Surface salt bridges contribute to the extreme thermal stability of an FN3-like domain from a thermophilic bacterium.

This study uses differential scanning calorimetry, X-ray crystallography, and molecular dynamics simulations to investigate the structural basis for the high thermal stability (melting temperature 97.5°C) of a FN3-like protein domain from thermophilic bacteria Thermoanaerobacter tengcongensis (FN3tt). FN3tt adopts a typical FN3 fold with a three-stranded beta sheet packing against a four-stranded beta sheet. We identified three solvent exposed arginine residues (R23, R25, and R72), which stabilize the protein through salt bridge interactions with glutamic acid residues on adjacent strands. Alanine mutation of the three arginine residues reduced melting temperature by up to 22°C. Crystal structures of the wild type (WT) and a thermally destabilized (∆Tm -19.7°C) triple mutant (R23L/R25T/R72I) were found to be nearly identical, suggesting that the destabilization is due to interactions of the arginine residues. Molecular dynamics simulations showed that the salt bridge interactions in the WT were stable and provided a dynamical explanation for the cooperativity observed between R23 and R25 based on calorimetry measurements. In addition, folding free energy changes computed using free energy perturbation molecular dynamics simulations showed high correlation with melting temperature changes. This work is another example of surface salt bridges contributing to the enhanced thermal stability of thermophilic proteins. The molecular dynamics simulation methods employed in this study may be broadly useful for in silico surface charge engineering of proteins.

Exome Sequencing of a Clinical Population for Autosomal Dominant Polycystic Kidney Disease.

Importance: Most studies of autosomal dominant polycystic kidney disease (ADPKD) genetics have used kidney specialty cohorts, focusing on PKD1 and PKD2. These can lead to biased estimates of population prevalence of ADPKD-associated gene variants and their phenotypic expression. Objective: To determine the prevalence of ADPKD and contributions of PKD1, PKD2, and other genes related to cystic kidney disease in a large, unselected cohort. Design, Setting, and Participants: This retrospective observational study used an unselected health system-based cohort in central and northeast Pennsylvania with exome sequencing (enrolled from 2004 to 2020) and electronic health record data (up to October 2021). The genotype-first approach included the entire cohort and the phenotype-first approach focused on patients with ADPKD diagnosis codes, confirmed by chart and imaging review. Exposures: Loss-of-function (LOF) variants in PKD1, PKD2, and other genes associated with cystic kidney disease (ie, ALG8, ALG9, DNAJB11, GANAB, HNF1B, IFT140, SEC61B, PKHD1, PRKCSH, SEC63); likely pathogenic missense variants in PKD1 and PKD2. Main Outcomes and Measures: Genotype-first analysis: ADPKD diagnosis code (Q61.2, Q61.3, 753.13, 753.12); phenotype-first analysis: presence of a rare variant in PKD1, PKD2, or other genes associated with cystic kidney disease. Results: Of 174 172 patients (median age, 60 years; 60.6% female; 93% of European ancestry), 303 patients had ADPKD diagnosis codes, including 235 with sufficient chart review data for confirmation. In addition to PKD1 and PKD2, LOF variants in IFT140, GANAB, and HNF1B were associated with ADPKD diagnosis after correction for multiple comparisons. Among patients with LOF variants in PKD1, 66 of 68 (97%) had ADPKD; 43 of 43 patients (100%) with LOF variants in PKD2 had ADPKD. In contrast, only 24 of 77 patients (31.2%) with a PKD1 missense variant previously classified as "likely pathogenic" had ADPKD, suggesting misclassification or variable penetrance. Among patients with ADPKD diagnosis confirmed by chart review, 180 of 235 (76.6%) had a potential genetic cause, with the majority being rare variants in PKD1 (127 patients) or PKD2 (34 patients); 19 of 235 (8.1%) had variants in other genes associated with cystic kidney disease. Of these 235 patients with confirmed ADPKD, 150 (63.8%) had a family history of ADPKD. The yield for a genetic determinant of ADPKD was higher for those with a family history of ADPKD compared with those without family history (91.3% [137/150] vs 50.6% [43/85]; difference, 40.7% [95% CI, 29.2%-52.3%]; P < .001). Previously unreported PKD1, PKD2, and GANAB variants were identified with pedigree data suggesting pathogenicity, and several PKD1 missense variants previously reported as likely pathogenic appeared to be benign. Conclusions and Relevance: This study demonstrates substantial genetic and phenotypic variability in ADPKD among patients within a regional health system in the US.

