Efficient tagging of endogenous proteins in human cell lines for structural studies by single-particle cryo-EM.

CRISPR/Cas9-based genome engineering has revolutionized our ability to manipulate biological systems, particularly in higher organisms. Here, we designed a set of homology-directed repair donor templates that enable efficient tagging of endogenous proteins with affinity tags by transient transfection and selection of genome-edited cells in various human cell lines. Combined with technological advancements in single-particle cryogenic electron microscopy, this strategy allows efficient structural studies of endogenous proteins captured in their native cellular environment and during different cellular processes. We demonstrated this strategy by tagging six different human proteins in both HEK293T and Jurkat cells. Moreover, analysis of endogenous glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in HEK293T cells allowed us to follow its behavior spatially and temporally in response to prolonged oxidative stress, correlating the increased number of oxidation-induced inactive catalytic sites in GAPDH with its translocation from cytosol to nucleus.

Mechanisms related to carbon nanotubes genotoxicity in human cell lines of respiratory origin.

Potential genotoxicity and carcinogenicity of carbon nanotubes (CNT), as well as the underlying mechanisms, remains a pressing topic. The study aimed to evaluate and compare the genotoxic effect and mechanisms of DNA damage under exposure to different types of CNT. Immortalized human cell lines of respiratory origin BEAS-2B, A549, MRC5-SV40 were exposed to three types of CNT: MWCNT Taunit-M, pristine and purified SWCNT TUBALL™ at concentrations in the range of 0.0006-200 µg/ml. Data on the CNT content in the workplace air were used to calculate the lower concentration limit. The genotoxic potential of CNTs was investigated at non-cytotoxic concentrations using a DNA comet assay. We explored reactive oxygen species (ROS) formation, direct genetic material damage, and expression of a profibrotic factor TGFB1 as mechanisms related to genotoxicity upon CNT exposure. An increase in the number of unstable DNA regions was observed at a subtoxic concentration of CNT (20 µg/ml), with no genotoxic effects at concentrations corresponding to industrial exposures being found. While the three test articles of CNTs exhibited comparable genotoxic potential, their mechanisms appeared to differ. MWCNTs were found to penetrate the nucleus of respiratory cells, potentially interacting directly with genetic material, as well as to enhance ROS production and TGFB1 gene expression. For A549 and MRC5-SV40, genotoxicity depended mainly on MWCNT concentration, while for BEAS-2B - on ROS production. Mechanisms of SWCNT genotoxicity were not so obvious. Oxidative stress and increased expression of profibrotic factors could not fully explain DNA damage under SWCNT exposure, and other mechanisms might be involved.

Evaluation of the Potential Cytoprotective Effect of Melatonin in Comparison with Vitamin E and Trolox against Cd2+-Induced Toxicity in SH-SY5Y, HCT 116, and HepG2 Cell Lines.

Cadmium (Cd) toxicity poses a significant threat to cellular health, leading to oxidative stress and cell damage. Antioxidant agents, particularly those of natural origin, have been studied as a potential alternative for mitigating heavy metal toxicity. This study aimed to evaluate the cytoprotective effects of the antioxidant melatonin (MLT) in comparison with Vitamin E (VitE) and Trolox against Cd2+-induced cellular toxicity. The MTT assay was employed to assess cell viability in neuronal SH-SY5Y, colorectal HCT 116, and hepatic HepG2 cell lines. The results showed that all three antioxidants offered some level of protection against Cd toxicity, with Vitamin E proving to be the most effective. MLT also demonstrated a substantial cytoprotective effect, especially at the highest Cd concentration of 30 µM. These findings suggest that MLT, alongside Vit E and Trolox, could be valuable in mitigating the detrimental effects of Cd exposure by reducing the oxidative stress in these cellular models.

Next-Gen Dual Transcriptomics for Adult Extrapulmonary Tuberculosis Biomarkers and Host-Pathogen Interplay in Human Cells: A Strategic Review.

