Dual indexed library design enables compatibility of in-Drop single-cell RNA-sequencing with exAMP chemistry sequencing platforms.

BACKGROUND: The increasing demand of single-cell RNA-sequencing (scRNA-seq) experiments, such as the number of experiments and cells queried per experiment, necessitates higher sequencing depth coupled to high data quality. New high-throughput sequencers, such as the Illumina NovaSeq 6000, enables this demand to be filled in a cost-effective manner. However, current scRNA-seq library designs present compatibility challenges with newer sequencing technologies, such as index-hopping, and their ability to generate high quality data has yet to be systematically evaluated. RESULTS: Here, we engineered a dual-indexed library structure, called TruDrop, on top of the inDrop scRNA-seq platform to solve these compatibility challenges, such that TruDrop libraries and standard Illumina libraries can be sequenced alongside each other on the NovaSeq. On scRNA-seq libraries, we implemented a previously-documented countermeasure to the well-described problem of index-hopping, demonstrated significant improvements in base-calling accuracy on the NovaSeq, and provided an example of multiplexing twenty-four scRNA-seq libraries simultaneously. We showed favorable comparisons in transcriptional diversity of TruDrop compared with prior inDrop libraries. CONCLUSIONS: Our approach enables cost-effective, high throughput generation of sequencing data with high quality, which should enable more routine use of scRNA-seq technologies.

HLA-A*02:01 allele is associated with tanshinone-induced cutaneous drug reactions in Chinese population.

Tanshinone, a widely used Chinese patent medicine, has been confirmed to have various kinds of pharmacological effects although frequently causing cutaneous adverse drug reactions (cADRs). We aim to identify whether human leukocyte antigen (HLA) class I alleles are associated with tanshinone-induced cADRs in Han Chinese. The association study including 18 patients with tanshinone-induced cADRs, 67 tanshinone-tolerant volunteers, and two general population databases consisted of 10,689 and 169,995 healthy subjects was performed. The frequency of tanshinone-induced cADRs patients carrying HLA-A*02:01 was significantly higher when compared with the general control groups (OR = 6.25, Pc = 7.20 × 10-5; OR = 7.14, Pc = 8.00 × 10-6), and with the tolerant group (OR = 5.09, Pc = 0.024). The molecular docking assay confirmed high affinity of the ingredients of tanshinone towards HLA-A*02:01 (≤-7.5 kcal/mol). The result suggested HLA-A*02:01 may work as a promisingly predictive marker for tanshinone personalized therapy in Han Chinese.

Discovery of tumor-reactive T cell receptors by massively parallel library synthesis and screening.

T cell receptor (TCR) gene therapy is a potent form of cellular immunotherapy in which patient T cells are genetically engineered to express TCRs with defined tumor reactivity. However, the isolation of therapeutic TCRs is complicated by both the general scarcity of tumor-specific T cells among patient T cell repertoires and the patient-specific nature of T cell epitopes expressed on tumors. Here we describe a high-throughput, personalized TCR discovery pipeline that enables the assembly of complex synthetic TCR libraries in a one-pot reaction, followed by pooled expression in reporter T cells and functional genetic screening against patient-derived tumor or antigen-presenting cells. We applied the method to screen thousands of tumor-infiltrating lymphocyte (TIL)-derived TCRs from multiple patients and identified dozens of CD4+ and CD8+ T-cell-derived TCRs with potent tumor reactivity, including TCRs that recognized patient-specific neoantigens.

Metabolomic profiling to identify potential serum biomarkers for schizophrenia and risperidone action.

