Partial Digestion Research Articles

Cloning and gene function of dicarboxylate-tricarboxylate carrier protein in Gastrodia elata

The gene GeDTC encoding the dicarboxylate-tricarboxylate carrier protein in Gastrodia elata was cloned by specific primers which were designed based on the transcriptome data of G. elata. Bioinformatics analysis on GeDTC gene was carried out by using ExPASY, ClustalW, MEGA, etc. Positive transgenic plants and potato minituber were obtained by virtue of the potato genetic transformation system. Agronomic characters, such as size, weight, organic acid content, and starch content, of potato minituber were tested and analyzed and GeDTC gene function was preliminarily investigated. The results showed that the open reading frame of GeDTC gene was 981 bp in length and 326 amino acid residues were encoded, with a relative molecular weight of 35.01 kDa. It was predicted that the theoretical isoelectric point of GeDTC protein was 9.83, the instability coefficient was 27.88, and the average index of hydrophilicity was 0.104, which was indicative of a stable hydrophilic protein. GeDTC protein had a transmembrane structure and no signal peptide and was located in the inner membrane of mitochondria. The phylogenetic tree showed that GeDTC was highly homologous with DTC proteins of other plant species, among which GeDTC had the highest homology with DcDTC(XP＿020675804.1) in Dendrobium candidum, reaching 85.89%. GeDTC overexpression vector pCambia1300-35Spro-GeDTC was constructed by double digests, and transgenic potato plants were obtained by Agrobacterium-mediated gene transformation. Compared with the wild-type plants, transgenic potato minituber harvested by transplanting had smaller size, lighter weight, lower organic acid content, and no significant difference in starch content. It is preliminarily induced that GeDTC is the efflux channel of tricarboxylate and related to the tuber development, which lays a foundation for further elucidating the molecular mechanism of G. elata tuber development.

Isolation and characterization of three bacteriophages infecting Erwinia amylovora and their potential as biological control agent

BackgroundFire Blight, incited by Erwinia amylovora, is one of the most damaging pear and apple diseases in the world. Fire blight was introduced to Egypt in the 1960 and threatens the Egypt’s costs for pear industry. Currently, Phage therapy is considered to be secured biological method for controlling plant bacterial diseases. This investigation aimed to isolate and identify molecularly for bacteria causing fire bright disease. As well as isolation and identification bacteriophages via spot and plaque assay techniques from pear fire blight lesions and soil. On the other hand, bacteriophages were identified based on plaque morphology, virion morphology, physical characters, profile of DNA restriction and protein.ResultsPathogenicity test revealed that healthy seedlings and pear fruits were responsive to fire blight E. amylovora. Considering the relatively wide host range and greatest protein and genetic variability, using restriction enzyme pattern, the three diversity phage isolates named, EAP1, EAP2 and EAP3 showed a lack of diversity out of five were fatherly characterized. The phages confirmed the close relation of EAP1, EAP2 to Siphoviridae (hexagonal head and long flexible non-contractile tail) and EAP3 to Myoviridae (icosahedral head and contractile tail). The phages retained higher lytic competence of 90.4; 92.68 and 95.25% for EAP1, EAP2 and EAP3, respectively. The phages were stable at strong alkaline (pH 10) 2% salt solution conditions and UV spectra. While EAP3 phage revealed the hexagonal head and very short tail that belongs to Myoviridae family. Bacteriophages were characterized by digestion of the phage DNA with three restriction endonucleases and were placed into three groups based on the patterns. Bacteriophages were 9 used for reducing bacterial infection populations and severity on pear. In a bioassay, the biocontrol of E. amylovora was evaluated using disks of immature pear fruit. On the pear disk surface, bacterial exudate was considerably suppressed by all phage isolates. According to measurements of the bacterial population still present on the disk surface, phage therapy could reduce it by up to 97%. Bacteriophages reduced pear fire blight disease severity on pear fruit trails.ConclusionThe results indicated that bacteriophage isolates may demonstrate variable reactivity against E. amylovora. Bacteriophages reduced pear fire blight disease severity on pear fruit trials. The results indicated that bacteriophage isolates may demonstrate variable reactivity against E. amylovora.

A method to generate capture baits for targeted sequencing.

