Preparation of Monoclonal Antibodies Against Prion Proteins With Full-length Hamster PrP

To prepare the PrP specific monoclonal antibodies （mAbs） that can be used for the detection of mammalian prions and study of pathogenesis of prion diseases. Methods Several BALB/c mice were immunized with recombinant hamster prion protein （HaPrP）. Three hybridoma cell lines designated as B7, B9, and B10, secreting monoclonal antibodies against HaPrP, were established by hybridoma technique. The mAbs reactivities were evaluated with ELISA, Western blot, and immunohistochemistry. Results The mAbs produced by these cell lines reacted well with different recombinant hamster PrP proteins. Western blot analyses showed that mAbs B7 and B9 reacted with PrP^Sc from the scrapie-infected animals after proteinase K digestion with three glycosylated forms. The mAbs exhibited cross-reactivity with various PrPc from several other mammalian species, including humans and cattles, lmmunohistochemistry assays confirmed that mAbs B7 and B9 could recognize not only extracellular but also intracellular PrPso. Conclusion The mAbs of prion protein are successfully generated by hybridoma technique and can be applied for the diagnosis of prion associated diseases.

Metabolism of minor isoforms of prion proteins Cytosolic prion protein and transmembrane prion protein

Transmissible spongiform encephalopathy or prion disease is triggered by the conversion from cellular prion protein to pathogenic prion protein. Growing evidence has concentrated on prion protein configuration changes and their correlation with prion disease transmissibility and patho- genicity. In vivo and in vitro studies have shown that several cytosolic forms of prion protein with specific topological structure can destroy intracellular stability and contribute to prion protein pathogenicity. In this study, the latest molecular chaperone system associated with endoplasmic re- ticulum-associated protein degradation, the endoplasmic reticulum resident protein quality-control system and the ubiquitination proteasome system, is outlined. The molecular chaperone system directly correlates with the prion protein degradation pathway. Understanding the molecular mechanisms will help provide a fascinating avenue for further investigations on prion disease treatment and prion protein-induced neurodegenerative diseases.

Topology of Prion Proteins

A conformal structure of a prion protein is thought to cause a prion disease by S.B. Prusiner＇s theory. Knot theory in mathematics is useful in studying a topological difference of topological objects. In this article, concerning this conjecture, a topological model of prion proteins （PrPc, PrPsc） called a prion-tangle is introduced to discuss a question of whether or not the prion proteins are easily entangled by an approach from the mathematical knot theory. It is noted that any prion-string with trivial loop which is a topological model of a prion protein can not be entangled topologically unless a certain restriction such as ＂Rotaxsane Property＂ is imposed on it. Nevertheless, it is shown that any two split prion-tangles can be changed by a one-crossing change into a non-split prion-tangle with the given prion-tangles contained while some attentions are paid to the loop systems. The proof is made by a mathematical argument on knot theory of spatial graphs. This means that the question above is answered affirmatively in this topological model of prion-tangles. Next, a question of what is the simplest topological situation of the non-split prion-tangles is considered. By a mathematical argument, it is determined for every n 〉 1 that the minimal crossing number of n-string non-split prion-tangles is 2n or 2n-2, respectively, according to whether or not the assumption that the loop system is a trivial link is counted.

Dynamic changes and surveillance function of prion protein expression in gastric cancer drug resistance

AIM:To explore the dynamic changes of prion protein (PrPc) in the process of gastric cancer drug resistance and the role of PrPc expression in the prognosis of gastric cancer patients receiving chemotherapy.METHODS:A series of gastric cancer cell lines resistant to different concentrations of adriamycin was established,and the expression of PrPc,Bcl-2 and Bax was detected in these cells.Apoptosis was determined using Annexin V staining.Western blotting and immunohisto-chemistry were performed to detect the expression of PrPc in patients receiving chemotherapy and to explore the role of PrPc expression in predicting the chemosensitivity and the outcome of gastric cancer patients receiving chemotherapy.Follow-up was performed for 2 years.RESULTS:PrPc expression was increased with the increase in drug resistance.Bcl-2,together with PrPc,increased the level of anti-apoptosis of cancer cells.Increased PrPc expression predicted the enhanced level of anti-apoptosis and resistance to anticancer drugs.PrPc expression could be used as a marker for predicting the efficacy of chemotherapy and the prognosis of gastric cancer.Increased PrPc expression predicted both poor chemosensitivity and a low 2-year survival rate.Contrarily,low PrPc expression predicted favorable chemosensitivity and a relatively high 2-year survival rate.CONCLUSION:PrPc expression is associated with histological types and differentiation of gastric cancer cells;The PrPc expression level might be a valuable marker in predicting the efficacy of chemotherapy and the prognosis of gastric cancer patients receiving chemotherapy.