The Scientific Consensus on When a Human's Life Begins.

Peer-reviewed journals in the biological and life sciences literature have published articles that represent the biological view that a human's life begins at fertilization ("the fertilization view"). As those statements are typically offered without explanation or citation, the fertilization view seems to be uncontested by the editors, reviewers, and authors who contribute to scientific journals. However, Americans are split on whether the fertilization view is a "philosophical or religious belief" (45%) or a "biological and scientific fact" (46%), and only 38% of Americans view fertilization as the starting point of a human's life. In the two studies that explored experts' views on the matter, the fertilization view was the most popular perspective held by public health and IVF professionals. Since a recent study suggested that 80% of Americans view biologists as the group most qualified to determine when a human's life begins, experts in biology were surveyed to provide a new perspective to the literature on experts' views on this matter. Biologists from 1,058 academic institutions around the world assessed survey items on when a human's life begins and, overall, 96% (5337 out of 5577) affirmed the fertilization view. The founding principles of the field Science Communication suggest that scientists have an ethical and professional obligation to inform Americans, as well as people around the world, about scientific developments so members of the public can be empowered to make life decisions that are consistent with the best information available. Given that perspective-and a recent study's finding that a majority of Americans believe they deserve to know when a human's life begins in order to make informed reproductive decisions-science communicators should work to increase the level of science awareness on the fertilization view, as it stands alone as the leading biological perspective on when a human's life begins.

General Anesthesia Versus Monitored Anesthesia Care for Transfemoral Transcatheter Aortic Valve Implantation: A Retrospective Study in a Single Belgian Referral Center.

OBJECTIVE: The objective of this study was to compare the effect of general anesthesia (GA) versus monitored anesthesia care (MAC) on post-interventional outcomes in patients undergoing transfemoral transcatheter aortic valve implantation (TAVI). DESIGN: Retrospective before-after study using a quasi-experimental design. SETTING: University Hospitals Leuven, a single Belgian referral center. PARTICIPANTS: Patients undergoing transfemoral TAVI from March 1, 2014, to December 31, 2017, were included. INTERVENTIONS: GA was switched to MAC at the authors' institution for transfemoral TAVI in April 2016, and outcomes of patients who received GA were compared with those who received MAC. MEASUREMENTS AND MAIN RESULTS: The primary outcomes were length of stay in the intensive care unit and hospital. Secondary outcomes included standardized endpoints for TAVI trials as suggested by the Valve Academic Research Consortium, the incidence of vasopressor use, maximum dose of vasopressors, procedural time, and total anesthesia time. In the period studied, 178 patients underwent transfemoral TAVI (GA: n = 85; MAC: n = 93). Implementation of MAC was associated with a significant reduction in total length of hospital stay (7.6 [6.05-9.15] v 5.48 [4.58-6.38] d; p = 0.02); vasopressor requirements; and procedural and total anesthesia time. Length of intensive care unit stay and Valve Academic Research Consortium endpoints were not significantly different. CONCLUSIONS: In the authors' institution, implementation of MAC for patients undergoing transfemoral TAVI resulted in a significant reduction of hospital length of stay and increased operating room efficiency. Results could be confounded by increasing operator experience and improved implantation techniques.

Constellation recovery and impairment evaluation through minimization of the blind EVM.

