Single Human Cells Research Articles

Ca2+-activated K+ channels modulate membrane potential in the human parathyroid cell: Possible role in exocytosis

The relationships between parathyroid hormone (PTH) secretion and parathyroid cell membrane potential, including the identities and roles of K+ channels that regulate and/or modulate membrane potential are not well defined. Here we have used Western blot/immunohistochemistry as well as patch-clamp and perifusion techniques to identify and localize specific K+ channels in parathyroid cells and to investigate their roles in the control of membrane potential and PTH secretion. We also re-investigated the relationship between membrane potential and exocytosis. We showed that in single human parathyroid cells K+ current is dependent on at least two types of Ca2+-activated K+ channels: a small-conductance Ca2+-activated K+ channel (KSK) and a large-conductance voltage and Ca2+-activated K+ channel (KBK). These channels were sensitive to specific peptide blocking toxins including apamin, charybdotoxin, and iberiotoxin. These channels confer sensitivity of the membrane potential in single cells to high extracellular K+, TEA, and peptide toxins. Blocking of KBK potently inhibited K+ channel current, and KBK was shown to be expressed in the plasma membrane of parathyroid cells. In addition, when using the capacitance technique as an indicator of exocytosis, clamping the parathyroid cell at −60 mV prevented exocytosis, whereas holding the membrane potential at 0 mV facilitated it. Taken together, the results show that human parathyroid cells have functional KBK and KSK channels but the data presented herein suggest that KBK/KSK channels likely contribute to the maintenance of the membrane potential, and that membrane potential, per se, modulates exocytosis independently of [Ca2+]i.

Combing of picogram level DNA equivalent to genomic DNA present in single human cell by self propelled droplet motion over a stable gradient surface

DNA combing is a powerful technique for studying replication profile, fork-directionality and fork velocity. At present, there is requirement of a methodology to comb DNA present in a single human cell for studying replication dynamics at early embryonic stage. In our study, a surface having dual characteristics i.e., affinity towards negatively charged single DNA molecules and a hydrophobic gradient for self propelled droplet motion of combing solution was developed. The surface was made by coating of TCOS (trichloro-octylsilane) by vapor diffusion on APTES (Aminopropyl-triethoxysilane) coated glass slides. A gradient surface having high deposition efficiency (DE) was developed on which 5 picogram DNA equivalent to genomic DNA present in one single human cell can be combed. The gradient surface was thermostable in nature having the ability to sustain boiling temperature for two hours and sustain anisotropy in 70 % ethanol for 80 h. Applicability for multiple runs was enhanced such that the surface can be used for 13–14 times. Factors associated with gradient surface are unidirectional movement of combing solution droplet over the gradient surface for combing straight DNA molecules and a longer gradient surface of more than 1 cm such that long size DNA molecules can be combed. Ellipsometry and contact angle hysteresis confirmed the presence of hydrophobic gradient. XPS (X-ray photoelectron spectroscopy) and FTIR (Fourier Transform Infrared Spectroscopy) confirmed the presence of characteristic affinity towards negatively charged DNA molecules on the gradient surface. Combing solution was optimized for increasing deposition efficiency and for increasing the applicability of gradient surface for multiple runs. High temperature of combing solution was found to increase Deposition Efficiency. Combing solution was also optimized for combing single DNA molecules over the gradient surface. Single DNA molecules were combed by reducing pH and lowering concentration of triton-X in the combing solution. Dye: bp ratio was optimized for high fluorescent intensity and low surface background.

Efficient detection of single circulating tumor cell in blood using Raman mapping based on Aptamer-SERS bio-probe coupled with micropore membrane filtration

