Implementation of whole-exome sequencing for pharmacogenomics profiling and exploring its potential clinical utilities.

Whole-exome sequencing (WES) is widely used in clinical settings; however, the exploration of its use in pharmacogenomic analysis remains limited. Our study compared the variant callings for 28 core absorption, distribution, metabolism and elimination genes by WES and array-based technology using clinical trials samples. The results revealed that WES had a positive predictive value of 0.71-0.92 and a sensitivity of single-nucleotide variants between 0.68 and 0.95, compared with array-based technology, for the variants in the commonly targeted regions of the WES and PhamacoScan™ assay. Besides the common variants detected by both assays, WES identified 200-300 exclusive variants per sample, totalling 55 annotated exclusive variants, including important modulators of metabolism such as rs2032582 (ABCB1) and rs72547527 (SULT1A1). This study highlights the potential clinical advantages of using WES to identify a wider range of genetic variations and enabling precision medicine.

XBB.1.5 monovalent booster improves antibody binding and neutralization against emerging SARS-CoV-2 Omicron variants.

The rapid emergence of divergent SARS-CoV-2 variants has led to an update of the COVID-19 booster vaccine to a monovalent version containing the XBB.1.5 spike. To determine the neutralization breadth following booster immunization, we collected blood samples from 24 individuals pre- and post-XBB.1.5 mRNA booster vaccination (∼1 month). The XBB.1.5 booster improved both neutralizing activity against the ancestral SARS-CoV-2 strain (WA1) and the circulating Omicron variants, including EG.5.1, HK.3, HV.1, XBB.1.5 and JN.1. Relative to the pre-boost titers, the XBB.1.5 monovalent booster induced greater total IgG and IgG subclass binding, particular IgG4, to the XBB.1.5 spike as compared to the WA1 spike. We evaluated antigen-specific memory B cells (MBCs) using either spike or receptor binding domain (RBD) probes and found that the monovalent booster largely increases non-RBD cross-reactive MBCs. These data suggest that the XBB.1.5 monovalent booster induces cross-reactive antibodies that neutralize XBB.1.5 and related Omicron variants.

Cleavage-intermediate Lassa virus trimer elicits neutralizing responses, identifies neutralizing nanobodies, and reveals an apex-situated site-of-vulnerability.

Lassa virus (LASV) infection is expanding outside its traditionally endemic areas in West Africa, posing a pandemic biothreat. LASV-neutralizing antibodies, moreover, have proven difficult to elicit. To gain insight into LASV neutralization, here we develop a prefusion-stabilized LASV glycoprotein trimer (GPC), pan it against phage libraries comprising single-domain antibodies (nanobodies) from shark and camel, and identify one, D5, which neutralizes LASV. Cryo-EM analyses reveal D5 to recognize a cleavage-dependent site-of-vulnerability at the trimer apex. The recognized site appears specific to GPC intermediates, with protomers lacking full cleavage between GP1 and GP2 subunits. Guinea pig immunizations with the prefusion-stabilized cleavage-intermediate LASV GPC, first as trimer and then as a nanoparticle, induce neutralizing responses, targeting multiple epitopes including that of D5; we identify a neutralizing antibody (GP23) from the immunized guinea pigs. Collectively, our findings define a prefusion-stabilized GPC trimer, reveal an apex-situated site-of-vulnerability, and demonstrate elicitation of LASV-neutralizing responses by a cleavage-intermediate LASV trimer.

Discovery, Optimization, and Evaluation of Novel Pyridin-2(1H)-one Analogues as Potent TRK Inhibitors for Cancer Treatment.

Tropomyosin receptor kinase (TRK) fusion, an oncogenic form of kinase with pan-tumor occurrence, is a clinically validated important antitumor target. In this study, we screened our in-house kinase inhibitor library against TRK and identified a promising hit compound 4 with a novel pyridin-2(1H)-one scaffold. Through a combination of structure-based drug design and structure-activity relationship (SAR) study, compound 14q was identified as a potent TRK inhibitor with good kinase selectivity. It also blocked cellular TRK signaling, thereby inhibiting TRK-dependent cell viability. Additionally, 14q displayed acceptable pharmacokinetic properties with 37.8% oral bioavailability in mice. Strong in vivo tumor growth inhibition of 14q was observed in subcutaneous M091 and KM12 tumor xenograft models with TRK fusion, causing significant tumor inhibition or even complete tumor regression.