We present a new method, blind EVM minimization, for constellation recovery and transmitter impairment evaluation of dual polarization optical signals with complex modulation formats. Using simulated data, for which transmitter impairments are known exactly, the method is shown to be accurate and robust. In addition, the method is successfully tested on measured QPSK and QAM16 data. Because of its relatively long run-time, the method might best be used for defining and measuring transmitter impairments and for judging the performance of faster constellation recovery methods that rely on serial parameter evaluation rather than optimization.

Induced conformational change in human IL-4 upon binding of a signal-neutralizing DARPin.

The crystal structure of DARPin 44C12V5 that neutralizes IL-4 signaling has been determined alone and bound to human IL-4. A significant conformational change occurs in the IL-4 upon DARPin binding. The DARPin binds to the face of IL-4 formed by the A and C α-helices. The structure of the DARPin remains virtually unchanged. The conformational changes in IL-4 include a reorientation of the C-helix Trp91 side chain and repositioning of CD-loop residue Leu96. Both side chains move by >9 Å, becoming buried in the central hydrophobic region of the IL-4:DARPin interface. This hydrophobic region is surrounded by a ring of hydrophilic interactions comprised of hydrogen bonds and salt bridges and represents a classical "hotspot." The structures also reveal how the DARPin neutralizes IL-4 signaling. Comparing the IL-4:DARPin complex structure with the structures of IL-4 bound to its receptors (Hage et al., Cell 1999; 97, 271-281; La Porte et al., Cell 2008, 132, 259-272), it is found that the DARPin binds to the same IL-4 face that interacts with the junction of the D1 and D2 domains of the IL-4Rα receptors. Signaling is blocked since IL-4 cannot bind to this receptor, which it must do first before initiating a productive receptor complex with either the IL-13α1 or the γc receptor.

C-terminal ß-strand swapping in a consensus-derived fibronectin Type III scaffold.

The crystal structures of six different fibronectin Type III consensus-derived Tencon domains, whose solution properties exhibit no, to various degrees of, aggregation according to SEC, have been determined. The structures of the five variants showing aggregation reveal 3D domain swapped dimers. In all five cases, the swapping involves the C-terminal ß-strand resulting in the formation of Tencon dimers in which the target-binding surface is blocked. All of the variants differ in sequence in the FG loop, which is the hinge loop in the ß-strand-swapped dimers. The six tencon variants have between 0 and 5 residues inserted between positions 77 and 78 in the FG loop. Analysis of the structures suggests that a non-glycine residue at position 77 and insertions of <4 residues may destabilize the ß-turn in the FG loop promoting ß-strand swapping. Swapped dimers with an odd number of inserted residues may be less stable, particularly if they contain proline residues, because they cannot form perfect ß-bridges in the FG regions that link the swapped dimers. The Tencon ß-swapped variants with the longest FG sequences are observed to form higher order hexameric or helical oligomeric structures in the crystal correlating well with the aggregation properties of these domains observed in solution. Understanding the structural basis for domain-swapped dimerization and oligomerization will support engineering efforts of the Tencon domain to produce variants with desired biophysical properties.

N-terminal ß-strand swapping in a consensus-derived alternative scaffold driven by stabilizing hydrophobic interactions.

The crystal structure of an N-terminal ß-strand-swapped consensus-derived tenascin FN3 alternative scaffold has been determined. A comparison with the unswapped structure reveals that the side chain of residue F88 orients differently and packs more tightly with the hydrophobic core of the domain. Dimer formation also results in the burial of a hydrophobic patch on the surface of the domain. Thus, it appears that tighter packing of F88 in the hydrophobic core and burial of surface hydrophobicity provide the driving forces for the N-terminal ß-strand swapping, leading to the formation of a stable compact dimer.

A ruptured omental aneurysm, a rare cause of intraabdominal bleeding.

Of all splanchnic artery aneurysms, the omental artery aneurysm is the most uncommon. In the published literature, there is no report of an incidental diagnosis, and all were discovered only after rupture. Until now, this is only the sixth case of a ruptured omental aneurysm published, and the first case with a concomitant nonruptured omental aneurysm.

