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Gastric cancer (GC) is one of the most common and lethal types of cancer affecting over one million people, leading to 768,793 deaths globally in 2020 alone. The key for improving the survival rate lies in reliable screening and early diagnosis. Existing techniques including barium-meal gastric photofluorography and upper endoscopy can be costly and time-consuming and are thus impractical for population screening. We look instead for small extracellular vesicles (sEVs, currently also referred as exosomes) sized ⌀ 30-150 nm as a candidate. sEVs have attracted a significantly higher level of attention during the past decade or two because of their potentials in disease diagnoses and therapeutics. Here, we report that the composition information of the collective Raman-active bonds inside sEVs of human donors obtained by surface-enhanced Raman spectroscopy (SERS) holds the potential for non-invasive GC detection. SERS was triggered by the substrate of gold nanopyramid arrays we developed previously. A machine learning-based spectral feature analysis algorithm was developed for objectively distinguishing the cancer-derived sEVs from those of the non-cancer sub-population. sEVs from the tissue, blood, and saliva of GC patients and non-GC participants were collected (

Liquid biopsy is a rapidly emerging field which involves the minimal/non-invasive assessment of signature somatic mutations through analysis of circulating tumor DNA (ctDNA) shed by tumor cells in bodily fluids. The broad, unmet need for liquid biopsy lung cancer detection is the lack of multiplex platform that can detect a mutation panel of lung cancer genes using minimum amount of sample, especially for ultra-short ctDNA (usctDNA). Here we developed a non-PCR and non-NGS-based single droplet based multiplexing microsensor technology “Electric Field-Induced Released and Measurement (EFIRM) Liquid Biopsy” (m-eLB) for lung can-cer-associated usctDNA. The m-eLB provides multiplexable assessment of usctDNA within a single droplet of biofluid in only one well of micro-electrodes, as each electrode coated with dif-ferent probes for the ctDNA. In this m-eLB prototype, it demonstrates accuracy for 3 tyrosine ki-nase inhibitor related EGFR target sequences in synthetic nucleotides. The accuracy for the multi-plexing assay has an AUC of 0.98 for L858R, Ex19del, AUC of 0.94 for Ex19 deletion and AUC of 0.93 for T790M. By combining the 3 EGFR assay together, the AUC was 0.97 for the multiplexing assay.

E-cadherin is an important tumor suppressor gene whose expression is lost when cells acquire a metastatic phenotype. We analyzed the role of E-cadherin missplicing as a mechanism of its downregulation by analyzing a misspliced E-cadherin transcript that lacks exon 11 of this gene. This results in a frameshift and a premature termination codon that targets this transcript for degradation. Tumor tissues, including breast (20%, n = 9), prostate (30%, n = 9) and head and neck (75%, n = 8) cancer, express the exon 11-skipped transcripts (vs. nonmalignant controls) and its levels inversely correlate with E-cadherin expression. This is a novel mechanism of E-cadherin downregulation by missplicing in tumor cells, which is observed in highly prevalent human tumors. In the head and neck cancer model, nontumorigenic keratinocytes express exon 11–skipped splice product two- to sixfold lower than the head and neck tumor cell lines. Mechanistic studies reveal that SFRS2 (SC35), a splicing factor, as one of the regulators that increases missplicing and downregulates E-cadherin expression. Furthermore, this splicing factor was found to be overexpressed in 5 of 7 head and neck cell lines and primary head and neck tumors. Also, methylation of E-cadherin gene acts as a regulator of this aberrant splicing process. In 2 head and neck cell lines, wild-type transcript expression increased 16- to 25-folds, whereas the percentage of exon 11-skipped transcripts in both the cell lines decreased five- to 30-folds when cells were treated with a hypomethylating agent, azacytidine. Our findings reveal that promoter methylation and an upregulated splicing factor (SFRS2) are involved in the E-cadherin missplicing in tumors. Mol Cancer Ther; 10(9); 1751–9. ©2011 AACR.

XLS file - 13K, List of 100 top most downregulated genes in the array. Average of 5 cell H&N cell lines compared to Tert Kert cells