Despite recent advances in understanding the pathophysiology of schizophrenia and the mechanisms of antipsychotic drug action, the development of biomarkers for diagnosis and therapeutic monitoring in schizophrenia remains challenging. Metabolomics provides a powerful approach to discover diagnostic and therapeutic biomarkers by analyzing global changes in an individual's metabolic profile in response to pathophysiological stimuli or drug intervention. In this study, we performed gas chromatography-mass spectrometry based metabolomic profiling in serum of unmedicated schizophrenic patients before and after an 8-week risperidone monotherapy, to detect potential biomarkers associated with schizophrenia and risperidone treatment. Twenty-two marker metabolites contributing to the complete separation of schizophrenic patients from matched healthy controls were identified, with citrate, palmitic acid, myo-inositol, and allantoin exhibiting the best combined classification performance. Twenty marker metabolites contributing to the complete separation between posttreatment and pretreatment patients were identified, with myo-inositol, uric acid, and tryptophan showing the maximum combined classification performance. Metabolic pathways including energy metabolism, antioxidant defense systems, neurotransmitter metabolism, fatty acid biosynthesis, and phospholipid metabolism were found to be disturbed in schizophrenic patients and partially normalized following risperidone therapy. Further study of these metabolites may facilitate the development of noninvasive biomarkers and more efficient therapeutic strategies for schizophrenia.

Association analysis of serotonin receptor 7 gene (HTR7) and risperidone response in Chinese schizophrenia patients.

Several lines of evidence suggest that the human 5-HT(7) receptor may be involved in the pharmacodynamics of risperidone and may influence clinical response of the drug. A pharmocogenetics study of this receptor may therefore be useful in developing individualized therapy. But few studies about it have been done. In this study, we genotyped ten single nucleotide polymorphisms (SNPs) distributed throughout the HTR7 gene and analyzed six of them for association with the reduction of Brief Psychiatric Rating Scale (BPRS) scores in drug-naive Chinese schizophrenia patients, following an eight-week period of risperidone monotherapy. The confounding effects of nongenetic factors were estimated and the baseline symptom score as well as the duration of illness were included as covariates for adjustment. No significant correlation of HTR7 with antipsychotic efficacy was detected in either genotype or haplotype analysis. These results demonstrate that variations in the HTR7 gene may not be good genetic markers for predicting the therapeutic efficacy of risperidone.

Immunomagnetic Capture of Big Six Shiga Toxin-Producing Escherichia coli Strains in Apple Juice with Detection by Multiplex Real-Time PCR Eliminates Interference from the Food Matrix.

Having reliable methods for detecting Shiga toxin-producing Escherichia coli (STEC) in foods is an important food safety goal. The majority of STEC outbreaks have involved either the O157:H7 serotype or one of six non-O157 serogroups, O26, O45, O103, O111, O121, and O145, termed "The Big Six." We have compared detection by PCR of the Shiga toxin genes stx1a and stx2a from STEC bacteria isolated from unclarified apple juice by simple centrifugation with the use of an immunocapture technique to minimize contaminants (such as pectin and polyphenols that may copurify with DNA) that may interfere with DNA amplification efficiencies and limit sensitivity. An internal control for successful immunocapture, DNA extraction, and PCR amplification was generated by introducing the pmRaspberry plasmid into an stx null strain, yielding an E. coli O45 pmRaspberry derivative that can be added to food samples directly. Using serial dilutions of a representative Big Six STEC in apple juice, our immunocapture method resulted in a 50% probability of detection value of 3.34, 2.25, and 4.25 CFU for detection by multiplex real-time PCR, growth on solid agar, and multiplex endpoint PCR, respectively. The time to result was 6.5 h, 9.5 h, and 1.5 days for immunocapture of Big Six STECs and detection by multiplex real-time PCR, endpoint PCR, and growth on solid agar, respectively. A set of 52 Big Six STEC isolates and 30 non-Big Six STEC strains was used to establish the inclusivity and exclusivity of the method. Finally, the ability to detect Big Six STEC contamination reliably was confirmed at 4.5 and 45 CFU/25-mL portions of refrigerated apple juice.

Effects of the dopamine D3 receptor (DRD3) gene polymorphisms on risperidone response: a pharmacogenetic study.

