Liquid biopsy testing in metastatic or advanced breast cancer patients during the COVID-19 pandemic

Objective: During the COVID-19 pandemic, cancer patients had restricted access to standard of care tissue biopsy. Liquid biopsy assays using next generation sequencing technology provides a less invasive method for determining circulating tumour mutations (ctDNA) associated with targeted treatments or prognosis. As part of deploying technology to help cancer patients obtain molecular testing, a clinical program was initiated to offer liquid biopsy testing for Canadian patients with advanced or metastatic breast cancer. Method(s): Blood was drawn in two 10 mL StreckTM DNA BCTs and sent to the CAP/CLIA/DAP accredited Imagia Canexia Health laboratory for testing using the clinically validated Follow ItTM liquid biopsy assay. Plasma was isolated using a double spin protocol and plasma cell-free DNA (cfDNA) extracted using an optimized Promega Maxwell RSC method. Extracted cfDNA was amplified using the multiplex amplicon-based hotspot 30 or 38 gene panel and sequenced. An inhouse developed bioinformatics pipeline and reporting platform were used to identify pathogenic single nucleotide variants (SNVs), indels (insertions and deletions), and gene amplification. Included in the panel are genes associated with metastatic breast cancer: AKT1, BRAF, ERBB2, ESR1, KRAS, PIK3CA, TP53. Result(s): To identify biomarkers, 1214 metastatic or advanced breast cancer patient cfDNA samples were tested. There were 15 cases sent for repeat testing. We reported 48% of samples harboring pathogenic ctDNA mutations in TP53 (22%), PIK3CA (19%), ESR1 (18%), AKT1 (2%), ERBB2 (1.5%). Co-occurring variants were identified in samples with ESR1/PIK3CA as well as TP53/PIK3CA (both p-values <0.001). Interestingly, 29% of samples with mutated ESR1 harbored >= 2 ESR1 ctDNA mutations. In 56% of cases, previous molecular testing indicated the cancer subtype as hormone receptor (ER, PR) positive with/without HER2 negative status. In this specific subgroup, 49% harbored ctDNA mutations with 63% of those being PIK3CA and/or ESR1 mutations. Conclusion(s): A population of Canadian women with metastatic breast cancer were tested using a liquid biopsy gene panel during the COVID-19 pandemic for identification of biomarkers for targeted therapeutic options. Over 50% of the samples were identified as hormone positive, with greater than 60% harboring PIK3CA and ESR1 ctDNA mutations. Studies have shown that metastatic PIK3CA mutated ER-positive/HER2-negative tumors are predictive to respond to alpelisib therapy and have FDA and Health Canada approval. Additionally, ESR1 mutations are associated with acquired resistance to antiestrogen therapies, and interestingly we identified 29% of ESR1 mutated samples with multiple mutations possibly indicating resistance subclones. In future studies, longitudinal monitoring for presence of multiple targetable and resistance mutations could be utilized to predict or improve clinical management.

Characterization of Genetic Mutations Using DNA Walks and Multifractal Detrended Fluctuation Analysis

Monitoring genetic mutations in DNA sequences and their subsequent characterisation provide the possibility for rapid development of diagnostics and therapeutic tools. Here, it is shown that the "DNA walk" (DNAW) representation together with multifractal detrended fluctuation analysis (MFDFA), i.e. DNAW/MFDFA, form a reliable characterization method for studying local and global properties of similar DNA sequences. The DNAW/MFDFA approach allows to study the stochastic properties of genetic sequences by constructing a one-to-one map of the sequence onto a walk, and is able to uncover the self-similarity properties of DNA walks. These features are illustrated on a set of similar DNA sequences of SARS-CoV-2 virus, in which the differences in nucleotide bases arise due to genetic mutations. The results show that DNAW/MFDFA can be used to extract long-range correlation information and type and degree of fractal complexity.

