[Poly-G for tumor matched samples chronicles the evolution of human colorectal cancer].

Objective: To analyze poly-guanine (poly-G) genotypes and construct the phylogenetic tree of colorectal cancer (CRC) and provide an efficient and convenient method for the study of intra-tumor heterogeneity and tumor metastasis pathway. Methods: The clinicopathological information of patients with primary colorectal cancer resection with regional lymph node metastases were retrospectively collected in the Department of General Surgery, General Hospital of Tianjin Medical University from January 2017 to December 2017. The paraffin sections of the paired tumor samples were performed consecutively, and multi-region microdissection was performed after histogene staining. The phenol-chloroform extraction and ethanol precipitation scheme was used to obtain DNA, and Poly-G multiplex PCR amplification and capillary electrophoresis detection were performed. The correlation between Poly-G mutation frequency and clinicopathological parameters was analyzed. Based on the difference of Poly-G genotypes between paired samples, the distance matrix was calculated, and the phylogenetic tree was constructed to clarify the tumor metastasis pathway. Results: A total of 237 paired samples were collected from 20 patients including 134 primary lesions, 66 lymph node metastases, 37 normal tissues, and Poly-G mutation was detected in 20 patients (100%). The mutation frequency of Poly-G in low and undifferentiated patients was (74.10±23.11)%, higher than that in high and medium differentiated patients [(31.36±12.04)%, P<0.001]. In microsatellite instability patients, the mutation frequency of Poly-G was (68.19±24.80)%, which was higher than that in microsatellite stable patients [(32.40±14.90)%, P=0.003]. The Poly-G mutation frequency was not correlated with age, gender, and pathological staging (all P>0.05). Based on Poly-G genotype difference of the paired samples, the phylogenetic trees of 20 patients were constructed, showing the evolution process of the tumor, especially the subclonal origins of lymph node metastasis. Conclusion: Poly-G mutations accumulate in the occurrence and development of CRC, and can be used as genetic markers to generate reliable maps of intratumor heterogeneity in large numbers of patients with minimal time and cost expenditure.

PolyG-DS: An ultrasensitive polyguanine tract-profiling method to detect clonal expansions and trace cell lineage.

Polyguanine tracts (PolyGs) are short guanine homopolymer repeats that are prone to accumulating mutations when cells divide. This feature makes them especially suitable for cell lineage tracing, which has been exploited to detect and characterize precancerous and cancerous somatic evolution. PolyG genotyping, however, is challenging because of the inherent biochemical difficulties in amplifying and sequencing repetitive regions. To overcome this limitation, we developed PolyG-DS, a next-generation sequencing (NGS) method that combines the error-correction capabilities of duplex sequencing (DS) with enrichment of PolyG loci using CRISPR-Cas9-targeted genomic fragmentation. PolyG-DS markedly reduces technical artifacts by comparing the sequences derived from the complementary strands of each original DNA molecule. We demonstrate that PolyG-DS genotyping is accurate, reproducible, and highly sensitive, enabling the detection of low-frequency alleles (<0.01) in spike-in samples using a panel of only 19 PolyG markers. PolyG-DS replicated prior results based on PolyG fragment length analysis by capillary electrophoresis, and exhibited higher sensitivity for identifying clonal expansions in the nondysplastic colon of patients with ulcerative colitis. We illustrate the utility of this method for resolving the phylogenetic relationship among precancerous lesions in ulcerative colitis and for tracing the metastatic dissemination of ovarian cancer. PolyG-DS enables the study of tumor evolution without prior knowledge of tumor driver mutations and provides a tool to perform cost-effective and easily scalable ultra-accurate NGS-based PolyG genotyping for multiple applications in biology, genetics, and cancer research.

Neuronal intranuclear inclusion disease: Polyglycine protein is the culprit.

In this issue of Neuron, Boivin et al. (2021) show that a polyglycine-expanded protein, uN2CpolyG, is translated from an expansion of GGC repeats in the 5' UTR of the NOTCH2NLC (Notch homolog 2 N-terminal-like C) gene, defining a new pathological mechanism for neuronal intranuclear inclusion diseases (NIID).

Inhibition of MARCO ameliorates silica-induced pulmonary fibrosis by regulating epithelial-mesenchymal transition.