Background: Oral cancer is the sixth most common cancer with a 5-year survival rate of approximately 60%. Presently, there are no scientifically credible early detection techniques beyond conventional clinical oral examination. The goal of this study is to validate whether the seven mRNAs and three proteins previously reported as biomarkers are capable of discriminating patients with oral squamous cell carcinomas (OSCC) from healthy subjects in independent cohorts and by a National Cancer Institute (NCI)-Early Detection Research Network (EDRN)-Biomarker Reference Laboratory (BRL).Methods: Three hundred and ninety-five subjects from five independent cohorts based on case controlled design were investigated by two independent laboratories, University of California, Los Angeles (Los Angeles, CA) discovery laboratory and NCI-EDRN-BRL.Results: Expression of all seven mRNA and three protein markers was increased in OSCC versus controls in all five cohorts. With respect to individual marker performance across the five cohorts, the increase in interleukin (IL)-8 and subcutaneous adipose tissue (SAT) was statistically significant and they remained top performers across different cohorts in terms of sensitivity and specificity. A previously identified multiple marker model showed an area under the receiver operating characteristic (ROC) curve for prediction of OSCC status ranging from 0.74 to 0.86 across the cohorts.Conclusions: The validation of these biomarkers showed their feasibility in the discrimination of OSCCs from healthy controls. Established assay technologies are robust enough to perform independently. Individual cutoff values for each of these markers and for the combined predictive model need to be further defined in large clinical studies.Impact: Salivary proteomic and transcriptomic biomarkers can discriminate oral cancer from control subjects. Cancer Epidemiol Biomarkers Prev; 21(4); 664–72. ©2012 AACR.

XLSX file - 13K, Fold change differences and P-values of miRNA.

XLSX file - 240K, Average Ct and average ΔCt values from the discovery phase. In the discovery phase, the expression of miRNAs in whole saliva from patients with a malignant parotid gland tumor (n=10) were compared to the expression of miRNAs in whole saliva from patients with a benign parotid gland tumor (n=10).

XLSX file - 22K, Average Ct and average ΔCt values from the validation phase. In the validation phase, the expression of 9 miRNAs were validated in whole saliva salmples from patients with a benign (n=19) or a malignant (n=28) parotid gland tumor.

PDF file - 65K

Purpose: This study was conducted to explore the differences in salivary microRNA (miRNA) profiles between patients with malignant or benign parotid gland tumors as a potential preoperative diagnostic tool of tumors in the salivary glands.Experimental Design: Whole saliva samples from patients with malignant (n = 38) or benign (n = 29) parotid gland tumors were obtained from the Salivary Gland Tumor Biorepository (SGTB). After total RNA isolation, human miRNA cards were used for miRNA profiling. The differential miRNA expression was analyzed using two-sided Wilcoxon test. Quantitative real-time PCR (qRT-PCR) was used to validate selected miRNAs in an independent sample set. Receiver-operating characteristics curve and probability of malignancy was exploited to evaluate the diagnostic power of the validated miRNAs.Results: With miRNA profiling, 57 of 750 investigated miRNAs were differently expressed, of which 54 showed higher miRNA expression in samples from patients with malignant tumors than those from patients with benign tumors. Validating the expression in an independent sample set of 9 miRNAs revealed indeed higher expression of miRNAs in malignant samples compared with benign samples. The expression of 6 validated miRNAs was statistically significantly different between the two groups (P < 0.05). A four miRNA combination was able to discriminate between saliva samples from patients with malignant tumors from those of patients with benign parotid gland tumors (sensitivity 69%, specificity 95%).Conclusions: Salivary miRNA profiles differ in saliva from patients with malignant from saliva from patients with a benign parotid gland tumor. These preliminary results are promising to develop a noninvasive diagnostic tool for diagnosing tumors in the salivary glands. Clin Cancer Res; 19(11); 3032–8. ©2013 AACR.

Business sustainability has received considerable attention in academia and industry. Accordingly, we use a multiple-case design to study the outcomes of business sustainability capability implementation. Using a two-phase data collection approach and resource-based view, we developed a conceptual model to illustrate the importance of business sustainability capability and business sustainability competence and their influence on firm performance. On the bases of the results from a case analysis of three Fortune 500 corporations in the fashion and textile industries, we show how three business sustainability capabilities (i.e., organizational, environmental, and economic competencies) affect business sustainability competence and consequently firm performance. Using in-depth case studies, we develop a set of propositions on how business sustainability competence associates with firm performance. This study was conducted over three years and demonstrates the importance of business sustainability capabilities by confirming the impact of economic competence in the context of market-driven competence and innovation, organizational competence in the context of managerial competence and social well-being, and environmental competence in the context of application of the five Rs (i.e., re-imagine, redesign, reuse, recycle, and reduce). This study thus provides valuable insights into how business sustainability capability and business sustainability competence enhance firm performance in the global fashion business.

The emergence of Omicron and Delta variants of SARS-CoV-2 has begun a number of discussions regarding breakthrough infection, waning immunity, need and timing for vaccine boosters and whether existing mRNA vaccines for the wildtype strain are adequate. Our work leverages a biosensor-based technique to evaluate the binding efficacy of SARS-CoV-2 S1 specific salivary antibodies to the Omicron and Delta variants using a cohort of mRNA vaccinated (n=109) and convalescent (n=19) subjects. We discovered a wide range of binding efficacies to the variant strains, with a mean reduction of 60.5%, 26.7%, and 14.7% in measurable signal to the Omicron strain and 13.4%, 2.4%, and −6.4% percent mean reduction to the Delta Variant for convalescent, Pfizer, and Moderna vaccinated groups respectively. This assay may be an important tool in determining susceptibility to infection or need for booster immunization as the pandemic evolves.Key PointsAMPERIAL assay developed to quantify salivary SARS-CoV-2 S1 IgG antibodies to Omicron and Delta variantsThere was a reduction in affinity to both Delta and Omicron VariantsThe reduction in affinity was more pronounced to Omicron than for Delta VariantsThere was a significant difference between IgG affinities in Individuals vaccinated with Pfizer versus Moderna Vaccines