Multiplex measurements of the impacts of ACE2 sequence and cell surface abundance during SARS-like coronavirus entry

SARS-like coronaviruses, including SARS-CoV and SARS-CoV-2, encode spike proteins that bind human ACE2 protein on the cell surface to enter target cells and cause infection. The efficiency of virus entry depends on ACE2 sequence and expression levels in target cells. A small fraction of humans encodes variants of ACE2, thus altering the biochemical properties at the protein interaction interface. All humans possess cells with vastly differing amounts of ACE2 on the cell surface, ranging from cell types with high expression in the gut and lungs to lower expression in the liver and pancreas. Mastering our understanding of spike-ACE2 interaction and infection requires experiments precisely perturbing both variables. Thus, we developed a synthetic cell engineering approach compatible with high throughput assays for pseudo-typed virus infection. Our assay system is capable of assessing both variables individually and in combination. We adapted an engineered HEK293T DNA recombinase landing pad cell line capable of expressing transgenic ACE2 sequences at highly precise levels. Infection with lentiviruses pseudotyped with the spikes of SARS-like coronaviruses revealed that high ACE2 abundance could mask the effects of impaired binding thereby making it challenging to know the role of affinity altering mutations during infection. We limited the ACE2 abundance on the cell surface by expressing transgenic ACE2 behind a suboptimal Kozak sequence, thereby altering its protein translation rate. This allowed us to understand how ACE2 sequence could impact its interaction with coronavirus spike proteins as two human ACE2 variants at the binding interface, K31D and D355N, exhibited reduced infection. Our experiments suggested that we need to better understand how ACE2 expression determines the susceptibility of cells for SARS-like coronavirus binding and infection. We thus created an ACE2 Kozak library consisting of ~4,096 Kozak variants, each conferring a different ACE2 protein translation rate thus resulting in a range of ACE2 steady-state abundances. Combining fluorescence-activated cell sorting and high-throughput DNA sequencing (FACS-seq) revealed the library to span two orders of magnitude of ACE2 abundance. Challenging this library of cells with spike pseudotyped lentiviruses revealed how ACE2 abundance correlated with infection rate. The library-based experiments yielded a dynamic range wider than traditional single sample infection assay, likely more representative of infection dynamics in vivo. Now that we have characterized the impacts of ACE2 abundance on infectivity in engineered cells, our next goal is to expand the comparison to physiologically relevant cells with endogenously expressed proteins. Modulating protein abundance levels will be key to creating maximally informative functional assays for any protein in cell-based assays, and we have laid the groundwork for being able to simultaneously test the impacts of protein abundance and sequence in combination for proteins involved in diverse cellular processes. This research was supported by a National Institute of Health (NIH) grant GM142886 (KAM).Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

Mechanisms of CRISPR-mediated immunity and applications beyond editing

Mammals, bacteria, and archaea have domesticated transposases (e.g., RAG1 and Cas1) to form adaptive immune systems. Bacteria and archaea acquire resistance to viruses and plasmids by preferentially integrating fragments of foreign DNA at one end of a CRISPR locus. DNA motifs upstream of the CRISPR (i.e., leader) facilitate integration at the first CRISPR repeat. But how do these upstream DNA motifs act over large distances of 130 bp, or roughly 440 A, to regulate integration allosterically? Here, we determine the structure of a 560 KDa integration complex that explains how the CRISPR leader DNA recruits Cas (i.e., Cas1-2/3) and non-Cas proteins (i.e., IHF). Cas1-2/3 and IHF cooperate to fold the genome into a successive U-shaped bend and a loop. The genomic U-bend traps foreign DNA against the integrase, whereas the genomic loop positions the leader-repeat junction at the Cas1 active site. The foreign DNA and the CRISPR repeat wrap around opposite faces of Cas2, poised for a Cas1-catalyzed strand-transfer reaction. The post-integration structure suggests that strand-transfer releases tension in the DNA loop. Therefore Cas1-2/3 may harness protein-induced DNA tension to favor the completion of the isoenergetic integration reaction. Cas1-2/3 interacts extensively with the leader and repeat without making sequence-specific contacts, and we demonstrate that protein-mediated folding of DNA drives integration into diverse sequences. These results reveal Cas1-2/3 and IHF strain DNA to enhance integration allosterically and suggest a mechanism for the de novo generation of new CRISPRs. Further, to address an urgent need for inexpensive and rapid detection of viruses, we recently repurposed a CRISPR immune signaling pathway to detect SARS-CoV-2 in patient samples. A.S-F. is a postdoctoral fellow of the Life Science Research Foundation, supported by the Simons Foundation. A.S-F. is supported by the PDEP award from the Burroughs Wellcome Fund, and by the National Institutes of Health, United States grant 1K99GM147842. This work was also supported by NSF (1828765), NIH (U24 GM129539, R35GM134867).Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

Using Synthetic Biology methods to construct a functional estrogen biosensor based on split Nanoluciferase activity

