Deep learning models to predict the editing efficiencies and outcomes of diverse base editors.

Applications of base editing are frequently restricted by the requirement for a protospacer adjacent motif (PAM), and selecting the optimal base editor (BE) and single-guide RNA pair (sgRNA) for a given target can be difficult. To select for BEs and sgRNAs without extensive experimental work, we systematically compared the editing windows, outcomes and preferred motifs for seven BEs, including two cytosine BEs, two adenine BEs and three C•G to G•C BEs at thousands of target sequences. We also evaluated nine Cas9 variants that recognize different PAM sequences and developed a deep learning model, DeepCas9variants, for predicting which variants function most efficiently at sites with a given target sequence. We then develop a computational model, DeepBE, that predicts editing efficiencies and outcomes of 63 BEs that were generated by incorporating nine Cas9 variants as nickase domains into the seven BE variants. The predicted median efficiencies of BEs with DeepBE-based design were 2.9- to 20-fold higher than those of rationally designed SpCas9-containing BEs.

Diameter and depth of femoral vessels by duplex ultrasound.

BACKGROUND: The anatomy of the femoral artery and vein plays an integral role in vascular access. Both technical feasibility and complication rates are associated with femoral vessel diameter and depth. The goal of this study is to establish normative values for common femoral artery (CFA) and vein (CFV) depth and diameter using a large, diverse patient population. METHODS: A retrospective review of all patients undergoing lower extremity venous duplex imaging over a 1 year period were reviewed. Patients with inadequate imaging or with evidence of deep vein thrombosis were excluded. The index image of all studies was a non-compressed view of the common femoral vein at the saphenous-femoral junction. All measurements were taken from this still. Vessel diameters were measured from intima to intima. Depth was measured from skin to intima. BMI and BSA were calculated using standard formulas. Chi square was used for univariate analysis. Linear regression was used to establish correlation. RESULTS: Over the 1 year period, 983 patients met criteria for inclusion. The majority were male (53%) with a mean age of 55. The patients were 47% white and 44% black. The majority had hypertension (53%). The mean BMI and BSA were 29 and 2, respectively. Mean CFA depth was 1.7 cm, while mean CFV depth was 1.8 cm. The mean CFA and CFV diameters were 0.9 and 1.1 cm, respectively. Amongst height, weight, BMI, and BSA, weight correlated best with CFA (R = 0.548) and CFV (R = 0.552) depth, while BSA correlated best for diameter for both CFA (R = 0.390) and CFV (R = 0.440). CONCLUSIONS: This study establishes mean diameters and depths for the common femoral artery and vein using a large, diverse patient group. BSA was most closely associated with vessel diameter, while weight was correlated with depth. This study provides normative diameter and depth values for the common femoral vasculature, which may assist in vascular access planning for providers.

Sniper2L is a high-fidelity Cas9 variant with high activity.

Although several high-fidelity SpCas9 variants have been reported, it has been observed that this increased specificity is associated with reduced on-target activity, limiting the applications of the high-fidelity variants when efficient genome editing is required. Here, we developed an improved version of Sniper-Cas9, Sniper2L, which represents an exception to this trade-off trend as it showed higher specificity with retained high activity. We evaluated Sniper2L activities at a large number of target sequences and developed DeepSniper, a deep learning model that can predict the activity of Sniper2L. We also confirmed that Sniper2L can induce highly efficient and specific editing at a large number of target sequences when it is delivered as a ribonucleoprotein complex. Mechanically, the high specificity of Sniper2L originates from its superior ability to avoid unwinding a target DNA containing even a single mismatch. We envision that Sniper2L will be useful when efficient and specific genome editing is required.

A pilot study of deep learning-based CT volumetry for traumatic hemothorax.