Tuberculosis (TB) is a major public health concern that results in significant morbidity and mortality, particularly in middle- to low-income countries. Extra-pulmonary tuberculosis (EPTB) in adults is a form of TB that affects organs other than the lungs and is challenging to diagnose and treat due to a lack of accurate early diagnostic markers and inadequate knowledge of host immunity. Next-generation sequencing-based approaches have shown potential for identifying diagnostic biomarkers and host immune responses related to EPTB. This strategic review discusses on the significance using primary human cells and cell lines for in vitro transcriptomic studies on common forms of EPTB, such as lymph node TB, brain TB, bone TB, and endometrial TB to derive potential insights. While organoids have shown promise as a model system, primary cell lines still remain a valuable tool for studying host-pathogen interplay due to their conserved immune system, non-iPSC origin, and lack of heterogeneity in cell population. This review outlines a basic workflow for researchers interested in performing transcriptomics studies in EPTB, and also discusses the potential of cell-line based dual RNA-Seq technology for deciphering comprehensive transcriptomic signatures, host-pathogen interplay, and biomarkers from the host and Mycobacterium tuberculosis. Thus, emphasizing the implementation of this technique which can significantly contribute to the global anti-TB effort and advance our understanding of EPTB.

Studying the Functional Potential of Ground Ivy (Glechoma hederacea L.) Extract Using an In Vitro Methodology.

Glechoma hederacea L., known as ground ivy, has a long history of use in folk medicine. The main bioactive compounds in ground ivy are polyphenolic compounds known for their potent antioxidant and antimicrobial activities and thus have high potential as functional ingredients against bacterial infections and the occurrence of chronic diseases associated with oxidative stress in the human body. The aim of the present study was to determine the biological activity of ground ivy extract on selected human cell lines, including hepatic (HepG2), tongue (CAL 27), gastric (AGS) and colon (Caco-2) cancer cell lines by evaluating cytotoxicity, formation of reactive oxygen species and genotoxicity. The antioxidant capacity of the extract was additionally evaluated using cellular model macromolecules of protein and DNA, bovine serum album and plasmid phiX174 RF1 DNA. The effect of ground ivy extract on representatives of human microflora, including L. plantarum, E. coli and S. aureus, was also studied. The cytotoxicity of the extract depended on the type of cells treated, and the pro-oxidant effect generally decreased with increasing exposure time. The most pronounced genoprotective effect against hydroxyl radical damage was monitored in model plasmid DNA and occurred at the highest tested concentration (0.25 mg mL-1), with 95.89% preservation of the supercoiled form of the plasmid. This concentration also had the most significant antioxidant activity on the model protein-14.01% more than the positive control prepared using Trolox. The ground ivy extract showed high antimicrobial potential against the pathogenic bacteria E. coli and S. aureus.

SARS-CoV-2 Disrupts Proximal Elements in the JAK-STAT Pathway.

SARS-CoV-2 can infect multiple organs, including lung, intestine, kidney, heart, liver, and brain. The molecular details of how the virus navigates through diverse cellular environments and establishes replication are poorly defined. Here, we generated a panel of phenotypically diverse, SARS-CoV-2-infectible human cell lines representing different body organs and performed longitudinal survey of cellular proteins and pathways broadly affected by the virus. This revealed universal inhibition of interferon signaling across cell types following SARS-CoV-2 infection. We performed systematic analyses of the JAK-STAT pathway in a broad range of cellular systems, including immortalized cells and primary-like cardiomyocytes, and found that SARS-CoV-2 targeted the proximal pathway components, including Janus kinase 1 (JAK1), tyrosine kinase 2 (Tyk2), and the interferon receptor subunit 1 (IFNAR1), resulting in cellular desensitization to type I IFN. Detailed mechanistic investigation of IFNAR1 showed that the protein underwent ubiquitination upon SARS-CoV-2 infection. Furthermore, chemical inhibition of JAK kinases enhanced infection of stem cell-derived cultures, indicating that the virus benefits from inhibiting the JAK-STAT pathway. These findings suggest that the suppression of interferon signaling is a mechanism widely used by the virus to evade antiviral innate immunity, and that targeting the viral mediators of immune evasion may help block virus replication in patients with COVID-19. IMPORTANCE SARS-CoV-2 can infect various organs in the human body, but the molecular interface between the virus and these organs remains unexplored. In this study, we generated a panel of highly infectible human cell lines originating from various body organs and employed these cells to identify cellular processes commonly or distinctly disrupted by SARS-CoV-2 in different cell types. One among the universally impaired processes was interferon signaling. Systematic analysis of this pathway in diverse culture systems showed that SARS-CoV-2 targets the proximal JAK-STAT pathway components, destabilizes the type I interferon receptor though ubiquitination, and consequently renders the infected cells resistant to type I interferon. These findings illuminate how SARS-CoV-2 can continue to propagate in different tissues even in the presence of a disseminated innate immune response.