The presence of estrogenic compounds (endocrine-disruptors, EDCs) in the water supply raises concerns about human and aquatic health. Current methods for detecting estrogen contamination require expensive, time-consuming techniques such as liquid chromatography-mass spectrometry and high-performance liquid chromatography. Previously reported estrogen biosensors required multiple cloning and transformation steps for successful detection in bacteria. Synthetic biology allows for the construction of genetic devises composed of DNA sequences modified to be interchangeable and provide novel functions. New tools and devices are constantly needed to enhance the already extensive list of novel genetic parts. Our approach to the design of an estrogen responsive element uses methodology developed in the Wells lab (Elledge et al, 2021) to detect SARS-CoV-2 antibodies. This methodology takes advantage of the split Nanoluciferase (spLUC) protein divided into two functional domains (designated SmBit and LgBit). Based on rational engineering design we express dimerization dependent LgBit and SmBit fused to the Estrogen Receptor alpha protein (ERalpha) in bacteria cells. These two monomeric proteins will dimerize in the presence of estrogen, reconstitute the split luciferase enzyme and reestablish enzyme activity. Cells can be lysed, and luminescence detected to quantify estrogen present in the sample. We present here the construction strategy and proof of concept data demonstrating the efficiency of this dual-functional biosensor and its effectiveness for detection of estrogenic compounds in contaminated water. NSF-REU-1852150, REU Site: A multisite REU in Synthetic Biology, 2019.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

Pathogenic mutations in ethnic Lebanese Arab patients with high risk for hereditary breast cancer

Background: Breast cancer accounts for 35-40% of cancer in women in Lebanese and Arab countries with 50% of patients (pts) diagnosed before age 50. Prevalence of pathogenic BRCA variants in high-risk pts is 5.6-20% (Abulkhair and El Saghir 2021). 7 BRCA1 and 7 BRCA2 pathogenic variants were found in 5.6% of 250 pts with high hereditary risk breast cancer using amplicon sequencing and MLPA (El Saghir 2015;Poulet 2016). We report results of Next Generation Sequencing (NGS) on selected cases based on Manchester Score. First report in ethnic Lebanese Arab pts. Method(s): Pts prospectively enrolled in 2009-2012. IRB approval secured. Pts signed informed consent. Data collected from medical records. Amplicon and MLPA was done on 250 patients. NGS was done on 100 cases with Manchester Score 14-56. DNAs of the 14 pts previously found to have a pathogenic variant (Manchester Score 10-59) were not re-sequenced. NGS on remaining 150 pts was not done due to Covid-19 pandemic and lack of additional funding. Result(s): NGS showed 7 pathogenic variants, 4 in PALB2 and 3 in ATM. No new BRCA variants were found. Two BRCA2 mutations noted by Amplicon/MLPA reported as VUS in 2015 are reclassified as pathogenic. Total BRCA2 pathogenic variants becomes 9. Total pathogenic variants 23. Risk of having hereditary breast cancer in pts with MS 10-59 is 20% (23/ 114), and at least 9.2% in the entire cohort (23/250). Age <=40 with family history (FH) carries 18.9% risk of harboring a pathogenic mutation while no FH, 1.4% (Table 1). All BRCA1 pts had triple negative and 7/9 BRCA2 pts had hormone receptor positive breast cancer. 4 unrelated pts shared the same c.1056_1057delGA PALB2 pathogenic variant thus we suggest this is a founder mutation in Lebanese Ethnic Arab population. Conclusion(s): Mutation rates in high hereditary risk pts with Manchester Score range 10-59 is 20%. Age <=40 with positive FH can be used to select pts for testing when resources are limited. Our data suggests that c.1056_1057delGA is a PALB2 founder mutation. No conflict of interest.Copyright © 2022 Elsevier Ltd. All rights reserved

CRISPR-Cas assisted diagnostics: A broad application biosensing approach

In addition to its remarkable genome editing capability, the CRISPR-Cas system has proven to be very effective in many fields of application, including the biosensing of pathogenic infections, mutagenic defects, or early cancer diagnosis. Thanks to their many advantages in terms of simplicity, efficiency, and reduced time, several CRISPR-Cas systems have been described for the design of sensitive and selective analytical tools, paving the way for the development and further commercialization of next-generation diagnostics. However, CRISPR-Cas-based biosensors still need further research efforts to improve some drawbacks, such as the need for target amplification, low reproducibility, and lack of knowledge of exploited element robustness. This review aims to describe the latest trends in the design of CRISPR-Cas biosensing technologies to better highlight the insights of their advantages and to point out the limitations that still need to be overcome for their future market entry as medical diagnostics.Copyright © 2023 Elsevier B.V.

