Liquid biopsy in patients with hepatocellular carcinoma: Circulating tumor cells and cell-free nucleic acids

Hepatocellular carcinoma(HCC), with its high incidence and mortality rate, is one of the most common malignant tumors. Despite recent development of a diagnostic and treatment method, the prognosis of HCC remains poor. Therefore, to provide optimal treatment for each patient with HCC, more precise and effective biomarkers are urgently needed which could facilitate a more detailed individualized decision-making during HCC treatment, including the following; risk assessment, early cancer detection, prediction of treatment or prognostic outcome. In the blood of cancer patients, accumulating evidence about circulating tumor cells and cell-free nucleic acids has suggested their potent clinical utilities as novel biomarker. This concept, so-called "liquid biopsy" is widely known as an alternative approach to cancer tissue biopsy. This method might facilitate a more sensitive diagnosis and better decision-making by obtaining genetic and epigenetic aberrations that are closely associated with cancer initiation and progression. In this article, we review recent developments based on the available literature on both circulating tumor cells and cell-free nucleic acids in cancer patients, especially focusing on Hepatocellular carcinoma.

Liquid biopsy of gastric cancer patients:Circulating tumor cells and cell-free nucleic acids

To improve the clinical outcomes of cancer patients, early detection and accurate monitoring of diseases are necessary. Numerous genetic and epigenetic alterations contribute to oncogenesis and cancer progression, and analyses of these changes have been increasingly utilized for diagnostic, prognostic and therapeutic purposes in malignant diseases including gastric cancer (GC). Surgical and/or biopsy specimens are generally used to understand the tumor-associated alterations; however, those approaches cannot always be performed because of their invasive characteristics and may fail to reflect current tumor dynamics and drug sensitivities, which may change during the therapeutic process. Therefore, the importance of developing a non-invasive biomarker with the ability to monitor real-time tumor dynamics should be emphasized. This concept, so called “liquid biopsy”, would provide an ideal therapeutic strategy for an individual cancer patient and would facilitate the development of “tailor-made” cancer management programs. In the blood of cancer patients, the presence and potent utilities of circulating tumor cells (CTCs) and cell-free nucleic acids (cfNAs) such as DNA, mRNA and microRNA have been recognized, and their clinical relevance is attracting considerable attention. In this review, we discuss recent developments in this research field as well as the relevance and future perspectives of CTCs and cfNAs in cancer patients, especially focusing on GC.

Liquid biopsy in patients with pancreatic cancer: Circulating tumor cells and cell-free nucleic acids

Despite recent advances in surgical techniques and perioperative management, the prognosis of pancreatic cancer(PCa) remains extremely poor. To provide optimal treatment for each patient with Pca, superior biomarkers are urgently needed in all phases of management from early detection to staging, treatment monitoring, and prognosis. In the blood of patients with cancer, circulating tumor cells(CTCs) and cell-free nucleic acids(cf NAs), such as DNA, m RNA, and noncoding RNA have been recognized. In the recent years, their presence in the blood has encouraged researchers to investigate their potential use as novel blood biomarkers, and numerous studies have demonstrated their potential clinical utility as a biomarker for certain types of cancer. This concept, called "liquid biopsy" has been focused on as a less invasive, alternative approach to cancer tissue biopsy for obtaining genetic and epigenetic aberrations that contribute to oncogenesis and cancer progression. In this article, we review the available literature on CTCs and cfN As in patients with cancer, particularly focusing on PCa, and discuss future perspectives in this field.

Circulating cell-free nucleic acids as prognostic and therapy predictive tools for metastatic castrate-resistant prostate cancer

Metastatic castrate-resistant prostate cancer remains a disease hard to cure,and for this reason predictive tools to monitor disease progression and therapy response are an urgent need.In this respect,liquid biopsy on circulating cell-free nucleic acids represents an interesting strategy based on robust data.The low invasiveness and the possibility to target circulating cell-free tumor deoxyribonucleic acid underline the high specificity,sensitivity and clinical usability of the technique.Moreover,it has been observed that the cell-free tumor deoxyribonucleic acid of metastatic castrate-resistant prostate cancer patients can be representative of the tumor heterogeneity.Cell-free tumor deoxyribonucleic acids express the same behaviors as mutations:Variation in gene copy number or the methylation rate of the tumor tissue.Recently,circulating cell-free ribonucleic acid molecules have emerged as interesting markers to stratify the disease.Due to high-throughput technologies,liquid biopsy on circulating cell-free nucleic acids will soon be utilized in the clinical management of metastatic castrate-resistant prostate cancer patients.