Pancreatic cancer is the fourth leading cause of cancer death in the United States. Pancreatic cancer presents dismal clinical outcomes in patients, and the incidence of pancreatic cancer has continuously increased to likely become the second most common cause of cancer-related deaths by as early as 2030. One of main reasons for the high mortality rate of pancreatic cancer is the lack of tools for early-stage detection. Current practice in detecting and monitoring therapeutic response in pancreatic cancer relies on imaging analysis and invasive endoscopic examination. Liquid biopsy-based analysis of genetic alterations in biofluids has become a fundamental component in the diagnosis and management of cancers. There is an urgent need for scientific and technological advancement to detect pancreatic cancer early and to develop effective therapies. The development of a highly sensitive and specific liquid biopsy tool will require extensive understanding on the characteristics of circulating tumor DNA in biofluids. Here, we have reviewed the current status of liquid biopsy in detecting and monitoring pancreatic cancers and our understanding of circulating tumor DNA that should be considered for the development of a liquid biopsy tool, which will greatly aid in the diagnosis and healthcare of people at risk.

Electric field–induced release and measurement (EFIRM) is a novel, plate-based, liquid biopsy platform capable of detecting circulating tumor DNA containing EGFR mutations directly from saliva and plasma in both early- and late-stage patients with non–small-cell lung cancer. We investigated the properties of the target molecule for EFIRM and determined that the platform preferentially detects single-stranded DNA molecules. We then investigated the properties of the EFIRM assay and determined the linearity, linear range, precision, and limit of detection for six different EGFR variants (the four most common g.Exon19del variants), p.T790M, and p.L858R). The limit of detection was in single-digit copy number for the latter two mutations, and the limit of detection for Exon19del was 5000 copies. Following these investigations, technical validations were performed for four separate EFIRM liquid biopsy assays, qualitative and quantitative assays for both saliva and plasma. We conclude that EFIRM liquid biopsy is an assay platform that interrogates a biomarker not targeted by any other extant platform (namely, circulating single-stranded DNA molecules). The assay has acceptable performance characteristics in both quantitative and qualitative assays on both saliva and plasma.

Background Hospital-acquired pressure injuries are a chronic phenomenon in health care, and their prevention is an ongoing challenge. This study aims to investigate whether the application of a silicone-bordered multilayered foam dressing during the initial trauma resuscitation reduces sacral hospital-acquired pressure injury occurrence in trauma patients. Methods This is a single-center quality improvement study using a nonequivalent control group posttest-only design to study the effect of silicone-bordered multilayered foam dressing on the incidence of hospital-acquired pressure injuries. The study population included admitted, highest tier trauma activations, age 18 years and older. Preimplementation 2014 data were compared with postimplementation 2018 data. Results The result showed no statistically significant reduction in hospital-acquired pressure injury occurrence between the control and intervention groups. Incident rates for sacral hospital-acquired pressure injuries were 0.23% (2014) compared with 0.21% (2018). No statistically significant difference was found in the hospital and intensive care lengths of stay or injury severity. Preventive dressing costs were $7,689 annually compared with the estimated treatment costs of $70,000 per hospital-acquired pressure injury. Conclusion Although this study's hospital-acquired pressure injury reduction rate was not significant, the inclusion of multidisciplinary team members in the quality improvement project led to the cultural hardwiring of hospital-acquired pressure injury prevention among all team members beyond that of just nursing.

Previous efforts to evaluate the detection of human papilloma viral (HPV) DNA in whole saliva as a diagnostic measure for HPV-associated oropharyngeal cancer (HPV-OPC) have not shown sufficient clinical performance. We hypothesize that salivary exosomes are packaged with HPV-associated biomarkers, and efficient enrichment of salivary exosomes through isolation can enhance diagnostic and prognostic performance for HPV-OPC. In this study, an acoustofluidic (the fusion of acoustics and microfluidics) platform was developed to perform size-based isolation of salivary exosomes. These data showed that this platform is capable of consistently isolating exosomes from saliva samples, regardless of viscosity variation and collection method. Compared with the current gold standard, differential centrifugation, droplet digital RT-PCR analysis showed that the average yield of salivary exosomal small RNA from the acoustofluidic platform is 15 times higher. With this high-yield exosome isolation platform, we show that HPV16 DNA could be detected in isolated exosomes from the saliva of HPV-associated OPC patients at 80% concordance with tissues/biopsies positive for HPV16. Overall, these data demonstrated that the acoustofluidic platform can achieve high-purity and high-yield salivary exosome isolation for downstream salivary exosome–based liquid biopsy applications. Additionally, HPV16 DNA sequences in HPV-OPC patients are packaged in salivary exosomes and their isolation will enhance the detection of HPV16 DNA.

