技术资料

PurposeIsolating circulating tumour cells (CTCs) from the blood is challenging due to their low abundance and heterogeneity. Limitations of conventional CTC detection methods highlight the need for improved strategies to detect and isolate CTCs. Currently,the Food and Drug Administration (FDA)-approved CellSearch™ and other RUO techniques are not available in India. Therefore,we wanted to develop a flexible CTC detection/isolation technique that addresses the limitation(s) of currently available techniques and is suitable for various downstream applications.MethodsWe developed a novel,efficient,user-friendly CTC isolation strategy combining density gradient centrifugation and immuno-magnetic hematogenous cell depletion with fluorescence-activated cell sorting (FACS)-based positive selection using multiple CTC-specific cell-surface markers. For FACS,a stringent gating strategy was optimised to exclude debris and doublets by side scatter/forward scatter (SSC/FSC) discriminator,remove dead cells by 4′,6-diamidino-2-phenylindole (DAPI) staining,and eliminate non-specific fluorescence using a “dump” channel. APC-labelled anti-CD45mAB was used to gate remaining hematogenous cells,while multiple epithelial markers (EpCAM,EGFR,and Pan-Cytokeratin) and an epithelial–mesenchymal transition (EMT) marker (Vimentin) labelled with fluorescein isothiocyanate (FITC) were used to sort cancer cells. The technique was initially developed by spiking Cal 27 cancer cells into the blood of healthy donors and then validated in 95 biopsy-proven oral squamous cell carcinoma (OSCC) patients. CTCs isolated from patients were reconfirmed by Giemsa staining,immuno-staining,and whole transcriptome amplification (WTA),followed by qRT-PCR. In vitro culture and RNA sequencing (RNA-Seq) were also performed to confirm their suitability for various downstream applications.ResultsThe mean detection efficiency for the Cal 27 tongue cancer cells spiked in the whole blood of healthy donors was 32.82% ± 12.71%. While ~75% of our patients (71/95) had detectable CTCs,the CTC positivity was independent of the TNM staging. The isolated potential cancer cells from OSCC patients were heterogeneous in size. They expressed different CTC-specific markers in various combinations as identified by qRT-PCR after WTA in different patients. Isolated CTCs were also found to be suitable for downstream applications like short-term CTC culture and RNA-Seq.ConclusionWe developed a sensitive,specific,flexible,and affordable CTC detection/isolation technique,which is scalable to larger patient cohorts,provides a snapshot of CTC heterogeneity,isolates live CTCs ready for downstream molecular analysis,and,most importantly,is suitable for developing countries. View Publication

Raman spectra often contain undesirable,randomly positioned,intense,narrow-bandwidth,positive,unidirectional spectral features generated when cosmic rays strike charge-coupled device cameras. These must be removed prior to analysis,but doing so manually is not feasible for large data sets. We developed a quick,simple,effective,semi-automated procedure to remove cosmic ray spikes from spectral data sets that contain large numbers of relatively homogenous spectra. Although some inhomogeneous spectral data sets can be accommodated—it requires replacing excessively modified spectra with the originals and removing their spikes with a median filter instead—caution is advised when processing such data sets. In addition,the technique is suitable for interpolating missing spectra or replacing aberrant spectra with good spectral estimates. The method is applied to baseline-flattened spectra and relies on fitting a third-order (or higher) polynomial through all the spectra at every wavenumber. Pixel intensities in excess of a threshold of 3× the noise standard deviation above the fit are reduced to the threshold level. Because only two parameters (with readily specified default values) might require further adjustment,the method is easily implemented for semi-automated processing of large spectral sets. View Publication

