CITOGENÉTICA HUMANA – UMA VISÃO FRACTAL

Introdução: Atualmente, existe um esforço crescente da comunidade científica em compreender a organização e funcionamento do genoma (i.e., nucleoma), com base no pressuposto de que a sua forma está relacionada às suas funções, e não com base apenas no sequenciamento linear do DNA(2). Um método atual e promissor é a geometria fractal, conceito do matemático francês Benoît Mandelbrot na década de 1970, que estuda as propriedades e comportamentos de objetos complexos que não seguem uma geometria euclidiana. Formas irregulares não euclidianas foram chamadas de “fractais”, por parecerem com estruturas “fraturadas”, com repetibilidade de padrões de forma, inclusive em diferentes escalas. Os fractais ocorrem por toda a natureza, desde ramificações das árvores à rede vascular e pulmonar, e têm sido aplicados em diferentes campos da biologia, a exemplo da mais recente elucidação da estrutura do glóbulo fractal da cromatina e dos territórios cromossômicos(3). Objetivos: Este resumo tem como objetivo discutir a possibilidade da utilização da dimensão fractal (DF) como método de investigação do dano à cromatina. Métodos: As buscas foram realizadas nas bases de dados do Google Acadêmico e do PubMed. Os artigos científicos foram encontrados por meio dos descritores: “fractal dimension”, “fractal analysis” e “fractal analysis and genetics”. Foram encontrados um total de 266 artigos que usaram a análise fractal, dos quais foram selecionados 4 artigos mais relacionados ao tema. Resultados: Observa-se que o uso dos métodos fractais na citogenética se mostrou capaz de distinguir, por exemplo, mudanças da cromatina quando as células foram irradiadas, evidenciando que danos induzidos radioinduzidos ao DNA já podem ser observados na cromatina interfásica. É possível que mutações radioinduzidas alterem a complexidade topológica física global da cromatina, e isso pode ser mensurável por ferramentas computacionais, como a dimensão fractal. O método também mostra ser de grande aplicabilidade para o diagnóstico e prognóstico de diversos tipos de câncer(3). Conclusões: A análise fractal da cromatina indica ser um promissor biomarcador morfométrico de exposições humanas às radiações ionizantes, radiações não-ionizantes e para o diagnóstico e prognóstico de diversos tipos de câncer, pois se baseia na análise da textura cromatina, como um sinal geral de que houve alterações estruturais na organização fractal do genoma humano. Este método pode vir a ser útil em triagens de pacientes ou de indivíduos expostos às radiações, bem como poderá melhor elucidar o processo de organização da cromatina.

Reconstructing mitochondrial genomes from ancient DNA through iterative mapping: an evaluation of software, parameters, and bait reference

(1) Within evolutionary biology, mitochondrial genomes (mitogenomes) provide useful insights at both population and species level. Several approaches are available to assemble mitogenomes. However, most are not suitable for divergent, extinct species, due to the requirement of a reference mitogenome from a conspecific or close relative, and relatively high-quality DNA. (2) Iterative mapping can overcome the lack of a close reference sequence, and has been applied to an array of extinct species. Despite its widespread use, the accuracy of the reconstructed assemblies are yet to be comprehensively assessed. Here, we investigated the influence of mapping software (BWA or MITObim), parameters, and bait reference phylogenetic distance on the accuracy of the reconstructed assembly using two simulated datasets: (i) spotted hyena and various mammalian bait references, and (ii) southern cassowary and various avian bait references. Specifically, we assessed the accuracy of results through pairwise distance (PWD) to the reference conspecific mitogenome, number of incorrectly inserted base pairs (bp), and total length of the reconstructed assembly. (3) We found large discrepancies in the accuracy of reconstructed assemblies using different mapping software, parameters, and bait references. PWD to the reference conspecific mitogenome, which reflected the level of incorrect base calls, was consistently higher with BWA than MITObim. The same was observed for the number of incorrectly inserted bp. In contrast, the total sequence length was lower. Overall, the most accurate results were obtained with MITObim using mismatch values of 3 or 5, and the phylogenetically closest bait reference sequence. Accuracy could be further improved by combining results from multiple bait references. (4) We present the first comprehensive investigation of how mapping software, parameters, and bait reference influence mitogenome reconstruction from ancient DNA through iterative mapping. Our study provides information on how mitogenomes are best reconstructed from divergent, short-read data. By obtaining the most accurate reconstruction possible, one can be more confident as to the reliability of downstream analyses, and the evolutionary inferences made from them.