A panel of monoclonal antibodies against the prion protein proves that there is no prion protein in human pancreatic ductal epithelial cells

Prion diseases are a group of neurodegenerative diseases that are fatal. The study of these unique diseases in China is hampered by a lack of resources. Amongst the most important resources for biological study are monoclonal antibodies. Here, we characterize a panel of monoclonal antibodies specific for cellular prion protein by enzyme-linked immunosorbent assay(ELISA), immunofluorescent staining, flow cytometry, and western blotting. We identify several antibodies that can be used for specific applications and we demonstrate that there is no prion protein expression in human pancreatic ductal epithelial cells(HPDC).

Astrocyte in prion disease: a double-edged sword

Prion diseases are infectious protein misfolding disorders of the central nervous system that result from misfolding of the cellular prion protein(PrPC)into the pathologic isoform PrPSc.Pathologic hallmarks of prion disease are depositions of pathological prion protein PrPSc,neuronal loss,spongiform degeneration and astrogliosis in the brain.Prion diseases affect human and animals,there is no effective therapy,and they invariably remain fatal.For a long time,neuronal loss was considered the sole reason for neurodegeneration in prion pathogenesis,and the contribution of non-neuronal cells like microglia and astrocytes was considered less important.Recent evidence suggests that neurodegeneration during prion pathogenesis is a consequence of a complex interplay between neuronal and non-neuronal cells in the brain,but the exact role of these non-neuronal cells during prion pathology is still elusive.Astrocytes are non-neuronal cells that regulate brain homeostasis under physiological conditions.However,astrocytes can deposit PrPSc aggregates and propagate prions in prion-infected brains.Additionally,sub-populations of reactive astrocytes that include neurotrophic and neurotoxic species have been identified,differentially expressed in the brain during prion infection.Revealing the exact role of astrocytes in prion disease is hampered by the lack of in vitro models of prion-infected astrocytes.Recently,we established a murine astrocyte cell line persistently infected with mouse-adapted prions,and showed how such astrocytes differentially process various prion strains.Considering the complexity of the role of astrocytes in prion pathogenesis,we need more in vitro and in vivo models for exploring the contribution of sub-populations of reactive astrocytes,their differential regulation of signaling cascades,and the interaction with neurons and microglia during prion pathogenesis.This will help to establish novel in vivo models and define new therapeutic targets against prion diseases.In this review,we will discuss the complex role of astrocytes in prion disease,the existing experimental resources,the challenges to analyze the contribution of astrocytes in prion disease pathogenesis,and future strategies to improve the understanding of their role in prion disease.

Saccharomyces cerevisiae in neuroscience:how unicellular organism helps to better understand prion protein?

The baker's yeast Saccharomyces(S.)cerevisiae is a single-celled eukaryotic model organism widely used in research on life sciences.Being a unicellular organism,S.cerevisiae has some evident limitations in application to neuroscience.However,yeast prions are extensively studied and they are known to share some hallmarks with mammalian prion protein or other amyloidogenic proteins found in the pathogenesis of Alzheimer's,Parkinson's,or Huntington's diseases.Therefore,the yeast S.cerevisiae has been widely used for basic research on aggregation properties of proteins in cellulo and on their propagation.Recently,a yeast-based study revealed that some regions of mammalian prion protein and amyloidβ1–42 are capable of induction and propagation of yeast prions.It is one of the examples showing that evolutionarily distant organisms share common mechanisms underlying the structural conversion of prion proteins making yeast cells a useful system for studying mammalian prion protein.S.cerevisiae has also been used to design novel screening systems for anti-prion compounds from chemical libraries.Yeastbased assays are cheap in maintenance and safe for the researcher,making them a very good choice to perform preliminary screening before further characterization in systems engaging mammalian cells infected with prions.In this review,not only classical red/white colony assay but also yeast-based screening assays developed during last year are discussed.Computational analysis and research carried out using yeast prions force us to expect that prions are widely present in nature.Indeed,the last few years brought us several examples indicating that the mammalian prion protein is no more peculiar protein–it seems that a better understanding of prion proteins nature-wide may aid us with the treatment of prion diseases and other amyloid-related medical conditions.

