Characterization and expression profiling of buffalo IFN-lambda family.

Interferon lambda (IFN-λ) is an important type III interferon triggered mainly by viral infection. IFN-λ binds to their heterodimeric receptors and signals through JAK-STAT pathways similar to type I IFN. In this study, we deduced the buffalo IFN-λ sequences through the polymerase chain reaction, and then studied IFN-λ's expression patterns in different tissues, and post induction with poly I:C and live MRSA using RT-qPCR. The full-length sequences of buffalo IFN-λ3, IFN-λ receptors, and a transcript variant of IFN-λ4 were determined. IFN-λ1 is identified as a pseudogene. Virus response elements and a recombination hotspot factor was observed in the regulatory region of IFN-λ. The IFN-λ3 expressed highest in lungs and monocytes but IFN-λ4 did not. The expression of Interferon Lambda Receptor 1 was tissue specific, while Interleukin 10 Receptor subunit beta was ubiquitous. Following poly I:C induction, IFN-λ3 expression was primarily observed in epithelial cells as opposed to fibroblasts, displaying cell type-dependent expression. The cytosolic RNA sensors were expressed highest in endometrial epithelial cells, whereas the endosomal receptor was higher in fibroblasts. 2',5'-oligoadenylate synthetase expressed higher in fibroblasts, myxoma resistance protein 1 and IFN-stimulated gene 56 in epithelial cells, displaying cell-specific antiviral response of the interferon stimulated genes (ISGs). The endometrial epithelial cells expressed IFN-λ3 after live S. aureus infection indicating its importance in bacterial infection. The induction of IFN-λ3 was S. aureus isolate specific at the same multiplicity of infection (MOI). This study elucidates the IFN-λ sequences, diverse expression patterns revealing tissue specificity, and specificity in response to poly I:C and bacterial stimuli, emphasising its crucial role in innate immune response modulation.

Antimicrobial and antibiofilm effect of cannabinoids from Cannabis sativa against methicillin-resistant Staphylococcus aureus (MRSA) causing bovine mastitis.

Antimicrobial resistance (AMR) poses a serious threat to human, animal, and plant health on a global scale. Search and elimination techniques should be used to effectively counter the spread of methicillin-resistant Staphylococcus aureus (MRSA) infections. With only a few novel drugs in clinical development, the quest for plant-based alternatives to prevent the spread of antibiotic resistance among bacteria has accelerated. Treatment of MRSA infections is challenging owing to rapidly emerging resistance mechanisms coupled with their protective biofilms. In the present research, we examined the antibacterial properties of ten plant-derived ethanolic leaf extracts. The most effective ethanolic leaf extract against MRSA in decreasing order of zone of inhibition, Cannabis sativa L. > Syzygium cumini > Murraya koenigii > Eucalyptus sp. > while Aloe barbadensis, Azadirachta indica, had very little impact. Mangifera indica, Curcuma longa, Tinospora cordifolia, and Carica papaya did not exhibit inhibitory effects against MRSA; hence, Cannabis was selected for further experimental study. The minimal inhibitory concentration (MIC) of Cannabis sativa L. extract was 0.25 mg ml-1 with 86% mortality. At a sub-MIC dosage of 0.125 mg ml-1, the biofilm formation was reduced by 71%. The two major cannabinoids detected were cannabidiol and delta-9-tetrahydrocannabinol (Δ9-THC), which were majorly attributed to substantial inhibitory action against MRSA. The time-kill kinetics demonstrated a bactericidal action at 4 MIC over an 8-20-h time window with a 90% reduction in growth rate. The results from SEM, and light microscopy Giemsa staining revealed a reduction in cells in the treated group with increased AKP activity, indicating bacterial cell membrane breakdown. These findings suggested cannabinoids may be a promising alternative to antibiotic therapy for bovine biofilm-associated MRSA.

Mucin and salt combination simulate typical fern-like pattern of buffalo saliva smear at estrus.