Estimating the cardiac signals of chimpanzees using a digital camera: validation and application of a novel non-invasive method for primate research.

Cardiac measures such as heart rate measurements are important indicators of both physiological and psychological states. However, despite their extraordinary potential, their use is restricted in comparative psychology because traditionally cardiac measures involved the attachment of sensors to the participant's body, which, in the case of undomesticated animals such as nonhuman primates, is usually only possible during anesthesia or after extensive training. Here, we validate and apply a camera-based system that enables contact-free detection of animals' heart rates. The system automatically detects and estimates the cardiac signals from cyclic change in the hue of the facial area of a chimpanzee. In Study 1, we recorded the heart rate of chimpanzees using the new technology, while simultaneously measuring heart rate using classic PPG (photoplethysmography) finger sensors. We found that both methods were in good agreement. In Study 2, we applied our new method to measure chimpanzees' heart rate in response to seeing different types of video scenes (groupmates in an agonistic interaction, conspecific strangers feeding, nature videos, etc.). Heart rates changed during video presentation, depending on the video content: Agonistic interactions and conspecific strangers feeding lead to accelerated heart rate relative to baseline, indicating increased emotional arousal. Nature videos lead to decelerated heart rate relative to baseline, indicating a relaxing effect or heightened attention caused by these stimuli. Our results show that the new contact-free technology can reliably assess the heart rate of unrestrained chimpanzees, and most likely other primates. Furthermore, our technique opens up new avenues of research within comparative psychology and facilitates the health management of captive individuals.

Structure-based drug discovery of novel fused-pyrazolone carboxamide derivatives as potent and selective AXL inhibitors.

As a novel and promising antitumor target, AXL plays an important role in tumor growth, metastasis, immunosuppression and drug resistance of various malignancies, which has attracted extensive research interest in recent years. In this study, by employing the structure-based drug design and bioisosterism strategies, we designed and synthesized in total 54 novel AXL inhibitors featuring a fused-pyrazolone carboxamide scaffold, of which up to 20 compounds exhibited excellent AXL kinase and BaF3/TEL-AXL cell viability inhibitions. Notably, compound 59 showed a desirable AXL kinase inhibitory activity (IC50: 3.5 nmol/L) as well as good kinase selectivity, and it effectively blocked the cellular AXL signaling. In turn, compound 59 could potently inhibit BaF3/TEL-AXL cell viability (IC50: 1.5 nmol/L) and significantly suppress GAS6/AXL-mediated cancer cell invasion, migration and wound healing at the nanomolar level. More importantly, compound 59 oral administration showed good pharmacokinetic profile and in vivo antitumor efficiency, in which we observed significant AXL phosphorylation suppression, and its antitumor efficacy at 20 mg/kg (qd) was comparable to that of BGB324 at 50 mg/kg (bid), the most advanced AXL inhibitor. Taken together, this work provided a valuable lead compound as a potential AXL inhibitor for the further antitumor drug development.

Relationship among DDR gene mutations, TMB and PD-L1 in solid tumour genomes identified using clinically actionable biomarker assays.

The DNA damage response (DDR) pathway regulates DNA repair and cell survival, and inactivating mutations in DDR genes can increase tumour mutational burden (TMB), a predictive biomarker of treatment benefit from anti-PD-1/PD-L1 immunotherapies. However, a better understanding of the relationship among specific DDR mutations, TMB and PD-L1 expression is needed to improve translational strategies. Here, we determined genomic alteration frequencies in selected DDR genes that are clinically actionable biomarkers and investigated their association with TMB and PD-L1 in bladder, colorectal, non-small cell lung, ovarian and prostate cancers using the FoundationInsights® web portal. Our results not only confirm known associations, such as mismatch repair and POLE gene mutations with high TMB, but also identify significant associations between mutations in the SWI/SNF chromatin remodelling genes ARID1A and SMARCA4 and high TMB in multiple tumour types. Mutations in the ATR gene were associated with high TMB in colorectal and prostate cancers; however, associations between individual DDR mutations and high PD-L1 expression were uncommon and tumour-type specific. Finally, we found that high TMB and high PD-L1 expression were poorly associated, emphasising their independence as predictive biomarkers for immune checkpoint inhibitor use.

