Search results for: primer sequences

Authors: Brianna M. Soule, Jesslyn A. Bryk-Lucy, Linda M. Ritchie

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Colic, defined as abdominal pain in the horse, has several known predisposing factors. Decreased water intake has been shown to predispose equines to impaction colic. The objective of this study was to determine if offering flavored water (sweet feed or banana extract) would increase voluntary water intake in horses to serve as an assessable, noninvasive method for farm managers, veterinarians, or owners to decrease the risk of impaction colic. An a priori power analysis, which was conducted using G*Power version 3.1.9.7, indicated that the minimum sample size required to achieve 80% power for detecting a large effect at a significance level of α = .05 was 19 horses for a one-way repeated measures ANOVA with three treatment levels and assuming a non-sphericity correction of ε=0.5. After a three-day control period, 21 horses were randomly divided into two sequences and offered either banana or sweet feed flavored water. Horses always had a bucket of unflavored water available. A repeated measure study design was used to measure water consumption of each horse over a 62-hour period. A one-way repeated measures ANOVA was conducted to determine whether there were statistically significant differences among the means for the three-day average water intake (ml/kg). Although not statistically significant (F(2, 38) = 1.28, p = .290, partial η2 = .063), the three-day average water intake was largest for banana flavored water (M = 53.51, SD = 9.25 ml/kg), followed by sweet feed (M = 52.93, SD = 11.99 ml/kg), and, finally, unflavored water (M = 50.40, SD = 10.82 ml/kg). Paired-samples t-tests were used to determine whether there was a statistically significant difference between the three-day average water intake (ml/kg) for flavored versus unflavored water. The average unflavored water intake (M = 29.3 ml/kg, SD = 8.9) over the measurement period was greater than the banana flavored water (M = 27.7 ml/kg, SD = 9.8), but the average consumption of the sweet feed flavored water (M = 30.4 ml/kg, SD = 14.6) was greater than unflavored water (M = 24.3 ml/kg, SD = 11.4). None of these differences in average intake were statistically significant (p > .244). Future research is warranted to determine if other flavors significantly increase voluntary water intake in horses.

Keywords: colic, equine, equine science, water intake, flavored water, horses, equine management, equine health, horse health, horse health care management, colic prevention

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Authors: Mukul Sharma, Madhusmita Das, Sundeep Chaitanya Vedithi

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Leprosy is a chronic human disease caused by Mycobacterium leprae, that cannot be cultured in vitro. Though treatable with multidrug therapy (MDT), recently, bacteria reported resistance to multiple antibiotics. Targeting DNA replication and repair pathways can serve as the foundation of developing new anti-leprosy drugs. Due to the absence of an axenic culture medium for the propagation of M. leprae, studying cellular processes, especially those belonging to DNA repair pathways, is challenging. Genomic understanding of M. Leprae harbors several protein-coding genes with no previously assigned function known as 'hypothetical proteins'. Here, we report identification and expression of known and hypothetical DNA repair genes from a human skin biopsy and mouse footpads that are involved in base excision repair, direct reversal repair, and SOS response. Initially, a bioinformatics approach was employed based on sequence similarity, identification of known protein domains to screen the hypothetical proteins in the genome of M. leprae, that are potentially related to DNA repair mechanisms. Before testing on clinical samples, pure stocks of bacterial reference DNA of M. leprae (NHDP63 strain) was used to construct standard graphs to validate and identify lower detection limit in the qPCR experiments. Primers were designed to amplify the respective transcripts, and PCR products of the predicted size were obtained. Later, excisional skin biopsies of newly diagnosed untreated, treated, and drug resistance leprosy cases from SIHR & LC hospital, Vellore, India were taken for the extraction of RNA. To determine the presence of the predicted transcripts, cDNA was generated from M. leprae mRNA isolated from clinically confirmed leprosy skin biopsy specimen across all the study groups. Melting curve analysis was performed to determine the integrity of the amplification and to rule out primer‑dimer formation. The Ct values obtained from qPCR were fitted to standard curve to determine transcript copy number. Same procedure was applied for M. leprae extracted after processing a footpad of nude mice of drug sensitive and drug resistant strains. 16S rRNA was used as positive control. Of all the 16 genes involved in BER, DR, and SOS, differential expression pattern of the genes was observed in terms of Ct values when compared to human samples; this was because of the different host and its immune response. However, no drastic variation in gene expression levels was observed in human samples except the nth gene. The higher expression of nth gene could be because of the mutations that may be associated with sequence diversity and drug resistance which suggests an important role in the repair mechanism and remains to be explored. In both human and mouse samples, SOS system – lexA and RecA, and BER genes AlkB and Ogt were expressing efficiently to deal with possible DNA damage. Together, the results of the present study suggest that DNA repair genes are constitutively expressed and may provide a reference for molecular diagnosis, therapeutic target selection, determination of treatment and prognostic judgment in M. leprae pathogenesis.

Keywords: DNA repair, human biopsy, hypothetical proteins, mouse footpads, Mycobacterium leprae, qPCR

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Authors: Maneesh Kumar, Roshan Kamal Topno, Manas Ranjan Dikhit, Vahab Ali, Ganesh Chandra Sahoo, Bhawana, Major Madhukar, Rishikesh Kumar, Krishna Pandey, Pradeep Das

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Chandipura vesiculovirus is an emerging (-) ssRNA viral entity belonging to the genus Vesiculovirus of the family Rhabdoviridae, associated with fatal encephalitis in tropical regions. The multi-functionally active viral RNA-dependent RNA polymerase (vRdRp) that has been incorporated with conserved amino acid residues in the pathogens, assigned to synthesize distinct viral polypeptides. The lack of proofreading ability of the vRdRp produces many mutated variants. Here, we have performed the evolutionary analysis of 20 viral protein sequences of vRdRp of different strains of Chandipura vesiculovirus along with other viral species from genus Vesiculovirus inferred in MEGA6.06, employing the Neighbour-Joining method. The p-distance algorithmic method has been used to calculate the optimum tree which showed the sum of branch length of about 1.436. The percentage of replicate trees in which the associated taxa are clustered together in the bootstrap test (1000 replicates), is shown next to the branches. No mutation was observed in the Indian strains of Chandipura vesiculovirus. In vRdRp, 1230(His) and 1231(Arg) are actively participated in catalysis and, are found conserved in different strains of Chandipura vesiculovirus. Both amino acid residues were also conserved in the other viral species from genus Vesiculovirus. Many isolates exhibited maximum number of mutations in catalytic regions in strains of Chandipura vesiculovirus at position 26(Ser→Ala), 47 (Ser→Ala), 90(Ser→Tyr), 172(Gly→Ile, Val), 172(Ser→Tyr), 387(Asn→Ser), 1301(Thr→Ala), 1330(Ala→Glu), 2015(Phe→Ser) and 2065(Thr→Val) which make them variants under different tropical conditions from where they evolved. The result clarifies the actual concept of RNA evolution using vRdRp to develop as an evolutionary marker. Although, a limited number of vRdRp protein sequence similarities for Chandipura vesiculovirus and other species. This might endow with possibilities to identify the virulence level during viral multiplication in a host.