We used a noninvasive electrochemical quantitative assay for IgG antibodies to SARS-CoV-2 S1 in saliva to investigate the kinetics of antibody response in a community-based population who had received either the Pfizer or Moderna mRNA-based vaccines. Samples were received from a total of 97 individuals including a subset of 42 individuals who collected samples twice-weekly for 3 months or longer. In all, 840 samples were collected and analyzed. In all individuals, salivary antibody levels rose sharply in the 2-week period following their second vaccination, with peak antibody levels being at 10–20 days post-vaccination. We observed that 20%, 10% and 2.4% of individuals providing serial samples had a 90%, 95%, and 99% drop respectively from peak levels during the duration of monitoring and two patients fell to pre-vaccination levels (5%). The use of non-invasive quantitative salivary antibody measurement can allow widespread, cost-effective monitoring of vaccine response., Article Summary Line: COVID-19 antibodies were measured in saliva and 20% of vaccinated subjects experienced a 90% drop in peak antibody levels over the course of monitoring.

IntroductionWe investigated whether network analysis revealed clusters of coregulated metabolites associated with prevalent type 2 diabetes (T2D) among Puerto Rican adults.Research design and methodsWe used liquid chromatography-mass spectrometry to measure fasting plasma metabolites (>600) among participants aged 40–75 years in the Boston Puerto Rican Health Study (BPRHS; discovery) and San Juan Overweight Adult Longitudinal Study (SOALS; replication), with (n=357; n=77) and without (n=322; n=934) T2D, respectively. Among BPRHS participants, we used unsupervised partial correlation network-based methods to identify and calculate metabolite cluster scores. Logistic regression was used to assess cross-sectional associations between metabolite clusters and prevalent T2D at the baseline blood draw in the BPRHS, and significant associations were replicated in SOALS. Inverse-variance weighted random-effect meta-analysis was used to combine cohort-specific estimates.ResultsSix metabolite clusters were significantly associated with prevalent T2D in the BPRHS and replicated in SOALS (false discovery rate (FDR) ConclusionsAmong Puerto Rican adults, we identified several known and novel metabolite clusters that associated with prevalent T2D.

Plasma cell-free DNA is a widely used biomarker for diagnostic screening. We introduce uscfDNA-seq, a single-stranded cell-free DNA NGS pipeline, which bypasses previous limitations to reveal a novel population of ultrashort single-stranded cell-free DNA in plasma with a modal size of 50nt. This species of cfDNA aligns predominantly to the nuclear genome and could potentially be used for novel biomarker discovery.

Simple Summary ctDNA assay is a promising non-invasive method to detect genomic alterations associated with lung cancer. In this prospective study of 25 patients with EGFR-mutant lung adenocarcinoma receiving osimertinib, ctDNA progression predated radiographic progression by 118 days in 11 of 20 patients with disease progression. Saliva-based ctDNA analysis and plasma NGS detected additional patients with ctDNA progression preceding clinical progression, suggesting the potential complementary roles of different ctDNA detection methodologies. Baseline mutant ctDNA level predicted progression-free survival while tumor volume measurements by volumetric CT did not. Serial ctDNA analysis of plasma and saliva is a clinically useful tool to monitor response and resistance to osimertinib. Abstract Background: We assessed whether serial ctDNA monitoring of plasma and saliva predicts response and resistance to osimertinib in EGFR-mutant lung adenocarcinoma. Three ctDNA technologies—blood-based droplet-digital PCR (ddPCR), next-generation sequencing (NGS), and saliva-based EFIRM liquid biopsy (eLB)—were employed to investigate their complementary roles. Methods: Plasma and saliva samples were collected from patients enrolled in a prospective clinical trial of osimertinib and local ablative therapy upon progression (NCT02759835). Plasma was analyzed by ddPCR and NGS. Saliva was analyzed by eLB. Results: A total of 25 patients were included. We analyzed 534 samples by ddPCR (n = 25), 256 samples by NGS (n = 24) and 371 samples by eLB (n = 22). Among 20 patients who progressed, ctDNA progression predated RECIST 1.1 progression by a median of 118 days (range: 61–272 days) in 11 (55%) patients. Of nine patients without ctDNA progression by ddPCR, two patients had an increase in mutant EGFR by eLB and two patients were found to have ctDNA progression by NGS. Levels of ctDNA measured by ddPCR and NGS at early time points, but not volumetric tumor burden, were associated with PFS. EGFR/ERBB2/MET/KRAS amplifications, EGFR C797S, PIK3CA E545K, PTEN V9del, and CTNNB1 S45P were key resistance mechanisms identified by NGS. Conclusion: Serial assessment of ctDNA in plasma and saliva predicts response and resistance to osimertinib, with each assay having supplementary roles.