Estrus detection in buffaloes primarily relies on behavioral and physiological signs. Especially during summer, these signs are less prominent to recognize. Thus, estrus detection is a pronounced challenge within the realm of buffalo husbandry, particularly in the summer. Therefore, a simple and accurate estrus detection method is required for buffalo farmers. The observation of fern-like salivary crystallization patterns is one such simple method to detect estrus in buffaloes, bactrian camels, beagle bitches, and cows. However, the exact mechanism for the formation of typical fern-like is not known. We hypothesized that it might be because of the estrus-specific mucins and salts. To test this hypothesis, we prepared the smears by combining different concentrations of mucin type -2 (MUC2) and -3 (MUC3) with sodium chloride (NaCl). Microscopic examination confirmed that fern-like patterns resulted from a combination of the MUC3 and NaCl produced more realistic fern patterns than that of MUC2 or BSA with salt. To predict possible mucin and salt concentration showing natural fern-like patterns at the estrus stage in buffalo saliva, we constructed a guide tree of artificially generated fern-like patterns using an image analysis online tool. This computation analysis revealed that most of the natural buffalo estrus saliva samples showing typical fern-like patterns clustered in the cluster 2 of the guide tree comprising of 13 clusters. In the cluster 2, MUC3 in combination with the salt concentrations of 100, 150, and 250 mM was commonly found in a close proximity to the natural typical fern-like patterns of saliva smear of buffaloes at estrus. Conclusively, the buffalo saliva at estrus is predicted to have a gel-forming heavily glycosylated protein such as mucin along with at least 100 mM of NaCl. RESEARCH HIGHLIGHTS: Glycoprotein and salts combination replicates fern-like pattern of buffalo saliva at estrus. MUC3 and NaCl salt combination produces more realistic fern-like patterns compared with MUC2 or BSA and salt combination. MUC3 with NaCl at 100, 150, and 250 mM consistently resembled natural estrus saliva fern-like patterns. During estrus, buffalo saliva is expected to contain heavily glycosylated mucin and at least of 100 mM NaCl.

Expression and characterization of novel chimeric endolysin CHAPk-SH3bk against biofilm-forming methicillin-resistant Staphylococcus aureus.

The continuous evolution of antibiotic resistance in methicillin-resistant Staphylococcus aureus (MRSA) due to the misuse of antibiotics lays out the need for the development of new antimicrobials with higher activity and lower resistance. In this study, we have expressed novel chimeric endolysin CHAPk-SH3bk derived from LysK to investigate its antibacterial activity against planktonic and biofilm-forming MRSA. The molecular docking and MD simulation results identified critical amino acids (ASP47, ASP56, ARG71, and Gly74) of CHAPk domain responsible for its catalytic activity. Chimeric endolysin CHAPk-SH3bk showed an effective binding to peptidoglycan fragment using 14 hydrogen bonds. The in-vitro antibacterial assays displayed higher activity of CHAPk against planktonic MRSA with 2-log10 reduction in 2 h. Both CHAPk and CHAPk-SH3bk displayed bactericidal activity against MRSA with ∼4log10 and ∼3.5log10 reduction in 24 h. Biofilm reduction activity displayed CHAPk-SH3bk reduced 33 % and 60 % of hospital-associated ATCC®BAA-44™ and bovine origin SA1 respectively. The CHAPk treatment reduced 47 % of the preformed biofilm formed by bovine-origin MRSA SA1. This study indicates an effective reduction of preformed MRSA biofilms of human and animal origin using novel chimeric construct CHAPk-SH3bk. Stating that the combination and shuffling of different domains of phage endolysin potentially increase its bacteriolytic effectiveness against MRSA.

Adaptive Selection in the Evolution of Aquaglyceroporins in Mammals.

