Amino Acid Sequence Analysis Research Articles

Single nucleotide polymorphism scanning and expression analysis of ACSL1 from different duck breeds

Accumulating studies have indicated that the long-chain fatty acyl-CoA1 (ACSL1) gene is related to fat deposition and meat quality in mammals. However, few studies have investigated the relationship between ACSL1 and lipid deposition in ducks. To examine this, we assessed the physicochemical property, homologous alignment, and phylogenetic analyses of the ACSL1 amino acid sequence using bioinformatics tools. The analysis indicated that the ACSL1 amino acid sequence varies in animals, and the duck ACSL1 protein is most closely related to that of chicken. Two single nucleotide polymorphism (SNP) sites were identified at 1749 and 1905 bp of the coding region of ACSL1 by sequencing. Quantitative real-time PCR and western blotting were used to measure mRNA and protein levels in abdominal fat, breast muscle, and liver tissue of Pekin duck (BD) and Cherry Valley duck (CD). mRNA and protein expression were significantly higher in BD than in CD in abdominal fat and liver tissue (P < 0.05). In breast muscle, the mRNA level of ACSL1 was also significantly higher in BD than in CD (P < 0.05), and protein expression in BD tended to be higher than that of CD. These results suggest that ACSL1 may contribute to lipid deposition and meat quality in ducks.

Molecular detection and characterization of highly pathogenic porcine reproductive and respiratory syndrome virus from a natural outbreak in wild pigs, Mizoram, India.

This study reports for the first time a natural outbreak of highly pathogenic porcine reproductive and respiratory syndrome (HP-PRRS) caused by HP-PRRS virus (HP-PRRSV) in wild pigs characterized by sudden onset of depression, anorexia, respiratory distress, and high fever. The disease has caused severe haemorrhagic pneumonia, haemorrhagic lymphadenitis, enlarged spleen with areas of infarction, and petechial haemorrhages on the myocardium and on the surface of kidneys. HP-PRRSV was detected in representative tissue samples by reverse transcription-PCR, and the field strain was isolated in the MA104 cell line. The phylogenetic analyses based on the whole genome sequences and nucleotide sequences of open reading frame 5 (ORF5) gene showed close grouping with the subtype IV of lineage 8/8.7 of PRRSV II, which represents the HP-PRRSV strains that predominate in the pig population of China since 2010. The amino acid sequence analysis of the ORF5 gene revealed the replacement of leucine (L) at position 39 to isoleucine (I) in the primary neutralizing epitope. Among the four potential N glycosylation sites, the N34 was mutated and found to be restricted to only three N glycosylation sites. The present findings have indicated that HP-PRRSV can cause fatal outbreaks and may emerge as a major threat to the wild pig population.

Isolation and molecular characterization of GP5 glycoprotein gene of Betaarterivirus suid 2 from Mizoram, India.

Porcine reproductive and respiratory syndrome (PRRS) is a serious swine disease causing great economic impact worldwide. The emergence of highly pathogenic strains in Asian countries is associated with large scale mortality in all age groups of pigs besides the classical presentation of severe respiratory distress, pneumonia, and a series of reproductive disorders in sows, like late-term abortion, premature farrowing, and an increased number of stillborn piglets. The present study was designed with the aim of isolation and characterization of the Betaarterivirus suid 2 from outbreaks in Mizoram in primary porcine alveolar macrophage and subsequently characterized the GP5 gene sequence of the isolate in terms of phylogenetic analysis and deduce amino acid sequence comparison. Virus propagation was performed in the porcine alveolar macrophage (PAM) primary cell culture and confirmed by immunoperoxidase test, FAT, and nested RT-PCR. The full-length GP5 gene (603nt) was amplified from the isolate and subsequently cloned and sequenced (MN928985). Phylogenetic analysis and sequence comparison of the present isolate was found to have similarity 98.7–98.8% with Myanmar HP-PRRS strains, 98–98.5% with Vietnam strains, 98.2–98.3% with China strains, indicating a close lineage with highly pathogenic PRRS strains. In deduced amino acid sequence analysis, one mutation was found in the primary neutralizing epitope (PNE) at position 39L → I39 and one more mutation was also found in the decoy epitope (DCE) at position 30 N → D30. The amino acid at this position is an N-linked glycosylation site, and mutation of the N-linked glycosylation is an immune escaped strategy adopted by this virus causing a persistent infection in the natural host.

