Genomic DNA Fragments Research Articles

OPTIMIZING ULTRASONIC gDNA FRAGMENTATION FOR NGS SEQUENCING

The fragmentation of genomic DNA (gDNA) is a crucial step in many Illumina library preparation protocols, including the TSO 500 assay, aimed at achieving fragment sizes ranging from 90 to 250 base pairs (bp) for next-generation sequencing (NGS). However, challenges arise in practice due to variations in fragmentation times, which are influenced by different extraction methods and sample sources. To optimize ultrasonic gDNA fragmentation across various extraction methods for NGS sequencing applications. Shearing of gDNA was performed using the Covaris S220 system, employing microtubes containing a total volume of 130 microliters of diluted Tris EDTA (pH 8.0) and 100 ng of gDNA, following the manufacturer's protocol. gDNA samples were extracted using three different kits: AllPrep DNA/RNA FFPE (Qiagen) from FFPE blocks, ReliaPrep FFPE gDNA Miniprep system (Promega) from glass slides, and Gentra Puregene (Qiagen) from cell lines. The shearing times varied depending on the extraction method and sample source. gDNA extracted using the AllPrep DNA/RNA FFPE kit from FFPE blocks required shearing times ranging from 300 to 400 seconds. In contrast, gDNA extracted using the ReliaPrep FFPE gDNA Miniprep system from glass slides exhibited shearing times of approximately 480 to 500 seconds. Lastly, gDNA extracted using the Gentra Puregene kit from cell lines showed shearing times ranging from 150 to 180 seconds. Our study demonstrates the influence of gDNA extraction methods and sample sources on ultrasonic fragmentation efficiency for NGS sequencing. The observed differences in shearing times highlight the need for method optimization to achieve consistent and optimal fragment sizes across diverse sample types. These findings contribute to the development of standardized protocols for gDNA fragmentation, facilitating reliable and reproducible NGS data generation in various research and clinical settings. Further investigations into the underlying factors affecting fragmentation efficiency are warranted to advance our understanding and improve NGS library preparation methodologies.

Efficient and accurate BmNPV bacmid editing system by two-step golden gate assembly

The silkworm-baculovirus expression vector system (silkworm-BEVS), using Bombyx mori nucleopolyhedrovirus (BmNPV) and silkworm larvae or pupae, has been used as a cost-effective expression system for the production of various recombinant proteins. Recently, several gene knockouts in baculoviruses have been shown to improve the productivity of recombinant proteins. However, the gene editing of the baculovirus genome (approximately 130 kb) remains challenging and time-consuming. In this study, we sought to further enhance the productivity of the silkworm-BEVS by synthesizing and gene editing the BmNPV bacmid from plasmids containing fragments of BmNPV genomic DNA using a two-step Golden Gate Assembly (GGA). The BmNPV genome, divided into 19 fragments, was amplified by PCR and cloned into the plasmids. From these initial plasmids, four intermediate plasmids containing the BmNPV genomic DNA were constructed by GGA with the type IIS restriction enzyme BsaI. Subsequently, the full-length bacmid was successfully synthesized from the four intermediate plasmids by GGA with another type IIS restriction enzyme PaqCI with a high efficiency of 97.2 %. Furthermore, this methodology enabled the rapid and straightforward generation of the BmNPV bacmid lacking six genes, resulting in the suppression of degradation of recombinant proteins expressed in silkworm pupae. These results indicate that the BmNPV bacmid can be quickly and efficiently edited using only simple cloning techniques and enzymatic reactions, marking a significant advancement in the improvement of the silkworm-BEVS.