Label-Free Study of the Global Cell Behavior during Exposure to Environmental Radiofrequency Fields in the Presence or Absence of Pro-Apoptotic or Pro-Autophagic Treatments.

It remains controversial whether exposure to environmental radiofrequency signals (RF) impacts cell status or response to cellular stress such as apoptosis or autophagy. We used two label-free techniques, cellular impedancemetry and Digital Holographic Microscopy (DHM), to assess the overall cellular response during RF exposure alone, or during co-exposure to RF and chemical treatments known to induce either apoptosis or autophagy. Two human cell lines (SH-SY5Y and HCT116) and two cultures of primary rat cortex cells (astrocytes and co-culture of neurons and glial cells) were exposed to RF using an 1800 MHz carrier wave modulated with various environmental signals (GSM: Global System for Mobile Communications, 2G signal), UMTS (Universal Mobile Telecommunications System, 3G signal), LTE (Long-Term Evolution, 4G signal, and Wi-Fi) or unmodulated RF (continuous wave, CW). The specific absorption rates (S.A.R.) used were 1.5 and 6 W/kg during DHM experiments and ranged from 5 to 24 W/kg during the recording of cellular impedance. Cells were continuously exposed for three to five consecutive days while the temporal phenotypic signature of cells behavior was recorded at constant temperature. Statistical analysis of the results does not indicate that RF-EMF exposure impacted the global behavior of healthy, apoptotic, or autophagic cells, even at S.A.R. levels higher than the guidelines, provided that the temperature was kept constant.

Wheat Germ Spermidine and Clove Eugenol in Combination Stimulate Autophagy In Vitro Showing Potential in Supporting the Immune System against Viral Infections.

Impaired autophagy, responsible for increased inflammation, constitutes a risk factor for the more severe COVID-19 outcomes. Spermidine (SPD) is a known autophagy modulator and supplementation for COVID-19 risk groups (including the elderly) is recommended. However, information on the modulatory effects of eugenol (EUG) is scarce. Therefore, the effects of SPD and EUG, both singularly and in combination, on autophagy were investigated using different cell lines (HBEpiC, SHSY5Y, HUVEC, Caco-2, L929 and U937). SPD (0.3 mM), EUG (0.2 mM) and 0.3 mM SPD + 0.2 mM EUG, significantly increased autophagy using the hallmark measure of LC3-II protein accumulation in the cell lines without cytotoxic effects. Using Caco-2 cells as a model, several crucial autophagy proteins were upregulated at all stages of autophagic flux in response to the treatments. This effect was verified by the activation/differentiation and migration of U937 monocytes in a three-dimensional reconstituted intestinal model (Caco-2, L929 and U937 cells). Comparable benefits of SPD, EUG and SPD + EUG in inducing autophagy were shown by the protection of Caco-2 and L929 cells against lipopolysaccharide-induced inflammation. SPD + EUG is an innovative dual therapy capable of stimulating autophagy and reducing inflammation in vitro and could show promise for COVID-19 risk groups.