Reproduction of continuous flow left ventricular assist device experimental data by means of a hybrid cardiovascular model with baroreflex control.

Long-term mechanical circulatory assistance opened new problems in ventricular assist device-patient interaction, especially in relation to autonomic controls. Modeling studies, based on adequate models, could be a feasible approach of investigation. The aim of this work is the exploitation of a hybrid (hydronumerical) cardiovascular simulator to reproduce and analyze in vivo experimental data acquired during a continuous flow left ventricular assistance. The hybrid cardiovascular simulator embeds three submodels: a computational cardiovascular submodel, a computational baroreflex submodel, and a hydronumerical interface submodel. The last one comprises two impedance transformers playing the role of physical interfaces able to provide a hydraulic connection with specific cardiovascular sites (in this article, the left atrium and the ascending/descending aorta). The impedance transformers are used to connect a continuous flow pump for partial left ventricular support (Synergy Micropump, CircuLite, Inc., Saddlebrooke, NJ, USA) to the hybrid cardiovascular simulator. Data collected from five animals in physiological, pathological, and assisted conditions were reproduced using the hybrid cardiovascular simulator. All parameters useful to characterize and tune the hybrid cardiovascular simulator to a specific hemodynamic condition were extracted from experimental data. Results show that the simulator is able to reproduce animal-specific hemodynamic status both in physiological and pathological conditions, to reproduce cardiovascular left ventricular assist device (LVAD) interaction and the progressive unloading of the left ventricle for different pump speeds, and to investigate the effects of the LVAD on baroreflex activity. Results in chronic heart failure conditions show that an increment of LVAD speed from 20 000 to 22 000 rpm provokes a decrement of left ventricular flow of 35% (from 2 to 1.3 L/min). Thanks to its flexibility and modular structure, the simulator is a platform potentially useful to test different assist devices, thus providing clinicians additional information about LVAD therapy strategy.

Transcatheter aortic valve implantation versus sutureless aortic valve replacement: a single-centre cost analysis.

AIMS: Sutureless aortic valve replacement (SU-AVR) and transcatheter aortic valve implantation (TAVI) are both viable therapeutic interventions for aortic stenosis in elderly patients. Meta-analyses show similar all-cause mortality for both techniques albeit with a different pattern of adverse effects. This study means to compare costs and, to a lesser extent, clinical outcomes of both techniques. METHODS: A retrospective single-centre analysis was performed for patients receiving SU-AVR or TAVI from 2008 to 2019. Perioperative clinical data were collected from patient files. Costs were assessed by a cost allocation tool. In an attempt to avoid confounding, propensity score matching was carried out. RESULTS: A total of 368 patients underwent either TAVI (n = 100) or SU-AVR (n = 268). After matching, there were 61 patients per treatment group. Length of stay was significantly longer in the SU-AVR group. Excluding device costs, total expenses for SU-AVR (median: €11,630) were significantly higher than TAVI (median: €9240). For both groups, these costs were mostly incurred on intensive care units, followed by nursing units. Non-medical staff was the largest contributor to expenses. Including device costs, SU-AVR (median: €14,683) was shown to be cost-saving compared to TAVI (median: €24,057). CONCLUSIONS: To conclude, we found SU-AVR to be cost-saving compared to TAVI, largely due to higher device costs associated with the latter. Excluding device costs, TAVI was associated with lower expenses and shorter length of stay. Non-medical staff was the largest source of costs, suggesting length of stay to be a major financial determinant.

Robot-Assisted MIDCAB Using Bilateral Internal Thoracic Artery: A Propensity Score-Matched Study With OPCAB Patients.