Aquaporins (AQPs) are integral membrane proteins responsible for water transport across cellular membranes in both prokaryotes and eukaryotes. A subfamily of AQPs, known as aquaglyceroporins (AQGPs), facilitate the transport of small solutes such as glycerol, water, and other solutes across cellular membranes. These proteins are involved in a variety of physiological processes, such as organogenesis, wound healing, and hydration. Although AQPs have been studied extensively in different species, their conservation patterns, phylogenetic relationships, and evolution in mammals remain unexplored. In the present study, 119 AQGP coding sequences from 31 mammalian species were analysed to identify conserved residues, gene organisation, and most importantly, the nature of AQGP gene selection. Repertoire analysis revealed the absence of AQP7, 9, and 10 genes in certain species of Primates, Rodentia, and Diprotodontia, although not all three genes were absent in a single species. Two Asparagine-Proline-Alanine (NPA) motifs located at the N- and C-terminal ends, aspartic acid (D) residues, and the ar/R region were conserved in AQP3, 9, and 10. Six exons encoding the functional MIP domain of AQGP genes were found to be conserved across mammalian species. Evolutionary analysis indicated signatures of positive selection in AQP7, 9, and 10 amongst different mammalian lineages. Furthermore, substitutions of certain amino acids located close to critical residues may alter AQGP functionality, which is crucial for substrate selectivity, pore formation, and transport efficiency required for the maintenance of homeostasis in different mammalian species.

Evaluation of Virulence, Antimicrobial Resistance and Biofilm Forming Potential of Methicillin-Resistant Staphylococcus aureus (MRSA) Isolates from Bovine Suspected with Mastitis.

Methicillin-resistant Staphylococcus aureus (MRSA) is a pathogen that poses a significant threat in cases of chronic mastitis in dairy animals. The ability of MRSA to persist in the host is attributed to various virulence factors, genes encoding surface adhesins, and determinants of antibiotic resistance, which provide it a survival advantage. This investigation focused to determine the virulence factors, antimicrobial resistance (AMR) profile and biofilm production potential of 46 MRSA isolates from 300 bovine mastitis milk samples. The AMR profile revealed a high level of resistance, with 46 and 42 isolates resistant to cefoxitin and oxacillin, respectively, followed by 24 and 12 isolates resistant to lomefloxacin and erythromycin, respectively. Only 2 isolates resistant to tetracycline and none were resistant to chloramphenicol. The study also evaluated various virulence factors such as coa (n = 46), nuc (n = 35) hlg (n = 36), pvl (n = 14), tsst-1(n = 28) spa (n = 39) and enterotoxin genes sea (n = 12) and seg (n = 28) and identified antibiotic resistance determinants mecA and blaZ in 46 and 27 isolates, respectively. Intercellular adhesion genes icaA and icaD were present in 40 and 43 isolates, respectively and surface adhesion genes ebps, fnbpA, eno, sasG, cna, and bap were found in 43, 40, 38, 26, 21 and 1 isolates, respectively. Microtiter plate (MTP) assay revealed that 29 MRSA isolates were capable of producing biofilms, whereas 17 were not. Biofilms producing MRSA isolates possessed adhesion genes, virulence factors, toxin genes and AMR genes that may act synergistically towards a chronic disease progression, illness and severe damage to the udder, which generally last for several months and very challenging to cure.

Novel aadA5 and dfrA17 variants of class 1 integron in multidrug-resistant Escherichia coli causing bovine mastitis.