HIV-1 neutralizing antibodies elicited in humans by a prefusion-stabilized envelope trimer form a reproducible class targeting fusion peptide.

Elicitation of antibodies that neutralize the tier-2 neutralization-resistant isolates that typify HIV-1 transmission has been a long-sought goal. Success with prefusion-stabilized envelope trimers eliciting autologous neutralizing antibodies has been reported in multiple vaccine-test species, though not in humans. To investigate elicitation of HIV-1 neutralizing antibodies in humans, here, we analyze B cells from a phase I clinical trial of the "DS-SOSIP"-stabilized envelope trimer from strain BG505, identifying two antibodies, N751-2C06.01 and N751-2C09.01 (named for donor-lineage.clone), that neutralize the autologous tier-2 strain, BG505. Though derived from distinct lineages, these antibodies form a reproducible antibody class that targets the HIV-1 fusion peptide. Both antibodies are highly strain specific, which we attribute to their partial recognition of a BG505-specific glycan hole and to their binding requirements for a few BG505-specific residues. Prefusion-stabilized envelope trimers can thus elicit autologous tier-2 neutralizing antibodies in humans, with initially identified neutralizing antibodies recognizing the fusion-peptide site of vulnerability.

Structural Optimization of Fibroblast Growth Factor Receptor Inhibitors for Treating Solid Tumors.

Small-molecule fibroblast growth factor receptor (FGFR) inhibitors have emerged as a promising antitumor therapy. Herein, by further optimizing the lead compound 1 under the guidance of molecular docking, we obtained a series of novel covalent FGFR inhibitors. After careful structure-activity relationship analysis, several compounds were identified to exhibit strong FGFR inhibitory activity and relatively better physicochemical and pharmacokinetic properties compared with those of 1. Among them, 2e potently and selectively inhibited the kinase activity of FGFR1-3 wildtype and high-incidence FGFR2-N549H/K-resistant mutant kinase. Furthermore, it suppressed cellular FGFR signaling, exhibiting considerable antiproliferative activity in FGFR-aberrant cancer cell lines. In addition, the oral administration of 2e in the FGFR1-amplified H1581, FGFR2-amplified NCI-H716, and SNU-16 tumor xenograft models demonstrated potent antitumor efficacy, inducing tumor stasis or even tumor regression.

Simulating cardiac signals on 3D human models for photoplethysmography development.

Introduction: Image-based heart rate estimation technology offers a contactless approach to healthcare monitoring that could improve the lives of millions of people. In order to comprehensively test or optimize image-based heart rate extraction methods, the dataset should contain a large number of factors such as body motion, lighting conditions, and physiological states. However, collecting high-quality datasets with complete parameters is a huge challenge. Methods: In this paper, we introduce a bionic human model based on a three-dimensional (3D) representation of the human body. By integrating synthetic cardiac signal and body involuntary motion into the 3D model, five well-known traditional and four deep learning iPPG (imaging photoplethysmography) extraction methods are used to test the rendered videos. Results: To compare with different situations in the real world, four common scenarios (stillness, expression/talking, light source changes, and physical activity) are created on each 3D human. The 3D human can be built with any appearance and different skin tones. A high degree of agreement is achieved between the signals extracted from videos with the synthetic human and videos with a real human-the performance advantages and disadvantages of the selected iPPG methods are consistent for both real and 3D humans. Discussion: This technology has the capability to generate synthetic humans within various scenarios, utilizing precisely controlled parameters and disturbances. Furthermore, it holds considerable potential for testing and optimizing image-based vital signs methods in challenging situations where real people with reliable ground truth measurements are difficult to obtain, such as in drone rescue.

Extraordinary Titer and Broad Anti-SARS-CoV-2 Neutralization Induced by Stabilized RBD Nanoparticles from Strain BA.5.

