Directionality of DNA mismatch repair in escherichia coli

Non-canonical base pairs that escape the proof-reading activity of the DNA polymerase emerge from DNA replication as DNA mismatches. To promote genomic integrity, these DNA mismatches are corrected by a secondary protection system, called DNA mismatch repair (MMR). Understanding the details of MMR is important for human health as defects in mismatch repair can result in cancer (e.g. hereditary nonpolyposis colorectal cancer, also known as Lynch syndrome). Being normally stochastic in nature, mismatches can emerge at random locations in a chromosome. Therefore, using a molecular tool to generate substrates for the MMR system at a defined locus has been particularly useful in my study of DNA mismatch repair in vivo. In this study, I have used a CTG•CAG repeat array, also called the “TNR array”, to generate frequent substrates for the MMR system in Escherichia coli. In E. coli, the MMR system searches for hemimethylated GATC motifs around a mismatch to initiate removal of the faulty nascent (un-methylated) strand. Analysing the usage of GATC motifs around the TNR array, I have found that the MMR system preferentially utilizes the GATC motifs on the origin distal side of the TNR array demonstrating that the bidirectionality of MMR in vitro is constrained in live cells. My results suggest that in vivo MMR operates by searching for the nearest hemimethylated GATC site located between the mismatch and the replication fork and excision of the nascent strand occurs directionally away from the fork towards the mismatch. Previous in vitro studies have established that the excision reaction during MMR terminates at a discrete point about 100 bp beyond a mismatch. However, in vivo recombination at a 275 bp tandem repeat, which has been proposed to be mediated by single stranded DNA generated during the excision reaction, has suggested that the end point of the excision reaction in live cells may extend much further from the mismatch than this. I have used this assay for extended excision to determine the influence of GATC sites on excision tracts. In this study, modification of the GATC motifs on the origin proximal side of the TNR has shown that the excision reaction does not stop at a GATC motif on the origin proximal side of the mismatch. In addition, sequential modifications of GATC motifs on the origin distal side of the TNR array, thereby shifting the start point of the excision reaction to a greater distance, have suggested that the length of an excision tract is a function of the distance it covers from the start point rather than from a mismatch. My observation of directionality with respect to DNA replication in the recognition of GATC sites suggested that MMR and DNA replication might be coupled in some way and that perhaps active (or blocked) MMR might impede the progress of the replication fork. However, no replication intermediates were detected using two-dimensional agarose gel electrophoresis of genomic DNA fragment containing the TNR array upon restriction digestion. I was therefore unable to support the hypothesis that active or blocked MMR led to a slowing down of DNA replication. Given my observation of a decrease in MMR by separating the mismatch from the closest origin distal GATC site, I set out to test whether MMR caused any selection pressure for the genomic distribution of GATC motifs. To do this, I generated artificial model genomes using a Markovian algorithm based on the nucleotide composition and codon usage in E. coli. Strikingly, the comparison of the distribution of GATC motifs in the E. coli genome with those from artificial sequences has shown that GATC motifs are distributed randomly in E. coli genome, except for a small clustering effect which has been detected for short spaced (0-40 basepairs) GATC motifs. The observed distribution of slightly over-represented GATC motifs in the E. coli genome appears to be a function of the total number of GATC motifs and it seems that the DNA mismatch repair system has evolved to utilize the natural distribution of GATC motifs to maintain genomic integrity

Modeling of Potato Shelf Life on Evaporative Cooling Storage

A model of evaporative cooling storage system was designed to increase potato shelf life for improving potato storage system. Two cultivars of potato ‘Diamant’ (100 gm and 51 gm per tuber) and ‘LalPakri (23 gm and 11 gm per tuber) were placed on four shelves of the bin. Each shelf holds 240 kg of potato from 23 march 2013 to December 2013. Potato spoilage, sprouting, shrinkage, moisture content, vitamin C and total sugar content of potato were measured. Experimental results revealed that potato spoilage progressively increased from April to November and sprouting of potato gradually increased from June to October, but stopped in November. The cumulative spoilage and sprouting were much lower in the improved bin compared to traditional farmer’s practices. Shrinkage of potato was found higher in farmer’s practice than that of storage bin from October to November. Moisture content of potato was higher during May and reduced gradually to the lowest value during November in both of practices. No significant difference was found in two practices on vitamin-C content. Sugar content of ‘Diamant; potato was lower in the storage bin during November. According to data analysis and regression curve storage bin model was more appropriate for both cultivars than farmer practice and significantly more appropriate for ‘LalPakri’ potato