Heterogeneity of telomerase reverse transcriptase mutation and expression, telomerase activity and telomere length across human cancer cell lines cultured in vitro.

Promoter region of the telomerase reverse transcriptase gene (TERTp) constitutes a regulatory element capable to affect TERT expression (TE), telomerase activity (TA) and telomere length (TL). TERTp mutation status, TL, TA and TE were assessed in 27 in vitro cultured human cell lines, including 11 solid tumour, 13 haematological and 3 normal cell lines. C228T and C250T TERTp mutations were detected in 5 solid tumour and none of haematological cell lines (p = 0.0100). As compared to other solid tumour cell lines, those with the presence of somatic mutations were characterized by: shorter TL, lower TA and TE. Furthermore, cell lines carrying TERTp mutations showed a linear correlation between TE and TA (R = 0.9708, p = 0.0021). Moreover, haematological cell lines exhibited higher TE compared to solid tumour cell lines (p = 0.0007). TL and TA were correlated in both solid tumour (R = 0.4875, p = 0.0169) and haematological (R = 0.4719, p = 0.0095) cell lines. Our results based on the in vitro model suggest that oncogenic processes may differ between solid tumours and haematological malignancies with regard to their TERT gene regulation mechanisms.

Time-related changes in cell morphology and biomarker immunoreactivity for cells stored in a buffer-based cell medium.

OBJECTIVE: Buffer-based cell media (BBCM) are a valuable tool in the post-collection processing of cytology samples, though with poorly defined effects on cell properties. In this study, time-related changes in cell morphology and biomarker immunoreactivity were evaluated for cells stored at room temperature in a BBCM prepared with bovine serum albumin (BSA) and ethylene diamine tetraacetic acid (EDTA). METHODS: Cytospins were prepared at five consecutive 24-hour intervals (0, 24, 48, 72, 96) from three human cell lines (MCF7, SK-MEL-28, FaDu) suspended and stored in BBCM. Preservation of cell morphology was evaluated on Papanicolaou-stained cytospins from the percentages of apoptotic cells. Preservation of immunoreactivity was evaluated for cytokeratins, oestrogen receptors, Ki67, and melanoma markers from the percentages of cells positive for the corresponding immunocytochemical reactions. RESULTS: Cell morphology was well preserved for the majority of cells of the three lines stored for 24 and 48 hours (93%, 97%, 98% and 62%, 81%, 88%, respectively), while the majority of cells were apoptotic after 72 and 96 hours (70%, 47%, 39% and 77%, 70%, 59%, respectively). The immunoreactivity of cytokeratins remained unchanged during the entire 96 hours, while that of melanoma markers (S100, HMB45, Melan-A) decreased by 27%, 2%, and 3%, respectively. The immunoreactivity of oestrogen receptors and Ki67 decreased by 29% and 17% after the first 24 hours, and was completely lost after 96 hours. CONCLUSIONS: A BBCM with the addition of BSA and EDTA facilitates good preservation of cell morphology and immunoreactivity of biomarkers for up to 48 hours at room temperature.

An amino acid change in nsP4 of chikungunya virus confers fitness advantage in human cell lines rather than in Aedes albopictus.

