Versatility of reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) from diagnosis of early pathological infection to mutation detection in organisms.

Loop-mediated isothermal amplification (LAMP) is a rapid, state-of-the-art DNA amplification technology, used primarily for the quick diagnosis and early identification of microbial infection, caused by pathogens such as virus, bacteria and malaria. A target DNA can be amplified within 30 min using the LAMP reaction, taking place at a steady temperature. The LAMP method uses four or six primers to bind eight regions of a target DNA and has a very high specificity. The devices used for conducting LAMP are usually simple since the LAMP method is an isothermal process. When LAMP is coupled with Reverse Transcription (RT), it allows direct detection of RNA in a sample. This greatly enhances the efficiency of diagnosis of RNA viruses in a sample. Recently, the rampant spread of COVID-19 demanded such a rapid, simple, and cost-effective Point of Care Test (PoCT) for the accurate diagnosis of this pandemic. Loop-mediated isothermal amplification (LAMP) assays are not only used for the detection of microbial pathogens, but there are various other applications such as detection of genetic mutations in food and various organisms. In this review, various implementations of RT-LAMP techniques would be discussed.

Application of Restriction Free (RF) Cloning in Circular Permutation.

The restriction free (RF) cloning has emerged as one of the highly efficient techniques in the area of genetic engineering. RF cloning has wide range of applications in plasmid DNA manipulation including cloning of a single gene, simultaneous assembly of multiple DNA fragments, and mutagenesis from single to multiple simultaneous alterations of a target DNA. Recently, we have developed a new technique of circular permutation using RF cloning. Circular permutation is widely used to investigate the mechanisms of protein folding and function. Previously, restriction enzyme based cloning was used to introduce circular permutation. Our RF cloning method made the protocol faster and more cost-effective. In this chapter, we describe a step-by-step protocol for generating circular permutants using RF methodology.

Molecular docking studies of phytochemicals from Terminalia chebula for identification of potential multi-target inhibitors of SARS-CoV-2 proteins.

Background: The COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has emerged as a global pandemic claiming more than 6 million lives worldwide as of 16 March 2022. Till date, no medicine has been developed which is proved to have 100% efficiency in combating against this deadly disease. We focussed on ayurvedic medicines to identify drug-like candidates for treatment and management of COVID-19. Among all ayurvedic medicines, we were interested in Terminalia chebula (T. chebula), as it is known to have antibacterial, antifungal, antiviral, antioxidant and anti-inflammatory properties. Objectives: In this study, we evaluated potential inhibitory effects of phytochemicals from T. chebula against eight structural and functional proteins of SARS-CoV-2. Material and methods: We performed blind molecular docking studies using fifteen phytochemicals from T. chebula against the proteins of SARS-CoV-2. The three-dimensional proteins structures were analysed and potential drug-binding sites were identified. The drug-likeness properties of the ligands were assessed as well. Results: Analysing the docking results by comparing Atomic Contact Energy (ACE) and intermolecular interactions along with assessment of ADME/T properties identified 1,3,6-Trigalloyl glucose (-332.14 ± 55.74 kcal/mol), Beta-Sitosterol (-324.75 ± 36.98 kcal/mol) and Daucosterol (-335.67 ± 104.79 kcal/mol) as most promising candidates which exhibit significantly high inhibition efficiency against all eight protein targets. Conclusions: We believe that our study has the potential to help the scientific communities to develop multi-target drugs from T. chebula to combat against the deadly pathogen of COVID-19, with the support of extensive wet lab analysis.

Portable sterilizer with microbe content detection device.