Cloning and Expression Analysis of a Prion Protein Encoding Gene in Guppy (Poecilia reticulata)

The full length cDNA of a prion protein (PrP) encoding gene of guppy (Poecilia reticulata) and the corresponding ge-nomic DNA were cloned. The cDNA was 2245 bp in length and contained an open reading frame (ORF) of 1545 bp encoding a pro-tein of 515 amino acids,which held all typical structural characteristics of the functional PrP. The cloned genomic DNA fragment corresponding to the cDNA was 3720 bp in length,consisting of 2 introns and 2 exons. The 5’ untranslated region of cDNA origi-nated from the 2 exons,while the ORF originated from the second exon. Although the gene was transcribed in diverse tissues in-cluding brain,eye,liver,intestine,muscle and tail,its transcript was most abundant in the brain. In addition,the transcription of the gene was enhanced by 5 salinity,implying that it was associated with the response of guppy to saline stress.

Peptide aptamer-mediated modulation of prion protein α-cleavage as treatment strategy for prion and other neurodegenerative diseases

Despite intensive research,most neurodegenerative diseases cannot be cured and for some of them no treatment is available to increase survival or quality of life.Among the latter are prion diseases,fatal and transmissible neurodegenerative diseases of humans and other animals.

Correlation between the Insertion/Deletion Mutations of Prion Protein Gene and BSE Susceptibility and Milk Performance in Dairy Cows

Objective To investigate the 23 bp and 12 bp insertion/deletion(indel)mutations within the bovine prion protein(PRNP)gene in Chinese dairy cows,and to detect the associations of two indel mutations with BSE susceptibility and milk performance.Methods Based on bovine PRNP gene sequence,two pairs of primers for testing the 23 bp and 12 bp indel mutations were designed.The PCR amplification and agarose electrophoresis were carried out to distinguish the different genotypes within the mutations.Moreover,based on previous data from other cattle breeds and present genotypic and allelic frequencies of two indels mutations in this study,the corrections between the two indel mutations and BSE susceptibility were tested,as well as the relationships between the mutations and milk performance traits were analyzed in this study based on the statistical analyses.Results In the analyzed Chinese Holstein population,the frequencies of two"del"alleles in 23 bp and 12 bp indel muations were more frequent.The frequency of haplotype of 23del-12del was higher than those of 23del-12ins and 23ins-12del.From the estimated r2and D’values,two indel polymorphisms were linked strongly in the Holstein population(D’=57.5%,r2=0.257).Compared with the BSE-affected cattle populations from the reported data,the significant differences of genotypic and allelic frequencies were found among present Holstein and some BSE-affected populations(P<0.05 or P<0.01).Similarly,there were significant frequency distribution differences of genotypes and alleles among Chinese Holstein and several previous reported healthy dairy cattle(P<0.05 or P<0.01).Moreover,association of genotype and combined genotypes of two indel polymorphisms with milk performance and resistant mastitis traits were analyzed in Holstein population,but no significant differences were found(P>0.05).Conclusions These observations revealed that the influence of two indel mutations within the bovine PRNP gene on BSE depended on the breed and they did not affect the milk production traits,which layed the foundation for future selection of resistant animals,and for improving health conditions for dairy breeding against BSE in China.

Cloning and expression of prion protein encoding gene of flounder (Paralichthys olivaceus)

The prion protein (PrP) encoding gene of flounder (Paralichthys olivaceus) was cloned. It was not interrupted by an intron. This gene has two promoters in its 5' upstream, indicating that its transcription may be intensive, and should have an important function. It was expressed in all 14 tissues tested, demonstrating that it is a house-keeping gene. Its expression in digestion and reproduction systems implies that the possible prions of fish may transfer horizontally.