Epithelial-mesenchymal transition (EMT) is linked to fibrosis following exposure to silica. The scavenger receptor, macrophage receptor with collagenous structure (MARCO) plays an important role in silica-induced inflammation, however, the effect of MARCO on silica-induced fibrosis has not been identified. We hypothesized that MARCO would regulate EMT and be involved in the development of silicosis. Herein, we found that MARCO was highly expressed in lung tissue after exposure to silica and a MARCO inhibitor PolyG could alleviate pulmonary fibrosis in vivo. Our results confirmed that the expression of epithelial marker such as E-cadherin decreased, while the expression of mesenchymal markers, including vimentin and α-SMA increased after silica treatment. Furthermore, PolyG administration efficiently blocked the mRNA and protein expression of EMT markers and decreased the level of fibrosis-related transcription factors and proteins, such as Col1a1, Col3a1, Collagen I and Collagen III in the lungs of silica-exposed rats. The findings demonstrate that the macrophage membrane receptor MARCO controls the fibrotic response through regulating EMT in experimental silicosis and suggest a novel target for preventive intervention.

No effect of AR polyG polymorphism on spinal and bulbar muscular atrophy phenotype.

BACKGROUND AND PURPOSE: Disease severity varies considerably among patients with Spinal and Bulbar Muscular Atrophy (SBMA). Our aim was to investigate the role of androgen receptor (AR) polymorphic repeats in SBMA phenotype. METHODS: We analyzed the length of AR polyQ and polyG tracts in 159 SBMA patients. RESULTS: No relationship between polyG size or polyG/polyQ haplotypes and clinical phenotype was found. An independent negative correlation between polyQ-length and onset of weakness was confirmed (P < 0.001). CONCLUSIONS: The negative results of our study prompt to continue the search for potential disease modifiers in SBMA outside the AR gene.

Delivery of cell-penetrating peptide-peptide nucleic acid conjugates by assembly on an oligonucleotide scaffold.

Delivery to intracellular target sites is still one of the main obstacles in the development of peptide nucleic acids (PNAs) as antisense-antigene therapeutics. Here, we designed a self-assembled oligonucleotide scaffold that included a central complementary region for self-assembly and lateral regions complementing the PNAs. Assembly of cell-penetrating peptide (CPP)-PNAs on the scaffold significantly promoted endocytosis of PNAs by at least 10-fold in cell cultures, particularly for scaffolds in which the central complementary region was assembled by poly(guanine) and poly(cytosine). The antisense activity of CPP-PNAs increased by assembly on the scaffold and was further enhanced after co-assembly with endosomolytic peptide (EP)-PNA. This synergistic effect was also observed following the assembly of antigene CPP-PNAs\EP-PNAs on the scaffold. However, antigene activity was only observed by targeting episomal viral DNA or transfected plasmids, but not the chromosome in the cell cultures. In conclusion, assembly on oligonucleotide scaffolds significantly enhanced the antisense-antigene activity of PNAs by promoting endocytosis and endosomal escape. This oligonucleotide scaffold provided a simple strategy for assembly of multiple functional peptide-PNA conjugates, expanding the applications of PNAs and demonstrating the potential of PNAs as antiviral therapeutics.

The frequency of poly(G) tracts in the human genome and their use as a sensor of DNA damage.

Tandem repeats of short DNA sequences are commonly found in human DNA. These simple sequence repeats or microsatellites are highly polymorphic in the human genome. Since the anti-tumour agent cisplatin preferentially forms DNA adducts at runs of consecutive guanine nucleotides (poly(G)), the position and frequency of occurrence of poly(G) sequences in the updated human genome was investigated. There are more runs of consecutive guanines than would be expected by random chance. This especially true for poly(G) sequences longer than approximately n=9. A plot of poly(G) length against log(observed/expected) frequency produced a straight line for n>9. A similar observation was also found for poly(A) DNA sequence repeats. This data implied that the increase in observed/expected frequency is directly related to length of DNA repeat. It was proposed that long runs of consecutive guanine nucleotides could be a sensitive sensor of cellular DNA damage since a number of DNA damaging agents cause lesions at poly(G) sequences.

Mathematical and live meningococcal models for simple sequence repeat dynamics - coherent predictions and observations.

