The TOX-RAGE axis mediates inflammatory activation and lung injury in severe pulmonary infectious diseases.

Thymocyte selection-associated high-mobility group box (TOX) is a transcription factor that is crucial for T cell exhaustion during chronic antigenic stimulation, but its role in inflammation is poorly understood. Here, we report that TOX extracellularly mediates drastic inflammation upon severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection by binding to the cell surface receptor for advanced glycation end-products (RAGE). In various diseases, including COVID-19, TOX release was highly detectable in association with disease severity, contributing to lung fibroproliferative acute respiratory distress syndrome (ARDS). Recombinant TOX-induced blood vessel rupture, similar to a clinical signature in patients experiencing a cytokine storm, further exacerbating respiratory function impairment. In contrast, disruption of TOX function by a neutralizing antibody and genetic removal of RAGE diminished TOX-mediated deleterious effects. Altogether, our results suggest an insight into TOX function as an inflammatory mediator and propose the TOX-RAGE axis as a potential target for treating severe patients with pulmonary infection and mitigating lung fibroproliferative ARDS.

Multiple transcription factors of Arabidopsis thaliana that are activated by LEAFY COTYLEDON 2 regulate triacylglycerol biosynthesis.

Plant triacylglycerols (TAG) are used in food and various industrial feedstocks. LEAFY COTYLEDON 2 (LEC2), a master positive regulator of TAG biosynthesis, regulates a complex network of transcription factors (TFs) during seed development. Aside from WRINKLED1 (WRI1), the TFs regulated by LEC2 related to TAG biosynthesis have not yet been identified. Previously, we identified 25 seed-expressing TFs that were upregulated in Arabidopsis leaves that overexpressed senescence-induced LEC2. In this study, each of the 25 TFs was transiently expressed in the leaves of Nicotiana benthamiana to identify unknown TFs that regulate TAG biosynthesis. The TAG content of the transformed leaves was analyzed using thin layer chromatography and gas chromatography. We observed that five TFs, ARABIDOPSIS RESPONSIVE REGULATOR 21 (ARR21), AINTEGUMENTA-LIKE 6 (AIL6), APETALA2/ETHYLENE RESPONSIVE FACTOR 55 (ERF55), WRKY DNA-BINDING PROTEIN 8 (WRKY8), and ARABIDOPSIS NAC DOMAIN CONTAINING PROTEIN 38 (ANAC038) increased TAG synthesis in the leaves. Among these, the promoters of AIL6, ERF55, WRKY8, and ANAC038 contain RY motifs, which are LEC2-binding sites activated by LEC2. AIL6 overexpression in Arabidopsis increased the total fatty acid (FA) content in seeds and altered the FA composition, with increases in 16:0, 18:1, and 18:2 and decreases in 18:0, 18:3, and 20:1 compared with those in the wild type (WT). AIL6 overexpression activates several FA and TAG biosynthesis genes. Therefore, our study successfully identified several new TFs regulated by LEC2 in TAG biosynthesis and showed that AIL6 increased the TAG content in seeds.

MCP-5 suppresses osteoclast differentiation through Ccr5 upregulation.

Human monocyte chemoattractant protein-1 (MCP-1) in mice has two orthologs, MCP-1 and MCP-5. MCP-1, which is highly expressed in osteoclasts rather than in osteoclast precursor cells, is an important factor in osteoclast differentiation. However, the roles of MCP-5 in osteoclasts are completely unknown. In this study, contrary to MCP-1, MCP-5 was downregulated during receptor activator of nuclear factor kappa B ligand (RANKL)-induced osteoclast differentiation and was considered an inhibitory factor in osteoclast differentiation. The inhibitory role of MCP-5 in osteoclast differentiation was closely related to the increase in Ccr5 expression and the inhibition of IκB degradation by RANKL. Transgenic mice expressing MCP-5 controlled by Mx-1 promoter exhibited an increased bone mass because of a decrease in osteoclasts. This result strongly supported that MCP-5 negatively regulated osteoclast differentiation. MCP-5 also prevented severe bone loss caused by RANKL.