Keywords: Chandipura, (-) ssRNA, viral RNA-dependent RNA polymerase, neighbour-joining method, p-distance algorithmic, evolutionary marker

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Authors: Fumihiro Ima, Shinichi Watanabe, Shingo Maeda, Haruna Imai, Hiroki Niimi

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It is important to know growth rate of brain tumors before surgery because it influences treatment planning including not only surgical resection strategy but also adjuvant therapy after surgery. Amide proton transfer (APT) imaging is an emerging molecular magnetic resonance imaging (MRI) technique based on chemical exchange saturation transfer without administration of contrast medium. The underlying assumption in APT imaging of tumors is that there is a close relationship between the proliferative activity of the tumor and mobile protein synthesis. We aimed to evaluate the diagnostic performance of APT imaging of pre-and post-treatment brain tumors. Ten patients with brain tumor underwent conventional and APT-weighted sequences on a 3.0 Tesla MRI before clinical intervention. The maximum and the minimum APT-weighted signals (APTWmax and APTWmin) in each solid tumor region were obtained and compared before and after clinical intervention. All surgical specimens were examined for histopathological diagnosis. Eight of ten patients underwent adjuvant therapy after surgery. Histopathological diagnosis was glioma in 7 patients (WHO grade 2 in 2 patients, WHO grade 3 in 3 patients and WHO grade 4 in 2 patients), meningioma WHO grade1 in 2 patients and primary lymphoma of the brain in 1 patient. High-grade gliomas showed significantly higher APTW-signals than that in low-grade gliomas. APTWmax in one huge parasagittal meningioma infiltrating into the skull bone was higher than that in glioma WHO grade 4. On the other hand, APTWmax in another convexity meningioma was the same as that in glioma WHO grade 3. Diagnosis of primary lymphoma of the brain was possible with APT imaging before pathological confirmation. APTW-signals in residual tumors decreased dramatically within one year after adjuvant therapy in all patients. APT imaging demonstrated excellent diagnostic performance for the planning of surgery and adjuvant therapy of brain tumors.

Keywords: amides, magnetic resonance imaging, brain tumors, cell proliferation

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Authors: Fumihiro Imai, Shinichi Watanabe, Shingo Maeda, Haruna Imai, Hiroki Niimi

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It is important to know the growth rate of brain tumors before surgery because it influences treatment planning, including not only surgical resection strategy but also adjuvant therapy after surgery. Amide proton transfer (APT) imaging is an emerging molecular magnetic resonance imaging (MRI) technique based on chemical exchange saturation transfer without the administration of a contrast medium. The underlying assumption in APT imaging of tumors is that there is a close relationship between the proliferative activity of the tumor and mobile protein synthesis. We aimed to evaluate the diagnostic performance of APT imaging of pre-and post-treatment brain tumors. Ten patients with brain tumor underwent conventional and APT-weighted sequences on a 3.0 Tesla MRI before clinical intervention. The maximum and the minimum APT-weighted signals (APTWmax and APTWmin) in each solid tumor region were obtained and compared before and after a clinical intervention. All surgical specimens were examined for histopathological diagnosis. Eight of ten patients underwent adjuvant therapy after surgery. Histopathological diagnosis was glioma in 7 patients (WHO grade 2 in 2 patients, WHO grade 3 in 3 patients, and WHO grade 4 in 2 patients), meningioma WHO grade 1 in 2 patients, and primary lymphoma of the brain in 1 patient. High-grade gliomas showed significantly higher APTW signals than that low-grade gliomas. APTWmax in one huge parasagittal meningioma infiltrating into the skull bone was higher than that in glioma WHO grade 4. On the other hand, APTWmax in another convexity meningioma was the same as that in glioma WHO grade 3. Diagnosis of primary lymphoma of the brain was possible with APT imaging before pathological confirmation. APTW signals in residual tumors decreased dramatically within one year after adjuvant therapy in all patients. APT imaging demonstrated excellent diagnostic performance for the planning of surgery and adjuvant therapy of brain tumors.

Keywords: amides, magnetic resonance imaging, brain tumors, cell proliferation

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Authors: Mohammad Nazri Abdul Bahari, Nurshafika Mohd Sakeh, Siti Nor Akmar Abdullah

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Basal stem rot (BSR) is the most devastating disease in oil palm. Among the oil palm pathogenic fungi, the most prevalent and virulent species associated with BSR is Ganoderma boninense. Early detection of G. boninense attack in oil palm wherein physical symptoms has not yet appeared can offer opportunities to prevent the spread of the necrotrophic fungus. However, poor understanding of molecular defense responses and roles of antifungal metabolites in oil palm against G. boninense has complicated the resolving measures. Hence, characterization of defense-related molecular responses and production of antifungal compounds during early interaction with G. boninense is of utmost important. Four month-old oil palm (Elaeis guineensis) seedlings were artificially infected with G. boninense-inoculated rubber wood block via sitting technique. RNA of samples were extracted from roots and leaves tissues at 0, 3, 7 and 11 days post inoculation (d.p.i) followed with sequencing using RNA-Seq method. Differentially-expressed genes (DEGs) of oil palm-G. boninense interaction were identified, while changes in metabolite profile will be scrutinized related to the DEGs. The RNA-Seq data generated a total of 113,829,376 and 313,293,229 paired-end clean reads from untreated (0 d.p.i) and treated (3, 7, 11 d.p.i) samples respectively, each with two biological replicates. The paired-end reads were mapped to Elaeis guineensis reference genome to screen out non-oil palm genes and subsequently generated 74,794 coding sequences. DEG analysis of phytohormone biosynthetic genes in oil palm roots revealed that at p-value ≤ 0.01, ethylene and jasmonic acid may act in antagonistic manner with salicylic acid to coordinate defense response at early interaction with G. boninense. Findings on metabolite profiling of G. boninense-infected oil palm roots and leaves are hoped to explain the defense-related compounds elicited by Elaeis guineensis in response to G. boninense colonization. The study aims to shed light on molecular defense response of oil palm at early interaction with G. boninense and promote prevention measures against Ganoderma infection.

Keywords: Ganoderma boninense, metabolites, phytohormones, RNA-Seq

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Authors: Wen Zhen

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Compared to content words, function words have been relatively overlooked by English learners especially articles. The article system, to a certain extent, becomes a resistance to know English better, driven by different elements. Three principal factors can be summarized in term of the nature of the articles when referring to the difficulty of the English article system. However, making the article system more complex are difficulties in the second acquisition process, for [-ART] learners have to create another category, causing even most non-native speakers at proficiency level to make errors. According to the sequences of acquisition of the English article, it is showed that the zero article is first acquired and in high inaccuracy. The zero article is often overused in the early stages of L2 acquisition. Although learners at the intermediate level move to underuse the zero article for they realize that the zero article does not cover any case, overproduction of the zero article even occurs among advanced L2 learners. The aim of the study is to investigate noun-phrase factors which give rise to incorrect usage or overuse of the zero article, thus providing suggestions for L2 English acquisition. Moreover, it enables teachers to carry out effective instruction that activate conscious learning of students. The research question will be answered through a corpus-based, data- driven approach to analyze the noun-phrase elements from the semantic context and countability of noun-phrases. Based on the analysis of the International Thurber Thesis corpus, the results show that: (1) Although context of [-definite,-specific] favored the zero article, both[-definite,+specific] and [+definite,-specific] showed less influence. When we reflect on the frequency order of the zero article , prototypicality plays a vital role in it .(2)EFL learners in this study have trouble classifying abstract nouns as countable. We can find that it will bring about overuse of the zero article when learners can not make clear judgements on countability altered from (+definite ) to (-definite).Once a noun is perceived as uncountable by learners, the choice would fall back on the zero article. These findings suggest that learners should be engaged in recognition of the countability of new vocabulary by explaining nouns in lexical phrases and explore more complex aspects such as analysis dependent on discourse.