Cloning of cytadhesin protein gene (pvpA) and expression analysis of recombinant fusion protein of Mycoplasma gallisepticum

Chronic respiratory disease (CRD) caused by Mycoplasma gallisepticum (MG) is one of the major respiratory tract infections of the poultry, resulting in significant economic loss to the poultry farmers. Diagnosis of such ailment is highly necessary for effective control measures. In addition, promising molecular tools are warranted for efficient epidemiological tracing of the outbreaks. The study was focused on the elucidation of phase variable cytadhesin protein gene (pvpA) of MG through cloning and expression analysis. A set of primers targeting the pvpA gene of MG was designed. The complete pvpA gene was amplified and cloned into pUC-derived expression vector pRSETA. Finally, the recombinant clones were examined through colony PCR and restriction endonuclease (RE) analysis with EcoR1 and BamH1 enzymes followed by sequencing. The expression of the recombinant pvpA gene was optimized at 1.4mM/μl concentration of Isopropyl-β-D-thiogalactoside induction at 30°C. The recombinant fusion protein was purified by immobilized metal affinity chromatography and characterized by SDS-PAGE followed by confirmation of recombinant cytadhesin fusion protein through western blot analysis. The pvpA gene was successfully cloned and expressed. The deduced amino acid sequence analysis had shown the presence of two direct repeats (DR1 and DR2) along with predicted PRP motifs repeatedly with high proline encoding regions at the carboxy-terminal of pvpA gene indicating its scope for epidemiological studies.

Delineating virulence of Vibrio campbellii: a predominant luminescent bacterial pathogen in Indian shrimp hatcheries

Luminescent vibriosis is a major bacterial disease in shrimp hatcheries and causes up to 100% mortality in larval stages of penaeid shrimps. We investigated the virulence factors and genetic identity of 29 luminescent Vibrio isolates from Indian shrimp hatcheries and farms, which were earlier presumed as Vibrio harveyi. Haemolysin gene-based species-specific multiplex PCR and phylogenetic analysis of rpoD and toxR identified all the isolates as V. campbellii. The gene-specific PCR revealed the presence of virulence markers involved in quorum sensing (luxM, luxS, cqsA), motility (flaA, lafA), toxin (hly, chiA, serine protease, metalloprotease), and virulence regulators (toxR, luxR) in all the isolates. The deduced amino acid sequence analysis of virulence regulator ToxR suggested four variants, namely A123Q150 (AQ; 18.9%), P123Q150 (PQ; 54.1%), A123P150 (AP; 21.6%), and P123P150 (PP; 5.4% isolates) based on amino acid at 123rd (proline or alanine) and 150th (glutamine or proline) positions. A significantly higher level of the quorum-sensing signal, autoinducer-2 (AI-2, p = 2.2e−12), and significantly reduced protease activity (p = 1.6e−07) were recorded in AP variant, whereas an inverse trend was noticed in the Q150 variants AQ and PQ. The pathogenicity study in Penaeus (Litopenaeus) vannamei juveniles revealed that all the isolates of AQ were highly pathogenic with Cox proportional hazard ratio 15.1 to 32.4 compared to P150 variants; PP (5.4 to 6.3) or AP (7.3 to 14). The correlation matrix suggested that protease, a metalloprotease, was positively correlated with pathogenicity (p > 0.05) and negatively correlated (p < 0.05) with AI-2 and AI-1. The syntenic organization of toxS-toxR-htpG operon in V. campbellii was found to be similar to pathogenic V. cholerae suggesting a similar regulatory role. The present study emphasizes that V. campbellii is a predominant pathogen in Indian shrimp hatcheries, and ToxR plays a significant role as a virulence regulator in the quorum sensing—protease pathway. Further, the study suggests that the presence of glutamine at 150th position (Q150) in ToxR is crucial for the pathogenicity of V. campbellii.

A Pigeon-Derived Sub-Genotype XXI.1.2 Newcastle Disease Virus from Bangladesh Induces High Mortality in Chickens.

