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.

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.

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.

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.

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.

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.

A brief review of novel nucleic acid test biosensors and their application prospects for salmonids viral diseases detection

Viral diseases represent one of the major threats for salmonids aquaculture.Early detection and identification of viral pathogens is the main prerequisite prior to undertaking effective prevention and control measures.Rapid,sensitive,efficient and portable detection method is highly essential for fish viral diseases detection.Biosensor strategies are highly prevalent and fulfill the expanding demands of on-site detection with fast response,cost-effectiveness,high sensitivity,and selectivity.With the development of material science,the nucleic acid biosensors fabricated by semiconductor have shown great potential in rapid and early detection or screening for diseases at salmonids fisheries.This paper reviews the current detection development of salmonids viral diseases.The present limitations and challenges of salmonids virus diseases surveillance and early detection are presented.Novel nucleic acid semiconductor biosensors are briefly reviewed.The perspective and potential application of biosensors in the on-site detection of salmonids diseases are discussed.

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.

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.

Non-exposed endoscopic wall-inversion surgery with one-step nucleic acid amplification for early gastrointestinal tumors:Personal experience and literature review

BACKGROUND Laparoscopic and endoscopic cooperative surgery is a safe,organ-sparing surgery that achieves full-thickness resection with adequate margins.Recent studies have demonstrated the safety and efficacy of these procedures.However,these techniques are limited by the exposure of the tumor and mucosa to the peritoneal cavity,which could lead to viable cancer cell seeding and the spillage of gastric juice or enteric liquids into the peritoneal cavity.Non-exposed endoscopic wallinversion surgery(NEWS)is highly accurate in determining the resection margins to prevent intraperitoneal contamination because the tumor is inverted into the visceral lumen instead of the peritoneal cavity.Accurate intraoperative assessment of the nodal status could allow stratification of the extent of resection.One-step nucleic acid amplification(OSNA)can provide a rapid method of evaluating nodal tissue,whilst nearinfrared laparoscopy together with indocyanine green can identify relevant nodal tissue intraoperatively.AIM To determine the safety and feasibility of NEWS in early gastric and colon cancers and of adding rapid intraoperative lymph node(LN)assessment with OSNA.METHODS The patient-based experiential portion of our investigations was conducted at the General and Oncological Surgery Unit of the St.Giuseppe Moscati Hospital(Avellino,Italy).Patients with early-stage gastric or colon cancer(diagnosed via endoscopy,endoscopic ultrasound,and computed tomography)were included.All lesions were treated by NEWS procedure with intraoperative OSNA assay between January 2022 and October 2022.LNs were examined intraoperatively with OSNA and postoperatively with conventional histology.We analyzed patient demographics,lesion features,histopathological diagnoses,R0 resection(negative margins)status,adverse events,and follow-up results.Data were collected prospectively and analyzed retrospectively.RESULTS A total of 10 patients(5 males and 5 females)with an average age of 70.4±4.5 years(range:62-78 years)were enrolled in this study.Five patients were diagnosed with gastric cancer.The remaining 5 patients were diagnosed with early-stage colon cancer.The mean tumor diameter was 23.8±11.6 mm(range:15-36 mm).The NEWS procedure was successful in all cases.The mean procedure time was 111.5±10.7 min(range:80-145 min).The OSNA assay revealed no LN metastases in any patients.Histologically complete resection(R0)was achieved in 9 patients(90.0%).There was no recurrence during the follow-up period.CONCLUSION NEWS combined with sentinel LN biopsy and OSNA assay is an effective and safe technique for the removal of selected early gastric and colon cancers in which it is not possible to adopt conventional endoscopic resection techniques.This procedure allows clinicians to acquire additional information on the LN status intraoperatively.

Certain sulfonylurea drugs increase serum free fatty acid in diabetic patients:A systematic review and meta-analysis

BACKGROUND Sulfonylurea(SU)is a commonly used antidiabetic drugs effective for type 2diabetes mellitus.Previous studies have reported that the SU treatment could alter the serum free fatty acid(FFA)concentration in diabetic patients;however,their exact effects remain unknown.AIM To assess the impact of SU on the FFA level in diabetic patients.METHODS A systematic literature search was conducted by consulting the PubMed,EMBASE,Cochrane Library,Reference Citation Analysis(https://www.referencecitationanalysis.com/),and Web of Science databases from January 1,1991 to July 30,2021.Either a fixed-effects model or random-effects model was applied to study the association between SU treatment and FFA concentration according to the heterogeneity test.Two investigators independently performed data extraction.The mean difference(MD)and corresponding 95%confidence interval(CI)were used to measure effect size.R3.5.1 software was utilized for conducting statistical analyses.RESULTS A total of 13 studies with 2273 individuals were selected.Results indicated that FFA concentration increased slightly after treatment with SU(MD=0.08,95%CI:0.03-0.12,P<0.01).In addition,we found that SU treatment combined with other antidiabetics could also increase the concentration of serum FFA(MD=0.14,95%CI:0.01-0.28,P<0.01).Regarding the type of SU,there was no significant difference in FFA concentration with glimepiride or glibenclamide.FFA concentration was higher at≥12 wk(MD=0.09,95%CI:0.04-0.13)but not at<12 wk(MD=0.01,95%CI:-0.07-0.09).CONCLUSION SU treatment could increase the serum FFA concentration in diabetic patients.The fundamental underlying mechanism still needs further investigation.