PURPOSE: We employ nnU-Net, a state-of-the-art self-configuring deep learning-based semantic segmentation method for quantitative visualization of hemothorax (HTX) in trauma patients, and assess performance using a combination of overlap and volume-based metrics. The accuracy of hemothorax volumes for predicting a composite of hemorrhage-related outcomes - massive transfusion (MT) and in-hospital mortality (IHM) not related to traumatic brain injury - is assessed and compared to subjective expert consensus grading by an experienced chest and emergency radiologist. MATERIALS AND METHODS: The study included manually labeled admission chest CTs from 77 consecutive adult patients with non-negligible (≥ 50 mL) traumatic HTX between 2016 and 2018 from one trauma center. DL results of ensembled nnU-Net were determined from fivefold cross-validation and compared to individual 2D, 3D, and cascaded 3D nnU-Net results using the Dice similarity coefficient (DSC) and volume similarity index. Pearson's r, intraclass correlation coefficient (ICC), and mean bias were also determined for the best performing model. Manual and automated hemothorax volumes and subjective hemothorax volume grades were analyzed as predictors of MT and IHM using AUC comparison. Volume cut-offs yielding sensitivity or specificity ≥ 90% were determined from ROC analysis. RESULTS: Ensembled nnU-Net achieved a mean DSC of 0.75 (SD: ± 0.12), and mean volume similarity of 0.91 (SD: ± 0.10), Pearson r of 0.93, and ICC of 0.92. Mean overmeasurement bias was only 1.7 mL despite a range of manual HTX volumes from 35 to 1503 mL (median: 178 mL). AUC of automated volumes for the composite outcome was 0.74 (95%CI: 0.58-0.91), compared to 0.76 (95%CI: 0.58-0.93) for manual volumes, and 0.76 (95%CI: 0.62-0.90) for consensus expert grading (p = 0.93). Automated volume cut-offs of 77 mL and 334 mL predicted the outcome with 93% sensitivity and 90% specificity respectively. CONCLUSION: Automated HTX volumetry had high method validity, yielded interpretable visual results, and had similar performance for the hemorrhage-related outcomes assessed compared to manual volumes and expert consensus grading. The results suggest promising avenues for automated HTX volumetry in research and clinical care.

How the COVID-19 Pandemic Has Changed Adolescent Health: Physical Activity, Sleep, Obesity, and Mental Health.

The purpose of this study is to provide essential data for the establishment of education and policy for the formation of healthy lifestyles of adolescents in the future by analyzing the patterns of changes in society due to the prolonged COVID-19 in the physical activities, sleeping habits, obesity, and mental health of Korean adolescents. To this end, a total of 147,346 adolescents were selected and analyzed according to the purpose of the study in the 2018 (14th), 2019 (15th), and 2020 (16th) raw data of the "Youth Health Behavior Online Survey," an annual national approval statistical survey conducted by a Korean government agency. The study examined changes in the physical activity, obesity, sleep, and mental health of Korean adolescents due to COVID-19. The physical activity rate of Korean adolescents in 2019 decreased by 5.3% from 2018. In addition, the physical activity rate in 2020 decreased by 2.1% compared to 2019. It was found that physical activity steadily decreased (p < 0.001). The obesity rate increased by 0.9% in 2019 compared to 2018 and by 1.8% in 2020 compared to 2019. Although the obesity rate steadily increased, it was found that it was accelerated due to COVID-19 (p < 0.001). Looking at the subjective sleep satisfaction rate of Korean adolescents, in 2019, it was 0.1% lower than in 2018, while in 2020, when COVID-19 began, it increased by 3.5% compared to 2019. It was found that satisfaction with sleep increased after COVID-19. Finally, the mental health characteristics of Korean adolescents by year were divided into stress and depression. Stress decreased by 1% compared to 2019 and 2018 and by 6.2% compared to 2020 and 2019. Depression increased by 1% in 2019 compared to 2018 and decreased by 3.4% in 2020 compared to 2019. In other words, stress and depression decreased after COVID-19. In 2020, when COVID-19 occurred, it was confirmed that there was a change in the health behavior of adolescents compared to 2018 and 2019. Therefore, active responses from schools, families, and communities are required to foster healthy lifestyle habits in social changes such as COVID-19.