Detailing the ultrastructure’s increase of prion protein in pancreatic adenocarcinoma

BACKGROUND Recent evidences have shown a relationship between prion protein(PrPc)expression and pancreatic ductal adenocarcinoma(PDAC).Indeed,PrPc could be one of the markers explaining the aggressiveness of this tumor.However,studies investigating the specific compartmentalization of increased PrPc expression within PDAC cells are lacking,as well as a correlation between ultrastructural evidence,ultrastructural morphometry of PrPc protein and clinical data.These data,as well as the quantitative stoichiometry of this protein detected by immuno-gold,provide a significant advancement in understanding the biology of disease and the outcome of surgical resection.AIM To analyze quantitative stoichiometry and compartmentalization of PrPc in PDAC cells and to correlate its presence with prognostic data METHODS Between June 2018 and December 2020,samples from pancreatic tissues of 45 patients treated with pancreatic resection for a preoperative suspicion of PDAC at our Institution were collected.When the frozen section excluded a PDAC diagnosis,or the nodules were too small for adequate sampling,patients were ruled out from the present study.Western blotting was used to detect,quantify and compare the expression of PrPc in PDAC and control tissues,such as those of non-affected neighboring pancreatic tissue of the same patient.To quantify the increase of PrPc and to detect the subcellular compartmentalization of PrPc within PDAC cells,immuno-gold stoichiometry within specific cell compartments was analyzed with electron microscopy.Finally,an analysis of quantitative PrPc expression according to prognostic data,such as cancer stage,recurrence of the disease at 12 mo after surgery and recurrence during adjuvant chemotherapy was made.RESULTS The amount of PrPc within specimen from 38 out of 45 patients was determined by semi-quantitative analysis by using Western blotting,which indicates that PrPc increases almost three-fold in tumor pancreatic tissue compared with healthy pancreatic regions[242.41±28.36 optical density(OD)vs 95±17.40 OD,P<0.0001].Quantitative morphometry carried out by using immuno-gold detection at transmission electron microscopy confirms an increased PrPc expression in PDAC ductal cells of all patients and allows to detect a specific compartmentalization of PrPc within tumor cells.In particular,the number of immuno-gold particles of PrPc was significantly higher in PDAC cells respect to controls,when considering the whole cell(19.8±0.79 particles vs 9.44±0.45,P<0.0001).Remarkably,considering PDAC cells,the increase of PrPc was higher in the nucleus than cytosol of tumor cells,which indicates a shift in PrPc compartmentalization within tumor cells.In fact,the increase of immuno-gold within nuclear compartment exceeds at large the augment of PrPc which was detected in the cytosol(nucleus:12.88±0.59 particles vs 5.12±0.32,P<0.0001;cytosol:7.74.±0.44 particles vs 4.3±0.24,P<0.0001).RESULTS In order to analyze the prognostic impact of PrPc,we found a correlation between PrPc expression and cancer stage according to pathology results,with a significantly higher expression of PrPc for advanced stages.Moreover,24 patients with a mean follow-up of 16.8 mo were considered.Immuno-blot analysis revealed a significantly higher expression of PrPc in patients with disease recurrence at 12 mo after radical surgery(360.71±69.01 OD vs 170.23±23.06 OD,P=0.023),also in the subgroup of patients treated with adjuvant CT(368.36±79.26 OD in the recurrence group vs 162.86±24.16 OD,P=0.028),which indicates a correlation with a higher chemo-resistance.CONCLUSION Expression of PrPc is significantly higher in PDAC cells compared with control,with the protein mainly placed in the nucleus.Preliminary clinical data confirm the correlation with a poorer prognosis.

Unraveling the molecular mechanism of prion disease:Insights fromα2 area mutations in human prion protein

Prion diseases are a class of fatal neurodegenerative diseases caused by misfolded prion proteins.The main reason is that pathogenic prion protein has a strong tendency to aggregate,which easily induces the damage to the central nervous system.Point mutations in the human prion protein gene can cause prion diseases such as Creutzfeldt-Jakob and Gerstmann's syndrome.To understand the mechanism of mutation-induced prion protein aggregation,the mutants in an aqueous solution are studied by molecular dynamics simulations,including the wild type,V180I,H187R and a double point mutation which is associated with CJD and GSS.After running simulations for 500 ns,the results show that these three mutations have different effects on the kinetic properties of PrP.The high fluctuations around the N-terminal residues of helix 2 in the V180I variant lead to a decrease in hydrogen bonding on helix 2,while an increase in the number of hydrogen bonds between the folded regions promotes the generation ofβ-sheet.Meanwhile,partial deletion of salt bridges in the H187R and double mutants allows the sub-structural domains of the prion protein to separate,which would accelerate the conversion from PrPC to PrPSc.A similar trend is observed in both SASA and Rg for all three mutations,indicating that the conformational space is reduced and the structure is compact.