Tb^(3+)-nucleic acid probe-based label-free and rapid detection of mercury pollution in food

Mercury is a threatening pollutant in food,herein,we developed a Tb^(3+)-nucleic acid probe-based label-free assay for mix-and-read,rapid detection of mercury pollution.The assay utilized the feature of light-up fluorescence of terbium ions(Tb^(3+))via binding with single-strand DNA.Mercury ion,Hg^(2+)induced thymine(T)-rich DNA strand to form a double-strand structure(T-Hg^(2+)-T),thus leading to fluorescence reduction.Based on the principle,Hg^(2+)can be quantified based on the fluorescence of Tb^(3+),the limit of detection was 0.0689μmol/L and the linear range was 0.1-6.0μmol/L.Due to the specificity of T-Hg^(2+)-T artificial base pair,the assay could distinguish Hg^(2+)from other metal ions.The recovery rate was ranged in 98.71%-101.34%for detecting mercury pollution in three food samples.The assay is low-cost,separation-free and mix-to-read,thus was a competitive tool for detection of mercury pollution to ensure food safety.

Design,delivery and efficacy testing of therapeutic nucleic acids used to inhibit hepatitis C virus gene expression in vitro and in vivo

Despite major achievements in the treatment ofchronic hepatitis C with the combination ofinterferons and the nucleoside analog ribavirin themajority of patients with chronic hepatitis C virus(HCV) infection cannot be treated effectively.Toimprove this response rate we used antisensetechnologies to inhibit HCV translation as possibleadditional option for experimental treatment.Antisense oligodeoxynucleotides(ODN) are

Mechanism of exogenous nucleic acids and their precursors improving the repair of intestinal epithelium after 7-irradiation in mice

AIM To clone expressed genes associated withrepair of irradiation-damaged mice intestinalgland cells treated by small intestinal RNA,andto explore the molecular mechanism ofexogenous nucleic acids improving repair ofintestinal crypt.METHODS The animal mode of test group andcontrol group was established,forty-five micebeing irradiated by γ ray were treated with smallintestinal RNA as test group,forty mice beingirradiated by γ ray were treated withphysiological saline as control group,five micewithout irradiation were used as normal control,their jejunal specimens were collectedrespectively at 6h,12h,24h,4d and 8d afterirradiation.Then by using LD-PCR based onsubtractive hybridization,these gene fragmentsdifferentially expressed between test group andcontrol group were obtained,and then werecloned into T vectors as well as beingsequenced.Obtained sequences were screenedagainst.GeneBank,if being new sequences,they were submitted to GeneBank.RESULTS Ninety clones were associated withrepair of irradiation-damaged intestinal glandcells treated by intestinal RNA.These clonesfrom test group of 6h,12h,24h,4d and 8dwere respectively 18,22,25,13,12.By screening against GeneBank,18 of which werenew sequences,the others were dramaticallysimilar to the known sequences,mainly similarto hsp,Nmi,Dutt1,alkaline phosphatase,homeobox,anti-CEA ScFv antibody,arginine/serine kinase and BMP-4,repA.Eighteen genefragments were new sequences,their acceptnumbers in GeneBank were respectivelyAF240164-AF240181.CONCLUSION Ninety clones were obtained tobe associated with repair of irradiation-damagedmice intestinal gland cells treated by smallintestinal RNA,which may be related toabnormal expression of genes and matchedproteins of hsp,Nmi,Duttl,Na,K-ATPase,alkalineph-osphatase,glkA,single strandedreplicative centromeric gene as well as 18 newsequences.

Application of cationic liposomes for delivery of nucleic acids

Nucleic acid-based bioactive substances have recently emerged as a new class of nextgeneration therapeutics, but their development has been limited by their relatively weakdelivery into target cells. Cationic liposomes have been studied as a means to enhance thestability of nucleic acid therapeutics in the bloodstream and improve their cellular delivery.As nucleic acid therapeutics, siRNA and plasmid DNA have been extensively tested fordelivery using cationic liposomes. This review discusses recent progress in the applicationof cationic liposomes for the delivery of nucleic acid therapeutics.