Association between smartphone overdependency and mental health in Korean adolescents during the COVID pandemic; Age-and gender-matched study.

Purpose: This study aimed to examine the relationship between smartphone dependency (SD) and mental health (MH) in adolescents in order to develop and implement plans pertaining to SD control. Methods: Raw data from the 16th Online Adolescent Health Behavior Survey in 2020 were analyzed. A total of 482 respondents were selected as study subjects based on their experience of smartphone overdependence (SO), specifically, 241 participants whose score for SO was 37 or higher (Group 2) and age- and gender-matched 241 participants whose score was lower than 10 (Group 1). Results: Frequency analysis, cross-tab analysis (χ2 test), and multinomial logistic regression were performed Analysis shows that the MH affecting the increase in SO is the subjective perception of happiness, subjective perception of stress, sadness and despair, and experience of Loneliness. But, the variable affecting the reduction is the subjective evaluation of sleep quality. The likelihood of SO increased as adolescents felt unhappier [Exp (ß) = 2.408] and more stressed [Exp (ß) = 4.453] and more often felt lonely [Exp (ß) = 8.149], but the likelihood decreased as they had neither sufficient nor insufficient sleep duration [Exp (ß) = 0.344]. The findings suggest that it is necessary to develop aggressive measures for the prevention and management of MH in adolescents showing SO because mental health is closely linked to SD. In developing the measures, realistic approaches to widely pervasive SO among adolescents should be explored by taking into account MH factors, that is, predictors of SO, and the characteristics of youths, such that they can self-control smartphone use and form desirable life habits.

Prediction of the sequence-specific cleavage activity of Cas9 variants.

Several Streptococcus pyogenes Cas9 (SpCas9) variants have been developed to improve an enzyme's specificity or to alter or broaden its protospacer-adjacent motif (PAM) compatibility, but selecting the optimal variant for a given target sequence and application remains difficult. To build computational models to predict the sequence-specific activity of 13 SpCas9 variants, we first assessed their cleavage efficiency at 26,891 target sequences. We found that, of the 256 possible four-nucleotide NNNN sequences, 156 can be used as a PAM by at least one of the SpCas9 variants. For the high-fidelity variants, overall activity could be ranked as SpCas9 ≥ Sniper-Cas9 > eSpCas9(1.1) > SpCas9-HF1 > HypaCas9 ≈ xCas9 >> evoCas9, whereas their overall specificities could be ranked as evoCas9 >> HypaCas9 ≥ SpCas9-HF1 ≈ eSpCas9(1.1) > xCas9 > Sniper-Cas9 > SpCas9. Using these data, we developed 16 deep-learning-based computational models that accurately predict the activity of these variants at any target sequence.

Assessment of Pharmacokinetics and Metabolism Profiles of SCH 58261 in Rats Using Liquid Chromatography-Mass Spectrometric Method.

5-Amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo(4,3-e)-1,2,4-triazolo(1,5-c) pyrimidine (SCH 58261) is one of the new chemical entities that has been developed as an adenosine A2A receptor antagonist. Although SCH 58261 has been reported to be beneficial, there is little information about SCH 58261 from a drug metabolism or pharmacokinetics perspective. This study describes the metabolism and pharmacokinetic properties of SCH 58261 in order to understand its behaviors in vivo. Rats were used as the in vivo model species. First, an LC-MS/MS method was developed for the determination of SCH 58261 in rat plasma. A GastroPlus™ simulation, in vitro microsomal metabolic stability, and bile duct-cannulated studies were also performed to understand its pharmacokinetic profile. The parameter sensitivity analysis of GastroPlus™ was used to examine the factors that influence exposure when the drug is orally administered. The factors are as follows: permeability, systemic clearance, renal clearance, and liver first-pass effect. In vitro microsomal metabolic stability indicates how much the drug is metabolized. The extrapolated hepatic clearance value of SCH 58261 was 39.97 mL/min/kg, indicating that the drug is greatly affected by hepatic metabolism. In vitro microsomal metabolite identification studies revealed that metabolites produce oxidized and ketone-formed metabolites via metabolic enzymes in the liver. The bile duct-cannulated rat study, after oral administration of SCH 58261, showed that a significant amount of the drug was excreted in feces. These results imply that the drug is not absorbed well in the body after oral administration. Taken together, SCH 58261 showed quite a low bioavailability when administered orally and this was likely due to significantly limited absorption, as well as high metabolism in vivo.