BACKGROUND: Infectious diseases, such as the latest COVID pandemic, caused by microorganisms like bacteria and virus, wreak havoc shaking human civilization with its rapid infection rate, and high number of mortalities. In case of a contagious disease, the virus can survive on any surface over a period of time and can be transferred to the human host through touching those surfaces unknowingly. Cleaning those possible surfaces to which these microorganisms can cling onto is one of the major ways to curb the spread. The aim of this study was to design a sterilizer which can clean such surfaces of daily used items easily within a certain period of time and can assess the cleaning efficacy by estimating the presence of microbes before and after sanitization. METHOD DEVELOPMENT: To achieve this goal, we propose a portable sterilization unit that contains a sterilization chamber fitted with a microbe content detector. The sterilization chamber will cleanse the surfaces off the microbes using ultraviolet radiation. The chamber can be portable and at the same time big enough to accommodate items of daily use, like watch, wallet, clothes, utensils to even foods for single-house application. The microbe content detector will detect the success of the sterilization procedure by examining the time-lapse laser speckle images captured by a high-speed camera by mean of image processing algorithm, such that the user can determine whether further sterilization is required. CONCLUSIONS: Microbe content detection device associated with the conventional sterilization procedure will give an assessment of the effectiveness of the sterilization. Successful implementation of sterilization for a wide variety of items of everyday use aided with microbe content detection technique is first of its kind and should be an effective tool for use in large communities, offices and public places for effective sterilization to help fight against the spread of infectious diseases.

Genomic and Proteomic Characterizations of Sfin-1, a Novel Lytic Phage Infecting Multidrug-Resistant Shigella spp. and Escherichia coli C.

Shigellosis is a public health threat in developed as well as developing countries like "India." While antibiotic therapy is the mainstay of treatment for shigellosis, current emergence of multidrug-resistant strains of Shigella spp. has posed the problem more challenging. Lytic bacteriophages which destroy antibiotic resistant Shigella spp. have great potential in this context and hence their identification and detailed characterization is necessary. In this study we presented the isolation and a detailed characterization of a novel bacteriophage Sfin-1, which shows potent lytic activity against multidrug-resistant isolates of Shigella flexneri, Shigella dysenteriae, Shigella sonnei obtained from clinical specimens from shigellosis patients. It is also active against Escherichia coli C. The purified phage is lytic in nature, exhibited absorption within 5-10 min, a latent period of 5-20 min and burst size of ∼28 to ∼146 PFU/cell. The isolated phage shows stability in a broad pH range and survives an hour at 50°C. Genome sequencing and phylogenetic analyses showed that Sfin-1 is a novel bacteriophage, which is very closely related to T1-like phages (89.59% identity with Escherichia virus T1). In silico analysis indicates that Sfin-1 genome consists of double stranded linear DNA of 50,403 bp (GC content of 45.2%) encoding 82 potential coding sequences, several potential promoters and transcriptional terminators. Under electron microscopy, Sfin-1 shows morphology characteristics of the family Siphoviridae with an isometric head (61 nm) and a non-contractile tail (155 nm). This is most likely the first report of a lytic bacteriophage that is active against three of the most virulent multidrug-resistant Shigella species and therefore might have a potential role in phage therapy of patients infected with these organisms.

Contact Order Is a Determinant for the Dependence of GFP Folding on the Chaperonin GroEL.

The GroE chaperonin system facilitates protein folding in an ATP-dependent manner. It has remained unclear why some proteins are obligate clients of the GroE system, whereas other closely related proteins are able to fold efficiently in its absence. Factors that cause folding to be slower affect kinetic partitioning between spontaneous folding and chaperone binding in favor of the latter. One such potential factor is contact order (CO), which is the average separation in sequence between residues that are in contact in the native structure. Here, we generated variants of enhanced green fluorescent protein with different COs using circular permutations. We found that GroE dependence in vitro and in vivo increases with increasing CO. Thus, our results show that CO is relevant not only for folding in vitro of relatively simple model systems but also for chaperonin dependence and folding in vivo.

Facilitating circular permutation using Restriction Free (RF) cloning.

Circular permutation is a powerful tool to test the role of topology in protein folding and function. Previous methods for generating circular permutants were based on rearranging gene elements using restriction enzymes-based cloning. Here, we present a Restriction Free (RF) approach to achieve circular permutation which is faster and more cost-effective.