Newcastle disease virus (NDV) is a significant pathogen of poultry; however, variants also affect other species, including pigeons. While NDV is endemic in Bangladesh, and poultry isolates have been recently characterized, information about viruses infecting pigeons is limited. Worldwide, pigeon-derived isolates are commonly of low to moderate virulence for chickens. Here, we studied a pigeon-derived NDV isolated in Bangladesh in 2010. To molecularly characterize the isolate, we sequenced its complete fusion gene and performed a comprehensive phylogenetic analysis. We further studied the biological properties of the virus by estimating mean death time (MDT) and by experimentally infecting 5-week-old naïve Sonali chickens. The studied virus clustered in sub-genotype XXI.1.2 with NDV from pigeons from Pakistan isolated during 2014–2018. Deduced amino acid sequence analysis showed a polybasic fusion protein cleavage site motif, typical for virulent NDV. The performed in vivo pathogenicity testing showed a MDT of 40.8 h, and along with previously established intracerebral pathogenicity index of 1.51, these indicated a velogenic pathotype for chickens, which is not typical for pigeon-derived viruses. The experimental infection of chickens resulted in marked neurological signs and high mortality starting at 7 days post infection (dpi). Mild congestion in the thymus and necrosis in the spleen were observed at an advanced stage of infection. Microscopically, lymphoid depletion in the thymus, spleen, and bursa of Fabricius were found at 5 dpi, which progressed to severe in the following days. Mild to moderate proliferation of glial cells was noticed in the brain starting at 2 dpi, which gradually progressed with time, leading to focal nodular aggregation. This study reports the velogenic nature for domestic chickens of a pigeon-derived NDV isolate of sub-genotype XXI.1.2. Our findings show that not all pigeon-derived viruses are of low virulence for chickens and highlight the importance of biologically evaluating the pathogenicity of NDV isolated from pigeons.

Identification of novel yolk ferritins unique to planarians: planarians supply aluminum rather than iron to vitellaria in egg capsules.

All animals, other than Platyhelminthes, produce eggs containing yolk, referred to as "entolecithal" eggs. However, only Neoophora, in the phylum Platyhelminthes, produce "ectolecithal" eggs (egg capsules), in which yolk is stored in the vitelline cells surrounding oocytes. Vitelline cells are derived from vitellaria (yolk glands). Vitellaria are important reproductive organs that may be studied to elucidate unique mechanisms that have been evolutionarily conserved within Platyhelminthes. Currently, only limited molecular level information is available on vitellaria. The current study identified major vitellaria-specific proteins in a freshwater planarian, Dugesia ryukyuensis, using peptide mass fingerprinting (PMF) and expression analyses. Amino acid sequence analysis and orthology analysis via OrthoFinder ver.2.3.8 indicated that the identified major vitellaria-specific novel yolk ferritins were conserved in planarians (Tricladida). Because ferritins play an important role in Fe (iron) storage, we examined the metal elements contained in vitellaria and ectolecithal eggs, using non-heme iron histochemistry, elemental analysis based on inductively coupled plasma mass spectrometry and transmission electron microscopy- energy-dispersive X-ray spectroscopy analysis. Interestingly, vitellaria and egg capsules contained large amounts of aluminum (Al), but not Fe. The knockdown of the yolk ferritin genes caused a decrease in the volume of egg capsules, abnormality in juveniles, and increase in Al content in vitellaria. Yolk ferritins of D. ryukyuensis may regulate Al concentration in vitellaria via their pooling function of Al and protect the egg capsule production and normal embryogenesis from Al toxicity.

Association of clusterin with the BRI2-derived amyloid molecules ABri and ADan

Familial British and Danish dementias (FBD and FDD) share striking neuropathological similarities with Alzheimer's disease (AD), including intraneuronal neurofibrillary tangles as well as parenchymal and vascular amyloid deposits. Multiple amyloid associated proteins with still controversial role in amyloidogenesis colocalize with the structurally different amyloid peptides ABri in FBD, ADan in FDD, and Aβ in AD. Genetic variants and plasma levels of one of these associated proteins, clusterin, have been identified as risk factors for AD. Clusterin is known to bind soluble Aβ in biological fluids, facilitate its brain clearance, and prevent its aggregation. The current work identifies clusterin as the major ABri- and ADan-binding protein and provides insight into the biochemical mechanisms leading to the association of clusterin with ABri and ADan deposits. Mirroring findings in AD, the studies corroborate clusterin co-localization with cerebral parenchymal and vascular amyloid deposits in both disorders. Ligand affinity chromatography with downstream Western blot and amino acid sequence analyses unequivocally identified clusterin as the major ABri- and ADan-binding plasma protein. ELISA highlighted a specific saturable binding of clusterin to ABri and ADan with low nanomolar Kd values within the same range as those previously demonstrated for the clusterin-Aβ interaction. Consistent with its chaperone activity, thioflavin T binding assays clearly showed a modulatory effect of clusterin on ABri and ADan aggregation/fibrillization properties. Our findings, together with the known multifunctional activity of clusterin and its modulatory activity on the complex cellular pathways leading to oxidative stress, mitochondrial dysfunction, and the induction of cell death mechanisms – all known pathogenic features of these protein folding disorders – suggests the likelihood of a more complex role and a translational potential for the apolipoprotein in the amelioration/prevention of these pathogenic mechanisms.