Keywords: noun phrase, zero article, corpus, second language acquisition

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Authors: Pattanapon Kayansamruaj, Ha Thanh Dong, Nopadon Pirarat, Channarong Rodkhum

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Streptococcus iniae (SI) is a devastating pathogenic bacteria causing heavy mortality in farmed fish. The application of commercialized bacterin vaccine has been reported failures as the outbreaks of the new serotype of SI were emerged in farms after vaccination and subsequently caused severe losses. In the present study, we attempted to develop effective DNA vaccines against SI infection using Nile tilapia (Oreochromis niloticus) as an animal model. Two monovalent DNA vaccines were constructed by the insertion of coding sequences of cell wall-associated virulence factors-encoding genes, comprised of eno (α-enolase) and mtsB (hydrophobic membrane protein), into cytomegalovirus expression vector (pCI-neo). In the animal trial, 30-g Nile tilapia were injected intramuscularly with 15 µg of each vaccine (mock vaccine group was injected by naked pCI-neo) and maintained for 35 days prior challenging with pathogenic SI at the dosage of 107 CFU/fish. At 13 days post-challenge, the relative percent survival of pEno, pMtsB and mock vaccine were 57%, 45% and 27%, respectively. The expression levels of immune responses-associated genes, namely, IL1β, TNF-α, TGF-β, COX2, IL-6, IL-12 and IL-13, were investigated from the spleen of experimental animal at 7 days post-vaccination (PV) and 7 days post-challenge (PC) using quantitative RT-PCR technique. Generally, at 7 days PV, the pEno vaccinated group exhibited highest level of up-regulation (1.7 to 2.9 folds) of every gene, but TGF-β, comparing to pMtsB and mock vaccine groups. However, at 7 days PC, pEno group showed significant up-regulation (1.4 to 8.5 folds) of immune-related genes as similar as mock vaccine group, while pMtsB group had lowest level of up-regulation (0.7 to 3.3 folds). Summarily, this study indicated that the pEno and pMtsB vaccines could elicit the immune responses of the fish and the magnitude of gene expression at 7 days PV was also consistent with the protection level conferred by the vaccine.

Keywords: gene expression, DNA vaccine, Nile tilapia, Streptococcus iniae

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Authors: Isabel Lopez-Oliva, Akshay Paropkari, Shweta Saraswat, Stefan Serban, Paola de Pablo, Karim Raza, Andrew Filer, Iain Chapple, Thomas Dietrich, Melissa Grant, Purnima Kumar

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Objective: We aimed to explicate the role of the subgingival microbiome in the causal link between rheumatoid arthritis (RA) and periodontitis (PD). Methods: Subjects with/without RA and with/without PD were randomized for treatment with scaling and root planing (SRP) or oral hygiene instructions. Subgingival biofilm, gingival crevicular fluid, and serum were collected at baseline and at 3- and 6-months post-operatively. Correlations were generated between 72 million 16S rDNA sequences, immuno-inflammatory mediators, circulating antibodies to oral microbial antigens, serum inflammatory molecules, and clinical metrics of RA. The dynamics of inter-microbial and host-microbial interactions were modeled using differential network analysis. Results: RA superseded periodontitis as a determinant of microbial composition, and DAS28 score superseded the severity of periodontitis as a driver of microbial assemblages (p=0.001, ANOSIM). RA subjects evidenced higher serum anti-PPAD (p=0.0013), anti-Pg-enolase (p=0.0031), anti-RPP3, anti- Pg-OMP and anti- Pi-OMP (p=0.001) antibodies than non-RA controls (with and without periodontitis). Following SRP, bacterial networks anchored by IL-1b, IL-4, IL-6, IL-10, IL-13, MIP-1b, and PDGF-b underwent ≥5-fold higher rewiring; and serum antibodies to microbial antigens decreased significantly. Conclusions: Our data suggest a circular relationship between RA and PD, beginning with an RA-influenced dysbiosis within the healthy subgingival microbiome that leads to exaggerated local inflammation in periodontitis and circulating antibodies to periodontal pathogens and positive correlation between severity of periodontitis and RA activity. Periodontal therapy restores host-microbial homeostasis, reduces local inflammation, and decreases circulating microbial antigens. Our data highlights the importance of integrating periodontal care into the management of RA patients.

Keywords: rheumatoid arthritis, periodontal, subgingival, DNA sequence analysis, oral microbiome

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Authors: Hsin Lee, Hsuan Lee

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Legal artificial intelligence (LegalAI) has been increasing applications within legal systems, propelled by advancements in natural language processing (NLP). Compared with general documents, legal case documents are typically long text sequences with intrinsic logical structures. Most existing language models have difficulty understanding the long-distance dependencies between different structures. Another unique challenge is that while the Judiciary of Taiwan has released legal judgments from various levels of courts over the years, there remains a significant obstacle in the lack of labeled datasets. This deficiency makes it difficult to train models with strong generalization capabilities, as well as accurately evaluate model performance. To date, models in Taiwan have yet to be specifically trained on judgment data. Given these challenges, this research proposes a Longformer-based pre-trained language model explicitly devised for retrieving similar judgments in Taiwanese legal documents. This model is trained on a self-constructed dataset, which this research has independently labeled to measure judgment similarities, thereby addressing a void left by the lack of an existing labeled dataset for Taiwanese judgments. This research adopts strategies such as early stopping and gradient clipping to prevent overfitting and manage gradient explosion, respectively, thereby enhancing the model's performance. The model in this research is evaluated using both the dataset and the Average Entropy of Offense-charged Clustering (AEOC) metric, which utilizes the notion of similar case scenarios within the same type of legal cases. Our experimental results illustrate our model's significant advancements in handling similarity comparisons within extensive legal judgments. By enabling more efficient retrieval and analysis of legal case documents, our model holds the potential to facilitate legal research, aid legal decision-making, and contribute to the further development of LegalAI in Taiwan.

Keywords: legal artificial intelligence, computation and language, language model, Taiwanese legal cases

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Authors: Ana Breda, Catarina Cruz

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Daily, we face unpredictable events, often attributed to chance, as there is no justification for such an occurrence. Chance, understood as a source of uncertainty, is present in several aspects of human life, such as weather forecasts, dice rolling, and lottery. Surprisingly, humans and some animals can quickly adjust their behavior to handle efficiently doubly stochastic processes (random events with two layers of randomness, like unpredictable weather affecting dice rolling). This adjustment ability suggests that the human brain has built-in mechanisms for perceiving, understanding, and responding to simple probabilities. It also explains why current trends in mathematics education include probability concepts in official curriculum programs, starting from the third year of primary education onwards. In the first years of schooling, children learn to use a certain type of (specific) vocabulary such as: never, always, rarely, perhaps, likely, and unlikely, to help them to perceive and understand the probability of some events. These are keywords of crucial importance for their perception and understanding of probabilities. The development of the probabilistic concepts comes from facts and cause-effect sequences resulting from the subject's actions, as well as the notion of chance and intuitive estimates based on everyday experiences. As part of a junior summer school program, which took place at a Portuguese university, a non-formal learning experiment was carried out with 18 children in the 5th and 6th grades. This experience was designed to be implemented in a dynamic of a serious ice-breaking game, to assess their levels of probabilistic, critical, and creative thinking in understanding impossible, certain, equally probable, likely, and unlikely events, and, also, to gain insight into how the non-formal learning context influenced their achievements. The data analysis employs a mixed-methods approach, based on students' written productions, audio recordings, and researchers' field notes. This methodology allowed us to conclude that such an approach is an appropriate and helpful formative assessment tool.