Pyrrole-Imidazole Polyamides - A Frontrunner in Nucleic Acid-Based Small Molecule Drugs

The remarkable success of messenger RNA vaccines against the ongoing coronavirus-2019 (COVID-19) pandemic renews attention toward nucleic acid therapeutics. While nucleic acid therapy using unmodified DNA or RNA is the primary focus in disease treatment, there is growing need to develop nucleic acid-based small molecules owing to their potential clinical benefits as drugs in terms of cost and scalability. While small molecules targeting protein-protein interactions are known to alter the transcriptional status of a cell, they can result in a transient effect and variation of bio-efficacy among patients. Small molecules targeting DNA and/or RNA are in demand in the precision medicine approach as they have consistent bioactivity among patients. This review details the progress of sequence-specific DNA-binding pyrrole-imidazole polyamides (PIPs) in modulating the transcriptional status of target gene(s) without altering the underlying DNA sequence. Here, the different versions of PIPs are listed, and also, how conjugating them with DNA alkylating agents, epigenetic modulators, and other drugs can improve their clinical utility as targeted transcription therapeutics. Owing to their specificity, functional diversity, and limited toxicity, PIP technology holds enormous promise as frontrunner in small-molecule-based nucleic acid drugs to precisely regulate therapeutically important genes on demand and treat intractable diseases.Copyright © 2023 Wiley-VCH GmbH.

Current pandemic COVID-19 vaccine strategies and development: a comprehensive review

The coronavirus disease-19 pandemic with the characteristics of asymptomatic condition, long incubation period and poor treatment has influenced the entire globe. Coronaviruses are important emergent pathogens, specifically, the recently emerged sever acute respiratory syndrome coronavirus 2, the causative virus of the current COVID-19 pandemic. To mitigate the virus and curtail the infection risk, vaccines are the most hopeful solution. The protein structure and genome sequence of SARS-CoV-2 were processed and provided in record time;providing feasibility to the development of COVID-19 vaccines. In an unprecedented scientific and technological effort, vaccines against SARS-CoV-2 have been developed in less than one year. This review addresses the approaches adopted for SARS-CoV-2 vaccine development and the effectiveness of the currently approved vaccines.Copyright © 2023, Finlay Ediciones. All rights reserved.

Metagenomic Next-Generation Sequencing of Nasopharyngeal Swabs in Acute Febrile Illness in Cambodia

Background. There is growing recognition of metagenomic next-generation sequencing (mNGS) as a valuable diagnostic tool capable of providing unbiased pathogen detection, but data on performance in low-resource settings remains scant. Here, we use mNGS of nasopharyngeal (NP) swabs taken from subjects in Cambodia to identify potential pathogens causing acute febrile illness. Methods. Febrile subjects aged 2 months to 65 years were enrolled in a crosssectional study conducted across 4 tertiary hospitals in Cambodia. NP swabs were collected at hospital presentation. Depending on reported symptom constellations, sera was also taken in a subset of subjects for comparison of mNGS results. RNA was isolated from biosamples, converted to cDNA libraries, and sequenced on a NextSeq2000 (Illumina). Raw sequence reads were stripped for host reads and aligned to NCBI nucleotide and protein databases using a cloud-based bioinformatics platform (CZID). Results. NP swabs were collected from 97 subjects between April 2020 and June 2021. Subjects were predominantly male (53.6%) and young (median age 3 years [IQR 1-25]). Pathogens were identified in 42 (43.2%) NP swabs;of these, 26 (61.9%) were respiratory viruses including 9 rhinovirus, 7 coronavirus (1 SARS-CoV-2), and 5 respirovirus cases. Co-infection was identified in 3 subjects with coronavirus and respirovirus (N=2) and coronavirus and rhinovirus (N=1). Of subjects with paired sera and NP samples (N=61), 18 (29.5%) had positive NP swabs but negative sera, 7 (11.5%) had negative NP swabs but positive sera, 12 (19.7%) had positive NP swabs and sera, and 24 (39.3%) had negative NP swabs and sera. Pathogen hits correlated in NP swabs and sera in 10 of 12 subjects, including six subjects with chikungunya. Conclusion. mNGS can be successfully implemented in low-resource settings to identify emerging pathogens and common respiratory pathogens, including coinfecting pathogens, from NP swabs of febrile patients. mNGS may also be able to detect chikungunya from NP swab alone, raising the possibility of non-invasive diagnostics for infections associated with high viremic states.