Isolation and Characterization of Endomycorrhizal Fungi Associated with Growth Promotion of Blueberry Plants.

Despite their notable root mutualism with blueberries (Vaccinium spp.), studies related to Ericoid mycorrhizal (ERM) are relatively limited. In this study, we report the isolation of 14 endomycorrhizal fungi and their identification by fungal colony morphology characterization combined with PCR-amplified fungal internal transcribed spacer (ITS) sequence analyses. Six of the isolated strains were confirmed as beneficial mycorrhizal fungi for blueberry plants following inoculation. We observed the formation of typical ERM hyphae coil structures—which promote and nutritionally support growth—in blueberry seedlings and significant nitrogen and phosphorous content increases in diverse tissues. QRT-PCRs confirmed changes in VcPHT1s expression patterns. After the formation of ERM, PHT1-1 transcription in roots was upregulated by 1.4- to threefold, whilst expression of PHT1-3 and PHT1-4 in roots were downregulated 72% and 60%, respectively. Amino acid sequence analysis of all four VcPHT1s genes from the blueberry variety “Sharpblue” revealed an overall structural similarity of 67% and predicted transmembrane domains. Cloning and overexpression of PHT1-1 and PHT1-3 genes in transgenic Arabidopsis thaliana plants significantly enriched total phosphorus and chlorophyll content, confirming that PHT1-1 and PHT1-3 gene functions are associated with the transport and absorption of phosphorus.

NATURAL INFECTION OF Tobacco mosaic virus ON BUTTERNUT SQUASH IN BALI, INDONESIA

Natural infection of Tobacco mosaic virus on butternut squash in Bali, Indonesia. Tobacco mosaic virus (TMV) was a newly emerging virus infecting cucumbers in Indonesia since 2017. The mosaic disease caused by TMV potentially caused yield loss cucumber in Java. In 2019, mosaic symptoms were observed in butternut squash plants in Bali and molecular detection using universal primer of Tobamovirus indicated the presence of TMV infection. Further research was conducted to determine molecular characteristics of TMV on butternut squash plants in Bali. Specific DNA bands of Tobamovirus were amplified using reverse transcription polymerase chain reaction method, followed by DNA sequencing. The DNA were successfully amplified from CP Tobamovirus using universal primers from several butternut squash samples, i.e. Denpasar, Gianyar, Buleleng, and Karangasem Districts. The homology analysis of nucleotide and amino acid sequences of TMV among isolates from Denpasar, Gianyar, Buleleng, and Karangasem Districts was ranged between 95.6 – 97.7% and 98.1 – 99.4%, respectively. This indicated that low genetic diversity of TMV among Bali isolates. The highest homology of corresponding sequences of TMV isolates from Denpasar, Gianyar, Buleleng, and Karangasem Districts was closely related to TMV Kediri-Indonesia isolate on cucumber plant. Correspondingly, the phylogenetic analysis showed that TMV Bali isolates were categorized into same cluster with Kediri-Indonesia isolates. This was the first report of TMV on butternut squash in Indonesia.

CLONING AND in silico STUDIES OF LIPASE GENE OF A THERMOPHILIC BACTERIA ISOLATED FROM THE HYDROTHERMAL VENT AREA OF KAWIO, NORTH SULAWESI

Underwater volcano is an example of a natural habitat of thermophilic bacteria, such as hydrothermal vent Kawio in North Sulawesi. The focus of this research was to screen the bacterial isolates originally from hydrothermal vent Kawio for the production of thermostable lipase. In this study, the lipase gene from the selected isolate was cloned, sequenced, and in silico analysis was performed to predict the characteristics of the lipase protein. One of three isolates examined, showed a positive reaction. Phylogenetic analysis of 16S rRNA gene showed that the isolate was a novel bacteria closely related to Bacillus safensis strain FO-26b with 96,87% homology. Amino acid sequence analysis of the lipase showed similarity between lipase of Bacillus pumilus strain B106 with 98% homology. Further analysis at the protein level indicated that the lipase isolated from marine water was a member of family I.4 of ‘true lipase’ group.