Nodal negatively regulates osteoclast differentiation by inducing STAT1 phosphorylation.

Several members of the transforming growth factor beta (TGF-ß) superfamily regulate the proliferation, differentiation, and function of bone-forming osteoblasts and bone-resorbing osteoclasts. However, it is still unknown whether Nodal, a member of the TGF-ß superfamily, serves a function in bone cells. In this study, we found that Nodal did not have any function in osteoblasts but instead negatively regulated osteoclast differentiation. Nodal inhibited RANKL-induced osteoclast differentiation by downregulating the expression of pro-osteoclastogenic genes, including c-fos, Nfatc1, and Blimp1, and upregulating the expression of antiosteoclastogenic genes, including Bcl6 and Irf8. Nodal activated STAT1 in osteoclast precursor cells, and STAT1 downregulation significantly reduced the inhibitory effect of Nodal on osteoclast differentiation. These findings indicate that Nodal activates STAT1 to downregulate or upregulate the expression of pro-osteoclastogenic or antiosteoclastogenic genes, respectively, leading to the inhibition of osteoclast differentiation. Moreover, the inhibitory effect of Nodal on osteoclast differentiation contributed to the reduction of RANKL-induced bone loss in vivo.

RBFOX2-regulated TEAD1 alternative splicing plays a pivotal role in Hippo-YAP signaling.

Alternative pre-mRNA splicing is key to proteome diversity; however, the biological roles of alternative splicing (AS) in signaling pathways remain elusive. Here, we focus on TEA domain transcription factor 1 (TEAD1), a YAP binding factor in the Hippo signaling pathway. Public database analyses showed that expression of YAP-TEAD target genes negatively correlated with the expression of a TEAD1 isoform lacking exon 6 (TEAD1ΔE6) but did not correlate with overall TEAD1 expression. We confirmed that the transcriptional activity and oncogenic properties of the full-length TEAD1 isoform were greater than those of TEAD1ΔE6, with the difference in transcription related to YAP interaction. Furthermore, we showed that RNA-binding Fox-1 homolog 2 (RBFOX2) promoted the inclusion of TEAD1 exon 6 via binding to the conserved GCAUG element in the downstream intron. These results suggest a regulatory mechanism of RBFOX2-mediated TEAD1 AS and provide insight into AS-specific modulation of signaling pathways.

Glioma Single-Cell Biomechanical Analysis by Cyclic Conical Constricted Microfluidics.

Determining the grade of glioma is a critical step in choosing patients' treatment plans in clinical practices. The pathological diagnosis of patient's glioma samples requires extensive staining and imaging procedures, which are expensive and time-consuming. Current advanced uniform-width-constriction-channel-based microfluidics have proven to be effective in distinguishing cancer cells from normal tissues, such as breast cancer, ovarian cancer, prostate cancer, etc. However, the uniform-width-constriction channels can result in low yields on glioma cells with irregular morphologies and high heterogeneity. In this research, we presented an innovative cyclic conical constricted (CCC) microfluidic device to better differentiate glioma cells from normal glial cells. Compared with the widely used uniform-width-constriction microchannels, the new CCC configuration forces single cells to deform gradually and obtains the biophysical attributes from each deformation. The human-derived glioma cell lines U-87 and U-251, as well as the human-derived normal glial astrocyte cell line HA-1800 were selected as the proof of concept. The results showed that CCC channels can effectively obtain the biomechanical characteristics of different 12-25 µm glial cell lines. The patient glioma samples with WHO grades II, III, and IV were tested by CCC channels and compared between Elastic Net (ENet) and Lasso analysis. The results demonstrated that CCC channels and the ENet can successfully select critical biomechanical parameters to differentiate the grades of single-glioma cells. This CCC device can be potentially further applied to the extensive family of brain tumors at the single-cell level.

