, this chapter will highlight cell surface antigens expressed in both tumor cells and immune cells. Particular focus will be on the development, biology and function of Glypican-3 (GPC3) and Mesothelin (MSLN) in the cancer progress of HCC and iCCA, respectively. By doing so, we will explore the prospects and applications of various immunotherapeutic strategies such as vaccines, monoclonal antibodies, immunotoxins, antibody-drug conjugates (ADCs) and chimeric antigen receptors (CARs) T cells that have been developed targeting GPC3 and MSLN.

Production and Purification of Shark and Camel Single-Domain Antibodies from Bacterial and Mammalian Cell Expression Systems.

Single-domain antibodies, including the antigen-binding variable domains of the shark immunoglobulin new antigen receptor and the camelid variable region of the heavy chain, are the smallest antigen recognition domains (∼15 kDa) and have unique characteristics compared to conventional antibodies. They are capable of binding epitopes that are hard to access for classical antibodies and can also be used for therapeutics or diagnostics or as modular building blocks for multi-domain constructs, antibody-drug conjugates, immunotoxins, or chimeric antigen receptor therapy. This article contains detailed procedures for the purification and validation of two single-domain antibodies (one shark and one camel), which bind to the S2 subunit of the SARS-CoV-2 spike protein, using both bacterial and mammalian cell expression systems. It provides a comprehensive reference for the production of single-domain antibodies with high yield, good quality, and purity. © Published 2022. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol: Production of single-domain antibodies from Escherichia coli Alternate Protocol: Production of single-domain antibodies using the mammalian cell line Expi293F Support Protocol 1: Production and purification of single-domain antibodies on a small scale with the polymyxin B method Support Protocol 2: Validation of single-domain antibodies by ELISA.

Development of Highly Effective Anti-Mesothelin hYP218 Chimeric Antigen Receptor T Cells With Increased Tumor Infiltration and Persistence for Treating Solid Tumors.

Mesothelin targeting CAR T cells have limited activity in patients. In this study, we sought to determine if efficacy of anti-mesothelin CAR T cells is dependent on the mesothelin epitopes that are recognized by them. To do so, we developed hYP218 (against membrane-proximal epitope) and SS1 (against membrane-distal epitope) CAR T cells. Their efficacy was assessed in vitro using mesothelin-positive tumor cell lines and in vivo in NSG mice with mesothelin-expressing ovarian cancer (OVCAR-8), pancreatic cancer (KLM-1), and mesothelioma patient-derived (NCI-Meso63) tumor xenografts. Persistence and tumor infiltration of CAR T cells was determined using flow cytometry. hYP218 CAR T cells killed cancer cells more efficiently than SS1 CAR T cells, with a two- to fourfold lower ET50 value (effector-to-target ratio for 50% killing of tumor cells). In mice with established tumors, single intravenous administration of hYP218 CAR T cells lead to improved tumor response and survival compared with SS1 CAR T cells, with complete regression of OVCAR-8 and NCI-Meso63 tumors. Compared with SS1 CAR T cells, there was increased peripheral blood expansion, persistence, and tumor infiltration of hYP218 CAR T cells in the KLM-1 tumor model. Persistence of hYP218 CAR T cells in treated mice led to antitumor immunity when rechallenged with KLM-1 tumor cells. Our results show that hYP218 CAR T cells, targeting mesothelin epitope close to cell membrane, are very effective against mesothelin-positive tumors and are associated with increased persistence and tumor infiltration. These results support its clinical development to treat patients with mesothelin-expressing cancers.