Previous observations of the anatomical distribution and pharmacological profile of the dopamine D(3) receptor (DRD3) have indicated its potential role in antipsychotic drug action. Risperidone, an effective first-line atypical antipsychotic agent, exhibits a relatively high affinity for this receptor. Recent studies have reported an association of the Ser9Gly polymorphism in the DRD3 gene with therapeutic response to risperidone, but the results were inconsistent. We therefore postulated that the Ser9Gly polymorphism might be in linkage disequilibrium with an undetected variant that exerts a direct influence on risperidone efficacy. The present study genotyped eight single nucleotide polymorphisms (SNPs) distributed throughout the DRD3 gene and examined five of these for association with treatment outcome, following an 8-week period of risperidone monotherapy in 130 schizophrenic patients from mainland China. Clinical symptoms were assessed before and after the treatment period, using the Brief Psychiatry Rating Scale (BPRS). The confounding effects of non-genetic factors were estimated and the baseline symptom score was included as a covariate for adjustment. Neither was any association observed between the five polymorphisms and improvement in total BPRS scores nor was any combined effect of these variants detected in the haplotype analysis. The current results indicate that genetic variations within the DRD3 gene may not contribute significantly to interindividual differences in the therapeutic efficacy of risperidone.

Mitochondrial dysfunction induced by leflunomide and its active metabolite.

Leflunomide, an anti-inflammatory drug used for the treatment of rheumatoid arthritis, has been marked with a black box warning regarding an increased risk of liver injury. The active metabolite of leflunomide, A771726, which also carries a boxed warning about potential hepatotoxicity, has been marketed as teriflunomide for the treatment of relapsing multiple sclerosis. Thus far, however, the mechanism of liver injury associated with the two drugs has remained elusive. In this study, cytotoxicity assays showed that ATP depletion and subsequent LDH release were induced in a time- and concentration-dependent manner by leflunomide in HepG2 cells, and to a lesser extent, by A77 1726. The decline of cellular ATP levels caused by leflunomide was dramatically exacerbated when galactose was substituted for glucose as the sugar source, indicating a potential mitochondrial liability of leflunomide. By measuring the activities of immuno-captured mitochondrial oxidative phosphorylation (OXPHOS) complexes, we found that leflunomide and A77 1726 preferentially targeted complex V (F1FO ATP synthase), with IC50 values of 35.0 and 63.7 µM, respectively. Bongkrekic acid, a mitochondrial permeability transition pore blocker that targets adenine nucleotide translocase, profoundly attenuated mitochondrial membrane depolarization, ATP depletion, and LDH leakage induced by leflunomide and A77 1726. Substantial alterations of mitochondrial function at the transcript level were observed in leflunomide-treated HepG2 cells, whereas the effects of A77 1726 on the cellular transcriptome were much less profound. Our results suggest that mitochondrial dysfunction may be implicated in the hepatotoxicity associated with leflunomide and A77 1726, with the former exhibiting higher toxicity potency.

A functional SNP in the 3'-UTR of TAP2 gene interacts with microRNA hsa-miR-1270 to suppress the gene expression.

The transporter associated with antigen processing 2 (TAP2) is involved in the development of multidrug resistance and the etiology of immunological diseases. In this study, we investigated whether the expression of TAP2 can be perturbed by single nucleotide polymorphisms (SNPs) located in 3'-untranslated region (3'-UTR) of the gene via interactions with microRNAs. Using a series of in silico assays, we selected the candidate microRNAs (miRNAs) with the potential to interact with functional SNPs of TAP2. The SNP rs241456-located in the 3'-UTR of TAP2-resides in a potential binding site for hsa-miR-1270 and hsa-miR-620. HEK 293 cells, from a human kidney cell line, were used to characterize the extent of binding of miRNAs to each polymorphic allele of the SNP by a luciferase reporter gene assay. RNA electrophoretic mobility shift assays were used to evaluate the interaction between the miRNAs and each allele sequence of the SNP. We found that hsa-miR-1270 inhibited luciferase activity by binding to the T allele of the SNP in an allele-specific manner. A negative correlation was also found between the expression of hsa-miR-1270 and the T allele of the SNP in kidney tissues. Our findings support the hypothesis that hsa-miR-1270 suppresses the production of TAP2 by binding to this SNP in the 3'-UTR of this gene. Environ. Mol. Mutagen. 59:134-143, 2018. © 2017 Wiley Periodicals, Inc.

Response of risperidone treatment may be associated with polymorphisms of HTT gene in Chinese schizophrenia patients.