Kernel-based hierarchical structural component models for pathway analysis.

MOTIVATION: Pathway analyses have led to more insight into the underlying biological functions related to the phenotype of interest in various types of omics data. Pathway-based statistical approaches have been actively developed, but most of them do not consider correlations among pathways. Because it is well known that there are quite a few biomarkers that overlap between pathways, these approaches may provide misleading results. In addition, most pathway-based approaches tend to assume that biomarkers within a pathway have linear associations with the phenotype of interest, even though the relationships are more complex. RESULTS: To model complex effects including non-linear effects, we propose a new approach, Hierarchical structural CoMponent analysis using Kernel (HisCoM-Kernel). The proposed method models non-linear associations between biomarkers and phenotype by extending the kernel machine regression and analyzes entire pathways simultaneously by using the biomarker-pathway hierarchical structure. HisCoM-Kernel is a flexible model that can be applied to various omics data. It was successfully applied to three omics datasets generated by different technologies. Our simulation studies showed that HisCoM-Kernel provided higher statistical power than other existing pathway-based methods in all datasets. The application of HisCoM-Kernel to three types of omics dataset showed its superior performance compared to existing methods in identifying more biologically meaningful pathways, including those reported in previous studies. AVAILABILITY AND IMPLEMENTATION: The HisCoM-Kernel software is freely available at http://statgen.snu.ac.kr/software/HisCom-Kernel/. The RNA-seq data underlying this article are available at https://xena.ucsc.edu/, and the others will be shared on reasonable request to the corresponding author. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Fibrillin2 in chloroplast plastoglobules participates in photoprotection and jasmonate-induced senescence.

Fibrillins (FBNs) are the major structural proteins of plastoglobules (PGs) in chloroplasts. PGs are associated with defense against abiotic and biotic stresses, as well as lipid storage. Although FBN2 is abundant in PGs, its independent function under abiotic stress has not yet been identified. In this study, the targeting of FBN2 to PGs was clearly demonstrated using an FBN2-YFP fusion protein. FBN2 showed higher expression in green photosynthetic tissues and was upregulated at the transcriptional level under high-light stress. The photosynthetic capacity of fbn2 knockout mutants generated using CRISPR/Cas9 technology decreased rapidly compared with that of wild-type (WT) plants under high-light stress. In addition to the photoprotective function of FBN2, fbn2 mutants had lower levels of plastoquinone-9 and plastochromanol-8. The fbn2 mutants were highly sensitive to methyl jasmonate (MeJA) and exhibited root growth inhibition and a pale-green phenotype due to reduced chlorophyll content. Consistently, upon MeJA treatment, the fbn2 mutants showed faster leaf senescence and more rapid chlorophyll degradation with decreased photosynthetic ability compared with the WT plants. The results of this study suggest that FBN2 is involved in protection against high-light stress and acts as an inhibitor of jasmonate-induced senescence in Arabidopsis (Arabidopsis thaliana).

Joint semiparametric kernel network regression.

Variable selection and graphical modeling play essential roles in highly correlated and high-dimensional (HCHD) data analysis. Variable selection methods have been developed under both parametric and nonparametric model settings. However, variable selection for nonadditive, nonparametric regression with high-dimensional variables is challenging due to complications in modeling unknown dependence structures among HCHD variables. Gaussian graphical models are a popular and useful tool for investigating the conditional dependence between variables via estimating sparse precision matrices. For a given class of interest, the estimated precision matrices can be mapped onto networks for visualization. However, the limitation of Gaussian graphical models is that they are only applicable to discretized response variables and for the case when p log ( p ) ≪ n $$ p\log (p)\ll n $$ , where p $$ p $$ is the number of variables and n $$ n $$ is the sample size. They are necessary to develop a joint method for variable selection and graphical modeling. To the best of our knowledge, the methods for simultaneously selecting variable selection and estimating networks among variables in the semiparametric regression settings are quite limited. Hence, in this paper, we develop a joint semiparametric kernel network regression method to solve this limitation and to provide a connection between them. Our approach is a unified and integrated method that can simultaneously identify important variables and build a network among those variables. We developed our approach under a semiparametric kernel machine regression framework, which can allow for nonlinear or nonadditive associations and complicated interactions among the variables. The advantages of our approach are that it can (1) simultaneously select variables and build a network among HCHD variables under a regression setting; (2) model unknown and complicated interactions among the variables and estimate the network among these variables; (3) allow for any form of semiparametric model, including non-additive, nonparametric model; and (4) provide an interpretable network that considers important variables and a response variable. We demonstrate our approach using a simulation study and real application on genetic pathway-based analysis.