Structural and dynamical mechanisms of a naturally occurring variant of the human prion protein in preventing prion conversion

Prion diseases are associated with the misfolding of the normal helical cellular form of prion protein (PrPC) into the β-sheet-rich scrapie form (PrPSc) and the subsequent aggregation of PrPSc into amyloid fibrils. Recent studies demonstrated that a naturally occurring variant V127 of human PrPC is intrinsically resistant to prion conversion and aggregation, and can completely prevent prion diseases. However, the underlying molecular mechanism remains elusive. Herein we perform multiple microsecond molecular dynamics simulations on both wildtype (WT) and V127 variant of human PrPC to understand at atomic level the protective effect of V127 variant. Our simulations show that G127V mutation not only increases the rigidity of the S2–H2 loop between strand-2 (S2) and helix-2 (H2), but also allosterically enhances the stability of the H2 C-terminal region. Interestingly, previous studies reported that animals with rigid S2–H2 loop usually do not develop prion diseases, and the increase in H2 C-terminal stability can prevent misfolding and oligomerization of prion protein. The allosteric paths from G/V127 to H2 C-terminal region are identified using dynamical network analyses. Moreover, community network analyses illustrate that G127V mutation enhances the global correlations and intra-molecular interactions of PrP, thus stabilizing the overall PrPC structure and inhibiting its conversion into PrPSc. This study provides mechanistic understanding of human V127 variant in preventing prion conversion which may be helpful for the rational design of potent anti-prion compounds.

Correlation between prion protein gene codon 129 polymorphism and late-onset Alzheimer's disease

BACKGROUND： Studies addressing the correlation between prion protein gene codon 129 polymorphism, Alzheimer＇s disease, and cognitive disorders have mainly focused on Caucasians. However, prion protein gene codon 129 polymorphism is thought to also affect the Chinese Han and Wei populations. OBJECTIVE： To analyze the differences of prion protein gene codon 129 distribution among the elderly Chinese Han, East Asian, and Caucasian populations, and to study the correlation between prion protein gene codon 129 distribution and late-onset Alzheimer＇s disease. DESIGN, TIME AND SETTING： A gene polymorphism analysis was performed in the Institute of Geriatrics, General Hospital of Chinese PLA between January 2006 and January 2007. PARTICIPANTS： A total of 152 elderly Chinese Han people were selected from the Beijing Troop Cadre＇s Sanitarium. Among them, 60 patients with late-onset Alzheimer＇s disease, with a mean age of （82 ± 7） years （range 67-94 years） and disease course of （5.9 ± 4.4） years, comprising 44 males with a mean age of （83 ± 7） years and 16 females with a mean age of （78 ±7） years, were selected for the case group. An additional 92 healthy elderly subjects, with a mean of （76 ± 9） years （range 60-94 years）, comprising 76 males with a mean age of （77 ± 9） years and 16 females with a mean age of （70 ± 8） years, were selected for the control group. There were no significant differences in age and gender between the two groups （P〉 0.05）. METHODS： DNA was extracted from peripheral blood leukocytes using routine phenol/chloroform methodology. Prion protein gene codon 129 potymorphism and ApoE polymorphism were measured using PCR-restriction fragment length polymorphism. The ApoEε allele was considered the standard for analyzing correlations between prion protein gene codon 129 polymorphism and late-onset Alzheimer＇s disease. MAIN OUTCOME MEASURES： Prion protein gene codon 129 distribution; correlation between genotypic frequency and allele frequency of prion protein gene codon 129 with Alzheimer＇s disease; relationship between methionine/methionine genotype of priori protein gene, ApoEε4 allele, gender, and age of Alzheimer＇s disease patients. RESULTS： Methionine/methionine genotypic frequency of prion protein gene codon 129 was 94.08% in the Chinese elderly population, and the methionine/valine genotypic frequency was 5.92%. However, valine/valine homozygotes were not determined. There was no significant difference in prion protein gene codon 129 polymorphism between the Chinese elderly and East Asian populations （P〉 0.05）. However, there was a significant difference between the Chinese elderly and the Caucasian population （P 〈 0.05）. The methionine/methionine genotype for the positive and negative ApoEε4 alleles was a risk factor for increased incidence of Alzheimer＇s disease, but there was no significant difference between the positives and the negatives （odds ratio = 1.33, 95% confidence interval = 0.32-6.49, P〉 0.05）. CONCLUSION： Prion protein gene codon 129 distribution in the Chinese elderly was different from the Caucasian population, which suggested that the methionine/methionine genotype of prion protein gene codon 129 negatively correlated with late-onset Alzheimer＇s disease.