Mobile genetic elements (MGEs) are associated with the emergence of multidrug resistance in extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae. This study explores the role of class 1 integrons and IS26 elements in breaching taxonomic barriers. A total of 110 E. coli bacteria were isolated from 300 clinical mastitis milk samples. The 98% E. coli isolates were extended-spectrum beta-lactamase- producers. About 83% of these isolates carried co-resistance for fluoroquinolones. The co-existence of (extended-spectrum beta-lactamase + quinolone resistance determining region mutations) and (extended-spectrum beta-lactamase + plasmid-mediated quinolone resistance genes) was found in 76% and 44% of isolates, respectively. The MGEs were detected in 88% of isolates with IS26 in 82% and class 1 integrase in 40% of isolates. The types of class 1 integron gene cassettes detected includes dfrA7, (dfrA17 + aadA5), and (dfrA1 + aadA1). We discovered 2 and 4 novel variants of the dfrA17 and aadA5 genes, respectively. We report a variant of aadA5 with mutation E235G in the Indian subcontinent earlier reported only in a human clinical isolate from Belgium. About 19 isolates carried IS26 linked to integrase gene intI1 with an internal deletion of 265 bp in the 5`CS of integrase gene intI1, earlier reported only in E. coli ST131 isolates from human clinical, wastewater samples. This study suggests intercontinental dissemination of antibiotic resistant genes (ARGs) across different microbiomes via mobile genetic elements. KEY POINTS: • The role of mobile genetic elements in the emergence of multidrug-resistant E. coli in bovine mastitis. • Novel variants of the aadA5 (aminoglycoside adenyl transferase) and dfrA17 (dihydrofolate reductase) genes were identified in pathogenic E. coli isolated from bovine mastitis in class 1 integron gene cassette. • Sequence analysis of mobile genetic components revealed the physical connection between IS26 and intI1 genes with an internal deletion in 5'CS of class 1 integrase.

Analysis of amplification and association polymorphisms in the bovine beta-defensin 129 (BBD129) gene revealed its function in bull fertility.

ß-defensins are adsorbable on the sperm surface in the male reproductive tract (MRT) and enhance sperm functional characteristics. The beta-defensin 129 (DEFB129) antimicrobial peptide is involved in sperm maturation, motility, and fertilization. However, its role in bovine fertility has not been well investigated. This study examines the relationship between the bovine BBD129 gene and Bos indicus x Bos taurus bull fertility. The complete coding sequence of BBD129 mRNA was identified by RNA Ligase Mediated-Rapid Amplification of cDNA End (RLM-RACE) and Sanger sequencing methodologies. It consisted of 582 nucleotides (nts) including 5' untranslated region (UTR) (46nts) and 3'UTR (23nts). It conserves all beta-defensin-like features. The expression level of BBD129 was checked by RT-qPCR and maximal expression was detected in the corpus-epididymis region compared to other parts of MRT. Polymorphism in BBD129 was also confirmed by Sanger sequencing of 254 clones from 5 high fertile (HF) and 6 low fertile (LF) bulls at two positions, 169 T > G and 329A > G, which change the S57A and N110S in the protein sequence respectively. These two mutations give rise to four types of BBD129 haplotypes. The non-mutated TA-BBD129 (169 T/329A) haplotype was substantially more prevalent among high-fertile bulls (P < 0.005), while the double-site mutated GG-BBD129 (169 T > G/329A > G) haplotype was significantly more prevalent among low-fertile bulls (P < 0.005). The in silico analysis confirmed that the polymorphism in BBD129 results in changes in mRNA secondary structure, protein conformations, protein stability, extracellular-surface availability, post-translational modifications (O-glycosylation and phosphorylation), and affects antibacterial and immunomodulatory capabilities. In conclusion, the mRNA expression of BBD129 in the MRT indicates its region-specific dynamics in sperm maturation. BBD129 polymorphisms were identified as the deciding elements accountable for the changed proteins with impaired functionality, contributing to cross-bred bulls' poor fertility.

Global epidemiology of CTX-M-type ß-lactam resistance in human and animal.

CTX-M ESBL are widely found in animal and human infections. For better understanding of CTX-M variations and epidemiology, a total of 2210 CTX-M sequences were retrieved from NCBI as at 20 December 2020. The maximum incidences of CTX-M were reported in China (n = 508), USA (n = 354) and Japan (n = 180). Single amino acid substitution in the domain region of CTX-M ESBL lead to survival benefits to the bacteria. A total of 31 different variations were found of which D240G was the most common followed by A77V and V103I substitutions. The variations in CTX-M enzymes were explained continent-wise revealing the maximum variation reported in America followed by Asia and Europe of which D240G substitution was the most prevalent. India contained only three variations (E166A, P167S D240G) found in New Delhi, Karnataka, West Bengal and Tamil Nadu. The P167 and D240 were under strong positive selection with dN/dS calculation.