1029. Torus Synestia Nucleic Acid Analysis Platform for Fast, High Multiplex Analysis of Nucleic Acids With Single-Nucleotide Discrimination Level

Background Nucleic acid amplification testing (NAAT) is an essential tool both for biomedical research and for clinical molecular diagnostics. Currently, there are multiple NAAT platforms available, each offering certain performance and utility advantages and disadvantages as compared to each other. Next generation NAAT platforms aim to deliver increased target detection sensitivity and specificity, low limits of target detection, quantitative high multiplex target capacity, rapid time to results, and simple sample-to-answer workflow. Methods Here we describe the Torus Synestia System, a NAAT platform capable of rapid, highly multiplexed amplification and detection of both DNA and RNA targets. The platform comprises a small, portable (~ 2kg) amplification and detection device and a disposable single-use cartridge housing a PCR amplification chamber with an integrated label-free microarray for real-time data acquisition and interpretation. The platform offers a 30-min turnaround time with a detection limit of 10 DNA/RNA molecules per assay and single nucleotide discrimination. Results We demonstrate the Synestia System performance and utility with three distinct molecular applications: 1) detection of 20 genetic loci and 30 single nucleotide polymorphisms in human genomic DNA; 2) detection and genotyping of 43 unique bacterial species associated with human urinary tract infections; and 3) detection and profiling human respiratory viral pathogens including SARS-CoV-1/2, seasonal coronaviruses, Influenza A/B, and human respiratory syncytial viruses. In addition, the single-nucleotide specificity of our label-free microarray probes allowed for robust identification and discrimination of newly emerging SARS-CoV-2 lineages, such as B.1.1.7 (a.k.a. UK), B.1.351 (a.k.a. South African), P.1 (a.k.a. Brazilian), and B.1.617 (a.k.a. Indian). Conclusion The Torus Synestia System has broad applicability in both clinical and research environments. We are confident that the Torus Synestia System will revolutionize syndromic diagnostics at the point of care (PoC) and lead to improved response times during future epidemic and pandemic pathogen outbreaks. Disclosures All Authors: No reported disclosures

New Insights on the Interaction of Phenanthroline Based Ligands and Metal Complexes and Polyoxometalates with Duplex DNA and G-Quadruplexes

This work provides new insights from our team regarding advances in targeting canonical and non-canonical nucleic acid structures. This modality of medical treatment is used as a form of molecular medicine specifically against the growth of cancer cells. Nevertheless, because of increasing concerns about bacterial antibiotic resistance, this medical strategy is also being explored in this field. Up to three strategies for the use of DNA as target have been studied in our research lines during the last few years: (1) the intercalation of phenanthroline derivatives with duplex DNA; (2) the interaction of metal complexes containing phenanthroline with G-quadruplexes; and (3) the activity of Mo polyoxometalates and other Mo-oxo species as artificial phosphoesterases to catalyze the hydrolysis of phosphoester bonds in DNA. We demonstrate some promising computational results concerning the favorable interaction of these small molecules with DNA that could correspond to cytotoxic effects against tumoral cells and microorganisms. Therefore, our results open the door for the pharmaceutical and medical applications of the compounds we propose.