Rapid and accurate detection of rare circulating tumor cells (CTCs) in human blood still remains a challenge. We present a surface enhanced Raman spectroscopy (SERS) method based on aptamer-SERS bio-probe recognition coupled with micropore membrane filtration capture for the detection of CTCs at single cell level. The parylene micropore membrane with optimized micropore size installed on a filtration holder could capture bio-probe labeled CTCs by gravity in less than 10 s, and only with very less white blood cells (WBCs) residual. In order to facilitate the synthesis of the aptamer-SERS bio-probe, ethyl acetate dehydration method was established. The bio-probe can be rapidly synthesized within 2 h by binding SH-aptamer to 4- mercaptobenzoic acid (4-MBA) modified AuNPs with the help of ethyl acetate. The SERS bio-probe with selected specific aptamer could distinguish single human non-small cell lung cancer A549 cells from residual WBCs on membrane efficiently and reliably based on their Raman signal intensity difference at 1075 cm−1. Through the filter membrane coupled with aptamer-SERS bio-probe system, even 20 A549 cells in blood solution simulating CTCs sample can be detected, which the recovery rate and recognition rate are more than 90%. This method is rapid, reliable and cost-effective, which indicates a good prospect in clinical application for CTCs detection.

Long-read whole-genome analysis of human single cells

Long-read sequencing has dramatically increased our understanding of human genome variation. Here, we demonstrate that long-read technology can give new insights into the genomic architecture of individual cells. Clonally expanded CD8+ T-cells from a human donor were subjected to droplet-based multiple displacement amplification (dMDA) to generate long molecules with reduced bias. PacBio sequencing generated up to 40% genome coverage per single-cell, enabling detection of single nucleotide variants (SNVs), structural variants (SVs), and tandem repeats, also in regions inaccessible by short reads. 28 somatic SNVs were detected, including one case of mitochondrial heteroplasmy. 5473 high-confidence SVs/cell were discovered, a sixteen-fold increase compared to Illumina-based results from clonally related cells. Single-cell de novo assembly generated a genome size of up to 598 Mb and 1762 (12.8%) complete gene models. In summary, our work shows the promise of long-read sequencing toward characterization of the full spectrum of genetic variation in single cells.

Functional Characterization of the A414G Loss-of-Function Mutation in HCN4 Associated with Sinus Bradycardia

Patients carrying the heterozygous A414G mutation in the HCN4 gene, which encodes the HCN4 protein, demonstrate moderate to severe bradycardia of the heart. Tetramers of HCN4 subunits compose the ion channels in the sinus node that carry the hyperpolarization-activated ‘funny’ current (If), also named the ‘pacemaker current’. If plays an essential modulating role in sinus node pacemaker activity. To assess the mechanism by which the A414G mutation results in sinus bradycardia, we first performed voltage clamp measurements on wild-type (WT) and heterozygous mutant HCN4 channels expressed in Chinese hamster ovary (CHO) cells. These experiments were performed at physiological temperature using the amphotericin-perforated patch-clamp technique. Next, we applied the experimentally observed mutation-induced changes in the HCN4 current of the CHO cells to If of the single human sinus node cell model developed by Fabbri and coworkers. The half-maximal activation voltage V1/2 of the heterozygous mutant HCN4 current was 19.9 mV more negative than that of the WT HCN4 current (p < 0.001). In addition, the voltage dependence of the heterozygous mutant HCN4 current (de)activation time constant showed a −11.9 mV shift (p < 0.001) compared to the WT HCN4 current. The fully-activated current density, the slope factor of the activation curve, and the reversal potential were not significantly affected by the heterozygous A414G mutation. In the human sinus node computer model, the cycle length was substantially increased, almost entirely due to the shift in the voltage dependence of steady-state activation, and this increase was more prominent under vagal tone. The introduction of a passive atrial load into the model sinus node cell further reduced the beating rate, demonstrating that the bradycardia of the sinus node was even more pronounced by interactions between the sinus node and atria. In conclusion, the experimentally identified A414G-induced changes in If can explain the clinically observed sinus bradycardia in patients carrying the A414G HCN4 gene mutation.

Cellular state landscape and herpes simplex virus type 1 infection progression are connected

Prediction, prevention and treatment of virus infections require understanding of cell-to-cell variability that leads to heterogenous disease outcomes, but the source of this heterogeneity has yet to be clarified. To study the multimodal response of single human cells to herpes simplex virus type 1 (HSV-1) infection, we mapped high-dimensional viral and cellular state spaces throughout the infection using multiplexed imaging and quantitative single-cell measurements of viral and cellular mRNAs and proteins. Here we show that the high-dimensional cellular state scape can predict heterogenous infections, and cells move through the cellular state landscape according to infection progression. Spatial information reveals that infection changes the cellular state of both infected cells and of their neighbors. The multiplexed imaging of HSV-1-induced cellular modifications links infection progression to changes in signaling responses, transcriptional activity, and processing bodies. Our data show that multiplexed quantification of responses at the single-cell level, across thousands of cells helps predict infections and identify new targets for antivirals.