Virulence and enterotoxin gene profile of methicillin-resistant Staphylococcus aureus isolates from bovine mastitis.

Bovine mastitis is a major infectious disease affecting dairy animals resulting in enormous economic losses, prolonged antibiotic treatment, reduced milk yield and death of livestock. Emergence of Methicillin-resistant Staphylococcus aureus (MRSA) among bovine mastitis is matter of concern for animal health and dairy industry. The present study was conducted to detect the distribution of virulence and enterotoxin genes among MRSA isolates from bovine mastitis. Out of 500 milk samples, 126 isolates were identified as Staphylococcus and from these only 56 were S. aureus. S.aureus were resistant to cefoxitin (75%), ceftazidime (75%), amoxicillin (71.4%), cefodaxime (67.8%), cefepime (66.1%), oxacillin (64.3%), norfloxacin (60.7%) and gentamicin (58.9%). Only 42 isolates were identified as MRSA strains among staphylococci isolates. MRSA were harbouring virulence genes; mecA (100%), coa (100%) and nuc (100%). The other virulence factors such as hlg (80.9%, 34/42), pvl (47.6%, 20/42) and spa (92.8%, 39/42) were also reported. Molecular characterisation of enterotoxin genes revealed that out of 42 tested isolates 11 were found negative (26%) for any enterotoxin gene whereas 7 (16.6%), 6 (14.3%), 18 (42.8%), 1 (2.3%), 26 (61.9%),27(64.2%),3 (7.1%) were found positive for sea, seb, sec, sed, seg, sei, and seq enterotoxin respectively.

Structural Elucidation of Inter-CARD Interfaces involved in NOD2 Tandem CARD Association and RIP2 Recognition.

Nucleotide-binding and oligomerization domain-containing protein 2 (NOD2) recognizes the muramyl dipeptide and activates the NF-κB signaling cascade following its interaction with receptor-interacting protein 2 (RIP2) via caspase recruitment domains (CARDs). The NOD2-RIP2 interaction is not understood well due to inadequate structural information. Using comparative modeling and multimicrosecond timescale molecular dynamics simulations, we have demonstrated the association of NOD2-CARDs (CARDa-CARDb) and their interaction with RIP2CARD. Our results suggest that a negatively charged interface of NOD2CARDa and positively charged type-Ia interface of NOD2CARDb are crucial for CARDa-CARDb association and the type-Ia interface of NOD2CARDa and type-Ib interface of RIP2CARD predicted to be involved in 1:1 CARD-CARD interaction. Moreover, the direct interaction of NOD2CARDb with RIP2CARD signifies the importance of both CARDs of NOD2 in RIP2-mediated CARD-CARD interaction. Altogether, the structural results could help in understanding the underlying molecular details of the NOD2-RIP2 association in higher and lower eukaryotes.

PU.1 is involved in the transcriptional up-regulation of RNA and DNA sensing pathway genes in buffalo fibroblasts.

PU.1, CEBPA, and CEBPB are Lineage Determining Transcription Factors (LDTFs) that play roles in biological processes such as cell differentiation and the immune system regulation including the innate immune pathways. The roles of these LDTFs in the innate RNA and DNA sensing pathways have received little attention. We show that in buffalo fibroblasts, PU.1 causes the mRNA up-regulation of the RNA and DNA sensors such as RIG-I (65.1 fold), MDA5 (20.4 fold), IFI16-l (8.0 fold), and cGAS (60.5 fold) while CEBPA does the same but to a lesser extent (RIG-I-26.4 fold, MDA5-10.8 fold, IFI16-l- 3.3 fold and cGAS-8.6 fold). CEBPB does not appear to have a role in the up-regulation of these genes. PU.1 expression also primes the cells to develop a strong immune response against the dsRNA virus mimic polyinosinic:polycytidylic acid (poly I:C) by significantly up-regulating Interferon-ß (14.9 fold change with p-value <0.0001). CEBPA up-regulates Interferon-ß to a lower level than PU.1 (4.7 fold change with p-value 0.0024), whereas CEBPB exhibits non-significant up-regulation (2.1 fold with p-value of 0.1449). As PU.1 robustly up-regulates the nucleic acid sensing pathways, it can prove to be useful in improving the defence against viruses that can cause losses to animal husbandry.