This study reports on the displacement of Alpha (B.1.1.7) by Delta (B.1.617.2 and its substrains AY.1, AY.2, and AY.3) in the United States. By analyzing RT-qPCR testing results and viral sequencing results of samples collected across the United States, we show that the percentage of SARS-CoV-2 positive cases caused by Alpha dropped from 67% in May 2021 to less than 3.0% in just 10 weeks. We also show that the Delta variant has outcompeted the Iota (B.1.526) variant of interest and Gamma (P.1) variant of concern. An analysis of the mean quantification cycles (Cq) values in positive tests over time also reveal that Delta infections lead to a higher viral load on average compared to Alpha infections, but this increase is only 2 to 3x on average for our study design. Our results are consistent with the hypothesis that the Delta variant is more transmissible than the Alpha variant, and that this could be due to the Delta variant's ability to establish a higher viral load earlier in the infection compared to the Alpha variant.

This paper aims to systematically review the supply chain innovation literature over the last 18 years. It examines the development and current state of supply chain innovation research in management and identifies research gaps. A literature review is conducted to identify and analyze publications in peer-reviewed academic journals that include contributions from different strands of management research. This paper analyzes the theoretical contributions of the supply chain innovation literature using Gregor's (2006) framework of theory classification. It also evaluates the levels of analysis of the literature using the structural view model proposed by Skinner, Han, and Chang (2006). This research identified and analyzed various topics related to the supply chain innovation construct and showed that supply chain innovations can be studied at multiple analytical levels. It also revealed that the field has largely relied on manufacturing firm-based samples and U.S. samples, limiting the generalizability of the findings. The identification and analysis of relevant articles highlighted the need to conceptualize the supply chain innovation construct and develop measurement scales to operationalize it. This literature review is the first to focus on supply chain innovations, summarizing the development of the last 18 years and providing fruitful opportunities for future research. The results presented can be applied to the decision-making process of managers regarding supply chain innovations.

To identify salivary metabolite biomarkers to differentiate patients with oral squamous cell carcinoma and oral epithelial dysplasia (OSCC/OED) from those with persistent suspicious oral mucosal lesions (PSOML). Whole unstimulated saliva samples were collected from age-, sex-, and race-matched patients who had a lesion in the oral cavity and for whom open biopsies were performed. The patients included OSCC (n = 6), OED (n = 10), and PSOML (n = 32). Hydrophilic metabolites in saliva samples were comprehensively analyzed using capillary electrophoresis mass spectrometry. To evaluate the discrimination ability of a combination of multiple markers, a multiple logistic regression (MLR) model was developed to differentiate OSCC/OED from PSOML. Six metabolites were significantly different in OSCC/OED compared with PSOML. From these six metabolites, ornithine, o-hydroxybenzoate, and ribose 5-phosphate (R5P) were used to develop the MLR model, which resulted in a high value for the area under receiver operating characteristic curve (AUC 0.871, 95% confidential interval (CI) 0.760–0.982; p

The highly transmissible B.1.1.7 variant of SARS-CoV-2, first identified in the United Kingdom, has gained a foothold across the world. Using S gene target failure (SGTF) and SARS-CoV-2 genomic sequencing, we investigated the prevalence and dynamics of this variant in the United States (U.S.), tracking it back to its early emergence. We found that while the fraction of B.1.1.7 varied by state, the variant increased at a logistic rate with a roughly weekly doubling rate and an increased transmission of 40-50%. We revealed several independent introductions of B.1.1.7 into the U.S. as early as late November 2020, with community transmission spreading it to most states within months. We show that the U.S. is on a similar trajectory as other countries where B.1.1.7 became dominant, requiring immediate and decisive action to minimize COVID-19 morbidity and mortality., Genomic epidemiology analyses explain the introduction and transmission of the B.1.1.7 variant of SARS-CoV-2 into the United States, with projections for it to soon be the dominant strain in the country.

Introduction Trauma is a major cause of morbidity and mortality amongst all populations in the United States. With the widespread increase of obesity in the United States, studies have been conducted to compare different body mass index (BMI) groups and their clinical outcomes for traumatic injuries. The goal of this study was to retrospectively compare mortality between adult trauma patients with a high BMI to those with a lower BMI as well as investigate whether the mechanism of trauma had an effect on the outcome. Methods This study was a retrospective review of all adult trauma patients presented to the emergency department at Arrowhead Regional Medical Center (ARMC) between January 2014 and October 2019. The outcome was all-cause mortality. Patients were grouped according to BMI and mechanisms of injury, including blunt trauma, low velocity penetrating trauma, and high velocity penetrating trauma. Patients were also stratified by injury severity scores (ISS). Results Among the 9642 patients assessed in this study, majority (88%) of patients sustained blunt trauma. The number of patients among the three different BMI groups was appropriately equal with 34.4% of normal BMI, 34.6% overweight, and 31.1% obese. The overall mortality of all patients studied was 2.6% (n=248). There was no statistically significant difference in mortality among the three different BMI groups for blunt trauma, penetrating trauma, and subgroup analyses stratified by ISS score (ISS