Hybridization capture approaches allow targeted high-throughput sequencing analysis at reduced costs compared to shotgun sequencing. Hybridization capture is particularly useful in analyses of genomic data from ancient, environmental, and forensic samples, where target content is low, DNA is fragmented and multiplex PCR or other targeted approaches often fail. Here, we describe a DNA bait synthesis approach for hybridization capture that we call Circular Nucleic acid Enrichment Reagent, or CNER (pronounced 'snare'). The CNER method uses rolling-circle amplification followed by restriction digestion to discretize microgram quantities of hybridization probes. We demonstrate the utility of the CNER method by generating probes for a panel of 23771 known sites of single nucleotide polymorphism in the horse genome. Using these probes, we capture and sequence from a panel of ten ancient horse DNA libraries, comparing CNER capture efficiency to a commercially available approach. With about one million read pairs per sample, CNERs captured more targets (90.5% versus 66.5%) at greater mean depth than an alternative commercial approach.

Biopanning, Heterologous Expression, and Characterization of a Shark-Derived Single-Domain Antibody Fusion Protein against Pancreatic Lipase.

Nowadays, obesity severely impacts human health and is the fifth leading risk factor that leads to death globally. Pancreatic lipase (PL) inhibitors have attracted extensive attention owing to their role in effective prevention and treatment of obesity. Here, a shark-derived single-domain antibody fusion protein was used to inhibit PL for the first time. After biopanning, the heterologous expression system pET28a-SUMO-D2 was constructed using the method of double restriction enzyme digestion and T4 ligase to achieve the soluble expression of the PL-specific antibody gene D2. According to the calculation of protein concentration, the final expression of fusion protein PL-D2S was 1.183 mg per liter of Luria Bertani broth. The binding ability of the soluble fusion protein PL-D2S to PL was identified. Enzyme-linked immunosorbent assay results showed that the fusion protein PL-D2S exhibited a strong binding affinity to PL. The experimental results of PL inhibition of PL-D2S in vitro showed that the fusion protein could significantly inhibit the activity of PL, with an IC50 of 404 μg/mL. Our study shows that the fusion protein PL-D2S is a potential PL inhibitor to prevent and treat obesity.

Photoelectrochemical biosensor for DNA demethylase detection based on enzymatically induced double-stranded DNA digestion by endonuclease-exonuclease system and Bi4O5Br2–Au/CdS photoactive material

A novel photoelectrochemical (PEC) biosensor for the detection of DNA demethylase MBD2 was developed based on Bi4O5Br2–Au/CdS photosensitive material. Bi4O5Br2 was firstly modified with gold nanoparticles (AuNPs), following with the modification onto the ITO electrode with CdS to realize the strong photocurrent response as a result of AuNPs had good conductibility and the matched energy between CdS and Bi4O5Br2. In the presence of MBD2, double-stranded DNA (dsDNA) on the electrode surface was demethylated, which triggered the digestion activity of endonuclease HpaII to cleave dsDNA and induced the further cleavage of the dsDNA fragment by exonuclease III (Exo III), causing the release of biotin labeled dsDNA and inhibiting the immobilization of streptavidin (SA) onto the electrode surface. As a results, the photocurrent was increased greatly. However, in the absence of MBD2, HpaII digestion activity was inhibited by DNA methylation modification, which further caused the failure in the release of biotin, leading to the successful immobilization of SA onto the electrode to realize a low photocurrent. The sensor had a detection of 0.3–200 ng/mL and a detection limit was 0.09 ng/mL (3σ). The applicability of this PEC strategy was assessed by studying the effect of environmental pollutants on MBD2 activity.

Endophytic Bacteria Showing Antioxidant Property from Periwinkle [Catharanthus roseus (L.) G. Don

The experiment was conducted in the year, April–December, 2020 at the Department of Microbiology, C. P. College of Agriculture, Sardarkrushinagar Dantiwada Agricultural University, Sardarkrushinagar, Gujarat, India to isolate and characterize promising antioxidant producing endophytic bacteria from periwinkle plant. Twenty four endophytic bacterial cultures derived from various parts of Catharanthus roseus (L.) G. Don were tested for total antioxidant capacity (TAC) and total phenolics content (TPC). The isolate R1 showed highest TAC (615.46 µg AAE mg-1 extract) followed by the isolate R2 (308.59 µg AAE mg-1 extract). Correlation coefficient observed between TAC and TPC was 0.7591, which shows that phenolic compounds were greatly responsible for antioxidant capacity of the bacterial isolates. 15 isolates showing higher TAC were studied for morphological and molecular characteristics. All the 15 isolates were rod shaped and were monobacillus. Nine isolates were Gram positive whereas 6 were Gram negative. 16S rDNA amplification using universal primers 27F and 27R produced a band of 1.5 kb. Restriction digestion of PCR products of all the isolates with tetracutter restriction enzymes AluI, TaqI, Hae III produced polymorphic diagnostic fingerprints. The dendrogram based on ARDRA profiling grouped the 15 bacteria into two groups (cluster A and cluster B) at a Jaccard’s similarity co-efficient of 0.83. Cluster A contained eleven bacterial isolates whereas cluster B had only four. The isolates R1 and R2 may serve as an excellent source of antioxidants and may be exploited for commercial production of antioxidants.

