Potential Use of CRISPR/Cas13 Machinery in Understanding Virus–Host Interaction

Prokaryotes have evolutionarily acquired an immune system to fend off invading mobile genetic elements, including viral phages and plasmids. Through recognizing specific sequences of the invading nucleic acid, prokaryotes mediate a subsequent degradation process collectively referred to as the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)–CRISPR-associated (Cas) (CRISPR–Cas) system. The CRISPR–Cas systems are divided into two main classes depending on the structure of the effector Cas proteins. Class I systems have effector modules consisting of multiple proteins, while class II systems have a single multidomain effector. Additionally, the CRISPR–Cas systems can also be categorized into types depending on the spacer acquisition components and their evolutionary features, namely, types I–VI. Among CRISPR/Cas systems, Cas9 is one of the most common multidomain nucleases that identify, degrade, and modulate DNA. Importantly, variants of Cas proteins have recently been found to target RNA, especially the single-effector Cas13 nucleases. The Cas13 has revolutionized our ability to study and perturb RNAs in endogenous microenvironments. The Cas13 effectors offer an excellent candidate for developing novel research tools in virological and biotechnological fields. Herein, in this review, we aim to provide a comprehensive summary of the recent advances of Cas13s for targeting viral RNA for either RNA-mediated degradation or CRISPR–Cas13-based diagnostics. Additionally, we aim to provide an overview of the proposed applications that could revolutionize our understanding of viral–host interactions using Cas13-mediated approaches.

Recombinant protein expression and purification of codon-optimized Pfu-Sso7d v2

This protocol has been optimized for the recombinant expression of a codon-optizimed Pfu-Sso7d DNA polymerase. This is a fusion protein composed of the Pfu enzyme from Pyrococcus furiosus for DNA amplification by PCR fused to a small 7 kDa protein from Sulfobulus solfataricus that binds to double-stranded DNA without any preference for specific sequences, thus enhancing polymerization processivity without affecting the catalytic activity or thermal stability of the enzyme. The goal of this protocol was to eliminate the use of large volumes for dyalisis and potential issues with the protein crashing out of the solution due to the use of concentrators for buffer exchange of this enzyme into storage conditions. We also eliminated the use of DTT, which is often found in other similar protocols. The sequence plasmid encoding the codon-optimized Pfu-Sso7d enzyme used here can be found at https://benchling.com/s/seq-2TcUPjO2uMbDG5ufTQN4

Mitochondrial Phylogenomics of Fagales Provides Insights Into Plant Mitogenome Mosaic Evolution

Fagales are an order of woody plants and comprise more than 1,100 species, most of which produce economically important timbers, nuts, and fruits. Their nuclear and plastid genomes are well-sequenced and provided valuable resources to study their phylogeny, breeding, resistance, etc. However, little is known about the mitochondrial genomes (mitogenomes), which hinder a full understanding of their genome evolution. In this study, we assembled complete mitogenomes of 23 species, covering five of the seven families of Fagales. These mitogenomes had similar gene sets but varied 2.4 times in size. The mitochondrial genes were highly conserved, and their capacity in phylogeny was challenging. The mitogenomic structure was extremely dynamic, and synteny among species was poor. Further analyses of the Fagales mitogenomes revealed extremely mosaic characteristics, with horizontal transfer (HGT)-like sequences from almost all seed plant taxa and even mitoviruses. The largest mitogenome, Carpinus cordata, did not have large amounts of specific sequences but instead contained a high proportion of sequences homologous to other Fagales. Independent and unequal transfers of third-party DNA, including nuclear genome and other resources, may partially account for the HGT-like fragments and unbalanced size expansions observed in Fagales mitogenomes. Supporting this, a mitochondrial plasmid-like of nuclear origin was found in Carpinus. Overall, we deciphered the last genetic materials of Fagales, and our large-scale analyses provide new insights into plant mitogenome evolution and size variation.