Elucidation of familial relationships using hair shaft proteomics

This study examines the potential of hair shaft proteomic analysis to delineate genetic relatedness. Proteomic profiling and amino acid sequence analysis provide information for quantitative and statistically-based analysis of individualization and sample similarity. Protein expression levels are a function of cell-specific transcriptional and translational programs. These programs are greatly influenced by an individual’s genetic background, and are therefore influenced by familial relatedness as well as ancestry and genetic disease. Proteomic profiles should therefore be more similar among related individuals than unrelated individuals. Likewise, profiles of genetically variant peptides that contain single amino acid polymorphisms, the result of non-synonymous SNP alleles, should behave similarly. The proteomically-inferred SNP alleles should also provide a basis for calculation of combined paternity and sibship indices. We test these hypotheses using matching proteomic and genetic datasets from a family of two adults and four siblings, one of which has a genetic condition that perturbs hair structure and properties. We demonstrate that related individuals, compared to those who are unrelated, have more similar proteomic profiles, profiles of genetically variant peptides and higher combined paternity indices and combined sibship indices. This study builds on previous analyses of hair shaft protein profiling and genetically variant peptide profiles in different real-world scenarios including different human hair shaft body locations and pigmentation status. It also validates the inclusion of proteomic information with other biomolecular substrates in forensic hair shaft analysis, including mitochondrial and nuclear DNA.

Expression and functional characterization of chemokine CXCL10 from Nile tilapia (Oreochromis niloticus) in response to bacterial infection and C1q-mediated regulation of monocytes/macrophages activity

Chemokines are a superfamily of chemotactic cytokines that regulate cell activation, inflammation, and immune defense. In this study, chemokine CXCL10 from Nile tilapia (Oreochromis niloticus) was identified and characterized. The open reading frame of OnCXCL10 is 408 bp encoding polypeptides of 135 amino acids. The amino acid sequence analysis of CXCL10 confirms homology with other species and contains four conserved cysteine residues. Spatial mRNA expression analysis revealed that OnCXCL10 was highly expressed in spleen, and widely exhibited in other tissues including liver, intestine and head kidney. Following in vivo challenges with lipopolysaccharide (LPS), Streptococcus agalactiae and, Poly I: C separately, the expressions of OnCXCL10 were observed significantly elevated in liver, spleen, and head kidney. The transcriptional up-regulation of OnCXCL10 was also demonstrated in head kidney monocytes/macrophages after in vitro stimulations with LPS, S. agalactiae and Poly I: C. Furthermore, mRNA expression analysis revealed that OnCXCL10 could involved in C1q-mediated intracellular signaling pathways. Taken together, these studies indicated that CXCL10 played an important role in response to pathogen infection and C1q-mediated immune regulation of monocytes/macrophages in Nile tilapia (Oreochromis niloticus).

Strength performance with buckling analysis of Intermediate filaments by consideration nonlocal parameters

Protein structures, that form intermediate filaments (IFs) was first found by an experiment known as the computerized analysis of amino acid sequence of a human epidermal keratin derived from cloned cDNAs. This study is made by the application of Euler beam theory. The buckling of intermediate filaments is studied keeping the nonlocal effects under consideration. It is observed that the nonlocal parameter has a great impact on the dynamics of intermediate filaments. The buckling behavior of intermediate filaments is investigated with different four conditions like as simply supported, clamped, cantilever and propped cantilever beam. Also the effect of critical bucking force is seen for different strengths of nonlocal parameter as 1,2,3,4.

