Search results for: first-step mutations

Authors: Kim Norgaard Helmersen, Jan Silberberger

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Questions of style and reproducibility in relation to architectural design are not only continuously debated; the very concepts can seem quite provocative to architects, who like to think of architectural design as depending on intuition, ideas, and individual personalities. This standpoint - dominant in architectural discourse - is challenged in the present paper presenting early findings from a comparative STS-inspired research study of architectural design teaching and research at different architecture schools in varying national contexts. In philosophy of science framework, the paper reflects empirical observations of design teaching at the Royal Academy of Fine Arts in Copenhagen and presents a tentative theoretical framework for the on-going research project. The framework suggests that architecture – as a field of knowledge production – is mainly dominated by three epistemological positions, which will be presented and discussed. Besides serving as a loosely structured framework for future data analysis, the proposed framework brings forth the argument that architecture can be roughly divided into different schools of thought, like the traditional science disciplines. Without reducing the complexity of the discipline, describing its main intellectual positions should prove fruitful for the future development of architecture as a theoretical discipline, moving an architectural critique beyond discussions of taste preferences. Unlike traditional science disciplines, there is a lack of a community-wide, shared pool of codified references in architecture, with architects instead referencing art projects, buildings, and famous architects, when positioning their standpoints. While these inscriptions work as an architectural reference system, to be compared to codified theories in academic writing of traditional research, they are not used systematically in the same way. As a result, architectural critique is often reduced to discussions of taste and subjectivity rather than epistemological positioning. Architects are often criticized as judges of taste and accused that their rationality is rooted in cultural-relative aesthetical concepts of taste closely linked to questions of style, but arguably their supposedly subjective reasoning, in fact, forms part of larger systems of thought. Putting architectural ‘styles’ under a loop, and tracing their philosophical roots, can potentially open up a black box in architectural theory. Besides ascertaining and recognizing the existence of specific ‘styles’ and thereby schools of thought in current architectural discourse, the study could potentially also point at some mutations of the conventional – something actually ‘new’ – of potentially high value for architectural design education.

Keywords: architectural theory, design research, science and technology studies (STS), sociology of architecture

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Authors: Nazish Badar

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In early 2009, Pandemic type A (H1N1) Influenza virus emerged globally. Since then, it has continued circulation causing considerable morbidity and mortality. The purpose of this study was to evaluate the evolutionary changes in Influenza A (H1N1) pdm09 viruses from 2009-15 and their relevance with the current vaccine viruses. Methods: Respiratory specimens were collected with influenza-like illness and Severe Acute Respiratory Illness. Samples were processed according to CDC protocol. Sequencing and phylogenetic analysis of Haemagglutinin (HA) and neuraminidase (NA) genes was carried out comparing representative isolates from Pakistan viruses. Results: Between Jan2009 - Feb 2016, 1870 (13.2%) samples were positive for influenza A out of 14086. During the pandemic period (2009–10), Influenza A/ H1N1pdm 09 was the dominant strain with 366 (45%) of total influenza positives. In the post-pandemic period (2011–2016), a total of 1066 (59.6%) cases were positive Influenza A/ H1N1pdm 09 with co-circulation of different Influenza A subtypes. Overall, the Pakistan A(H1N1) pdm09 viruses grouped in two genetic clades. Influenza A(H1N1)pdm09 viruses only ascribed to Clade 7 during the pandemic period whereas viruses belong to clade 7 (2011) and clade 6B (2015) during the post-pandemic years. Amino acid analysis of the HA gene revealed mutations at positions S220T, I338V and P100S specially associated with outbreaks in all the analyzed strains. Sequence analyses of post-pandemic A(H1N1)pdm09 viruses showed additional substitutions at antigenic sites; S179N,K180Q (SA), D185N, D239G (CA), S202A (SB) and at receptor binding sites; A13T, S200P when compared with pandemic period. Substitution at Genetic markers; A273T (69%), S200P/T (15%) and D239G (7.6%) associated with severity and E391K (69%) associated with virulence was identified in viruses isolated during 2015. Analysis of NA gene revealed outbreak markers; V106I (23%) among pandemic and N248D (100%) during post-pandemic Pakistan viruses. Additional N-Glycosylation site; HA S179N (23%), NA I23T(7.6%) and N44S (77%) in place of N386K(77%) were only found in post-pandemic viruses. All isolates showed histidine (H) at position 275 in NA indicating sensitivity to neuraminidase inhibitors. Conclusion: This study shows that the Influenza A(H1N1)pdm09 viruses from Pakistan clustered into two genetic clades, with co-circulation of some variants. Certain key substitutions in the receptor binding site and few changes indicative of virulence were also detected in post-pandemic strains. Therefore, it is imperative to continue monitoring of the viruses for early identification of potential variants of high virulence or emergence of drug-resistant variants.

Keywords: Influenza A (H1N1) pdm09, evolutionary analysis, post pandemic period, Pakistan

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Authors: Showkat Ahmad Bhat, Iqra Reyaz, Falaque ul Afshan, Ahmad Arif Reshi, Muneeb U. Rehman, Manzoor R. Mir, Sabhiya Majid, Sonallah, Sheikh Bilal, Ishraq Hussain

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Background: Gastric cancer is the fourth most common cancer and the second leading cause of worldwide cancer-related deaths, with a wide variation in incidence rates across different geographical areas. The current view of cancer is that a malignancy arises from a transformation of the genetic material of a normal cell, followed by successive mutations and by chain of alterations in genes such as DNA repair genes, oncogenes, Tumor suppressor genes. Minerals are necessary for the functioning of several transcriptional factors, proteins that recognize certain DNA sequences and have been found to play a role in gastric cancer. Material Methods:The present work was a case control study and its aim was to ascertain the role of minerals and promoter hypermethylation of CpG islands of DAP-K gene in Gastric cancer patients among the Kashmiri population. Serum was extracted from all the samples and mineral estimation was done by AAS from serum, DNA was also extracted and was modified using bisulphite modification kit. Methylation-specific PCR was used for the analysis of the promoter hypermethylation status of DAP-K gene. The epigenetic analysis revealed that unlike other high risk regions, Kashmiri population has a different promoter hypermethylation profile of DAP-K gene and has different mineral profile. Results: In our study mean serum copper levels were significantly different for the two genders (p<0.05), while as no significant differences were observed for iron and zinc levels. In Methylation-specific PCR the methylation status of the promoter region of DAP-K gene was as 67.50% (27/40) of the gastric cancer tissues showed methylated DAP-K promoter and 32.50% (13/40) of the cases however showed unmethylated DAP-K promoter. Almost all 85% (17/20) of the histopathologically confirmed normal tissues showed unmethylated DAP-K promoter except only in 3 cases where DAP-K promoter was found to be methylated. The association of promoter hypermethylation with gastric cancer was evaluated by χ2 (Chi square) test and was found to be significant (P=0.0006). Occurrence of DAP-K methylation was found to be unequally distributed in males and females with more frequency in males than in females but the difference was not statistically significant (P =0.7635, Odds ratio=1.368 and 95% C.I=0.4197 to 4.456). When the frequency of DAP-K promoter methylation was compared with clinical staging of the disease, DAP-K promoter methylation was found to be certainly higher in Stage III/IV (85.71%) compared to Stage I/ II (57.69%) but the difference was not statistically significant (P =0.0673). These results suggest that DAP-K aberrant promoter hypermethylation in Kashmiri population contributes to the process of carcinogenesis in Gastric cancer and is reportedly one of the commonest epigenetic changes in the development of Gastric cancer.

Keywords: gastric cancer, minerals, AAS, hypermethylation, CpG islands, DAP-K gene

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Authors: Edison Ramos, John Peter V. Dacanay, Milwida Josefa Villanueva

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Soil-Transmitted Helminth (STH) infections caused by helminths are the most prevalent neglected tropical diseases (NTDs). They are commonly found in warm, humid regions and developing countries, particularly in rural areas with poor hygiene. Occasionally, human hosts exposed to pig manure may harbor Ascaris suum parasites without experiencing any symptoms. To address the significant issue of helminth infections, an effective anthelmintic is necessary. However, the effectiveness of various medications as anthelmintics can be reduced due to mutations. In recent years, there has been a growing interest in using plants as a source of medicine due to their natural origin, accessibility, affordability, and potential lack of complications. Herbal medicine has been advocated as an alternative treatment for helminth infections, especially in underdeveloped countries, considering the numerous adverse effects and drug resistance associated with commercially available anthelmintics. Medicinal plants are considered suitable replacements for current anthelmintics due to their historical usage in treating helminth infections. The objective of this research was to investigate the effects of aqueous extraction of pomegranate peel (Punica granatum L.) as an anthelmintic on female Ascaris suum in vitro. The in vitro assay involved observing the motility of Ascaris suum in different concentrations (25%, 50%, 75%, and 100%) of pomegranate peel aqueous extraction, along with mebendazole as a positive control. The results indicated that as the concentration of the extract increased, the time required to paralyze the worms decreased. At 25% concentration, the average time for paralysis was 362.0 minutes, which decreased to 181.0 minutes at 50% concentration, 122.7 minutes at 75% concentration, and 90.0 minutes at 100% concentration. The time of death for the worms was directly proportional to the concentration of the pomegranate peel extract. Death was observed at an average time of 240.7 minutes at 75% concentration and 147.7 minutes at 100% concentration. The findings suggest that as the concentration of pomegranate peel extract increases, the time required for paralysis and death of Ascaris suum decreases. This indicates a concentration-dependent relationship, where higher concentrations of the extract exhibit greater effectiveness in inducing paralysis and causing the death of the worms. These results emphasize the potential anthelmintic properties of pomegranate peel extract and its ability to effectively combat Ascaris suum infestations. There was no significant difference in the anthelmintic effectiveness between the pomegranate peel extract and Mebendazole. These findings highlight the potential of pomegranate peel extract as an alternative anthelmintic treatment for Ascaris suum infections. The researchers recommend determining the optimal dose and administration route to maximize the effectiveness of pomegranate peel as an anthelmintic therapeutic against Ascaris suum.