Author(s): Washington, Nicole L; Gangavarapu, Karthik; Zeller, Mark; Bolze, Alexandre; Cirulli, Elizabeth T; Schiabor Barrett, Kelly M; Larsen, Brendan B; Anderson, Catelyn; White, Simon; Cassens, Tyler; Jacobs, Sharoni; Levan, Geraint; Nguyen, Jason; Ramirez, Jimmy M; Rivera-Garcia, Charlotte; Sandoval, Efren; Wang, Xueqing; Wong, David; Spencer, Emily; Robles-Sikisaka, Refugio; Kurzban, Ezra; Hughes, Laura D; Deng, Xianding; Wang, Candace; Servellita, Venice; Valentine, Holly; De Hoff, Peter; Seaver, Phoebe; Sathe, Shashank; Gietzen, Kimberly; Sickler, Brad; Antico, Jay; Hoon, Kelly; Liu, Jingtao; Harding, Aaron; Bakhtar, Omid; Basler, Tracy; Austin, Brett; Isaksson, Magnus; Febbo, Phil; Becker, David; Laurent, Marc; McDonald, Eric; Yeo, Gene W; Knight, Rob; Laurent, Louise C; de Feo, Eileen; Worobey, Michael; Chiu, Charles; Suchard, Marc A; Lu, James T; Lee, William; Andersen, Kristian G | Abstract: As of January of 2021, the highly transmissible B.1.1.7 variant of SARS-CoV-2, which was first identified in the United Kingdom (U.K.), has gained a strong foothold across the world. Because of the sudden and rapid rise of B.1.1.7, we investigated the prevalence and growth dynamics of this variant in the United States (U.S.), tracking it back to its early emergence and onward local transmission. We found that the RT-qPCR testing anomaly of S gene target failure (SGTF), first observed in the U.K., was a reliable proxy for B.1.1.7 detection. We sequenced 212 B.1.1.7 SARS-CoV-2 genomes collected from testing facilities in the U.S. from December 2020 to January 2021. We found that while the fraction of B.1.1.7 among SGTF samples varied by state, detection of the variant increased at a logistic rate similar to those observed elsewhere, with a doubling rate of a little over a week and an increased transmission rate of 35-45%. By performing time-aware Bayesian phylodynamic analyses, we revealed several independent introductions of B.1.1.7 into the U.S. as early as late November 2020, with onward community transmission enabling the variant to spread to at least 30 states as of January 2021. Our study shows that the U.S. is on a similar trajectory as other countries where B.1.1.7 rapidly became the dominant SARS-CoV-2 variant, requiring immediate and decisive action to minimize COVID-19 morbidity and mortality.

Head and neck cancer remains one of the leading causes of death worldwide. Liquid biopsy has emerged as a promising tool for detecting cancer and monitoring the disease status. Circulating tumor DNA (ctDNA), circulating tumor cells (CTCs), and exosomal microRNAs (miRNAs) warrant close attention because of their apparent clinical implications. Circulating biomarkers have led to a paradigm shift and opened up novel therapeutic avenues, starting a new era of precision medicine. An increasing number of studies suggest that circulating biomarkers can be used to detect cancer, plan treatment, monitor treatment response, and assess the prognosis. However, no validated circulating biomarkers have been integrated into clinical practice for head and neck cancer despite their potential clinical utilities. This chapter focuses on the recent advances in studying and utilizing circulating biomarkers in the context of head and neck cancer, together with the limitations and key clinical applications.

Author(s): Bouchareychas, L; Duong, P; Covarrubias, S; Alsop, E; Q Phu, TA; Chung, A; Gomes, M; Wong, D; Meechoovet, B; Capili, A; Yamamoto, R; Nakauchi, H; Mcmanus, M; Carpenter, S; Van Keuren-Jensen, K; Raffai, RL

Amperial™ is a novel assay platform that uses immobilized antigen in a conducting polymer gel followed by detection via electrochemical measurement of oxidation-reduction reaction between H2O2/Tetrametylbenzidine and peroxidase enzyme in a completed assay complex. A highly specific and sensitive assay was developed to quantify levels of IgG antibodies to SARS-CoV-2 in saliva. After establishing linearity and limit of detection we established a reference range of 5 standard deviations above the mean. There were no false positives in 667 consecutive saliva samples obtained prior to 2019. Saliva was obtained from 34 patients who had recovered from documented COVID-19 or had documented positive serologies. All of the patients with symptoms severe enough to seek medical attention had positive antibody tests and 88% overall had positive results. We obtained blinded paired saliva and plasma samples from 14 individuals. The plasma was analyzed using an EUA-FDA cleared ELISA kit and the saliva was analyzed by our Amperial™ assay. All 5 samples with negative plasma titers were negative in saliva testing. Eight of the 9 positive plasma samples were positive in saliva and 1 had borderline results. A CLIA validation was performed as a laboratory developed test in a high complexity laboratory. A quantitative non-invasive saliva based SARS-CoV-2 antibody test was developed and validated with sufficient specificity to be useful for population-based monitoring and monitoring of individuals following vaccination.