Split application of phosphorus fertilizer increased wheat grain weight under post-anthesis drought stress by delaying programmed cell death of endosperm

Drought is one of the important factors affecting wheat yield. However, the effect of split application of phosphorus fertilizer on the programmed cell death (PCD) of wheat endosperm cells under different post-anthesis water treatments is still unclear. In this study, three phosphorus fertilization methods (P1, all phosphorus fertilizer was applied at the spring green-up stage; P2, 50 % and 50 % of phosphorus fertilizer were applied at the spring green-up stage and jointing stage, respectively; P3, 40 %, 30 %, and 30 % of phosphorus fertilizer were applied at the spring green-up, jointing, and grain filling stages, respectively) were designed. Besides, two post-anthesis water treatments were designed: WT, full irrigation treatment; DT, drought stress treatment. Then, the effects of P1, P2, and P3 treatments on the endosperm cell development, nuclear number, nuclease activity, DNA fragmentation, DNA content, grain weight, and phosphorus content of winter wheat variety “Xindong 23” under WT and DT conditions were determined at different stages after anthesis. The results showed that under WT and DT conditions, the degree of nuclear deformation and nuclear membrane depression in P3 treatment was milder than that in P1 and P2 treatments. The ACPase amount in P3 treatment was more than that in P1 and P2 treatments. The number of endosperm nuclei in P3 treatment was higher than that in P1 and P2 treatments. Under DT condition, the endosperm DNA content of wheat grains collected 14–35 days post anthesis (DPA) in P3 treatment increased, and the peak time of genomic DNA fragmentation in P3 treatment was later, compared with those in P1 treatment. Under DT condition, the grain weight and grouting rate in P3 treatment were higher than those of P1 treatment at 7–28 DPA, and the 1000-grain weight of P3 was the highest under both WT and DT conditions, reaching 47.03 and 40.57 g, respectively. Under WT and DT conditions, compared with P1 treatment, the protein content change rate (3.16 % and 5.59 %, respectively) and the total phosphorus content change rate (34.36 % and 35.08 %, respectively) of wheat grains in P3 treatment were the highest. In summary, under the DT condition, split application of phosphorus fertilizer (P3, spring green-up stage stage: jointing stage: filling stage = 4: 3: 3) could increase the phosphorus uptake and utilization of wheat plants and grains, suppress the DNA fragmentation of endosperm cells, and delay the deformation and disintegration of nuclei, which ultimately delayed the PCD in endosperm and realized high grain weight and protein content.

Association of ACTN4 Gene Mutation with Primary Nephrotic Syndrome in Children in Guangxi Autonomous Region, China.

Objective: To investigate the association between ACTN4 gene mutation and primary nephrotic syndrome (PNS) in children in Guangxi Autonomous Region, China. Methods: The high-throughput sequencing technology was used to sequence ACTN4 gene in 155 children with PNS in Guangxi Autonomous Region in China, with 98 healthy children serving as controls. Twenty-three exon-specific capture probes targeting ACTN4 were designed and used to hybridize with the genomic DNA library. The targeted genomic region DNA fragments were enriched and sequenced. The protein levels of ACTN4 in both case and control groups were quantified using ELISA method. Results: Bioinformatics analysis revealed five unique ACTN4 mutations exclusively in patients with PNS, including c.1516G>A (p.G506S) on one exon in 2 patients, c.1442 + 10G>A at the splice site in 1 patient, c.1649A>G (p.D550G) on exon in 1 patient, c.2191-4G>A at the cleavage site in 2 patients, and c.2315C>T (p.A772V) on one exon in 1 patient. The c.1649A>G (p.D550G) and c.2315C>T (p.A772V) were identified from the same patient. Notably, c.1649A>G (p.D550G) represents a novel mutation in ACTN4. In addition, three other ACTN4 polymorphisms occurred in both case and control groups, including c.162 + 6C>T (1 patient in case group and 2 patients in control group), c.572 + 11G>A (1 patient in case group and 2 patients in control group), and c.2191-5C>T (4 patients in the case group and 3 patients in control group). The serum ACTN4 concentration in the case group was markedly higher, averaging 544.7 ng/mL (range: 264.6-952.6 ng/mL), compared with 241.20 ng/mL (range: 110.75-542.35 ng/mL) in the control group. Conclusion: Five ACTN4 polymorphisms were identified among children with PNS in Guangxi Autonomous Region, China, including the novel mutation c.1649A>G. The lower serum levels of α-actinin-4 in the case group suggest that this protein might play a protective role in PNS.