Keywords: pomegranate peel, aqueous extract, anthelmintic, in vitro

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Authors: Yakup Ulusu, Numan Eczacıoğlu, İsa Gökçe, Helen Waller, Jeremy H. Lakey

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Besides having the appropriate amino acid sequence to perform the function of proteins, it is important to have correct conformation after this sequence to process. To consist of this conformation depends on the amino acid sequence at the primary structure, hydrophobic interaction, chaperones and enzymes in charge of folding etc. Misfolded proteins are not functional and tend to be aggregated. Cysteine originating disulfide cross-links make stable this conformation of functional proteins. When two of the cysteine amino acids come side by side, disulfide bond is established that forms a cystine bridge. Due to this feature cysteine plays an important role on the formation of three-dimensional structure of many proteins. There are two cysteine amino acids (C44, C69) in the Tol-A-III protein. Unlike protein disulfide bonds from within his own, any non-specific cystine bridge causes a change in the three dimensional structure of the protein. Proteins can be expressed in various host cells as directly or fusion (chimeric). As a result of overproduction of the recombinant proteins, aggregation of insoluble proteins in the host cell can occur by forming a crystal structure called inclusion body. In general fusion proteins are produced for provide affinity tags to make proteins more soluble and production of some toxic proteins via fusion protein expression system like pTolT. Proteins can be modified by using a site-directed mutagenesis. By this way, creation of non-specific disulfide crosslinks can be prevented at fusion protein expression system via the present cysteine replaced by another amino acid such as serine, glycine or etc. To do this, we need; a DNA molecule that contains the gene that encodes for the target protein, required primers for mutation to be designed according to site directed mutagenesis reaction. This study was aimed to be replaced cysteine encoding codon TGT with serine encoding codon AGT. For this sense and reverse primers designed (given below) and used site-directed mutagenesis reaction. Several new copy of the template plasmid DNA has been formed with above mentioned mutagenic primers via polymerase chain reaction (PCR). PCR product consists of both the master template DNA (wild type) and the new DNA sequences containing mutations. Dpn-l endonuclease restriction enzyme which is specific for methylated DNA and cuts them to the elimination of the master template DNA. E. coli cells obtained after transformation were incubated LB medium with antibiotic. After purification of plasmid DNA from E. coli, the presence of the mutation was determined by DNA sequence analysis. Developed this new plasmid is called PtolT-δ.

Keywords: site directed mutagenesis, Escherichia coli, pTolT, protein expression

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Authors: Maria E. V. Amarante, Carolina L. Cerdeira, Julia V. Cortes, Fiorita G. L. Mundim

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Basal cell carcinoma (BCC) is the most prevalent type of skin cancer in humans. It arises from the basal cells of the epidermis and cutaneous appendages. The role of sunlight exposure as a risk factor for BCC is very well defined due to its power to influence genetic mutations, in addition to having a suppressor effect on the skin immune system. Despite showing low metastasis and mortality rates, the tumor is locally infiltrative, aggressive, and destructive. Considering the high prevalence rate of this carcinoma and the importance of early detection, a retrospective study was carried out in order to correlate the clinical data available on BBC, characterize it epidemiologically, and thus enable effective prevention measures for the population. Data on the period from January 2015 to December 2019 were collected from the medical records of patients registered at one pathology service located in the southeast region of Brazil, known as SVO, which delivers skin biopsy results. The study was aimed at correlating the variables, sex, age, and subtypes found. Data analysis was performed using the chi-square test at a nominal significance level of 5% in order to verify the independence between the variables of interest. Fisher's exact test was applied in cases where the absolute frequency in the cells of the contingency table was less than or equal to five. The statistical analysis was performed using the R® software. Ninety-three basal cell carcinoma were analyzed, and its frequency in the 31-to 45-year-old age group was 5.8 times higher in men than in women, whereas, from 46 to 59 years, the frequency was found 2.4 times higher in women than in men. Between the ages of 46 to 59 years, it should be noted that the sclerodermiform subtype appears more than the solid one, with a difference of 7.26 percentage points. Reversely, the solid form appears more frequently in individuals aged 60 years or more, with a difference of 8.57 percentage points. Among women, the frequency of the solid subtype was 9.93 percentage points higher than the sclerodermiform frequency. In males, the same percentage difference is observed, but sclerodermiform is the most prevalent subtype. It is concluded in this study that, in general, there is a predominance of basal cell carcinoma in females and in individuals aged 60 years and over, which demonstrates the tendency of this tumor. However, when rarely found in younger individuals, the male gender prevailed. The most prevalent subtype was the solid one. It is worth mentioning that the sclerodermiform subtype, which is more aggressive, was seen more frequently in males and in the 46-to 59-year-old range.

Keywords: basal cell carcinoma, epidemiology, sclerodermiform basal cell carcinoma, skin cancer, solar radiation, solid basal cell carcinoma

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Authors: Debjit Ray

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Horizontal gene transfer (HGT) and recombination leads to the emergence of bacterial antibiotic resistance and pathogenic traits. HGT events can be identified by comparing a large number of fully sequenced genomes across a species or genus, define the phylogenetic range of HGT, and find potential sources of new resistance genes. In-depth comparative phylogenomics can also identify subtle genome or plasmid structural changes or mutations associated with phenotypic changes. Comparative phylogenomics requires that accurately sequenced, complete and properly annotated genomes of the organism. Assembling closed genomes requires additional mate-pair reads or “long read” sequencing data to accompany short-read paired-end data. To bring down the cost and time required of producing assembled genomes and annotating genome features that inform drug resistance and pathogenicity, we are analyzing the performance for genome assembly of data from the Illumina NextSeq, which has faster throughput than the Illumina HiSeq (~1-2 days versus ~1 week), and shorter reads (150bp paired-end versus 300bp paired end) but higher capacity (150-400M reads per run versus ~5-15M) compared to the Illumina MiSeq. Bioinformatics improvements are also needed to make rapid, routine production of complete genomes a reality. Modern assemblers such as SPAdes 3.6.0 running on a standard Linux blade are capable in a few hours of converting mixes of reads from different library preps into high-quality assemblies with only a few gaps. Remaining breaks in scaffolds are generally due to repeats (e.g., rRNA genes) are addressed by our software for gap closure techniques, that avoid custom PCR or targeted sequencing. Our goal is to improve the understanding of emergence of pathogenesis using sequencing, comparative genomics, and machine learning analysis of ~1000 pathogen genomes. Machine learning algorithms will be used to digest the diverse features (change in virulence genes, recombination, horizontal gene transfer, patient diagnostics). Temporal data and evolutionary models can thus determine whether the origin of a particular isolate is likely to have been from the environment (could it have evolved from previous isolates). It can be useful for comparing differences in virulence along or across the tree. More intriguing, it can test whether there is a direction to virulence strength. This would open new avenues in the prediction of uncharacterized clinical bugs and multidrug resistance evolution and pathogen emergence.

Keywords: genomics, pathogens, genome assembly, superbugs

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Authors: Rachael Bentley, Callahan Gergen, Brodie Thiede

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Roadways have a significant impact on the environment in so far as they function as barriers to wildlife movement, both through road mortality and through resultant road avoidance. Roads have an im-mense presence worldwide, and it is predicted to increase substantially in the next thirty years. As roadways become even more common, it is important to consider their environmental impact, and to mitigate the negative effects which they have on wildlife and wildlife mobility. In a thorough analysis of several related studies, a common conclusion was that roads cause habitat fragmentation, which can lead split populations to evolve differently, for better or for worse. Though some populations adapted positively to roadways, becoming more resistant to road mortality, and more tolerant to noise and chemical contamination, many others experienced maladaptation, either due to chemical contamination in and around their environment, or because of genetic mutations from inbreeding when their population was fragmented too substantially to support a large enough group for healthy genetic exchange. Large mammals were especially susceptible to maladaptation from inbreed-ing, as they require larger areas to roam and therefore require even more space to sustain a healthy population. Regardless of whether a species evolved positively or negatively as a result of their proximity to a road, animals tended to avoid roads, making the genetic diversity from habitat fragmentation an exceedingly prevalent issue in the larger discussion of road ecology. Additionally, the consideration of solu-tions, such as overpasses and underpasses, is crucial to ensuring the long term survival of many wildlife populations. In studies addressing the effectiveness of overpasses and underpasses, it seemed as though animals adjusted well to these sorts of solutions, but strategic place-ment, as well as proper sizing, proper height, shelter from road noise, and other considerations were important in construction. When an underpass or overpass was well-built and well-shielded from human activity, animals’ usage of the structure increased significantly throughout its first five years, thus reconnecting previously divided populations. Still, these structures are costly and they are often unable to fully address certain issues such as light, noise, and contaminants from vehicles. Therefore, the need for further discussion of new, crea-tive solutions remains paramount. Roads are one of the most consistent and prominent features of today’s landscape, but their environmental impacts are largely overlooked. While roads are useful for connecting people, they divide landscapes and animal habitats. Therefore, further research and investment in possible solutions is necessary to mitigate the negative effects which roads have on wildlife mobility and to pre-vent issues from resultant habitat fragmentation.

