Amphotericin B release rate is the link between drug status in the liposomal bilayer and toxicity

Amphotericin B(AmB)is an amphiphilic drug commonly formulated in liposomes and administered intravenously to treat systemic fungal infections.Recent studies on the liposomal drug product have shed light on the AmB aggregation status in the bilayer,which heat treatment(curing)modifies.Although toxicity was found related to aggregation status-loose aggregates significantly more toxic than tight aggregates-the precise mechanism linking aggregation and toxicitywas notwell understood.This study directlymeasured drug release rate fromvarious AmB liposomal preparations made with modified curing protocols to evaluate correlations among drug aggregation state,drug release,and in vitro toxicity.UV–Vis spectroscopy of these products detected unique curing-induced changes in the UV spectral features:a∼25nm blue-shift of the main absorption peak(λ_(max))in aqueous buffer and a decrease in the OD_(346)/OD_(322) ratio upon thermal curing,reflecting tighter aggregation.In vitro release testing(IVRT)data showed,by applying and fitting first-order release kinetic models for one or two pools,that curing impacts two significant changes:a 3–5-fold drop in the overall drug release rate and a ten-fold decrease in the ratio between the loosely aggregated and the tightly aggregated,more thermodynamically stable drug pool.The kinetic data thus corroborated the trend independently deduced from the UV–Vis spectral data.The in vitro toxicity assay indicated a decreased toxicity with curing,as shown by the significantly increased concentration,causing half-maximal potassium release(TC50).The data suggest that the release of AmB requires dissociation of the tight complexes within the bilayer and that the reduced toxicity relates to this slower rate of dissociation.This study demonstrates the relationship between AmB aggregation status within the lipid bilayer and drug release(directly measured rate constants),providing a mechanistic link between aggregation status and in vitro toxicity in the liposomal formulations.

Single-cell RNA-sequencing and subcellular spatial transcriptomics facilitate the translation of liver microphysiological systems for regulatory application

Reducing the use of animal models in drug development and safety assessment has long been supported by the U.S.Food and Drug Administration(FDA).The report by Royal Society for the Prevention of Cruelty to Animals indicates that in 2020,experiments involved the use of over 100 million animals,with the United States leading the list by utilizing 20 million animals.Beyond ethical considerations associated with animal testing and the costs in terms of time and money,animal models are not always effective in predicting human reactions to drug exposure.While animal testing has been the traditional method for assessing the safety and efficacy of drugs.

Levels of Chemical Toxicants in Waterpipe Tobacco and Waterpipe Charcoal Solid Waste

This work provides insights on waterpipe tobacco and waterpipe charcoal as potential sources of environmental toxicants. Selected harmful and potentially harmful constituents (HPHCs) from ten U.S. commercial waterpipe tobacco filler products (before and after electric heating) and five waterpipe charcoal products (before and after burning) were investigated. The differences in quantities of HPHCs between the evaluated products appear to be affected by raw material properties and/or the manufacturing processes involved in product production. Trace metal quantities in waterpipe tobacco and charcoal products were observed after heating or burning conditions compared to unheated or unburned conditions, which could impact the environment through the generation of toxic tobacco product waste. This study demonstrates that waterpipe tobacco and waterpipe charcoal contain substantial quantities of benzo[a]pyrene (B[a]P) and trace metals (i.e., selenium, arsenic, cadmium, chromium, cobalt, lead, nickel) before use and that extensive and varied changes in trace metal quantities take place as a result of heating, and more studies are needed to estimate the magnitude of the environmental impact of waterpipe tobacco use.