Oral Porphyromonas gingivalis infection affects intestinal microbiota and promotes atherosclerosis.

AIM: The link between periodontitis and intestinal dysbiosis, two factors that contribute to atherosclerosis, has not been clearly defined. We investigated the integrative effects of oral infection with Porphyromonas gingivalis (PG), the major pathogen for periodontitis, on intestinal microbiota and atherosclerosis. MATERIALS AND METHODS: ApoE-/- mice were fed a normal chow diet (NC), a Western diet (WD) or a WD with oral PG infection (PG). The PG infection was investigated by placing a total of 109 CFUs of live PG into the oral cavity of each mouse using a feeding needle five times a week for 3 weeks. Atherosclerotic lesions of the aortae were measured, and blood lipoproteins and the expression of molecules related to lipid metabolism in the liver were analysed. We also performed 16S RNA sequencing and a microbiome analysis using faeces. RESULTS: En face bloc preparation of the aortae showed that the PG group had a 1.7-fold increase in atherosclerotic lesions compared with the WD group (p < .01). Serum analyses showed that oral PG infection induced a significant decrease in high-density lipoprotein (HDL) and triglyceride. Western blots of hepatic tissue lysates revealed that PG infection reduced the expression of scavenger receptor class B type 1 (SR-B1) in the liver by 50%. Faecal microbiota analysis revealed that species richness estimates (Chao1, ACE) decreased immediately after PG infection. PG infection also induced a significant decrease in Shannon diversity and an increase in Simpson's indices in the WD-fed mice. PG infection significantly increased the phyla Actinobacteria and Deferribacteres, along with the species Mucispirillum schaedleri and Lactobacillus gasseri, in the mice. The functional study showed that PG infection increased the expression of proteins that function in carbohydrate and glucose metabolism, including phosphotransferase system (PTS) proteins and the GntR family transcriptional regulator. CONCLUSIONS: Oral PG infection promotes atherosclerosis and induces significant metabolic changes, including reduced serum HDL and reduced hepatic SR-B1 and ABCA1 expression, as well as changes in intestinal microbiota. Our study suggests that intestinal dysbiosis accompanies periodontitis and could play a role in atherosclerosis.

From the perspective of pseudo-progression rather than treatment failure, how long should we wait before considering treatment failure if large cystic enlargement occurs after Gamma Knife radiosurgery for vestibular schwannoma?: insight into pseudoprogression based on two case reports.