Keywords: fences, habitat fragmentation, roadways, wildlife mobility

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Authors: Mia Bahar, Özlem Bozkurt

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Obsessive-compulsive disorder (OCD) is a typical, persistent, and long-lasting mental health condition in which a person experiences uncontrollable, recurrent thoughts (or "obsessions") and/or activities (or "compulsions") that they feel compelled to engage in repeatedly. Obsessive-compulsive disorder is both underdiagnosed and undertreated. It frequently manifests in a variety of medical settings and is persistent, expensive, and burdensome. Obsessive-compulsive neurosis was long believed to be a condition that offered valuable insight into the inner workings of the unconscious mind. Obsessive-compulsive disorder is now recognized as a prime example of a neuropsychiatric condition susceptible to particular pharmacotherapeutic and psychotherapy therapies and mediated by pathology in particular neural circuits. An obsessive-compulsive disorder which is called OCD, usually has two components, one cognitive and the other behavioral, although either can occur alone. Obsessions are often repetitive and intrusive thoughts that invade consciousness. These obsessions are incredibly hard to control or dismiss. People who have OCD often engage in rituals to reduce anxiety associated with intrusive thoughts. Once the ritual is formed, the person may feel extreme relief and be free from anxiety until the thoughts of contamination intrude once again. These thoughts are strengthened through a manifestation of negative reinforcement because they allow the person to avoid anxiety and obscurity. These thoughts are described as autogenous, meaning they most likely come from nowhere. These unwelcome thoughts are related to actions which we can describe as Thought Action Fusion. The thought becomes equated with an action, such as if they refuse to perform the ritual, something bad might happen, and so people perform the ritual to escape the intrusive thought. In almost all cases of OCD, the person's life gets extremely disturbed by compulsions and obsessions. Studies show OCD is an estimated 1.1% prevalence, making it a challenging issue with high co-morbidities with other issues like depressive episodes, panic disorders, and specific phobias. The first to reveal brain anomalies in OCD were numerous CT investigations, although the results were inconsistent. A few studies have focused on the orbitofrontal cortex (OFC), anterior cingulate gyrus (AC), and thalamus, structures also implicated in the pathophysiology of OCD by functional neuroimaging studies, but few have found consistent results. However, some studies have found abnormalities in the basal ganglion. There have also been some discussions that OCD might be genetic. OCD has been linked to families in studies of family aggregation, and findings from twin studies show that this relationship is somewhat influenced by genetic variables. Some Research has shown that OCD is a heritable, polygenic condition that can result from de novo harmful mutations as well as common and unusual variants. Numerous studies have also presented solid evidence in favor of a significant additive genetic component to OCD risk, with distinct OCD symptom dimensions showing both common and individual genetic risks.

Keywords: compulsions, obsessions, neuropsychiatric, genetic

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Authors: Leonard Lipovich, Pattaraporn Thepsuwan, Anton-Scott Goustin, Juan Cai, Donghong Ju, James B. Brown

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Two-thirds of human genes do not encode any known proteins. Aside from long non-coding RNA (lncRNA) genes with recently-discovered functions, the ~40,000 non-protein-coding human genes remain poorly understood, and a role for their transcripts as de-facto unconventional messenger RNAs has not been formally excluded. Ribosome profiling (Riboseq) predicts translational potential, but without independent evidence of proteins from lncRNA open reading frames (ORFs), ribosome binding of lncRNAs does not prove translation. Previously, we mass-spectrometrically documented translation of specific lncRNAs in human K562 and GM12878 cells. We now examined lncRNA translation in human MCF7 cells, integrating strand-specific Illumina RNAseq, Riboseq, and deep mass spectrometry in biological quadruplicates performed at two core facilities (BGI, China; City of Hope, USA). We excluded known-protein matches. UCSC Genome Browser-assisted manual annotation of imperfect (tryptic-digest-peptides)-to-(lncRNA-three-frame-translations) alignments revealed three peptides hypothetically explicable by 'stop-to-nonstop' in-frame replacement of stop codons by amino acids in two ORFs of the lncRNA MMP24-AS1. To search for this phenomenon genomewide, we designed and implemented a novel pipeline, matching tryptic-digest spectra to wildcard-instead-of-stop versions of repeat-masked, six-frame, whole-genome translations. Along with singleton putative stop-to-nonstop events affecting four other lncRNAs, we identified 24 additional peptides with stop-to-nonstop in-frame substitutions from multiple positive-strand MMP24-AS1 ORFs. Only UAG and UGA, never UAA, stop codons were impacted. All MMP24-AS1-matching spectra met the same significance thresholds as high-confidence known-protein signatures. Targeted resequencing of MMP24-AS1 genomic DNA and cDNA from the same samples did not reveal any mutations, polymorphisms, or sequencing-detectable RNA editing. This unprecedented apparent gene-specific violation of the genetic code highlights the importance of matching peptides to whole-genome, not known-genes-only, ORFs in mass-spectrometry workflows, and suggests a new mechanism enhancing the combinatorial complexity of the proteome. Funding: NIH Director’s New Innovator Award 1DP2-CA196375 to LL.

Keywords: genetic code, lncRNA, long non-coding RNA, mass spectrometry, proteogenomics, ribo-seq, ribosome, RNAseq

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Authors: Christine P. Gibson, Ramona Alex, Michael Farney, Johana Vallejo-Elias, Mitra Esfandiarei

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Aortic aneurysm is the leading cause of death in Marfan syndrome (MFS), a connective tissue disorder caused by mutations in fibrillin-1 gene (FBN1). MFS aneurysm is characterized by weakening of the aortic wall due to elastin fibers fragmentation and disorganization. The above-average height and distinct physical features make young adults with MFS desirable candidates for competitive sports; but little is known about the exercise limit at which they will be at risk for aortic rupture. On the other hand, aerobic cardiovascular exercise has been shown to have protective effects on the heart and aorta. We have previously reported that mild aerobic exercise can delay the formation of aortic aneurysm in a mouse model of MFS. In this study, we aimed to investigate the effects of various levels of exercise intensity on the progression of aortic aneurysm in the mouse model. Starting at 4 weeks of age, we subjected control and MFS mice to different levels of exercise intensity (8m/min, 10m/min, 15m/min, and 20m/min, corresponding to 55%, 65%, 75%, and 85% of VO2 max, respectively) on a treadmill for 30 minutes per day, five days a week for the duration of the study. At 24 weeks of age, aortic tissue were isolated and subjected to structural and functional studies using histology and wire myography in order to evaluate the effects of different exercise routines on elastin fragmentation and organization and aortic wall elasticity/stiffness. Our data shows that exercise training at the intensity levels between 55%-75% significantly reduces elastin fragmentation and disorganization, with less recovery observed in 85% MFS group. The reversibility of elasticity was also significantly restored in MFS mice subjected to 55%-75% intensity; however, the recovery was less pronounced in MFS mice subjected to 85% intensity. Furthermore, our data shows that smooth muscle cells (SMCs) contractilion in response to vasoconstrictor agent phenylephrine (100nM) is significantly reduced in MFS aorta (54.84 ± 1.63 mN/mm2) as compared to control (95.85 ± 3.04 mN/mm2). At 55% of intensity, exercise did not rescue SMCs contraction (63.45 ± 1.70 mN/mm2), while at higher intensity levels, SMCs contraction in response to phenylephrine was restored to levels similar to control aorta [65% (81.88 ± 4.57 mN/mm2), 75% (86.22 ± 3.84 mN/mm2), and 85% (83.91 ± 5.42 mN/mm2)]. This study provides the first time evidence that high intensity exercise (e.g. 85%) may not provide the most beneficial effects on aortic function (vasoconstriction) and structure (elastin fragmentation, aortic wall elasticity) during the progression of aortic aneurysm in MFS mice. On the other hand, based on our observations, medium intensity exercise (e.g. 65%) seems to provide the utmost protective effects on aortic structure and function in MFS mice. These findings provide new insights into the potential capacity, in which MFS patients could participate in various aerobic exercise routines, especially in young adults affected by cardiovascular complications particularly aortic aneurysm. This work was funded by Midwestern University Research Fund.

Keywords: aerobic exercise, aortic aneurysm, aortic wall elasticity, elastin fragmentation, Marfan syndrome

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Authors: Heiko Jessen, Laura Tanus, Slobodan Ruzicic

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Objectives: The efficacy of Stribild®, an integrase strand transfer inhibitor (INSTI) -based STR, has been evaluated in randomized clinical trials and it has demonstrated durable capability in terms of achieving sustained suppression of HIV-1 RNA-levels. However, differences in monitoring frequency, existing selection bias and profile of patients enrolled in the trials, may all result in divergent efficacy of this regimen in routine clinical settings. The aim of this study was to assess the virologic outcomes, safety and tolerability profile of Stribild® in a routine clinical setting. Methods: This was a retrospective monocentric analysis on HIV-1-infected patients, who started with or were switched to Stribild®. Virological failure (VF) was defined as confirmed HIV-RNA>50 copies/ml. The minimum time of follow-up was 24 weeks. The percentage of patients remaining free of therapeutic failure was estimated using the time-to-loss-of-virologic-response (TLOVR) algorithm, by intent-to-treat analysis. Results: We analyzed the data of 197 patients (56 ART-naïve and 141 treatment-experienced patients), who fulfilled the inclusion criteria. Majority (95.9%) of patients were male. The median time of HIV-infection at baseline was 2 months in treatment-naïve and 70 months in treatment-experienced patients. Median time [IQR] under ART in treatment-experienced patients was 37 months. Among the treatment-experienced patients 27.0% had already been treated with a regimen consisting of two NRTIs and one INSTI, whereas 18.4% of them experienced a VF. The median time [IQR] of virological suppression prior to therapy with Stribild® in the treatment-experienced patients was 10 months [0-27]. At the end of follow-up (median 33 months), 87.3% (95% CI, 83.5-91.2) of treatment-naïve and 80.3% (95% CI, 75.8-84.8) of treatment-experienced patients remained free of therapeutic failure. Considering only treatment-experienced patients with baseline VL<50 copies/ml, 83.0% (95% CI, 78.5-87.5) remained free of therapeutic failure. A total of 17 patients stopped treatment with Stribild®, 5.4% (3/56) of them were treatment-naïve and 9.9% (14/141) were treatment-experienced patients. The Stribild® therapy was discontinued in 2 (1.0%) because of VF, loss to follow-up in 4 (2.0%), and drug-drug interactions in 2 (1.0%) patients. Adverse events were in 7 (3.6%) patients the reason to switch from therapy with Stribild® and further 2 (1.0%) patients decided personally to switch. The most frequently observed adverse events were gastrointestinal side effects (20.0%), headache (8%), rash events (7%) and dizziness (6%). In two patients we observed an emergence of novel resistances in integrase-gene. The N155H evolved in one patient and resulted in VF. In another patient S119R evolved either during or shortly upon switch from therapy with Stribild®. In one further patient with VF two novel mutations in the RT-gene were observed when compared to historical genotypic test result (V106I/M and M184V), whereby it is not clear whether they evolved during or already before the switch to Stribild®. Conclusions: Effectiveness of Stribild® for treatment-naïve patients was consistent with data obtained in clinical trials. The safety and tolerability profile as well as resistance development confirmed clinical efficacy of Stribild® in a daily practice setting.