Cloning and Molecular Characterization of the Recombinant CVB4E2 Immunogenic Viral Protein (rVP1), as a Potential Subunit Protein for Vaccine and Immunodiagnostic Reagent Candidate.

The aim of the present study was, first, to clone the VP1 gene of the human coxsackievirus B4 strain E2 (CVB4E2) in the prokaryotic pUC19 plasmid expression vector then to compare it with the structural capsid proteins of the same strain using bioinformatic tools. PCR colony amplification followed through a restriction digestion analysis and sequencing process which affirmed the success of the cloning process. SDS-PAGE and Western Blotting were used to characterize the purified recombinant viral protein expressed in bacteria cells. The BLASTN tool revealed that the nucleotide sequence of the recombinant VP1 (rVP1) expressed by pUC19 highly matched the target nucleotide sequence of the diabetogenic CVB4E2 strain. Secondary structure and three-dimension structure prediction suggested that rVP1, such as wild-type VP1, is chiefly composed of random coils and a high percentage of exposed amino acids. Linear B-cell epitope prediction showed that several antigenic epitopes are likely present in rVP1 and CVB4E2 VP1 capsid protein. Additionally, phosphorylation site prediction revealed that both proteins may affect the signal transduction of host cells and can be involved in virus virulence. The present work highlights the usefulness of cloning and bioinformatics characterizations for gene investigation. Furthermore, the collected data are helpful for future experimental research related to the development of immunodiagnostic reagents and subunit vaccines based on the expression of immunogenic viral capsid proteins.

Determination of Genetic variation among Beauveria bassiana and Metarhizium anisopliae Isolates from Palestine and other Geographic regions using ITS and β-tubulin Sequences, RFLP and ISSR Data

In this work we report the genetic variation of ten isolates of entomopathogenic fungi, four of which were isolated from Palestine while the remaining six were from other geographic regions. The variation was determined using molecular-based techniques including; sequence analysis of β-tubulin (BT) gene, the Internal Transcribed Spacers (ITS) 1 & 2 of the rDNA, Restriction Fragment Length Polymorphism (RFLP), and Inter-Simple Sequence Repeat (ISSR) analysis. The sequencing data of ITS regions confirmed the taxonomic identity of six isolates as Metarhizium anisopliae while the identity of the other four as Beauveria bassiana. These data also revealed that the two Palestinian Beauveria b. isolates are probably new strains since theyshowed 99% homology with the best match sequences reported in the gene bank. The restriction digestion of the ITS2 sequences produced a fingerprint for the PAL-M01 isolate. The ITS and BT sequences were able to distinguish the B-Bot genotype from the other Beauveria b. isolates. In contrast, the ITS could discriminate the three Metarhizium isolates: Ma-7, PAL-M01, and PPRC. Moreover, the ISSR analysis detected high levels of polymorphism among the Beauveria b. (80%) and the Metarhizium a. (90%) isolates. The fact that the PPRC and B-Bot isolates areobviously genetically different from their Metarhizium a. and Beauveria b. counterparts could be attributed to their distant geographic origins. The molecular data obtained in this work can be utilized for specific identification of each ofthese isolates which, would facilitate the local use of these fungi as biocontrol agents. To the best of our knowledge, this is the first study, in Palestine, attempting to discriminate between EPF isolates using advanced molecular techniques.