An assessment of the moral value of neuronal cell models and brain organoids

Advances in stem cell technology enable neuroscientists to develop induced pluripotent stem cell (iPSC)-based neuronal models of varying complexity, ranging from single human brain cells to two-dimensional neuronal cell models and three-dimensional brain organoids. While the discussion on the moral status of brain organoids is taking center stage in the bioethical literature and is invariably linked to the presumed capacity of future brain organoids to develop some form of consciousness, analyses of the moral status of other – less complex – iPSC-based neuronal models are lacking. In this paper we aim to clarify the moral value of various types of existing neuronal models, including brain organoids. We show how it is made up of several layers that may encompass various sorts of considerations, including moral values, the results of empirical research, and biological characteristics. We identify four such layers – instrumental, intrinsic, symbolic, and relational – that are relevant for the assessment of the moral value of neuronal models. We demonstrate that it lies not in a capacity to develop some form of consciousness (which is absent in current iPSC-based neuronal models, including brain organoids), but in other considerations, including the genetic links between models and donors, the ability of models to mimic brain (dys)function, and their symbolic value, all of which are often overlooked in the bioethical literature. Also, we demonstrate that the 'thickness' of the layers (i.e., their moral weight) increases when the neuronal model is more complex. Finally, we discuss the practical-ethical implications of our analysis for the use of neuronal models in research settings, for instance in relation to informed consent and biobank governance. Our four-layer framework can be applied also in moral assessments of other iPSC-based models, including emerging and future cell models.

Thermo-Mechanical Fluid-Structure Interaction Numerical Modelling and Experimental Validation of MEMS Electrothermal Actuators for Aqueous Biomedical Applications.

Recent developments in MEMS technologies have made such devices attractive for use in applications that involve precision engineering and scalability. In the biomedical industry, MEMS devices have gained popularity in recent years for use as single-cell manipulation and characterisation tools. A niche application is the mechanical characterisation of single human red blood cells, which may exhibit certain pathological conditions that impart biomarkers of quantifiable magnitude that are potentially detectable via MEMS devices. Such applications come with stringent thermal and structural specifications wherein the potential device candidates must be able to function with no exceptions. This work presents a state-of-the-art numerical modelling methodology that is capable of accurately predicting MEMS device performance in various media, including aqueous ones. The method is strongly coupled in nature, whereby thermal as well as structural degrees of freedom are transferred to and from finite element and finite volume solvers at every iteration. This method therefore provides MEMS design engineers with a reliable tool that can be used in design and development stages and helps to avoid total reliability on experimental testing. The proposed numerical model is validated via a series of physical experiments. Four MEMS electrothermal actuators with cascaded V-shaped drivers are presented. With the use of the newly proposed numerical model as well as the experimental testing, the MEMS devices' suitability for biomedical applications is confirmed.

Neuropharmacology of human TERA2.cl.SP12 stem cell-derived neurons in ultra-long-term culture for antiseizure drug discovery.

Modeling the complex and prolonged development of the mammalian central nervous system in vitro remains a profound challenge. Most studies of human stem cell derived neurons are conducted over days to weeks and may or may not include glia. Here we have utilized a single human pluripotent stem cell line, TERA2.cl.SP12 to derive both neurons and glial cells and determined their differentiation and functional maturation over 1 year in culture together with their ability to display epileptiform activity in response to pro-convulsant agents and to detect antiseizure drug actions. Our experiments show that these human stem cells differentiate in vitro into mature neurons and glia cells and form inhibitory and excitatory synapses and integrated neural circuits over 6-8 months, paralleling early human neurogenesis in vivo; these neuroglia cultures display complex electrochemical signaling including high frequency trains of action potentials from single neurons, neural network bursts and highly synchronized, rhythmical firing patterns. Neural activity in our 2D neuron-glia circuits is modulated by a variety of voltage-gated and ligand-gated ion channel acting drugs and these actions were consistent in both young and highly mature neuron cultures. We also show for the first time that spontaneous and epileptiform activity is modulated by first, second and third generation antiseizure agents consistent with animal and human studies. Together, our observations strongly support the value of long-term human stem cell-derived neuroglial cultures in disease modeling and neuropsychiatric drug discovery.