ArVirInd - a database of arboviral antigenic proteins from the Indian subcontinent

Studies on antigenic proteins for arboviruses are important for providing diagnostics and vaccine development. India and its neighbouring countries have huge burden of arboviral diseases. Data mining for country-specific sequences from existing databases is cumbersome and time-consuming. This necessitated the development of a database of antigenic proteins from arbo-viruses isolated from the countries of the Indian subcontinent. Arboviral antigenic protein sequences were obtained from the NCBI and other databases. In silico antigenic characterization was performed (Epitope predictions) and data incorporated in the database. The front end is designed and developed using HTML, CSS and PHP. For the backend of the database, we have used MySQL. A database, named ArVirInd, is created as a repository of information on antigenic proteins. This enlists sequences by country and year of outbreak or origin of the viral strain. For each entry antigenic information is provided along with functional sites, etc. Researchers can search this database by virus/protein name, country and year of collection (or in combination). It is available publicly via Internet at http://www.arvirind.co.in. ArVirInd will be useful in the study of immuno-informatics, diagnostics and vaccinology for arboviruses.

Widespread bacterial diversity within the bacteriome of fungi

Knowledge of associations between fungal hosts and their bacterial associates has steadily grown in recent years as the number and diversity of examinations have increased, but current knowledge is predominantly limited to a small number of fungal taxa and bacterial partners. Here, we screened for potential bacterial associates in over 700 phylogenetically diverse fungal isolates, representing 366 genera, or a tenfold increase compared with previously examined fungal genera, including isolates from several previously unexplored phyla. Both a 16 S rDNA-based exploration of fungal isolates from four distinct culture collections spanning North America, South America and Europe, and a bioinformatic screen for bacterial-specific sequences within fungal genome sequencing projects, revealed that a surprisingly diverse array of bacterial associates are frequently found in otherwise axenic fungal cultures. We demonstrate that bacterial associations with diverse fungal hosts appear to be the rule, rather than the exception, and deserve increased consideration in microbiome studies and in examinations of microbial interactions.

Adenine Quadruplexes Show Surprising Stability: Potential Implications for SARS-CoV-2

SARS-CoV-2 is an endemic positive-sense RNA virus naturally transmissible between numerous species with notable infectivity and associated mortality. It is characterized by a poly-adenylated structure capping the genomic terminus. This poly(A) tail is crucial to a cascade of viral replicative activity occurring both extra- and intra-cellular during infection. As a route to proposing potential chemotherapy, this study suggests simple biplanar adenine quadruplexes (A4s) which may fold in specific sequences of the viral genome. To the best of our knowledge, uniquely biplanar A4s have not been previously described in any context. Using molecular modeling techniques and molecular dynamics simulations, some of these non-canonical structures show reasonable stability in a biological context. Notably, mRNA configured as a biplanar A4, shows less dynamic activity than DNA equivalents. This observation may be especially relevant in a physiological context. Furthermore, in contrast to well-characterized guanine quadruplexes, co-ordination with cations appears not to impact on stability. Our molecular dynamics simulations and analyses demonstrate that some A4s are stable in biologically relevant terms. These conclusions may apply to SARS-CoV-2, its variants and other pathogenic RNA viruses.

Validation of the Tactual Span in individuals with congenital and acquired blindness

Neuropsychological assessment tools for individuals with blindness are relatively scarce. In the current study, we assessed the validity of the Tactual Span, a task aimed at evaluating tactile working memory. During the task, the fingers of both hands are touched in specific sequences of ascending difficulty, which participants are asked to repeat in exact and reverse order. Twelve participants with congenital blindness and 13 with acquired blindness were examined alongside 18 sighted controls, matched to the experimental group with respect to age and education. Participants performed the Tactual Span and three additional tasks assessing working memory in the auditory modality, as well as a Semantic Fluency test. Results showed that the Tactual Span was significantly correlated with most of the other working memory measures, in all groups, but not with the Semantic Fluency test. In addition, the congenital and acquired blindness groups performed similarly to one another and better than sighted controls on most working memory tasks, but not on the Semantic Fluency test. Findings suggest that the Tactual Span is a feasible task for measuring tactile working memory in individuals with congenital and acquired blindness. Therefore, it can expand the cognitive assessment toolbox of professionals working with blind individuals and increase the strength of conclusions drawn from cognitive assessments in educational and vocational settings.