Keywords: ART, HIV, integrase inhibitor, stribild

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Authors: Wu Liching

Abstract:

Aim This case aims to discuss the pathogenesis, clinical manifestations and medical treatment of strokes caused by mitochondrial gene mutations. Methods Diagnosis of ischemic stroke caused by mitochondrial gene defect by means of "next-generation sequencing mitochondrial DNA gene variation detection", imaging examination, neurological examination, and medical history; this study took samples from the neurology ward of a medical center in northern Taiwan cases diagnosed with acute cerebral infarction as the research objects. Result This case is a 49-year-old married woman with a rare disease, mitochondrial gene mutation inducing ischemic stroke. She has severe hearing impairment and needs to use hearing aids, and has a history of diabetes. During the patient’s hospitalization, the blood test showed that serum Lactate: 7.72 mmol/L, Lactate (CSF) 5.9 mmol/L. Through the collection of relevant medical history, neurological evaluation showed changes in consciousness and cognition, slow response in language expression, and brain magnetic resonance imaging examination showed subacute bilateral temporal lobe infarction, which was an atypical type of stroke. The lineage DNA gene has m.3243A>G known pathogenic mutation point, and its heteroplasmic level is 24.6%. This pathogenic point is located in MITOMAP and recorded as Mitochondrial Encephalopathy, Lactic Acidosis, and Stroke-like episodes (MELAS) , Leigh Syndrome and other disease-related pathogenic loci, this mutation is located in ClinVar and recorded as Pathogenic (dbSNP: rs199474657), so it is diagnosed as a case of stroke caused by a rare disease mitochondrial gene mutation. After medical treatment, there was no more seizure during hospitalization. After interventional rehabilitation, the patient's limb weakness, poor language function, and cognitive impairment have all improved significantly. Conclusion Mitochondrial disorders can also be associated with abnormalities in psychological, neurological, cerebral cortical function, and autonomic functions, as well as problems with internal medical diseases. Therefore, the differential diagnoses cover a wide range and are not easy to be diagnosed. After neurological evaluation, medical history collection, imaging and rare disease serological examination, atypical ischemic stroke caused by rare mitochondrial gene mutation was diagnosed. We hope that through this case, the diagnosis of rare disease mitochondrial gene variation leading to cerebral infarction will be more familiar to clinical medical staff, and this case report may help to improve the clinical diagnosis and treatment for patients with similar clinical symptoms in the future.

Keywords: acute stroke, MELAS, lactic acidosis, mitochondrial disorders

Procedia PDF Downloads 43

Authors: Julia Gontar, Natalia Buderatskaya, Igor Ilyin, Olga Parnitskaya, Sergey Lavrynenko, Eduard Kapustin, Ekaterina Ilyina, Yana Lakhno

Abstract:

Introduction: It is well known that oocytes obtained from older reproductive women have accumulated mitochondrial DNA mutations, which negatively affects the morphology of a developing embryo and may lead to the birth of a child with mitochondrial disease. Special techniques have been developed to allow a donor oocyte cytoplasmic transfer with the parents’ biological nuclear DNA retention. At the same time, it is important to understand whether the procedure affects the future embryonic chromosome sets as the nuclear DNA is the transfer subject in this new complex procedure. Material and Methods: From July 2015 to July 2016, the investigation was carried out in the Medical Centre IGR. 34 donor oocytes (group A) were used for the manipulation with the aim of donating cytoplasm: 21 oocytes were used for zygotes pronuclear transfer and oocytes 13 – for the spindle transfer. The mean age of the oocyte donors was 28.4±2.9 years. The procedure was performed using Nikon Ti Eclipse inverted microscope equipped with the micromanipulators Narishige system (Japan), Saturn 3 laser console (UK), Oosight imaging systems (USA). For the preimplantation genetic screening (PGS) blastocyst biopsy was performed, trophectoderm samples were diagnosed using fluorescent in situ hybridization on chromosomes 9, 13, 15, 16, 17, 18, 21, 22, X, Y. For comparison of morphological characteristics and euploidy, was chosen a group of embryos (group B) with the amount of 121 blastocysts obtained from 213 oocytes, which were gotten from the donor programs of assisted reproductive technologies (ART). Group B was not subjected to donor oocyte cytoplasmic transfer procedure and studied on the above mentioned chromosomes. Statistical analysis was carried out using the criteria t, x^2 at a significance levels p<0.05, p<0.01, p<0.001. Results: After the donor cytoplasm transfer process the amount of the third day developing embryos was 27 (79.4%). In this stage, the group B consisted of 189 (88.7%) developing embryos, and there was no statistically significant difference (SSD) between the two groups (p>0.05). After a comparative analysis of the morphological characteristics of the embryos on the fifth day, we also found no SSD among the studied groups (p>0.05): from 34 oocytes exposed to manipulation, 14 (41.2%) blastocysts was obtained, while the group B blastocyst yield was 56.8% (n=121) from 213 oocytes. The following results were obtained after PGS performing: in group A euploidy in studied chromosomes were 28.6%(n=4) blastocysts, whereas in group B this rate was 40.5%(n=49), 28.6%(n=4) and 21.5%(n=26) of mosaic embryos and 42.8%(n=6) and 38.0%(n=46) aneuploid blastocysts respectively were identified. None of these specified parameters had an SSD (p>0.05). But attention was drawn by the blastocysts in group A with identified mosaicism, which was chaotic without any cell having euploid chromosomal set, in contrast to the mosaic embryos in group B where identified chaotic mosaicism was only 2.5%(n=3). Conclusions: According to the obtained results, there is no direct procedural effect on the chromosome in embryos obtained following donor oocyte cytoplasmic transfer. Thus, the technology introduction will enhance the infertility treating effectiveness as well as avoiding having a child with mitochondrial disease.

Keywords: donor oocyte cytoplasmic transfer, embryos’ chromosome set, oocyte spindle transfer, pronuclear transfer

Procedia PDF Downloads 297

Authors: Sandra Smieszek, Jonathan Haines

Abstract:

Introduction: Numerous research efforts have focused on searching for ‘longevity genes’. However, attempting to decipher the genetic component of the longevous phenotype have resulted in limited success and the mechanisms governing longevity remain to be explained. We conducted a genome-wide homozygosity analysis (GWHA) of the founder population of the Amish community in central Ohio. While genome-wide association studies using unrelated individuals have revealed many interesting longevity associated variants, these variants are typically of small effect and cannot explain the observed patterns of heritability for this complex trait. The Amish provide a large cohort of extended kinships allowing for in depth analysis via family-based approach excellent population due to its. Heritability of longevity increases with age with significant genetic contribution being seen in individuals living beyond 60 years of age. In our present analysis we show that the heritability of longevity is estimated to be increasing with age particularly on the paternal side. Methods: The present analysis integrated both phenotypic and genotypic data and led to the discovery of a series of variants, distinct for stratified populations across ages and distinct for paternal and maternal cohorts. Specifically 5437 subjects were analyzed and a subset of 893 successfully genotyped individuals was used to assess CHIP heritability. We have conducted the homozygosity analysis to examine if homozygosity is associated with increased risk of living beyond 90. We analyzed AMISH cohort genotyped for 614,957 SNPs. Results: We delineated 10 significant regions of homozygosity (ROH) specific for the age group of interest (>90). Of particular interest was ROH on chromosome 13, P < 0.0001. The lead SNPs rs7318486 and rs9645914 point to COL4A2 and our lead SNP. COL25A1 encodes one of the six subunits of type IV collagen, the C-terminal portion of the protein, known as canstatin, is an inhibitor of angiogenesis and tumor growth. COL4A2 mutations have been reported with a broader spectrum of cerebrovascular, renal, ophthalmological, cardiac, and muscular abnormalities. The second region of interest points to IRS2. Furthermore we built a classifier using the obtained SNPs from the significant ROH region with 0.945 AUC giving ability to discriminate between those living beyond to 90 years of age and beyond. Conclusion: In conclusion our results suggest that a history of longevity does indeed contribute to increasing the odds of individual longevity. Preliminary results are consistent with conjecture that heritability of longevity is substantial when we start looking at oldest fifth and smaller percentiles of survival specifically in males. We will validate all the candidate variants in independent cohorts of centenarians, to test whether they are robustly associated with human longevity. The identified regions of interest via ROH analysis could be of profound importance for the understanding of genetic underpinnings of longevity.

Keywords: regions of homozygosity, longevity, SNP, Amish

Procedia PDF Downloads 206

Authors: P.P. Rupasinghe, A.H. Farid

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The Aleutian mink disease virus (AMDV, Carnivore amdoparvovirus 1) causes persistent infection, plasmacytosis, and formation and deposition of immune complexes in various organs in adult mink, leading to glomerulonephritis, arteritis and sometimes death. The disease has no cure nor an effective vaccine, and identification and culling of mink positive for anti-AMDV antibodies have not been successful in controlling the infection in many countries. The failure to eradicate the virus from infected farms may be caused by keeping false-negative individuals on the farm, virus transmission from wild animals, or neighboring farms. The identification of sources of infection, which can be performed by comparing viral sequences, is important in the success of viral eradication programs. High mutation rates could cause inaccuracies when viral sequences are used to trace back an infection to its origin. There is no published information on the mutation rate of AMDV either in vivo or in vitro. The in vivo estimation is the most accurate method, but it is difficult to perform because of the inherent technical complexities, namely infecting live animals, the unknown numbers of viral generations (i.e., infection cycles), the removal of deleterious mutations over time and genetic drift. The objective of this study was to determine the mutation rate of AMDV on which no information was available. A homogenate was prepared from the spleen of one naturally infected American mink (Neovison vison) from Nova Scotia, Canada (parental template). The near full-length genome of this isolate (91.6%, 4,143 bp) was bidirectionally sequenced. A group of black mink was inoculated with this homogenate (descendant mink). Spleen sampled were collected from 10 descendant mink after 16 weeks post-inoculation (wpi) and from anther 10 mink after 176 wpi, and their near-full length genomes were bi-directionally sequenced. Sequences of these mink were compared with each other and with the sequence of the parental template. The number of nucleotide substitutions at 176 wpi was 3.1 times greater than that at 16 wpi (113 vs 36) whereas the estimates of mutation rate at 176 wpi was 3.1 times lower than that at 176 wpi (2.85×10-3 vs 9.13×10-4 substitutions/ site/ year), showing a decreasing trend in the mutation rate per unit of time. Although there is no report on in vivo estimate of the mutation rate of DNA viruses in animals using the same method which was used in the current study, these estimates are at the higher range of reported values for DNA viruses determined by various techniques. These high estimates are logical based on the wide range of diversity and pathogenicity of AMDV isolates. The results suggest that increases in the number of nucleotide substitutions over time and subsequent divergence make it difficult to accurately trace back AMDV isolates to their origin when several years elapsed between the two samplings.