Assembly and Comparative Analysis of the Complete Mitochondrial Genome of Ilex rotunda Thunb.

Ilex rotunda Thunb. stands as a representative tree species in subtropical evergreen broad-leaved forests, widely distributed across southeast Asia. This species holds significant value in forestry due to its ecological resilience and adaptability. Although researchers have conducted in-depth research on the plastid genome (plastome) of I. rotunda, the mitochondrial genome (mitogenome) of this species has remained undocumented. In the present study, we successfully sequenced and assembled the I. rotunda mitogenome. The mitogenome has a circular structure and is 567,552 bp in total length, with a GC content of 45.47%. The composition of the mitogenome encompasses 40 protein-coding genes, along with 3 rRNA genes and 19 tRNA genes. Notably, the mitogenome exhibits a universal distribution of repetitive sequences, but the total length of repeats contributes to a relatively small proportion (4%) of the whole mitogenome, suggesting that repeats do not serve as the primary cause of the amplification of the Ilex mitogenomes. Collinear analysis indicates that the I. rotunda mitogenome is very conservative within Aquifoliales species. Additionally, our research identified 51 fragments of plastid genomic DNA, which have migrated from the plastome into the mitogenome, with five genes from the plastome remaining intact. Eventually, the phylogenetic analyses based on the plastomes and mitogenomes of 36 angiosperms determine the Aquifoliales to be the basal group in the campanulids. This study establishes the bedrock for prospective investigations in molecular breeding research.

Biotinylated cyclic naphthalene diimide as a searching tool for G4 sites on the genome

A biotinyl cyclic naphthalene diimide (biotinyl cNDI) (1), in which biotin is introduced on the cyclic linker chain of cNDI with high G-quadruplex (G4) specificity, was synthesized. 1 was used for binding analysis to G4 DNAs such as c-myc, c-kit, CEGF, or TA-core. The results showed that 1 bind to G4 DNAs with high affinity and, especially, two molecules of 1 bind to c-myc DNA from top and bottom of G4 site at K = 3.9 × 10−6 M−1 without changing the G4 structure. As a pulldown assay, 1 and streptavidin magnetic beads could be used to recover a c-myc DNA or 120-mer DNA fragment having single c-myc sequence. The qPCR results for the 120-meric DNAs showed that more than 50% of genomic DNA fragments could be recovered by this pulldown assay. The results obtained here might allow the recovery of G4-containing DNA fragments from genomic DNA to analyze the true G4 present in the genome.Graphic abstract

Abstract 3750: Dual isolation and profiling of circulating tumor cells and circulating tumor DNA from a single liquid sample using the Genesis Cell Isolation System and Droplet Digital PCR