OBJECTIVE: Robot-assisted minimally invasive direct coronary artery bypass (RA-MIDCAB) is an attractive strategy for coronary revascularization. Growing evidence supports the use of total arterial grafting in coronary surgery. We evaluated total arterial left-sided coronary revascularization with bilateral internal thoracic artery (BITA) using RA-MIDCAB and compared it with a propensity score-matched (PSM) off-pump CAB (OPCAB) surgery population. METHODS: We retrospectively included all isolated OPCAB and RA-MIDCAB surgery using BITA without saphenous vein graft from January 1, 2015, to October 31, 2022. We analyzed all our RA-MIDCAB patients and performed PSM to compare them with our OPCAB population. Primary outcomes were major adverse cardiovascular and cerebrovascular events (MACCE) and mortality. Secondary outcomes were surgical parameters, length of hospital stay, and learning curve. RESULTS: We included 601 OPCAB and 77 RA-MIDCAB procedures, which resulted in 2 cohorts of 54 patients after PSM. Mortality and MACCE survival analysis showed no significant difference. There was less blood transfusion in the RA-MIDCAB (16.7%) compared with the OPCAB group (38.9%; P = 0.02). We observed fewer intensive care unit (ICU) admissions (24.1% vs 96.6%), shorter ICU stay (0.78 ± 1.7 vs 1.91 ± 1.01 days), and shorter hospital stay (6.78 ± 2.4 vs 8.01 ± 2.5 days) in the RA-MIDCAB versus OPCAB group (P < 0.01). Surgery time decreased from 400.0 ± 70.8 to 325.0 ± 38.0 min with more experience in RA-MIDCAB BITA harvesting (P < 0.01). CONCLUSIONS: This is a first publication of 77 consecutive RA-MIDCAB BITA harvesting for left coronary artery system revascularization. This technique is safe in terms of MACCE and mortality. Additional advantages are shorter length of hospital stay, fewer ICU admissions, and less blood transfusion.

Coronary Physiological Indices to Evaluate Myocardial Ischemia in Patients with Aortic Stenosis Undergoing Valve Replacement.

BACKGROUND: Evaluation of myocardial ischemia in patients with aortic valve stenosis (AS) with concomitant coronary artery disease (CAD) and possible microvascular dysfunction (MVD) is challenging because fractional flow reserve (FFR) and resting full-cycle ratio (RFR) have not been validated in this clinical setting. OBJECTIVES: The objectives of this study in patients with AS and CAD are: 1. to describe the relationship between hyperemic and resting indices. 2. to investigate the acute and long-term effects of aortic valve replacement (AVR) on epicardial indices and microvascular function. 3. To assess the impact of these changes on clinical decision making. 4. To determine FFR/RFR ischemia cut-off points in AS. METHODS: In this prospective multicentric study, we performed serial measurements of FFR, RFR, and evaluated MVD by means of coronary flow reserve (CFR), the index of microvascular resistance (IMR) and microvascular resistance reserve (MRR) in patients with severe AS and intermediate-to-severe CAD, before and six months after AVR. Patients underwent myocardial perfusion single-photon emission computed tomography (SPECT) before AVR. RESULTS: In total, 146 coronary lesions in 116 patients were included. Before AVR, we observed high FFR/RFR discordance according to standard cut-off values: FFR-negative (>0.80)/RFR-positive (≤0.89) in 42.3% (68/137) of these lesions. Acutely after AVR, FFR decreased significantly (-0.0120 ± 0.0192, p=0.0045) while RFR remained stable (0.0140 ± 0.0673, p=0.3089). Six months after AVR, FFR decreased (-0.0279±0.0368) while RFR increased significantly (+0.0410±0.0487) (p<0.0001 for both), resulting in 21.5% (21/98) and 39.8% (39/98) of lesions crossing traditional FFR and RFR cut-off lines, respectively. LV-mass decreased significantly (153.68g ± 44.22 before vs 134.66g ± 37.26 after, p<0.0001). MVD was frequently observed at baseline (32.1% abnormal IMR; 68.6% abnormal MRR) with all microvascular parameters improving after AVR. Most accurate cut-offs to predict ischemia were FFR ≤0.83 and RFR ≤0.85 with comparable accuracy (75-80%). CONCLUSIONS: In patients with severe AS and CAD, FFR ≤0.83 and RFR ≤0.85 appear to predict myocardial ischemia more accurately. Six months after AVR, FFR decreases while RFR increases significantly, with simultaneous decrease of LV mass and improvement of microvascular function.