Chikungunya virus (CHIKV) has caused large-scale epidemics of fever, rash and arthritis since 2004. This unprecedented re-emergence has been associated with mutations in genes encoding structural envelope proteins, providing increased fitness in the secondary vector Aedes albopictus. In the 2008-2013 CHIKV outbreaks across Southeast Asia, an R82S mutation in non-structural protein 4 (nsP4) emerged early in Malaysia or Singapore and quickly became predominant. To determine whether this nsP4-R82S mutation provides a selective advantage in host cells, which may have contributed to the epidemic, the fitness of infectious clone-derived CHIKV with wild-type nsP4-82R and mutant nsP4-82S were compared in Ae. albopictus and human cell lines. Viral infectivity, dissemination and transmission in Ae. albopictus were not affected by the mutation when the two variants were tested separately. In competition, the nsP4-82R variant showed an advantage over nsP4-82S in dissemination to the salivary glands, but only in late infection (10 days). In human rhabdomyosarcoma (RD) and embryonic kidney (HEK-293T) cell lines coinfected at a 1 : 1 ratio, wild-type nsP4-82R virus was rapidly outcompeted by nsP4-82S virus as early as one passage (3 days). In conclusion, the nsP4-R82S mutation provides a greater selective advantage in human cells than in Ae. albopictus, which may explain its apparent natural selection during CHIKV spread in Southeast Asia. This is an unusual example of a naturally occurring mutation in a non-structural protein, which may have facilitated epidemic transmission of CHIKV.

Joint analyses of multi-tissue Hi-C and eQTL data demonstrate close spatial proximity between eQTLs and their target genes.

BACKGROUND: Gene regulation is important for cells and tissues to function. It has been studied from two aspects at the genomic level, the identification of expression quantitative trait loci (eQTLs) and identification of long-range chromatin interactions. It is important to understand their relationship, such as whether eQTLs regulate their target genes through physical chromatin interaction. Although chromatin interactions have been widely believed to be one of the main mechanisms underlying eQTLs, most evidence came from studies of cell lines and yet no direct evidence exists for tissues. RESULTS: We performed various joint analyses of eQTL and high-throughput chromatin conformation capture (Hi-C) data from 11 human primary tissue types and 2 human cell lines. We found that chromatin interaction frequency is positively associated with the number of genes that have eQTLs and that eQTLs and their target genes tend to fall into the same topologically associating domain (TAD). These results are consistent across all tissues and cell lines we evaluated. Moreover, in 6 out of 11 tissues (aorta, dorsolateral prefrontal cortex, hippocampus, pancreas, small bowel, and spleen), tissue-specific eQTLs are significantly enriched in tissue-specific frequently interacting regions (FIREs). CONCLUSIONS: Our data have demonstrated the close spatial proximity between eQTLs and their target genes among multiple human primary tissues.

Mapping human N-linked glycoproteins and glycosylation sites using mass spectrometry.

N-linked glycoprotein is a highly interesting class of proteins for clinical and biological research. Over the last decade, large-scale profiling of N-linked glycoproteins and glycosylation sites from biological and clinical samples has been achieved through mass spectrometry-based glycoproteomic approaches. In this paper, we reviewed the human glycoproteomic profiles that have been reported in more than 80 individual studies, and mainly focused on the N-glycoproteins and glycosylation sites identified through their deglycosylated forms of glycosite-containing peptides. According to our analyses, more than 30,000 glycosite-containing peptides and 7,000 human glycoproteins have been identified from five different body fluids, twelve human tissues (or related cell lines), and four special cell types. As the glycoproteomic data is still missing for many organs and tissues, a systematical glycoproteomic analysis of various human tissues and body fluids using a uniform platform is still needed for an integrated map of human N-glycoproteomes.

Fundamental insights into autosomal dominant polycystic kidney disease from human-based cell models.

Several animal- and human-derived models are used in autosomal dominant polycystic kidney disease (ADPKD) research to gain insight in the disease mechanism. However, a consistent correlation between animal and human ADPKD models is lacking. Therefore, established human-derived models are relevant to affirm research results and translate findings into a clinical set-up. In this review, we give an extensive overview of the existing human-based cell models. We discuss their source (urine, nephrectomy and stem cell), immortalisation procedures, genetic engineering, kidney segmental origin and characterisation with nephron segment markers. We summarise the most studied pathways and lessons learned from these different ADPKD models. Finally, we issue recommendations for the derivation of human-derived cell lines and for experimental set-ups with these cell lines.

Castanea sativa Mill. Shells Aqueous Extract Exhibits Anticancer Properties Inducing Cytotoxic and Pro-Apoptotic Effects.