Keywords: critical and creative thinking, non-formal mathematics learning, probabilistic thinking, serious game

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Authors: Caspar S. Carson, Gabriel W. Prather, Nicholas E. Wong, Jeffery R. Anton, William H. McCoy

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Cutibacterium acnes is a commensal bacterium found on human skin that has been linked to acne. C. acnes can also be an opportunistic pathogen when it infiltrates the body during surgery. This pathogen can cause dangerous infections of medical implants, such as shoulder replacements, leading to life-threatening blood infections. Compounding this issue, C. acnes resistance to many antibiotics has become an increasing problem worldwide, creating a need for special forms of treatment. C. acnes expresses the protein RoxP, and it requires this protein to colonize human skin. Though this protein is required for C. acnes skin colonization, its function is not yet understood. Inhibition of RoxP function might be an effective treatment for C. acnes infections. To develop such reagents, the McCoy Laboratory generated four unique anti-RoxP antibodies. Preliminary studies in the McCoy Lab have established that each antibody binds a distinct site on RoxP. To assess the potential of these antibodies as therapeutics, it is necessary to specifically characterize these antibody epitopes and evaluate them in assays that assess their ability to inhibit RoxP-dependent C. acnes growth. To provide material for these studies, an antibody expression construct, Fv-clasp(v2), was adapted to encode anti-RoxP antibody sequences. The author hypothesizes that this expression strategy can produce sufficient amounts of >95% pure antibody fragments for further characterization of these antibodies. Four anti-RoxP Fv-clasp(v2) expression constructs (pET vector-based) were transformed into E. coli BL21-Gold(DE3) cells and a small-scale expression and purification trial was performed for each construct to evaluate anti-RoxP Fv-clasp(v2) yield and purity. Successful expression and purification of these antibody constructs will allow for their use in structural studies, such as protein crystallography and cryogenic electron microscopy. Such studies would help to define the antibody binding sites on RoxP, which could then be leveraged in the development of certain methods to treat C. acnes infection through RoxP inhibition.

Keywords: structural biology, protein expression, infectious disease, antibody, therapeutics, E. coli

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Authors: Suharni Mohamad Suharni Mohamad, Nurul Izzati Hamzan Nurul Izzati Hamzan, Norhayu Abdul Rahman Norhayu Abdul Rahman, Siti Suraiya Md Noor Siti Suraiya Md Noor

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Human papillomavirus (HPV) is an important risk factor for development of oral cancer. HPV16 is the most common type found in HPV-positive squamous cell carcinoma. In the present study, we established a real-time loop-mediated isothermal amplification (real-time LAMP) for detection of HPV16. A set of six primers was specially designed to recognize eight distinct sequences of HPV16-E6. Detection and quantification was achieved by real-time monitoring using a real-time turbidimeter based on threshold time required for turbidity in the LAMP reaction. LAMP reagents (MgSO4, dNTPs, Bst polymerase concentrations) and various incubation times and temperatures were optimized. The sensitivity was determined using 10-fold serial dilutions of HPV16 standard strain. The specificity of was evaluated using other HPV genotypes. The optimized method was established with specifically designed primers by real-time detection in approximately 30 min at 65°C. The limit of detection of HPV16 using the LAMP assay was 10 pg/ml that could be detected in 30 min. The LAMP assay was 10 times more sensitive than the conventional PCR in detecting HPV16. No cross-reactivity with other HPV genotypes was observed. This quantitative real-time LAMP assay may improve diagnostic potential for the detection and quantification of HPV16 in clinical samples and epidemiological studies due to its rapidity, simplicity, high sensitivity and specificity. This assay will be further evaluated with HPV DNAs of saliva from patients with oral squamous cell carcinoma. Acknowledgement: This study was financially supported by the ScienceFund Grant, Ministry of Science, Technology and Innovation (305/PPSG/6113219).

Keywords: Oral Squamous Cell Carcinoma (OSCC), Human Papillomavirus 16 (HPV16), Loop-Mediated Isothermal Amplification (LAMP), rapid detection

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Authors: Irena Maus, Katharina Gabriella Cibis, Andreas Bremges, Yvonne Stolze, Geizecler Tomazetto, Daniel Wibberg, Helmut König, Alfred Pühler, Andreas Schlüter

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Within the agricultural sector, the production of biogas from organic substrates represents an economically attractive technology to generate bioenergy. Complex consortia of microorganisms are responsible for biomass decomposition and biogas production. Recently, species belonging to the phylum Thermotogae were detected in thermophilic biogas-production plants utilizing renewable primary products for biomethanation. To analyze adaptive genome features of representative Thermotogae strains, Defluviitoga tunisiensis L3 was isolated from a rural thermophilic biogas plant (54°C) and completely sequenced on an Illumina MiSeq system. Sequencing and assembly of the D. tunisiensis L3 genome yielded a circular chromosome with a size of 2,053,097 bp and a mean GC content of 31.38%. Functional annotation of the complete genome sequence revealed that the thermophilic strain L3 encodes several genes predicted to facilitate growth of this microorganism on arabinose, galactose, maltose, mannose, fructose, raffinose, ribose, cellobiose, lactose, xylose, xylan, lactate and mannitol. Acetate, hydrogen (H2) and carbon dioxide (CO2) are supposed to be end products of the fermentation process. The latter gene products are metabolites for methanogenic archaea, the key players in the final step of the anaerobic digestion process. To determine the degree of relatedness of dominant biogas community members within selected digester systems to D. tunisiensis L3, metagenome sequences from corresponding communities were mapped on the L3 genome. These fragment recruitments revealed that metagenome reads originating from a thermophilic biogas plant covered 95% of D. tunisiensis L3 genome sequence. In conclusion, availability of the D. tunisiensis L3 genome sequence and insights into its metabolic capabilities provide the basis for biotechnological exploitation of genome features involved in thermophilic fermentation processes utilizing renewable primary products.

Keywords: genome sequence, thermophilic biogas plant, Thermotogae, Defluviitoga tunisiensis

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Authors: Ahilya Singh, Brad Carte, Ramesh Subramani, William Aalbersberg

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A total of 37 actinomycete strains were purified from 25 Solomon Islands marine sediments using four different types of isolation media. Among them, 54% of the strains had obligate requirement of seawater for growth. The ethyl acetate extract of 100 ml fermentation product of each strain was screened for antimicrobial activity against multidrug resistant human pathogens and cytotoxic activity against brine shrimps. A total of 67% of the ethyl acetate extracts showed antimicrobial and/or cytotoxic activities. A strain F-1915 was selected for isolation and evaluation of bioactive compound(s) based on its bioactive properties and chemical profile analysis using the LC-MS. The strain F-1915 was identified to have 96% sequence similarity to Streptomyces violaceusniger on the basis of 16S rDNA sequences using BLAST analysis. The 16S rDNA revealed that the strain F-1915 is a new member of MAR4 clade of actinomycetes. The MAR4 clade is an interesting clade of actinomycetes known for the production of pharmaceutically important hybrid isoprenoid compounds. The ethyl acetate extract of the fermentation product of this strain was purified by silica gel column chromatography and afforded the isolation of one bioactive pure compound. Based on the 1D and 2D NMR spectral data of compound 1 it was identified as a new mono-brominated phenazinone, Marinophenazimycin A, a structure which has already been studied by external collaborators at Scripps Institution of Oceanography but is yet to be published. Compound 1 displayed significant antimicrobial activity against drug resistant human pathogens. The minimum inhibitory concentration (MIC) of compound 1 was against Methicillin Resistant Staphylococcus aureus (MRSA) was about 1.9 μg/ml and MIC recorded against Amphotericin Resistant Candida albicans (ARCA) was about 0.24 μg/ml. The bioactivity of compound 1 against ARCA was found to be better than the standard antifungal agent amphotericin B. Compound 1 however did not show any cytotoxic activity against brine shrimps.