Local energetic frustration affects the dependence of green fluorescent protein folding on the chaperonin GroEL.

The GroE chaperonin system in Escherichia coli comprises GroEL and GroES and facilitates ATP-dependent protein folding in vivo and in vitro Proteins with very similar sequences and structures can differ in their dependence on GroEL for efficient folding. One potential but unverified source for GroEL dependence is frustration, wherein not all interactions in the native state are optimized energetically, thereby potentiating slow folding and misfolding. Here, we chose enhanced green fluorescent protein as a model system and subjected it to random mutagenesis, followed by screening for variants whose in vivo folding displays increased or decreased GroEL dependence. We confirmed the altered GroEL dependence of these variants with in vitro folding assays. Strikingly, mutations at positions predicted to be highly frustrated were found to correlate with decreased GroEL dependence. Conversely, mutations at positions with low frustration were found to correlate with increased GroEL dependence. Further support for this finding was obtained by showing that folding of an enhanced green fluorescent protein variant designed computationally to have reduced frustration is indeed less GroEL-dependent. Our results indicate that changes in local frustration also affect partitioning in vivo between spontaneous and chaperonin-mediated folding. Hence, the design of minimally frustrated sequences can reduce chaperonin dependence and improve protein expression levels.

DnaK dependence of the mycobacterial stress-responsive regulator HspR is mediated through its hydrophobic C-terminal tail.

HspR is a repressor known to control expression of heat shock operons in a number of Eubacteria. In mycobacteria and in several other actinobacteria, this protein is synthesized from the dnaKJE-hspR operon. Previous investigations revealed that HspR binds to the operon promoter, thereby controlling its expression in an autoregulatory manner. DnaK, which is a product of the same operon, further aids this autoregulatory process by stimulating the operator binding activity of HspR. The molecular mechanism by which DnaK assists HspR in executing its function is not clearly understood. In this study, it has been shown that DnaK can augment DNA binding activity of HspR by two mechanisms: (i) DnaK can restore the activity of completely denatured HspR by forming a complex with it, and (ii) DnaK can prevent thermal instability of HspR renatured by other means. Unlike the first mechanism, the latter function does not involve complex formation. The C-terminal hydrophobic tail of HspR was found to play a significant role in determining its thermal stability and DnaK dependence properties. A deletion mutant in which this region is removed does not respond to thermal stress and functions independent of DnaK. The hydrophobic C-terminal tails of HspRs of Mycobacterium tuberculosis and related Actinomycetales therefore may have evolved to make these HspRs more sensitive to thermal stress and, at the same time, subject to regulation by DnaK.

Modulation of DNA-binding activity of Mycobacterium tuberculosis HspR by chaperones.

In Mycobacterium tuberculosis, hspR is the last gene of the dnaKJE operon. It encodes the repressor HspR, which regulates the expression from this operon by binding to a consensus upstream sequence known as HAIR (HspR-associated inverted repeats). Previous investigations in the related Gram-positive bacterium Streptomyces coelicolor have revealed that DnaK acts as a co-repressor for HspR. In this investigation, a similar situation was encountered using the corresponding mycobacterial pair. However, the novel feature unearthed in this study is that the mycobacterial GroELs, GroEL1 and GroEL2, considerably stimulate the HAIR-binding activity of the HspR-DnaK combination. That these GroELs play a role in the folding process was evident from the observation that when heat- or chemically denatured HspR was renatured, the protein gained optimal activity only if one of these GroEL class chaperones was present along with DnaK. The renaturation process was found to be dependent on ATP hydrolysis. The DnaK-dependent DNA-binding activity of HspR could also be stimulated by DnaJ, but GrpE, which is known to release DnaK-bound substrates, was found to be inhibitory. The results of this study suggest that protein folding plays a substantial role in the activation of HspR following heat shock and that DnaK may be involved in two ways -- first, as a chaperone acting in concert with GroEL and/or DnaJ and second, as a co-repressor bound to HspR.