Dromedary camel nanobodies broadly neutralize SARS-CoV-2 variants.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike is a trimer of S1/S2 heterodimers with three receptor-binding domains (RBDs) at the S1 subunit for human angiotensin-converting enzyme 2 (hACE2). Due to their small size, nanobodies can recognize protein cavities that are not accessible to conventional antibodies. To isolate high-affinity nanobodies, large libraries with great diversity are highly desirable. Dromedary camels (Camelus dromedarius) are natural reservoirs of coronaviruses like Middle East respiratory syndrome CoV (MERS-CoV) that are transmitted to humans. Here, we built large dromedary camel VHH phage libraries to isolate nanobodies that broadly neutralize SARS-CoV-2 variants. We isolated two VHH nanobodies, NCI-CoV-7A3 (7A3) and NCI-CoV-8A2 (8A2), which have a high affinity for the RBD via targeting nonoverlapping epitopes and show broad neutralization activity against SARS-CoV-2 and its emerging variants of concern. Cryoelectron microscopy (cryo-EM) complex structures revealed that 8A2 binds the RBD in its up mode with a long CDR3 loop directly involved in the ACE2 binding residues and that 7A3 targets a deeply buried region that uniquely extends from the S1 subunit to the apex of the S2 subunit regardless of the conformational state of the RBD. At a dose of ≥5 mg/kg, 7A3 efficiently protected transgenic mice expressing hACE2 from the lethal challenge of variants B.1.351 or B.1.617.2, suggesting its therapeutic use against COVID-19 variants. The dromedary camel VHH phage libraries could be helpful as a unique platform ready for quickly isolating potent nanobodies against future emerging viruses.

A novel PD-L1-targeted shark VNAR single-domain-based CAR-T cell strategy for treating breast cancer and liver cancer.

Chimeric antigen receptor (CAR)-T cell therapy shows excellent potency against hematological malignancies, but it remains challenging to treat solid tumors, mainly because of a lack of appropriate antigenic targets and an immunosuppressive tumor microenvironment (TME). The checkpoint molecule programmed death-ligand 1 (PD-L1) is widely overexpressed in multiple tumor types, and the programmed death-ligand 1 (PD-1)/PD-L1 interaction is a crucial mediator of immunosuppression in the TME. Here we constructed a semi-synthetic shark VNAR phage library and isolated anti-PD-L1 single-domain antibodies. Among these VNARs, B2 showed cross-reactivity to human, mouse, and canine PD-L1, and it partially blocked the interaction of human PD-1 with PD-L1. CAR (B2) T cells specifically lysed human breast cancer and liver cancer cells by targeting constitutive and inducible expression of PD-L1 and hindered tumor metastasis. Combination of PD-L1 CAR (B2) T cells with CAR T cells targeted by GPC3 (a liver cancer-specific antigen) regresses liver tumors in mice. We concluded that PD-L1-targeted shark VNAR single-domain-based CAR-T therapy is a novel strategy to treat breast and liver cancer. This study provides a rationale for potential use of PD-L1 CAR-T cells as a monotherapy or in combination with a tumor-specific therapy in clinical studies.

ProtSeq: Toward high-throughput, single-molecule protein sequencing via amino acid conversion into DNA barcodes.

We demonstrate early progress toward constructing a high-throughput, single-molecule protein sequencing technology utilizing barcoded DNA aptamers (binders) to recognize terminal amino acids of peptides (targets) tethered on a next-generation sequencing chip. DNA binders deposit unique, amino acid-identifying barcodes on the chip. The end goal is that, over multiple binding cycles, a sequential chain of DNA barcodes will identify the amino acid sequence of a peptide. Toward this, we demonstrate successful target identification with two sets of target-binder pairs: DNA-DNA and Peptide-Protein. For DNA-DNA binding, we show assembly and sequencing of DNA barcodes over six consecutive binding cycles. Intriguingly, our computational simulation predicts that a small set of semi-selective DNA binders offers significant coverage of the human proteome. Toward this end, we introduce a binder discovery pipeline that ultimately could merge with the chip assay into a technology called ProtSeq, for future high-throughput, single-molecule protein sequencing.

Camel nanobodies broadly neutralize SARS-CoV-2 variants.

