Nanoporous silica-based protocells at multiple scales for designs of life and nanomedicine.

Various protocell models have been constructed de novo with the bottom-up approach. Here we describe a silica-based protocell composed of a nanoporous amorphous silica core encapsulated within a lipid bilayer built by self-assembly that provides for independent definition of cell interior and the surface membrane. In this review, we will first describe the essential features of this architecture and then summarize the current development of silica-based protocells at both micro- and nanoscale with diverse functionalities. As the structure of the silica is relatively static, silica-core protocells do not have the ability to change shape, but their interior structure provides a highly crowded and, in some cases, authentic scaffold upon which biomolecular components and systems could be reconstituted. In basic research, the larger protocells based on precise silica replicas of cells could be developed into geometrically realistic bioreactor platforms to enable cellular functions like coupled biochemical reactions, while in translational research smaller protocells based on mesoporous silica nanoparticles are being developed for targeted nanomedicine. Ultimately we see two different motivations for protocell research and development: (1) to emulate life in order to understand it; and (2) to use biomimicry to engineer desired cellular interactions

Calcium Signaling in Live Cells on Elastic Gels under Mechanical Vibration at Subcellular Levels

A new device was designed to generate a localized mechanical vibration of flexible gels where human umbilical vein endothelial cells (HUVECs) were cultured to mechanically stimulate these cells at subcellular locations. A Fluorescence Resonance Energy Transfer (FRET)-based calcium biosensor (an improved Cameleon) was used to monitor the spatiotemporal distribution of intracellular calcium concentrations in the cells upon this mechanical stimulation. A clear increase in intracellular calcium concentrations over the whole cell body (global) can be observed in the majority of cells under mechanical stimulation. The chelation of extracellular calcium with EGTA or the blockage of stretch-activated calcium channels on the plasma membrane with streptomycin or gadolinium chloride significantly inhibited the calcium responses upon mechanical stimulation. Thapsigargin, an endoplasmic reticulum (ER) calcium pump inhibitor, or U73122, a phospholipase C (PLC) inhibitor, resulted in mainly local calcium responses occurring at regions close to the stimulation site. The disruption of actin filaments with cytochalasin D or inhibition of actomyosin contractility with ML-7 also inhibited the global calcium responses. Therefore, the global calcium response in HUVEC depends on the influx of calcium through membrane stretch-activated channels, followed by the release of inositol trisphosphate (IP3) via PLC activation to trigger the ER calcium release. Our newly developed mechanical stimulation device can also provide a powerful tool for the study of molecular mechanism by which cells perceive the mechanical cues at subcellular levels

Multi-scale cellular engineering: From molecules to organ-on-a-chip.

Recent technological advances in cellular and molecular engineering have provided new insights into biology and enabled the design, manufacturing, and manipulation of complex living systems. Here, we summarize the state of advances at the molecular, cellular, and multi-cellular levels using experimental and computational tools. The areas of focus include intrinsically disordered proteins, synthetic proteins, spatiotemporally dynamic extracellular matrices, organ-on-a-chip approaches, and computational modeling, which all have tremendous potential for advancing fundamental and translational science. Perspectives on the current limitations and future directions are also described, with the goal of stimulating interest to overcome these hurdles using multi-disciplinary approaches

Differential RhoA Dynamics in Migratory and Stationary Cells Measured by FRET and Automated Image Analysis

Genetically-encoded biosensors based on fluorescence resonance energy transfer (FRET) have been widely applied to study the spatiotemporal regulation of molecular activity in live cells with high resolution. The efficient and accurate quantification of the large amount of imaging data from these single-cell FRET measurements demands robust and automated data analysis. However, the nonlinear movement of live cells presents tremendous challenge for this task. Based on image registration of the single-cell movement, we have developed automated image analysis methods to track and quantify the FRET signals within user-defined subcellular regions. In addition, the subcellular pixels were classified according to their associated FRET signals and the dynamics of the clusters analyzed. The results revealed that the EGF-induced reduction of RhoA activity in migratory HeLa cells is significantly less than that in stationary cells. Furthermore, the RhoA activity is polarized in the migratory cells, with the gradient of polarity oriented toward the opposite direction of cell migration. In contrast, there is a lack of consistent preference in RhoA polarity among stationary cells. Therefore, our image analysis methods can provide powerful tools for high-throughput and systematic investigation of the spatiotemporal molecular activities in regulating functions of live cells with their shapes and positions continuously changing in time