AbstractHiBiT is an engineered luciferase’s 11-amino-acid component that can be introduced as a tag at either terminus of a protein of interest. When the LgBiT component and a substrate are present,HiBiT and LgBiT dimerize forming a functional luciferase. The HiBiT technology has been extensively used for high-throughput protein turnover studies in cells. Here,we have adapted the use of the HiBiT technology to quantify messenger RNA (mRNA) translation temporally in vitro in the rabbit reticulocyte system and in cellulo in HEK293 cells constitutively expressing LgBiT. The assay system can uniquely detect differences in cap,5?UTR,modified nucleotide composition,coding sequence optimization and poly(A) length,and their effects on mRNA translation over time. Importantly,using these assays we established the optimal mRNA composition varied depending on the encoded protein of interest,highlighting the importance of screening methods tailored to the protein of interest,and not reliant on reporter proteins. Our findings demonstrated that HiBiT can be easily and readily adapted to monitor real-time mRNA translation in live cells and offers a novel and highly favourable method for the development of mRNA-based therapeutics. Graphical Abstract Graphical Abstract View Publication

The amino acid sequence of a human placental bFGF was determined by a combination of protein and cDNA sequencing. The placental bFGF consists of 157 amino acid residues with a calculated molecular weight of 17,464 and is highly homologous to bovine pituitary bFGF. The human protein contains an amino terminal extension when compared to the sequence established for bovine bFGF (Esch et al.,1985) and to the sequence of the predicted translation product based on human bFGF cDNA clones (Abraham et al.,1986). View Publication

Human pluripotent stem cells (hPSCs),including human embryonic stem cells and induced pluripotent stem cells,are promising for numerous biomedical applications,such as cell replacement therapies,tissue and whole-organ engineering,and high-throughput pharmacology and toxicology screening. Each of these applications requires large numbers of cells of high quality; however,the scalable expansion and differentiation of hPSCs,especially for clinical utilization,remains a challenge. We report a simple,defined,efficient,scalable,and good manufacturing practice-compatible 3D culture system for hPSC expansion and differentiation. It employs a thermoresponsive hydrogel that combines easy manipulation and completely defined conditions,free of any human- or animal-derived factors,and entailing only recombinant protein factors. Under an optimized protocol,the 3D system enables long-term,serial expansion of multiple hPSCs lines with a high expansion rate (∼20-fold per 5-d passage,for a 1072-fold expansion over 280 d),yield (∼2.0 × 107 cells per mL of hydrogel),and purity (∼95% Oct4+),even with single-cell inoculation,all of which offer considerable advantages relative to current approaches. Moreover,the system enabled 3D directed differentiation of hPSCs into multiple lineages,including dopaminergic neuron progenitors with a yield of ∼8 × 107 dopaminergic progenitors per mL of hydrogel and ∼80-fold expansion by the end of a 15-d derivation. This versatile system may be useful at numerous scales,from basic biological investigation to clinical development. View Publication

While a third of the world carries the burden of tuberculosis,disease control has been hindered by a lack of tools,including a rapid,point-of-care diagnostic and a protective vaccine. In many infectious diseases,antibodies (Abs) are powerful biomarkers and important immune mediators. However,in Mycobacterium tuberculosis (Mtb) infection,a discriminatory or protective role for humoral immunity remains unclear. Using an unbiased antibody profiling approach,we show that individuals with latent tuberculosis infection (Ltb) and active tuberculosis disease (Atb) have distinct Mtb-specific humoral responses,such that Ltb infection is associated with unique Ab Fc functional profiles,selective binding to FcγRIII,and distinct Ab glycosylation patterns. Moreover,compared to Abs from Atb,Abs from Ltb drove enhanced phagolysosomal maturation,inflammasome activation,and,most importantly,macrophage killing of intracellular Mtb. Combined,these data point to a potential role for Fc-mediated Ab effector functions,tuned via differential glycosylation,in Mtb control. View Publication

Nonsense mutations are present in 10{\%} of patients with CF,produce a premature termination codon in CFTR mRNA causing early termination of translation,and lead to lack of CFTR function. There are no currently available animal models which contain a nonsense mutation in the endogenous Cftr locus that can be utilized to test nonsense mutation therapies. In this study,we create a CF mouse model carrying the G542X nonsense mutation in Cftr using CRISPR/Cas9 gene editing. The G542X mouse model has reduced Cftr mRNA levels,demonstrates absence of CFTR function,and displays characteristic manifestations of CF mice such as reduced growth and intestinal obstruction. Importantly,CFTR restoration is observed in G542X intestinal organoids treated with G418,an aminoglycoside with translational readthrough capabilities. The G542X mouse model provides an invaluable resource for the identification of potential therapies of CF nonsense mutations as well as the assessment of in vivo effectiveness of these potential therapies targeting nonsense mutations. View Publication

