Retinoic acid modulation guides human-induced pluripotent stem cell differentiation towards left or right ventricle-like cardiomyocytes.

BACKGROUND: Cardiomyocytes (CMs) derived from human induced pluripotent stem cells (hiPSCs) by traditional methods are a mix of atrial and ventricular CMs and many other non-cardiomyocyte cells. Retinoic acid (RA) plays an important role in regulation of the spatiotemporal development of the embryonic heart. METHODS: CMs were derived from hiPSC (hi-PCS-CM) using different concentrations of RA (Control without RA, LRA with 0.05µM and HRA with 0.1 µM) between day 3-6 of the differentiation process. Engineered heart tissues (EHTs) were generated by assembling hiPSC-CM at high cell density in a low collagen hydrogel. RESULTS: In the HRA group, hiPSC-CMs exhibited highest expression of contractile proteins MYH6, MYH7 and cTnT. The expression of TBX5, NKX2.5 and CORIN, which are marker genes for left ventricular CMs, was also the highest in the HRA group. In terms of EHT, the HRA group displayed the highest contraction force, the lowest beating frequency, and the highest sensitivity to hypoxia and isoprenaline, which means it was functionally more similar to the left ventricle. RNAsequencing revealed that the heightened contractility of EHT within the HRA group can be attributed to the promotion of augmented extracellular matrix strength by RA. CONCLUSION: By interfering with the differentiation process of hiPSC with a specific concentration of RA at a specific time, we were able to successfully induce CMs and EHTs with a phenotype similar to that of the left ventricle or right ventricle.

Wearable Aptalyzer Integrates Microneedle and Electrochemical Sensing for In Vivo Monitoring of Glucose and Lactate in Live Animals.

Continuous monitoring of clinically relevant biomarkers within the interstitial fluid (ISF) using microneedle (MN)-based assays, has the potential to transform healthcare. This study introduces the Wearable Aptalyzer, an integrated system fabricated by combining biocompatible hydrogel MN arrays for ISF extraction with an electrochemical aptamer-based biosensor for in situ monitoring of blood analytes. The use of aptamers enables continuous monitoring of a wide range of analytes, beyond what is possible with enzymatic monitoring. The Wearable Aptalyzer is used for real-time and multiplexed monitoring of glucose and lactate in ISF. Validation experiments using live mice and rat models of type 1 diabetes demonstrate strong correlation between the measurements collected from the Wearable Aptalyzer in ISF and those obtained from gold-standard techniques for blood glucose and lactate, for each analyte alone and in combination. The Wearable Aptalyzer effectively addresses the limitations inherent in enzymatic detection methods as well as solid MN biosensors and the need for reliable and multiplexed bioanalytical monitoring in vivo.

Linking Aging to Cancer: The Role of Chromatin Biology.

Epigenetic changes have been established to be a hallmark of aging, which implies that aging science requires collaborating with the field of chromatin biology. DNA methylation patterns, changes in relative abundance of histone post-translational modifications, and chromatin remodeling are the central players in modifying chromatin structure. Aging is commonly associated with an overall increase in chromatin instability, loss of homeostasis, and decondensation. However, numerous publications have highlighted that the link between aging and chromatin changes is not nearly as linear as previously expected. This complex interplay of these epigenetic elements during the lifetime of an organism likely contributes to cellular senescence, genomic instability, and disease susceptibility. Yet, the causal links between these phenomena still need to be fully unraveled. In this perspective article, we discuss potential future directions of aging chromatin biology.

Protocol for the genomic analysis of salt-fractionated chromatin from frozen murine liver.

Salt fractionation is a classical approach for separating chromatin based on its differential salt solubility and physical properties. Here, we present a protocol to apply salt fractionation for genome-scale profiling of chromatin isolated from livers at different stages of aging in mice. We elaborate on the steps to isolate nuclei, digest with micrococcal nuclease, sequentially salt fractionate, purify DNA, and construct libraries for genome profiling. We also include information on a computational pipeline for data analysis. For complete details on the use and execution of this protocol, please refer to Yang et al.1 This protocol is an adaptation of the salt fractionation method of Teves and Henikoff.2.

High-Precision Viral Detection Using Electrochemical Kinetic Profiling of Aptamer-Antigen Recognition in Clinical Samples and Machine Learning.