In this study, chestnut shells (CS) were used in order to obtain bioactive compounds through different extraction procedures. The aqueous extracts were chemically characterized. The highest extraction yield and total phenolic content was obtained by conventional liquid extraction (CLE). Gallic and protocatechuic acids were the main simple phenols in the extract, with 86.97 and 11.20 mg/g chestnut shells dry extract (CSDE), respectively. Six tumor cell lines (DU 145, PC-3, LNCaP, MDA-MB-231, MCF-7, and HepG2) and one normal prostate epithelial cell line (PNT2) were exposed to increasing concentration of CSDE (1-100 µg/mL) for 24 h, and cell viability was evaluated using 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide MTT assay. A reduced rate in cell viability was observed in DU 145, PC-3, LNCaP, and MCF-7 cells, while viability of the other assessed cells was not affected, except for PNT2 cells at a concentration of 100 µg/mL. Furthermore, CSDE-at concentrations of 55.5 and 100 µg/mL-lead to a significant increase of apoptotic cells in DU 145 cells of 28.2% and 61%, respectively. In conclusion, these outcomes suggested that CS might be used for the extraction of several polyphenols that may represent good candidates for alternative therapies or in combination with current chemotherapeutics.

Duckweed for Human Nutrition: No Cytotoxic and No Anti-Proliferative Effects on Human Cell Lines.

Duckweeds (Lemnaceae) possess good qualitative and quantitative profiles of nutritional components for its use as human food. However, no studies have been conducted on the probable presence or absence of any adverse effects. The extracts from seven duckweed species (Spirodela polyrhiza, Landoltia punctata, Lemna gibba, Lemna minor, Wolffiella hyalina, Wolffia globosa, and Wolffia microscopica) covering all five genera of the plant family were herewith tested for cytotoxic effects on the human cell lines HUVEC, K-562, and HeLa and for anti-proliferative activity on HUVEC and K-562 cell lines. From these assays, it is evident that duckweeds do not possess any detectable anti-proliferative or cytotoxic effects, thus, the high nutritional value is not diminished by such detrimental factors. The present result is a first step to exclude any harmful effects of highly nutritious duckweed for human.

Authentication of M14 melanoma cell line proves misidentification of MDA-MB-435 breast cancer cell line.

A variety of analytical approaches have indicated that melanoma cell line UCLA-SO-M14 (M14) and breast carcinoma cell line MDA-MB-435 originate from a common donor. This indicates that at some point in the past, one of these cell lines became misidentified, meaning that it ceased to correspond to the reported donor and instead became falsely identified (through cross-contamination or other means) as a cell line from a different donor. Initial studies concluded that MDA-MB-435 was the misidentified cell line and M14 was the authentic cell line, although contradictory evidence has been published, resulting in further confusion. To address this question, we obtained early samples of the melanoma cell line (M14), a lymphoblastoid cell line from the same donor (ML14), and donor serum preserved at the originator's institution. M14 samples were cryopreserved in December 1975, before MDA-MB-435 cells were established in culture. Through a series of molecular characterizations, including short tandem repeat (STR) profiling and cytogenetic analysis, we demonstrated that later samples of M14 and MDA-MB-435 correspond to samples of M14 frozen in 1975, to the lymphoblastoid cell line ML14, and to the melanoma donor's STR profile, sex and blood type. This work demonstrates conclusively that M14 is the authentic cell line and MDA-MB-435 is misidentified. With clear provenance information and authentication testing of early samples, it is possible to resolve debates regarding the origins of problematic cell lines that are widely used in cancer research.

Improved Method for Determining Absolute Phosphorylation Stoichiometry Using Bayesian Statistics and Isobaric Labeling.