Movement and Genomic Methods Reveal Mechanisms Promoting Connectivity in a Declining Shorebird: The Lesser Yellowlegs

Integrating tracking technology and molecular approaches provides a comprehensive picture of contemporary and evolutionary mechanisms promoting connectivity. We used mitochondrial DNA and double digest restriction-site associated DNA (ddRAD) sequencing combined with satellite telemetry to investigate the connectivity of geographically disparate breeding populations of a declining boreal shorebird, the lesser yellowlegs (Tringa flavipes). We were able to track 33 individuals on their round-trip migrations to Central and South America and back to the boreal wetlands of North America. Nearly all (93%) adults captured on the breeding grounds returned to within 5 km of the original capture site, with a median dispersal distance of 629 m. While our telemetry data revealed limited breeding dispersal in adults, genetic data uncovered significant interconnectedness across the species’ range. Very little genetic structure was estimated at ddRAD autosomal (ΦST = 0.001), Z-linked (ΦST = 0.001), and mtDNA loci (ΦST = 0.020), and maximum likelihood-based clustering methods placed all individuals in a single cluster regardless of capture location, indicating the species is panmictic. Our data indicate that large-scale juvenile dispersal is the main mechanism maintaining connectivity in this species, resulting in the absence of genomic structure.

Novel Method for Detection of PIK3CA Mutations in Circulating Tumor DNA of Patients with Colorectal Cancer.

The PIK3CA mutation is considered a potential target for treatment of colorectal cancer. We evaluated a PIK3CA mutation assay on plasma cell-free DNA (cfDNA) using a newly developed PCR with restriction digestion integrated and followed by Sanger's sequencing. We analyzed PIK3CA mutation in plasma with our newly developed assays and in matching tumor tissues by routine methods. We detected the PIK3CA gene mutation status by both methods in samples from 40 colorectal cancer patients. Three H1047R mutations of PIK3CA gene were detected in the cfDNA of the 40 patients by restriction digestion PCR. Neither E545K nor H1047R mutations were detected in the cfDNA by routine PCR/sequencing. The PIK3CA H1047R and E545K mutations in cfDNA can be sensitively detected with our newly developed assays. The colorectal cancer has been used as a clinical example in testing our new assays, which indicates that the new assays may have wider applications in detecting mutations in precision oncology. Trial registration: Current Controlled Trials ChiCTR-DDT-12002848, 8 October 2012.

Factors controlling tungsten mobility in W[sbnd]Cu skarn tailings

Few studies have addressed the mobility of tungsten in mine waste, which could act as a point source for metal leaching of this potentially toxic metal. This study examines the behavior of tungsten in acidic floodplain tailings and circumneutral impoundment tailings at the Cantung Mine in the Northwest Territories, Canada. In the Cantung deposit, tungsten is present as scheelite (CaWO4), copper is hosted in chalcopyrite (CuFeS2), and gangue mineralogy consists of abundant pyrrhotite (Fe1-xS), silicate minerals, and carbonate minerals. Samples of surface waters, tailings porewaters, and solid tailings samples were collected from Cantung Mine. For surface waters, both filtered (0.45 μm) and unfiltered aliquots were collected to compare dissolved and particulate element concentrations.Tungsten concentrations range from 5.8 to 30.0 μg/L in water samples with pH values between 7.1 and 8.1, whereas samples below pH 7.1 have concentrations below the detection limit of 0.1 μg/L. Tungsten and iron concentrations are both on average 1.7 times higher in unfiltered aliquots compared to filtered aliquots, suggesting that tungsten is transported as dissolved species but is also adsorbed to suspended Fe-oxyhydroxide minerals at neutral pH. Tailings were analyzed by scanning electron microscopy (SEM) paired with automated mineralogy software (MLA), synchrotron-based μX-ray diffraction and fluorescence (μXRD-XRF), and partial leach digestions with inductively coupled plasma mass spectrometry (cold and hot hydroxylamine hydrochloride, aqua regia, Li-metaborate fusion, and 4-acid digestion with HNO3-HClO4-HF-HCl). Samples from the impounded tailings and the highly oxidized Flat River Tailings distributed on the floodplain (FRT) have similar scheelite content (0.15 wt% and 0.21 wt%, respectively), and scheelite shows no evidence of alteration. Synchrotron-based μXRD of Fe-oxyhydroxide minerals in the FRT identified goethite (FeOOH) and lepidocrocite (γ-FeOOH) as products of pyrrhotite oxidation. Rare rims on pyrrhotite from the tailings impoundment have μXRD spectra consistent with hematite (Fe2O3) and maghemite (γ-Fe2O3). The μXRF maps of the hematite-maghemite rims have prominent tungsten peaks, which represent included scheelite grains and possibly structurally incorporated tungsten. The hydroxylamine leaches yield higher tungsten concentrations in tailings samples from the circumneutral impoundments than samples from the acidic FRT, suggesting the tailings impoundments have more tungsten that is associated with poorly crystalline and amorphous Fe-oxyhydroxide phases than the FRT. Over time, labile-hosted tungsten in the FRT may have been washed down the Flat River during Fe-oxyhydroxide recrystallization and high energy flooding events. These results indicate that scheelite has limited solubility in neutral or acid mine waters, and the mobility of the small amount of tungsten released is governed by the present of iron oxyhydroxide minerals, the transport of colloidal material in surface water, and the stability of the depositional environment.