Homology Model and Ligand Binding Interactions of the Extracellular Domain of the Human α4β2 Nicotinic Acetylcholine Receptor

Addiction to nicotine, and possibly other tobacco constituents, is a major factor that contributes to the difficulties smokers face when attempting to quit smoking. Amongst the various subtypes of nicotinic acetylcholine receptors (nAChRs), the α4β2 subtype plays an important role in mediating the addiction process. The characterization of human α4β2-ligand binding interactions provides a molecular framework for understanding ligand-receptor interactions, rendering insights into mechanisms of nicotine addiction and may furnish a tool for efficiently identifying ligands that can bind the nicotine receptor. Therefore, we constructed a homology model of human α4β2 nAChR and performed molecular docking and molecular dynamics (MD) simulations to elucidate the potential human α4β2-ligand binding modes for eleven compounds known to bind to this receptor. Residues V96, L97 and F151 of the α4 subunit and L111, F119 and F121 of the β2 subunit were found to be involved in hydrophobic interactions while residues S153 and W154 of the α4 subunit were involved in the formation of hydrogen bonds between the receptor and respective ligands. The homology model and its eleven ligand-bound structures will be used to develop a virtual screening program for identifying tobacco constituents that are potentially addictive.

食品中农药残留的高通量分析之展望(英文)

The screening of pesticide residues plays a vital role in food safety.Applications of high throughput analytical procedures are desirable for screening a large number of pesticides and food samples in a time-efficient and cost-effective manner.This review discusses how sample throughput of pesticide analysis could be improved with an emphasis on sample preparation,instrumentation and data analysis.

AB039.Peptidyl-prolyl cis-trans isomerase A(PPIA)-a novel biomarker of multi-episodic(recurrent)ocular toxoplasmosis

Background:Ocular toxoplasmosis(OT)is the most common etiology of posterior uveitis.The high incidence of macular scarring associated with OT is a leading cause of visual morbidity.Serum biomarkers of the disease would aid in its diagnosis.This work was designed as a pilot study to detect OT potential biomarkers.Methods:Blood samples were collected from four groups of nine patients each;toxoplasmosis IgG-with no history of uveitis,non-toxoplasmosis uveitic,first episode OT,and symptomatic recurrent OT.Plasma serum was isolated and subjected to proteomics analysis using 2D gel electrophoresis(GE)and surface-enhanced laser desorption ionization mass spectrometry(SELDI-MS).Selected proteins were separated by GE and sequenced using tandem MS.Results:Fifty markers of OT and 46 markers of recurrent disease were discovered by MS;47%were cross-validated;14 biomarkers were selected for verification by 1D-GE.2D-GE analysis yielded 57 differentially expressed bands,20 of which were excised and identified.One serum protein,peptidyl-prolyl cis-trans isomerase A,was validated to be a biomarker of multi-episodic OT by immunoblotting in patient and control samples.Conclusions:This pilot study sought,for the first time,to elucidate plasma serum biomarkers for OT.This study demonstrates the potential for SELDI-MS and well as other MS technologies to identify novel disease biomarkers.

Functional Analysis of Semi-conserved Transit Peptide Motifs and Mechanistic Implications in Precursor Targeting and Recognition

Over 95% of plastid proteins are nuclear-encoded as their precursors containing an N-terminal extension known as the transit peptide （TP）. Although highly variable, TPs direct the precursors through a conserved, posttranslational mechanism involving translocons in the outer （TOC） and inner envelope （TOC）. The organelle import specificity is mediated by one or more components of the Toc complex. However, the high TP diversity creates a paradox on how the sequences can be specifically recognized. An emerging model of TP design is that they contain multiple loosely conserved motifs that are recognized at different steps in the targeting and transport process. Bioinformatics has demonstrated that many TPs contain semiconserved physicochemical motifs, termed FGLK. In order to characterize FGLK motifs in TP recognition and import, we have analyzed two well-studied TPs from the precursor of RuBisCO small subunit （SStp） and ferredoxin （Fdtp）. Both SStp and Fdtp contain two FGLK motifs. Analysis of large set mutations （-85） in these two motifs using in vitro, in organello, and in vivo approaches support a model in which the FGLK domains mediate interaction with TOC34 and possibly other TOC components. In vivo import analysis suggests that multiple FGLK motifs are functionally redundant. Furthermore, we discuss how FGLK motifs are required for efficient precursor protein import and how these elements may permit a convergent function of this highly variable class of targeting sequences.