In Vitro, In Silico, and In Vivo Assessments of Pharmacokinetic Properties of ZM241385.

Parkinson's disease is one of the most common neurodegenerative diseases. Adenosine regulates the response to other neurotransmitters in the brain regions related to motor function. In the several subtypes of adenosine receptors, especially, adenosine 2A receptors (A2ARs) are involved in neurodegenerative conditions. ZM241385 is one of the selective non-xanthine A2AR antagonists with high affinity in the nanomolar range. This study describes the in vitro and in vivo pharmacokinetic properties of ZM241385 in rats. A liquid chromatography-quadrupole time-of-flight mass spectrometric (LC-qToF MS) method was developed for the determination of ZM241385 in rat plasma. In vivo IV administration studies showed that ZM241385 was rapidly eliminated in rats. However, the result of in vitro metabolic stability studies showed that ZM241385 had moderate clearance, suggesting that there is an extra clearance pathway in addition to hepatic clearance. In addition, in vivo PO administration studies demonstrated that ZM241385 had low exposure in rats. The results of semi-mass balance studies and the in silico PBPK modeling studies suggested that the low bioavailability of ZM241385 after oral administration in rats was due to the metabolism and by liver, kidney, and gut.

Quantitative Analysis of Tozadenant Using Liquid Chromatography-Mass Spectrometric Method in Rat Plasma and Its Human Pharmacokinetics Prediction Using Physiologically Based Pharmacokinetic Modeling.

Tozadenant is one of the selective adenosine A2a receptor antagonists with a potential to be a new Parkinson's disease (PD) therapeutic drug. In this study, a liquid chromatography-mass spectrometry based bioanalytical method was qualified and applied for the quantitative analysis of tozadenant in rat plasma. A good calibration curve was observed in the range from 1.01 to 2200 ng/mL for tozadenant using a quadratic regression. In vitro and preclinical in vivo pharmacokinetic (PK) properties of tozadenant were studied through the developed bioanalytical methods, and human PK profiles were predicted using physiologically based pharmacokinetic (PBPK) modeling based on these values. The PBPK model was initially optimized using in vitro and in vivo PK data obtained by intravenous administration at a dose of 1 mg/kg in rats. Other in vivo PK data in rats were used to validate the PBPK model. The human PK of tozadenant after oral administration at a dose of 240 mg was simulated by using an optimized and validated PBPK model. The predicted human PK parameters and profiles were similar to the observed clinical data. As a result, optimized PBPK model could reasonably predict the PK in human.

3D printable hyaluronic acid-based hydrogel for its potential application as a bioink in tissue engineering.

BACKGROUND: After recognition of 3D printing and injectable hydrogel as a critical issue in tissue/organ engineering and regenerative medicine society, many hydrogels as bioinks have been developed worldwide by using polymeric biomaterials such as gelatin, alginate, hyaluronic acid and others. Even though some gels have shown good performances in 3D bioprinting, still their performances do not meet the requirements enough to be used as a bioink in tissue engineering. METHOD: In this study, a hydrogel consisting of three biocompatible biomaterials such as hyaluronic acid (HA), hydroxyethyl acrylate (HEA) and gelatin-methacryloyl, i.e. HA-g-pHEA-gelatin gel, has been evaluated for its possibility as a bioprinting gel, a bioink. Hydrogel synthesis was obtained by graft polymerization of HEA to HA and then grafting of gelatin- methacryloyl via radical polymerization mechanism. Physical and biological properties of the HA-based hydrogels fabricated with different concentrations of methacrylic anhydride (6 and 8%) for gelatin-methacryloylation have been evaluated such as swelling, rheology, morphology, cell compatibility, and delivery of small molecular dimethyloxalylglycine. Printings of HA-g-pHEA-Gelatin gel and its bioink with bone cell loaded in lattice forms were also evaluated by using home-built multi-material (3D bio-) printing system. CONCLUSION: The experimental results demonstrated that the HA-g-pHEA-gelatin hydrogel showed both stable rheology properties and excellent biocompatibility, and the gel showed printability in good shape. The bone cells in bioinks of the lattice-printed scaffolds were viable. This study showed HA-g-pHEA-Gelatin gel's potential as a bioink or its tissue engineering applications in injectable and 3D bioprinting forms.