An Improved Method for Efficient DNA Extraction from Grapevine

Grapevine (Vitis vinifera L.) is one of the oldest and most important perennial crops worldwide which has been the subject of extensive genetic studies including gene mapping, genetic transformation, and DNA fingerprinting. Grapevines are rich in polysaccharides, polyphenolic compounds, and various secondary metabolites, many of which have significant importance in food, agrochemical, and pharmaceutical industries. While metabolites are one of the indicators of quality of grapevines, the presence of them makes grapevine one of the most difficult plants to extract DNA from. These metabolites not only affect DNA extraction procedures but also downstream reactions such as restriction digestion and PCR. Development of new genotyping techniques based on sequencing such as genotyping by sequencing (GBS) requires high-quality DNA for digestion and sequencing. To date, several protocols have been developed for DNA extraction from grapevine. In this study, three different protocols with modifications were compared for DNA extraction performance from grapevine leaves from four different cultivars. Efficiencies of these methods were determined by extracted DNA’s quantity and quality. To confirm the suitability for GBS, extracted DNA was digested with restriction enzymes. Although all protocols were based on the traditional CTAB method, they resulted in different DNA yield and restriction digestion efficiency. The modified protocol including PVP-40 and ß-mercaptoethanol was found to be the most efficient method to obtain high quality and quantity grapevine DNA that is amenable to restriction digestion.

The therapeutic effect of PEI-Fe3O4/pYr-ads-8-5HRE-cfosp-IFNG albumin nanospheres combined with magnetic fluid hyperthermia on hepatoma.

Hepatocellular carcinoma (HCC) is one of the most prevalent and deadly malignant tumors with serious clinical and socioeconomic consequences. Although gene therapy holds great promise in the treatment of hepatoma, its clinical applications are hindered by uncontrolled gene transmission and transcription. The pY-ads-8-5HRE-cfosp-IFNG plasmid was constructed and identified by double enzyme digestion and gene sequencing. The expression of pYr-ads-8-5HRE-cfosp-IFNG in HepG2 cells was detected by quantitative PCR. PEI-Fe3O4/pYr-ads-8-5HRE-cfosp-IFNG albumin nanospheres were prepared and characterized. In vitro heating test of magnetic albumin nanospheres in an alternating magnetic field (AMF) was carried out. The therapeutic effect of PEI-Fe3O4/pYr-ads-8-5HRE-cfosp-IFNG albumin nanospheres on hepatocellular carcinoma was investigated by cell and animal experiments. After treatment, mice blood was collected for clinical biochemical analysis and histopathological evaluation of major organs was performed to assess potential adverse effects of treatment. Double enzyme digestion and gene sequencing showed that the pY-ads-8-5HRE-cfosp-IFNG plasmid was constructed successfully. QPCR results showed that the IFNγ transcript level in the PEI-Fe3O4/pYr-ads-8-5HRE-cfosp-IFNG group was higher than that in the PEI-Fe3O4/pYr-ads-8-cfosp-IFNG group after being treated with hypoxia (P<0.05). TEM revealed that the self-prepared PEI-Fe3O4/pYr-ads-8-5HRE-cfosp-IFNG albumin nanospheres exhibit an approximately spherical or elliptical shape. The hydrodynamic size of the albumin nanospheres was 139.7 nm. The maximum temperature of 0.25 mg/mL solution is stable at about 44°C, which is suitable for tumor thermal therapy without damaging normal tissues. The relative cell inhibition rate of the radiation-gene therapy and MFH combination group was higher than that of other control groups in CCK8 experiment. (P<0.05) Flow cytometry showed that the apoptosis rate and necrosis rate of the combined treatment group were 42.32% and 35.73%, respectively, higher than those of the other groups. (P<0.05) In animal experiments, the mass and volume inhibition rates of the combined treatment group were 66.67% and 72.53%, respectively, higher than those of other control groups. (P<0.05) Clinical biochemical analysis and histopathological evaluation showed no abnormality. The results indicated the successful construction of the radiation-induced plasmid and demonstrated that the hypoxia enhancer could augment the expression of INFγ in a hypoxia environment. Gene therapy combined with magnetic fluid hyperthermia (MFH) has exhibited excellent outcomes in both cell and animal studies. Our experiments demonstrated that the PEI-Fe3O4/pYr-ads-8-5HRE-cfosp-IFNG albumin nanospheres system is a comprehensive treatment method for hepatoma, which can effectively combine immune genre therapy with hyperthermia.