With the emergence of SARS-CoV-2 variants, there is urgent need to develop broadly neutralizing antibodies. Here, we isolate two V H H nanobodies (7A3 and 8A2) from dromedary camels by phage display, which have high affinity for the receptor-binding domain (RBD) and broad neutralization activities against SARS-CoV-2 and its emerging variants. Cryo-EM complex structures reveal that 8A2 binds the RBD in its up mode and 7A3 inhibits receptor binding by uniquely targeting a highly conserved and deeply buried site in the spike regardless of the RBD conformational state. 7A3 at a dose of ≥5 mg/kg efficiently protects K18-hACE2 transgenic mice from the lethal challenge of B.1.351 or B.1.617.2, suggesting that the nanobody has promising therapeutic potentials to curb the COVID-19 surge with emerging SARS-CoV-2 variants. ONE-SENTENCE SUMMARY: Dromedary camel ( Camelus dromedarius ) V H H phage libraries were built for isolation of the nanobodies that broadly neutralize SARS-CoV-2 variants.

Fe-Ion Bolted VOPO4 ∙2H2 O as an Aqueous Fe-Ion Battery Electrode.

Iron ion batteries using Fe2+ as a charge carrier have yet to be widely explored, and they lack high-performing Fe2+ hosting cathode materials to couple with the iron metal anode. Here, it is demonstrated that VOPO4 ∙2H2 O can reversibly host Fe2+ with a high specific capacity of 100 mAh g-1 and stable cycling performance, where 68% of the initial capacity is retained over 800 cycles. In sharp contrast, VOPO4 ∙2H2 O's capacity of hosting Zn2+ fades precipitously over tens of cycles. VOPO4 ∙2H2 O stores Fe2+ with a unique mechanism, where upon contacting the electrolyte by the VOPO4 ∙2H2 O electrode, Fe2+ ions from the electrolyte get oxidized to Fe3+ ions that are inserted and trapped in the VOPO4 ∙2H2 O structure in an electroless redox reaction. The trapped Fe3+ ions, thus, bolt the layered structure of VOPO4 ∙2H2 O, which prevents it from dissolution into the electrolyte during (de)insertion of Fe2+ . The findings offer a new strategy to use a redox-active ion charge carrier to stabilize the layered electrode materials.

VITT following Ad26.COV2.S vaccination presenting without radiographically demonstrable thrombosis.

We report a case of vaccine-induced immune thrombotic thrombocytopenia (VITT) in a young man diagnosed 13 days after Ad26.COV2.S COVID-19 (Johnson & Johnson/Janssen) vaccination. He presented to us with 5 days of progressive left leg pain, thrombocytopenia, hypofibrinogenemia, and markedly elevated d-dimers, but without radiographically demonstrable thrombosis. Despite negative imaging, we initiated treatment of presumptive VITT given the striking clinical picture that included the timing of his recent adenovirus-based COVID-19 vaccine, leg symptoms, marked thrombocytopenia, and consumptive coagulopathy. He received intravenous immune globulin, prednisone, and argatroban and was discharged 7 days later much improved. His positive platelet factor 4 enzyme-linked immunosorbent assay antibody test returned after treatment was initiated. To our knowledge, this is the first reported case of VITT following Ad26.COV2.S vaccination presenting without radiographically demonstrable thrombosis. Our patient highlights the importance of knowing vaccine status and initiating treatment as soon as possible in the right clinical setting, even in the absence of radiographic evidence of thrombus. Early VITT recognition and treatment provide an opportunity to prevent serious thrombotic complications.

Hypothalamic primary cilium: A hub for metabolic homeostasis.

Obesity is a global health problem that is associated with adverse consequences such as the development of metabolic disorders, including cardiovascular disease, neurodegenerative disorders, and type 2 diabetes. A major cause of obesity is metabolic imbalance, which results from insufficient physical activity and excess energy intake. Understanding the pathogenesis of obesity, as well as other metabolic disorders, is important in the development of methods for prevention and therapy. The coordination of energy balance takes place in the hypothalamus, a major brain region that maintains body homeostasis. The primary cilium is an organelle that has recently received attention because of its role in controlling energy balance in the hypothalamus. Defects in proteins required for ciliary function and formation, both in humans and in mice, have been shown to cause various metabolic disorders. In this review, we provide an overview of the critical functions of primary cilia, particularly in hypothalamic areas, and briefly summarize the studies on the primary roles of cilia in specific neurons relating to metabolic homeostasis.