Recent large-scale genomics efforts to characterize the cis-regulatory sequences that orchestrate genome-wide expression patterns have produced impressive catalogues of putative regulatory elements. Most of these sequences have not been functionally tested,and our limited understanding of the non-coding genome prevents us from predicting which sequences are bona fide cis-regulatory elements. Recently,massively parallel reporter assays (MPRAs) have been deployed to measure the activity of putative cis-regulatory sequences in several biological contexts,each with specific advantages and distinct limitations. We developed LV-MPRA,a novel lentiviral-based,massively parallel reporter gene assay,to study the function of genome-integrated regulatory elements in any mammalian cell type; thus,making it possible to apply MPRAs in more biologically relevant contexts. We measured the activity of 2,600 sequences in U87 glioblastoma cells and human neural progenitor cells (hNPCs) and explored how regulatory activity is encoded in DNA sequence. We demonstrate that LV-MPRA can be applied to estimate the effects of local DNA sequence and regional chromatin on regulatory activity. Our data reveal that primary DNA sequence features,such as GC content and dinucleotide composition,accurately distinguish sequences with high activity from sequences with low activity in a full chromosomal context,and may also function in combination with different transcription factor binding sites to determine cell type specificity. We conclude that LV-MPRA will be an important tool for identifying cis-regulatory elements and stimulating new understanding about how the non-coding genome encodes information. View Publication

Host proteins are essential for HIV entry and replication and can be important nonviral therapeutic targets. Large-scale RNA interference (RNAi)-based screens have identified nearly a thousand candidate host factors,but there is little agreement among studies and few factors have been validated. Here we demonstrate that a genome-wide CRISPR-based screen identifies host factors in a physiologically relevant cell system. We identify five factors,including the HIV co-receptors CD4 and CCR5,that are required for HIV infection yet are dispensable for cellular proliferation and viability. Tyrosylprotein sulfotransferase 2 (TPST2) and solute carrier family 35 member B2 (SLC35B2) function in a common pathway to sulfate CCR5 on extracellular tyrosine residues,facilitating CCR5 recognition by the HIV envelope. Activated leukocyte cell adhesion molecule (ALCAM) mediates cell aggregation,which is required for cell-to-cell HIV transmission. We validated these pathways in primary human CD4(+) T cells through Cas9-mediated knockout and antibody blockade. Our findings indicate that HIV infection and replication rely on a limited set of host-dispensable genes and suggest that these pathways can be studied for therapeutic intervention. View Publication

The complete array of genes required for terminal erythroid differentiation remains unknown. To address this knowledge gap,we perform a genome-scale CRISPR knock-out screen in the human erythroid progenitor cell line HUDEP-2 and validate candidate regulators of erythroid differentiation in a custom secondary screen. Comparison of sgRNA abundance in the CRISPR library,proerythroblasts,and orthochromatic erythroblasts,resulted in the identification of genes that are essential for proerythroblast survival and genes that are required for terminal erythroid differentiation. Among the top genes identified are known regulators of erythropoiesis,underscoring the validity of this screen. Notably,using a Log2 fold change of <−1 and false discovery rate of <0.01,the screen identified 277 genes that are required for terminal erythroid differentiation,including multiple genes not previously nominated through GWAS. NHLRC2,which was previously implicated in hemolytic anemia,was a highly ranked gene. We suggest that anemia due to NHLRC2 mutation results at least in part from a defect in erythroid differentiation. Another highly ranked gene in the screen is VAC14,which we validated for its requirement in erythropoiesis in vitro and in vivo. Thus,data from this CRISPR screen may help classify the underlying mechanisms that contribute to erythroid disorders. Subject terms: Erythropoiesis,CRISPR-Cas9 genome editing,Haematopoietic stem cells View Publication