A terpolymeric hydrogel of hyaluronate-hydroxyethyl acrylate-gelatin methacryloyl with tunable properties as biomaterial.

Here, we report synthesis of a terpolymeric covalently crosslinked hydrogel of hyaluronate (HA) as biomaterial with elasticity, mechanical properties and cell interactions via conventional free radical polymerization technique. To provide elasticity and mechanical properties, 2-hydroxyethyl acrylate (HEA) was grafted in HA, while to tune cellular interactions, gelatin methacryloyl (GM) was used as crosslinker. The composition and probable structure of the terpolymer (HA-g-pHEA-x-GM) were analysed by FTIR, 1H HR-MAS-NMR, and TGA analyses. The SEM and texture analyses of hydrogel showed interconnected micro-porous network and high mechanical properties, respectively. In vitro biocompatibility was studied against human chondrocytes, whereas, in vivo biocompatibility and tissue regeneration were confirmed using mouse model. The hydrogel releases model protein-bovine serum albumin, and corticosteroid drug-dexamethasone in a sustain way at pH 7.4 and 37 °C. Overall, the tunable mechanical properties, micro-porous network, and cytocompatibility of the HA-g-pHEA-x-GM hydrogel highlights its potential applicability in cartilage tissue engineering and drug delivery.

Analysis of Vipadenant and Its In Vitro and In Vivo Metabolites via Liquid Chromatography-Quadrupole-Time-of-Flight Mass Spectrometry.

A simple and sensitive liquid chromatography⁻quadrupole-time-of-flight⁻mass spectrometric (LC-QTOF-MS) assay has been developed for the evaluation of drug metabolism and pharmacokinetics (PK) properties of vipadenant in rat, a selective A2a receptor antagonist as one of the novel immune checkpoint inhibitors. A simple protein precipitation method using acetonitrile was used for the sample preparation and the pre-treated samples were separated by a reverse-phase C18 column. The calibration curve was evaluated in the range of 3.02 ~ 2200 ng/mL and the quadratic regression (weighted 1/concentration) was used for the best fit of the curve with a correlation coefficient ≥0.997. The in vivo PK studies in rats showed that vipadenant bioavailability was 30.4 ± 8.9% with a low to moderate drug clearance. In addition, in vitro/in vivo metabolite profiles in rat were also explored. Five different metabolites were observed in our experimental conditions and the major metabolites were different between in vitro and in vivo conditions. As far as we know, there has been no report on the development of quantitative methods for its PK samples nor the identification of its metabolites since vipadenant was developed. Therefore, this paper would be very useful to better understand the pharmacokinetic and drug metabolism properties of vipadenant in rat as well as other species.

Qualification and Application of a Liquid Chromatography-Quadrupole Time-of-Flight Mass Spectrometric Method for the Determination of Adalimumab in Rat Plasma.