Background: The longitudinal monitoring of actionable oncogenes in circulating tumor DNA (ctDNA) of non-small cell lung cancer (NSCLC) is crucial for clinicians to evaluate current therapeutic response and adjust therapeutic strategies. Saliva-based electric field-induced release and measurement (EFIRM) is liquid biopsy platform to that can directly detect mutation genes with a small volume of samples. Herein, we compared the effectiveness of longitudinal monitoring for the combination of epidermal growth factor receptor (EGFR) and phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) mutations between saliva-based EFIRM and plasma-based platforms (ddPCR and NGS) in two advanced NSCLC patients undergoing the treatment with osimertinib before and after local ablative therapy (LAT). Patients and Methods: Two patients with unresectable advanced NSCLC were enrolled into the National Institutes of Health Clinical Center (NIHCC) Study (ClinicalTrials.gov: 16-C-0092; local ablative therapy for the treatment of oligoprogressive, EGFR-mutated, non-small cell lung cancer after treatment with osimertinib). Serial collections of saliva, plasma, and metastatic tumor volume measurement by computed tomography (CT) were performed. Longitudinal paired saliva and plasma samples were analyzed for p.L858R EGFR, exon19 del EGFR, and p.E545K PIK3CA ctDNA using EFIRM (saliva) and ddPCR and NGS (plasma). Results: In Case 1, the saliva ctDNA curve of exon19 del EGFR by EFIRM demonstrated a strong similarity to those of tumor volume (R = 0.78, P = 0.00) and exon19 del EGFR in ddPCR (R = 0.53, P = 0.01). Moreover, the curve of p.E545K PIK3CA in EFIRM showed similarity to those of tumor volume (R = 0.70, P = 0.00) and p.E545K PIK3CA in NGS (R = 0.72, P = 0.00). In Case 2, the curve of p.E545K PIK3CA in EFIRM revealed a reverse relationship to that of tumor volume (R = -0.65, P = 0.01). Conclusion: In these two case reports, saliva-based EFIRM platform demonstrates a high level of concordance to plasma-based platforms (ddPCR and NGS) for longitudinally monitoring the combination of EGFR and PIK3CA ctDNA and can be a useful platform to monitor tumor progression and response to targeted therapy in NSCLC patients.

Acoustofluidic centrifugation enables the spinning of a fluidic droplet and nanoparticle enrichment and separation., Liquid droplets have been studied for decades and have recently experienced renewed attention as a simplified model for numerous fascinating physical phenomena occurring on size scales from the cell nucleus to stellar black holes. Here, we present an acoustofluidic centrifugation technique that leverages an entanglement of acoustic wave actuation and the spin of a fluidic droplet to enable nanoparticle enrichment and separation. By combining acoustic streaming and droplet spinning, rapid (

Alcohol (ethanol, EtOH) consumption during pregnancy can result in fetal alcohol spectrum disorders (FASDs), which are characterized by prenatal and postnatal growth restriction and craniofacial dysmorphology. Recently, cell-derived extracellular vesicles, including exosomes and microvesicles containing several species of RNAs (exRNAs), have emerged as a mechanism of cell-to-cell communication. However, EtOH’s effects on the biogenesis and function of non-coding exRNAs during fetal development have not been explored. Therefore, we studied the effects of maternal EtOH exposure on the composition of exosomal RNAs in the amniotic fluid (AF) using rat fetal alcohol exposure (FAE) model. Through RNA-Seq analysis we identified and verified AF exosomal miRNAs with differential expression levels specifically associated with maternal EtOH exposure. Uptake of purified FAE AF exosomes by rBMSCs resulted in significant alteration of molecular markers associated with osteogenic differentiation of rBMSCs. We also determined putative functional roles for AF exosomal miRNAs (miR-199a-3p, miR-214-3p and let-7g) that are dysregulated by FAE in osteogenic differentiation of rBMSCs. Our results demonstrate that FAE alters AF exosomal miRNAs and that exosomal transfer of dysregulated miRNAs has significant molecular effects on stem cell regulation and differentiation. Our results further suggest the usefulness of assessing molecular alterations in AF exRNAs to study the mechanisms of FAE teratogenesis that should be further investigated by using an in vivo model.