Serotonin transporter (5-HTT) is a key component of the serotonergic neurotransmitter system. Few studies have focused on polymorphisms of the serotonin transporter and antipsychotic response and, in particular, there have so far been no published studies on the association between the serotonin transporter and response to risperidone. This study examined the relationship between two polymorphisms of the serotonin transporter and the efficacy of risperidone treatment in 129 patients with schizophrenia. Our results revealed that patients with l allele of HTTRLP showed a greater improvement than those without l allele on the overall brief psychiatric rating scale (BPRS) (P=0.025). But no such relationship was found for the HTTVNTR. In haplotype analysis, the frequency of L-12 haplotype showed a significant difference between the responder group and the non-responder group (P=0.005). Our study has, for the first time, produced evidence that the potential for therapy in patients with schizophrenia is related to the HTTRLP polymorphism in the HTT gene and haplotype L-12 may help to predict risperidone treatment efficiency.

Endoplasmic reticulum stress and MAPK signaling pathway activation underlie leflunomide-induced toxicity in HepG2 Cells.

Leflunomide, used for the treatment of rheumatoid arthritis, has been reported to cause severe liver problems and liver failure; however, the underlying mechanisms are not clear. In this study, we used multiple approaches including genomic analysis to investigate and characterize the possible molecular mechanisms of the cytotoxicity of leflunomide in hepatic cells. We found that leflunomide caused endoplasmic reticulum (ER) stress and activated an unfolded protein response, as evidenced by increased expression of related genes including CHOP and GADD34; and elevated protein levels of typical ER stress markers including CHOP, ATF-4, p-eIF2α, and spliced XBP1. The secretion of Gaussia luciferase was suppressed in cells treated with leflunomide in an ER stress reporter assay. Inhibition of ER stress with an ER stress inhibitor 4-phenylbutyrate, and knockdown of ATF-4 and CHOP genes partially protected cells upon leflunomide exposure. In addition, both genomic and biochemical analyses revealed that JNK and ERK1/2 of MAPK signaling pathways were activated, and both contributed to the leflunomide-induced cytotoxicity. Inhibiting JNK activation using a JNK inhibitor attenuated the ER stress and cytotoxicity of leflunomide, whereas inhibiting ERK1/2 using an ERK1/2 inhibitor or ERK1/2 siRNA increased the adverse effect caused by leflunomide, suggesting opposite roles for the two pathways. In summary, our data indicate that both ER stress and the activation of JNK and ERK1/2 contribute to leflunomide-induced cytotoxicity.

Development of HepG2-derived cells expressing cytochrome P450s for assessing metabolism-associated drug-induced liver toxicity.

The generation of reactive metabolites from therapeutic agents is one of the major mechanisms of drug-induced liver injury (DILI). In order to evaluate metabolism-related toxicity and improve drug efficacy and safety, we generated a battery of HepG2-derived cell lines that express 14 cytochrome P450s (CYPs) (1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 3A4, 3A5 and 3A7) individually using a lentiviral expression system. The expression/production of a specific CYP in each cell line was confirmed by an increased abundance of the CYP at both mRNA and protein levels. Moreover, the enzymatic activities of representative CYPs in the corresponding cell lines were also measured. Using our CYP-expressed HepG2 cells, the toxicity of three drugs that could induce DILI (amiodarone, chlorpromazine and primaquine) was assessed, and all of them showed altered (increased or decreased) toxicity compared to the toxicity in drug-treated wild-type HepG2 cells. CYP-mediated drug toxicity examined in our cell system is consistent with previous reports, demonstrating the potential of these cells for assessing metabolism-related drug toxicity. This cell system provides a practical in vitro approach for drug metabolism screening and for early detection of drug toxicity. It is also a surrogate enzyme source for the enzymatic characterization of a particular CYP that contributes to drug-induced liver toxicity.

The role of CYP 3A4 and 1A1 in amiodarone-induced hepatocellular toxicity.