SEC-seq: association of molecular signatures with antibody secretion in thousands of single human plasma cells

The secreted products of cells drive many functions in vivo; however, methods to link this functional information to surface markers and transcriptomes have been lacking. By accumulating secretions close to secreting cells held within cavity-containing hydrogel nanovials, we demonstrate workflows to analyze the amount of IgG secreted from single human B cells and link this information to surface markers and transcriptomes from the same cells. Measurements using flow cytometry and imaging flow cytometry corroborate the association between IgG secretion and CD38/CD138. By using oligonucleotide-labeled antibodies we find that upregulation of pathways for protein localization to the endoplasmic reticulum and mitochondrial oxidative phosphorylation are most associated with high IgG secretion, and uncover surrogate plasma cell surface markers (e.g., CD59) defined by the ability to secrete IgG. Altogether, this method links quantity of secretion with single-cell sequencing (SEC-seq) and enables researchers to fully explore the links between genome and function, laying the foundation for discoveries in immunology, stem cell biology, and beyond.

Multimodal single cell analysis infers widespread enhancer co-activity in a lymphoblastoid cell line

Non-coding regulatory elements such as enhancers are key in controlling the cell-type specificity and spatio-temporal expression of genes. To drive stable and precise gene transcription robust to genetic variation and environmental stress, genes are often targeted by multiple enhancers with redundant action. However, it is unknown whether enhancers targeting the same gene display simultaneous activity or whether some enhancer combinations are more often co-active than others. Here, we take advantage of recent developments in single cell technology that permit assessing chromatin status (scATAC-seq) and gene expression (scRNA-seq) in the same single cells to correlate gene expression to the activity of multiple enhancers. Measuring activity patterns across 24,844 human lymphoblastoid single cells, we find that the majority of enhancers associated with the same gene display significant correlation in their chromatin profiles. For 6944 expressed genes associated with enhancers, we predict 89,885 significant enhancer-enhancer associations between nearby enhancers. We find that associated enhancers share similar transcription factor binding profiles and that gene essentiality is linked with higher enhancer co-activity. We provide a set of predicted enhancer-enhancer associations based on correlation derived from a single cell line, which can be further investigated for functional relevance.

Preclinical Models of Adrenocortical Cancer.

Adrenocortical cancer is an aggressive endocrine malignancy with an incidence of 0.72 to 1.02 per million people/year, and a very poor prognosis with a five-year survival rate of 22%. As an orphan disease, clinical data are scarce, meaning that drug development and mechanistic research depend especially on preclinical models. While a single human ACC cell line was available for the last three decades, over the last five years, many new in vitro and in vivo preclinical models have been generated. Herein, we review both in vitro (cell lines, spheroids, and organoids) and in vivo (xenograft and genetically engineered mouse) models. Striking leaps have been made in terms of the preclinical models of ACC, and there are now several modern models available publicly and in repositories for research in this area.

Single-cell intracellular pH dynamics regulate the cell cycle by timing the G1 exit and G2 transition.

Transient changes in intracellular pH (pHi) regulate normal cell behaviors, but roles for spatiotemporal pHi dynamics in single-cell behaviors remain unclear. Here, we mapped single-cell spatiotemporal pHi dynamics during mammalian cell cycle progression both with and without cell cycle synchronization. We found that single-cell pHi is dynamic throughout the cell cycle: pHi decreases at G1/S, increases in mid-S, decreases at late S, increases at G2/M and rapidly decreases during mitosis. Importantly, although pHi is highly dynamic in dividing cells, non-dividing cells have attenuated pHi dynamics. Using two independent pHi manipulation methods, we found that low pHi inhibits completion of S phase whereas high pHi promotes both S/G2 and G2/M transitions. Our data also suggest that low pHi cues G1 exit, with decreased pHi shortening G1 and increased pHi elongating G1. Furthermore, dynamic pHi is required for S phase timing, as high pHi elongates S phase and low pHi inhibits S/G2 transition. This work reveals that spatiotemporal pHi dynamics are necessary for cell cycle progression at multiple phase transitions in single human cells.