Rapid Activation of Rac GTPase in Living Cells by Force Is Independent of Src

It is well known that mechanical forces are crucial in regulating functions of every tissue and organ in a human body. However, it remains unclear how mechanical forces are transduced into biochemical activities and biological responses at the cellular and molecular level. Using the magnetic twisting cytometry technique, we applied local mechanical stresses to living human airway smooth muscle cells with a magnetic bead bound to the cell surface via transmembrane adhesion molecule integrins. The temporal and spatial activation of Rac, a small guanosine triphosphatase, was quantified using a fluorescent resonance energy transfer (FRET) method that measures changes in Rac activity in response to mechanical stresses by quantifying intensity ratios of ECFP (enhanced cyan fluorescent protein as a donor) and YPet (a variant yellow fluorescent protein as an acceptor) of the Rac biosensor. The applied stress induced rapid activation (less than 300 ms) of Rac at the cell periphery. In contrast, platelet derived growth factor (PDGF) induced Rac activation at a much later time (>30 sec). There was no stress-induced Rac activation when a mutant form of the Rac biosensor (RacN17) was transfected or when the magnetic bead was coated with transferrin or with poly-L-lysine. It is known that PDGF-induced Rac activation depends on Src activity. Surprisingly, pre-treatment of the cells with specific Src inhibitor PP1 or knocking-out Src gene had no effects on stress-induced Rac activation. In addition, eliminating lipid rafts through extraction of cholesterol from the plasma membrane did not prevent stress-induced Rac activation, suggesting a raft-independent mechanism in governing the Rac activation upon mechanical stimulation. Further evidence indicates that Rac activation by stress depends on the magnitudes of the applied stress and cytoskeletal integrity. Our results suggest that Rac activation by mechanical forces is rapid, direct and does not depend on Src activation. These findings suggest that signaling pathways of mechanical forces via integrins might be fundamentally different from those of growth factors

The Systemic Immune Inflammatory Index Predicts No-Reflow Phenomenon after Primary Percutaneous Coronary Intervention in Older Patients with STEMI

Purpose: Coronary no-reflow phenomenon (NRP), a common adverse complication in patients with ST-segment elevation myocardial infarction (STEMI) treated by percutaneous coronary intervention (PCI), is associated with poor patient prognosis. In this study, the correlation between the systemic immune-inflammation index (SII) and NRP in older patients with STEMI was studied, to provide a basis for early identification of high-risk patients and improve their prognosis.Materials and methods: Between January 2017 and June 2020, 578 older patients with acute STEMI admitted to the Department of Cardiology of Hebei General Hospital for direct PCI treatment were selected for this retrospective study. Patients were divided into an NRP group and normal-flow group according to whether NRP occurred during the operation. Clinical data and the examination indexes of the two groups were collected. Logistic regression was used to analyze the independent predictors of NRP, and the receiver operating characteristic curve was used to further analyze the ability of SII to predict NRP in older patients with STEMI.Results: Multivariate logistic analysis indicated that hypertension (OR=2.048, 95% CI:1.252–3.352, P=0.004), lymphocyte count (OR=0.571, 95% CI:0.368–0.885, P=0.012), platelet count (OR=1.009, 95% CI:1.005–1.013, P<0.001), hemoglobin (OR=1.015, 95% CI:1.003–1.028, P=0.018), multivessel disease (OR=2.237, 95% CI:1.407–3.558, P=0.001), and SII≥1814 (OR=3.799, 95% CI:2.190–6.593, P<0.001) were independent predictors of NRP after primary PCI in older patients with STEMI. Receiver operating characteristic curve analysis demonstrated that SII had a high predictive value for NRP (AUC=0.738; 95% CI:0.686–0.790), with the best cut-off value of 1814, a sensitivity of 52.85% and a specificity of 85.71%.Conclusion: For older patients with STEMI undergoing primary PCI, SII is a valid predictor of NRP

