Method of generating speckle patterns for digital image correlation based on modified Conway's Game of Life.

The measurement accuracy of digital image correlation (DIC) is influenced by the quality of the speckle pattern. Although various models for generating random speckle patterns have been well discussed, obtaining appropriate speckle images with isotropic quality and performance could be a challenging issue in DIC. In this paper, we propose a novel (to our knowledge) method for generating speckle patterns based on modified Conway's game of life (GoL). By sequentially assembling the speckle patterns generated from the modified GoL, we produced the GoL speckle image. Then, verification and comparison experiments were conducted through pure in-plane translations. The results show that the generated speckle image which was resized with k s=6& k r=2 processing and subsequently fuzzified using a Gaussian filter, produces the best accuracy for DIC measurement. Furthermore, based on the rigid body in-plane rotation displacement tests in the physical experimental results of three different speckle images, the GoL speckle generated from our proposed method shows the smallest measurement error. This indicates that the proposed speckle patterns generating method could provide a new type of speckle pattern with better quality and accuracy.

Characterization of N-glycosylation and its functional role in SIDT1-Mediated RNA uptake.

The mammalian SID-1 transmembrane family members, SIDT1 and SIDT2, are multipass transmembrane proteins that mediate the cellular uptake and intracellular trafficking of nucleic acids, playing important roles in the immune response and tumorigenesis. Previous work has suggested that human SIDT1 and SIDT2 are N-glycosylated, but the precise site-specific N-glycosylation information and its functional contribution remain unclear. In this study, we use high-resolution liquid chromatography tandem mass spectrometry to comprehensively map the N-glycosites and quantify the N-glycosylation profiles of SIDT1 and SIDT2. Further molecular mechanistic probing elucidates the essential role of N-linked glycans in regulating cell surface expression, RNA binding, protein stability, and RNA uptake of SIDT1. Our results provide crucial information about the potential functional impact of N-glycosylation in the regulation of SIDT1-mediated RNA uptake and provide insights into the molecular mechanisms of this promising nucleic acid delivery system with potential implications for therapeutic applications.

Phase separations in oncogenesis, tumor progressions and metastasis: a glance from hallmarks of cancer.

Liquid-liquid phase separation (LLPS) is a novel principle for interpreting precise spatiotemporal coordination in living cells through biomolecular condensate (BMC) formation via dynamic aggregation. LLPS changes individual molecules into membrane-free, droplet-like BMCs with specific functions, which coordinate various cellular activities. The formation and regulation of LLPS are closely associated with oncogenesis, tumor progressions and metastasis, the specific roles and mechanisms of LLPS in tumors still need to be further investigated at present. In this review, we comprehensively summarize the conditions of LLPS and identify mechanisms involved in abnormal LLPS in cancer processes, including tumor growth, metastasis, and angiogenesis from the perspective of cancer hallmarks. We have also reviewed the clinical applications of LLPS in oncologic areas. This systematic summary of dysregulated LLPS from the different dimensions of cancer hallmarks will build a bridge for determining its specific functions to further guide basic research, finding strategies to intervene in LLPS, and developing relevant therapeutic approaches.

Mechanism of a rabbit monoclonal antibody broadly neutralizing SARS-CoV-2 variants.

Due to the continuous evolution of SARS-CoV-2, the Omicron variant has emerged and exhibits severe immune evasion. The high number of mutations at key antigenic sites on the spike protein has made a large number of existing antibodies and vaccines ineffective against this variant. Therefore, it is urgent to develop efficient broad-spectrum neutralizing therapeutic drugs. Here we characterize a rabbit monoclonal antibody (RmAb) 1H1 with broad-spectrum neutralizing potency against Omicron sublineages including BA.1, BA.1.1, BA.2, BA.2.12.1, BA.2.75, BA.3 and BA.4/5. Cryo-electron microscopy (cryo-EM) structure determination of the BA.1 spike-1H1 Fab complexes shows that 1H1 targets a highly conserved region of RBD and avoids most of the circulating Omicron mutations, explaining its broad-spectrum neutralization potency. Our findings indicate 1H1 as a promising RmAb model for designing broad-spectrum neutralizing antibodies and shed light on the development of therapeutic agents as well as effective vaccines against newly emerging variants in the future.

Mechanism of an RBM-targeted rabbit monoclonal antibody 9H1 neutralizing SARS-CoV-2.