High-precision viral detection at point of need with clinical samples plays a pivotal role in the diagnosis of infectious diseases and the control of a global pandemic. However, the complexity of clinical samples that often contain very low viral concentrations makes it a huge challenge to develop simple diagnostic devices that do not require any sample processing and yet are capable of meeting performance metrics such as very high sensitivity and specificity. Herein we describe a new single-pot and single-step electrochemical method that uses real-time kinetic profiling of the interaction between a high-affinity aptamer and an antigen on a viral surface. This method generates many data points per sample, which when combined with machine learning, can deliver highly accurate test results in a short testing time. We demonstrate this concept using both SARS-CoV-2 and Influenza A viruses as model viruses with specifically engineered high-affinity aptamers. Utilizing this technique to diagnose COVID-19 with 37 real human saliva samples results in a sensitivity and specificity of both 100 % (27 true negatives and 10 true positives, with 0 false negative and 0 false positive), which showcases the superb diagnostic precision of this method.

Surfaceome analysis of extracellular vesicles from senescent cells uncovers uptake repressor DPP4.

Senescent cells are beneficial for repairing acute tissue damage, but they are harmful when they accumulate in tissues, as occurs with advancing age. Senescence-associated extracellular vesicles (S-EVs) can mediate cell-to-cell communication and export intracellular content to the microenvironment of aging tissues. Here, we studied the uptake of EVs from senescent cells (S-EVs) and proliferating cells (P-EVs) and found that P-EVs were readily taken up by proliferating cells (fibroblasts and cervical cancer cells) while S-EVs were not. We thus investigated the surface proteome (surfaceome) of P-EVs relative to S-EVs derived from cells that had reached senescence via replicative exhaustion, exposure to ionizing radiation, or treatment with etoposide. We found that relative to P-EVs, S-EVs from all senescence models were enriched in proteins DPP4, ANXA1, ANXA6, S10AB, AT1A1, and EPHB2. Among them, DPP4 was found to selectively prevent uptake by proliferating cells, as ectopic overexpression of DPP4 in HeLa cells rendered DPP4-expressing EVs that were no longer taken up by other proliferating cells. We propose that DPP4 on the surface of S-EVs makes these EVs refractory to internalization by proliferating cells, advancing our knowledge of the impact of senescent cells in aging-associated processes.

The AT-hook is an evolutionarily conserved auto-regulatory domain of SWI/SNF required for cell lineage priming.

The SWI/SNF ATP-dependent chromatin remodeler is a master regulator of the epigenome, controlling pluripotency and differentiation. Towards the C-terminus of the catalytic subunit of SWI/SNF is a motif called the AT-hook that is evolutionary conserved. The AT-hook is present in many chromatin modifiers and generally thought to help anchor them to DNA. We observe however that the AT-hook regulates the intrinsic DNA-stimulated ATPase activity aside from promoting SWI/SNF recruitment to DNA or nucleosomes by increasing the reaction velocity a factor of 13 with no accompanying change in substrate affinity (KM). The changes in ATP hydrolysis causes an equivalent change in nucleosome movement, confirming they are tightly coupled. The catalytic subunit's AT-hook is required in vivo for SWI/SNF remodeling activity in yeast and mouse embryonic stem cells. The AT-hook in SWI/SNF is required for transcription regulation and activation of stage-specific enhancers critical in cell lineage priming. Similarly, growth assays suggest the AT-hook is required in yeast SWI/SNF for activation of genes involved in amino acid biosynthesis and metabolizing ethanol. Our findings highlight the importance of studying SWI/SNF attenuation versus eliminating the catalytic subunit or completely shutting down its enzymatic activity.

A universal bacterial sensor created by integrating a light modulating aptamer complex with photoelectrochemical signal readout.