Amiodarone is a widely used potent antiarrhythmic for the treatment of cardiac disease; however, its use is often discontinued due to numerous adverse effects, including hepatotoxicity. To investigate the role of drug metabolism in this liver toxicity, amiodarone and its major metabolite desethylamiodarone were incubated with HepG2 cells overexpressing a series of cytochrome P450 (CYP) isoforms. Significantly higher cytotoxicity of amiodarone was observed in HepG2 cells overexpressing CYP3A4 or CYP1A1, compared with that observed in empty vector transduced control cells. Further, higher levels of the more potent hepatotoxic metabolite desethylamiodarone were detected in CYP3A4 or CYP1A1 expressed cells. The CYP3A4 inhibitor ketoconazole and the CYP1A1 inhibitor α-naphthoflavone drastically inhibited the metabolism of amiodarone to desethylamiodarone. Along with the inhibition of CYP1A1 or CYP3A4, the cytotoxicity of amiodarone was significantly reduced. These data indicate that the metabolism of amiodarone to desethylamiodarone by CYP1A1 or CYP3A4 plays an important role in the hepatocellular toxicity of amiodarone.

HLADR: a database system for enhancing the discovery of biomarkers for predicting human leukocyte antigen-mediated idiosyncratic adverse drug reactions.

AIM: To establish a database for the associations between idiosyncratic drug reactions (IDRs) and human leukocyte antigens (HLAs) and to systematically assess the characteristics of the drug-HLA associations. MATERIALS & METHODS: Electronic databases were searched to extensively identify drug-HLA association studies from 1966 to present. RESULTS: A drug-HLA-IDR database, HLADR, was created. The drug-HLA relationship network clearly reflected an ethnicity dependency of the associations. The positive predictive values and the negative predictive values demonstrated that other potential factors may also regulate the occurrence of HLA-specific IDRs. CONCLUSIONS: Constructing studies with samples from homogeneous ethnic groups and identifying cofactors that affect negative predictive values and positive predictive values will become necessary to enhance the predictability of HLA biomarkers for future research on IDRs.

Ginkgo biloba leaf extract induces DNA damage by inhibiting topoisomerase II activity in human hepatic cells.

Ginkgo biloba leaf extract has been shown to increase the incidence in liver tumors in mice in a 2-year bioassay conducted by the National Toxicology Program. In this study, the DNA damaging effects of Ginkgo biloba leaf extract and many of its constituents were evaluated in human hepatic HepG2 cells and the underlying mechanism was determined. A molecular docking study revealed that quercetin, a flavonoid constituent of Ginkgo biloba, showed a higher potential to interact with topoisomerase II (Topo II) than did the other Ginkgo biloba constituents; this in silico prediction was confirmed by using a biochemical assay to study Topo II enzyme inhibition. Moreover, as measured by the Comet assay and the induction of γ-H2A.X, quercetin, followed by keampferol and isorhamnetin, appeared to be the most potent DNA damage inducer in HepG2 cells. In Topo II knockdown cells, DNA damage triggered by Ginkgo biloba leaf extract or quercetin was dramatically decreased, indicating that DNA damage is directly associated with Topo II. DNA damage was also observed when cells were treated with commercially available Ginkgo biloba extract product. Our findings suggest that Ginkgo biloba leaf extract- and quercetin-induced in vitro genotoxicity may be the result of Topo II inhibition.

Sertraline induces endoplasmic reticulum stress in hepatic cells.

Sertraline is used for the treatment of depression, and is also used for the treatment of panic, obsessive-compulsive, and post-traumatic stress disorders. Previously, we have demonstrated that sertraline caused hepatic cytotoxicity, with mitochondrial dysfunction and apoptosis being underlying mechanisms. In this study, we used microarray and other biochemical and molecular analyses to identify endoplasmic reticulum (ER) stress as a novel molecular mechanism. HepG2 cells were exposed to sertraline and subjected to whole genome gene expression microarray analysis. Pathway analysis revealed that ER stress is among the significantly affected biological changes. We confirmed the increased expression of ER stress makers by real-time PCR and Western blots. The expression of typical ER stress markers such as PERK, IRE1α, and CHOP was significantly increased. To study better ER stress-mediated drug-induced liver toxicity; we established in vitro systems for monitoring ER stress quantitatively and efficiently, using Gaussia luciferase (Gluc) and secreted alkaline phosphatase (SEAP) as ER stress reporters. These in vitro systems were validated using well-known ER stress inducers. In these two reporter assays, sertraline inhibited the secretion of Gluc and SEAP. Moreover, we demonstrated that sertraline-induced apoptosis was coupled to ER stress and that the apoptotic effect was attenuated by 4-phenylbutyrate, a potent ER stress inhibitor. In addition, we showed that the MAP4K4-JNK signaling pathway contributed to the process of sertraline-induced ER stress. In summary, we demonstrated that ER stress is a mechanism of sertraline-induced liver toxicity.