Characterization of Anti-Müllerian Hormone (AMH) Gene in Buffaloes and Goats.

The Anti-Müllerian Hormone (AMH) is a member of the transforming growth factor beta (TGF-ß) superfamily, playing a significant role in cell proliferation, differentiation and apoptosis. In females, AMH is secreted throughout their reproductive life span from ovaries, whereas in males it is secreted by gonadal cells at a very early stage of testicular development. AMH is a promising marker of ovarian reserve in women and can be used to measure the female reproductive lifespan. In the present study, we cloned and sequenced the GC rich AMH gene from Indian riverine buffalo (Bubalus bubalis) and goat (Capra hircus). Obtained sequences were compared to the AMH sequences of other mammals, and corresponding amino acid sequences revealed that the caprine and bovine AMH sequences are more closely related to each other than to those of other mammals. Furthermore, we analyzed the chromosomal localization of AMH genes in mammalian species to understand potential syntenic relationship. The AMH gene is localized between the sequences for the SF3A and JSRP1 genes and maintains this precise location in relation to other nearby genes. The dN/dS ratio of AMH gene did not indicate any pressure for either positive or negative selection; thus, the physiological function of the AMH gene in the reproduction of these two ruminant species remains very vital. Similar to other mammals, the AMH gene may be an important indicator for regulating female reproductive biology function in bovine, cetacean, caprine, and camelidae.

Poly I:C stimulation in-vitro as a marker for an antiviral response in different cell types generated from Buffalo (Bubalus bubalis).

The innate immune system is activated upon virus invasion of a host cell by recognizing viral component, such as dsRNA through specific receptors, resulting in the production of type- I IFNs, which confer an antiviral state within the invaded as well as surrounding cells. In the present study, fibroblast, monocyte and macrophage cells derived from water Buffalo (Bubalus bubalis) were exposed to a synthetic dsRNA analogue, poly I:C to mimic viral invasion in each cell type. Recognition of poly I:C through cytosolic helicase receptors RIG-I and MDA5 molecule lead to the activation of the RLR pathway, subsequently activating the MAVS-IRF3/7 cascade and the production of antiviral effector molecule like IFNß and ISGs. Within the different cell types, we identified variability in RLR receptor and IFNß expression after poly I:C administration. Fibroblasts responded quickly and strongly with IFNß production, followed by macrophages and monocytes. Despite absolute expression variability among different cell types the expression trend of RLRs pathway genes were similar. Length of poly I:C molecule also influence IFNß expression in response of RLR pathway. Short (LMW) poly I:C induce stronger IFN-ß expression in myeloid (macrophage and monocyte) cells. In contrast long (HMW) poly I:C preferably elicit higher IFNß expression in non-myeloid (fibroblast) cell. Therefore, MDA5 and RIG-1 plays an indispensable role in eliciting antiviral response in non- immune (fibroblast) host cell. Thus, stimulation of RLR pathway with suitable and potentially cell-type specific agonist molecules successfully elicit antiviral state in the host animal, with fibroblasts conferring a stronger antiviral state compared with the monocytes and macrophages.

Effect of betaine supplementation on growth performance, nutrient intake and expression of IGF-1 in Karan Fries heifers during thermal stress.