The receptor-binding domain (RBD) of the SARS-CoV-2 spike is a primary target of neutralizing antibodies and a key component of licensed vaccines. Substantial mutations in RBD, however, enable current variants to escape immunogenicity generated by vaccination with the ancestral (WA1) strain. Here, we produce and assess self-assembling nanoparticles displaying RBDs from WA1 and BA.5 strains by using the SpyTag:SpyCatcher system for coupling. We observed both WA1- and BA.5-RBD nanoparticles to degrade substantially after a few days at 37 °C. Incorporation of nine RBD-stabilizing mutations, however, increased yield ~five-fold and stability such that more than 50% of either the WA1- or BA.5-RBD nanoparticle was retained after one week at 37 °C. Murine immunizations revealed that the stabilized RBD-nanoparticles induced ~100-fold higher autologous neutralization titers than the prefusion-stabilized (S2P) spike at a 2 µg dose. Even at a 25-fold lower dose where S2P-induced neutralization titers were below the detection limit, the stabilized BA.5-RBD nanoparticle induced homologous titers of 12,795 ID50 and heterologous titers against WA1 of 1767 ID50. Assessment against a panel of ß-coronavirus variants revealed both the stabilized BA.5-RBD nanoparticle and the stabilized WA1-BA.5-(mosaic)-RBD nanoparticle to elicit much higher neutralization breadth than the stabilized WA1-RBD nanoparticle. The extraordinary titer and high neutralization breadth elicited by stabilized RBD nanoparticles from strain BA.5 make them strong candidates for next-generation COVID-19 vaccines.

Current State of Pleural-Directed Adjuncts Against Malignant Pleural Mesothelioma.

Multimodality therapy including surgical resection is the current paradigm in treating malignant pleural mesothelioma (MPM), a thoracic surface cancer without cure. The main limitation of all surgical approaches is the lack of long-term durability because macroscopic complete resection (R1 resection) commonly predisposes to locoregional relapse. Over the years, there have been many studies that describe various intrapleural strategies that aim to extend the effect of surgical resection. The majority of these approaches are intraoperative adjuvants. Broadly, there are three therapeutic classes that employ diverse agents. The most common, widely used group of adjuvants are comprised of direct therapeutics such as intracavitary chemotherapy (± hyperthermia). By comparison, the least commonly employed intrathoracic adjuvant is the class comprised of drug-device combinations like photodynamic therapy (PDT). But the most rapidly evolving (new) class with much potential for improved efficacy are therapeutics delivered by specialized drug vehicles such as a fibrin gel containing cisplatin. This review provides an updated perspective on pleural-directed adjuncts in the management of MPM as well as highlighting the most promising near-term technology breakthroughs.

Comparison of Two Illumina Whole Transcriptome RNA Sequencing Library Preparation Methods Using Human Cancer FFPE Specimens.

Objective: RNA extraction and library preparation from formalin-fixed, paraffin-embedded (FFPE) samples are crucial pre-analytical steps towards achieving optimal downstream RNA sequencing (RNASeq) results. In this study, we assessed 2 Illumina library preparation methods for RNA-Seq analysis using archived FFPE samples from human cancer indications at 2 independent vendors. Methods: Twenty-five FFPE samples from 5 indications (non-small cell lung cancer, colorectal cancer, renal carcinoma, breast cancer, and hepatocellular carcinoma) were included, covering a wide range of sample storage durations (3-25 years-old), sample qualities, and specimen types (resection vs core needle biopsy). Each sample was processed independently by both vendors. Total RNA was isolated using the Qiagen miRNeasy FFPE kit followed by library construction using either TruSeq Stranded Total RNA library preparation kit with Ribo-Zero Gold, or TruSeq RNA Access library preparation kit. Libraries were normalized to 20 pM and sequenced on an Illumina HiSeq 2500 using V3 chemistry in paired-end mode with a read length of 2 × 50 bp. The data were processed through a standard RNASeq pipeline to produce counts and transcripts per millions for each gene in each sample to compare 2 library kits at 2 different vendors. Results: Our data showed that TruSeq RNA Access libraries yield over 80% exonic reads across different quality samples, indicating higher selectivity of the exome pull down by the capture approach compared to the random priming of the TruSeq Stranded Total kit. The overall QC data for FFPE RNA extraction, library preparation, and sequencing generated by the 2 vendors are comparable, and downstream gene expression quantification results show high concordance as well. With the TruSeq Stranded Total kit, the mean Spearman correlation between vendors was 0.87 and the mean Pearson correlation was 0.76. With the TruSeq RNA Access kit, the mean Spearman correlation between vendors was 0.89 and the mean Pearson correlation was 0.73. Interestingly, examination of the cross-vendor correlations compared to various common QC statistics suggested that library concentration is better correlated with consistency between vendors than is the RNA quantity. Conclusions: Our analyses provide evidence to guide selection of sequencing methods for FFPE samples in which the sample quality may be severely compromised.