Peptide inhibition of neutrophil-mediated injury after in vivo challenge with supernatant of Pseudomonas aeruginosa and immune-complexes

Neutrophils are recognized for their role in host defense against pathogens as well as inflammatory conditions mediated through many mechanisms including neutrophil extracellular trap (NET) formation and generation of reactive oxygen species (ROS). NETs are increasingly appreciated as a major contributor in autoimmune and inflammatory diseases such as cystic fibrosis. Myeloperoxidase (MPO), a key neutrophil granule enzyme mediates generation of hypochlorous acid which, when extracellular, can cause host tissue damage. To better understand the role played by neutrophils in inflammatory diseases, we measured and modulated myeloperoxidase activity and NETs in vivo, utilizing a rat peritonitis model. RLS-0071 is a 15 amino acid peptide that has been shown to inhibit myeloperoxidase activity and NET formation in vitro. The rat model of inflammatory peritonitis was induced with intraperitoneal injection of either P. aeruginosa supernatant or immune-complexes. After euthanasia, a peritoneal wash was performed and measured for myeloperoxidase activity and free DNA as a surrogate for measurement of NETs. P. aeruginosa supernatant caused a 2-fold increase in MPO activity and free DNA when injected IP. Immune-complexes injected IP increased myeloperoxidase activity and free DNA 2- fold. RLS-0071 injection decreased myeloperoxidase activity and NETs in the peritoneal fluid generally to baseline levels in the presence of P. aeruginosa supernatant or immune-complexes. Taken together, RLS-0071 demonstrated the ability to inhibit myeloperoxidase activity and NET formation in vivo when initiated by different inflammatory stimuli including shed or secreted bacterial constituents as well as immune-complexes.

Colorectal cancer screening using a stool DNA-based SDC2 methylation test: a multicenter, prospective trial

Background Prevention and early detection of colorectal cancer (CRC) is a global priority, with many countries conducting population-based CRC screening programs. Although colonoscopy is the most accurate diagnostic method for early CRC detection, adherence remains low because of its invasiveness and the need for extensive bowel preparation. Non-invasive fecal occult blood tests or fecal immunochemical tests are available; however, their sensitivity is relatively low. Syndecan-2 (SDC2) is a stool-based DNA methylation marker used for early detection of CRC. Using the EarlyTect™-Colon Cancer test, the sensitivity and specificity of SDC2 methylation in stool DNA for detecting CRC were previously demonstrated to be greater than 90%. Therefore, a larger trial to validate its use for CRC screening in asymptomatic populations is now required. Methods All participants will collect their stool (at least 20 g) before undergoing screening colonoscopy. The samples will be sent to a central laboratory for analysis. Stool DNA will be isolated using a GT Stool DNA Extraction kit, according to the manufacturer’s protocol. Before performing the methylation test, stool DNA (2 µg per reaction) will be treated with bisulfite, according to manufacturer’s instructions. SDC2 and COL2A1 control reactions will be performed in a single tube. The SDC2 methylation test will be performed using an AB 7500 Fast Real-time PCR system. CT values will be calculated using the 7500 software accompanying the instrument. Results from the EarlyTect™-Colon Cancer test will be compared against those obtained from colonoscopy and any corresponding diagnostic histopathology from clinically significant biopsied or subsequently excised lesions. Based on these results, participants will be divided into three groups: CRC, polyp, and negative. The following clinical data will be recorded for the participants: sex, age, colonoscopy results, and clinical stage (for CRC cases). Discussion This trial investigates the clinical performance of a device that allows quantitative detection of a single DNA marker, SDC2 methylation, in human stool DNA in asymptomatic populations. The results of this trial are expected to be beneficial for CRC screening and may help make colonoscopy a selective procedure used only in populations with a high risk of CRC. Trial registration: This trial (NCT04304131) was registered at ClinicalTrials.gov on March 11, 2020 and is available at https://clinicaltrials.gov/ct2/show/NCT04304131?cond=NCT04304131&draw=2&rank=1.