Abstract Introduction: Recent advances in technology support the use of liquid biopsy as a noninvasive method for cancer monitoring and diagnosis. Circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) are both regarded as key liquid biopsy biomarkers, offering significant disease-related molecular insights. Despite their potential, dual isolation and profiling have been challenged in a clinical setting by the lack of established platforms and optimized protocols that support this approach. The Genesis System with Celselect SlidesTM captures CTCs based on size and deformability, then the enriched cells can be recovered for subsequent downstream analysis. Concurrently, Droplet Digital PCR (ddPCRTM) can be utilized to interrogate cell-free DNA (cfDNA) to determine the presence of a range of DNA aberrations present in ctDNA. This study, using a model lung CTC-ctDNA sample, illustrates the feasibility of this dual-biomarker approach to detect cancer-associated nucleic acid mutations simultaneously with CTC enumeration, using commercially available kits and instruments. Methods: To model lung cancer samples, blood samples were prepared by spiking in two model circulating markers, cultured cancer cells (model CTCs, human ㅣlung cancer cell line A549) and fragmented genomic DNA containing EGFR mutations (model ctDNA, multiplexed 23 ctDNA [~145 bp band > 95%]), into whole blood from a healthy donor prior to enrichment/extraction. Plasma isolation from the whole blood model samples was performed using either two-step centrifugation or Ficoll-Paque PLUS solution for plasma/buffy coat separation. The extracted plasma layer was used for ctDNA separation, while the remaining cell layer was used for CTC isolation on the Genesis System with a modified protocol. Results: The Genesis System isolated 78-79% of spiked cancer cells in whole blood at a concentration of 40 cells/ml. Despite additional plasma separation steps and liquid handling, the system isolated CTCs spiked in with minimal cell loss (<6%). With an optimized ctDNA extraction, more than 70% of spiked ctDNA in blood samples in the size range of 120-220 bp was extracted. Using the Bio-Rad QX200 ddPCR System and ddPCR EGFR T790M Mutation Detection Assay, blood samples with the model ctDNA spiked in showed positive droplets (17 copies) for EGFR T790M while control blood samples without model ctDNA spiked in showed no positive droplets. Discussion and Conclusions: This work illustrates an optimized protocol for the dual isolation and profiling of CTCs and ctDNA from a single blood sample by using an automated CTC isolation platform and high-sensitivity ddPCR based ctDNA mutation detection. This protocol enables comprehensive liquid biopsy studies using a single blood sample from cancer patients with readily available instruments and kits, which can be applied to many other cancers. Citation Format: Yoon-Tae Kang, Abiodun Bodunrin, Errile Pusod, Kris Simonyi, Adam Corner, David Coe, Michelle Racey, Elizabeth J. Dreskin. Dual isolation and profiling of circulating tumor cells and circulating tumor DNA from a single liquid sample using the Genesis Cell Isolation System and Droplet Digital PCR [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3750.

Abstract 5014: Analytical validation of a complete system for detection and characterization of genomic aberrations in circulating tumor DNA

Abstract Cell-free DNA (cfDNA) fragments are readily detected at a low concentration in the human blood stream. In healthy patients cfDNA is primarily derived from apoptosis of hematopoietic cells. In cancer patients circulating DNA derived from tumor cells (ctDNA) undergoing apoptosis, shedding, and necrosis contribute to total cfDNA. Next generation sequencing (NGS) of cfDNA isolated from patient plasma can be used to fully characterize cancer driven changes in the tumor DNA including the relative contribution of cfDNA, single nucleotide variants (SNVs), small insertions and deletions (Indels), larger deletions, regions of amplified DNA, and structural rearrangements. Frontage has developed a complete lab system for analysis of circulating cfDNA including automated sample extraction, preparation and sequencing of libraries targeting exon sequences of 293 oncology-related genes, and a bioinformatic reporting system. The Frontage sample extraction process uses a post-extraction size selection step to remove high molecular weight DNA derived from lysed blood cells in the plasma sample to increase the sensitivity of detection of ctDNA. The NGS platform couples hybridization-based selection of genomic DNA fragments derived from protein-coding exons with deep sequencing to ~4,000X gross coverage on the NextSeq 2000. The bioinformatic pipeline integrates unique molecular barcode-based generation of consensus reads for each template molecule, somatic variant calling with GATK Mutect2 and FreeBayes, and removal of false positive variants by comparison to a 60 samples healthy patient data set. Analytical validation was performed for each component of the system. For verification of the sample extraction process, cfDNA was extracted in duplicate from four lots of pooled K2 EDTA plasma of healthy patients as well as individual K2 EDTA plasma of colorectal cancer (CRC) patients in four independent extraction runs. The mean yields of cfDNA from healthy samples ranged from 2.3 - 4.2 nanograms per ml plasma and all 32 of the isolated cfDNA aliquots from healthy patient plasma showed a prominent peak around 167 nt with minimal contamination from high molecular weight DNA. For validation of the NGS and bioinformatics platform a set of 8 different reference cfDNAs were processed through four independent runs of library preparation, sequencing, and analysis. For each of the validation runs, greater than 97% of 208 SNVs and Indels with expected allele frequencies (AF) between 2-3% were detected by the platform with higher levels of accuracy for variants at above 3% AF. Two or less false positive variants per run were detected out of 278,000 nucleotides of verified non-mutated sequence that overlapped with the 293 gene panel. Four known DNA amplification events were detected in each of the validation runs. Data from characterization of CRC and other clinical samples with the system will be presented. Citation Format: Ericca Stamper, Weiming Shen, Aman Pruthi, Casey Nagel, Marcin Piechota, Katarzyna Tomala, Qi Wei, David Willoughby. Analytical validation of a complete system for detection and characterization of genomic aberrations in circulating tumor DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5014.

