MS2 RNA aptamer enhances prime editing in rice

Prime editing is a universal and very promising precise genome editing technology. However, optimization of prime editor (PE) from different aspects remains vital for its use as a routine tool in plant basic research and crop molecular breeding. In this report, we tested MS2-based prime editor (MS2PE). We fused the M-MLV reverse transcriptase (RT) gene variant to the MS2 RNA binding protein gene, MCP, and allowed the MCP-RT fusion gene to co-express with the SpCas9 nickase gene, SpCas9H840A, and various engineered pegRNAs harboring MS2 RNA (MS2pegR). Compared with control PEs, MS2PEs significantly enhanced editing efficiency at four of six targets in rice protoplasts, and achieved 1.2~10.1-fold increase in editing efficiency at five of six targets in transgenic rice lines. Furthermore, we tested total 22 different MS2pegR scaffolds, 3 RT variants or genes, 2 MCP variants, and various combinations of the Cas9 nickase, RT, and MCP modules. Our results demonstrated an alternative strategy for enhancing prime editing.

Loss of Aly/ALYREF Suppresses Toxicity in Both Tau and TDP-43 Models of Neurodegeneration

Background Neurodegenerative diseases with tau pathology, or tauopathies, include Alzheimer’s Disease and related dementia disorders. Previous work has shown that loss of the poly(A) RNA-binding protein gene sut2/MSUT2 strongly suppressed tauopathy in C. elegans, human cell culture, and mouse models of tauopathy. However, the mechanism of suppression is still unclear. Recent work has shown that MSUT2 protein interacts with the THO complex and ALYREF, which are components of the mRNA nuclear export complex. Additionally, previous work showed ALYREF homolog Ref1 modulates TDP-43 and G 4 C 2 toxicity in D. melanogaster models. Methods We used transgenic C. elegans models of tau or TDP-43 toxicity to investigate the effects of loss of ALYREF function on tau and TDP-43 toxicity. In C. elegans, there are three genes that are homologous to human ALYREF: aly-1, aly-2, and aly-3. Results We found that loss of C. elegans aly gene function, especially loss of both aly-2 and aly-3, suppressed tau-induced toxic phenotypes. Loss of aly-2 and aly-3 was also able to suppress TDP-43-induced behavioral deficits. However, loss of aly-2 and aly-3 had divergent effects on mRNA and protein levels as total tau protein levels were reduced while mRNA levels were increased, but no significant effects were seen on total TDP-43 protein or mRNA levels. Conclusions Our results suggest that although aly genes modulate both tau and TDP-43-induced toxicity phenotypes, the molecular mechanisms of suppression are different and separated from impacts on mRNA and protein levels. Altogether this study highlights the importance of elucidating RNA-related mechanisms in both tau and TDP-43-induced toxicity.

MS2 RNA aptamer greatly enhances prime editing in rice

Prime editing is a universal and very promising precise genome editing technology. However, optimization of prime editor (PE) from different aspects remains vital for its use as a routine tool in plant basic research and crop molecular breeding. In this report, we tested MS2-based prime editor (MS2PE). We fused the M-MLV reverse transcriptase (RT) gene variant to the MS2 RNA binding protein gene, MCP, and allowed the MCP-RT fusion gene to co-express with the SpCas9 nickase gene, SpCas9H840A, and various engineered pegRNAs harboring MS2 RNA (MS2pegR). Compared with control PEs, MS2PEs significantly enhanced editing efficiency at four of six targets in rice protoplasts, and achieved 1.2~10.1-fold increase in editing efficiency at five of six targets in transgenic rice lines. Furthermore, we tested total 22 different MS2pegR scaffolds, 3 RT variants or genes, 2 MCP variants, and various combinations of the Cas9 nickase, RT, and MCP modules. Our results demonstrated a new strategy for more efficient prime editing and provide a platform for further directed evolution of PEs.

​Cloning and Sequence Analysis of Transferrin Binding Protein Gene A of Field and Vaccine Strains of Pasteurella multocida B:2

Background: Pasteurella multocida is an important bacterial pathogen that causes many major diseases of which haemorrhagic septiciemia (HS) in cattle and buffalo is responsible for catastrophic epizootics in India and South Asia. In India, the disease haemorrhagic septiciemia is considered as the most dreaded bacterial disease. Various host- and pathogen- specific determinants are responsible for disease outcome. Various bacterial virulence genes (tbpA, pfhA, toxA, hgbB, hgbA, nanH, nanB, sodA, sodC, oma87 and ptfA) have been proposed to play a key role in this interaction. Methods: The present study was done to compare the gene and deduced amino acid sequence of transferrin binding protein gene (tbpA) gene of field isolates and vaccine strain of P. multocida B: 2. Result: It was observed that tbpA gene of field and vaccine strains have similar nucleotide sequence except at positions 574 and 620. The sequence of tbpA gene was used for prediction of matured TbpA protein characteristics. The deduced amino acid sequences of 242 amino acids revealed 99% homology with TbpA of P. multocida and with a variety of other TonB-dependent receptor proteins, indicating that it belongs to the family of outer membrane receptors. Deduced amino acid sequence was found to be similar in field and vaccine strains except at 207th amino acid. In field isolates Leucine was there while in vaccine strain Phenyl alanine was found. These both amino acids are hydrophobic in nature so no change in physico-chemical property of TbpA is expected. From this study it is concluded that single amino acid difference between field isolate and vaccine strain might not cause change in its binding and physico-chemical property.