Sertraline, an antidepressant, induces apoptosis in hepatic cells through the mitogen-activated protein kinase pathway.

Sertraline is generally used for the treatment of depression and is also approved for the treatment of panic, obsessive-compulsive, and posttraumatic stress disorders. Previously, using rat primary hepatocytes and isolated mitochondria, we demonstrated that sertraline caused hepatic cytotoxicity and mitochondrial impairment. In the current study, we investigated and characterized molecular mechanisms of sertraline toxicity in human hepatoma HepG2 cells. Sertraline decreased cell viability and induced apoptosis in a dose- and time-dependent manner. Sertraline activated the intrinsic checkpoint protein caspase-9 and caused the release of cytochrome c from mitochondria to cytosol; this process was Bcl-2 family dependent because antiapoptotic Bcl-2 family proteins were decreased. Pretreatment of the HepG2 cells with caspase-3, caspase-8, and caspase-9 inhibitors partially but significantly reduced the release of lactate dehydrogenase, indicating that sertraline-induced apoptosis is mediated by both intrinsic and extrinsic apoptotic pathways. Moreover, sertraline markedly increased the expression of tumor necrosis factor (TNF) and the phosphorylation of JNK, extracellular signal-regulated kinase (ERK1/2), and p38. In sertraline-treated cells, the induction of apoptosis and cell death was shown to be the result of activation of JNK, but not ERK1/2 or p38 in the mitogen-activated protein kinase (MAPK) pathway. Furthermore, silencing MAP4K4, the upstream kinase of JNK, attenuated both apoptosis and cell death caused by sertraline. Taken together, our findings suggest that sertraline induced apoptosis in HepG2 cells at least partially via activation of the TNF-MAP4K4-JNK cascade signaling pathway.

HLA-B*13:01 is associated with salazosulfapyridine-induced drug rash with eosinophilia and systemic symptoms in Chinese Han population.

AIM: Salazosulfapyridine (SASP) frequently causes several adverse reactions, such as drug rash with eosinophilia and systemic symptoms (DRESS). This study aims to assess whether there is an association between SASP-induced DRESS and HLA-A, -B and -C alleles in the Chinese Han population. SUBJECTS & METHODS: We performed an association study of six subjects with SASP-induced DRESS, 30 SASP-tolerant patients and 283 general subjects from the human MHC database, all of whom are Han Chinese. RESULTS: The frequency of the SASP-induced DRESS patients carrying the HLA-B*13:01 allele is 66.67% (4/6). It is significantly higher compared with the general Chinese Han population (15.19%, 43/283; odds ratio: 11.16; p = 0.007) or with the SASP-tolerant patients (13.33%, 4/30; odds ratio: 13.00; p = 0.004). CONCLUSION: These findings show for the first time that in the Chinese Han population, HLA-B*13:01 is associated with SASP-induced DRESS. HLA-B*13:01 might serve as a potential genetic marker for reducing the prevalence of SASP-induced DRESS.

Next-generation sequencing in the clinic: promises and challenges.

The advent of next generation sequencing (NGS) technologies has revolutionized the field of genomics, enabling fast and cost-effective generation of genome-scale sequence data with exquisite resolution and accuracy. Over the past years, rapid technological advances led by academic institutions and companies have continued to broaden NGS applications from research to the clinic. A recent crop of discoveries have highlighted the medical impact of NGS technologies on Mendelian and complex diseases, particularly cancer. However, the ever-increasing pace of NGS adoption presents enormous challenges in terms of data processing, storage, management and interpretation as well as sequencing quality control, which hinder the translation from sequence data into clinical practice. In this review, we first summarize the technical characteristics and performance of current NGS platforms. We further highlight advances in the applications of NGS technologies towards the development of clinical diagnostics and therapeutics. Common issues in NGS workflows are also discussed to guide the selection of NGS platforms and pipelines for specific research purposes.