Quantitative Aspects of the Human Cell Proteome.

The number and identity of proteins and proteoforms presented in a single human cell (a cellular proteome) are fundamental biological questions. The answers can be found with sophisticated and sensitive proteomics methods, including advanced mass spectrometry (MS) coupled with separation by gel electrophoresis and chromatography. So far, bioinformatics and experimental approaches have been applied to quantitate the complexity of the human proteome. This review analyzed the quantitative information obtained from several large-scale panoramic experiments in which high-resolution mass spectrometry-based proteomics in combination with liquid chromatography or two-dimensional gel electrophoresis (2DE) were used to evaluate the cellular proteome. It is important that even though all these experiments were performed in different labs using different equipment and calculation algorithms, the main conclusion about the distribution of proteome components (proteins or proteoforms) was basically the same for all human tissues or cells. It follows Zipf's law and has a formula N = A/x, where N is the number of proteoforms, A is a coefficient, and x is the limit of proteoform detection in terms of abundance.

The Influence of Spirodi(Iminohydantoin) on Charge Transfer through ds-DNA Containing 8-OXO-dG: A Theoretical Approach.

Genetic information stored in a DNA base sequence is continuously exposed to harmful factors. It has been determined that 9 × 104 different DNA damage events occur in a single human cell every 24 h. Of these, 7,8-dihydro-8-oxo-guanosine (OXOG) is one of the most abundant and can undergo further transformations towards spirodi(iminohydantoin) (Sp). Sp is highly mutagenic in comparison to its precursor if not repaired. In this paper, the influence of both Sp diastereomers 4R and 4S as well as their anti and syn conformers on charge transfer through the double helix was taken into theoretical consideration. In addition, the electronic properties of four modelled double-stranded oligonucleotides (ds-oligos) were also discussed, i.e., d[A1Sp2A3oxoG4A5] * [T5C4T3C2T1]. Throughout the study, the M06-2X/6-31++G** level theory was used. Solvent-solute non-equilibrated and equilibrated interactions were also considered. The subsequent results elucidated that the 7,8-dihydro-8-oxo-guanosine:cytidine (OXOGC) base pair is the settled point of a migrated radical cation in each of the discussed cases, due to its low adiabatic ionization potential, i.e., ~5.55 [eV]. The opposite was noted for excess electron transfer through ds-oligos containing anti (R)-Sp or anti (S)-Sp. The radical anion was found on the OXOGC moiety, whereas in the presence of syn (S)-Sp or syn (R)-Sp, an excess electron was found on the distal A1T5 or A5T1 base pair, respectively. Furthermore, a spatial geometry analysis of the discussed ds-oligos revealed that the presence of syn (R)-Sp in the ds-oligo caused only a slight deformation to the double helix, while syn (S)-Sp formed an almost ideal base pair with a complementary dC. The above results are in strong agreement with the final charge transfer rate constant, as calculated according to Marcus' theory. In conclusion, DNA damage such as spirodi(iminohydantoin), especially when becoming part of clustered DNA damage, can affect the effectiveness of other lesion recognition and repair processes. This can lead to the acceleration of undesired and deleterious processes such as carcinogenesis or aging. However, in terms of anticancer radio-/chemo- or combined therapy, the slowing down of the repair machinery can result in increased effectiveness. With this in mind, the influence of clustered damage on charge transfer and its subsequent effect on single-damage recognition by glycosylases justifies future investigation.

PITX2 Knockout Induces Key Findings of Electrical Remodeling as Seen in Persistent Atrial Fibrillation.