Keywords: actinomycetes, antimicrobial activity, brominated phenazine, MAR4 clade, marine natural products, multidrug resistent, 1D and 2D NMR

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Authors: M. Y. Abubakar, Ipinjolu J. K., Yuzine B. Esa, Magawata I., Hassan W. A., Turaki A. A.

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Catfish (Heterobranchus spp.) is a major freshwater fish that are widely distributed in Nigeria waters and are gaining rapid aquaculture expansion. However, indiscriminate artificial crossbreeding of the species with others poses a threat to their biodiversity. There is a paucity of information about the genetic variability, hence this insight on the genetic variability is badly needed, not only for the species conservation but for aquaculture expansion. In this study, we tested the level of Genetic diversity, population differentiation and phylogenetic relationship analysis on 35 individuals of two populations of Heterobranchus bidorsalis and 29 individuals of three populations of Heterobranchus longifilis using the mitochondrial cytochrome c oxidase subunit I (mtDNA COI) gene sequence. Nucleotide sequences of 650 bp fragment of the COI gene of the two species were compared. In the whole 4 and 5 haplotypes were distinguished in the populations of H. bidorsalis & H. longifilis with accession numbers (MG334168 - MG334171 & MG334172 to MG334176) respectively. Haplotypes diversity indices revealed a range of 0.59 ± 0.08 to 0.57 ± 0.09 in H. bidorsalis and 0.000 to 0.001051 ± 0.000945 in H. longifilis population, respectively. Analysis of molecular variance (AMOVA) revealed no significant variation among H. bidorsalis population of the Niger & Benue Rivers, detected significant genetic variation was between the Rivers of Niger, Kaduna and Benue population of H. longifilis. Two main clades were recovered, showing a clear separation between H. bidorsalis and H. longifilis in the phylogenetic tree. The mtDNA COI genes studied revealed high gene flow between populations with no distinct genetic differentiation between the populations as measured by the fixation index (FST) statistic. However, a proportion of population-specific haplotypes was observed in the two species studied, suggesting a substantial degree of genetic distinctiveness for each of the population investigated. These findings present the description of the species character and accessions of the fish’s genetic resources, through gene sequence submitted in Genetic database. The data will help to protect their valuable wild resource and contribute to their recovery and selective breeding in Nigeria.

Keywords: AMOVA, genetic diversity, Heterobranchus spp., mtDNA COI, phylogenetic tree

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Authors: Suchithra V., Shreedhanya, Kavya Menon, Vidya Niranjan

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Skin metagenomics has a wide range of applications with direct relevance to the health of the organism. It gives us insight to the diverse community of microorganisms (the microbiome) harbored on the skin. In the recent years, it has become increasingly apparent that the interaction between skin microbiome and the human body plays a prominent role in immune system development, cancer development, disease pathology, and many other biological implications. Next Generation Sequencing has led to faster and better understanding of environmental organisms and their mutual interactions. This project is studying the human skin microbiome of different individuals having varied skin conditions. Bacterial 16S rRNA data of skin microbiome is downloaded from SRA toolkit provided by NCBI to perform metagenomics analysis. Twelve samples are selected with two controls, and 3 different categories, i.e., sex (male/female), skin type (moist/intermittently moist/sebaceous) and occlusion (occluded/intermittently occluded/exposed). Quality of the data is increased using Cutadapt, and its analysis is done using FastQC. USearch, a tool used to analyze an NGS data, provides a suitable platform to obtain taxonomy classification and abundance of bacteria from the metagenome data. The statistical tool used for analyzing the USearch result is METAGENassist. The results revealed that the top three abundant organisms found were: Prevotella, Corynebacterium, and Anaerococcus. Prevotella is known to be an infectious bacterium found on wound, tooth cavity, etc. Corynebacterium and Anaerococcus are opportunist bacteria responsible for skin odor. This result infers that Prevotella thrives easily in sebaceous skin conditions. Therefore it is better to undergo intermittently occluded treatment such as applying ointments, creams, etc. to treat wound for sebaceous skin type. Exposing the wound should be avoided as it leads to an increase in Prevotella abundance. Moist skin type individuals can opt for occluded or intermittently occluded treatment as they have shown to decrease the abundance of bacteria during treatment.

Keywords: bacterial 16S rRNA , next generation sequencing, skin metagenomics, skin microbiome, taxonomy

Procedia PDF Downloads 157

Authors: Saima Saleem, Zubair Abbasi, Abdul Hameed, Mansoor Ahmed Khan, Navid Rashid Qureshi, Abid Azhar

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Oral Squamous Cell Carcinoma (OSCC) is the leading cause of death in the developing countries like Pakistan. This problem aggravates because of the excessive use of available chewing products. In spite of widespread information on their use and purported legislations against their use the Pakistani markets are classical examples of selling chewable carcinogenic mutagens. Reported studies indicated that these products are rich in reactive oxygen species (ROS) and polyphenols. TP53 gene is involved in the suppression of tumor. It has been reported that somatic mutations caused by TP53 gene are the foundation of the cancer. This study aims to find the loss of TP53 functions due to mutation/polymorphism caused by genomic alteration and interaction with tobacco and its related ingredients. Total 260 tissues and blood specimens were collected from OSCC patients and compared with age and sex matched controls. Mutations in exons 2-11 of TP53 were examined by PCR-SSCP. Samples showing mobility shift were directly sequenced. Two mutations were found in exon 4 at nucleotide position 108 and 215 and one in exon 7 at nucleotide position 719 of the coding sequences in patient’s tumor samples. These results show that substitution of proline with arginine at codon 72 and serine with threonine at codon 240 of p53 protein. These polymorphic changes, found in tumor samples of OSCC, could be involved in loss of heterozygocity and apoptotic activity in the binding domain of TP53. The model of the mutated TP53 gene elaborated a nonfunctional unfolded p53 protein, suggesting an important role of these mutations in p53 protein inactivation and malfunction. This nonfunctional 3D model also indicates that exogenous tobacco related carcinogens may act as DNA-damaging agents affecting the structure of DNA. The interpretations could be helpful in establishing the pathways responsible for tumor formation in OSCC patients.

Keywords: TP53 mutation/polymorphism, OSCC, PCR-SSCP, direct DNA sequencing, 3D structure

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Authors: Y. B. Sharma, K. B. Biswas

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This research project examines a 5000 cal yr BP sediment core record to reveal the consequences of human impact and climate variability on the tropical dry forests of the Mahi river basin, western India. To date there has been little research to assess the impact of climate variability and human impact on the vegetation dynamics of this region. There has also been little work to link changes in vegetation cover to documented changes in the basin hydrology over the past 100 years – although it is assumed that the two are closely linked. The key objective of this research project therefore is to understand the driving mechanisms responsible for the abrupt changes in the Mahi river basin as detailed in historical documentation and its impact on water resource management. The Mahi river basin is located in western India (22° 11’-24° 35’ N 72° 46’-74° 52’ E). Mahi river arises in the Malwa Plateau, Madhya Pradesh near Moripara and flows through the uplands and alluvial plain of Rajasthan and Gujarat provinces before draining into the Gulf of Cambay. Palaeoecological procedures (sedimentology, geochemical analysis, C&N isotopes and fossil pollen evidences) have been applied on sedimentary sequences collected from lakes in the Mahi basin. These techniques then facilitate to reconstruct the soil erosion, nutrient cycling, vegetation changes and climatic variability over the last 5000 years. Historical documentation detailing changes in demography, climate and landscape use over the past 100 years in this region will also be collated to compare with the most recent palaeoecological records. The results of the research work provide a detailed record of vegetation change, soil erosion, changes in aridity, and rainfall patterns in the region over the past 5000 years. This research therefore aims to determine the drivers of change and natural variability in the basin. Such information is essential for its current and future management including restoration.