Decoding the complexity of on-target integration: characterizing DNA insertions at the CRISPR-Cas9 targeted locus using nanopore sequencing

BackgroundCRISPR-Cas9 technology has advanced in vivo gene therapy for disorders like hemophilia A, notably through the successful targeted incorporation of the F8 gene into the Alb locus in hepatocytes, effectively curing this disorder in mice. However, thoroughly evaluating the safety and specificity of this therapy is essential. Our study introduces a novel methodology to analyze complex insertion sequences at the on-target edited locus, utilizing barcoded long-range PCR, CRISPR RNP-mediated deletion of unedited alleles, magnetic bead-based long amplicon enrichment, and nanopore sequencing.ResultsWe identified the expected F8 insertions and various fragment combinations resulting from the in vivo linearization of the double-cut plasmid donor. Notably, our research is the first to document insertions exceeding ten kbp. We also found that a small proportion of these insertions were derived from sources other than donor plasmids, including Cas9-sgRNA plasmids, genomic DNA fragments, and LINE-1 elements.ConclusionsOur study presents a robust method for analyzing the complexity of on-target editing, particularly for in vivo long insertions, where donor template integration can be challenging. This work offers a new tool for quality control in gene editing outcomes and underscores the importance of detailed characterization of edited genomic sequences. Our findings have significant implications for enhancing the safety and effectiveness of CRISPR-Cas9 gene therapy in treating various disorders, including hemophilia A.

Functional Analysis of the HbREF1 Promoter from Hevea brasiliensis and Its Response to Phytohormones

The rubber elongation factor (REF) is the most abundant protein in the latex of Hevea brasiliensis, which is closely related to natural rubber biosynthesis. In order to gain a deeper understanding of the transcriptional regulation mechanism of HbREF1, a 1758 bp genomic DNA fragment of the HbREF1 promoter was isolated. Promoter sequence analysis revealed several transcription factor binding sites in the HbREF1 promoter, such as bZIP, bHLH, EIL, AP2/ERF, MYB, and Trihelix. To assess the promoter activity, a series of HbREF1 promoter deletion derivatives were created and fused with firefly luciferase (LUC). The LUC image demonstrated that all of the HbREF1 promoters exhibited transcriptional activity. Furthermore, the assay revealed the presence of multiple regulatory elements within the promoter region that negatively regulate the transcriptional activity. Subsequent analysis of the transcriptional activity following treatment with phytohormones identified an ABA-responsive element located between −583 bp and −200 bp, an ET-responsive element between −718 bp and −583 bp, a JA-responsive element between −1758 bp and −1300 bp, and a SA-responsive element between −1300 bp and −718 bp. These results were largely consistent with the predictions of cis-acting elements. This study has established significant groundwork for future investigations into the regulatory mechanism of HbREF1.

Genomic SELEX Screening of Regulatory Targets of Transcription Factors.

The genome of Escherichia coli K-12 is transcribed by a single species of RNA polymerase. The selectivity of transcriptional targets is determined via interaction with one of seven species of the sigma subunit and a total of approximately 300 species of transcription factor (TFs). For comprehensive identification of the regulatory targets of these two groups of regulatory proteins on the genome, we developed an in vitro approach, "Genomic SELEX" (gSELEX) screening. Here we describe a detailed protocol of the gSELEX screening system, which uses purified regulatory proteins and fragments of genomic DNA from E. coli. Moreover, we describe methods and examples of results using cell-free synthetic proteins.