Electrical remodeling in human persistent atrial fibrillation is believed to result from rapid electrical activation of the atria, but underlying genetic causes may contribute. Indeed, common gene variants in an enhancer region close to PITX2 (paired-like homeodomain transcription factor 2) are strongly associated with atrial fibrillation, but the mechanism behind this association remains unknown. This study evaluated the consequences of PITX2 deletion (PITX2-/-) in human induced pluripotent stem cell-derived atrial cardiomyocytes. CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9) was used to delete PITX2 in a healthy human iPSC line that served as isogenic control. Human induced pluripotent stem cell-derived atrial cardiomyocytes were differentiated with unfiltered retinoic acid and cultured in atrial engineered heart tissue. Force and action potential were measured in atrial engineered heart tissues. Single human induced pluripotent stem cell-derived atrial cardiomyocytes were isolated from atrial engineered heart tissue for ion current measurements. PITX2-/- atrial engineered heart tissue beats slightly slower than isogenic control without irregularity. Force was lower in PITX2-/- than in isogenic control (0.053±0.015 versus 0.131±0.017 mN, n=28/3 versus n=28/4, PITX2-/- versus isogenic control; P<0.0001), accompanied by lower expression of CACNA1C and lower L-type Ca2+ current density. Early repolarization was weaker (action potential duration at 20% repolarization; 45.5±13.2 versus 8.6±5.3 ms, n=18/3 versus n=12/4, PITX2-/- versus isogenic control; P<0.0001), and maximum diastolic potential was more negative (-78.3±3.1 versus -69.7±0.6 mV, n=18/3 versus n=12/4, PITX2-/- versus isogenic control; P=0.001), despite normal inward rectifier currents (both IK1 and IK,ACh) and carbachol-induced shortening of action potential duration. Complete PITX2 deficiency in human induced pluripotent stem cell-derived atrial cardiomyocytes recapitulates some findings of electrical remodeling of atrial fibrillation in the absence of fast beating, indicating that these abnormalities could be primary consequences of lower PITX2 levels.

Dysregulation of innate cell types in the hepatic immune microenvironment of alcoholic liver cirrhosis.

The risk of alcoholic cirrhosis increases in a dose- and time-dependent manner with alcohol consumption and ethanol metabolism in the liver. Currently, no effective antifibrotic therapies are available. We aimed to obtain a better understanding of the cellular and molecular mechanisms involved in the pathogenesis of liver cirrhosis. We performed single-cell RNA-sequencing to analyze immune cells from the liver tissue and peripheral blood form patients with alcoholic cirrhosis and healthy controls to profile the transcriptomes of more than 100,000 single human cells and yield molecular definitions for non-parenchymal cell types. In addition, we performed single-cell RNA-sequencing analysis to reveal the immune microenvironment related to alcoholic liver cirrhosis. Hematoxylin and eosin, Immunofluorescence staining and Flow cytometric analysis were employed to study the difference between tissues and cells with or without alcoholic cirrhosis. We identified a fibrosis-associated M1 subpopulation of macrophages that expands in liver fibrosis, differentiates from circulating monocytes, and is pro-fibrogenic. We also define mucosal-associated invariant T (MAIT) cells that expand in alcoholic cirrhosis and are topographically restricted to the fibrotic niche. Multilineage modeling of ligand and receptor interactions between the fibrosis-associated macrophages, MAIT, and NK cells revealed the intra-fibrotic activity of several pro-fibrogenic pathways, including responses to cytokines and antigen processing and presentation, natural killer cell-mediated cytotoxicity, cell adhesion molecules, Th1/Th2/Th17 cell differentiation, IL-17 signaling pathway, and Toll-like receptor signaling pathway. Our work dissects unanticipated aspects of the cellular and molecular basis of human organ alcoholic fibrosis at the single-cell level and provides a conceptual framework for the discovery of rational therapeutic targets in liver alcoholic cirrhosis.

Sarcomere dynamics simulations to uncover mechanisms in hypertrophic cardiomyopathy.