Study on the Relationship Between Hepatitis B Virus Infection in Pregnant Women and Adverse Pregnancy Outcomes

Objective: Aim to the relationship between adverse pregnant outcomes with chronic hepatitis B virus (HBV) infection in pregnant women. Simultaneously, assess the incidence of adverse pregnancy outcomes (APO) among different serum HBV status in pregnant women. Method: From 2017 to 2019, we studied HBsAg (+) pregnant women and HBsAg (-) who gave birth at our hospital in Guangzhou City, China. We compared of the incidence of pregnant women with HBsAg(+) or HBsAg(-). Further, among HBsAg(+) pregnant women, We compared of the incidence of pregnant women with HBeAg(+) group or HBeAg(-) group, high HBV DNA loads (HBV DNA≥2×10^5IU/mL) group or low HBV DNA loads (HBV DNA＜2×10^5IU/mL) group, respectively. Finally, multivariate logistic regression analysis was used to evaluate the independent association between HBV infection and the risk of developing APO.Result: First, Our research Indicates that the rates of gestational diabetes mellitus (GDM), intrahepatic cholestasis of pregnancy (ICP), premature rupture of membrane (PROM), Fetal distress (FD), Oligohydramnios, Premature delivery (PD), Low birth weight (LBW), Meconium contamination (MC), Neonatal hyperbilirubinemia(NH) in HBsAg(+) group were higher than those in HBsAg(-) group (P<0.05). Second, among 711 HBsAg(+) pregnant women, the rates of GDM and ICP in high loads of HBV DNA were higher than those in low loads of HBV DNA group (P<0.05). Similarly, The rates of ICP in HBeAg(+) group were higher than those in HBeAg(-) group. Further, through multivariable logistical regression model analysis, we observed maternal HBsAg carrier (OR, 6.758; 95% CI, 2.358-19.369) had an independent risk for ICP. Similarly, HBsAg carrier(OR, 1.101; 95% CI, 1.066-1.137) ,advanced age (OR, 1.407; 95% CI,1.016-1.137) and abortion(OR,1.446; 95% CI, 1.062-1.969) had independent risk for GDM. Conclusions: Chronic HBV infection can increase the rate of host adverse pregnancy outcomes (APO). The maternal viral load and HBeAg status were significantly associated with the incidence of GDM and ICP. Maternal HBsAg carrier had an independent risk for GDM and ICP.

Comparison of Tenofovir with Telbivudine in Preventing Hepatitis B Transmission in Mothers with High Viral Load: A Retrospective Cohort Study

Background: Little data exist regarding comparison of efficacy and safety between tenofovir disoproxil fumarate (TDF) and Telbivudine (LdT) in late pregnancy to prevent hepatitis B mother-to-child transmission (MTCT) in real-world settings.Methods: We retrospectively included HB-s antigen (HBsAg) positive mothers with HBV DNA ≥2*105IU/mL to receive TDF or LdT after gestational weeks 24～32 weeks. All infants received standard immunoprophylaxis. Primary outcomes were MTCT rates at infants’ age of 52 weeks and safety of TDF or LdT use. Secondary outcomes were the decline of HBV-DNA levels at delivery and rates of on-treatment and off-treatment alanine aminotransferase (ALT) elevation>2 upper limits of normal (ULN) during the study.Results: Of 1407 women, 209 received TDF and 1198 received LdT treatment. There were no differences between mean duration of TDF and LdT treatment (TDF vs. LdT: 11.76±2.20 weeks vs 11.64±2.79 weeks, p=0.47). At birth, 213 (9.8%) infants in the TDF-group were HBsAg positive, lower than 1180 (20.8%) in the LdT-group (p<0.001). Among 1405 infants (TDF/LdT=213/1192) of the 1385 (TDF/LdT=205/1180) women completed the 52-weeks study, intention‐to‐treat analysis indicated one infant (0.5 %) was lost to follow-up in TDF treated mothers and three (0.3 %) in LDT treated mothers (p=0.48). On-treatment analysis indicated no HBsAg positive infants in the two groups. Levels of HBV-DNA decline in TDF-treated mothers were observed comparable to LdT-treated mothers (4.05±0.93 log10IU/mlvs.3.99±1.30 log10IU/ml, p=0.50). TDF-treated mothers had complained more symptoms of the digestive system and less arthralgia than LdT-treated mothers. All adverse events in the two groups were grade I-II. Alanine aminotransferase (ALT) elevation(>2ULN) in TDF-treated mothers were lower than in LdT-treated mothers (7.3% vs.15.7%, p<0.05).Conclusions: TDF and LdT use in late pregnancy for highly viremic mothers was equally effective in reducing MTCT. However, TDF has fewer ALT abnormalities than LdT during treatment and is the preferred choice.