A hypermutator strain of Pseudomonas aeruginosa in a COVID-19 patient with ventilator-associated pneumonia

Background. P. aeruginosa is a cause of hospital-acquired and ventilatorassociated pneumonia. Hypermutator P. aeruginosa strains have been described in patients with cystic fibrosis and chronic respiratory infections but are rare in patients with acute P. aeruginosa infection. This case describes a hypermutator strain of P. aeruginosa found in a patient with COVID-19-associated acute respiratory distress syndrome (ARDS). Methods. Serial respiratory and blood cultures were collected. Short-read sequencing libraries were prepared using the Illumina Nextera XT kit, and wholegenome sequencing was performed using the Illumina NextSeq platform. Long-read sequencing libraries were prepared from unsheared genomic DNA using ligation sequencing kit SQK-LSK109 and sequenced on the Oxford MinION platform. Single nucleotide variants were identified by aligning reads from each isolate to the complete genome of the first available clinical isolate. Hypermutator assays were performed by measuring the mutation frequency rate for rifampin resistance. Antibiotic minimal inhibitory concentrations (MICs) were performed. Growth curves were performed with a starting OD600 of 0.1 with measurements taken every 30 minutes for 24 hours. Results. Seventeen respiratory and five blood isolates were obtained throughout 62 days of hospitalization. Fourteen of the 22 isolates exhibited hypermutator phenotypes by rifampin resistance assays, which demonstrated rapid accumulation of mutations. All five bloodstream isolates were hypermutators. MIC testing noted increased resistance to aminoglycosides, fluoroquinolones, and aztreonam in the hypermutator isolates. All bloodstream isolates descended from a single progenitor noted on whole-genome sequencing. Each hypermutator strain contained a mutation in the mismatch repair gene mutL, previously associated with the hypermutator phenotype. Genetic Tree of Patient Isolates The genetic tree highlights hypermutator versus non-hypermutator single nucleotide variants Conclusion. This case was notable for multiple isolates of hypermutator P. aeruginosa that persisted over weeks. The patient's COVID-19 infection and acute respiratory distress syndrome may have facilitated persistence of the P. aeruginosa lineage, allowing a hypermutator lineage to emerge.

Reverse Genetics of Avian Coronavirus Infectious Bronchitis Virus

We have developed a reverse genetics system for the avian coronavirus infectious bronchitis virus (IBV) in which a full-length cDNA corresponding to the IBV genome is inserted into the vaccinia virus genome under the control of a T7 promoter sequence. Vaccinia virus as a vector for the full-length IBV cDNA has the advantage that modifications can be introduced into the IBV cDNA using homologous recombination, a method frequently used to insert and delete sequences from the vaccinia virus genome. Here, we describe the use of transient dominant selection as a method for introducing modifications into the IBV cDNA;that has been successfully used for the substitution of specific nucleotides, deletion of genomic regions, and the exchange of complete genes. Infectious recombinant IBVs are generated in situ following the transfection of vaccinia virus DNA, containing the modified IBV cDNA, into cells infected with a recombinant fowlpox virus expressing T7 DNA dependant RNA polymerase. Copyright © Springer Science+Business Media New York 2016.

SARS-CoV-2 surveillance: Nanopore sequencing as potential rapid tool for novel pathogen variants characterization

Genomic epidemiology is a crucial weapon in the public health fight against infectious diseases.Whole- Genome Sequencing (WGS) of SARS-CoV-2 provides critical insight into viral transmission and evolution.Oxford Nanopore Technology (ONT) works by monitoring changes to an electrical current as a strand of nucleic acid passes through a protein nanopore and the resulting signal from multiple nucleotides is decoded to provide the specific sequence.Illumina sequencing technology, sequencing by synthesis (SBS), detects base-by-base as they are incorporated into growing DNA strands enabling accurate data. In the present study a comparison between ONT performance on MinION and Illumina ISeq was evaluated.Library preparation was performed on 25 SARS-CoV-2-positive specimens collected at Hygiene Laboratory, San Martino Hospital (Genoa, Italy) by COVID panel CE-IVD kit developed at 4Bases and CleanPlex SARS-CoV-2 NGS Panel by Paragon Genomics, respectively to be sequenced on ONT MinION MK1B and Illumina iSeq100 platforms.Data analysis was carried out using the 4eVAR (4Bases) and Sophia DDM Software (Sophia Genetics) platforms and consensus sequence was analyzed using Nextclade webservice. Each variant was classified according to World Health Organization (WHO), Clade and Lineage.Both technologies correctly identified 7 Delta and 18 Omicron variants. Mean coverage on MinION was 763x, after 3 hours of run, covering 98% of the genome. There was perfect match for the WHO, Clade and the Lineage classification except for one difference along the sub-lineage classification of one omicron variant (BA.2 in MinION, BA.2.3 in Paragon).Single mutations analysis showed 96% overlap on average in mutation classification considering all mutations, 98% considering mutations on Spike protein.The two different technologies show a comparable analytical performance, with small differences concerning precise mutation classification. MinION enables real-time analysis, therefore is faster, cheaper and more flexible than standard sequencing techniques. By this analysis it is confirmed to be a promising platform, with the potential to represent a convenient portable and rapid tool for the SARS-COV2 surveillance.