A highly sensitive signal-on biosensor based on restriction enzyme-mediated molecular switch for detection of TET1

Ten-eleven translocation 1 (TET1) is a member of the TET enzyme family of dioxygenases, which plays an important role in active DNA demethylation. Therefore, the sensitive TET1 detection could help us better understand DNA methylation-demethylation in epigenetics. Here we report a detection method that consists of electrode fabrication, TET1 modification, DNA digestion, signal-on oxidoreduction, and current peak monitoring. An exquisitely designed 5′end-G-rich oligodeoxynucleotide was synthesized bearing a methylated cytosine (5-mC), which formed into hairpin dsDNA with the MspI recognition sequence (CmCGG/GGCC). Then hairpin dsDNA was fabricated onto gold nanoparticles modified glassy carbon electrode (DNA/AuNPs/GCE) via Au-S bond. The combination uses of restriction enzyme MspI and hemin converted fabricated-dsDNA into peroxidase-mimicking DNAzyme, thereby promoting the reduction of H2O2 with a current peak. However, the current peak was extremely decreased once TET1 and T4 β-GT were used in advance. We confirmed a delicately linear relationship matching between the current difference and TET1 activity from 0.7 to 10.5 ng μL−1 with a detection limit of 0.027 ng μL−1, which outcompeted the former methods at least one order of magnitudes. The TET1 activity evaluation in the existence of Bobcat339 was also tested as the proof of concept of inhibitors screening. Our strategy provides a novel, label-free, and sensitive electrochemical approach that enables us to complete both TET1 activity evaluation and potential TET1 inhibitors screening.

Abstract 48: Increasing the racial diversity of lung adenocarcinoma cell lines

Abstract Lung cancer is a prominent source of cancer health disparities. Black males tend to smoke less, but have a 37% higher chance of developing lung cancer. The three most common histological subtypes of lung cancer are lung adenocarcinoma (LUAD), squamous cell lung cancer (LUSQ), and small cell lung cancer (SCLC). To study lung cancer development, progression, and treatment, numerous tools are needed, including lung cancer cell lines. We undertook a literature and web-based investigation of lung cancer cell line availability from different racial/ethnic groups. We identified 188 cell lines from White individuals, 374 cell lines from Asian subjects, but only 26 cell lines from Blacks and none from Hispanic patients. We conclude that Black and Hispanic lung cancer cell lines are strongly underrepresented. Here we focus on LUAD cell lines since LUAD is the most common histological subtype in all racial/ethnic groups. While 67 White LUAD cell lines have been established, we only identified 5 Black LUAD lines. To remedy this lack of Black LUAD cell lines, we are collecting LUAD samples from Black patients to establish new cell lines (with full consent). In addition, we aim to increase the representation of Black cell lines by creating isogenic cell line variants of existing cell lines, like NCI-H23. NCI-H23 was derived from a Black male patient and carries a KRASG12C heterozygous mutation. We are using CRISPR/CAS9-based genome editing to introduce amino acid substitutions, including KRASG12V and KRASG12A. Our gene editing DNA includes a silent restriction site to help identify edited clones. A fluorescently labeled structured tracrRNA is included during the genomic editing process, allowing the electroporated cells to undergo fluorescence-activated cell sorting. We use the polymerase chain reaction to amplify the relevant genomic region, followed by restriction digestion to identify potential clones and sequencing to confirm successful editing. Several clones are currently under evaluation. Once new hetero- and homozygous KRAS mutants have been identified, we will test the efficacy of polyisoprenylated cysteinyl amide inhibitors (PCAIs). PCAIs have been shown to disrupt the RAS-mediated signaling pathway in racially/ethnically diverse cell lines (manuscript submitted). By generating newly-established and isogenic Black LUAD cell lines, we intend to ensure that potential therapeutics are tested on cell lines with the appropriate racial/ethnic genetic background. In the future, we hope to establish similar cell lines from Hispanic LUAD subjects. Citation Format: Christopher Leon, Matthew A. Gladstone, Chunli Yan, Diana I. Romero, Yong Huang, Nazarius S. Lamango, Ite A. Offringa. Increasing the racial diversity of lung adenocarcinoma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 48.