The Impact of Acute Exercise on Hemostasis and Angiogenesis Mediators in Patients With Continuous-Flow Left Ventricular Assist Devices: A Prospective Observational Pilot Study.

Impaired primary hemostasis and dysregulated angiogenesis, known as a two-hit hypothesis, are associated with gastrointestinal (GI) bleeding in patients with continuous-flow left ventricular assist devices (CF-LVADs). Exercise is known to influence hemostasis and angiogenesis in healthy individuals; however, little is known about the effect in patients with CF-LVADs. The objective of this prospective observational study was to determine whether acute exercise modulates two-hit hypothesis mediators associated with GI bleeding in patients with a CF-LVAD. Twenty-two patients with CF-LVADs performed acute exercise either on a cycle ergometer for approximately 10 minutes or on a treadmill for 30 minutes. Blood samples were taken pre- and post-exercise to analyze hemostatic and angiogenic biomarkers. Acute exercise resulted in an increased platelet count (p < 0.00001) and platelet function (induced by adenosine diphosphate, p = 0.0087; TRAP-6, p = 0.0005; ristocetin, p = 0.0009). Additionally, high-molecular-weight vWF multimers (p < 0.00001), vWF collagen-binding activity (p = 0.0012), factor VIII (p = 0.034), angiopoietin-1 (p = 0.0026), and vascular endothelial growth factor (p = 0.0041) all increased after acute exercise. This pilot work demonstrates that acute exercise modulated two-hit hypothesis mediators associated with GI bleeding in patients with CF-LVADs.

Switch to a bicarbonate-based Impella CP™ purge solution in a patient with haemothorax: a case report.

Background: Percutaneous mechanical circulatory support (pMCS) by an Impella™ device implies the initiation of systemic anticoagulation to prevent systemic thrombotic complications and a purge fluid to prevent device blockage. Traditionally, unfractionated heparin (UFH) was used for both. In April 2022, the use of bicarbonate-based purge solution (BBPS) as an alternative to UFH in dextrose solution was approved by the Food and Drug Administration in case of contraindications for UFH. Case summary: We present the case of a 73-year-old female that was admitted to the cardiac intensive care unit with cardiogenic shock, requiring upgrade with pMCS by an axillary Impella CP™. When she developed a severe haemothorax, all UFH was stopped and the purge was switched to BBPS-dextrose solution without increase in purge pressures nor development of haemolysis. The bleeding stagnated and the patient could be weaned from the Impella™ after 2 days. Discussion: Here, we present the first case report of the switch to BBPS in an Impella CP™ supported patient with major bleeding since the FDA approval in April 2022. The switch to BBSP in addition with the administration of platelets and protamine resulted in cessation of the bleeding in this case.

Postoperative delirium and quality of life after transcatheter and surgical aortic valve replacement: A prospective observational study.