Analysis on the factors associated with COVID-19 infection among Chinese residents after the implementation of the 10 new rules to optimize COVID-19 response: a cross-sectional study

IntroductionThis study aimed to investigate the status of COVID-19 infection and the associated factors among Chinese residents after the implementation of the 10 New Rules to optimize COVID response.MethodsParticipants were recruited using convenience sampling. The study used self-filled questionnaires to examine COVID-19 infection and associated factors among Chinese residents, from December 29, 2022, to January 2, 2023. For the statistical analysis, descriptive and quantitative analyses were used. The potential risk factors for COVID-19 infection were identified by multivariable logistic regression analysis.ResultsAfter the adjustments in control strategies against COVID-19, the infection rate of COVID-19 was high among respondents, and 98.4% of individuals who tested positive showed symptoms including cough, fever, fatigue, headache, sore throat, nasal congestion, sputum production, muscle and joint pain, and runny nose. The main problems respondents reported were the shortage of drugs and medical supplies, the increased burden on families, and the unreliable information source of COVID-19 infection. Logistic regression showed that isolating patients with COVID-19 at home was associated with a lower risk of COVID-19 infection (OR = 0.58, 95%CI: 0.42–0.81).ConclusionCOVID-19 infection among residents is closely related to age, gender, and epidemic prevention measures. The government needs to strengthen education for individuals and centrally manage and properly address difficulties that may arise during COVID-19

Direct and sustained intracellular delivery of exogenous molecules using acoustic-transfection with high frequency ultrasound

Controlling cell functions for research and therapeutic purposes may open new strategies for the treatment of many diseases. An efficient and safe introduction of membrane impermeable molecules into target cells will provide versatile means to modulate cell fate. We introduce a new transfection technique that utilizes high frequency ultrasound without any contrast agents such as microbubbles, bringing a single-cell level targeting and size-dependent intracellular delivery of macromolecules. The transfection apparatus consists of an ultrasonic transducer with the center frequency of over 150 MHz and an epi-fluorescence microscope, entitled acoustic-transfection system. Acoustic pulses, emitted from an ultrasonic transducer, perturb the lipid bilayer of the cell membrane of a targeted single-cell to induce intracellular delivery of exogenous molecules. Simultaneous live cell imaging using HeLa cells to investigate the intracellular concentration of Ca2+ and propidium iodide (PI) and the delivery of 3 kDa dextran labeled with Alexa 488 were demonstrated. Cytosolic delivery of 3 kDa dextran induced via acoustic-transfection was manifested by diffused fluorescence throughout whole cells. Short-term (6 hr) cell viability test and long-term (40 hr) cell tracking confirmed that the proposed approach has low cell cytotoxicity.1

CRISPR-Cas9 screens in human cells and primary neurons identify modifiers of C9ORF72 dipeptide-repeat-protein toxicity.

Hexanucleotide-repeat expansions in the C9ORF72 gene are the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (c9ALS/FTD). The nucleotide-repeat expansions are translated into dipeptide-repeat (DPR) proteins, which are aggregation prone and may contribute to neurodegeneration. We used the CRISPR-Cas9 system to perform genome-wide gene-knockout screens for suppressors and enhancers of C9ORF72 DPR toxicity in human cells. We validated hits by performing secondary CRISPR-Cas9 screens in primary mouse neurons. We uncovered potent modifiers of DPR toxicity whose gene products function in nucleocytoplasmic transport, the endoplasmic reticulum (ER), proteasome, RNA-processing pathways, and chromatin modification. One modifier, TMX2, modulated the ER-stress signature elicited by C9ORF72 DPRs in neurons and improved survival of human induced motor neurons from patients with C9ORF72 ALS. Together, our results demonstrate the promise of CRISPR-Cas9 screens in defining mechanisms of neurodegenerative diseases