One-pot green synthesis of acridine alkaloid derivatives and screening of in vitro anticancer activity against Cdc25b and SHP1

Aims: To develop of anticancer active pharmaceutical intermediates. Background: Acridone derivatives possesses wide range of pharmacological activities:1) intercalate DNA 2) form covalent bond with DNA. Objective: To Screening of in vitro anticancer activity against Cdc25b and SHP1 of new acridone derivatives and preliminary study on structure-activity relationship. Materials and Methods: Synthesis of new acridone derivatives and in vitro evaluation of their anticancer activity on Cdc25b and SHP1. Natural products that contain acridine structures, such as cystodytin A and acronycine, are isolated from certain marine (tunicates & ascidians, sponges, sea anemones) and plant (bark of Australian scrub ash tree) species. Herein, we report the efficient one-pot green synthesis of twelve novel 3,4-dihydro-1 (2H) acridone derivatives, using montmorillonite K10 as the catalyst and iron/citric acid in water. Also, their inhibitory activity against Cdc25B and SHP1 is examined, in which specific derivatives show enhanced inhibitory activity compared to others. Results and Discussion: Starting from 2-nitrobenzaldehyde derivatives and 1, 3-cyclohexanedione derivatives, twelve new acridone derivatives were prepared and exhibited substantial anticancer activity against Cdc25b and SHP1 cells. Conclusion: Preliminary studies of the structure-activity relationship have shown the influence of the structural parameters and, in particular, the nature of the substituent on aromatic ring structure and cyclohexanone. Other: Further study on structure-activity relationship.

Molecular Characterization of the Begomovirus Associated Satellites Infecting Spinacia Oleracea and Capsicum Annum Plants in Kohat, Pakistan

Begomovirus is a major and economically important genus of the Geminiviridae family. It comprises a wide range of viruses that infect a number of dicot plants including the horticulture crops, cereal crops, aromatic plants, vegetable crops, medicinal plants and weeds in various regions of the world. This study aims to investigate and correlate the various symptoms of begomovirus / satellites in different plants grown in the vicinity of Kohat, Pakistan. Furthermore, the characterization of the selected virus-associated satellites at the molecular level is also studied. Samples of suspected plants showing begomoviral infection were collected from the Kohat District. Genomic DNA was extracted from the infected plants and subjected to PCR using DNA-1/DNA-2 and Beta01/Beta02 for alpha satellites and beta satellites, respectively. The amplified PCR products were cloned and sequenced commercially. After sequencing, in silico sequence and phylogenetic analysis was also performed. Our study discovered that many plants in the Kohat District display begomovirus and satellite disease symptoms with mild to extreme disease severity. Disease incidence is especially high in okra. Beta satellites were isolated and sequenced from Spinacia oleracea and Capsicum annum plants and they showed more than 90% sequence similarity with chilli leaf curl and tomato leaf curl beta satellites. The existence of betasatellites in spinach and chilli plants was discovered for the first time in the Kohat region. Moreover, the distribution of these highly pathogenic variants of chilli leaf curl and tomato leaf curl betasatellites in the district Kohat has been reported previously.