Paraspinal Muscle Activity Differences in Various Subtypes of Idiopathic Scoliosis

Background: Scoliosis refers to deviation of spinal alignment in three dimensional planes. In response to functional demands Muscle fibers change in motor unit function. Knowledge of difference in muscle fiber activity in functional situations allows us to plan curve specific effective physical therapy. Methodology: All study subjects were categorised based on Rigo Classification and assessed for para spinal muscle activity by Surface Electro Myography at cervical, thoracic, lumbar, concave- convex sides of apex and lumbar /lumbo sacral regions of spine in general sequence and curve specific sequence. Results: Total number of subjects were 25 (16 female and 9 male). Age ranging from 12 to 39 years and Risser from 1-5. Overall maximum amplitudes recorded in antigravity positions. Amplitudes of as low as (M±σ = mean± standard deviation) M±σ: 0.92±0.79µv to M± σ: 1411.6±734.9µv were found. Conclusion: Differences in right and left paraspinal activity was observed in general and specific sequences, but this difference was not consistent in general and specific sequence surface electro myography. Maximum amplitudes found at lumbar and lumbo sacral regions on antigravity positions than other positions. Convex side has more activity than concave sides at apex regions. Differences were not similar in all positions, this difference in left and right side was varying with curve type and type of movement. Results of paraspinal activity may be used for planning of suitable exercises after achieving best possible correction in sagittal and frontal planes. Key words: Paraspinal muscle activity differences, idiopathic scoliosis, deviation of spinal alignment.

Evidences of Z- and W-Linked Regions on the Genome of Fenneropenaeus chinensis

Fenneropenaeus chinensis is a commercially cultured shrimp in China. F. chinensis adults show significant sexual dimorphism, with larger females than males. However, sex determination (SD) of F. chinensis has not yet been elucidated. Clarification of the sex-determining system of F. chinensis could enrich our knowledge of the sex differentiation mechanism in crustaceans and facilitate the study of sex-controlling technologies. Here, we studied the sex-determining system of F. chinensis using the fixation index (FST) between the sexes to detect the genetic differentiation in resequencing data of multiple males and females. We located the candidate sex chromosome in the genome of F. chinensis and concluded the female heterogametic (ZW) SD system. We also assembled female-specific sequences, which could be used as molecular markers to identify the sex of F. chinensis. However, the differentiation of the F. chinensis Z and W chromosome is limited. RNA-seq data detected many genes with male-biased expression in the Z-specific region, which possibly could further intensify the divergency between the Z and W chromosomes.

Succinct Encoding of Binary Strings Representing Triangulations

We consider the problem of designing a succinct data structure for representing the connectivity of planar triangulations. The main result is a new succinct encoding achieving the information-theory optimal bound of 3.24 bits per vertex, while allowing efficient navigation. Our representation is based on the bijection of Poulalhon and Schaeffer (Algorithmica, 46(3):505–527, 2006) that defines a mapping between planar triangulations and a special class of spanning trees, called PS-trees. The proposed solution differs from previous approaches in that operations in planar triangulations are reduced to operations in particular parentheses sequences encoding PS-trees. Existing methods to handle balanced parentheses sequences have to be combined and extended to operate on such specific sequences, essentially for retrieving matching elements. The new encoding supports extracting the d neighbors of a query vertex in O(d) time and testing adjacency between two vertices in O(1) time. Additionally, we provide an implementation of our proposed data structure. In the experimental evaluation, our representation reaches up to 7.35 bits per vertex, improving the space usage of state-of-the-art implementations for planar embeddings.