Sequencing XMET genes to promote genotype-guided risk assessment and precision medicine

High-throughput next generation sequencing (NGS) is a shotgun approach applied in a parallel fashion by which the genome is fragmented and sequenced through small pieces and then analyzed either by aligning to a known reference genome or by de novo assembly without reference genome.This technology has led researchers to conduct an explosion of sequencing related projects in multidisciplinary fields of science.However,due to the limitations of sequencing-based chemistry,length of sequencing reads and the complexity of genes,it is difficult to determine the sequences of some portions of the human genome,leaving gaps in genomic data that frustrate further analysis.Particularly,some complex genes are difficult to be accurately sequenced or mapped because they contain high GC-content and/or low complexity regions,and complicated pseudogenes,such as the genes encoding xenobiotic metabolizing enzymes and transporters (XMETs).The genetic variants in XMET genes are critical to predicate interindividual variability in drug efficacy,drug safety and susceptibility to environmental toxicity.We summarized and discussed challenges,wet-lab methods,and bioinformatics algorithms in sequencing "complex" XMET genes,which may provide insightful information in the application of NGS technology for implementation in toxicogenomics and pharmacogenomics.

QuaPra: Efficient transcript assembly and quantification using quadratic programming with Apriori algorithm

RNA sequencing(RNA-seq) has greatly facilitated the exploring of transcriptome landscape for diverse organisms.However,transcriptome reconstruction is still challenging due to various limitations of current tools and sequencing technologies.Here,we introduce an efficient tool,QuaPra(Quadratic Programming combined with Apriori),for accurate transcriptome assembly and quantification.QuaPra could detect at least 26.5% more low abundance(0.1–1 FPKM) transcripts with over 2.7% increase of sensitivity and precision on simulated data compared to other currently popular tools.Moreover,around one-quarter more known transcripts were correctly assembled by QuaPra than other assemblers on real sequencing data.QuaPra is freely available at http://www.megabionet.org/QuaPra/.

Antimicrobial susceptibility testing in veterinary medicine:performance,interpretation of results,best practices and pitfalls

The performance of antimicrobial susceptibility testing(AST)of bacteria and the interpretation of AST results for bacteria isolated from animals are complex tasks which must be performed using standard published methodology and overseen by experts in clinical microbiology and in consultation with clinical pharmacologists.Otherwise,AST has significant potential for errors and mistakes.In this review,we provide guidance on how to correctly perform AST of bacteria isolated from animals and interpret the AST results.Particular emphasis is placed on the various approved or published methodologies for the different bacteria as well as the application of interpretive criteria,including clinical breakpoints and epidemiological cut-off values(ECVs/ECOFFs).Application of approved interpretive criteria and definitions of susceptible,susceptible dose-dependent,nonsusceptible,intermediate,and resistant for clinical breakpoints as well as wild-type and non-wildtype for ECVs,are explained and the difficulties resulting from the lack of approved clinical breakpoints for other bacteria,indications,and animal species is discussed.The requirement of quality controls in any AST approach is also emphasized.In addition,important parameters,often used in monitoring and surveillance studies,such as MIC50,MIC90,and testing range,are explained and criteria for the classification of bacteria as multidrug-resistant,extensively drug-resistant or pandrug-resistant are provided.Common mistakes are presented and the means to avoid them are described.To provide the most accurate AST,one must strictly adhere to approved standards or validated methodologies,like those of the Clinical and Laboratory Standards Institute or other internationally accepted AST documents and the detailed information provided therein.