Recent advances in living cell nucleic acid probes based on nanomaterials for early cancer diagnosis

The early diagnosis of cancer is vital for effective treatment and improved prognosis. Tumor biomarkers, which can be used for the early diagnosis, treatment, and prognostic evaluation of cancer, have emerged as a topic of intense research interest in recent years. Nucleic acid, as a type of tumor biomarker, contains vital genetic information, which is of great significance for the occurrence and development of cancer. Currently, living cell nucleic acid probes, which enable the in situ imaging and dynamic monitoring of nucleic acids, have become a rapidly developing field. This review focuses on living cell nucleic acid probes that can be used for the early diagnosis of tumors. We describe the fundamental design of the probe in terms of three units and focus on the roles of different nanomaterials in probe delivery.

Development of an Integrated Disposable Device for SARSCoV-2 Nucleic Acid Extraction and Detection

Objective To develop a highly sensitive and rapid nucleic acid detection method for the severe acute respiratory syndrome coronavirus 2(SARS-CoV-2).Methods We designed,developed,and manufactured an integrated disposable device for SARS-CoV-2 nucleic acid extraction and detection.The precision of the liquid transfer and temperature control was tested.A comparison between our device and a commercial kit for SARS-Cov-2 nucleic acid extraction was performed using real-time fluorescence reverse transcription polymerase chain reaction(RT-PCR).The entire process,from SARS-CoV-2 nucleic acid extraction to amplification,was evaluated.Results The precision of the syringe transfer volume was 19.2±1.9μL(set value was 20),32.2±1.6(set value was 30),and 57.2±3.5(set value was 60).Temperature control in the amplification tube was measured at 60.0±0.0℃(set value was 60)and 95.1±0.2℃(set value was 95)respectively.SARS-Cov-2 nucleic acid extraction yield through the device was 7.10×10^(6) copies/mL,while a commercial kit yielded 2.98×10^(6) copies/mL.The mean time to complete the entire assay,from SARS-CoV-2 nucleic acid extraction to amplification detection,was 36 min and 45 s.The detection limit for SARS-CoV-2 nucleic acid was 250 copies/mL.Conclusion The integrated disposable devices may be used for SARS-CoV-2 Point-of-Care test(POCT).

Study on the correlation between abdominal infection and psychological stress in children based on nucleic acid detection

BACKGROUND Diagnosing and treating abdominal infection in children remains a challenge.Nucleic acid detection,as a rapid and accurate diagnosis tool,has great significance in this field.AIM To investigate the diagnosis and treatment of abdominal infection by nucleic acid detection and its possible correlation with psychological stress in children.METHODS A total of 50 pediatric patients diagnosed with abdominal infections between September 2020 and July 2021 were included in this study.Intra-abdominal pus samples were collected for pathogen culture,drug susceptibility testing,and broad-spectrum bacterial nucleic acid testing.Psychological stress,anxiety,depression,and coping styles were assessed using the coping with a disease(CODI)scale.RESULTS Based on susceptibility testing,a regimen of cefazoxime,piperacillin/tazobactam,and metronidazole or ornidazole achieved 100%effectiveness in treating appendicitis.Psychological assessments revealed a positive correlation between pressure level and both anxiety(r=0.324,P=0.001)and depressive disorders(r=0.325,P<0.001).Acceptance and distancing as coping strategies were negatively correlated with anxiety and depression,while negative emotional responses were strongly associated with increased anxiety(r=0.574,P<0.001)and depression(r=0.511,P=0.001).Coping strategies such as illusion and escape showed no significant correlation with emotional outcomes.CONCLUSION Nucleic acid testing helps in the diagnosis of abdominal infections in children,and also focuses on children's mental health.

Electronic Structures and Spectra of the Bases and Base Pairs of Nucleic Acids

The present paper covers electronic structures and spectra of the bases and the base pairs of nucleic acids calculated by using the INDO/S method. For free bases we give the energy levels of ground states and transition energies of low-lying excited states and discuss the band characters. The results indicate that the calculated spectra are in good agreement with experimental values. On the other hand, our calculations for A-T and G-C pairs are very beneficial to understanding hydrogen bond properties of these pairs.