Gamma knife radiosurgery (GKRS) has been accepted as a safe and effective treatment for vestibular schwannoma (VS). However, during follow-up, tumor expansion induced by irradiation can occur, and diagnosis of failure in radiosurgery for VS is still controversial. Tumor expansion with cystic enlargement causes some confusion regarding whether further treatment should be performed. We analyzed more than 10 years of clinical findings and imaging of patients with VS with cystic enlargement after GKRS. A 49-year-old male with hearing impairment was treated with GKRS (12 Gy; isodose, 50%) for a left VS with a preoperative tumor volume of 0.8 cc. The tumor size increased with cystic changes from the third year after GKRS, reaching a volume of 10.8 cc at 5 years after GKRS. At the 6th year of follow-up, the tumor volume started to decrease, up to 0.3 cc by the 14th year of follow-up. A 52-year-old female with hearing impairment and left facial numbness was treated with GKRS for a left VS (13 Gy; isodose, 50%). The preoperative tumor volume was 6.3 cc, which started to increase with cystic enlargement from the first year after GKRS, and reaching 18.2 cc by 5 years after GKRS. The tumor maintained a cystic pattern with slight changes in size, but no other neurologic symptoms developed during the follow-up period. After 6 years of GKRS, tumor regression was observed, eventually reaching a volume of 3.2 cc by the 13th year of follow-up. In both cases, persistent cystic enlargement in VS was observed at 5 years after GKRS, after which the tumors began to stabilize. After more than 10 years of GKRS, the tumor volume was less than that before GKRS. Enlargement with large cystic formation in the first 3-5 years after GKRS has been considered as treatment failure. However, our cases show that further treatment for cystic enlargement should be deferred for at least 10 years, especially in patients without neurological deterioration, as inadequate surgery can be prevented within that period.

Method of Evaluating Multiple Scenarios in a Single Simulation Run for Automated Vehicle Assessment.

With advances in the technology applied to automated driving systems (ADSs), active efforts have been made to evaluate the safety of ADS in various complex situations using simulations. In accordance with these efforts, numerous institutions have developed single-scenario pools that reflect a variety of road and traffic characteristics and ADS performances. However, a single scenario has limitations in comprehensively evaluating the performance of complex ADS. Therefore, this study proposed a methodology that combines and transforms single scenarios into multiple scenarios. This aided in continuously evaluating the ADS performance over entire road segments and implemented this methodology in the simulations.

The mysterious role of fibrillin in plastid metabolism: current advances in understanding.

Fibrillins (FBNs) are a family of genes in cyanobacteria, algae, and plants. The proteins they encode possess a lipid-binding motif, exist in various types of plastids, and are associated with lipid bodies called plastoglobules, implicating them in lipid metabolism. FBNs present in the thylakoid and stroma are involved in the storage, transport, and synthesis of lipid molecules for photoprotective functions against high-light stress. In this review, the diversity of subplastid locations in the evolution of FBNs, regulation of FBNs expression by various stresses, and the role of FBNs in plastid lipid metabolism are comprehensively summarized and directions for future research are discussed.

Neural Mechanisms of Facial Emotion Recognition in Autism: Distinct Roles for Anterior Cingulate and dlPFC.

OBJECTIVE: The present study sought to measure and internally validate neural markers of facial emotion recognition (FER) in adolescents and young adults with ASD to inform targeted intervention. METHOD: We utilized fMRI to measure patterns of brain activity among individuals with ASD (N = 21) and matched controls (CON; N = 20) 2 s prior to judgments about the identity of six distinct facial emotions (happy, sad, angry, surprised, fearful, disgust). RESULTS: Predictive modeling of fMRI data (support vector classification; SVC) identified mechanistic roles for brain regions that forecasted correct and incorrect identification of facial emotion as well as sources of errors over these decisions. BOLD signal activation in bilateral insula, anterior cingulate (ACC) and right dorsolateral prefrontal cortex (dlPFC) preceded accurate FER in both controls and ASD. Predictive modeling utilizing SVC confirmed the utility of ACC in forecasting correct decisions in controls but not ASD, and further indicated that a region within the right dlPFC was the source of a type 1 error signal in ASD (i.e. neural marker reflecting an impending correct judgment followed by an incorrect behavioral response) approximately two seconds prior to emotion judgments during fMRI. CONCLUSIONS: ACC forecasted correct decisions only among control participants. Right dlPFC was the source of a false-positive signal immediately prior to an error about the nature of a facial emotion in adolescents and young adults with ASD, potentially consistent with prior work indicating that dlPFC may play a role in attention to and regulation of emotional experience.