A liquid chromatography⁻quadrupole time-of-flight (Q-TOF) mass spectrometric method was developed for early-stage research on adalimumab in rats. The method consisted of immunoprecipitation followed by tryptic digestion for sample preparation and LC-QTOF-MS/MS analysis of specific signature peptides of adalimumab in the positive ion mode using electrospray ionization. This specific signature peptide is derived from the complementarity-determining region (CDR) of adalimumab. A quadratic regression (weighted 1/concentration), with an equation y = ax² + bx + c, was used to fit calibration curves over the concentration range of 1⁻100 μg/mL for adalimumab. The qualification run met the acceptance criteria of ±25% accuracy and precision values for quality control (QC) samples. This qualified LC-QTOF-MS/MS method was successfully applied to a pharmacokinetic study of adalimumab in rats as a case study. This LC-QTOF-MS/MS approach would be useful as a complementary method for adalimumab or its biosimilars at an early stage of research.

A single liquid chromatography-quadrupole time-of-flight mass spectrometric method for the quantification of total antibody, antibody-conjugated drug and free payload of antibody-drug conjugates.

A single hybrid affinity-captured-LC-TOF-MS/MS method was developed and applied for the quantification of total antibody, antibody conjugated drug and free payload of antibody drug conjugate (ADC). Adcetris®, a valine-citrulline monomethyl auristatin E conjugated ADC, was used as a model ADC compound. A quadratic regression (weighted 1/concentration) was used to fit calibration curves over the concentration range 30.65-613.00 ng/mL with an equation y = ax2 + bx + c for the antibody-conjugated drug of Adcetris®. The qualification run met the acceptance criteria of ±25% accuracy and precision values for quality control samples. For the analysis of total antibody, a signature peptide (TTPPVLDSDGSFFLYSK, molecular weight 1874) was used after affinity capture using magnetic beads and on-bead trypsin digestion. A quadratic regression (weighted 1/concentration) was used to fit calibration curves over the concentration range 5.00-100.00 µg/mL with an equation y = ax2 + bx + c for total antibody. For free payload analysis of monomethyl auristatin E, a protein precipitation method followed by LC-TOF-MS/MS analysis was used. A quadratic regression (weighted 1/concentration) was used to fit calibration curves over the concentration range 1.01-2200 ng/mL with an equation y = ax2 + bx + c for free payload. Pharmacokinetic study samples and in vitro stability samples in rat were successfully analyzed by this a hybrid affinity-captured-LC-TOF-MS/MS method. This single platform method is a useful complementary method for the pharmacokinetics study of ADC with valine-citrulline linker at the early drug discovery stage.

Rapid and simultaneous quantification of a mixture of biopharmaceuticals by a liquid chromatography/quadrupole time-of-flight mass spectrometric method in rat plasma following cassette-dosing.

RATIONALE: The cassette-dosing technique is a technique that administers various drugs to a single animal at once and quantitated simultaneously. The purpose of this study was to evaluate the feasibility of cassette-dosing as a means of increasing throughput and decreasing animal usage for pharmacokinetic studies of biopharmaceuticals using liquid chromatography/time-of-flight mass spectrometric (LC/TOF-MS) analysis. METHODS: Brentuximab, trastuzumab, cetuximab and adalimumab were used as model biopharmaceuticals. The method consisted of immunoprecipitation followed by tryptic digestion for sample preparation and LC/TOF-MS analysis of specific signature peptides in the positive ion mode using electrospray ionization. The specific signature peptides used for quantification were from the complementarity-determining regions of each mAb. All rats received a single intravenous bolus injection containing either a single mAb or a mixture of four mAbs. RESULTS: The proposed method has been qualified in linearity range of 1-100 µg/mL with correlation coefficients higher than 0.990. The qualification run met the acceptance criteria of ±25% accuracy and precision values for quality control (QC) samples. This qualified LC/TOF-MS method was successfully applied to a pharmacokinetic study in the rat. The PK properties of mAbs administered as a cassette-dosage were similar to the pharmacokinetics of each antibody drug when administered as a single entity. CONCLUSIONS: These findings suggest that the cassette-dosing approach could be used to evaluate the PK properties of biopharmaceuticals in the early drug discovery stage. Also, this method would be useful for other preclinical sample analysis without developing new reagents for sample preparation.