The COVID-19 pandemic, caused by SARS-CoV-2, has led to over 750 million infections and 6.8 million deaths worldwide since late 2019. Due to the continuous evolution of SARS-CoV-2, many significant variants have emerged, creating ongoing challenges to the prevention and treatment of the pandemic. Therefore, the study of antibody responses against SARS-CoV-2 is essential for the development of vaccines and therapeutics. Here we perform single particle cryo-electron microscopy (cryo-EM) structure determination of a rabbit monoclonal antibody (RmAb) 9H1 in complex with the SARS-CoV-2 wild-type (WT) spike trimer. Our structural analysis shows that 9H1 interacts with the receptor-binding motif (RBM) region of the receptor-binding domain (RBD) on the spike protein and by directly competing with angiotensin-converting enzyme 2 (ACE2), it blocks the binding of the virus to the receptor and achieves neutralization. Our findings suggest that utilizing rabbit-derived mAbs provides valuable insights into the molecular interactions between neutralizing antibodies and spike proteins and may also facilitate the development of therapeutic antibodies and expand the antibody library.

Microneedle-Coupled Epidermal Sensors for In-Situ-Multiplexed Ion Detection in Interstitial Fluids.

Maintaining the concentrations of various ions in body fluids is critical to all living organisms. In this contribution, we designed a flexible microneedle patch coupled electrode array (MNP-EA) for the in situ multiplexed detection of ion species (Na+, K+, Ca2+, and H+) in tissue interstitial fluid (ISF). The microneedles (MNs) are mechanically robust for skin or cuticle penetration (0.21 N/needle) and highly swellable to quickly extract sufficient ISF onto the ion-selective electrochemical electrodes (∼6.87 µL/needle in 5 min). The potentiometric sensor can simultaneously detect these ion species with nearly Nernstian response in the ranges wider enough for diagnosis purposes (Na+: 0.75-200 mM, K+: 1-128 mM, Ca2+: 0.25-4.25 mM, pH: 5.5-8.5). The in vivo experiments on mice, humans, and plants demonstrate the feasibility of MNP-EA for timely and convenient diagnosis of ion imbalances with minimal invasiveness. This transdermal sensing platform shall be instrumental to home-based diagnosis and health monitoring of chronic diseases and is also promising for smart agriculture and the study of plant biology.

Construction and properties detection of 3D micro-structure scaffolds base on decellularized sheep kidney before and after crosslinking.

Decellularized extracellular matrix is one form of natural material in tissue engineering. The process of dECM retains the tissue microstructure, provides good cell adhesion sites, maintains most of biological signals that promotes the survival and differentiation ability of cells. In this study, sheep kidney was decellularized followed by histochemical staining, elemental analysis and scanning electron microscopy characterizations. The dECM scaffold was prepared with different sequences of freeze drying technology, crosslinking and the water absorption, porosity, mechanical strength with subsequent thermogravimetric analysis, Infrared spectroscopy and biocompatibility tests. Our results indicated that these decellularized treatments of sheep kidney can effectively remove DNA and retain uniform pore size distribution. After crosslinking the scaffold's water absorption decreased from 987.56 ± 40.21% to 934.39 ± 39.61%, the porosity decreased from 89.64 ± 3.2% to 85.09 ± 17.63%, and the compression modulus increased from 304.32 ± 25.43 kPa to 459.53 ± 38.92 kPa, with thermal process the percentage of weight loss decreased from 66.57% to 44.731%, in addition, the composition didn't change significantly, crosslinking could also promote the stability. In terms of biocompatibility, the number of viable cells increased significantly with the days. In conclusion, the crosslinked decellularized sheep kidney extracellular matrix scaffold reduced water absorption and porosity slightly, but has a significant increase in mechanical properties, and presented excellent biocompatibility which are beneficial to cell adhesion, growth and differentiation.

Structural Insight Into hnRNP A2/B1 Homodimerization and DNA Recognition.

Heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1) has been identified as a nuclear DNA sensor. Upon viral infection, hnRNP A2/B1 recognizes pathogen-derived DNA as a homodimer, which is a prerequisite for its translocation to the cytoplasm to activate the interferon response. However, the DNA binding mechanism inducing hnRNP A2/B1 homodimerization is unknown. Here, we show the crystal structure of the RNA recognition motif (RRM) of hnRNP A2/B1 in complex with a U-shaped ssDNA, which mediates the formation of a newly observed protein dimer. Our biochemical assays and mutagenesis studies confirm that the hnRNP A2/B1 homodimer forms in solution by binding to pre-generated ssDNA or dsDNA with a U-shaped bulge. These results depict a potential functional state of hnRNP A2/B1 in antiviral immunity and other cellular processes.

MiR-302c-5p affects the stemness and cisplatin resistance of nasopharyngeal carcinoma cells by regulating HSP90AA1.