Correlation of polymorphism in Toll-Like Receptor (TLR1 and TLR2) genes with susceptibility of pulmonary tuberculosis in Doda region of India

BackgroundPulmonary tuberculosis has emerged as one of the leading causes of deaths across the globe. The prevalence of Mycobacterium tuberculosis has also shown an increasing trend over the time which may be attributed to the increase in multidrug resistant strains and HIV epidemics. There are several factors like change in the gene structure and cellular activities of the host and the bacterium which may have changed the host response towards tuberculosis. Additionally, the recent reports have suggested that Toll-Like Receptors (TLRs) play an important role in the activation of immune responses against various pathogens. Therefore, this study has been designed to investigate the possible correlation of TLR gene polymorphism and prevalence of pulmonary tuberculosis. MethodThis study investigates 300 samples collected from patients with pulmonary tuberculosis (150) and healthy controls (150) from the Doda region of Jammu, India. For analysis purpose, DNA from the collected samples were isolated and subjected to sequence specific PCR amplification of TLR-1 and TLR-2 genes. The amplicons of TLR-1 and TLR-2 were further digested with restriction enzymes PvuII and Xbal, respectively, and visualized on agarose gel, subsequently. ResultThe results suggest that frequency of TLR2 gene polymorphism (73.9%) is high in the patients below the age of 50 years, whereas, frequency of TLR-1 gene polymorphism is high (71%) in the patients above 50 years of age (p = 0.005). Further, the restriction digestion analysis of TLR1 genes has shown that nearly 78% of the confirmed normal cases exhibit homozygous normal conditions followed by 12% cases with heterozygous conditions and 10% cases of homozygous mutants. Similarly for TLR2 genes, nearly 78.6% of the confirmed normal cases have shown homozygous normal conditions followed heterozygous conditions (12.6%) and homozygous mutants (8.6%). ConclusionThis study establishes a preliminary correlation between TLR polymorphism and tuberculosis.

The Use of Cell-penetrating Peptide for Delivery of Recombinant Transcription Factor DNA into Primary Human Fibroblast

Background: Reprogrammed cell therapy has not been applied for clinical purposes due to the malignancy issue. The aim of this study was to design the recombinant vector of the transcription factors and analyze the effectiveness of cell-penetrating peptide delivering system for human primary fibroblast transfection to avoid the malignancy issue.Materials and methods: The constructions of CCAT/enhancer binding protein alpha (CEBPA), hepatocyte nuclear factor 4 alpha (HNF4A), nuclear receptor subfamily 1 group I member 2 (NR1I2) were confirmed with DNA digestion and sequencing. Breast reduction (BRED) and palate (PAL) tissue were used as human primary fibroblast sources. The transcription factors were delivered into BRED and PAL with recombination of avian leukosis sarcoma virus (ALSV), human immunodeficiency virus (HIV) matrix, and regulator of expression of virion proteins (Rev) (ALMR), tagged with enhanced green fluorescence protein (eGFP). Post-transfection cells were then cultivated with optimized medium. Gene expression was measured with quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR).Results: Gene expression levels of CEBPA, HNF4A, NR1I2, glutamate-ammonia ligase (GLUL), albumin (ALB), and cytochrome P450 (CYP) were increased. Transfection with ALMR, which were more efficient in BRED than PAL fibroblasts may have the advantage in autologous cell therapy for elderly patients.Conclusion: Transfection of transcription factors to human primary fibroblast may be performed by using constructions of plasmid as designed in this study.Keywords: recombinant plasmid, hepatocyte-like cells, primary fibroblasts, recombinant peptide, cell reprogramming, autologous cells therapy

A quick and cost-effective method for DNA-free total RNA isolation using magnetic silica beads