Photoelectrochemical (PEC) signal transduction is of great interest for ultrasensitive biosensing; however, signal-on PEC assays that do not require target labeling remain elusive. In this work, we developed a signal-on biosensor that uses nucleic acids to modulate PEC currents upon target capture. Target presence removes a biorecognition probe from a DNA duplex carrying a gold nanoparticle, bringing the gold nanoparticle in direct contact to the photoelectrode and increasing the PEC current. This assay was used to develop a universal bacterial detector by targeting peptidoglycan using an aptamer, demonstrating a limit-of-detection of 82 pg/mL (13 pM) in buffer and 239 pg/mL (37 pM) in urine for peptidoglycan and 1913 CFU/mL forEscherichia coliin urine. When challenged with a panel of unknown targets, the sensor identified samples with bacterial contamination versus fungi. The versatility of the assay was further demonstrated by analyzing DNA targets, which yielded a limit-of-detection of 372 fM.

A hyper-quiescent chromatin state formed during aging is reversed by regeneration.

Epigenetic alterations are a key hallmark of aging but have been limitedly explored in tissues. Here, using naturally aged murine liver as a model and extending to other quiescent tissues, we find that aging is driven by temporal chromatin alterations that promote a refractory cellular state and compromise cellular identity. Using an integrated multi-omics approach and the first direct visualization of aged chromatin, we find that globally, old cells show H3K27me3-driven broad heterochromatinization and transcriptional suppression. At the local level, site-specific loss of H3K27me3 over promoters of genes encoding developmental transcription factors leads to expression of otherwise non-hepatocyte markers. Interestingly, liver regeneration reverses H3K27me3 patterns and rejuvenates multiple molecular and physiological aspects of the aged liver.

Spurious intragenic transcription is a feature of mammalian cellular senescence and tissue aging.

Mammalian aging is characterized by the progressive loss of tissue function and increased risk for disease. Accumulation of senescent cells in aging tissues partly contributes to this decline, and targeted depletion of senescent cells in vivo ameliorates many age-related phenotypes. The fundamental molecular mechanisms responsible for the decline of cellular health and fitness during senescence and aging are largely unknown. In this study, we investigated whether chromatin-mediated loss of transcriptional fidelity, known to contribute to fitness and survival in yeast and worms, also occurs during human cellular senescence and mouse aging. Our findings reveal aberrant transcription initiation inside genes during senescence and aging that co-occurs with changes in the chromatin landscape. Interventions that alter these spurious transcripts have profound consequences on cellular health, primarily affecting intracellular signal transduction pathways. We propose that age-related spurious transcription promotes a noisy transcriptome and degradation of coherent transcriptional networks.

Integrating Water Purification with Electrochemical Aptamer Sensing for Detecting SARS-CoV-2 in Wastewater.

Wastewater analysis of pathogens, particularly SARS-CoV-2, is instrumental in tracking and monitoring infectious diseases in a population. This method can be used to generate early warnings regarding the onset of an infectious disease and predict the associated infection trends. Currently, wastewater analysis of SARS-CoV-2 is almost exclusively performed using polymerase chain reaction for the amplification-based detection of viral RNA at centralized laboratories. Despite the development of several biosensing technologies offering point-of-care solutions for analyzing SARS-CoV-2 in clinical samples, these remain elusive for wastewater analysis due to the low levels of the virus and the interference caused by the wastewater matrix. Herein, we integrate an aptamer-based electrochemical chip with a filtration, purification, and extraction (FPE) system for developing an alternate in-field solution for wastewater analysis. The sensing chip employs a dimeric aptamer, which is universally applicable to the wild-type, alpha, delta, and omicron variants of SARS-CoV-2. We demonstrate that the aptamer is stable in the wastewater matrix (diluted to 50%) and its binding affinity is not significantly impacted. The sensing chip demonstrates a limit of detection of 1000 copies/L (1 copy/mL), enabled by the amplification provided by the FPE system. This allows the integrated system to detect trace amounts of the virus in native wastewater and categorize the amount of contamination into trace (<10 copies/mL), medium (10-1000 copies/mL), or high (>1000 copies/mL) levels, providing a viable wastewater analysis solution for in-field use.

Proteomics- and Metabolomics-Based Analysis of Metabolic Changes in a Swine Model of Pulmonary Hypertension.