Phosphorylation stoichiometry, or occupancy, is one element of phosphoproteomics that can add useful biological context (Gerber et al. Proc. Natl. Acad. Sci. U. S. A. 2003, 100, 6940-5). We previously developed a method to assess phosphorylation stoichiometry on a proteome-wide scale (Wu et al. Nat. Methods 2011, 8, 677-83). The stoichiometry calculation relies on identifying and measuring the levels of each nonphosphorylated counterpart peptide with and without phosphatase treatment. The method, however, is problematic in that low stoichiometry phosphopeptides can return negative stoichiometry values if measurement error is larger than the percent stoichiometry. Here, we have improved the stoichiometry method through the use of isobaric labeling with 10-plex TMT reagents. In this way, five phosphatase treated and five untreated samples are compared simultaneously so that each stoichiometry is represented by five ratio measurements with no missing values. We applied the method to determine basal stoichiometries of HCT116 cells growing in culture. With this method, we analyzed five biological replicates simultaneously with no need for phosphopeptide enrichment. Additionally, we developed a Bayesian model to estimate phosphorylation stoichiometry as a parameter confined to an interval between 0 and 1 implemented as an R/Stan script. Consequently, both point and interval estimates are consistent with the plausible range of values for stoichiometry. Finally, we report absolute stoichiometry measurements with credible intervals for 6772 phosphopeptides containing at least a single phosphorylation site.

Production of coagulation factor VII in human cell lines Sk-Hep-1 and HKB-11.

Recombinant factor VII (rFVII) is the main therapeutic choice for hemophilia patients who have developed inhibitory antibodies against conventional treatments (FVIII and FIX). Because of the post-translational modifications, rFVII needs to be produced in mammalian cell lines. In this study, for the first time, we have shown efficient rFVII production in HepG2, Sk-Hep-1, and HKB-11 cell lines. Experiments in static conditions for a period of 96 h showed that HepG2-FVII produced the highest amounts of rhFVII, with an average of 1843 ng/mL. Sk-hep-1-FVII cells reached a maximum protein production of 1432 ng/mL and HKB-11-FVII cells reached 1468 ng/mL. Sk-Hep-1-rFVII and HKB-11-rFVII were selected for the first step of scale-up. Over 10 days of spinner flask culture, HKB-11 and SK-Hep-1 cells showed a cumulative production of rFVII of 152 µg and 202.6 µg in 50 mL, respectively. Thus, these human cell lines can be used for an efficient production of recombinant FVII. With more investment in basic research, human cell lines can be optimized for the commercial production of different bio therapeutic proteins.

Interrogating the variation of element masses and distribution patterns in single cells using ICP-MS with a high efficiency cell introduction system.

Cellular heterogeneity is an inherent condition of cell populations, which results from stochastic expression of genes, proteins, and metabolites. The heterogeneity of individual cells can dramatically influence cellular decision-making and cell fate. So far, our knowledge about how the variation of endogenous metals and non-metals in individual eukaryotic cells is limited. In this study, ICP-MS equipped with a high efficiency cell introduction system (HECIS) was developed as a method of single-cell ICP-MS (SC-ICP-MS). The method was applied to the single-cell analysis of Mn, Fe, Co, Cu, Zn, P, and S in human cancer cell lines (HeLa and A549) and normal human bronchial epithelial cell line (16HBE). The analysis showed obvious variation of the masses of Cu, Fe, Zn, and P in individual HeLa cells, and variation of Fe, Zn, and P in individual A549 cells. On the basis of the single-cell data, a multimodal distribution of the elements in the cell population was fitted, which showed marked differences among the various cell lines. Importantly, subpopulations of the elements were found in the cell populations, especially in the HeLa cancer cells. This study demonstrates that SC-ICP-MS is able to unravel the extent of variation of endogenous elements in individual cells, which will help to improve our fundamental understanding of cellular biology and reveal novel insights into human biology and medicine. Graphical abstract The variations of masses and distribution patterns of elements Mn, Fe, Co, Cu, Zn, P, and S in single cells were successfully detected by ICP-MS coupled with a high efficiency cell introduction system (HECIS).