Keywords: human impact, climate variability, vegetation cover, hydrology, water resource management, Mahi river basin, sedimentology, geochemistry, fossil pollen, nutrient cycling, vegetation changes, palaeoecology, aridity, rainfall, drivers of change

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Authors: Jacques Lamarche, Juhani Dickinson

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This paper introduces a computational model of a Grammar as Logic of Labeling (GLL), where the lexical primitives of morphosyntax are phonological matrixes, the form of words, understood as labels that apply to realities (or targets) assumed to be outside of grammar altogether. The hypothesis is that even though a lexical label relates to its target arbitrarily, this label in a complex (constituent) label is part of a labeling pattern which, depending on its value (i.e., N, V, Adj, etc.), imposes language-specific restrictions on what it targets outside of grammar (in the world/semantics or in cognitive knowledge). Lexical forms categorized as nouns, verbs, adjectives, etc., are effectively targets of labeling patterns in use. The paper illustrates GLL through a computer model of basic patterns in English NPs. A constituent label is a binary object that encodes: i) alignment of input forms so that labels occurring at different points in time are understood as applying at once; ii) endocentric structuring - every grammatical constituent has a head label that determines the target of the constituent, and a limiter label (the non-head) that restricts this target. The N or A values are restricted to limiter label, the two differing in terms of alignment with a head. Consider the head initial DP ‘the dog’: the label ‘dog’ gets an N value because it is a limiter that is evenly aligned with the head ‘the’, restricting application of the DP. Adapting a traditional analysis of ‘the’ to GLL – apply label to something familiar – the DP targets and identifies one reality familiar to participants by applying to it the label ‘dog’ (singular). Consider next the DP ‘the large dog’: ‘large dog’ is nominal by even alignment with ‘the’, as before, and since ‘dog’ is the head of (head final) ‘large dog’, it is also nominal. The label ‘large’, however, is adjectival by narrow alignment with the head ‘dog’: it doesn’t target the head but targets a property of what dog applies to (a property or value of attribute). In other words, the internal composition of constituents determines that a form targets a property or a reality: ‘large’ and ‘dog’ happen to be valid targets to realize this constituent. In the presentation, the computer model of the analysis derives the 8 possible sequences of grammatical values with three labels after the determiner (the x y z): 1- D [ N [ N N ]]; 2- D [ A [ N N ] ]; 3- D [ N [ A N ] ]; 4- D [ A [ A N ] ]; 5- D [ [ N N ] N ]; 5- D [ [ A N ] N ]; 6- D [ [ N A ] N ] 7- [ [ N A ] N ] 8- D [ [ Adv A ] N ]. This approach that suggests that a computer model of these grammatical patterns could be used to construct ontologies/knowledge using speakers’ judgments about the validity of lexical meaning in grammatical patterns.

Keywords: syntactic theory, computational linguistics, logic and grammar, semantics, knowledge and grammar

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Authors: Aymen Arfaoui, Abdelkader Soumaya, Ali Kadri, Noureddine Ben Ayed

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The major contractional events characterized by strike-slip faulting, folding, and thrusting occurred in the Eocene, Late Miocene, and Quaternary along with the NE Tunisian domain between Bou Kornine-Ressas- Msella and Cap Bon Peninsula. During the Plio-Quaternary, the Grombalia and Mornag grabens show a maximum of collapse in parallelism with the NNW-SSE SHmax direction and developed as 3rd order extensive regions within a regional compressional regime. Using available tectonic and geophysical data supplemented by new fault-kinematic observations, we show that Cenozoic deformations are dominated by first order N-S faults reactivation, this sinistral wrench system is responsible for the formation of strike-slip duplexes, thrusts, folds, and grabens. Based on our new structural interpretation, the major faults of N-S Axis, Bou Kornine-Ressas-Messella (MRB), and Hammamet-Korbous (HK) form an N-S first order restraining stepover within a left-lateral strike-slip duplex. The N-S master MRB fault is dominated by contractional imbricate fans, while the parallel HK fault is characterized by a trailing of extensional imbricate fans. The Eocene and Miocene compression phases in the study area caused sinistral strike-slip reactivation of pre-existing N-S faults, reverse reactivation of NE-SW trending faults, and normal-oblique reactivation of NW-SE faults, creating a NE-SW to N-S trending system of east-verging folds and overlaps. Seismic tomography images reveal a key role for the lithospheric subvertical tear or STEP fault (Slab Transfer Edge Propagator) evidenced below this region on the development of the MRB and the HK relay zone. The presence of extensive syntectonic Pliocene sequences above this crustal scale fault may be the result of a recent lithospheric vertical motion of this STEP fault due to the rollback and lateral migration of the Calabrian slab eastward.

Keywords: Tunisia, strike-slip fault, contractional duplex, tectonic stress, restraining stepover, STEP fault

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Authors: Guna Raj Dhungana, Roshan Nepal, Apshara Parajuli, , Archana Maharjan, Shyam K. Mishra, Pramod Aryal, Rajani Malla

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Introduction: Klebsiella pneumoniae is a well-known opportunistic human pathogen, primarily causing healthcare-associated infections. The global emergence of carbapenemase-producing K. pneumoniaeis a major public health burden, which is often extensively multidrug resistant.Thus, because of the difficulty to treat these ‘superbug’ and menace and some term as ‘apocalypse’ of post antibiotics era, an alternative approach to controlling this pathogen is prudent and one of the approaches is phage mediated control and/or treatment. Objective: In this study, we aimed to isolate novel bacteriophage against carbapenemase-producing K. pneumoniaeand characterize for potential use inphage therapy. Material and Methods: Twenty lytic phages were isolated from river water using double layer agar assay and purified. Biological features, physiochemical characters, burst size, host specificity and activity spectrum of phages were determined. One most potent phage: Phage TU_Kle10O was selected and characterized by electron microscopy. Whole genome sequences of the phage were analyzed for presence/absence of virulent factors, and other lysin genes. Results: Novel phage TU_Kle10O showed multiple host range within own genus and did not induce any BIM up to 5th generation of host’s life cycle. Electron microscopy confirmed that the phage was tailed and belonged to Caudovirales family. Next generation sequencing revealed its genome to be 166.2 Kb. bioinformatical analysis further confirmed that the phage genome ‘did not’ contain any ‘bacterial genes’ within phage genome, which ruled out the concern for transfer of virulent genes. Specific 'lysin’ enzyme was identified phages which could be used as 'antibiotics'. Conclusion: Extensively multidrug resistant bacteria like carbapenemase-producing K. pneumoniaecould be treated efficiently by phages.Absence of ‘virulent’ genes of bacterial origin and presence of lysin proteins within phage genome makes phages an excellent candidate for therapeutics.