Keywords: Aleutian mink disease virus, American mink, mutation rate, nucleotide substitution

Procedia PDF Downloads 96

Authors: Ioannis Panagiotopoulos, Aristidis Anastasakis, Konstantinos Toutouzas, Ioannis Iakovou, Charalampos Vlachopoulos, Vasilis Voudris, Georgios Tziomalos, Konstantinos Tsioufis, Efstathios Kastritis, Alexandros Briassoulis, Kimon Stamatelopoulos, Alexios Antonopoulos, Paraskevi Exadaktylou, Evanthia Giannoula, Anastasia Katinioti, Maria Kalantzi, Evangelos Leontiadis, Eftychia Smparouni, Ioannis Malakos, Nikolaos Aravanis, Argyrios Doumas, Maria Koutelou

Abstract:

Introduction: Cardiac amyloidosis from Transthyretin (ATTR-CA) is an infiltrative disease characterized by the deposition of pathological transthyretin complexes in the myocardium. This study describes the characteristics of patients diagnosed with ATTR-CA from 2019 until present at the Nuclear Medicine Department of Onassis Cardiac Surgery Center and AHEPA Hospital. These centers have extensive experience in amyloidosis and modern technological equipment for its diagnosis. Materials and Methods: Records of consecutive patients (N=73) diagnosed with any type of amyloidosis were collected, analyzed, and prospectively followed. The diagnosis of amyloidosis was made using specific myocardial scintigraphy with Tc-99m DPD. Demographic characteristics, including age, gender, marital status, height, and weight, were collected in a database. Clinical characteristics, such as amyloidosis type (ATTR and AL), serum biomarkers (BNP, troponin), electrocardiographic findings, ultrasound findings, NYHA class, aortic valve replacement, device implants, and medication history, were also collected. Some of the most significant results are presented. Results: A total of 73 cases (86% male) were diagnosed with amyloidosis over four years. The mean age at diagnosis was 82 years, and the main symptom was dyspnea. Most patients suffered from ATTR-CA (65 vs. 8 with AL). Out of all the ATTR-CA patients, 61 were diagnosed with wild-type and 2 with two rare mutations. Twenty-eight patients had systemic amyloidosis with extracardiac involvement, and 32 patients had a history of bilateral carpal tunnel syndrome. Four patients had already developed polyneuropathy, and the diagnosis was confirmed by DPD scintigraphy, which is known for its high sensitivity. Among patients with isolated cardiac involvement, only 6 had left ventricular ejection fraction below 40%. The majority of ATTR patients underwent tafamidis treatment immediately after diagnosis. Conclusion: In conclusion, the experiences shared by the two centers and the continuous exchange of information provide valuable insights into the diagnosis and management of cardiac amyloidosis. Clinical suspicion of amyloidosis and early diagnostic approach are crucial, given the availability of non-invasive techniques. Cardiac scintigraphy with DPD can confirm the presence of the disease without the need for a biopsy. The ultimate goal still remains continuous education and awareness of clinical cardiologists so that this systemic and treatable disease can be diagnosed and certified promptly and treatment can begin as soon as possible.

Keywords: amyloidosis, diagnosis, myocardial scintigraphy, Tc-99m DPD, transthyretin

Procedia PDF Downloads 38

Authors: Simran Baweja, Bhavika Kalal, Surajit Maity

Abstract:

Photoinduced tautomerization reactions have been the centre of attention among the scientific community over the past several decades because of their significance in various biological systems. 7-azaindole (7AI) is considered a model system for DNA base pairing and to understand the role of such tautomerization reactions in mutations. To the best of our knowledge, extensive studies have been carried out on 7-azaindole and its solvent clusters exhibiting proton/ hydrogen transfer in both solution as well as gas phases. Derivatives of the above molecule, like 2,7- and 2,6-diazaindoles are proposed to have even better photophysical properties due to the presence of -aza group on the 2nd position. However, there are studies in the solution phase that suggest the relevance of these molecules, but there are no experimental studies reported in the gas phase yet. In our current investigation, we present the first gas phase spectroscopic data of 2,7-diazaindole (2,7-DAI) and its solvent cluster (2,7-DAI-H2O). In this, we have employed state-of-the-art laser spectroscopic methods such as fluorescence excitation (LIF), dispersed fluorescence (DF), resonant two-photon ionization-time of flight mass spectrometry (2C-R2PI), photoionization efficiency spectroscopy (PIE), IR-UV double resonance spectroscopy, i.e., fluorescence-dip infrared spectroscopy (FDIR) and resonant ion-dip infrared spectroscopy (IDIR) to understand the electronic structure of the molecule. The origin band corresponding to the S1 ← S0 transition of the bare 2,7-DAI is found to be positioned at 33910 cm-1, whereas the origin band corresponding to S1 ← S0 transition of the 2,7-DAI-H2O is positioned at 33074 cm-1. The red-shifted transition in the case of solvent cluster suggests the enhanced feasibility of excited state hydrogen/ proton transfer. The ionization potential for the 2,7-DAI molecule is found to be 8.92 eV which is significantly higher than the previously reported 7AI (8.11 eV) molecule, making it a comparatively complex molecule to study. The ionization potential is reduced by 0.14 eV in the case of 2,7-DAI-H2O (8.78 eV) cluster compared to that of 2,7-DAI. Moreover, on comparison with the available literature values of 7AI, we found the origin band of 2,7-DAI and 2,7-DAI-H2O to be red-shifted by -729 and -280 cm-1 respectively. The ground and excited state N-H stretching frequencies of the 27DAI molecule were determined using fluorescence-dip infrared spectra (FDIR) and resonant ion dip infrared spectroscopy (IDIR), obtained at 3523 and 3467 cm-1, respectively. The lower value of vNH in the electronically excited state of 27DAI implies the higher acidity of the group compared to the ground state. Moreover, we have done extensive computational analysis, which suggests that the energy barrier in the excited state reduces significantly as we increase the number of catalytic solvent molecules (S= H2O, NH3) as well as the polarity of solvent molecules. We found that the ammonia molecule is a better candidate for hydrogen transfer compared to water because of its higher gas-phase basicity. Further studies are underway to understand the excited state dynamics and photochemistry of such N-rich chromophores.

Keywords: excited state hydrogen transfer, supersonic expansion, gas phase spectroscopy, IR-UV double resonance spectroscopy, laser induced fluorescence, photoionization efficiency spectroscopy

Procedia PDF Downloads 41

Authors: Yana Posmitnaya, Galina Rudnitskaya, Tatyana Lukashenko, Anton Bukatin, Anatoly Evstrapov

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An important area of biological and medical research is the study of genetic mutations and polymorphisms that can alter gene function and cause inherited diseases and other diseases. The following methods to analyse DNA fragments are used: capillary electrophoresis and electrophoresis on microfluidic chip (MFC), mass spectrometry with electrophoresis on MFC, hybridization assay on microarray. Electrophoresis on MFC allows to analyse small volumes of samples with high speed and throughput. A soft lithography in polydimethylsiloxane (PDMS) was chosen for operative fabrication of MFCs. A master-form from silicon and photoresist SU-8 2025 (MicroChem Corp.) was created for the formation of micro-sized structures in PDMS. A universal topology which combines T-injector and simple cross was selected for the electrophoretic separation of the sample. Glass K8 and PDMS Sylgard® 184 (Dow Corning Corp.) were used for fabrication of MFCs. Electroosmotic flow (EOF) plays an important role in the electrophoretic separation of the sample. Therefore, the estimate of the quantity of EOF and the ways of its regulation are of interest for the development of the new methods of the electrophoretic separation of biomolecules. The following methods of surface modification were chosen to change EOF: high-frequency (13.56 MHz) plasma treatment in oxygen and argon at low pressure (1 mbar); 1% aqueous solution of polyvinyl alcohol; 3% aqueous solution of Kolliphor® P 188 (Sigma-Aldrich Corp.). The electroosmotic mobility was evaluated by the method of Huang X. et al., wherein the borate buffer was used. The influence of physical and chemical methods of treatment on the wetting properties of the PDMS surface was controlled by the sessile drop method. The most effective way of surface modification of MFCs, from the standpoint of obtaining the smallest value of the contact angle and the smallest value of the EOF, was the processing with aqueous solution of Kolliphor® P 188. This method of modification has been selected for the treatment of channels of MFCs, which are used for the separation of mixture of oligonucleotides fluorescently labeled with the length of chain with 10, 20, 30, 40 and 50 nucleotides. Electrophoresis was performed on the device MFAS-01 (IAI RAS, Russia) at the separation voltage of 1500 V. 6% solution of polydimethylacrylamide with the addition of 7M carbamide was used as the separation medium. The separation time of components of the mixture was determined from electropherograms. The time for untreated MFC was ~275 s, and for the ones treated with solution of Kolliphor® P 188 – ~ 220 s. Research of physical-chemical methods of surface modification of MFCs allowed to choose the most effective way for reducing EOF – the modification with aqueous solution of Kolliphor® P 188. In this case, the separation time of the mixture of oligonucleotides decreased about 20%. The further optimization of method of modification of channels of MFCs will allow decreasing the separation time of sample and increasing the throughput of analysis.