Heat stress hampers nutrient utilisation and production of animals, and dietary betaine supplementation can mitigate the adverse effects of heat stress on animals and improve their productivity. The present study was conducted to explore the effects of betaine supplementation on the growth performance of eighteen growing Karan Fries (KF) heifers having similar age and body conditions. The experiment was carried out on three groups (n = 6) of KF heifers viz. control, treatment I (betaine supplemented at 25  g/d/animal), and treatment II (betaine supplemented at 50  g/d/animal). The experiment lasted for eight months covering the three major seasons of Indian tropical conditions viz. hot-dry (temperature humidity index, THI = 83), hot-humid (THI = 85) and thermoneutral season (THI = 73). Blood samples were collected at fortnightly intervals and analysed for plasma growth hormone (GH; competitive ELISA) and total insulin-like growth factor 1 (IGF-1; Sandwich ELISA), as well as expression of IGF-I in peripheral blood mononuclear cells (PBMC) using real-time polymerase chain reaction. Betaine supplementation resulted in significant (p < 0.05) increase in dry matter intake, feed conversion efficiency, body weight gain, plasma GH and IGF-1 levels during all seasons. The concentrations of plasma IGF-1 and the mRNA expression of IGF-1 were higher (p < 0.01) in treatment I as compared to other groups during all seasons. Betaine supplementation at 25  g/d/animal was more cost-effective in improving growth performance of heat-stressed heifers as compared to 50  g/d/animal. The study suggests that the betaine protects intestinal integrity, enhances nutrient utilisation during heat stress and improves growth performance of growing heifers.

Nature of selection varies on different domains of IFI16-like PYHIN genes in ruminants.

BACKGROUND: ALRs (AIM2-like Receptors) are germline encoded PRRs that belong to PYHIN gene family of cytokines, which are having signature N-terminal PYD (Pyrin, PAAD or DAPIN) domain and C-terminal HIN-200 (hematopoietic, interferon-inducible nuclear protein with 200 amino acid repeat) domain joined by a linker region. The positively charged HIN-200 domain senses and binds with negatively charged phosphate groups of single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) purely through electrostatic attractions. On the other hand, PYD domain interacts homotypically with a PYD domain of other mediators to pass the signals to effector molecules downwards the pathways for inflammatory responses. There is remarkable inter-specific diversity in the numbers of functional PYHIN genes e.g. one in cow, five in human, thirteen in mice etc., while there is a unique loss of PYHIN genes in the bat genomes which was revealed by Ahn et al. (2016) by studying genomes of ten different bat species belonging to sub-orders yinpterochiroptera and yangochiroptera. The conflicts between host and pathogen interfaces are compared with "Red queen's arms race" which is also described as binding seeking dynamics and binding avoidance dynamics. As a result of this never-ending rivalry, eukaryotes developed PRRs as antiviral mechanism while viruses developed counter mechanisms to evade host immune defense. The PYHIN receptors are directly engaged with pathogenic molecules, so these should have evolved under the influence of selection pressures. In the current study, we investigated the nature of selection pressure on different domain types of IFI16-like (IFI16-L) PYHIN genes in ruminants. RESULTS: Three transcript variants of the IFI16-like gene were found in PBMCs of ruminant animals-water buffalo, zebu cattle, goat, and sheep. The IFI16-like gene has one N-terminal PYD domain and one C-terminal HIN-200 domain, separated by an inter-domain linker region. HIN domain and inter-domain region are positively selected while the PYD domain is under the influence of purifying selection. CONCLUSION: Herein, we conclude that the nature of selection pressure varies on different parts (PYD domain, HIN domain, and inter-domain linker region) of IFI16-like PYHIN genes in the ruminants. This data can be useful to predict the molecular determinants of pathogen interactions.

Generation of Genomic Deletions (of Rig-I GENE) in Goat Primary Cell Culture Using CRISPR/CAS9 Method.

CRISPR/Cas9 system is a natural immune system in prokaryotes protecting them from infectious viral or plasmid DNA invading the cells. This RNA-guided system can act as powerful tool for introducing genomic alterations in eukaryotic cells with high efficiency. In the present study, Rig-Igene is taken as model gene to study the efficiency of CRISPR/Cas9 system induced gene deletion in primary fibroblast cell culture. Rig-I(retinoic acid-inducible gene-1) is involved in regulating immune response in mammals. In this study, we optimized the CRISPR/Cas9 method for knocking out Rig-Igene in Goat primary fibroblasts by using a NHEJ pathway. Cells were screened for inactivation of the Rig-Igene and two positive clones were found out of thirty colonies screened. Thus, cells containing Rig-Igene inactivation could be achieved by CRISPR/Cas9 in goat fibroblast cells.