Characterization of Genetic Mutations Using DNA Walks and Multifractal Detrended Fluctuation Analysis

Monitoring genetic mutations in DNA sequences and their subsequent characterisation provide the possibility for rapid development of diagnostics and therapeutic tools. Here, it is shown that the "DNA walk" (DNAW) representation together with multifractal detrended fluctuation analysis (MFDFA), i.e. DNAW/MFDFA, form a reliable characterization method for studying local and global properties of similar DNA sequences. The DNAW/MFDFA approach allows to study the stochastic properties of genetic sequences by constructing a one-to-one map of the sequence onto a walk, and is able to uncover the self-similarity properties of DNA walks. These features are illustrated on a set of similar DNA sequences of SARS-CoV-2 virus, in which the differences in nucleotide bases arise due to genetic mutations. The results show that DNAW/MFDFA can be used to extract long-range correlation information and type and degree of fractal complexity.

Identification and classification of coronavirus genomic signals based on linear predictive coding and machine learning methods.

Corona disease has become one of the problems and challenges of humankind over the past two years. One of the problems that existed from the first days of this epidemic was clinical symptoms similar to other infectious viruses such as colds and influenza. Therefore, diagnosis of this disease and its coping and treatment approaches is also been difficult. In this study, Attempts has been made to investigate the origin of this disease and the genetic structure of the virus leading to it. For this purpose, signal processing and linear predictive coding approaches were used which are widely used in data compression. A pattern recognition model was presented for the detection and separation of covid samples from the influenza virus case studies. This model, which was based on support vector machine classifier, was tested successfully on several datasets collected from different countries. The obtained results performed on all datasets by more than 98% accuracy. The proposed model, in addition to its good performance accuracy, can be a step forward in quantifying and digitizing medical big data information.

kmer2vec: A Novel Method for Comparing DNA Sequences by word2vec Embedding.

The comparison of DNA sequences is of great significance in genomics analysis. Although the traditional multiple sequence alignment (MSA) method is popularly used for evolutionary analysis, optimally aligning k sequences becomes computationally intractable when k increases due to the intrinsic computational complexity of MSA. Despite numerous k-mer alignment-free methods being proposed, the existing k-mer alignment-free methods may not truly capture the contextual structures of the sequences. In this study, we present a novel k-mer contextual alignment-free method (called kmer2vec), in which the sequence k-mers are semantically embedded to word2vec vectors, an essential technique in natural language processing. Consequently, the method converts each DNA/RNA sequence into a point in the word2vec high-dimensional space and compares DNA sequences in the space. Because the word2vec vectors are trained from the contextual relationship of k-mers in the genomes, the method may extract valuable structural information from the sequences and reflect the relationship among them properly. The proposed method is optimized on the parameters from word2vec training and verified in the phylogenetic analysis of large whole genomes, including coronavirus and bacterial genomes. The results demonstrate the effectiveness of the method on phylogenetic tree construction and species clustering. The method running speed is much faster than that of the MSA method, especially the phylogenetic relationships constructed by the kmer2vec method are more accurate than the conventional k-mer alignment-free method. Therefore, this approach can provide new perspectives for phylogeny and evolution and make it possible to analyze large genomes. In addition, we discuss special parameterization in the k-mer word2vec embedding construction. An effective tool for rapid SARS-CoV-2 typing can also be derived when combining kmer2vec with clustering methods.