Evolvability by means of simple sequence repeat (SSR) instability is a feature under the constant influence of opposing selective pressures to expand and compress the repeat tract and is mechanistically influenced by factors that affect genetic instability. In addition to direct selection for protein expression and structural integrity, other factors that influence tract length evolution were studied. The genetic instability of SSRs that switch the expression of antibiotic resistance ON and OFF was modelled mathematically and monitored in a panel of live meningococcal strains. The mathematical model showed that the SSR length of a theoretical locus in an evolving population may be shaped by direct selection of expression status (ON or OFF), tract length dependent (α) and tract length independent factors (ß). According to the model an increase in α drives the evolution towards shorter tracts. An increase in ß drives the evolution towards a normal distribution of tract lengths given that an upper and a lower limit are set. Insertion and deletion biases were shown to skew allelic distributions in both directions. The meningococcal SSR model was tested in vivo by monitoring the frequency of spectinomycin resistance OFF→ON switching in a designed locus. The instability of a comprehensive panel of the homopolymeric SSRs, constituted of a range of 5-13 guanine nucleotides, was monitored in wildtype and mismatch repair deficient backgrounds. Both the repeat length itself and mismatch repair deficiency were shown to influence the genetic instability of the homopolymeric tracts. A possible insertion bias was observed in tracts ≤G10. Finally, an inverse correlation between the number of tract-encoded amino acids and growth in the presence of ON-selection illustrated a limitation to SSR expansion in an essential gene associated with the designed model locus and the protein function mediating antibiotic resistance.

Hypermutable DNA chronicles the evolution of human colon cancer.

Intratumor genetic heterogeneity reflects the evolutionary history of a cancer and is thought to influence treatment outcomes. Here we report that a simple PCR-based assay interrogating somatic variation in hypermutable polyguanine (poly-G) repeats can provide a rapid and reliable assessment of mitotic history and clonal architecture in human cancer. We use poly-G repeat genotyping to study the evolution of colon carcinoma. In a cohort of 22 patients, we detect poly-G variants in 91% of tumors. Patient age is positively correlated with somatic mutation frequency, suggesting that some poly-G variants accumulate before the onset of carcinogenesis during normal division in colonic stem cells. Poorly differentiated tumors have fewer mutations than well-differentiated tumors, possibly indicating a shorter mitotic history of the founder cell in these cancers. We generate poly-G mutation profiles of spatially separated samples from primary carcinomas and matched metastases to build well-supported phylogenetic trees that illuminate individual patients' path of metastatic progression. Our results show varying degrees of intratumor heterogeneity among patients. Finally, we show that poly-G mutations can be found in other cancers than colon carcinoma. Our approach can generate reliable maps of intratumor heterogeneity in large numbers of patients with minimal time and cost expenditure.

Frequency and distribution of simple and compound microsatellites in forty-eight Human papillomavirus (HPV) genomes.

Simple sequence repeats (SSRs) are tandem-repeated sequences ubiquitously present but differentially distributed across genomes. Present study is a systematic analysis for incidence, composition and complexity of different microsatellites in 48 representative Human papillomavirus (HPV) genomes. The analysis revealed a total of 1868 SSRs and 120 cSSRs. However, four genomes (HPV-60, HPV-92, HPV-112 and HPV-136) lacked any cSSR content; while HPV-31 accounted for a maximum of 10 cSSRs. An overall increase in cSSR% with higher dMAX was observed. The SSRs and cSSRs were prevalent in coding regions. Poly(A/T) repeats were significantly more abundant than poly(G/C) repeats possibly due to high (A/T) content of the HPV genomes. Further, higher prevalence of di-nucleotide repeats over tri-nucleotide repeats may be attributed to instability of former because of higher slippage rate. An in-depth study of the satellite sequences would provide an insight into the imperfections and evolution of microsatellites.

Clonal expansions and short telomeres are associated with neoplasia in early-onset, but not late-onset, ulcerative colitis.