Anti-Melanoma efficacy of traditional multi-herbal extracts from mongolian ethnomedicine on B16F10 murine cells

Multi-herbal formulation is an attractive approach to developing novel therapeutic strategies to manage advanced forms of melanoma. This research aims to evaluate the anti-melanoma potential of Traditional Multi-Herbal (G4) Extracts sourced from Mongolian Ethnomedicine utilizing both cellular and xenograft models. In vitro and ex vivo experiments employing B16F10 melanoma cells were conducted to evaluate the anti-cancer effect of the G4 extract. Furthermore, in vivo experiments utilizing BALB/C nu/nu mice xenograft models were carried out to gauge the extract's effectiveness. A comprehensive analysis encompassing various assays, such as cell viability, migration and invasion assays, cellular phase analysis, and key indicators of apoptosis, was performed. These indicators included activation of the caspase-3 cascade, genomic DNA fragmentation, nuclear staining alterations, and levels of cell cycle and apoptotic regulatory markers analysis. Our Results showed that the G4 extract exhibited potent anti-cancer effects on B16F10 melanoma cells, notably inhibiting cell migration and vascular sprouting in a concentration-dependent manner, suggesting its potential to impede melanoma metastasis. This investigation underscores the promising anti-cancer potential of the G4 extract against melanoma cells through the modulation of apoptotic pathways and suppression of tumor xenograft growth. Ultimately, our findings suggest that the G4 extract holds promise as a candidate for the development of future melanoma chemotherapeutics.

<i>NeuroD2/6</i> regulate expression balance of transcription factors controlling neurocortical cytoarchitecture

BACKGROUND: Genes of the NeuroD family, including NeuroD1, NeuroD2, and NeuroD6, control neuronal survival, differentiation, maturation, and neurite specification in the nervous system. Deletion of NeuroD1 in the mouse brain results in complete loss of dentate gyrus because of neuronal apoptosis. NeuroD2 is required for neuron survival in the cerebellum and integration of thalamo-cortical connections into neocortex and formation of somatosensory whisker barrel cortex. In NeuroD2/6 double deficient (DKO) mice, callosal axon projections are defective due to abnormal EfnA4 signaling. In order to investigate the NeuroD2/6 controlled molecular cascade, we explored the expression of key transcription factors that control various aspects of cortical development in brains of NeuroD2 and NeuroD6 deficient mutants. AIM: To investigate possible changes in differentiation programs downstream of NeuroD2/6 transcription factors. METHODS: Embryos with NeuroD2/6 double deficiency were used in the experiments, and pregnant mice carrying E13.5 embryos were operated for in utero electroporation. We performed in situ hybridization at various stages of embryonic development to study the expression pattern of target genes. Analyzing the activity of a gene promoter, genomic DNA fragments containing NeuroD2/6 motifs were cloned into pMCS-Gaussia Luc vector for luciferase assays. Charts were made with GraphPad Prism software and data were presented as mean ± standard error. RESULTS: Our findings showed that NeuroD1 expression is ectopically upregulated in postmitotic neurons of NeuroD2/6 DKO neocortex and hippocampus. We detected changes in expression of key transcription factors, Cux1, Tbr1, Lhx2, and Id2. Additionally, Cux1 was shown to be direct target of NeuroD2/6. Moreover, Olig2+ progenitors were increased in NeuroD2/6 DKO neocortex and expression of NeuroD2/6 and Olig2 was mutually exclusive. Thus, NeuroD2/6 regulates the expression of transcription factors in the developing brain. CONCLUSION: Our findings indicate that cumulative action of NeuroD2 and NeuroD6 is required to initiate and maintain the expression of transcription factors Cux1, Tbr1, Lhx2, and Id2. Additionally, both genes are required to prevent premature differentiation of Olig2 positive glial precursors.