Keywords: bacteriophage, Klebsiella pneumoniae, MDR, phage therapy, carbapenemase,

Procedia PDF Downloads 171

Authors: M. Gogniashvili, I. Maisaia, A. Kotorashvili, N. Kotaria, T. Beridze

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Three types of plasmon (A, B and G) is typical for genus Triticum. In polyploid species - Triticum turgidum L. and Triticum aestivum L. plasmon B is detected. In the forthcoming paper, complete nucleotide sequence of chloroplast DNA of 11 representatives of Georgian wheat polyploid species, carrying plasmon B was determined. Sequencing of chloroplast DNA was performed on an Illumina MiSeq platform. Chloroplast DNA molecules were assembled using the SOAPdenovo computer program. All contigs were aligned to the reference chloroplast genome sequence using BLASTN. For detection of SNPs and Indels and phylogeny tree construction computer programs Mafft and Blast were used. Using Triticum aestivum L. subsp. macha (Dekapr. & Menabde) Mackey var. paleocolchicum Dekapr. et Menabde as a reference, 5 SNPs can be identified in chloroplast DNA of Georgian endemic polyploid wheat. The number of noncoding substitutions is 2, coding substitutions - 3. In comparison with reference DNA two - 38 bp and 56 bp inversions were observed in paleocolchicum subspecies. There were six 1 bp indels detected in Georgian polyploid wheats, all of them at microsatellite stretches. The phylogeny tree shows that subspecies macha, carthlicum and paleocolchicum occupy different positions. According to the simplified scheme based on SNP and indel data, the ancestral, female parent of the all studied polyploid wheat is unknown X predecesor, from which four lines were formed. 1 SNP and two inversions (38 bp and 56 bp) caused the formation of subsp. paleocolchicum. Three other lines are macha, durum and carthlicum lines. Macha line is further divided into two sublines (M_1 and M_4). Carthlicum line includes subsp.carthlicum and T.aestivum - C_1 - C_2 - A_1. One of the central question of wheat domestication is which people(s) participated in wheat domestication? It is proposed that the predecessors of Georgian peoples (Proto-Kartvelians) must be placed, on the evidence of archaic lexical and toponymic data, in the mountainous regions of the western and central part of the Little Caucasus (the Transcaucasian foothills) at least 4,000 years ago. One of the possibility to explain the ‘wheat puzzle’ is that Kartvelian speakers brought domesticated wheat species and subspecis from Fertile Crescent further north to South Caucasus.

Keywords: chloroplast DNA, sequencing, SNP, triticum

Procedia PDF Downloads 139

Authors: Narayan Moger, Divya B., Preethi Jambagi, Krishnaveni C. K., Apsana M. R., B. R. Patil, Basvaraj Bagewadi

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Salinity is one of the major threats to world food security. Considering the requirement for salt tolerant crop plants in the present study was undertaken to clone and transferred the salt tolerant ectA gene from marine ecosystem into agriculture crop system to impart salinity tolerance. Ectoine is the compatible solute which accumulates in the cell membrane, is known to be involved in salt tolerance activity in most of the Halophiles. The present situation is insisting to development of salt tolerant transgenic lines to combat abiotic stress. In this background, the investigation was conducted to develop transgenic tomato lines by cloning and transferring of ectA gene is an ectoine derivative capable of enzymatic action for the production of acetyl-diaminobutyric acid. The gene ectA is involved in maintaining the osmotic balance of plants. The PCR amplified ectA gene (579bp) was cloned into T/A cloning vector (pTZ57R/T). The construct pDBJ26 containing ectA gene was sequenced by using gene specific forward and reverse primers. Sequence was analyzed using BLAST algorithm to check similarity of ectA gene with other isolates. Highest homology of 99.66 per cent was found with ectA gene sequences of isolates Halomonas elongata with the available sequence information in NCBI database. The ectA gene was further sub cloned into pRI101-AN plant expression vector and transferred into E. coli DH5α for its maintenance. Further pDNM27 was mobilized into A. tumefaciens LBA4404 through tri-parental mating system. The recombinant Agrobacterium containing pDNM27 was transferred into tomato plants through In planta plant transformation method. Out of 300 seedlings, co-cultivated only twenty-seven plants were able to well establish under the greenhouse condition. Among twenty-seven transformants only twelve plants showed amplification with gene specific primers. Further work must be extended to evaluate the transformants at T1 and T2 generations for ectoine accumulation, salinity tolerance, plant growth and development and yield.

Keywords: salinity, computable solutes, ectA, transgenic, in planta transformation

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Authors: S. Behnia, S. Fathizadeh, A. Akhshani

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In the recent decades, DNA has increasingly interested in the potential technological applications that not directly related to the coding for functional proteins that is the expressed in form of genetic information. One of the most interesting applications of DNA is related to the construction of nanostructures of high complexity, design of functional nanostructures in nanoelectronical devices, nanosensors and nanocercuits. In this field, DNA is of fundamental interest to the development of DNA-based molecular technologies, as it possesses ideal structural and molecular recognition properties for use in self-assembling nanodevices with a definite molecular architecture. Also, the robust, one-dimensional flexible structure of DNA can be used to design electronic devices, serving as a wire, transistor switch, or rectifier depending on its electronic properties. In order to understand the mechanism of the charge transport along DNA sequences, numerous studies have been carried out. In this regard, conductivity properties of DNA molecule could be investigated in a simple, but chemically specific approach that is intimately related to the Su-Schrieffer-Heeger (SSH) model. In SSH model, the non-diagonal matrix element dependence on intersite displacements is considered. In this approach, the coupling between the charge and lattice deformation is along the helix. This model is a tight-binding linear nanoscale chain established to describe conductivity phenomena in doped polyethylene. It is based on the assumption of a classical harmonic interaction between sites, which is linearly coupled to a tight-binding Hamiltonian. In this work, the Hamiltonian and corresponding motion equations are nonlinear and have high sensitivity to initial conditions. Then, we have tried to move toward the nonlinear dynamics and phase space analysis. Nonlinear dynamics and chaos theory, regardless of any approximation, could open new horizons to understand the conductivity mechanism in DNA. For a detailed study, we have tried to study the current flowing in DNA and investigated the characteristic I-V diagram. As a result, It is shown that there are the (quasi-) ohmic areas in I-V diagram. On the other hand, the regions with a negative differential resistance (NDR) are detectable in diagram.

Keywords: DNA conductivity, Landauer resistance, negative dierential resistance, Chaos theory, mean Lyapunov exponent

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Authors: Alexander Rych

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Piql Preservation Services (“Piql”) is a turnkey solution designed for secure, migration-free long- term preservation of digital data. Piql sets an open standard for long- term preservation for the future. It consists of equipment and processes needed for writing and retrieving digital data. Exponentially growing amounts of data demand for logistically effective and cost effective processes. Digital storage media (hard disks, magnetic tape) exhibit limited lifetime. Repetitive data migration to overcome rapid obsolescence of hardware and software bears accelerated risk of data loss, data corruption or even manipulation and adds significant repetitive costs for hardware and software investments. Piql stores any kind of data in its digital as well as analog form securely for 500 years. The medium that provides this is a film reel. Using photosensitive film polyester base, a very stable material that is known for its immutability over hundreds of years, secure and cost-effective long- term preservation can be provided. The film reel itself is stored in a packaging capable of protecting the optical storage medium. These components have undergone extensive testing to ensure longevity of up to 500 years. In addition to its durability, film is a true WORM (write once- read many) medium. It therefore is resistant to editing or manipulation. Being able to store any form of data onto the film makes Piql a superior solution for long-term preservation. Paper documents, images, video or audio sequences – all of those file formats and documents can be preserved in its native file structure. In order to restore the encoded digital data, only a film scanner, a digital camera or any appropriate optical reading device will be needed in the future. Every film reel includes an index section describing the data saved on the film. It also contains a content section carrying meta-data, enabling users in the future to rebuild software in order to read and decode the digital information.