Spectroscopic Study of the Interactionbetween New Fluorescent Dyes with Nucleic Acids

A series of new dansylamide derivatives have been synthesized and the specific bindingaffinity of such fluorophores to nucleic acids has been investigated by using absorption, circulardichroism (CD), fluorescence and atomic force microscopy (AFM). The results indicate that thepositive charge of the ligand and the stacking between the dansy1 part of the ligand and theDNA base pair may play an important role when binding to polynucleotides.

Effects of Peptide Nucleic Acids against Ki-67 Gene on the Proliferation and Apoptosis of Human Renal Carcinoma Cell Line

To investigate the effects of anti-sense peptide nucleic acids （PNAs） targeting Ki-67 gene on modulation of the proliferation and apoptosis of human renal carcinoma cell lines, human renal carcinoma cell line 786-0 cells were treated with anti-sense PNAs at different concentrations （1.0 μmol/L, 2.0 μmol/L, 10.0 μmol/L）. The Ki-67 expression of 786-0 cells was detected by immunohistochemical technique and Western blot method respectively. The proliferation of 786-0 cells was studied by cell growth curves and ^3H-thymidine incorporation. The apoptosis of 786-0 cells was detected by TUNEL assay. The control groups were treated with anti-sense oligonucleotide （ASODNs） targeting Ki-67 gene. Our results showed that the Ki-67 expression of 786-0 cells treated with anti-sense PNAs （16.9±0.7） was significantly inhibited as compared with that of the control groups （28.6±0.4） （P〈0.01）. The Ki-67 protein rate of 786-0 cells treated with anti-sense PNAs （42.1 ±2.2） was significantly reduced when compared with that of the control groups （83.6± 1.4） （P〈0.01）. Proliferation of 786-0 cells treated with anti-sense PNAs （20.7 ± 1.5） was significantly inhibited as compared with that of the control groups （58.6± 1.4） （P〈0.01）. The apoptosis rate of 786-0 cells treated with anti-sense PNAs （28.7 ± 2.3） was significantly increased higher compared with that of the control groups （13.8 ±1.0） （P〈0.01）. From these finds we are led to conclude that anti-sense PNAs targeting Ki-67 gene have stronger effects on the inhibition of the proliferation and induction of apoptosis of human renal carcinoma cells than ASODNs targeting Ki-67 gene. The strategies using anti-sense PNAs targeting Ki-67 gene may be a promising approach for the treatment of renal cell carcinoma.

Validation of the CHARMM27 force field for nucleic acids using 2D nuclear overhauser effect spectroscopy

Nuclear magnetic resonance spectroscopy offers a powerful method for validation of molecular dynamics simulations as it provides information on the molecular structure and dynamics in solution. We performed 10 ns MD simulations using the CHARMM27 force field of four palindromic oligonucleotides and compared the results with experimental NOESY data using the full relaxation matrix formalism. The correlation coefficients between theoretical and experimental data for the four molecular species under study ranged from 0.82 to 0.98 confirming the high quality of the selected force field and providing a valid basis for the identification of force field imperfections. Hence, we observed an unsatisfactory treatment of deoxyribose conformational equilibrium, which resulted in the overrepresentation of the energetically favorable C3'-endo conformation in the MD trajectory. Our developed approach for force field validation based on NMR NOESY spectral data is applicable to a wide range of molecular systems and appropriate force fields.

Essential Gene(s) Targeted by Peptide Nucleic Acids Kills <i>Mycobacterium smegmatis</i>in Culture and in Infected Macrophages