Polymorphism of FABP3 gene and its effect on litter size and milk production of synthetic population bulgarian milk ewes

Aim. The purpose of this work was to study polymorphism of FABP3 (heart-type fatty acid binding protein) gene and its effect on litter size and milk production. The experiment included 30 ewes of Synthetic Population Bulgarian Milk breed from Institute of Animal Science – Kostinbrod. Methods. By PCR-RFLP method with endonuclease BseDI in SNP3 of FABP3 gene were detected two genotypes – GG and AG. Results. In SNP3 of FABP3 gene the frequency of allele G was 0.85 and the frequency of allele A was 0.15. The genotype GG was with frequency 0.70 and AG – with 0.30. Conclusions. In this study of ewes from SPBM breed, the presence of heterozygous genotype AG in SNP3 of the FABP3 was associated with increased litter size, while the presence of the homozygous genotype GG led to increased milk productivity during lactation. Keywords: sheep, FABP3 gene, litter size, milk production.

ScFT6: A Putative Candidate for Sugarcane Floral Inducer

One of the factors that can decrease sugarcane productivity is the flowering, because it affects the quantity and quality of feedstock, due to sucrose consumption from the stem during inflorescence emission. Photoperiodicity is the main environmental factor involved in sugarcane floral induction, which occurs by the integration of gene regulatory networks in response to environmental and endogenous stimuli. One of the genes involved in those regulatory networks is the FLOWERING LOCUS T (FT), which is considered a phloem-mobile signal that stimulates floral induction in the shoot apical meristem. This work aimed to identify and characterize homologs of the FT gene in sugarcane, as well as to determine the putative function of these genes during floral induction. From this perspective, we have conducted in silico analyses of putative FT orthologs in sugarcane, as well as the expression levels in different photoperiodic conditions in a 24-hours-day-cycle of ScFT6 in different plant tissues in contrasting cultivars in terms of flowering time. Three new possible FT orthologs were found with high similarity to FT homologs in other species. Among three genes identified, we highlighted ScFT6, which has a conserved domain and amino acids at characteristic positions for the flowering inducer phosphatidylethanolamine-binding protein gene family. Additionally, its expression occurs according to coincidental model, possibly being controlled by the circadian clock. Cultivars with distinct flowering time behavior display variable expression for the ScFT6 gene, suggesting a possible genotypic relationship for its expression. Therefore, sugarcane has at least one putative orthologous gene in relation to FT that promotes floral induction.

Study on the Anti-Aging Physiological Characteristics and Molecular Mechanism of Camellia Oleifera

To study the physiological and molecular regulating mechanism of ancient Camellia oleifera which kept a exuberant vitality for more than one hundred years, leaves of 30a year old and > 100 year old Camellia oleifera were selected as targets. On the basis of the study of the generation and the clearance of reactive oxygen species, sequencing analysis of the transcriptome and expression profiling by high throughput sequencing analysis technique was conducted to study differentially expressed functional genes related to the tree age. It showed that the chlorophyll content and enzyme activities increased in ancient Camellia oleifera leaves. Expression of chlorophyll a/b binding protein gene, auxin related gene, the signal transduction factor and the transcription factor gene in ancient trees were all higher than mature tree. The down regulated gene expression of inductive genes related to protein degradation in ancient tree. Under the comprehensive function of those factors, ancient Camellia oleifera leaves still kept an exuberant vitality which was very useful for studies of stress resistance molecular biology and genetic improvement of Camellia oleifera.

Congenital hypofibrinogenemia: a case report

Fibrinogen disorders are rare bleeding disorders. Fibrinogen is also called factor I which is involved in last step of coagulation cascade. Congenital hypofibrinogenemia is usually caused by mutation of FIB (fibrinogen-binding protein) gene. These disorders should be suspected when Thrombin time is prolonged in well look child with history of bleeding manifestations. We are describing a female child who is having pallor with history of recurrent ecchymosis and minor post traumatic bleeding. Based on coagulation screening profile, we made the diagnosis of hypofibrinogenemia.

First detection of Leishmania of the subgenus viannia in Alipiopsitta xanthops, endemic bird of South America

We report the first molecular detection of Leishmania infection (subgenus Viannia) in the yellow-faced parrot (Alipiopsitta xanthops), at a wildlife rehabilitation center located in the city of Campo Grande, Brazil, an endemic area for leishmaniasis. PCRs targeting kinetoplast DNA (kDNA) and the small subunit of ribosomal RNA of Leishmania spp. were performed, both positive, followed by the sequencing of the amplified region of the SSU rDNA gene, which confirmed the identity of the parasite. This is the first report of success obtained in the use of PCR targeting the IRBP (Interphotoreceptor retinoid-binding protein) gene as an internal control in the molecular diagnosis of pathogens in bird species.