Nasopharyngeal carcinoma (NPC) is one of the most frequent malignant tumors diagnosed in China. Cisplatin is one of the most commonly used anticancer drugs containing platinum in combined chemotherapy. The molecular mechanism of NPC is still largely unknown, and we aim to spare no effort to elucidate it. Normal human nasopharyngeal epithelial cells and NPC cell lines were cultured. The expression levels of miR-302c-5p and HSP90AA1 were detected with quantitative real-time PCR. Western blotting was used to analyze levels of the HSP90AA1, protein kinase B (AKT), p-AKT, CD44 and SOX2 proteins. The interaction between miR-302c-5p and HSP90AA1 was detected using a luciferase reporter assay. The bicinchoninic acid assay was used to observe cisplatin resistance in NPC cells. Our records confirmed that the expression of miR-302c-5p was substantially reduced and HSP90AA1 was increased in NPC cells. Additionally, miR-302c-5p inhibited cisplatin resistance and the traits of stem cells in NPC. A luciferase assay confirmed that miR-302c-5p is bound to HSP90AA1. Overexpression of HSP90AA1 may reverse the effects of overexpressed miR-302c-5p and inhibit cisplatin resistance and stem cell traits of NPC. This study investigated whether miR-302c-5p inhibited the AKT pathway by regulating HSP90AA1 expression and altered the resistance of NPC cells to cisplatin and the traits of tumor stem cells, which has not yet been reported.

Brain-specific loss of Abcg1 disturbs cholesterol metabolism and aggravates pyroptosis and neurological deficits after traumatic brain injury.

Based on accumulating evidence, cholesterol metabolism dysfunction has been suggested to contribute to the pathophysiological process of traumatic brain injury (TBI) and lead to neurological deficits. As a key transporter of cholesterol that efflux from cells, the ATP-binding cassette (ABC) transporter family exerts many beneficial effects on central nervous system (CNS) diseases. However, there is no study regarding the effects and mechanisms of ABCG1 on TBI. As expected, TBI resulted in the different time-course changes of cholesterol metabolism-related molecules in the injured cortex. Considering ABCG1 is expressed in neuron and glia post-TBI, we generated nestin-specific Abcg1 knockout (Abcg1-KO) mice using the Cre/loxP recombination system. These Abcg1-KO mice showed reduced plasma high-density lipoprotein cholesterol levels and increased plasma lower-density lipoprotein cholesterol levels under the base condition. After TBI, these Abcg1-KO mice were susceptible to cholesterol metabolism turbulence. Moreover, Abcg1-KO exacerbated TBI-induced pyroptosis, apoptosis, neuronal cell insult, brain edema, neurological deficits, and brain lesion volume. Importantly, we found that treating with retinoid X receptor (RXR, the upstream molecule of ABCG1) agonist, bexarotene, in Abcg1-KO mice partly rescued TBI-induced neuronal damages mentioned above and improved functional deficits versus vehicle-treated group. These data show that, in addition to regulating brain cholesterol metabolism, Abcg1 improves neurological deficits through inhibiting pyroptosis, apoptosis, neuronal cell insult, and brain edema. Moreover, our findings demonstrate that the cerebroprotection of Abcg1 on TBI partly relies on the activation of the RXRalpha/PPARgamma pathway, which provides a potential therapeutic target for treating TBI.

A Scalable CMOS Molecular Electronics Chip for Single-Molecule Biosensing.

This work reports the first CMOS molecular electronics chip. It is configured as a biosensor, where the primary sensing element is a single molecule "molecular wire" consisting of a ∼100 GΩ, 25 nm long alpha-helical peptide integrated into a current monitoring circuit. The engineered peptide contains a central conjugation site for attachment of various probe molecules, such as DNA, proteins, enzymes, or antibodies, which program the biosensor to detect interactions with a specific target molecule. The current through the molecular wire under a dc applied voltage is monitored with millisecond temporal resolution. The detected signals are millisecond-scale, picoampere current pulses generated by each transient probe-target molecular interaction. Implemented in a 0.18 µm CMOS technology, 16k sensors are arrayed with a 20 µm pitch and read out at a 1 kHz frame rate. The resulting biosensor chip provides direct, real-time observation of the single-molecule interaction kinetics, unlike classical biosensors that measure ensemble averages of such events. This molecular electronics chip provides a platform for putting molecular biosensing "on-chip" to bring the power of semiconductor chips to diverse applications in biological research, diagnostics, sequencing, proteomics, drug discovery, and environmental monitoring.

Colorimetric microneedle patches for multiplexed transdermal detection of metabolites.