Background: Antisense peptide nucleic acids (PNAs) exhibit growth inhibitory effects on bacteria by inhibiting the expression of essential genes and could be promising therapeutic agents for treating bacterial infections. A study was carried out to determine the efficacy of several antisense PNAs in inhibiting extracellular and intracellular growth of Mycobacterium smegmatis. Methods: Six PNAs obtained from a commercial supplier were tested to evaluate the inhibitory effect on bacterial growth by inhibiting the expression of the following essential genes: inhA (a fatty acid elongase), rpsL (ribosomal S12 protein), gyrA (DNA gyrase), pncA (pyrazinamidase), polA (DNA polymerase I) and rpoC (RNA polymerase β subunit) of M. smegmatis. Each PNA was tested at 20 μM, 10 μM, 5 μM and 2.5 μM concentrations to determine whether they caused a dose dependent killing of M. smegmatis cultured in Middlebrook 7H9 broth or in a J774A.1 murine macrophage cell line. Results: In Middlebrook broth, the strong growth inhibitory effect against M. smegmatis was observed by PNAs targeting the inhA and rpsL genes at all four concentrations. The PNAs targeting the pncA, polA and rpoC genes were found to exhibit strong growth inhibition against M. smegmatis but only at 20 μM concentration. No growth inhibition of M. smegmatis was seen in pure culture when treated with PNAs targeting gyrA and a mismatch PNA targeting dnaG (DNA primase). All six PNAs showed killing of M. smegmatis in J774A.1 macrophage cell line that were statistically significant (p < 0.05). Conclusion: It may be concluded from this study that PNAs could be potential therapeutics for mycobacterial infections.

Thermodynamics of the long range assembly of safranine T on molecular surfaces of nucleic acids

By making use of the fluorescence quenching properties of safranine T(ST) in its long range assembly on the molecular surfaces of nucleic acids, the assembly number and constant of ST with calf thymus DNA, fish sperm DNA and yeast RNA were determined at 12℃. The corresponding free energy change, enthalpy change and entropy change of the long range assembly were calculated at the same temperature. It was found the assembly complexes are very stable and the assembly is a spontaneous process characterized an entropy increase.

Comparison of Nucleic Acid Content among Non-diapause Pupae, Diapause Pupae and Eclosion-adult from Diapause Pupae of Papilio memnon

[Objective] The aim of this study was to provide basis for deeply understanding the diapause mechanism of Papilio memnon L. [Method] RNA and DNA content of non-diapause pupae, diapause pupae and eclosion-adult from diapause pupae at different development stages were detected by the colorimetry. [Result] RNA content of non-diapause pupae was 4.614 0-7.946 3 μg/mg, while diapause pupae was 4.326 0-5.885 3 μg/mg and eclosion-adult from diapause pupae was 20.779 3 μg/mg at initial stage. DNA content of non-diapause pupae was 0.448 7-0.535 0 μg/mg, while diapause pupae was 0.452 0-0.828 3 μg/mg and eclosion-adult from diapause pupae was 1.727 0 μg/mg at initial stage. [Conclusion] The nucleic acid content and change is related to the development stage.

Real-Time PCR Technique and Its Application in Quantification of Plant Nucleic Acid Molecules

Real-time PCR is a closed DNA amplification system that skillfully integrates biochemical, photoelectric and computer techniques. Fluorescence data acquired once per cycle provides rapid absolute quantification of initial template copy numbers as PCR products are generated. This technique significantly simplifies and accelerates the process of producing reproducible quantification of nucleic acid molecules. It not only is a sensitive, accurate and rapid quantitative method, but it also provides an easier way to calculate the absolute starting copy number of nucleic acid molecules to be tested. Together with molecular bio-techniques, like microarray, real-time PCR will play a very important role in many aspects of molecular life science such as functional gene analysis and disease molecular diagnostics. This review introduces the detailed principles and application of the real-time PCR technique, describes a recently developed system for exact quantification of AUX/IAA genes In Arabidopsis, and discusses the problems with the real-time PCR process.

Recent advances and perspectives of nucleic acid detection for coronavirus

The recent pneumonia outbreak caused by a novel coronavirus(SARS-CoV-2)is posing a great threat to global public health.Therefore,rapid and accurate identification of pathogenic viruses plays a vital role in selecting appropriate treatments,saving people's lives and preventing epidemics.It is important to establish a quick standard diagnostic test for the detection of the infectious disease(COVID-19)to prevent subsequent secondary spread.Polymerase chain reaction(PCR)is regarded as a gold standard test for the molecular diagnosis of viral and bacterial infections with high sensitivity and specificity.Isothermal nucleic acid amplification is considered to be a highly promising candidate method due to its fundamental advantage in quick procedure time at constant temperature without thermocycler opera-tion.A variety of improved or new approaches also have been developed.This review summarizes the currently available detection methods for coronavirus nucleic acid.It is anticipated that this will assist researchers and clinicians in developing better techniques for timely and effective detection of coro-navirus infection.