Mutations in the lamin A/C gene (LMNA) cause several disorders referred to as laminopathies, which include premature aging syndromes, lipodystrophy, and striated muscle disorders. There is evidence that lamin A/C plays a role in gene expression. MicroRNAs (miRNAs) are short noncoding RNAs regulating mRNAs involved in various biological processes, including the pathophysiology of striated muscles. Here, we profiled the expression of the miRNA transcriptome in skeletal muscle from patients with LMNA-related muscular dystrophy. Results show that control and patient groups can be distinguished based on their miRNA expression profile. Sixteen miRNAs are significantly dysregulated in patients compared with controls. Pathway enrichment analysis in the predicted targets of these miRNAs revealed pathways involved in muscle repair, such as MAPK, transforming growth factor-β, and Wnt signaling. Interestingly, 9 of these miRNAs (hsa-miR-100, -127-3p, -148a, -136*, -192, -335, -376c, -489, and -502-3p) are highly expressed in fetal muscle, suggesting that the fetal miRNA gene program mediates a regenerative process. Overexpression of these miRNAs in C2C12 mouse myoblasts revealed that 3 of them (miR-100, -192, and -335) participate in muscle proliferation and differentiation. We identified target genes that likely mediate this effect, which include the calcineurin gene PPP3CA. Our findings are the first to demonstrate that miRNA expression is affected in laminopathies.
Education acts as a soul in the overall societal development, in one way or the other. Aspirants, who gain their degrees genuinely, will help society with their knowledge and skills. But, on the other side of the coin, the problem of fake certificates is alarming and worrying. It has been prevalent in different forms from paper-based dummy certificates to replicas backed with database tampering and has increased to astronomic levels in this digital era. In this regard, an overlay mechanism using blockchain technology is proposed to store the genuine certificates in digital form and verify them firmly whenever needed without delay. The proposed system makes sure that the certificates, once verified, can be present online in an immutable form for further reference and provides a tamper-proof concealment to the existing certification system. To confirm the credibility of the proposed method, a prototype of blockchain-based credential securing and verification system is developed in ethereum test network. The implementation and test results show that it is a secure and feasible solution to online credential management system.
Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic mouse models using this method necessitates the construction of a 'targeting' vector, which contains homologous DNA sequences to the target gene, and has for many years been a limiting step in the process. Vector construction can be performed in vivo in Escherichia coli cells using homologous recombination mediated by phage recombinases using a technique termed recombineering. Recombineering is the preferred technique to subclone the long homology sequences (>4kb) and various targeting elements including selection markers that are required to mediate efficient allelic exchange between a targeting vector and its cognate genomic locus. Typical recombineering protocols follow an iterative scheme of step-wise integration of the targeting elements and require intermediate purification and transformation steps. Here, we present a novel recombineering methodology of vector assembly using a multiplex approach. Plasmid gap repair is performed by the simultaneous capture of genomic sequence from mouse Bacterial Artificial Chromosome libraries and the insertion of dual bacterial and mammalian selection markers. This subcloning plus insertion method is highly efficient and yields a majority of correct recombinants. We present data for the construction of different types of conditional gene knockout, or knock-in, vectors and BAC reporter vectors that have been constructed using this method. SPI vector construction greatly extends the repertoire of the recombineering toolbox and provides a simple, rapid and cost-effective method of constructing these highly complex vectors.
The lambda phage Red recombination system can mediate efficient homologous recombination in Escherichia coli, which is the basis of the DNA engineering technique termed recombineering. Red mediated insertion of DNA requires DNA replication, involves a single-stranded DNA intermediate and is more efficient on the lagging strand of the replication fork. Lagging strand recombination has also been postulated to explain the Red mediated repair of gapped plasmids by an Okazaki fragment gap filling model. Here, we demonstrate that gap repair involves a different strand independent mechanism. Gap repair assays examining the strand asymmetry of recombination did not show a lagging strand bias. Directly testing an ssDNA plasmid showed lagging strand recombination is possible but dsDNA plasmids did not employ this mechanism. Insertional recombination combined with gap repair also did not demonstrate preferential lagging strand bias, supporting a different gap repair mechanism. The predominant recombination route involved concerted insertion and subcloning though other routes also operated at lower frequencies. Simultaneous insertion of DNA resulted in modification of both strands and was unaffected by mutations to DNA polymerase I, responsible for Okazaki fragment maturation. The lower efficiency of an alternate Red mediated ends-in recombination pathway and the apparent lack of a Holliday junction intermediate suggested that gap repair does not involve a different Red recombination pathway. Our results may be explained by a novel replicative intermediate in gap repair that does not involve a replication fork. We exploited these observations by developing a new recombineering application based on concerted insertion and gap repair, termed SPI (subcloning plus insertion). SPI selected against empty vector background and selected for correct gap repair recombinants. We used SPI to simultaneously insert up to four different gene cassettes in a single recombineering reaction. Consequently, our findings have important implications for the understanding of E. coli replication and Red recombination.
Biometric systems are the most advanced access technology developed so far in the 21st century. It does not even require to carry key cards or passwords in mind. Today most of the commercial and private entries are protected by biometric recognition systems like fingerprint scans facial recognition, retina scans, voice matching, etc. Even our phones, laptops, and daily access devices are equipped with biometric systems. In banks, the PCs are secured by the combination of passwords and fingerprint scans. Biometric scans are considered the most secure access technology so far. Our paper is to examine whether they are secure? Should we rely on them with our hard-earned money and social identity? Is there any way we can use these services without actually compromising our data and security? Our observation is on our digital identity. Promoting digitization in every department brings our topic in the picture. All our information is saved in our phones, our daily routine, whom we talk, what we purchase, whom we chat, where we travel, etc. Almost every smartphone has biometric fingerprint locks which means our phones have our fingerprint scans in database and with internet blend it’s tethered worldwide. Our fingerprints are connected to our bank accounts, PAN Cards, Passport, and SIM Cards using Aadhar Cards. If someone has our fingerprint they can easily reach our Aadhar Card and through that to all our personal information. Most of the phone companies are Chinese, Korean, German, and American. As per their country policies, they must share their data with the governing authorities. We aim to create a security system without actually using the biometric scans. The system is an advancement of the biometric system but with better accuracy and intelligence. We interface image acquisition tools to live track the red color things. The web camera or inbuilt system lens can be used as the acquisition system. When the red color object is moved in front of the lens it shows the corresponding coordinate of the object shown. We use these x and y coordinate of the objects as the authentication points. If the correct value grant access is 120 ⩽ x ⩽ 122 means the system grants permission only if the value of x=120,121 or 122 is obtained. Now, this is tricky. Even if you know the correct value also, it is very difficult to bring the correct point. Think about if you don’t know the point and it is also possible to make it much difficult by adding y coordinate so if the desired point is x=10, y=12 (10, 12) it is way more difficult. Each point is a possible password candidate and the screen of any device have megapixels where 1 Megapixel=106 pixels. Each pixel is a possible key or password entry. It can keep all our information safe and secure. We use a microcontroller and motor driver connected gate to demonstrate the result.
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