OBJECTIVE: In older patients, postoperative delirium is a frequently occurring complication after surgical aortic valve replacement, leading to an excess in postoperative morbidity and mortality. It remains controversial whether transcatheter aortic valve implantation and minimally invasive surgical aortic valve replacement can reduce the risk of postoperative delirium. This study aimed to compare the incidence of postoperative delirium after transcatheter aortic valve implantation and surgical aortic valve replacement and the impact on long-term outcomes. METHODS: Between September 2018 and January 2020, we conducted an observational, prospective cohort study in patients aged 70 years or more undergoing transcatheter aortic valve implantation or surgical aortic valve replacement. The primary end point was the incidence of in-hospital postoperative delirium during 5 postoperative days assessed with the Confusion Assessment Method. Secondary end points included perioperative inflammation, postoperative complications, health status (EuroQol 5-dimensional questionnaire 5 levels), and mortality up to 6 months. Transcatheter aortic valve implantation and surgical aortic valve replacement were compared using propensity weighting to account for important baseline differences (European System for Cardiac Operative Risk Evaluation II, age, and frailty). RESULTS: We included 250 patients with a mean (standard deviation) age of 80 (±5.8) years and a European System for Cardiac Operative Risk Evaluation score of 5 (±4.7). In the propensity-weighted analysis, those undergoing surgical aortic valve replacement (N = 166) had a higher incidence of postoperative delirium compared with transcatheter aortic valve implantation (N = 84) (51% vs 15%: P < .0001). Furthermore, patients undergoing surgical aortic valve replacement experienced more inflammation, a greater depth of anesthesia, and more intraoperative hypotension. After surgical aortic valve replacement, 41% of patients experienced an improved health status compared with 12% after transcatheter aortic valve implantation (P < .0001). No outcome differences were noted within the surgical aortic valve replacement groups. CONCLUSIONS: Transcatheter aortic valve implantation is associated with a lower risk for postoperative delirium. Nevertheless, patients undergoing surgical aortic valve replacement experience the greatest improvement in quality of life. Heart teams should consider these outcomes in shared decision-making in the choice of transcatheter aortic valve implantation or surgical aortic valve replacement.

Development of an icIEF assay for monitoring AAV capsid proteins and application to gene therapy products.

Adeno-associated virus (AAV) gene therapy vectors, which contain a DNA transgene packaged into a protein capsid, have shown tremendous therapeutic potential in recent years. Methods traditionally used in quality control labs, such as high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE), do not provide a complete understanding of capsid viral protein (VP) charge heterogeneity. In the present study, we developed simple, one-step sample preparation and charge-based VP separation using imaged capillary isoelectric focusing (icIEF) for monitoring AAV products. The robustness of the method was confirmed through a design of experiments (DoE) exercise. An orthogonal reverse-phase (RP) HPLC method coupled with mass spectrometry was developed to separate and identify charge species. Additionally, capsid point mutants demonstrate the capability of the method to resolve deamidation at a single site on the viral proteins. Finally, case studies using two different AAV serotype vectors establish the icIEF method as stability indicating and demonstrate that increases in acidic species measured by icIEF correlate with increased deamidation, which, we show, results in decreased transduction efficiency. The addition of a rapid and robust icIEF method to the AAV capsid analytical toolkit enables development and consistent manufacturing of well-characterized gene therapy products.

"Stapling" scFv for multispecific biotherapeutics of superior properties.

Single-chain fragment variable (scFv) domains play an important role in antibody-based therapeutic modalities, such as bispecifics, multispecifics and chimeric antigen receptor T cells or natural killer cells. However, scFv domains exhibit lower stability and increased risk of aggregation due to transient dissociation ("breathing") and inter-molecular reassociation of the two domains (VL and VH). We designed a novel strategy, referred to as stapling, that introduces two disulfide bonds between the scFv linker and the two variable domains to minimize scFv breathing. We named the resulting molecules stapled scFv (spFv). Stapling increased thermal stability (Tm) by an average of 10°C. In multiple scFv/spFv multispecifics, the spFv molecules display significantly improved stability, minimal aggregation and superior product quality. These spFv multispecifics retain binding affinity and functionality. Our stapling design was compatible with all antibody variable regions we evaluated and may be widely applicable to stabilize scFv molecules for designing biotherapeutics with superior biophysical properties.

Heartmate 3 implantation in small patients: CT-guided chest diameter assessment.

In recent years, the Heartmate 3 (HM3) has largely replaced the use of other intracorporeal left ventricular assist devices in the adult field. Because the HM3 is larger than the Heartware Ventricular Assist Device, the general consensus was that for small patients, the Heartware Ventricular Assist Device was the most appropriate implantable device option. Our goal was to describe our experiences with the successful implantation of the HM3 in 2 children, aged 9 and 11. We report on the chest cavity dimensions, as measured on computed tomography, that can be used to assess the feasibility of HM3 implantation in small patients.