AACR President's initiative - 2020 by 2020: Democratizing precision cancer medicine and advancing health equity in the black belt

African Americans (AA) have higher incidence and mortality rates for several cancer types in comparison to their European American (EA) counterparts. Increasing participation in clinical research and patient registries, related to precision cancer medicine, will significantly improve cancer health equity. Many AA cancer patients are treated in community oncology clinics. Unfortunately, these health systems have limited access to Clinical Laboratory Improvement Amendments (CLIA) next generation sequence (NGS) germline and somatic DNA and RNA testing that are used to inform oncologists on the best treatment and/or clinical trial options for cancer patients. Indeed, AA CLIA NGS sample sets are poorly represented, which could presumably result in incomplete knowledge of genomic variants that could affect their treatment and overall outcomes. Hence, it is crucial to implement CLIA NGS efforts for all cancer patients. To address these disparities, Morehouse School of Medicine has formed the Comprehensive Approach to Reimagine health Equity Solutions (CARhES) consortium with Tuskegee University that has engaged community oncology practices in Alabama and Georgia - two of five Black Belt states. The CARhES consortium aims to implement precision cancer medicine to underserved and underrepresented communities that will improve the standard of cancer care by providing access to CLIA NGS testing, clinical trials, and personalized cancer care. Here we describe the first proof of concept of this approach with community oncology partners, i.e., Grady Health System, Wellstar Health System, Georgia Urology, Midtown Urology, and Maui Memorial Medical Center. At the time of consent, saliva, buccal, and tumor samples were collected from participants. Germline and somatic CLIA NGS was performed, and medical reports were returned to practitioners within 14 days. Prior to the COVID pandemic, the study enrolled over 880 patients with a 88% consent rate (n = 1000) in the first 11months of the program. At the start of the COVID pandemic, recruitment efforts were suspended for four months with a slow restart by June 2020. A decrease in the number of staff, office visits (67% reduction), and increase in COVID cases significantly limited recruitment efforts. During this slowdown, we established and improved eConsenting capabilities, which exist today. Community anxiety, due to the pandemic and SARS-CoV-19 vaccine efforts, resulted in a significant reduction in consent rates (88% to 60%). Nevertheless, this study began in April of 2019 and consented 1,750 participants in less than 2 years. Taken together, our study shows that a community-focused precision medicine approach requires meeting people where they are and providing them with access and understanding the benefit of clinical trial participation. The approximate 2,000 clinically annotated genomic AA datasets will greatly contribute to our understanding of cancer health disparities and among the first steps to democratize precision medicine.

Aberrant DNA 5mC and 6mA methylations increase ACE2 expression in intestinal cancer cells susceptible to SARS-CoV-2 infection

Angiotensin converting enzyme II (ACE2) is the cellular receptor of SARS-CoV-2. At present, ACE2 receptor is considered to be the key component in the SARS-CoV-2 infection and transmitting in the host. Among the cancer patients with COVID-19, the gastrointestinal cancer is the second most prevalent. The MethyLight and QASM assays were used to evaluated the genomic DNA 5mC methylation, while the CviAII enzyme-based 6mA-RE-qPCR was applied to determine motif-specific DNA 6mA methylation. The 6mA and 5mC methylation analyses of the long interspersed nuclear elements 1 (LINE1) were used to evaluate the global level of genomic 6mA and 5mC methylations, respectively. To investigate the role of ACE2 DNA methylation in regulating ACE2 expression, we performed a genome-wide methylation analysis in colorectal cancer samples collected at the Sixth Affiliated Hospital of Sun Yat-sen University. The DNA 5mC methylation of ACE2 promoter in tumor tissues were significantly lower than that in normal tissues, while the DNA 6mA methylation of ACE2 promoter in tumor tissues was significantly higher than that in normal tissues. In addition, the mRNA and protein expression of ACE2 in tumor tissues were lower than that in normal tissues. To explore the epigenetic regulation on ACE2 expression, we treated colon cancer cell lines with 5-Azacytidine and found ACE2 expression was upregulated after lowering the DNA 5mC methylation. The correlation analysis in patient cohort samples showed that ACE2 mRNA expression was positively correlated with DNA 5mC and negatively associated with DNA 6mA methylation. Next, a novel CRISPR-based tool was developed for sequence-specific 6mA editing on ACE2 promoter region, and it was applied in HCT116 cell to further confirm the regulatory role of DNA 6mA methylation in ACE2 mRNA expression. This tool was proved to be reliable with our findings that the CRISPR/dCas9-METTL3 tool could dramatically upregulate DNA 6mA methylation in ACE2 promoter, while the global level of genomic 6mA methylation remained unchanged. Both the mRNA and protein expression of ACE2 were significantly increased following a sequence-specific DNA 6mA editing in ACE2 promoter. In conclusion, we revealed the aberrant DNA 5mC and 6mA methylations in colorectal cancer, which upregulate ACE2 expression in colorectal cancer cells that may confer the susceptibility to SARS-CoV-2 infection. We developed a novel CRISPR-based tool that could realize site-directed 6mA methylation editing. Notably, the epigenetic regulation of DNA 6mA methylation on ACE2 expression provides an insight into the intersection of the biology of cancer, SARS-CoV-2 infection and organ-specific complication in COVID-19. Aberrant ACE2 methylation may serve as a biomarker and treatment target in these patients.