Implication of MAPK, Lipocalin-2, and Fas in the protective action of liposomal resveratrol against isoproterenol-induced kidney injury

Background and ObjectiveIsoproterenol (ISO) is a non-selective β-adrenergic receptor agonist. It can be used to treat bradycardia and cardiogenic shock. Despite its usefulness, the overstimulation of β-receptors by ISO can cause “cardiorenal syndrome,” a term used to describe heart and kidney damage. Resveratrol (RES), a natural polyphenol, has marked anti-inflammatory and antioxidant activities. The present work was designed to study the protective efficacy of liposomal resveratrol (L-RES) against ISO-induced kidney injury. Materials and MethodsThe kidney injury was induced in rats by administering ISO (50 mg/kg, s.c.) twice a week for 2 weeks. RES and L-RES were administered at a dose (20 mg/kg/ day, p.o.) along with ISO for 2 weeks. Inflammatory and apoptotic biomarkers were analyzed, which were validated using histochemical analysis. ResultsISO caused renal dysfunction, which manifested as elevated urea, creatinine and uric acid, besides cystatin c and MAPK protein overexpression. In addition, ISO induced gene expression of Fas and lipocalin-2 and provoked genomic DNA fragmentation in renal tissues as compared with the control group. Histological examination confirmed morphological alterations of the kidney tissues obtained from the ISO group. Concurrent treatment of either RES or L-RES with ISO significantly ameliorated kidney damage as demonstrated by the improvement of all measured parameters with the best results for L-RES. The histopathological findings were correlated with the above biochemical parameters. ConclusionL-RES could be a promising approach for the prevention of kidney injury induced by ISO, most likely via the downregulation of MAPK, cystatin c, Fas, and lipocalin-2.

Caspase-9 inhibition triggers Hsp90-based chemotherapy-mediated tumor intrinsic innate sensing and enhances antitumor immunity

BackgroundAntineoplastic chemotherapies are dramatically efficient when they provoke immunogenic cell death (ICD), thus inducing an antitumor immune response and even tumor elimination. However, activated caspases, the hallmark of most cancer...

Hereditary Colorectal Cancer Diagnosis by Next-Generation Sequencing.

Pathogenic germline variants causally contribute to the etiology of colorectal cancer (CRC) and polyposis. The era of massively parallel sequencing, also known as next-generation sequencing (NGS), make it highly possible, effective, and efficient to offer rapid and cost-effective diagnosis for CRC. To aid clinical laboratories in testing the most clinically significant genes, along with the published ACMG CRC technical standard guidelines, this protocol aims to provide a step-by-step technical workflow for carrying out the NGS-panel based CRC molecular diagnosis focusing on the wet lab portion of library preparation and massively parallel sequencing. Using the most popular pull-down-based target enrichment, the chapter particularly encompasses genomic DNA (gDNA) fragmentation, adapter ligation, indexing, hybridization, and capture, which is the most variable and technically challenging part of NGS testing involving at least 3 quality control (QC) checkpoints plus the pre- and post-capture PCR. The gDNA extraction and sequencing is less covered because they are relatively standard technologies with little variations and choices. Although this protocol also introduces pertinent testing algorithms and a brief guideline for pre- and post-testing genetic counselling, the audiences are required to refer to National Comprehensive Cancer Network (NCCN) clinical practice guidelines to determine the most appropriate testing strategies. Since NGS panel-based testing is a highly complex and dynamic platform with multiple choices from different technology and commercial resources, this technical benchtop-based protocol also aims to cover some of the key ramification points for decision-making by each laboratory at the discretion of the directors. © 2023 Wiley Periodicals LLC. Basic Protocol: Hereditary colorectal cancer (CRC) diagnosis by next-generation sequencing.

Megakaryocyte- and erythroblast-specific cell-free DNA patterns in plasma and platelets reflect thrombopoiesis and erythropoiesis levels

Circulating cell-free DNA (cfDNA) fragments are a biological analyte with extensive utility in diagnostic medicine. Understanding the source of cfDNA and mechanisms of release is crucial for designing and interpreting cfDNA-based liquid biopsy assays. Using cell type-specific methylation markers as well as genome-wide methylation analysis, we determine that megakaryocytes, the precursors of anuclear platelets, are major contributors to cfDNA (~26%), while erythroblasts contribute 1–4% of cfDNA in healthy individuals. Surprisingly, we discover that platelets contain genomic DNA fragments originating in megakaryocytes, contrary to the general understanding that platelets lack genomic DNA. Megakaryocyte-derived cfDNA is increased in pathologies involving increased platelet production (Essential Thrombocythemia, Idiopathic Thrombocytopenic Purpura) and decreased upon reduced platelet production due to chemotherapy-induced bone marrow suppression. Similarly, erythroblast cfDNA is reflective of erythrocyte production and is elevated in patients with thalassemia. Megakaryocyte- and erythroblast-specific DNA methylation patterns can thus serve as biomarkers for pathologies involving increased or decreased thrombopoiesis and erythropoiesis, which can aid in determining the etiology of aberrant levels of erythrocytes and platelets.