Using Mobile-Based Formative Assessment in ESL/EFL Speaking

With the widespread application of smartphones in and outside the classroom, mobile-based teaching and learning is drawing much attention and hence being extensively practised nowadays across the globe. Recently, using smartphones for assessment purposes has been a new phenomenon and the researchers are still examining what processes the use of mobile-based assessment tools may include and what outcomes and challenges they can cause to teachers and students in terms of learning/teaching performance, motivation and attitudes. There have been a good number of research studies on the use of Mobile Assisted Language Learning (MALL) or Mobile Learning (ML) in EFL or ESL classroom but not much literature is known about the mobile-based language assessment, especially mobile-based formative assessment (MBFA). Hence, this study attempts to shed light on MBFA and review the recent literature available on it and its effective utilization in developing ESL/EFL speaking skill. This paper uses a qualitative research method that exclusively uses the relevant secondary references/works available on the topic. The literature revealed that MBFA practices in ESL/EFL speaking classes are effective to a certain extent and some tools and procedures seem to be more effective than others depending on the design principles and strategies used by teachers or app developers

Word level Bangla Sign Language Dataset for Continuous BSL Recognition

An robust sign language recognition system can greatly alleviate communication barriers, particularly for people who struggle with verbal communication. This is crucial for human growth and progress as it enables the expression of thoughts, feelings, and ideas. However, sign recognition is a complex task that faces numerous challenges such as same gesture patterns for multiple signs, lighting, clothing, carrying conditions, and the presence of large poses, as well as illumination discrepancies across different views. Additionally, the absence of an extensive Bangla sign language video dataset makes it even more challenging to operate recognition systems, particularly when utilizing deep learning techniques. In order to address this issue, firstly, we created a large-scale dataset called the MVBSL-W50, which comprises 50 isolated words across 13 categories. Secondly, we developed an attention-based Bi-GRU model that captures the temporal dynamics of pose information for individuals communicating through sign language. The proposed model utilizes human pose information, which has shown to be successful in analyzing sign language patterns. By focusing solely on movement information and disregarding body appearance and environmental factors, the model is simplified and can achieve a speedier performance. The accuracy of the model is reported to be 85.64%

RecBCD coordinates repair of two ends at a DNA double-strand break, preventing aberrant chromosome amplification

DNA double-strand break (DSB) repair is critical for cell survival. A diverse range of organisms from bacteria to humans rely on homologous recombination for accurate DSB repair. This requires both coordinate action of the two ends of a DSB and stringent control of the resultant DNA replication to prevent unwarranted DNA amplification and aneuploidy. In Escherichia coli, RecBCD enzyme is responsible for the initial steps of homologous recombination. Previous work has revealed recD mutants to be nuclease defective but recombination proficient. Despite this proficiency, we show here that a recD null mutant is defective for the repair of a two-ended DSB and that this defect is associated with unregulated chromosome amplification and defective chromosome segregation. Our results demonstrate that RecBCD plays an important role in avoiding this amplification by coordinating the two recombining ends in a manner that prevents divergent replication forks progressing away from the DSB site

Accumulation of copy number alterations and clinical progression across advanced prostate cancer