Spherical nucleic acids for biomedical applications

Spherical nucleic acids(SNAs)are a 3D spherical nanostructure composed of highly oriented,dense layers of oligonucleotides conjugated to a hollow or solid core.This structure allows SNAs to show resistance to nuclease degradation,enter into nearly all cells without transfection agents and enable precise interactions with target molecules.Based on superior biological properties,SNAs can be tailored for diverse biological applications,rendering them a flexible and biosafe tool for biological applications as well as an enabling platform for therapy.In this review,we mainly discuss the structure and conjugation mode of SNAs and focus on recent advances in their applications,such as biomedical detection,imaging,and drug delivery.Finally,the remaining challenges and future directions of SNAs are also discussed and proposed.

Current molecular methods for the detection of hepatitis C virus in high risk group population:A systematic review

Hepatitis C virus(HCV) is an emerging infection worldwide and the numbers of persons infected are increasing every year. Poor blood transfusion methods along with unsafe injection practices are potential sources for the rapid spread of infection. Early detection of HCV is the need of the hour especially in high riskgroup population as these individuals are severely immunocompromised. Enzyme Immunoassays are the most common detection techniques but they provide no evidence of active viremia or identification of infected individuals in the antibody-negative phase and their efficacy is limited in individuals within high risk group population. Molecular virological techniques have an important role in detecting active infection with utmost specificity and sensitivity. Technologies for assessment of HCV antibody and RNA levels have improved remarkably, as well as our understanding of how to best use these tests in patient management. This review aims to give an overview of the different serological and molecular methods employed in detecting HCV infection used nowadays. Additionally, the review gives an insight in the new molecular techniques that are being developed to improve the detection techniques particularly in High Risk Group population who are severely immunocompromised.

Therapeutic nucleic acids in regenerative medicine and tissue repair

In the vanguard of biomedical innovation,regenerative medicine emerges as a transformative paradigm,concentrating its efforts on rectifying tissue deficits and functional aberrations in patients through the strategic augmentation of endogenous cellular processes.Central to this approach is nucleic acid therapy,which offers a novel pathway for tissue regeneration by modulating key signaling pathways.As one of the most effective ways to regulate cell function,nucleic acids are crucial for various tissue regeneration;however,their in vivo therapeutic applications face substantial challenges,including nuclease degradation,cell membrane impermeability,and targeted intracellular transport.Biomaterial-based gene delivery systems offer a solution for stable and localized drug delivery by enabling the controlled overexpression of therapeutic nucleic acids,producing functional regulatory agents.This review presents examples of nucleic acid applications in regenerative medicine,highlighting the synergy between nucleic acids and biomaterial technologies.It underlines the importance of nucleic acid delivery techniques,the choice of therapeutic nucleic acids,and biomaterials for advancing tissue repair.The latest developments in designing nucleic acid biomaterial-based delivery vehicles are explored,the mechanism of nucleic acids in tissue regeneration is elucidated,and their limitations are discussed while considering future directions for the clinical translation of nucleic acid-based therapeutics.

A novel mesenchymal stem cell-targeting dual-miRNA delivery system based on aptamer-functionalized tetrahedral framework nucleic acids:Application to endogenous regeneration of articular cartilage

Articular cartilage injury(ACI)remains one of the key challenges in regenerative medicine,as current treatment strategies do not result in ideal regeneration of hyaline-like cartilage.Enhancing endogenous repair via micro-RNAs(miRNAs)shows promise as a regenerative therapy.miRNA-140 and miRNA-455 are two key and promising candidates for regulating the chondrogenic differentiation of mesenchymal stem cells(MSCs).In this study,we innovatively synthesized a multifunctional tetrahedral framework in which a nucleic acid(tFNA)-based targeting miRNA codelivery system,named A-T-M,was used.With tFNAs as vehicles,miR-140 and miR-455 were connected to and modified on tFNAs,while Apt19S(a DNA aptamer targeting MSCs)was directly integrated into the nanocomplex.The relevant results showed that A-T-M efficiently delivered miR-140 and miR-455 into MSCs and subsequently regulated MSC chondrogenic differentiation through corresponding mechanisms.Interestingly,a synergistic effect between miR-140 and miR-455 was revealed.Furthermore,A-T-M successfully enhanced the endogenous repair capacity of articular cartilage in vivo and effectively inhibited hypertrophic chondrocyte formation.A-T-M provides a new perspective and strategy for the regeneration of articular cartilage,showing strong clinical application value in the future treatment of ACI.

Tetrahedral framework nucleic acids promote the proliferation and differentiation potential of diabetic bone marrow mesenchymal stem cell

Diabetes mellitus considerably affects bone marrow mesenchymal stem cells(BMSCs),for example,by inhibiting their proliferation and differentiation potential,which enhances the difficulty in endogenous bone regeneration.Hence,effective strategies for enhancing the functions of BMSCs in diabetes have farreaching consequences for bone healing and regeneration in diabetes patients.Tetrahedral framework nucleic acids(tFNAs)are nucleic acid nanomaterials that can autonomously enter cells and regulate their behaviors.In this study,we evaluated the effects of tFNAs on BMSCs from diabetic rats.We found that tFNAs could promote the proliferation,migration,and osteogenic differentiation of BMSCs from rats with type 2 diabetes mellitus,and inhibited cell senescence and apoptosis.Furthermore,tFNAs effectively scavenged the accumulated reactive oxygen species and activated the suppressed protein kinase B(Akt)signaling pathway.Overall,we show that tFNAs can recover the proliferation and osteogenic potential of diabetic BMSCs by alleviating oxidative stress and activating Akt signaling.The study provides a strategy for endogenous bone regeneration in diabetes and also paves the way for exploiting DNA-based nanomaterials in regenerative medicine.