Overexpression of Neurogenin 1 Negatively Regulates Osteoclast and Osteoblast Differentiation.

Neurogenin 1 (Ngn1) belongs to the basic helix-loop-helix (bHLH) transcription factor family and plays important roles in specifying neuronal differentiation. The present study aimed to determine whether forced Ngn1 expression contributes to bone homeostasis. Ngn1 inhibited the p300/CREB-binding protein-associated factor (PCAF)-induced acetylation of nuclear factor of activated T cells 1 (NFATc1) and runt-related transcription factor 2 (Runx2) through binding to PCAF, which led to the inhibition of osteoclast and osteoblast differentiation, respectively. In addition, Ngn1 overexpression inhibited the TNF-α- and IL-17A-mediated enhancement of osteoclast differentiation and IL-17A-induced osteoblast differentiation. These findings indicate that Ngn1 can serve as a novel therapeutic agent for treating ankylosing spondylitis with abnormally increased bone formation and resorption.

The ATF3-OPG Axis Contributes to Bone Formation by Regulating the Differentiation of Osteoclasts, Osteoblasts, and Adipocytes.

Activating transcription factor 3 (ATF3) has been identified as a negative regulator of osteoblast differentiation in in vitro study. However, it was not associated with osteoblast differentiation in in vivo study. To provide an understanding of the discrepancy between the in vivo and in vitro findings regarding the function of ATF3 in osteoblasts, we investigated the unidentified roles of ATF3 in osteoblast biology. ATF3 enhanced osteoprotegerin (OPG) production, not only in osteoblast precursor cells, but also during osteoblast differentiation and osteoblastic adipocyte differentiation. In addition, ATF3 increased nodule formation in immature osteoblasts and decreased osteoblast-dependent osteoclast formation, as well as the transdifferentiation of osteoblasts to adipocytes. However, all these effects were reversed by the OPG neutralizing antibody. Taken together, these results suggest that ATF3 contributes to bone homeostasis by regulating the differentiation of various cell types in the bone microenvironment, including osteoblasts, osteoclasts, and adipocytes via inducing OPG production.

Transcription Factor Lmx1b Negatively Regulates Osteoblast Differentiation and Bone Formation.

The LIM-homeodomain transcription factor Lmx1b plays a key role in body pattern formation during development. Although Lmx1b is essential for the normal development of multiple tissues, its regulatory mechanism in bone cells remains unclear. Here, we demonstrated that Lmx1b negatively regulates bone morphogenic protein 2 (BMP2)-induced osteoblast differentiation. Overexpressed Lmx1b in the osteoblast precursor cells inhibited alkaline phosphatase (ALP) activity and nodule formation, as well as the expression of osteoblast maker genes, including runt-related transcription factor 2 (Runx2), alkaline phosphatase (Alpl), bone sialoprotein (Ibsp), and osteocalcin (Bglap). Conversely, the knockdown of Lmx1b in the osteoblast precursors enhanced the osteoblast differentiation and function. Lmx1b physically interacted with and repressed the transcriptional activity of Runx2 by reducing the recruitment of Runx2 to the promoter region of its target genes. In vivo analysis of BMP2-induced ectopic bone formation revealed that the knockdown of Lmx1b promoted osteogenic differentiation and bone regeneration. Our data demonstrate that Lmx1b negatively regulates osteoblast differentiation and function through regulation of Runx2 and provides a molecular basis for therapeutic targets for bone diseases.

Prognostic Impact of In-Hospital Use of Mechanical Cardiopulmonary Resuscitation Devices Compared with Manual Cardiopulmonary Resuscitation: A Nationwide Population-Based Observational Study in South Korea.