BACKGROUND: Patients with ulcerative colitis (UC) are at risk of developing colorectal cancer. We have previously reported that cancer progression is associated with the presence of clonal expansions and shorter telomeres in nondysplastic mucosa. We sought to validate these findings in an independent case-control study. METHODS: This study included 33 patients with UC: 14 progressors (patients with high-grade dysplasia or cancer) and 19 nonprogressors. For each patient, a mean of 5 nondysplastic biopsies from proximal, mid, and distal colon were assessed for clonal expansions, as determined by clonal length altering mutations in polyguanine tracts, and telomere length, as measured by quantitative PCR. Both parameters were compared with individual clinicopathological characteristics. RESULTS: Clonal expansions and shorter telomeres were more frequent in nondysplastic biopsies from UC progressors than nonprogressors, but only for patients with early-onset of UC (diagnosis at younger than 50 years of age). Late-onset progressor patients had very few or no clonal expansions and longer telomeres. A few nonprogressors exhibited clonal expansions, which were associated with older age and shorter telomeres. In progressors, clonal expansions were associated with proximity to dysplasia. The mean percentage of clonally expanded mutations distinguished early-onset progressors from nonprogressors with 100% sensitivity and 80% specificity. CONCLUSIONS: Early-onset progressors develop cancer in a field of clonally expanded epithelium with shorter telomeres. The detection of these clones in a few random nondysplastic colon biopsies is a promising cancer biomarker in early-onset UC. Curiously, patients with late-onset UC seem to develop cancer without the involvement of such fields.

3'poly-G-tailed ODNs inhibit F-spondin to induce cell death and neurite retraction in rat embryonic neurons.

The effects and mechanism of action of oligodeoxyribonucleotides containing CpG motif (CpG-ODNs) on neuron cells are largely unexamined. Here, we found that CpG-A ODNs but not other types of CpG-ODNs induced neurite retraction and cell apoptosis of rat embryonic neurons in a TLR9-independent manner. These effects of CpG-A ODNs were primarily due to the poly-guanosine at the 3' terminus (3'G-ODNs). Pull-down analysis showed that 3'G-ODNs associated with transcription factor Y-BOX1 (YB-1) to facilitate the translocation of YB-1 into the nucleus via the nuclear localizing sequence of YB-1. YB-1 then interacted with the promoter of F-spondin directly at -45 and -1,375 sites as demonstrated by chromatin immunoprecipitation (ChIP) analysis. Binding of YB-1 to F-spondin promoter resulted in downregulation of F-spondin expression. Overexpression of F-spondin rescued the cell death and neurite retraction induced by 3'G-ODNs in embryonic neuron cells. Taken together, these findings suggest that 3'G-ODNs enhance nucleus YB-1 to inhibit F-spondin leading to cell death and neurite retraction of embryonic neuron cells.

Nucleoside analogue electrochemical behaviour and in situ evaluation of DNA-clofarabine interaction.

Nucleoside analogues have had a substantial impact on the treatment of cancer, especially haematological malignancies. The electrochemical behaviour of the nucleoside analogue clofarabine (CLF) was investigated at a glassy carbon electrode using cyclic, differential pulse and square wave voltammetry in different pH supporting electrolytes. The oxidation process of CLF is irreversible and pH-dependent with transfer of two protons and two electrons, following a diffusion controlled mechanism. The oxidation mechanism of CLF involves deprotonation and leads to the formation of a hydroxylated species that undergoes reversible redox reactions. The interaction of DNA and the antileukemia drug CLF was investigated using a dsDNA-electrochemical biosensor in incubated solutions by differential pulse voltammetry. The CLF-DNA interaction leads to changes in the DNA morphological structure, confirmed using the purinic homo-polynucleotide single stranded sequences of guanosine and adenosine, poly[G] and poly[A]-electrochemical biosensors.

A novel ribonuclease with potent HIV-1 reverse transcriptase inhibitory activity from cultured mushroom Schizophyllum commune.

A 20-kDa ribonuclease (RNase) was purified from fresh fruiting bodies of cultured Schizophyllum commune mushrooms. The RNase was not adsorbed on Affi-gel blue gel but adsorbed on DEAE-cellulose and CM-cellulose. It exhibited maximal RNase activity at pH 6.0 and 70°C. It demonstrated the highest ribonucleolytic activity toward poly (U) (379.5 µ/mg), the second highest activity toward poly (C) (244.7 µ/mg), less activity toward poly (A) (167.4 µ/mg), and much weaker activity toward poly (G) (114.5 µ/mg). The RNase inhibited HIV-1 reverse transcriptase with an IC(50) of 65 µM. No effect on [(3)H-methyl]-thymidine uptake by lymphoma MBL2 cells and leukemia L1210 cells was observed at 100 µM concentration of the RNase. A comparison of RNases from S. commune and Volvariella volvacea revealed that they demonstrated some similarities in N-terminal amino acid sequence, optimum pH and polyhomoribonucleotide specificity. However, some differences in chromatographic behavior and molecular mass were observed.