Validation of a Molecular Diagnostic Test for Circulating Tumor DNA by Next-Gen Sequencing.

A modified version of the PGDx elioTM Plasma Resolve assay was validated as a laboratory-developed test (LDT) for clinical use in the Molecular Diagnostics Laboratory at Fox Chase Cancer Center. The test detects single nucleotide variants (SNVs) and small insertions and deletions (indels) in 33 target genes using fragmented genomic DNA extracted from plasma. The analytical performance of this assay was assessed with reference standard DNA and 29 samples from cancer patients and detected 66 SNVs and 23 indels. Using 50 ng of input DNA, the sensitivity was 95.5% to detect SNVs at 0.5% allele frequency, and the specificity was 92.3%. The sensitivity to detect indels at 1% allele frequency was 70.4%. A cutoff of 0.25% variant allele frequency (VAF) was set up for diagnostic reporting. An inter-laboratory study of concordance with an orthologous test resulted in a positive percent agreement (PPA) of 91.7%.

Skim exome capture genotyping in wheat.

Next-generation sequencing (NGS) technology advancements continue to reduce the cost of high-throughput genome-wide genotyping for breeding and genetics research. Skim sequencing, which surveys the entire genome at low coverage, has become feasible for quantitative trait locus (QTL) mapping and genomic selection in various crops. However, the genome complexity of allopolyploid crops such as wheat (Triticum aestivum L.) still poses a significant challenge for genome-wide genotyping. Targeted sequencing of the protein-coding regions (i.e., exome) reduces sequencing costs compared to whole genome re-sequencing and can be used for marker discovery and genotyping. We developed a method called skim exome capture (SEC) that combines the strengths of these existing technologies and produces targeted genotyping data while decreasing the cost on a per-sample basis compared to traditional exome capture. Specifically, we fragmented genomic DNA using a tagmentation approach, then enriched those fragments for the low-copy genic portion of the genome using commercial wheat exome baits and multiplexed the sequencing at different levels to achieve desired coverage. We demonstrated that for a library of 48 samples, ∼7-8× target coverage was sufficient for high-quality variant detection. For higher multiplexing levels of 528 and 1056 samples per library, we achieved an average coverage of 0.76× and 0.32×, respectively. Combining these lower coverage SEC sequencing data with genotype imputation using a customized wheat practical haplotype graph database that we developed, we identified hundreds of thousands of high-quality genic variants across the genome. The SEC method can be used for high-resolution QTL mapping, genome-wide association studies, genomic selection, and other downstream applications.

Leafhopper transmits soybean stay-green associated virus to leguminous plants

A novel geminivirus, soybean stay-green associated virus (SoSGV), was previously shown to cause soybean delayed senescence and is associated with the incidence of soybean stay-green syndrome. The modes of SoSGV transmission were not yet known. We captured insects belonging to 24 distinct species in a soybean field with the SoSGV outbreak and detected the presence of SoSGV only in leafhoppers and bean bugs (Riptortus pedestris). Caged feeding experiments using captured leafhoppers and bean bugs from soybean fields showed that leafhoppers, but not bean bugs, are vectors transmitting SoSGV. The common brown leafhopper (Orosius orientalis) is identified as the dominant leafhopper species and can establish colonies feeding on soybean plants in experimental conditions. An investigation of SoSGV defective DNA revealed that soybean genomic DNA fragments could be inserted into the SoSGV genome, while sequences from wild soybean, red bean, and cowpea were also identified. We further showed that the common brown leafhopper could transmit SoSGV to wild soybean and red bean plants, emphasizing a vector’s role of the leafhopper in the transmission of SoSGV in the field.