Skin Interstitial Fluid (ISF) is an alternative source for biomarkers. Herein, a highly swellable microneedle patch (MNP) to rapidly extract ISF painlessly and bloodlessly is presented. The MNP is made of crosslinked methacrylated hyaluronic acid (MeHA) and dissolvable hyaluronic acid (HA) with the optimal balance of mechanical strength (0.6 N/MN) and absorption capability (16.22 µL in 20 min). Incorporated with wax-patterned and sensing-reagent-decorated test paper (TP) for multiplexed colorimetric detection of metabolites (glucose, lactate, cholesterol, and pH), this TP-MNP biosensor gives rapid color change in biomarker concentration-dependent manner based on specific enzymatic reactions, whereby allowing diagnosis by the naked eye or quantitative RGB analysis. Both the in vitro and in vivo experiments demonstrate the feasibility of TP-MNPs to detect multiple biomarkers in skin interstitial fluid within minutes. Such convenient and self-administrable profiling of metabolites shall be instrumental for home-based long-term monitoring and management of metabolic diseases.

Mid-infrared optical switches enabled by metal-organic frameworks for compact high-power nanosecond laser sources at 3 µm.

Pulsed lasers operating in the mid-infrared are of great importance for numerous applications in spectroscopy, medical surgery, laser processing, and communications. In spite of recent advances with mid-infrared gain platforms, the lack of a capable pulse generation mechanism hinders the development of compact mid-infrared pulsed laser source. Here we show that MIL-68(Al) and MIL-68(Fe), which are aluminum- and iron- based metal-organic frameworks (MOFs) with ordered atoms distribution and periodic mesoporous structure, constitute exceptional optical switches for the mid-infrared. We fabricated the MIL-68(Al) and MIL-68(Fe) via hydrothermal method and prepared reflection-type MIL-68(Al)- and MIL-68(Fe)- saturable absorber mirrors (SAMs). By employing the as-prepared SAMs in the laser cavities, we achieved high-power nanosecond Q-switched fiber lasers at 2.8 µm. Especially, the average output power and pulse duration of the MIL-68(Al) Q-switched fiber laser reached 809.1 mW and 567 ns, respectively. To the best of our knowledge, this is the first time to demonstrate that MIL-68(M) can be efficient optical switches for 3-µm mid-IR laser pulses generation. Our findings reveal that MIL-68(M) is promising saturable absorber for compact and high-performance mid-infrared pulsed lasers.

Structures of Omicron spike complexes and implications for neutralizing antibody development.

The emergence of the SARS-CoV-2 Omicron variant is dominant in many countries worldwide. The high number of spike mutations is responsible for the broad immune evasion from existing vaccines and antibody drugs. To understand this, we first present the cryo-electron microscopy structure of ACE2-bound SARS-CoV-2 Omicron spike. Comparison to previous spike antibody structures explains how Omicron escapes these therapeutics. Secondly, we report structures of Omicron, Delta, and wild-type spikes bound to a patient-derived Fab antibody fragment (510A5), which provides direct evidence where antibody binding is greatly attenuated by the Omicron mutations, freeing spike to bind ACE2. Together with biochemical binding and 510A5 neutralization assays, our work establishes principles of binding required for neutralization and clearly illustrates how the mutations lead to antibody evasion yet retain strong ACE2 interactions. Structural information on spike with both bound and unbound antibodies collectively elucidates potential strategies for generation of therapeutic antibodies.

Decellularized Pig Kidney with a Micro-Nano Secondary Structure Contributes to Tumor Progression in 3D Tumor Model.

In spite of many anti-cancer drugs utilized in clinical treatment, cancer is still one of the diseases with the highest morbidity and mortality worldwide, owing to the complexity and heterogeneity of the tumor microenvironment. Compared with conventional 2D tumor models, 3D scaffolds could provide structures and a microenvironment which stimulate native tumor tissues more accurately. The extracellular matrix (ECM) is the main component of the cell in the microenvironment that is mainly composed of three-dimensional nanofibers, which can form nanoscale fiber networks, while the decellularized extracellular matrix (dECM) has been widely applied to engineered scaffolds. In this study, pig kidney was used as the source material to prepare dECM scaffolds. A chemical crosslinking method was used to improve the mechanical properties and other physical characteristics of the decellularized pig kidney-derived scaffold. Furthermore, a human breast cancer cell line (MCF-7) was used to further investigate the biocompatibility of the scaffold to fabricate a tumor model. The results showed that the existence of nanostructures in the scaffold plays an important role in cell adhesion, proliferation, and differentiation. Therefore, the pig kidney-derived matrix scaffold prepared by decellularization could provide more cell attachment sites, which is conducive to cell adhesion and proliferation, physiological activities, and tumor model construction.