The concept of liquid biopsy has emerged into the cancer lexicon to describe detected alterations of tumor biomarkers within body fluids which reflect the presence and the biology of cancer. This is typically performed by assessing circulating tumor cells (CTCs), circulating tumor DNAs (ctDNAs), tumor-derived extracellular vesicles (EVs), microRNAs (miRNAs), and proteins. Although plasma, urine, and cerebral spinal fluid (CSF) are all viable biofluids, growing attention has recently been cast on saliva. Saliva is readily available, can be obtained noninvasively, is easily collected and stored, and also demonstrates compelling pathophysiological association with systemic diseases. Importantly, saliva liquid biopsy delivers the best clinical performance to detect ctDNAs in lung cancer patients. In conjunction with validated biomarkers, and reliable and robust analytical detection tools, saliva has the potential to pioneer a new landscape of real-time point-of-care testing in personalized medicine. This chapter will review the salivaomics, the disease-related biomarker properties of saliva, and the scientific advances in the pathophysiological foundations which make saliva an ideal candidate for noninvasive liquid biopsy. Moreover, a novel liquid biopsy technology termed electric field-induced release and measurement (EFIRM), which works in tandem with the technological advances to extract crucial disease information in saliva, is reviewed.

BACKGROUND It was recently discovered that abundant and stable extracellular RNA (exRNA) species exist in bodily fluids. Saliva is an emerging biofluid for biomarker development for noninvasive detection and screening of local and systemic diseases. Use of RNA-Sequencing (RNA-Seq) to profile exRNA is rapidly growing; however, no single preparation and analysis protocol can be used for all biofluids. Specifically, RNA-Seq of saliva is particularly challenging owing to high abundance of bacterial contents and low abundance of salivary exRNA. Given the laborious procedures needed for RNA-Seq library construction, sequencing, data storage, and data analysis, saliva-specific and optimized protocols are essential. METHODS We compared different RNA isolation methods and library construction kits for long and small RNA sequencing. The role of ribosomal RNA (rRNA) depletion also was evaluated. RESULTS The miRNeasy Micro Kit (Qiagen) showed the highest total RNA yield (70.8 ng/mL cell-free saliva) and best small RNA recovery, and the NEBNext library preparation kits resulted in the highest number of detected human genes [5649–6813 at 1 reads per kilobase RNA per million mapped (RPKM)] and small RNAs [482–696 microRNAs (miRNAs) and 190–214 other small RNAs]. The proportion of human RNA-Seq reads was much higher in rRNA-depleted saliva samples (41%) than in samples without rRNA depletion (14%). In addition, the transfer RNA (tRNA)-derived RNA fragments (tRFs), a novel class of small RNAs, were highly abundant in human saliva, specifically tRF-4 (4%) and tRF-5 (15.25%). CONCLUSIONS Our results may help in selection of the best adapted methods of RNA isolation and small and long RNA library constructions for salivary exRNA studies.

Amperial™ is a novel assay platform that uses immobilized antigen in a conducting polymer gel followed by detection via electrochemical measurement of oxidation-reduction reaction between H2O2/Tetrametylbenzidine and peroxidase enzyme in a completed assay complex. A highly specific and sensitive assay was developed to quantify levels of IgG antibodies to SARS-CoV-2 in saliva. After establishing linearity and limit of detection we established a reference range of 5 standard deviations above the mean. There were no false positives in 667 consecutive saliva samples obtained prior to 2019. Saliva was obtained from 34 patients who had recovered from documented COVID-19 or had documented positive serologies. All of the patients with symptoms severe enough to seek medical attention had positive antibody tests and 88% overall had positive results. We obtained blinded paired saliva and plasma samples from 14 individuals. The plasma was analyzed using an EUA-FDA cleared ELISA kit and the saliva was analyzed by our Amperial™ assay. All 5 samples with negative plasma titers were negative in saliva testing. Eight of the 9 positive plasma samples were positive in saliva and 1 had borderline results. A CLIA validation was performed as a laboratory developed test in a high complexity laboratory. A quantitative non-invasive saliva based SARS-CoV-2 antibody test was developed and validated with sufficient specificity to be useful for population-based monitoring and monitoring of individuals following vaccination.

AimsInsulin resistance (IR) detection is challenging and no test is currently used in clinical practice. We developed salivary biomarkers that could be used for IR detection.MethodsWe collected saliva from 186 healthy and 276 pre-diabetic participants, divided them into high and low IR groups based on a HOMA cutoff of 2.5. We profiled extracellular transcriptome by microarray in saliva supernatant from 23 high IR and 15 low IR participants, and pre-validated the top ten extracellular mRNA (exRNA) markers in a new cohort of 40 high and 40 low IR participants. A prediction panel was then built and validated in an independent cohort of 149 high and 195 low IR participants.ResultsTranscriptomic analyses identified 42 exRNA candidates differentially present in saliva of high and low IR participants. From the top ten candidates, six were individually validated (PRKCB, S100A12, IL1R2, CAMP, VPS4B, CAP1) (p