Analysis of the Electrophoretic Profiles of Prion Protein in Carcinous and Pericarcinous Lysates of Six Different Types of Cancers

Objective To find the different electrophoretic profiles of prion protein in carcinous and individual pericarcinous tissues in lysates of gastric,colon,liver,lung,thyroid,and laryngeal cancers.Methods Sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE)and Western blot were used to test the amounts and electrophoretic patterns of total Pr P and the tolerance of PK(protease K)digestion among six various cancer tissue types.Results A mass of Pr P signals with a large molecular weight were identified in the homogenates of peripheral tissues.The amounts and electrophoretic patterns of total Pr P did not differ significantly between carcinous and pericarcinous tissues.Pr Ps in all types of the tested cancer samples were PK sensitive but showed diversity in the tolerance of PK digestion among various tissue types.Conclusions The study revealed that the included electrophoretic patterns of carcinous and pericarcinous tissues were almost similar.Unlike Pr P-specific immunohistochemical assay,evaluation of Pr P electrophoretic patterns in the peripheral organs and tissues by Western blot does not reflect tumor malignancy.

Expression of Recombinant Protein Bovine Prion pCIp264 in COS-7 Cells and Its Detection

Bovine spongiform encephalopathy （BSE） is thought to be caused by prions initially derived from sheep scrapie, and epidemiological studies suggest that new viriant form of CJD manifest in Great Britain may be caused by BSE prions. PrP^Sc is thought to pathogenic factor of transmissible spongiform encephalopathy （TSE）, which invariably involve a post-translational modification process of PrPc encoded by the host euchromosome PrP gene during the period it converted into the pathogenic form （PrP^Sc）, PrP is nomal cellular protein which has been found in both neuronal and nonneuronal tissues. Since the crucial infectious event in protein-transmitted diseases is an induced misfolding of prion proteins （PrP^c） catalyzed by already misfolded PrP^Sc, it is of high importance that such collisions are enhanced by two-dimensional diffusion in cell membranes is of high importance compared to three-dimensional diffusion in solution. The level of PrP mRNA in brain is higher than other tissue, but purification of PrPc from rodent has been difficult. To understand the formation of PrP^Sc, it seemed useful to develop a system for produced a large quantities of PrPc since there is no nature source of PrP^c. The pCI-neo mammalian expression vector contains the neomycin phosphtransferase gene which serves as a marker for the selection of stable transfected cells with G418. COS-7 cells constitutively express simian viruse 40 （SV40） T-antigen and support replication of expression plasmids containing the SV40 origin of replication, amplifying the introduced expression cassettes, now become important routine of expression a large number of heterologous gene products. In this paper, the authors used pCI-neo vector to construct a recombnant pCIp264 （cotains mPrP, N-signalpeptide and C-GPI anchor） plasmid to express it in the COS-7 cells and meanwhile detect the expression fusion using IN-ELISA, IN-IFA and western blot, and obtain some approximative nature PrPc.