Exploring the Role of 2D-Graphdiyne as a Charge Carrier Layer in Field-Effect Transistors for Non-Covalent Biological Immobilization against Human Diseases.

Graphdiyne's (GDY's) outstanding features have made it a novel 2D nanomaterial and a great candidate for electronic gadgets and optoelectronic devices, and it has opened new opportunities for the development of highly sensitive electronic and optical detection methods as well. Here, we testified a non-covalent grafting strategy in which GDY serves as a charge carrier layer and a bioaffinity substrate to immobilize biological receptors on GDY-based field-effect transistor (FET) devices. Firm non-covalent anchoring of biological molecules via pyrene groups and electrostatic interactions in addition to preserved electrical properties of GDY endows it with features of an ultrasensitive and stable detection mechanism. With emerging new forms and extending the subtypes of the already existing fatal diseases, genetic and biological knowledge demands more details. In this regard, we constructed simple yet efficient platforms using GDY-based FET devices in order to detect different kinds of biological molecules that threaten human health. The resulted data showed that the proposed non-covalent bioaffinity assays in GDY-based FET devices could be considered reliable strategies for novel label-free biosensing platforms, which still reach a high on/off ratio of over 10⁴. The limits of detection of the FET devices to detect DNA strands, the CA19-9 antigen, microRNA-155, the CA15-3 antigen, and the COVID-19 antigen were 0.2 aM, 0.04 pU mL^-1, 0.11 aM, 0.043 pU mL^-1, and 0.003 fg mL^-1, respectively, in the linear ranges of 1 aM to 1 pM, 1 pU mL^-1 to 0.1 µU mL^-1, 1 aM to 1 pM, 1 pU mL^-1 to 10 µU mL^-1, and 1 fg mL^-1 to 10 ng mL^-1, respectively. Finally, the extraordinary performance of these label-free FET biosensors with low detection limits, high sensitivity and selectivity, capable of being miniaturized, and implantability for in vivo analysis makes them a great candidate in disease diagnostics and point-of-care testing.

RAPID POINT-OF-CARE DIAGNOSIS OF CT/NG USING EXPAR DNA AMPLIFICATION COUPLED TO A NOVEL OPTICAL DETECTION SYSTEM

Introduction The Covid-19 pandemic has dramatically accelerated the point of care (POC) landscape, increasing awareness and demand for rapid diagnostics of other diseases. STIs are a major current health issue, with Neisseria gonorrhoea (NG) and Chlamydia trachomatis (CT) being highly prevalent. Current diagnostic methods are not POC and the most rapid takes around 20 mins. We have developed a new molecular assay, taking < 10 minutes for a diagnostic result, and combined it with a novel, rapid detection mechanism to produce a fully integrated POC device. Methods Our assay will make use of the exponential amplification reaction (EXPAR), a rapid isothermal DNA amplification technique, to produce an output detectable by Linear Dichroism (LD). LD is a highly sensitive optical detection technique, relying on exploiting structural properties of a scaffold such as M13 bacteriophage. Adapting EXPAR and combining it with a DNA sensitive LD assay allows detection of specific DNA sequences, signalling the presence of CT and/or NG. A principal advantage of our system is it allows multiplexing on the same detection scaffold. Results Initial clinical trials using EXPAR show detection of CT/NG patient samples within 10 minutes of DNA amplification at a constant temperature. The sequences detected represent specific and well conserved regions of CT and NG. Large signal changes give M13 a high analytical sensitivity. Discussion Our systems will ensure faster and more accurate diagnosis and ultimately better patient health outcomes. We are currently focused on an expansion of the scope of diseases that we can detect, using our in-house sequence selection process. (Figure Presented).