Novel (S)-Selective Hydrolase from Arthrobacter sp. K5 for Kinetic Resolution of Cyclic Amines

Chiral 2-methylpiperidine (2-MPI) is an important building block that has potential for applications in pharmaceuticals and pesticides. In this study, we observed that the hydrolase in Arthrobacter sp. K5 exhibits high (S)-selectivity toward rac-N-pivaloyl-2-MPI to yield (S)-2-MPI with 80.2% enantiomeric excess (ee) in a 38.2% conversion. The hydrolase, which was identified by analyses of partial amino acid sequences of the purified enzyme and genome sequence of Arthrobacter sp. K5, exhibited moderate homology with amidohydrolases up to 67% (molinate hydrolase from Gulosibacter molinativorax). The hydrolase gene was overexpressed in Rhodococcus erythropolis. The recombinant cells produced (S)-2-MPI with 83.5% ee in a 48.4% conversion (E = 26.3) from 100 mM rac-N-pivaloyl-2-MPI. These results suggest the possibility of an efficient preparation of chiral 2-MPI in kinetic resolution.

Variation in UV-Mediated Damage Recovery Among Pseudoidium neolycopersici Isolates: Possible Mechanisms

The cryptochrome/photolyase family (CPF) consists of a diversified class of flavoproteins that are evolutionarily related. Although their domain architecture is highly conserved, they perform entirely different physiological functions. Previous studies have confirmed the presence of a functional photolyase in Pseudoidium neolycopersici, which repairs UV-C-induced DNA damage by using near UV-A/blue light as an energy source. Similar doses of UV-C treatment followed by incubation with dark or blue light was tested on conidia germination of five isolates of P. neolycopersici collected from different regions of Norway and the Netherlands. The results showed variations in the effect of UV on germination and germination recovery under blue light incubation after UV treatment. Evolutionary studies confirmed that P. neolycopersici photolyase is highly conserved among different isolates of P. neolycopersici and among different species. All CPF members have a core domain consisting of an identical cofactor, flavin adenine dinucleotide (FAD), and an additional photoantenna chromophore. An amino acid sequence analysis demonstrated that CPF members have highly conserved C terminals compared with their N terminals, because FAD binds in the C-terminal region. When compared with other CPF members, varying lengths of N and C terminals were noticed in P. neolycopersici photolyase and putative cryptochrome, respectively. Further research using comparative genomics targeting pyrimidine base composition, the role of regulatory elements, including promotor architecture, and the characterization of optical properties of native photolyase among isolates may help to explain the differences in the biological responses of conidia germination of P. neolycopersici treated with UV. [Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Genome-wide Identification and Abiotic Stress Response Pattern Analysis of NF-Y Gene Family in Peanut (Arachis Hypogaea L.)

The nuclear factor Y (NF-Y) transcription factor (TF) family consists of three subfamilies NF-YA, NF-YB and NF-YC. Many studies have proven that NF-Y complex plays multiple essential roles in stress response in Arabidopsis and other plant species. However, little attention has been given to these genes in peanut. In this study, thirty-three AhNF-Y genes were identified in cultivated peanut and they were distributed on 16 chromosomes. A phylogenetic analysis of the NF-Y amino acid sequences indicated that the peanut NF-Y proteins were clustered in pairs at the end of the branches and showed high conservation with previous reported plant NF-Ys. Evolutionary history analysis showed that only segmental duplication contributed to expansion of this gene family. Analysis of the 1500-bp regulatory regions upstream the start codon showed that, except for AhNF-YB6, peanut NF-Ys contained at least one abiotic stress response element in their regulatory region. Expression patterns of peanut NF-Ys in 22 tissues and developmental stages were analyzed. A few NF-Ys showed universal expression patterns, while most NF-Ys showed specific expression patterns. Through RNA-seq and qRT-PCR analyses, expression of six AhNF-Y genes was induced under salt stress in leaves or roots. In addition, AhNF-YA4/8/11, NF-YB4 and NF-YC2/8 also responded to osmotic stress, ABA (abscisic acid) and salicylic acid (SA) treatment.

Roles of constitutive and signal-dependent protein phosphatase 2A docking motifs in burst attenuation of the cyclic AMP response element-binding protein