Current RNA purification methods widely use silica-based columns that allow quick isolation of high-quality and good quantities of RNA. However, the major limitations include high cost, the requirement of different kits for small RNA isolation, genomic DNA contamination, and not being flexible. Here, we used the in-house RNA isolation reagent (RIR) for cell lysis, followed by RNA precipitation using isopropanol. RNA isolated using the in-house RIR resulted in a similar quantity and quality compared to the commercial TRIzol. Furthermore, the commercial RNA isolation kits with silica-based columns recommend genomic DNA digestion during or after RNA purification, adding time and cost to RNA purification. Here, we developed an optimized in-house protocol for isolating high-quality RNA free of genomic DNA contamination using magnetic silica beads without needing DNase digestion. Additionally, our method purifies total RNA along with the small RNA fraction, including miRNAs, which usually require a separate kit for extraction. Additionally, the RNA prepared with our method was equally suitable for mRNA and miRNA expression analysis using RT-qPCR. Together, the in-house method of RNA isolation using the magnetic silica beads has exhibited comparable or better total RNA extraction compared to commercial kits at a fraction of the cost and across various cells and tissues.

Effective use of limestones to reconstruct seawater Li isotope compositions - A community standard proposal

Past variations in lithium concentration and isotope ratio of seawater recorded in marine carbonates have shown great potential in tracing carbon cycles during critical climate transitions, especially coupled with the radiogenic strontium isotope system. However, acquiring reliable seawater Li isotope compositions from marine carbonates can be technically challenging because non‑carbonate phases co-existed in ancient marine carbonates can easily contaminate carbonate Li signal during inappropriate sample dissolution. Each contaminant phase, including Mn oxides, clays, and silicate minerals, can have different Li isotope compositions than that preserved in the carbonate lattice. More importantly, a significant portion of Li is adsorbed on the Mn oxides and clay minerals. This fraction is considered exchangeable and can be liberated using weak acid without destruction of mineral structure. Cleaning procedures and partial digestion have been applied to remove the exchangeable Li fraction in contaminative phases and selectively dissolve the carbonates. However, previous methods cannot be directly applied to ancient marine carbonates of various carbonate purity, which lack systematic testing on the threshold of sample purity to maintain the efficiency of the precleaning and partial digestion. Here we report an efficient sequential leaching protocol to extract primary seawater Li and Sr isotope compositions from bulk marine carbonates. The procedure consists of pre-cleaning steps using 1 M ammonium acetate and 1 M ammonium carbonate to completely remove the exchangeable fraction, followed by partial digestion using 0.3 M acetic acid to selectively target carbonate-bounded Li. We validate this method by mixing carbonate and contaminant endmembers in different weight percentages. We found this protocol works effectively for carbonates of >80% purity. Additionally, we demonstrate filtration is necessary when separating leachates from the solids to quantitatively avoid contaminations from non‑carbonate phases. We also suggest NIST-SRM-1d to be a cross-lab carbonate standard for Li isotope analyses and report its long-term external precision (two years). To sum, we propose an effective community standard procedure to reconstruct δ7Lisw records using ancient bulk carbonate samples.

Cloning, expression, purification, and immunoblotting analysis of recombinant type III fibronectin domains of human oncostatin M receptor.

The human oncostatin M receptor subunit , commonly known as the oncostatin M receptor (OSMR), is a cell surface protein and belongs to the family of type I cytokine receptors. It is highly expressed in several cancers and is a potential therapeutic target. Structurally, OSMR consists of three major domains: the extracellular, transmembrane, and cytoplasmic domains. The extracellular domain further comprises four Type III fibronectin subdomains. The functional relevance of these type III fibronectin domains is not known yet, and it is of great interest to us to understand their role in OSMR-mediated interactions with other oncogenic proteins. The four type III fibronectin domains of hOSMR were amplified by PCR using the pUNO1-hOSMR construct as a template. The molecular size of the amplified products was confirmed by agarose gel electrophoresis. The amplicons were then cloned into a pGEX4T3 vector containing GST as an N-terminal tag. Positive clones with domain inserts were identified by restriction digestion and overexpressed in E. coli Rosetta (DE3) cells. The optimum conditions for overexpression were found to be 1 mM IPTG and an incubation temperature of 37°C. The overexpression of the fibronectin domains was confirmed by SDS-PAGE, and they are affinity purified by using glutathione agarose beads in three repetitive steps. The purity of the isolated domains analyzed by SDS-PAGE and western blotting showed that they were exactly at their corresponding molecular weights as a single distinct band. In this study, we have successfully cloned, expressed, and purified four Type III fibronectin subdomains of hOSMR.