Background and Objectives: This study analyzed the prognostic impact of mechanical cardiopulmonary resuscitation (CPR) devices in out-of-hospital cardiac arrest (OHCA) patients, in comparison to manual CPR. Materials and Methods: This study was a nationwide population-based observational study in South Korea. Data were retrospectively collected from 142,905 OHCA patients using the South Korean Out-of-Hospital Cardiac Arrest Surveillance database. We included adult OHCA patients who received manual or mechanical CPR in the emergency room. The primary outcome was survival at discharge and the secondary outcome was sustained return of spontaneous circulation (ROSC). Statistical analysis included propensity score matching and multivariate logistic regression. Results: A total of 19,045 manual CPR and 1125 mechanical CPR cases (671 AutoPulseTM vs. 305 ThumperTM vs. 149 LUCASTM) were included. In the matched multivariate analyses, all mechanical CPR devices were associated with a lower ROSC than that of manual CPR. AutoPulseTM was associated with lower survival in the multivariate analysis after matching (aOR with 95% CI: 0.57 (0.33-0.96)), but the other mechanical CPR devices were associated with similar survival to discharge as that of manual CPR. Witnessed arrest was commonly associated with high ROSC, but the use of mechanical CPR devices and cardiac origin arrest were associated with low ROSC. Only target temperature management was the common predictor for high survival. Conclusions: The mechanical CPR devices largely led to similar survival to discharge as that of manual CPR in OHCA patients; however, the in-hospital use of the AutoPulseTM device for mechanical CPR may significantly lower survival compared to manual CPR.

Discriminatory Detection of ssDNA by Surface-Enhanced Raman Spectroscopy (SERS) and Tree-Based Support Vector Machine (Tr-SVM).

We report label-free detection of 86-base single-stranded DNA (ssDNA) gene segments by surface-enhanced Raman spectroscopy (SERS). The use of a slippery liquid infused porous (SLIP) membrane induced aggregation of 43 nm gold nanoparticles and ssDNA upon pin-free droplet evaporation. The combined SLIPSERS approach generates significant numbers of SERS hot-spots and enabled detection at the 100 nM level of mecA and intI1 gene segments-two genes of interest in the context of antibiotic resistance. Tree-based multiclass support vector machine (Tr-SVM) classifiers were built to discriminate SERS spectra of 12 different gene sequences obtained by SLIPSERS: mecA, intI1, as well as analogues of mecA and intI1, respectively, with 2-10 base mismatches, and two random sequences. The trained predictive Tr-SVM classifiers correctly identified each gene sequence with a prediction accuracy of ∼90%. This study illustrates a novel means for discriminatory label-free SERS detection of ssDNA enabled by Tr-SVM.

Plasmonically Calibrated Label-Free Surface-Enhanced Raman Spectroscopy for Improved Multivariate Analysis of Living Cells in Cancer Subtyping and Drug Testing.

Plasmonic nanostructure-enabled label-free surface-enhanced Raman spectroscopy (SERS) emerges as a rapid nondestructive molecular fingerprint characterization technique for complex biological samples. However, label-free SERS bioanalysis faces challenges in reliability and reproducibility due to SERS signals' high susceptibility to local optical field variations at plasmonic hotspots, which can bias correlations between the measured spectroscopic features and the actual molecular concentration profiles of complex biochemical matrices. Herein, we report that plasmonically enhanced electronic Raman scattering (ERS) signals from metal nanostructures can serve as a SERS calibration internal standard to improve multivariate analysis of living biological systems. Through side-by-side comparisons with noncalibrated SERS datasets, we demonstrate that the ERS-based SERS calibration can enhance supervised learning classification of label-free living cell SERS spectra in (1) subtyping breast cancer cells with different degrees of malignancy and (2) assessing cancer cells' drug responses at different dosages. Notably, the ERS-based SERS calibration has the advantages of excellent photostability under laser excitation, no spectral interference with biomolecule Raman signatures, and no occupation competition with biomolecules at hotspots. Therefore, we envision that the ERS-based SERS calibration can significantly boost the multivariate analysis performance in label-free SERS measurements of living biological systems and other complex biochemical matrices.