Binding of the anticancer alkaloid sanguinarine to double stranded RNAs: insights into the structural and energetics aspects.

Elucidation of the molecular aspects of small molecule-RNA complexation is of prime importance for rational RNA targeted drug design strategies. Towards this, the interaction of the cytotoxic plant alkaloid sanguinarine to three double stranded ribonucleic acids, poly (A).poly(U), poly(I).poly(C) and poly(C).poly(G) was studied using various biophysical and thermodynamic techniques. Absorbance and fluorescence studies showed that the alkaloid bound cooperatively to these RNAs with binding affinities of the order 10(4) M(-1). Fluorescence quenching and hydrodynamic studies gave evidence for intercalation of sanguinarine to these RNA duplexes. Isothermal titration calorimetric studies revealed that the binding was characterized by negative enthalpy and positive entropy changes and the affinity constants derived were in agreement with the overall binding affinity values obtained from spectroscopic data. The binding of sanguinarine stabilized the melting of poly(A). poly(U) and poly(I).poly(C) and the binding data evaluated from the melting data were in agreement with that obtained from other techniques. The overall binding affinity of sanguinarine to these double stranded RNAs varied in the order, poly(A).poly(U) > poly(I).poly(C) >> poly(C).poly(G). The temperature dependence of the enthalpy changes afforded negative values of heat capacity changes for the binding of sanguinarine to poly(A).poly(U) and poly(I).poly(C), suggesting substantial hydrophobic contribution in the binding process. Further, enthalpy-entropy compensation phenomena was also seen in poly(A).poly(U) and poly(I).poly(C) systems that correlated to the strong binding involving a multiplicity of weak noncovalent interactions compared to the weak binding with poly(C).poly(G). These results further advance our understanding on the binding of small molecules that are specific binders to double stranded RNA sequences.

Effect of quadruplex conformation on radiation-induced formation of 8-hydroxyguanine and unaltered base release in polyguanylic acid.

The ability of guanine-rich sequences to form quadruplex structures in telomeres for example is important in a number of biological processes such as aging, carcinogenesis and gene regulation. Ionizing radiation can cause damage to guanine moieties that can affect the stability or formation of the guanine quadruplex structures. In addition, the mechanisms of formation of these radiation damages in quadruplex structures may be different from those that occur in single- or double-stranded conformations. We have studied the quantitative aspects of the radiation induced formation of 8-hydroxy-2'-guanine base modifications and unaltered guanine base release in single-, double- and four-stranded conformations of polyriboguanylic acid as a model of guanine-rich sequences in telomere-like structures. The results show that the strandedness of guanine-rich sequences is an important variable in the observed yields of these base damages and suggests that telomere-like structures with G-quadruplexes will be relatively more radiosensitive than the other regions of duplex DNA. Hydroxyl radicals are the major reactive species that produce the DNA damage, although the presence of oxygen significantly reduces their radiation yields for all conformations of polyriboguanylic acid and changes the proportions of the yields of the various damages among the polymer conformations.

Dysregulation of kisspeptin and neurogenesis at adolescence link inborn immune deficits to the late onset of abnormal sensorimotor gating in congenital psychological disorders.