Molecular electronics sensors on a scalable semiconductor chip: A platform for single-molecule measurement of binding kinetics and enzyme activity.

For nearly 50 years, the vision of using single molecules in circuits has been seen as providing the ultimate miniaturization of electronic chips. An advanced example of such a molecular electronics chip is presented here, with the important distinction that the molecular circuit elements play the role of general-purpose single-molecule sensors. The device consists of a semiconductor chip with a scalable array architecture. Each array element contains a synthetic molecular wire assembled to span nanoelectrodes in a current monitoring circuit. A central conjugation site is used to attach a single probe molecule that defines the target of the sensor. The chip digitizes the resulting picoamp-scale current-versus-time readout from each sensor element of the array at a rate of 1,000 frames per second. This provides detailed electrical signatures of the single-molecule interactions between the probe and targets present in a solution-phase test sample. This platform is used to measure the interaction kinetics of single molecules, without the use of labels, in a massively parallel fashion. To demonstrate broad applicability, examples are shown for probe molecule binding, including DNA oligos, aptamers, antibodies, and antigens, and the activity of enzymes relevant to diagnostics and sequencing, including a CRISPR/Cas enzyme binding a target DNA, and a DNA polymerase enzyme incorporating nucleotides as it copies a DNA template. All of these applications are accomplished with high sensitivity and resolution, on a manufacturable, scalable, all-electronic semiconductor chip device, thereby bringing the power of modern chips to these diverse areas of biosensing.

Identification of patients at risk of new onset heart failure: Utilizing a large statewide health information exchange to train and validate a risk prediction model.

BACKGROUND: New-onset heart failure (HF) is associated with poor prognosis and high healthcare utilization. Early identification of patients at increased risk incident-HF may allow for focused allocation of preventative care resources. Health information exchange (HIE) data span the entire spectrum of clinical care, but there are no HIE-based clinical decision support tools for diagnosis of incident-HF. We applied machine-learning methods to model the one-year risk of incident-HF from the Maine statewide-HIE. METHODS AND RESULTS: We included subjects aged ≥ 40 years without prior HF ICD9/10 codes during a three-year period from 2015 to 2018, and incident-HF defined as assignment of two outpatient or one inpatient code in a year. A tree-boosting algorithm was used to model the probability of incident-HF in year two from data collected in year one, and then validated in year three. 5,668 of 521,347 patients (1.09%) developed incident-HF in the validation cohort. In the validation cohort, the model c-statistic was 0.824 and at a clinically predetermined risk threshold, 10% of patients identified by the model developed incident-HF and 29% of all incident-HF cases in the state of Maine were identified. CONCLUSIONS: Utilizing machine learning modeling techniques on passively collected clinical HIE data, we developed and validated an incident-HF prediction tool that performs on par with other models that require proactively collected clinical data. Our algorithm could be integrated into other HIEs to leverage the EMR resources to provide individuals, systems, and payors with a risk stratification tool to allow for targeted resource allocation to reduce incident-HF disease burden on individuals and health care systems.

Evaluation of Development Datasets for Hermetia illucens (L.) (Diptera: Stratiomyidae) for Estimating the Time of Placement of Human and Swine Remains in Texas, USA.

A basic tenet of forensic entomology is development data of an insect can be used to predict the time of colonization (TOC) by insect specimens collected from remains, and this prediction is related to the time of death and/or time of placement (TOP). However, few datasets have been evaluated to determine their accuracy or precision. The black soldier fly, Hermetia illucens (L.) (Diptera: Stratiomyidae) is recognized as an insect of forensic importance. This study examined the accuracy and precision of several development datasets for the black soldier fly by estimating the TOP of five sets of human and three sets of swine remains in San Marcos and College Station, TX, respectively. Data generated from this study indicate only one of these datasets consistently (time-to-prepupae 52%; time-to-eclosion 75%) produced TOP estimations that occurred within a day of the actual TOP of the remains. It is unknown if the precolonization interval (PreCI) of this species is long, but it has been observed that the species can colonize within 6 d after death. This assumption remains untested by validation studies. Accounting for this PreCI improved accuracy for the time-to-prepupae group, but reduced accuracy in the time-to-eclosion group. The findings presented here highlight a need for detailed, forensic-based development data for the black soldier fly that can reliably and accurately be used in casework. Finally, this study outlines the need for a basic understanding of the timing of resource utilization (i.e., duration of the PreCI) for forensically relevant taxa so that reasonable corrections may be made to TOC as related to minimum postmortem interval (mPMI) estimates.