Mutations in the beta myosin heavy chain (MYH7) can cause hypercontractility and cardiac remodeling in hypertrophic cardiomyopathy (HCM), but the mechanisms of specific mutations can be difficult to predict. Many mutations change intrinsic rates of myosin activity and their force sensitivity, which can dynamically alter chemo-mechanical cycle events and resultant force generation during cardiac contraction. Our team's multiscale approach enables measurement of the kinetics and force generation in purified myosin proteins, isolated myofibrils, and single and multicellular human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) platforms. Computational modeling can integrate results across these scales and provide additional insights into the individual and combinatorial effects of different changes in myosin kinetics. We have previously used a continuum model to highlight the significance of dysregulation of the super relaxed state of myosin driving hypercontractility in hiPSC-CMs with the P710R mutation. Sensitivity analysis also supported variable myofibril density and sarcomeric organization as a source of population heterogeneity in the force generating capacity of individual hiPSC-CMs. In our ongoing studies, we are leveraging computational modeling to compare the predicted and measured effects of HCM mutations in our different experiments. We have incorporated the measured changes in unloaded myosin to predict force generation after isometric maximal calcium activation (myofibril measurements) and twitch stimulation in single hiPSC-CMs on gels of physiologic stiffness. Additional simulations with spatially explicit models enable more direct prediction of the impact of heterogeneous mixtures of myosins with different molecular profiles and on cooperativity effects associated with the transmission of tension along the length of a sarcomere. Finally, we have visualized the strain profiles of individual sarcomeres in contracting myofibrils in live cells and are correlating these measurements to our simulations. These studies will give new insights into the key biophysical drivers of HCM.

Time-of-Flight Fragmentation Spectra Generated by the Proteomic Analysis of Single Human Cells Do Not Exhibit Atypical Fragmentation Patterns.

Recent work detailed the unique characteristics of fragmentation spectra derived from peptides from single human cells. This valuable report utilized an ultrahigh-field Orbitrap and directly compared the spectra obtained from high-concentration bulk cell HeLa lysates to those obtained from nanogram dilutions of the same and from nanowell-processed single HeLa cells. The analysis demonstrated marked differences between the fragmentation spectra generated at high and single-cell loads, most strikingly, the loss of high-mass y-series fragment ions. As significant differences exist in the physics of Orbitrap and time-of-flight mass analyzers, a comparison appeared warranted. A similar analysis was performed using isolated single pancreatic cancer cells compared to pools consisting of 100 cells. While a reanalysis of the prior Orbitrap data supports the author's original findings, the same trends are not observed in time-of-flight mass spectra of peptides from single human cells. The results are particularly striking when directly comparing the matched intensity fragment values between bulk and single-cell data generated on the same mass analyzers. Instrument acquisition files, processed data, and spectrum libraries are publicly available on MASSIVE via accession MSV000090635.

A new modality for cholesterol impact tracking in colon cancer development - Raman imaging, fluorescence and AFM studies combined with chemometric analysis.

Colorectal cancer (CRC) is the third most common cancer worldwide. Obesity, alcohol consumption, smoking, high consumption of red or processed meat and a diet with low fibre, fruit, and vegetable intake increase CRC risk. Despite advances in surgery (the basic treatment for recovery), chemotherapy, and radiotherapy, CRC remains the second leading cause of cancer-related deaths in the world. Therefore the social importance of this problem stimulates research aimed at developing new tools for rapid CRC diagnosis and analysis of CRC risk factors. Considering the association between the cholesterol level and CRC, we hypothesize that cholesterol spectroscopic and AFM (atomic force microscopy) studies combined with chemometric analysis can be new, powerful tools used to visualize the cholesterol distribution, estimate cholesterol content and determine its influence on the biochemical and nanomechanical properties of colon cells. Our paper presents the analysis of human colon tissues: normal and cancer and human colon single cells normal CCD18-Co and cancer CaCo-2 in the physiological state and CaCo-2 upon mevastatin supplementation. Based on vibrational features we have shown that Raman spectroscopy and imaging allow cholesterol content in human colon tissues and human colon single cells of both types to be tracked and allow the effectiveness of mevastatin in the mevalonate pathway modulation and disruption of the cholesterol level to be proven. All observations have been confirmed by chemometric analysis including principal component analysis (PCA) and partial least squares discriminant analysis (PLSDA). The positive impact of statins on cholesterol content has also been studied by using fluorescence microscopy and atomic force microscopy (AFM). A significant increase in Young modulus as a mechanomarker for CaCo-2 human cancer colon cells upon mevastatin supplementation compared to CCD18-Co human normal colon cells was observed. This paper is one of the first reports about the use of Raman spectroscopic techniques in cholesterol investigations and the first one about cholesterol investigation using Raman spectroscopy (RS) on human cells ex vivo in the context of colon cancer development.