Neuropsychological syndromes including schizophrenia often do not manifest until late adolescence or early adulthood. Studies attributing a role in brain maintenance to the immune system led us to propose that malfunction of immune-dependent regulation of brain functions at adolescence underlies the late onset of such diseases/syndromes. One such function is sensorimotor gating, the ability to segregate a continuous stream of sensory and cognitive information, and to selectively allocate attention to a significant event by silencing the background (measured by prepulse inhibition; PPI). This activity is impaired in schizophrenia, as well as in several other neuropsychological diseases. Using a model of prenatal immune activation (maternal polyriboinosinic-polyribocytidylic acid (poly I:C) injection), often used as a model for schizophrenia, and in which abnormal PPI has a delayed appearance, we demonstrated a form of immune deficit in the adult offspring. Similar abnormal PPI with a delayed appearance was found in congenitally immune-deficient mice (severe combined immune deficient, SCID), and could be reversed by immune reconstitution. This functional deficit correlated with impairment of both hippocampal neurogenesis and expression of the gene encoding kisspeptin (Kiss1) that manifested at adulthood. Moreover, exogenous administration of a kisspeptin-derived peptide partially reversed the gating deficits in the SCID mice. Our results suggest that a form of congenital immune deficiency may be a key factor that determines manifestation of developmental neuropsychological disorders with onset only at early adulthood.

Production of poly-gama-glutamate (PGA) biopolymer by batch and semicontinuous cultures of immobilized Bacilluslicheniformis strain-R

Production of Polyglutamate (PGA) biopolymer by immobilized Bacilluslicheniformis strain-R was intensively investigated. Preliminary experiments were carried out to address the most suitable immobilization methodology. Entrapment of Bacillus cells in alginate-agar led optimal PGA production (36.75 g/l), with 1.32- and 2.18-fold increase in comparison with alginate- or K-carrageenan-immobilized cells, respectively. During semicontinuous cultivation of agar-alginate gel-cell mixture, production of PGA by 10 ml mixture was increased from 2nd to 3rd run whereas, increased till the 4th run using 15ml mixture. Adsorption was the most suitable immobilization technique for production of PGA and the sponge cubes was the preferred matrix recording 43.2 g/l of PGA with the highest cell adsorption. Furthermore, no PGA was detected when B. licheniformis cells were adsorbed on wood and pumice. Although luffa pulp-adsorbed cells recorded the highest PGA production (50.4 g/l), cell adsorption was the lowest. Semicontinuous cultivation of B. licheniformis cells adsorbed on sponge led to increase of PGA production till the 3rd run and reached 55.5 g/l then slightly decreased in the 4th run. The successful use of fixed-bed bioreactor for semicontinuous cultivation of B. licheniformis cells held on sponge cubes (3 runs, 96 hours/run) provides insight for the potential biotechnological production of PGA by immobilized cells.

Human DNA topoisomerase inhibitors from Potentilla argentea and their cytotoxic effect against MCF-7.

Two polyphenolics, kaempferol 3-O-beta-D-(6"-E-p-coumaroyl)-glucopyranoside (tiliroside) (1) and methyl brevifolincarboxylate (2) isolated from aerial parts of Potentilla argentea L. (Rosaceae) were evaluated for their cytotoxicities against human breast carcionoma cell line (MCF-7) and their DNA-binding ability. The DNA-binding ability of these compounds was studied by means of the human DNA topoisomerase I and II inhibition assay and ethidium displacement assay using calf thymus DNA, poly(dA-dT)2 and poly(dG-dC)2. Compound 2 was much more active and showed a higher level of cytotoxic potency than compound 1, with IC50 values of 1.11 +/- 2 microM and 21.60 +/- 2 microM, respectively. In DNA topoisomerase I and II inhibition in vitro assays both investigated compounds 1 and 2 were more effective against topoisomerase II than I. The results of DNA binding studies reveal that methyl brevifolincarboxylate had a greater DNA binding affinity that tiliroside, which correlates with its greater potency as a topoisomerase I/II inhibitor.

Double-stranded RNA homopolymer poly(rC).poly(rG) for a new pH-sensitive drug carrier.

Double-stranded RNA homopolymer poly(rC).poly(rG) has been used as a new pH-sensitive drug carrier. The poly(rC).poly(rG) had proton buffering capacity around pH 6, owing to protonation of cytosine, as determined by acid-base titration. By circular dichroism measurement, the protonation caused conformational change of the RNA. The poly(rC).poly(rG) and doxorubicin (Dox), as an anticancer drug, formed the complexes which released the drugs at endosomal pH. The resulting complex exhibited higher anticancer activity than the Dox alone. These results result suggest that the poly(rC).poly(rG) is a promising biopolymer for a new class of pH-sensitive drug carriers.