POP1 might be recruiting its type-Ia interface for NLRP3-mediated PYD-PYD interaction: Insights from MD simulation.

Inflammasomes are multiprotein caspase-activating complexes that enhance the maturation and release of proinflammatory cytokines (IL-1ß and IL-18) in response to the invading pathogen and/or host-derived cellular stress. These are assembled by the sensory proteins (viz NLRC4, NLRP1, NLRP3, and AIM-2), adaptor protein (ASC), and effector molecule procaspase-1. In NLRP3-mediated inflammasome activation, ASC acts as a mediator between NLRP3 and procaspase-1 for the transmission of signals. A series of homotypic protein-protein interactions (NLRP3PYD :ASCPYD and ASCCARD :CASP1CARD ) propagates the downstream signaling for the production of proinflammatory cytokines. Pyrin-only protein 1 (POP1) is known to act as the regulator of inflammasome. It modulates the ASC-mediated inflammasome assembly by interacting with pyrin domain (PYD) of ASC. However, despite similar electrostatic surface potential, the interaction of POP1 with NLRP3PYD is obscured till date. Herein, to explore the possible PYD-PYD interactions between NLRP3PYD and POP1, a combined approach of protein-protein docking and molecular dynamics simulation was adapted. The current study revealed that POP1's type-Ia interface and type-Ib interface of NLRP3PYD might be crucial for 1:1 PYD-PYD interaction. In addition to type-I mode of interaction, we also observed type-II and type-III interaction modes in two different dynamically stable heterotrimeric complexes (POP1-NLRP3-NLRP3 and POP1-NLRP3-POP1). The inter-residual/atomic distance calculation exposed several critical residues that possibly govern the said interaction, which need further investigation. Overall, the findings of this study will shed new light on hitherto concealed molecular mechanisms underlying NLRP3-mediated inflammasome, which will have strong future therapeutic implications.

Computational studies on receptor-ligand interactions between novel buffalo (Bubalus bubalis) nucleotide-binding oligomerization domain-containing protein 2 (NOD2) variants and muramyl dipeptide (MDP).

Nucleotide binding and oligomerization domain 2 (NOD2), a member of intracellular NOD-like receptors (NLRs) family, recognizes the bacterial peptidoglycan, muramyl dipeptide (MDP) and initiates host immune response. The precise ligand recognition mechanism of NOD2 has remained elusive, although studies have suggested leucine rich repeat (LRR) region of NOD2 as the possible binding site of MDP. In this study, we identified multiple transcripts of NOD2 gene in buffalo (buNOD2) and at least five LRR variants (buNOD2-LRRW (wild type), buNOD2-LRRV1-V4) were found to be expressed in buffalo peripheral blood mononuclear cells. The newly identified buNOD2 transcripts were shorter in lengths as a result of exon-skipping and frame-shift mutations. Among the variants, buNOD2-LRRW, V1, and V3 were expressed more frequently in the animals studied. A comparative receptor-ligand interaction study through modeling of variants, docking, and molecular dynamics simulation revealed that the binding affinity of buNOD2-LRRW towards MDP was greater than that of the shorter variants. The absence of a LRR segment in the buNOD2 variants had probably affected their affinity toward MDP. Notwithstanding a high homology among the variants, the amino acid residues that interact with MDP were located on different LRR motifs. The binding free energy calculation revealed that the amino acids Arg850(LRR4) and Glu932(LRR7) of buNOD2-LRRW, Lys810(LRR3) of buNOD2-LRRV1, and Lys830(LRR3) of buNOD2-LRRV3 largely contributed towards MDP recognition. The knowledge of MDP recognition and binding modes on buNOD2 variants could be useful to understand the regulation of NOD-mediated immune response as well as to develop next generation anti-inflammatory compounds.