Thermal impacts on transcriptome of Pectoralis major muscle collected from commercial broilers, Thai native chickens and its crossbreeds

Objective The main objective of this study was to define molecular mechanisms associated with thermal stress responses of chickens from commercial broilers (BR, Ross 308), Thai native chickens (NT) and crossbreeds between BR×NT (H75). Methods Twenty days before reaching specific market age, chickens from each breed were divided into control and thermal-stressed groups. The stressed groups were exposed to a cyclic thermal challenge (35°C±1°C for 6 h, followed by 26°C±1°C for 18 h) for 20 days. Control group was raised under a constant temperature of 26°C±1°C. Pectoralis major (n = 4) from each group was collected for transcriptome analysis using HiSeq Illumina and analysis of glycogen and lactate. Gene expression patterns between control and thermal-stressed groups were compared within the same breeds. Results Differentially expressed transcripts of 65, 59, and 246 transcripts for BR, NT, and H75, respectively, were revealed by RNA-Seq and recognized by Kyoto encyclopedia of genes and genomes database. Pathway analysis underlined altered glucose homeostasis and protein metabolisms in all breeds. The signals centered around phosphatidylinositol 3-kinase (PI3K)/Akt signaling, focal adhesion, and MAPK signaling in all breeds with slight differences in molecular signal transduction patterns among the breeds. An extensive apoptosis was underlined for BR. Roles of AMPK, MAPK signaling and regulation of actin cytoskeleton in adaptive response were suggested for H75 and NT chickens. Lower glycogen content was observed in the breast muscles of BR and NT (p<0.01) compared to their control counterparts. Only BR muscle exhibited increased lactate (p<0.01) upon exposure to the stress. Conclusion The results provided a better comprehension regarding the associated biological pathways in response to the cyclic thermal stress in each breed and in chickens with different growth rates

Insights Into Transcriptome Profiles Associated With Wooden Breast Myopathy in Broilers Slaughtered at the Age of 6 or 7 Weeks

open9siThis research was financially supported by Cluster and Program Management, National Science and Technology Development Agency (Thailand; project number P15-50668), and by Office of the Permanent Secretary, Ministry of Higher Education, Science, Research and Innovation (Thailand; P20-50946 and P21-50165).Transcriptomes associated with wooden breast (WB) were characterized in broilers at two different market ages. Breasts (Pectoralis major) were collected, 20-min postmortem, from male Ross 308 broilers slaughtered at 6 and 7 weeks of age. The breasts were classified as “non-WB” or “WB” based on palpation hardness scoring (non-WB = no abnormal hardness, WB = consistently hardened). Total RNA was isolated from 16 samples (n = 3 for 6 week non-WB, n = 3 for 6 week WB; n = 5 for 7 week non-WB, n = 5 for 7 week WB). Transcriptome was profiled using a chicken gene expression microarray with one-color hybridization technique, and compared between non-WB and WB samples of the same age. Among 6 week broilers, 910 transcripts were differentially expressed (DE) (false discovery rate, FDR &lt; 0.05). Pathway analysis underlined metabolisms of glucose and lipids along with gap junctions, tight junction, and focal adhesion (FA) signaling as the top enriched pathways. For the 7 week broilers, 1,195 transcripts were identified (FDR &lt; 0.05) with regulation of actin cytoskeleton, mitogen-activated protein kinase (MAPK) signaling, protein processing in endoplasmic reticulum and FA signaling highlighted as the enriched affected pathways. Absolute transcript levels of eight genes (actinin-1 – ACTN1, integrin-linked kinase – ILK, integrin subunit alpha 8 – ITGA8, integrin subunit beta 5 – ITGB5, protein tyrosine kinase 2 – PTK2, paxillin – PXN, talin 1 – TLN1, and vinculin – VCL) of FA signaling pathway were further elucidated using a droplet digital polymerase chain reaction. The results indicated that, in 6 week broilers, ITGA8 abundance in WB was greater than that of non-WB samples (p &lt; 0.05). Concerning 7 week broilers, greater absolute levels of ACTN1, ILK, ITGA8, and TLN1, accompanied with a reduced ITGB5 were found in WB compared with non-WB (p &lt; 0.05). Transcriptional modification of FA signaling underlined the potential of disrupted cell-cell communication that may incite aberrant molecular events in association with development of WB myopathy.openMalila, Yuwares; Uengwetwanit, Tanaporn; Thanatsang, Krittaporn V.; Arayamethakorn, Sopacha; Srimarut, Yanee; Petracci, Massimiliano; Soglia, Francesca; Rungrassamee, Wanilada; Visessanguan, WonnopMalila, Yuwares; Uengwetwanit, Tanaporn; Thanatsang, Krittaporn V.; Arayamethakorn, Sopacha; Srimarut, Yanee; Petracci, Massimiliano; Soglia, Francesca; Rungrassamee, Wanilada; Visessanguan, Wonno

Transcriptional Profiles of Skeletal Muscle Associated With Increasing Severity of White Striping in Commercial Broilers