Noncontact Sensing of Contagion.

The World Health Organization (WHO) has declared COVID-19 a pandemic. We review and reduce the clinical literature on diagnosis of COVID-19 through symptoms that might be remotely detected as of early May 2020. Vital signs associated with respiratory distress and fever, coughing, and visible infections have been reported. Fever screening by temperature monitoring is currently popular. However, improved noncontact detection is sought. Vital signs including heart rate and respiratory rate are affected by the condition. Cough, fatigue, and visible infections are also reported as common symptoms. There are non-contact methods for measuring vital signs remotely that have been shown to have acceptable accuracy, reliability, and practicality in some settings. Each has its pros and cons and may perform well in some challenges but be inadequate in others. Our review shows that visible spectrum and thermal spectrum cameras offer the best options for truly noncontact sensing of those studied to date, thermal cameras due to their potential to measure all likely symptoms on a single camera, especially temperature, and video cameras due to their availability, cost, adaptability, and compatibility. Substantial supply chain disruptions during the pandemic and the widespread nature of the problem means that cost-effectiveness and availability are important considerations.

Development of a liquid biopsy based purely quantitative digital droplet PCR assay for detection of MLH1 promoter methylation in colorectal cancer patients.

BACKGROUND: MutL Homolog 1 (MLH1) promotor methylation is associated with microsatellite instability high colorectal cancer (CRC). The strong correlation between methylation status and cancer development and progression has led to a growing interest in the use of methylation markers in circulating tumor DNA (ctDNA) for early cancer detection and longitudinal monitoring. As cancer-specific DNA methylation changes in body fluids are limited, it is particularly challenging to develop clinically applicable liquid biopsy methodologies with high sensitivity and specificity. The purpose of this study was to develop a fit-for-purpose methylation sensitive restriction enzyme (MSRE) based digital droplet PCR (ddPCR) assay to examine MLH1 promoter methylation in ctDNA in advanced CRC. METHODS: Primers and probes were designed to amplify CpG sites of the MLH1 promoter. Methylated and unmethylated control genomic DNA were sheared to mimic ctDNA and subjected to MSRE HpaII digestion. Plasma samples from 20 healthy donors and 28 CRC patients were analyzed with the optimized MSRE procedure using ddPCR. RESULTS: Using methylated and unmethylated controls, we optimized the conditions for HpaII enzyme digestion to ensure complete digestion and avoid false positives. Based on the results from the ddPCR assay using 1 ng circulating cell-free DNA (cfDNA) input from healthy donors or CRC samples, ROC curves were generated with an area under the curve (AUC) value of 0.965 (95% CI: 0.94, 0.99). The statistically optimal assay sensitivity and specificity was achieved when 8 positive droplets were used as acceptance criteria (78% sensitivity and 100% specificity, 95% CI: 0.45, 0.95). A tiered-based cutoff (20, 50, 80% percentile based) was applied to distinguish CRC samples with different methylation level. CONCLUSIONS: Our study demonstrated that the liquid biopsy assay for MLH1 promoter methylation detection using purely quantitative ddPCR is a simple and highly sensitive procedure that provides reliable methylation detection in ctDNA. The MSRE ddPCR approach can also be applied to other genes of interest where methylation patterns could reveal clinically relevant information for future clinical biomarker and/or companion diagnostic development.

Fit-for-purpose quantitative liquid biopsy based droplet digital PCR assay development for detection of programmed cell death ligand-1 (PD-L1) RNA expression in PAXgene blood samples.