Background: Genomic copy number alterations commonly occur in prostate cancer and are one measure of genomic instability. The clinical implication of copy number change in advanced prostate cancer, which defines a wide spectrum of disease from high-risk localised to metastatic, is unknown. Methods: We performed copy number profiling on 688 tumour regions from 300 patients, who presented with advanced prostate cancer prior to the start of long-term androgen deprivation therapy (ADT), in the control arm of the prospective randomised STAMPEDE trial. Patients were categorised into metastatic states as follows; high-risk non-metastatic with or without local lymph node involvement, or metastatic low/high volume. We followed up patients for a median of 7 years. Univariable and multivariable Cox survival models were fitted to estimate the association between the burden of copy number alteration as a continuous variable and the hazard of death or disease progression. Results: The burden of copy number alterations positively associated with radiologically evident distant metastases at diagnosis (P=0.00006) and showed a non-linear relationship with clinical outcome on univariable and multivariable analysis, characterised by a sharp increase in the relative risk of progression (P=0.003) and death (P=0.045) for each unit increase, stabilising into more modest increases with higher copy number burdens. This association between copy number burden and outcome was similar in each metastatic state. Copy number loss occurred significantly more frequently than gain at the lowest copy number burden quartile (q=4.1 × 10−6). Loss of segments in chromosome 5q21-22 and gains at 8q21-24, respectively including CHD1 and cMYC occurred more frequently in cases with higher copy number alteration (for either region: Kolmogorov–Smirnov distance, 0.5; adjusted P<0.0001). Copy number alterations showed variability across tumour regions in the same prostate. This variance associated with increased risk of distant metastases (Kruskal-Wallis test P=0.037). Conclusions: Copy number alteration in advanced prostate cancer associates with increased risk of metastases at diagnosis. Accumulation of a limited number of copy number alterations associates with most of the increased risk of disease progression and death. The increased likelihood of involvement of specific segments in high copy number alteration burden cancers may suggest an order underlying the accumulation of copy number changes

RecG directs DNA synthesis during double-strand break repair

Homologous recombination provides a mechanism of DNA double-strand break repair (DSBR) that requires an intact, homologous template for DNA synthesis. When DNA synthesis associated with DSBR is convergent, the broken DNA strands are replaced and repair is accurate. However, if divergent DNA synthesis is established, over-replication of flanking DNA may occur with deleterious consequences. The RecG protein of Escherichia coli is a helicase and translocase that can re-model 3-way and 4-way DNA structures such as replication forks and Holliday junctions. However, the primary role of RecG in live cells has remained elusive. Here we show that, in the absence of RecG, attempted DSBR is accompanied by divergent DNA replication at the site of an induced chromosomal DNA double-strand break. Furthermore, DNA double-stand ends are generated in a recG mutant at sites known to block replication forks. These double-strand ends, also trigger DSBR and the divergent DNA replication characteristic of this mutant, which can explain over-replication of the terminus region of the chromosome. The loss of DNA associated with unwinding joint molecules previously observed in the absence of RuvAB and RecG, is suppressed by a helicase deficient PriA mutation (priA300), arguing that the action of RecG ensures that PriA is bound correctly on D-loops to direct DNA replication rather than to unwind joint molecules. This has led us to put forward a revised model of homologous recombination in which the re-modelling of branched intermediates by RecG plays a fundamental role in directing DNA synthesis and thus maintaining genomic stability

The impact of ecological conditions on the prevalence of malaria among orangutans.

Contemporary human land use patterns have led to changes in orangutan ecology, such as the loss of habitat. One management response to orangutan habitat loss is to relocate orangutans into regions of intact, protected habitat. Young orangutans are also kept as pets and have at times been a valuable commodity in the illegal pet trade. In response to this situation, government authorities have taken law enforcement action by removing these animals from private hands and attempted to rehabilitate and release these orangutans. In relocating free-ranging orangutans, the animals are typically held isolated or with family members for &lt;48 h and released, but during the course of rehabilitation, orangutans often spend some time in captive and semicaptive group settings. Captive/semicaptive groups have a higher density of orangutans than wild populations, and differ in other ways that may influence susceptibility to infectious disease. In order to determine the impact of these ecological settings on malaria, the prevalence of malaria was compared between 31 captive and semicaptive orangutans in a rehabilitation program at the Sepilok Orangutan Rehabilitation Centre and 43 wild orangutans being moved in a translocation project. The prevalence of malaria parasites, as determined by blood smear and Plasmodium genus-specific nested-polymerase chain reaction, was greater in the captive/semicaptive population (29 of 31) than in the wild population (5 of 43) even when accounting for age bias. This discrepancy is discussed in the context of population changes associated with the management of orangutans in captive/semicaptive setting, in particular a 50-fold increase in orangutan population density. The results provide an example of how an ecological change can influence pathogen prevalence.</p