Mechanisms of immune checkpoint inhibitormediated liver injury

The immune checkpoints,cytotoxic T-lymphocyte-associated antigen 4(CTLA-4) and programmed cell death protein-1/ligand-1(PD-1/PD-L1) are vital contributors to immune resulation and tolerance.Recently immune checkpoint inhibitors(ICIs) have revolutionized cancer therapy;however,they come with the cost of immune related adverse events involving multiple organs such as the liver.Due to its constant expo sure to foreign antigens,the liver has evolved a high capacity for immune tolerance,therefore,blockade of the immune checkpoints can result in aberrant immune activation affecting the liver in up to 20% of patients depending on the agent(s) used and underlying factors.This type of hepatotoxicity is termed immune mediated liver injury from checkpoint inhibitors(ILICI) and is more common when CTLA4 and PD-1/PDL1 are used in combination.The underlying mechanisms of this unique type of hepatotoxicity are not fully understood;however,the contribution of CD8^(+) cytotoxic T lymphocytes,various CD4^(+) T cells populations,cytokines,and the secondary activation of the innate immune system leading to liver injury have all been suggested.This review summarizes our current understanding of the underlying mechanisms of liver injury in immunotherapy using animal models of ILICI and available patient data from clinical studies.

Harnessing the collective properties of nanoparticle ensembles for cancer theranostics

Individual inorganic nanoparticles （NPs） have been widely used in the fields of drug delivery, cancer imaging and therapy. There are still many hurdles that limit the performance of individual NPs for these applications. The utilization of highly ordered NP ensembles opens a door to resolve these problems, as a result of their new or advanced collective properties. The assembled NPs show several advantages over individual NP-based systems, such as improved cell internalization and tumor targeting, enhanced multimodality imaging capability, superior combination therapy arising from synergistic effects, possible complete clearance from the whole body by degradation of assemblies into original small NP building blocks, and so on. In this review, we discuss the potential of utilizing assembled NP ensembles for cancer imaging and treatment by taking plasmonic vesicular assemblies of Au NPs as an example. We first summarize the recent developments in the self-assembly of plasmonic vesicular structures of NPs from amphiphilic polymer-tethered NP building blocks. We further review the utilization of plasmonic vesicles of NPs for cancer imaging （e.g. multi-photon induced luminescence, photothermal, and photoacoustic imaging）, and cancer therapy （e.g., photothermal therapy, and chemotherapy）. Finally, we outline current challenges and our perspectives along this line.

FDA's Opioids Action Plan: A Midyear Checkup

As FDA works to address the opioid epidemic of abuse, misuse and addiction, it＇s valuable to see firsthand some of the ways the crisis is affecting our communities.

Innovative confocal laser method for exact dioptric power measurement of intraocular lens implants Invited Paper

We present a novel confocal laser method （CLM） for precise testing of the dioptric power of both positive and negative intraocular lens （IOL） implants. The CLM principle is based on a simple fiber-optic confocal laser design including a single-mode fiber coupler that serves simultaneously as a point light source used for formation of a collimated Gaussian laser beam, and as a highly sensitive confocal point receiver. The CLM approach provides an accurate, repeatable, objective, and fast method for IOL dioptric power measurement over the range from 0 D to greater than ±30 D under both dry and in-situ simulated conditions.

Application of genome analysis strategies in the clinical testing for pediatric diseases

Next-generation sequencing (NGS) is being used in clinical testing.Government authorities in both China and the United States are overseeing the clinical application of NGS instruments and reagents.In addition,the US Association for Molecular Pathology and the College of American Pathologists have jointly released a guidance to standardize the analysis and interpretation of NGS data involved in clinical testing.At present,the analysis strategies and pipelines for NGS data related to the clinical detection of pediatric disease are similar to those used for adult diseases.However,for rare pediatric diseases without linkage to known genetic variants,it is currently difficult to detect the relevant pathogenic genes using NGS technology.Additionally,it is challenging to identify novel pathogenic genes of familial pediatric tumors.Therefore,characterization of the pathogenic genes associated with above diseases is important for the diagnosis and treatment of rare diseases in children.This article introduces the general pipelines for NGS data analyses of diseases and elucidates data analysis strategies for the pathogenic genes of rare pediatric diseases and familial pediatric tumors.