Keywords: electrophoresis, microfluidic chip, modification, nucleic acid, polydimethylsiloxane, soft lithography

Procedia PDF Downloads 384

Authors: Simran Baweja, Bhavika Kalal, Surajit Maity

Abstract:

Photoinduced tautomerization reactions have been the centre of attention among scientific community over past several decades because of their significance in various biological systems. 7-azaindole (7AI) is considered as a model system for DNA base pairing and to understand the role of such tautomerization reactions in mutations. To the best of our knowledge, extensive studies have been carried on 7-azaindole and its solvent clusters exhibiting proton/ hydrogen transfer in both solution as well as gas phase. Derivatives of above molecule, like 2,7- and 2,6-diazaindoles are proposed to have even better photophysical properties due to the presence of -aza group on the 2nd position. However, there are a few studies in the solution phase which suggest the relevance of these molecules, but there are no experimental studies reported in the gas phase yet. In our current investigation, we present the first gas phase spectroscopic data of 2,7-diazaindole (2,7-DAI) and its solvent cluster (2,7-DAI-H2O). In this, we have employed state-of-the-art laser spectroscopic methods such as fluorescence excitation (LIF), dispersed fluorescence (DF), resonant two-photon ionization time of flight mass spectrometry (2C-R2PI), photoionization efficiency spectroscopy (PIE), IR-UV double resonance spectroscopy i.e. fluorescence-dip infrared spectroscopy (FDIR) and resonant ion-dip infrared spectroscopy (IDIR) to understand the electronic structure of the molecule. The origin band corresponding to S1 ← S0 transition of the bare 2,7-DAI is found to be positioned at 33910 cm-1 whereas the origin band corresponding to S1 ← S0 transition of the 2,7-DAI-H2O is positioned at 33074 cm-1. The red shifted transition in case of solvent cluster suggests the enhanced feasibility of excited state hydrogen/ proton transfer. The ionization potential for the 2,7-DAI molecule is found to be 8.92 eV, which is significantly higher that the previously reported 7AI (8.11 eV) molecule, making it a comparatively complex molecule to study. The ionization potential is reduced by 0.14 eV in case of 2,7-DAI-H2O (8.78 eV) cluster compared to that of 2,7-DAI. Moreover, on comparison with the available literature values of 7AI, we found the origin band of 2,7-DAI and 2,7-DAI-H2O to be red shifted by -729 and -280 cm-1 respectively. The ground and excited state N-H stretching frequencies of the 27DAI molecule were determined using fluorescence-dip infrared spectra (FDIR) and resonant ion dip infrared spectroscopy (IDIR), obtained at 3523 and 3467 cm-1, respectively. The lower value of vNH in the electronic excited state of 27DAI implies the higher acidity of the group compared to the ground state. Moreover, we have done extensive computational analysis, which suggests that the energy barrier in excited state reduces significantly as we increase the number of catalytic solvent molecules (S= H2O, NH3) as well as the polarity of solvent molecules. We found that the ammonia molecule is a better candidate for hydrogen transfer compared to water because of its higher gas-phase basicity. Further studies are underway to understand the excited state dynamics and photochemistry of such N-rich chromophores.

Keywords: photoinduced tautomerization reactions, gas phse spectroscopy, ), IR-UV double resonance spectroscopy, resonant two-photon ionization time of flight mass spectrometry (2C-R2PI)

Procedia PDF Downloads 52

Authors: Nourhan Hussein Fanaki, Hoda Mohamed Gamal El-Din Omar, Nihal Kadry Moussa, Eva Adel Edward Farid

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The resistance of staphylococci to various antibiotics has become a major concern for health care professionals. The efficacy of the combinations of selected glycopeptides (vancomycin and teicoplanin) with gentamicin or rifampicin, as well as that of gentamicin/rifampicin combination, was studied against selected pathogenic staphylococcus isolated from Egypt. The molecular distribution of genes conferring resistance to these four antibiotics was detected among tested clinical isolates. Antibiotic combinations were studied using the checkerboard technique and the time-kill assay (in both the stationary and log phases). Induction of resistance to glycopeptides in staphylococci was tried in the absence and presence of diclofenac sodium as inducer. Transmission electron microscopy was used to study the effect of glycopeptides on the ultrastructure of the cell wall of staphylococci. Attempts were made to cure gentamicin resistance plasmids and to study the transfer of these plasmids by conjugation. Trials for the transformation of the successfully isolated gentamicin resistance plasmid to competent cells were carried out. The detection of genes conferring resistance to the tested antibiotics was performed using the polymerase chain reaction. The studied antibiotic combinations proved their efficacy, especially when tested during the log phase. Induction of resistance to glycopeptides in staphylococci was more promising in presence of diclofenac sodium, compared to its absence. Transmission electron microscopy revealed the thickening of bacterial cell wall in staphylococcus clinical isolates due to the presence of tested glycopeptides. Curing of gentamicin resistance plasmids was only successful in 2 out of 9 tested isolates, with a curing rate of 1 percent for each. Both isolates, when used as donors in conjugation experiments, yielded promising conjugation frequencies ranging between 5.4 X 10-2 and 7.48 X 10-2 colony forming unit/donor cells. Plasmid isolation was only successful in one out of the two tested isolates. However, low transformation efficiency (59.7 transformants/microgram plasmid DNA) of such plasmids was obtained. Negative regulators of autolysis, such as arlR, lytR and lrgB, as well as cell-wall associated genes, such as pbp4 and/or pbp2, were detected in staphylococcus isolates with reduced susceptibility to the tested glycopeptides. Concerning rifampicin resistance genes, rpoBstaph was detected in 75 percent of the tested staphylococcus isolates. It could be concluded that in vitro studies emphasized the usefulness of the combination of vancomycin or teicoplanin with gentamicin or rifampicin, as well as that of gentamicin with rifampicin, against staphylococci showing varying resistance patterns. However, further in vivo studies are required to ensure the safety and efficacy of such combinations. Diclofenac sodium can act as an inducer of resistance to glycopeptides in staphylococci. Cell-wall thickness is a major contributor to such resistance among them. Gentamicin resistance in these strains could be chromosomally or plasmid mediated. Multiple mutations in the rpoB gene could mediate staphylococcus resistance to rifampicin.

Keywords: glycopeptides, combinations, induction, diclofenac, transmission electron microscopy, polymerase chain reaction

Procedia PDF Downloads 253

Authors: Binder Hans

Abstract:

Cancer is no longer seen as solely a genetic disease where genetic defects such as mutations and copy number variations affect gene regulation and eventually lead to aberrant cell functioning which can be monitored by transcriptome analysis. It has become obvious that epigenetic alterations represent a further important layer of (de-)regulation of gene activity. For example, aberrant DNA methylation is a hallmark of many cancer types, and methylation patterns were successfully used to subtype cancer heterogeneity. Hence, unraveling the interplay between different omics levels such as genome, transcriptome and epigenome is inevitable for a mechanistic understanding of molecular deregulation causing complex diseases such as cancer. This objective requires powerful downstream integrative bioinformatics methods as an essential prerequisite to discover the whole genome mutational, transcriptome and epigenome landscapes of cancer specimen and to discover cancer genesis, progression and heterogeneity. Basic challenges and tasks arise ‘beyond sequencing’ because of the big size of the data, their complexity, the need to search for hidden structures in the data, for knowledge mining to discover biological function and also systems biology conceptual models to deduce developmental interrelations between different cancer states. These tasks are tightly related to cancer biology as an (epi-)genetic disease giving rise to aberrant genomic regulation under micro-environmental control and clonal evolution which leads to heterogeneous cellular states. Machine learning algorithms such as self organizing maps (SOM) represent one interesting option to tackle these bioinformatics tasks. The SOMmethod enables recognizing complex patterns in large-scale data generated by highthroughput omics technologies. It portrays molecular phenotypes by generating individualized, easy to interpret images of the data landscape in combination with comprehensive analysis options. Our image-based, reductionist machine learning methods provide one interesting perspective how to deal with massive data in the discovery of complex diseases, gliomas, melanomas and colon cancer on molecular level. As an important new challenge, we address the combined portrayal of different omics data such as genome-wide genomic, transcriptomic and methylomic ones. The integrative-omics portrayal approach is based on the joint training of the data and it provides separate personalized data portraits for each patient and data type which can be analyzed by visual inspection as one option. The new method enables an integrative genome-wide view on the omics data types and the underlying regulatory modes. It is applied to high and low-grade gliomas and to melanomas where it disentangles transversal and longitudinal molecular heterogeneity in terms of distinct molecular subtypes and progression paths with prognostic impact.

Keywords: integrative bioinformatics, machine learning, molecular mechanisms of cancer, gliomas and melanomas

Procedia PDF Downloads 117

Authors: Siddhant Sethi, Yasuharu Takashima, Shigetaka Nakamura, Kenzo Fujimoto

Abstract:

Site-directed mutagenesis is a renowned technique to introduce specific mutations in the genome. To achieve site-directed mutagenesis, many chemical and enzymatic approaches have been reported in the past like disulphite induced genome editing, CRISPR-Cas9, TALEN etc. The chemical methods are invasive whereas the enzymatic approaches are time-consuming and expensive. Most of these techniques are unusable in the cellular application due to their toxicity and other limitations. Photo-chemical cytosine deamination, introduced in 2010, is one of the major technique for enzyme-free single-point mutation of cytosine to uracil in DNA and RNA, wherein, 3-cyanovinylcarbazole nucleoside (CNVK) containing oligodeoxyribonucleotide (ODN) having CNVK at -1 position to that of target cytosine is reversibly crosslinked to target DNA strand using 366 nm and then incubated at 90ºC to accommodate deamination. This technique is superior to enzymatic methods of site-directed mutagenesis but has a disadvantage that it requires the use of high temperature for the deamination step which restricts its applicability in the in vivo applications. This study has been focused on improving the technique by reducing the temperature required for deamination. Firstly, the photo-cross-linker, CNVK has been modified by replacing cyano group attached to vinyl group with methyl ester (OMeVK), amide (NH2VK), and carboxylic acid (OHVK) to observe the acceleration in the deamination of target cytosine cross-linked to vinylcarbazole derivative. Among the derivatives, OHVK has shown 2 times acceleration in deamination reaction as compared to CNVK, while the other two derivatives have shown deceleration towards deamination reaction. The trend of rate of deamination reaction follows the same order as that of hydrophilicity of the vinylcarbazole derivatives. OHVK being most hydrophilic has shown highest acceleration while OMeVK is least hydrophilic has proven to be least active for deamination. Secondly, in the related study, the counter-base of the target cytosine, guanine has been replaced by inosine, 2-aminopurine, nebularine, and 5-nitroindole having distinct hydrogen bonding patterns with target cytosine. Among the ODNs with these counter bases, ODN with inosine has shown 12 fold acceleration towards deamination of cytosine cross-linked to CNVK at physiological conditions as compared to guanosine. Whereas, when 2-aminopurine, nebularine, and 5-nitroindole were used, no deamination reaction took place. It can be concluded that inosine has potential to be used as the counter base of target cytosine for the CNVK mediated photo-cross-linking induced deamination of cytosine. The increase in rate of deamination reaction has been attributed to pattern and number of hydrogen bonding between the cytosine and counter base. One of the important factor is presence of hydrogen bond between exo-cyclic amino group of cytosine and the counter base. These results will be useful for development of more efficient technique for site-directed mutagenesis for C → U transformations in the DNA/RNA which might be used in the living system for treatment of various genetic disorders and genome engineering for making designer and non-native proteins.