Pulmonary vein stenosis (PVS) causes a rare type of pulmonary hypertension (PH) by impacting the flow and pressure within the pulmonary vasculature, resulting in endothelial dysfunction and metabolic changes. A prudent line of treatment in this type of PH would be targeted therapy to relieve the pressure and reverse the flow-related changes. We used a swine model in order to mimic PH after PVS using pulmonary vein banding (PVB) of the lower lobes for 12 weeks to mimic the hemodynamic profile associated with PH and investigated the molecular alterations that provide an impetus for the development of PH. Our current study aimed to employ unbiased proteomic and metabolomic analyses on both the upper and lower lobes of the swine lung to identify regions with metabolic alterations. We detected changes in the upper lobes for the PVB animals mainly pertaining to fatty acid metabolism, reactive oxygen species (ROS) signaling and extracellular matrix (ECM) remodeling and small, albeit, significant changes in the lower lobes for purine metabolism.

A hyper-quiescent chromatin state formed during aging is reversed by regeneration.

Epigenetic alterations are a key hallmark of aging but have been limitedly explored in tissues. Here, using naturally aged murine liver as a model and extending to other quiescent tissues, we find that aging is driven by temporal chromatin alterations that promote a refractory cellular state and compromise cellular identity. Using an integrated multi-omics approach, and the first direct visualization of aged chromatin we find that globally, old cells show H3K27me3-driven broad heterochromatinization and transcription suppression. At the local level, site-specific loss of H3K27me3 over promoters of genes encoding developmental transcription factors leads to expression of otherwise non-hepatocyte markers. Interestingly, liver regeneration reverses H3K27me3 patterns and rejuvenates multiple molecular and physiological aspects of the aged liver.

Gene body DNA hydroxymethylation restricts the magnitude of transcriptional changes during aging.

DNA hydroxymethylation (5hmC) is the most abundant oxidative derivative of DNA methylation (5mC) and is typically enriched at enhancers and gene bodies of transcriptionally active and tissue-specific genes. Although aberrant genomic 5hmC has been implicated in many age-related diseases, the functional role of the modification in aging remains largely unknown. Here, we report that 5hmC is stably enriched in multiple aged organs. Using the liver and cerebellum as model organs, we show that 5hmC accumulates in gene bodies associated with tissue-specific function and thereby restricts the magnitude of gene expression changes during aging. Mechanistically, we found that 5hmC decreases binding affinity of splicing factors compared to unmodified cytosine and 5mC, and is correlated with age-related alternative splicing events, suggesting RNA splicing as a potential mediator of 5hmC's transcriptionally restrictive function. Furthermore, we show that various age-related contexts, such as prolonged quiescence and senescence, are partially responsible for driving the accumulation of 5hmC with age. We provide evidence that this age-related function is conserved in mouse and human tissues, and further show that the modification is altered by regimens known to modulate lifespan. Our findings reveal that 5hmC is a regulator of tissue-specific function and may play a role in regulating longevity.

Improved Macrophage Enrichment from Mouse Skeletal Muscle.

Macrophages are a heterogeneous class of innate immune cells that offer a primary line of defense to the body by phagocytizing pathogens, digesting them, and presenting the antigens to T and B cells to initiate adaptive immunity. Through specialized pro-inflammatory or anti-inflammatory activities, macrophages also directly contribute to the clearance of infections and the repair of tissue injury. Macrophages are distributed throughout the body and largely carry out tissue-specific functions. In skeletal muscle, macrophages regulate tissue repair and regeneration; however, the characteristics of these macrophages are not yet fully understood, and their involvement in skeletal muscle aging remains to be elucidated. To investigate these functions, it is critical to efficiently isolate macrophages from skeletal muscle with sufficient purity and yield for various downstream analyses. However, methods to prepare enriched skeletal muscle macrophages are scarce. Here, we describe in detail an optimized method to isolate skeletal muscle macrophages from mice. This method has allowed the isolation of CD45 + /CD11b + macrophage-enriched cells from young and old mice, which can be further used for flow cytometric analysis, fluorescence-activated cell sorting (FACS), and single-cell RNA sequencing. This protocol was validated in: eLife (2022), DOI: 10.7554/eLife.77974.

Single-cell analysis of skeletal muscle macrophages reveals age-associated functional subpopulations.