Keywords: digital data, long-term preservation, migration-free, photosensitive film

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Authors: Thomas Bryan, Veton Kepuska, Ivica Kostanic

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Speaker recognition is performed in high Additive White Gaussian Noise (AWGN) environments using principals of Computational Auditory Scene Analysis (CASA). CASA methods often classify sounds from images in the time-frequency (T-F) plane using spectrograms or cochleargrams as the image. In this paper atomic decomposition implemented by matching pursuit performs a transform from time series speech signals to the T-F plane. The atomic decomposition creates a sparsely populated T-F vector in “weight space” where each populated T-F position contains an amplitude weight. The weight space vector along with the atomic dictionary represents a denoised, compressed version of the original signal. The arraignment or of the atomic indices in the T-F vector are used for classification. Unsupervised feature learning implemented by a sparse autoencoder learns a single dictionary of basis features from a collection of envelope samples from all speakers. The approach is demonstrated using pairs of speakers from the TIMIT data set. Pairs of speakers are selected randomly from a single district. Each speak has 10 sentences. Two are used for training and 8 for testing. Atomic index probabilities are created for each training sentence and also for each test sentence. Classification is performed by finding the lowest Euclidean distance between then probabilities from the training sentences and the test sentences. Training is done at a 30dB Signal-to-Noise Ratio (SNR). Testing is performed at SNR’s of 0 dB, 5 dB, 10 dB and 30dB. The algorithm has a baseline classification accuracy of ~93% averaged over 10 pairs of speakers from the TIMIT data set. The baseline accuracy is attributable to short sequences of training and test data as well as the overall simplicity of the classification algorithm. The accuracy is not affected by AWGN and produces ~93% accuracy at 0dB SNR.

Keywords: time-frequency plane, atomic decomposition, envelope sampling, Gabor atoms, matching pursuit, sparse dictionary learning, sparse autoencoder

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Authors: Jui-Hsueh Hu

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This study investigated various models of refusal speech acts among three target groups: French learners of Mandarin Chinese (FM), Taiwanese native Mandarin speakers (TM), and native French speakers (NF). The refusal responses were analyzed in terms of their options, frequencies, and sequences and the contents of their semantic formulas. This study also examined differences in refusal strategies, as determined by social status and social distance, among the three groups. The difficulties of refusal speech acts encountered by FM were then generalized. The results indicated that Mandarin instructors of NF should focus on the different reasons for the pragmatic failure of French learners and should assist these learners in mastering refusal speech acts that rely on abundant cultural information. In this study, refusal policies were mainly classified according to the research of Beebe et al. (1990). Discourse completion questionnaires were collected from TM, FM, and NF, and their responses were compared to determine how refusal policies differed among the groups. This study not only emphasized the dissimilarities of refusal strategies between native Mandarin speakers and second-language Mandarin learners but also used NF as a control group. The results of this study demonstrated that regarding overall strategies, FM were biased toward NF in terms of strategy choice, order, and content, resulting in pragmatic transfer under the influence of social factors such as 'social status' and 'social distance,' strategy choices of FM were still closer to those of NF, and the phenomenon of pragmatic transfer of FM was revealed. Regarding the refusal difficulties among the three groups, the F-test in the analysis of variance revealed statistical significance was achieved for Role Playing Items 13 and 14 (P < 0.05). A difference was observed in the average number of refusal difficulties between the participants. However, after multiple comparisons, it was found that item 13 (unrecognized heterosexual junior colleague requesting contacts) was significantly more difficult for NF than for TM and FM; item 14 (contacts requested by an unrecognized classmate of the opposite sex) was significantly more difficult to refuse for NF than for TM. This study summarized the pragmatic language errors that most FM often perform, including the misuse or absence of modal words, hedging expressions, and empty words at the end of sentences, as the reasons for pragmatic failures. The common social pragmatic failures of FM include inaccurately applying the level of directness and formality.

Keywords: French Mandarin, interlanguage refusal, pragmatic transfer, speech acts

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Authors: Saeed Ur Rehman Chaudhry

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The objectives of the study are to reconstruct sequence stratigraphic framework and petrophysical analysis of the reservoir marked by using sequence stratigraphy of Sawan Gas Field. The study area lies in Central Indus Basin, District Khairpur, Sindh province, Pakistan. The study area lies tectonically in an extensional regime. Lower Goru Formation and Sembar Formation act as a reservoir and source respectively. To achieve objectives, data set of seismic lines, consisting of seismic lines PSM96-114, PSM96-115, PSM96-133, PSM98-201, PSM98-202 and well logs of Sawan-01, Sawan-02 and Gajwaro-01 has been used. First of all interpretation of seismic lines has been carried out. Interpretation of seismic lines shows extensional regime in the area and cut entire Cretaceous section. Total of seven reflectors has been marked on each seismic line. Lower Goru Formation is thinning towards west. Seismic lines also show eastward tilt of stratigraphy due to uplift at the western side. Sequence stratigraphic reconstruction has been done by integrating seismic and wireline log data. Total of seven sequence boundaries has been interpreted between the top of Chiltan Limestone to Top of Lower Goru Formation. It has been observed on seismic lines that Sembar Formation initially generated shelf margin profile and then ramp margin on which Lower Goru deposition took place. Shelf edge deltas and slope fans have been observed on seismic lines, and signatures of slope fans are also observed on wireline logs as well. Total of six sequences has been interpreted. Stratigraphic and sequence stratigraphic correlation has been carried out by using Sawan 01, Sawan 02 and Gajwaro 01 and a Low Stand Systems tract (LST) within Lower Goru C sands has been marked as a zone of interest. The petrophysical interpretation includes shale volume, effective porosity, permeability, saturation of water and hydrocarbon. On the basis of good effective porosity and hydrocarbon saturation petrophysical analysis confirms that the LST in Sawan-01 and Sawan-02 has good hydrocarbon potential.

Keywords: petrophysical analysis, reservoir potential, Sawan Gas Field, sequence stratigraphy

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Authors: Iman Farasat, Howard M. Salis

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The CRISPR/Cas9 system has revolutionized genome engineering by enabling site-directed and high-throughput genome editing, genome insertion, and gene knockdowns in several species, including bacteria, yeast, flies, worms, and human cell lines. This technology has the potential to enable human gene therapy to treat genetic diseases and cancer at the molecular level; however, the current CRISPR/Cas9 system suffers from seemingly sporadic off-target genome mutagenesis that prevents its use in gene therapy. A comprehensive mechanistic model that explains how the CRISPR/Cas9 functions would enable the rational design of the guide-RNAs responsible for target site selection while minimizing unexpected genome mutagenesis. Here, we present the first quantitative model of the CRISPR/Cas9 genome mutagenesis system that predicts how guide-RNA sequences (crRNAs) control target site selection and cleavage activity. We used statistical thermodynamics and law of mass action to develop a five-step biophysical model of cas9 cleavage, and examined it in vivo and in vitro. To predict a crRNA's binding specificities and cleavage rates, we then compiled a nearest neighbor (NN) energy model that accounts for all possible base pairings and mismatches between the crRNA and the possible genomic DNA sites. These calculations correctly predicted crRNA specificity across 5518 sites. Our analysis reveals that cas9 activity and specificity are anti-correlated, and, the trade-off between them is the determining factor in performing an RNA-mediated cleavage with minimal off-targets. To find an optimal solution, we first created a scheme of safe-design criteria for Cas9 target selection by systematic analysis of available high throughput measurements. We then used our biophysical model to determine the optimal Cas9 expression levels and timing that maximizes on-target cleavage and minimizes off-target activity. We successfully applied this approach in bacterial and mammalian cell lines to reduce off-target activity to near background mutagenesis level while maintaining high on-target cleavage rate.

Keywords: biophysical model, CRISPR, Cas9, genome editing

Procedia PDF Downloads 387