Keywords: C to U transformation, DNA editing, genome engineering, ultra-fast photo-cross-linking

Procedia PDF Downloads 209

Authors: S. Najafi, E. Bertini, M. Pezzotti, G.B. Tornielli, S. Zenoni

Abstract:

Grapevine is an important agricultural fruit crop plant consumed worldwide and with a key role in the global economy. Grapevine is strongly affected by both biotic and abiotic stresses, which impact grape growth at different stages, such as during plant and berry development and pre- and post-harvest, consequently causing significant economic losses. Recently global warming has propelled the anticipation of the onset of berry ripening, determining the reduction of a grape color and increased volatilization of aroma compounds. Climate change could negatively alter the physiological characteristics of the grape and affect the berry and wine quality. Modern plant breeding can provide tools such as genome editing for improving grape resilience traits while maintaining intact the viticultural and oenological quality characteristics of the genotype. This study aims at developing a platform for genome editing application in grapevine plants with the final goal to improve berry quality, biotic, and abiotic resilience traits. We chose to directly deliver ribonucleoproteins (RNP, preassembled Cas protein and guide RNA) into plant protoplasts, and, from these cell structures, regenerate grapevine plants edited in specific selected genes controlling traits of interest. Edited plants regenerated by somatic embryogenesis from protoplasts will then be sequenced and molecularly characterized. Embryogenic calli of Sultana and Shiraz cultivars were initiated from unopened leaves of in-vitro shoot tip cultures and from stamens, respectively. Leaves were placed on NB2 medium while stamens on callus initiation medium (PIV) medium and incubated in the dark at 28 °C for three months. Viable protoplasts, tested by FDA staining, isolated from embryogenic calli were cultured by disc method at 1*105 protoplasts/ml. Mature well-shaped somatic embryos developed directly in the protoplast culture medium two months later and were transferred in the light into to shooting medium for further growth. Regenerated plants were then transferred to the greenhouse; no phenotypic alterations were observed when compared to non in-vitro cultured plants. The performed experiments allowed to established an efficient protocol of embryogenic calli production, protoplast isolation, and regeneration of the whole plant through somatic embryogenesis in both Sultana and Shiraz. Regenerated plants, through direct somatic embryogenesis deriving from a single cell, avoid the risk of chimerism during the regeneration process, therefore improving the genome editing process. As pre-requisite of genome editing, an efficient method for transfection of protoplast by yellow fluorescent protein (YFP) marker genes was also established and experiments of direct delivery of CRISPR–Cas9 ribonucleoproteins (RNPs) in protoplasts to achieve efficient DNA-free targeted mutations are in progress.

Keywords: CRISPR-cas9, plant regeneration, protoplast isolation, Vitis vinifera

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Authors: Igor A. Krichtafovitch

Abstract:

The evolutionary processes are not linear. Long periods of quiet and slow development turn to rather rapid emergences of new species and even phyla. During Cambrian explosion, 22 new phyla were added to the previously existed 3 phyla. Contrary to the common credence the natural selection or a survival of the fittest cannot be accounted for the dominant evolution vector which is steady and accelerated advent of more complex and more intelligent living organisms. Neither Darwinism nor alternative concepts including panspermia and intelligent design propose a satisfactory solution for these phenomena. The proposed hypothesis offers a logical and plausible explanation of the evolutionary processes in general. It is based on two postulates: a) the Biosphere is a single living organism, all parts of which are interconnected, and b) the Biosphere acts as a giant biological supercomputer, storing and processing the information in digital and analog forms. Such supercomputer surpasses all human-made computers by many orders of magnitude. Living organisms are the product of intelligent creative action of the biosphere supercomputer. The biological evolution is driven by growing amount of information stored in the living organisms and increasing complexity of the biosphere as a single organism. Main evolutionary vector is not a survival of the fittest but an accelerated growth of the computational complexity of the living organisms. The following postulates may summarize the proposed hypothesis: biological evolution as a natural life origin and development is a reality. Evolution is a coordinated and controlled process. One of evolution’s main development vectors is a growing computational complexity of the living organisms and the biosphere’s intelligence. The intelligent matter which conducts and controls global evolution is a gigantic bio-computer combining all living organisms on Earth. The information is acting like a software stored in and controlled by the biosphere. Random mutations trigger this software, as is stipulated by Darwinian Evolution Theories, and it is further stimulated by the growing demand for the Biosphere’s global memory storage and computational complexity. Greater memory volume requires a greater number and more intellectually advanced organisms for storing and handling it. More intricate organisms require the greater computational complexity of biosphere in order to keep control over the living world. This is an endless recursive endeavor with accelerated evolutionary dynamic. New species emerge when two conditions are met: a) crucial environmental changes occur and/or global memory storage volume comes to its limit and b) biosphere computational complexity reaches critical mass capable of producing more advanced creatures. The hypothesis presented here is a naturalistic concept of life creation and evolution. The hypothesis logically resolves many puzzling problems with the current state evolution theory such as speciation, as a result of GM purposeful design, evolution development vector, as a need for growing global intelligence, punctuated equilibrium, happening when two above conditions a) and b) are met, the Cambrian explosion, mass extinctions, happening when more intelligent species should replace outdated creatures.

Keywords: supercomputer, biological evolution, Darwinism, speciation

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Authors: T. C. C. Soo, S. Bhassu

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Unlike the common presence of both innate and adaptive immunity in vertebrates, crustaceans, in particular, shrimps, have been discovered to possess only innate immunity. This further emphasizes the importance of innate immunity within shrimps in pathogenic resistance. Under the study of pathogenic immune challenge, different shrimp species actually exhibit varying degrees of immune resistance towards the same pathogen. Furthermore, even within the same shrimp species, different batches of challenged shrimps can have different strengths of immune defence. Several important pathways are activated within shrimps during pathogenic infection. One of them is JAK-STAT pathway that is activated during bacterial, viral and fungal infections by which STAT(Signal Transducer and Activator of Transcription) gene is the core element of the pathway. Based on theory of Central Dogma, the genomic information is transmitted in the order of DNA, RNA and protein. This study is focused in uncovering the important evolutionary patterns present within the DNA (non-coding region) and RNA (coding region). The three shrimp species involved are Macrobrachium rosenbergii, Penaeus monodon and Litopenaeus vannamei which all possess commercial significance. The shrimp species were challenged with a famous penaeid shrimp virus called white spot syndrome virus (WSSV) which can cause serious lethality. Tissue samples were collected during time intervals of 0h, 3h, 6h, 12h, 24h, 36h and 48h. The DNA and RNA samples were then extracted using conventional kits from the hepatopancreas tissue samples. PCR technique together with designed STAT gene conserved primers were utilized for identification of the STAT coding sequences using RNA-converted cDNA samples and subsequent characterization using various bioinformatics approaches including Ramachandran plot, ProtParam and SWISS-MODEL. The varying levels of immune STAT gene activation for the three shrimp species during WSSV infection were confirmed using qRT-PCR technique. For one sample, three biological replicates with three technical replicates each were used for qRT-PCR. On the other hand, DNA samples were important for uncovering the structural variations within the genomic region of STAT gene which would greatly assist in understanding the STAT protein functional variations. The partially-overlapping primers technique was used for the genomic region sequencing. The evolutionary inferences and event predictions were then conducted through the Bayesian Inference method using all the acquired coding and non-coding sequences. This was supplemented by the construction of conventional phylogenetic trees using Maximum likelihood method. The results showed that adaptive evolution caused STAT gene sequence mutations between different shrimp species which led to evolutionary divergence event. Subsequently, the divergent sites were correlated to the differing expressions of STAT gene. Ultimately, this study assists in knowing the shrimp species innate immune variability and selection of disease resistant shrimps for breeding purpose. The deeper understanding of STAT gene evolution from the perspective of both purifying and adaptive approaches not only can provide better immunological insight among shrimp species, but also can be used as a good reference for immunological studies in humans or other model organisms.

Keywords: gene evolution, JAK-STAT pathway, immunology, STAT gene

Procedia PDF Downloads 116

Authors: Ved Vrat Verma, Rani Gupta, Manisha Goel

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γ-glutamyltranspeptidase (GGT: EC 2.3.2.2) is an N-terminal nucleophile hydrolase conserved in all three domains of life. GGT plays a key role in glutathione metabolism where it catalyzes the breakage of the γ-glutamyl bonds and transfer of γ-glutamyl group to water (hydrolytic activity) or amino acids or short peptides (transpeptidase activity). GGTs from bacteria, archaea, and eukaryotes (human, rat and mouse) are homologous proteins sharing >50% sequence similarity and conserved four layered αββα sandwich like three dimensional structural fold. These proteins though similar in their structure to each other, are quite diverse in their enzyme activity: some GGTs are better at hydrolysis reactions but poor in transpeptidase activity, whereas many others may show opposite behaviour. GGT is known to be involved in various diseases like asthma, parkinson, arthritis, and gastric cancer. Its inhibition prior to chemotherapy treatments has been shown to sensitize tumours to the treatment. Microbial GGT is known to be a virulence factor too, important for the colonization of bacteria in host. However, all known inhibitors (mimics of its native substrate, glutamate) are highly toxic because they interfere with other enzyme pathways. However, a few successful efforts have been reported previously in designing species specific inhibitors. We aim to leverage the diversity seen in GGT family (pathogen vs. eukaryotes) for designing specific inhibitors. Thus, in the present study, we have used DIVERGE software to identify sites in GGT proteins, which are crucial for the functional and structural divergence of these proteins. Since, type II divergence sites vary in clade specific manner, so type II divergent sites were our focus of interest throughout the study. Type II divergent sites were identified for pathogen vs. eukaryotes clusters and sites were marked on clade specific representative structures HpGGT (2QM6) and HmGGT (4ZCG) of pathogen and eukaryotes clade respectively. The crucial divergent sites within 15 A radii of the binding cavity were highlighted, and in-silico mutations were performed on these sites to delineate the role of these sites on the mechanism of catalysis and protein folding. Further, the amino acid network (AAN) analysis was also performed by Cytoscape to delineate assortative mixing for cavity divergent sites which could strengthen our hypothesis. Additionally, molecular dynamics simulations were performed for wild complexes and mutant complexes close to physiological conditions (pH 7.0, 0.1 M ionic strength and 1 atm pressure) and the role of putative divergence sites and structural integrities of the homologous proteins have been analysed. The dynamics data were scrutinized in terms of RMSD, RMSF, non-native H-bonds and salt bridges. The RMSD, RMSF fluctuations of proteins complexes are compared, and the changes at protein ligand binding sites were highlighted. The outcomes of our study highlighted some crucial divergent sites which could be used for novel inhibitors designing in a species-specific manner. Since, for drug development, it is challenging to design novel drug by targeting similar protein which exists in eukaryotes, so this study could set up an initial platform to overcome this challenge and help to deduce the more effective targets for novel drug discovery.