The cAMP response element-binding protein (CREB) is an important regulator of cell growth, metabolism, and synaptic plasticity. CREB is activated through phosphorylation of an evolutionarily conserved Ser residue (S133) within its intrinsically disordered kinase-inducible domain (KID). Phosphorylation of S133 in response to cAMP, Ca2+, and other stimuli triggers an association of the KID with the KID-interacting (KIX) domain of the CREB-binding protein (CBP), a histone acetyl transferase (HAT) that promotes transcriptional activation. Here we addressed the mechanisms of CREB attenuation following bursts in CREB phosphorylation. We show that phosphorylation of S133 is reversed by protein phosphatase 2A (PP2A), which is recruited to CREB through its B56 regulatory subunits. We found that a B56-binding site located at the carboxyl-terminal boundary of the KID (BS2) mediates high-affinity B56 binding, while a second binding site (BS1) located near the amino terminus of the KID mediates low affinity binding enhanced by phosphorylation of adjacent casein kinase (CK) phosphosites. Mutations that diminished B56 binding to BS2 elevated both basal and stimulus-induced phosphorylation of S133, increased CBP interaction with CREB, and potentiated the expression of CREB-dependent reporter genes. Cells from mice harboring a homozygous CrebE153D mutation that disrupts BS2 exhibited increased S133 phosphorylation stoichiometry and elevated transcriptional bursts to cAMP. These findings provide insights into substrate targeting by PP2A holoenzymes and establish a new mechanism of CREB attenuation that has implications for understanding CREB signaling in cell growth, metabolism, synaptic plasticity, and other physiologic contexts.

Genetic characterisation of the influenza viruses circulating in Bulgaria during the 2019\u20132020 winter season

Influenza viruses have a high potential for genetic changes. The objectives of this study were to analyse influenza virus circulation in Bulgaria during the 2019/2020 season, to perform a phylogenetic and molecular analyses of the haemagglutinin (HA) and neuraminidase (NA) sequences of representative influenza strains, and to identify amino acid substitutions compared to the current vaccine strains. Seasonal influenza viruses A(H3N2), A(H1N1)pdm09 and B/Victoria-lineage were detected using a real-time RT-PCR in 323 (23.3%), 149 (10.7%) and 138 (9.9%) out of 1387 patient samples studied, respectively. The HA genes of A(H3N2) viruses analysed belonged to clades 3C.3a (21 strains) and 3C.2a (5 strains): subclades 3C.2a1b + T131K, 3C.2a1b + T135K-B and 3C.2a1b + T135K-A. The clade 3C.3a and subclade 3C.2a1b viruses carried 5 and 14–17 substitutions in HA, as well as 3 and 9 substitutions in NA, respectively, in comparison with the A/Kansas/14/2017 vaccine virus, including some substitutions in the HA antigenic sites A, B, C and E. All 21 A(H1N1)pdm09 viruses sequenced fell into 6B.1A5A subclade. Amino acid sequence analysis revealed the presence of 7–11 substitutions in HA, compared to the A/Brisbane/02/2018 vaccine virus, three of which occurred in antigenic site Sb, along with 6–9 changes at positions in NA. All 10 B/Victoria-lineage viruses sequenced belonged to clade 1A with a triple deletion in HA1 (genetic group 1A(Δ3)B) and carried 7 and 3 substitutions in HA and NA, respectively, with respect to the B/Colorado/06/2017 vaccine virus. The results of this study confirm the rapid evolution of influenza viruses and the need for continuous antigenic and genetic surveillance.

Target of rapamycin (TOR) gene is vital for whitefly survival and reproduction

The whitefly Bemisia tabaci (Gennadius) is a devastating polyphagous pest that causes significant losses in several economically important crops worldwide. Although chemical control is the fastest management strategy, insecticide resistance development poses a greater constraint in whitefly management. In this study, we used RNA interference (RNAi) technique to understand the role of target of rapamycin (TOR) gene in whitefly. In insects, TOR plays a crucial role in cell growth, metabolism, proliferation, and reproduction. Although sequence analyses of TOR revealed 95 nucleotides different in TOR gene sequence compared to predicted sequence (GenBank accession no. XM019060193.1), but this led to synthesis of only two different amino acids at positions 1448 and 1768. Phylogenetic analysis of amino acid sequence revealed that TOR gene of B. tabaci, Acrythosiphum pisum and Nilaparvata lugens were more closely related to each other and were clustered together. Oral feeding of artificial diet containing dsRNA viz 2.0, 1.0, 0.5, 0.1 μg/μl corresponding to TOR gene to adult whitefly resulted in 42.5% adult mortality, reduced fecundity i.e. 36.57 eggs per female, and 62.50% total nymphal mortality in the next generation. The qRT-PCR analysis confirmed the downregulation of TOR gene expression in adult whiteflies upon dstor feeding. These results suggest that TOR gene significantly regulates the survival and reproduction in whitefly and can be a potential RNAi target for insect management.