Upregulated lncRNA PRNT promotes progression and oxaliplatin resistance of colorectal cancer cells by regulating HIPK2 transcription

BACKGROUND Colorectal cancer(CRC)is the third most common cancer and a significant cause of cancer-related mortality globally.Resistance to chemotherapy,especially during CRC treatment,leads to reduced effectiveness of drugs and poor patient outcomes.Long noncoding RNAs(lncRNAs)have been implicated in various pathophysiological processes of tumor cells,including chemotherapy resistance,yet the roles of many lncRNAs in CRC remain unclear.AIM To identify and analyze the lncRNAs involved in oxaliplatin resistance in CRC and to understand the underlying molecular mechanisms influencing this resistance.METHODS Gene Expression Omnibus datasets GSE42387 and GSE30011 were reanalyzed to identify lncRNAs and mRNAs associated with oxaliplatin resistance.Various bioinformatics tools were employed to elucidate molecular mechanisms.The expression levels of lncRNAs and mRNAs were assessed via quantitative reverse transcription-polymerase chain reaction.Functional assays,including MTT,wound healing,and Transwell,were conducted to investigate the functional implications of lncRNA alterations.Interactions between lncRNAs and trans-cription factors were examined using RIP and luciferase reporter assays,while Western blotting was used to confirm downstream pathways.Additionally,a xenograft mouse model was utilized to study the in vivo effects of lncRNAs on chemotherapy resistance.RESULTS LncRNA prion protein testis specific(PRNT)was found to be upregulated in oxaliplatin-resistant CRC cell lines and negatively correlated with homeodomain interacting protein kinase 2(HIPK2)expression.PRNT was demonstrated to sponge transcription factor zinc finger protein 184(ZNF184),which in turn could regulate HIPK2 expression.Altered expression of PRNT influenced CRC cell sensitivity to oxaliplatin,with overexpression leading to decreased sensitivity and decreased expression reducing resistance.Both RIP and luciferase reporter assays indicated that ZNF184 and HIPK2 are targets of PRNT.The PRNT/ZNF184/HIPK2 axis was implicated in promoting CRC progression and oxaliplatin resistance both in vitro and in vivo.CONCLUSION The study concludes that PRNT is upregulated in oxaliplatin-resistant CRC cells and modulates the expression of HIPK2 by sponging ZNF184.This regulatory mechanism enhances CRC progression and resistance to oxaliplatin,positioning PRNT as a promising therapeutic target for CRC patients undergoing oxaliplatin-based chemotherapy.

High cellular prion protein expression in cholangiocarcinoma:A marker for early postoperative recurrence and unfavorable prognosis

BACKGROUND The cellular prion protein(PrPC),traditionally associated with neurodegenerative disorders,plays an important role in cancer progression and metastasis by inhibiting apoptosis.AIM To investigate the influence of PrPC expression in cholangiocarcinoma(CCA)on patient outcomes following surgical resection.METHODS Patients who underwent curative surgical resection for either intrahepatic or hilar CCA were enrolled in this retrospective study.Based on the immunohistochemical staining results of the surgical specimens,patients were categorized into two groups:The low PrPC group(negative or 1+)and the high PrPC group(2+or 3+).Survival analyses,including overall survival and recurrence-free survival,were conducted using the Kaplan-Meier method and compared using the log-rank test.RESULTS In total,seventy-six patients diagnosed with CCA(39 with intrahepatic and 37 with hilar CCA)underwent curative hepatectomy from January 2011 to November 2021.Among these patients,38(50%)demonstrated high PrPC expression,whereas the remaining 38(50%)showed low expression of PrPC.During a median follow-up period of 31.2 months(range:1 to 137 months),the high PrPC group had a significantly shorter median overall survival than the low PrPC group(40.4 months vs 137.9 months,respectively;P=0.041).Moreover,the high PrPC group had a significantly shorter median recurrence-free survival than the low PrPC group(13.3 months vs 23.8 months,respectively;P=0.026).CONCLUSION PrPC expression is significantly associated with early recurrence and decreased survival period in CCA patients following surgical resection.Thus,PrPC may be used as a prognostic factor in treatment planning.

Using Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to analyze differentially expressed brain polypeptides in scrapie strain 22L-infected BALB/c mice

Differentialiy expressed polypeptides in the brain of a BALB/c mouse model infected with scrapie strain 22L were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The results showed that 21 peptides were down-regulated, with peptides of mass-to-charge ratio 758.772 5 and mass-to-charge ratio 5 432.206 9, demonstrating the most significant decreases. These finding suggest that these peptides are candidate biomarkers and may play an important role in the pathogenesis of prion disease.