Keywords: γ-glutamyltranspeptidase, divergence, species-specific, drug design

Procedia PDF Downloads 236

Authors: Bianca Peterson, Tomasz J. Sańko, Carlos C. Bezuidenhout, Johnnie Van Den Berg

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Busseola fusca (Fuller) (Lepidoptera: Noctuidae), the maize stem borer, is a major pest in sub-Saharan Africa. It causes economic damage to maize and sorghum crops and has evolved non-recessive resistance to genetically modified (GM) maize expressing the Cry1Ab insecticidal toxin. Since B. fusca is a non-model organism, very little genomic information is publicly available, and is limited to some cytochrome c oxidase I, cytochrome b, and microsatellite data. The biology of B. fusca is well-described, but still poorly understood. This, in combination with its larval-specific behavior, may pose problems for limiting the spread of current resistant B. fusca populations or preventing resistance evolution in other susceptible populations. As part of on-going research into resistance evolution, B. fusca larvae were collected from Bt and non-Bt maize in South Africa, followed by RNA isolation (15 specimens) and sequencing on the Illumina HiSeq 2500 platform. Quality of reads was assessed with FastQC, after which Trimmomatic was used to trim adapters and remove low quality, short reads. Trinity was used for the de novo assembly, whereas TransRate was used for assembly quality assessment. Transcript identification employed BLAST (BLASTn, BLASTp, and tBLASTx comparisons), for which two libraries (nucleotide and protein) were created from 3.27 million lepidopteran sequences. Several transcripts that have previously been implicated in Cry toxin resistance was identified for B. fusca. These included aminopeptidase N, cadherin, alkaline phosphatase, ATP-binding cassette transporter proteins, and mitogen-activated protein kinase. MEGA7 was used to align these transcripts to reference sequences from Lepidoptera to detect mutations that might potentially be contributing to Cry toxin resistance in this pest. RSEM and Bioconductor were used to perform differential gene expression analysis on groups of B. fusca larvae challenged and unchallenged with the Cry1Ab toxin. Pairwise expression comparisons of transcripts that were at least 16-fold expressed at a false-discovery corrected statistical significance (p) ≤ 0.001 were extracted and visualized in a hierarchically clustered heatmap using R. A total of 329,194 transcripts with an N50 of 1,019 bp were generated from the over 167.5 million high-quality paired-end reads. Furthermore, 110 transcripts were over 10 kbp long, of which the largest one was 29,395 bp. BLAST comparisons resulted in identification of 157,099 (47.72%) transcripts, among which only 3,718 (2.37%) were identified as Cry toxin receptors from lepidopteran insects. According to transcript expression profiles, transcripts were grouped into three subclusters according to the similarity of their expression patterns. Several immune-related transcripts (pathogen recognition receptors, antimicrobial peptides, and inhibitors) were up-regulated in the larvae feeding on Bt maize, indicating an enhanced immune status in response to toxin exposure. Above all, extremely up-regulated arylphorin genes suggest that enhanced epithelial healing is one of the resistance mechanisms employed by B. fusca larvae against the Cry1Ab toxin. This study is the first to provide a resource base and some insights into a potential mechanism of Cry1Ab toxin resistance in B. fusca. Transcriptomic data generated in this study allows identification of genes that can be targeted by biotechnological improvements of GM crops.

Keywords: epithelial healing, Lepidoptera, resistance, transcriptome

Procedia PDF Downloads 164

Authors: Maddalena Arigoni, Maria Luisa Ratto, Raffaele A. Calogero, Luca Alessandri

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The presence of tumor heterogeneity, where distinct cancer cells exhibit diverse morphological and phenotypic profiles, including gene expression, metabolism, and proliferation, poses challenges for molecular prognostic markers and patient classification for targeted therapies. Understanding the causes and progression of cancer requires research efforts aimed at characterizing heterogeneity, which can be facilitated by evolving single-cell sequencing technologies. However, analyzing single-cell data necessitates computational methods that often lack objective validation. Therefore, the establishment of benchmarking datasets is necessary to provide a controlled environment for validating bioinformatics tools in the field of single-cell oncology. Benchmarking bioinformatics tools for single-cell experiments can be costly due to the high expense involved. Therefore, datasets used for benchmarking are typically sourced from publicly available experiments, which often lack a comprehensive cell annotation. This limitation can affect the accuracy and effectiveness of such experiments as benchmarking tools. To address this issue, we introduce omics benchmark experiments designed to evaluate bioinformatics tools to depict the heterogeneity in single-cell tumor experiments. We conducted single-cell RNA sequencing on six lung cancer tumor cell lines that display resistant clones upon treatment of EGFR mutated tumors and are characterized by driver genes, namely ROS1, ALK, HER2, MET, KRAS, and BRAF. These driver genes are associated with downstream networks controlled by EGFR mutations, such as JAK-STAT, PI3K-AKT-mTOR, and MEK-ERK. The experiment also featured an EGFR-mutated cell line. Using 10XGenomics platform with cellplex technology, we analyzed the seven cell lines together with a pseudo-immunological microenvironment consisting of PBMC cells labeled with the Biolegend TotalSeq™-B Human Universal Cocktail (CITEseq). This technology allowed for independent labeling of each cell line and single-cell analysis of the pooled seven cell lines and the pseudo-microenvironment. The data generated from the aforementioned experiments are available as part of an online tool, which allows users to define cell heterogeneity and generates count tables as an output. The tool provides the cell line derivation for each cell and cell annotations for the pseudo-microenvironment based on CITEseq data by an experienced immunologist. Additionally, we created a range of pseudo-tumor tissues using different ratios of the aforementioned cells embedded in matrigel. These tissues were analyzed using 10XGenomics (FFPE samples) and Curio Bioscience (fresh frozen samples) platforms for spatial transcriptomics, further expanding the scope of our benchmark experiments. The benchmark experiments we conducted provide a unique opportunity to evaluate the performance of bioinformatics tools for detecting and characterizing tumor heterogeneity at the single-cell level. Overall, our experiments provide a controlled and standardized environment for assessing the accuracy and robustness of bioinformatics tools for studying tumor heterogeneity at the single-cell level, which can ultimately lead to more precise and effective cancer diagnosis and treatment.

Keywords: single cell omics, benchmark, spatial transcriptomics, CITEseq

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Authors: Jaco Oosthuizen, Nerina C. Van Der Merwe

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The National Health Laboratory Services in Bloemfontien has been the diagnostic testing facility for 1830 patients for familial breast cancer since 1997. From the cohort, 540 were comprehensively screened using High-Resolution Melting Analysis or Next Generation Sequencing for the presence of point mutations and/or indels. Approximately 90% of these patients stil remain undiagnosed as they are BRCA1/2 negative. Multiplex ligation-dependent probe amplification was initially added to screen for copy number variation detection, but with the introduction of next generation sequencing in 2017, was substituted and is currently used as a confirmation assay. The aim was to investigate the viability of utilizing internationally designed copy number variation detection assays based on mostly European/Caucasian genomic data for use within a South African context. The multiplex ligation-dependent probe amplification technique is based on the hybridization and subsequent ligation of multiple probes to a targeted exon. The ligated probes are amplified using conventional polymerase chain reaction, followed by fragment analysis by means of capillary electrophoresis. The experimental design of the assay was performed according to the guidelines of MRC-Holland. For BRCA1 (P002-D1) and BRCA2 (P045-B3), both multiplex assays were validated, and results were confirmed using a secondary probe set for each gene. The next generation sequencing technique is based on target amplification via multiplex polymerase chain reaction, where after the amplicons are sequenced parallel on a semiconductor chip. Amplified read counts are visualized as relative copy numbers to determine the median of the absolute values of all pairwise differences. Various experimental parameters such as DNA quality, quantity, and signal intensity or read depth were verified using positive and negative patients previously tested internationally. DNA quality and quantity proved to be the critical factors during the verification of both assays. The quantity influenced the relative copy number frequency directly whereas the quality of the DNA and its salt concentration influenced denaturation consistency in both assays. Multiplex ligation-dependent probe amplification produced false positives due to ligation failure when ligation was inhibited due to a variant present within the ligation site. Next generation sequencing produced false positives due to read dropout when primer sequences did not meet optimal multiplex binding kinetics due to population variants in the primer binding site. The analytical sensitivity and specificity for the South African population have been proven. Verification resulted in repeatable reactions with regards to the detection of relative copy number differences. Both multiplex ligation-dependent probe amplification and next generation sequencing multiplex panels need to be optimized to accommodate South African polymorphisms present within the genetically diverse ethnic groups to reduce the false copy number variation positive rate and increase performance efficiency.

Keywords: familial breast cancer, multiplex ligation-dependent probe amplification, next generation sequencing, South Africa

Procedia PDF Downloads 187