Development of a clinically applicable liquid biopsy-based test for PD-L1 mRNA expression would be beneficial in providing complementary evidence to current immunohistochemistry assays. Hence, we report the development of a fit-for-purpose assay for detection of blood PD-L1 mRNA expression using droplet digital polymerase chain reaction (ddPCR). TaqMan® assays were selected based on coverage of the PD-L1 gene and were tested for linearity and efficiency using real-time quantitative PCR. Four reference genes were analyzed in positive control cell line (A549 treated with interferon gamma, [IFN γ]) genomic DNA. The PD-L1 primer/probe sets were also evaluated in ddPCR for limit of blank, limit of detection, and precision. Finally, thirty-five healthy volunteer samples were evaluated to establish a baseline level of PD-L1 expression. In ddPCR, the limit of blank was determined to be 0 copies and the limit of detection was determined to be less than or equal to 19 copies of PD-L1. The average intra-run coefficient of variation in the ddPCR assay was 7.44% and average inter-run CV was 7.70%. Treatment of A549 cells with IFN γ resulted in a 6.7-fold increase in PD-L1 expression (21,580 copies in untreated cDNA versus 145,000 copies in treated cDNA). Analysis of healthy human samples yielded a median value of 1659 PD-L1 copies/µL with a range of 768-7510 copies/µL. The assay was transferred to an external service provider and results from our in-house experiments and those conducted externally shows a correlation of 0.994. In conclusion, a fit-for-purpose liquid biopsy-based, purely quantitative ddPCR assay for the detection of PD-L1 mRNA expression was developed and validated using PAXgene RNA blood samples. Linearity, reproducibility, limit of blank and limit of detection were measured and deemed suitable for clinical application. This ultra-sensitive liquid biopsy ddPCR assay has promising clinical potential in screening, longitudinal monitoring and disease progression detection.

Veronicastrum axillare Alleviates Ethanol-Induced Injury on Gastric Epithelial Cells via Downregulation of the NF-kB Signaling Pathway.

We used human gastric epithelial cells (GES-1) line in an ethanol-induced cell damage model to study the protective effect of Veronicastrum axillare and its modulation to NF-κB signal pathway. The goal was to probe the molecular mechanism of V. axillare decoction in the prevention of gastric ulcer and therefore provide guidance in the clinical application of V. axillare on treating injuries from chronic nephritis, pleural effusion, gastric ulcer, and other ailments. The effects of V. axillare-loaded serums on cell viability were detected by MTT assays. Enzyme-linked immunosorbent assay (ELISA) and Real-Time PCR methods were used to analyze the protein and mRNA expression of TNF-α, NF-κB, IκBα, and IKKß. The results showed that V. axillare-loaded serum partially reversed the damaging effects of ethanol and NF-κB activator (phorbol-12-myristate-13-acetate: PMA) and increased cell viability. The protein and mRNA expressions of TNF-α, NF-κB, IκBα, and IKKß were significantly upregulated by ethanol and PMA while they were downregulated by V. axillare-loaded serum. In summary, V. axillare-loaded serum has significantly protective effect on GES-1 against ethanol-induced injury. The protective effect was likely linked to downregulation of TNF-α based NF-κB signal pathway.

An elastography analytical method for the rapid detection of endotoxin.

We present a flexible analytical method for the study of coagulation systems by monitoring elastography (EG). The rapid detection of endotoxin is achieved by the EG analysis of endotoxin-induced limulus amebocyte lysate coagulation. This method is superior to other methods using the same reagents in not only sensitivity but also detecting time.

Relative specificity of acupuncture reinforcing and reducing methods.

OBJECTIVE: To probe the effects of acupuncture reinforcing and reducing methods on the human body. METHODS: Studied acupuncture reinforcing and reducing actions in the ancient literature, based on modern clinical and experimental studies, in combination with experience of acupuncture teaching and clinical treatment. RESULTS: The key to the generation of reinforcing and reducing actions is not the acupuncture reinforcing and reducing methods themselves, but instead the functional state of the patient during the acupuncture. When reinforcing and reducing methods act on the human body, a reinforcing-reducing effect is produced through regulation of Qi, indicating that the production of the reinforcing-reducing effect requires a certain condition. Specifically, acupuncture does not produce the reinforcing-reducing effect under all conditions, but can produce a reinforcing effect in patients with deficiency syndrome and a reducing effect in patients with excess syndrome. CONCLUSION: Reinforcing and reducing methods each have therapeutic effects on both deficiency syndrome and excess syndrome, but a reinforcing method should be selected first for deficiency syndrome and a reducing method should be selected first for excess syndrome.

WITHDRAWN: Reprint of: Mechanisms of retinal ganglion cell injury and defense in glaucoma.

The Publisher regrets that this article is an accidental duplication of an article that has already been published, doi:10.1016/j.exer.2010.04.002. The duplicate article has therefore been withdrawn.