Tissue-resident macrophages represent a group of highly responsive innate immune cells that acquire diverse functions by polarizing toward distinct subpopulations. The subpopulations of macrophages that reside in skeletal muscle (SKM) and their changes during aging are poorly characterized. By single-cell transcriptomic analysis with unsupervised clustering, we found 11 distinct macrophage clusters in male mouse SKM with enriched gene expression programs linked to reparative, proinflammatory, phagocytic, proliferative, and senescence-associated functions. Using a complementary classification, membrane markers LYVE1 and MHCII identified four macrophage subgroups: LYVE1-/MHCIIhi (M1-like, classically activated), LYVE1+/MHCIIlo (M2-like, alternatively activated), and two new subgroups, LYVE1+/MHCIIhi and LYVE1-/MHCIIlo. Notably, one new subgroup, LYVE1+/MHCIIhi, had traits of both M2 and M1 macrophages, while the other new subgroup, LYVE1-/MHCIIlo, displayed strong phagocytic capacity. Flow cytometric analysis validated the presence of the four macrophage subgroups in SKM and found that LYVE1- macrophages were more abundant than LYVE1+ macrophages in old SKM. A striking increase in proinflammatory markers (S100a8 and S100a9 mRNAs) and senescence-related markers (Gpnmb and Spp1 mRNAs) was evident in macrophage clusters from older mice. In sum, we have identified dynamically polarized SKM macrophages and propose that specific macrophage subpopulations contribute to the proinflammatory and senescent traits of old SKM.

Development of Nucleic-Acid-Based Electrochemical Biosensors for Clinical Applications.

Nucleic acids are remarkable molecules. In addition to Watson-Crick base pairing, the different structural motifs of these molecules can bind non-nucleic acid targets or catalyze chemical reactions. Additionally, nucleic acids are easily modified with different molecules or functional groups. These properties make nucleic acids, particularly DNA, ideally suited for use in electrochemical biosensors, both as biorecognition elements and redox reporter probes. In this Minireview, we will review the historical evolution of nucleic acids as probes in electrochemical biosensors. We will then review the specific examples of nucleic-acid-based biosensors that have been evaluated for clinical use in the areas of infectious disease, cancer, or cardiovascular health.

Loss of lung microvascular endothelial Piezo2 expression impairs NO synthesis, induces EndMT, and is associated with pulmonary hypertension.

Mechanical forces are translated into biochemical stimuli by mechanotransduction channels, such as the mechanically activated cation channel Piezo2. Lung Piezo2 expression has recently been shown to be restricted to endothelial cells. Hence, we aimed to investigate the role of Piezo2 in regulation of pulmonary vascular function and structure, as well as its contribution to development of pulmonary arterial hypertension (PAH). The expression of Piezo2 was significantly reduced in pulmonary microvascular endothelial cells (MVECs) from patients with PAH, in lung tissue from mice with a Bmpr2+/R899X knock-in mutation commonly found in patients with pulmonary hypertension, and in lung tissue of monocrotaline (MCT) and sugen-hypoxia-induced PH (SuHx) PAH rat models, as well as from a swine model with pulmonary vein banding. In MVECs, Piezo2 expression was reduced in response to abnormal shear stress, hypoxia, and TGFß stimulation. Functional studies in MVECs exposed to shear stress illustrated that siRNA-mediated Piezo2 knockdown impaired endothelial alignment, calcium influx, phosphorylation of AKT, and nitric oxide production. In addition, siPiezo2 reduced the expression of the endothelial marker PECAM-1 and increased the expression of vascular smooth muscle markers ACTA2, SM22a, and calponin. Thus, Piezo2 acts as a mechanotransduction channel in pulmonary MVECs, stimulating shear-induced production of nitric oxide and is essentially involved in preventing endothelial to mesenchymal transition. Its blunted expression in pulmonary hypertension could impair the vasodilator capacity and stimulate vascular remodeling, indicating that Piezo2 might be an interesting therapeutic target to attenuate progression of the disease.NEW & NOTEWORTHY The mechanosensory ion channel Piezo2 is exclusively expressed in lung microvascular endothelial cells (MVECs). Patient MVECs as well as animal models of pulmonary (arterial) hypertension showed lower expression of Piezo2 in the lung. Mechanistically, Piezo2 is required for calcium influx and NO production in response to shear stress, whereas stimuli known to induce endothelial to mesenchymal transition (EndMT) reduce Piezo2 expression in MVECs, and Piezo2 knockdown induces a gene and protein expression pattern consistent with EndMT.