Development of the white striping (WS) abnormality adversely impacts overall quality of broiler breast meat. Its etiology remains unclear. This study aimed at exploring transcriptional profiles of broiler skeletal muscles exhibiting different WS severity to elucidate molecular mechanisms underlying the development and progression of WS. Total RNA was isolated from pectoralis major of male 7-week-old Ross 308 broilers. The samples were classified as mild (n = 6), moderate (n = 6), or severe (n = 4), based on number and thickness of the white striations on the meat surface. The transcriptome was profiled using a chicken gene expression microarray with one-color hybridization technique. Gene expression patterns of each WS severity level were compared against each other; hence, there were three comparisons: moderate vs. mild (C1), severe vs. moderate (C2), and severe vs. mild (C3). Differentially expressed genes (DEGs) were identified using the combined criteria of false discovery rate 64 0.05 and absolute fold change 651.2. Differential expression of 91, 136, and 294 transcripts were identified in C1, C2, and C3, respectively. There were no DEGs in common among the three comparisons. Based on pathway analysis, the enriched pathways of C1 were related with impaired homeostasis of macronutrients and small biochemical molecules with disrupted Ca2+-related pathways. Decreased abundance of the period circadian regulator suggested the shifted circadian phase when moderate WS developed. The enriched pathways uniquely obtained in C2 were RNA degradation, Ras signaling, cellular senescence, axon guidance, and salivary secretion. The DEGs identified in those pathways might play crucial roles in regulating cellular ion balances and cell-cycle arrest. In C3, the pathways responsible for phosphatidylinositol 3-kinase-Akt signaling, p53 activation, apoptosis, and hypoxia-induced processes were modified. Additionally, pathways associated with a variety of diseases with the DEGs involved in regulation of [Ca2+], collagen formation, microtubule-based motor, and immune response were identified. Eight pathways were common to all three comparisons (i.e., calcium signaling, Ras-associated protein 1 signaling, ubiquitin-mediated proteolysis, vascular smooth muscle contraction, oxytocin signaling, and pathway in cancer). The current findings support the role of intracellular ion imbalance, particularly Ca2+, oxidative stress, and impaired programmed cell death on WS progression

Monitoring of white striping and wooden breast cases and impacts on quality of breast meat collected from commercial broilers ()

Objective This study aimed at investigating white striping (WS) and wooden breast (WB) cases in breast meat collected from commercial broilers. Methods A total of 183 breast samples were collected from male Ross 308 broilers slaughtered at the age of 6 weeks (n = 100) and 7 weeks (n = 83). The breasts were subjected to meat defect inspection, meat quality determination and histology evaluation. Results Of 183, 4 breasts from 6-week-old broilers were classified as non-defective while the others exhibited the WS lesion. Among the 6-week-old birds, the defective samples from the medium size birds (carcass weight ≤2.5 kg) showed mild to moderate WS degree with no altered meat quality. Some of the breasts from the 6-week-old birds with carcass weight above 2.5 kg exhibited WB in accompanied with the WS condition. Besides of a reduction of protein content, increases in collagen matter and pH values in the defective samples (p<0.05), no other impaired quality indices were detected within this group. All 7-week-old broilers yielded carcasses weighing above 2.5 kg and showed abnormal characteristics with progressive severity. The breasts affected with severe WS and WB showed the greatest cook loss, hardness, springiness and chewiness (p<0.05). Development of WB induced significantly increased drip loss in the samples (p<0.05). Histology indicated necrotic events in the defective myofibers. Based on logistic regression, increasing percent breast weight by one unit enhanced the chance of WS and WB development with advanced severity by 50.9% and 61.0%, respectively. Delayed slaughter age from 6 to 7 weeks increased the likelihood of obtaining increased WS severity by 56.3%. Conclusion Cases of WS and WB defects in Southeast Asia have been revealed. Despite few cases of the severe WS and WB, such abnormal conditions significantly impaired technological properties and nutritional quality of broiler breasts

Metagenomic discovery of lipases with predicted structural similarity to Candida antarctica lipase B.

Here we employed sequence-based and structure-based screening for prospecting lipases that have structural homolog to Candida antarctica lipase B (CalB). CalB, a widely used biocatalyst, was used as structural template reference because of its enzymatic properties. Structural homolog could aid in the discovery of novel wild-type enzymes with desirable features and serve as a scaffold for further biocatalyst design. The available metagenomic data isolated from various environments was leveraged as a source for bioprospecting. We identified two bacteria lipases that showed high structural similarity to CalB with <40% sequence identity. Partial purification was conducted. In comparison to CalB, the enzymatic characteristics of two potential lipases were examined. A candidate exhibited optimal pH of 8 and temperature of 50°C similar to CalB. The second lipase candidate demonstrated an optimal pH of 8 and a higher optimal temperature of 55°C. Notably, this candidate sustained considerable activity at extreme conditions, maintaining high activity at 70°C or pH 9, contrasting with the diminished activity of CalB under similar conditions. Further comprehensive experimentation is warranted to uncover and exploit these novel enzymatic properties for practical biotechnological purposes

Identification of novel SARS-CoV-2 RNA dependent RNA polymerase (RdRp) inhibitors:From in silico screening to experimentally validated inhibitory activity

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2019 has posed a serious threat to global health and the economy for over two years, prompting the need for development of antiviral inhibitors. Due to its vital role in viral replication, RNA-dependent RNA polymerase (RdRp) is a promising therapeutic target. Herein, we analyzed amino acid sequence conservation of RdRp across coronaviruses. The conserved amino acids at the catalytic binding site served as the ligand-contacting residues for in silico screening to elucidate possible resistant mutation. Molecular docking was employed to screen inhibitors of SARS-CoV-2 from the ZINC ChemDiv database. The top-ranked compounds selected from GOLD docking were further investigated for binding modes at the conserved residues of RdRp, and ten compounds were selected for experimental validation. Of which, three compounds exhibited promising antiviral activity. The most promising candidate showed a half-maximal effective concentration (EC(50)) of 5.04 µM. Molecular dynamics simulations, binding free-energy calculation and hydrogen bond analysis were performed to elucidate the critical interactions providing a foundation for developing lead compounds effective against SARS-CoV-2

Comparative Analysis of PacBio and Oxford Nanopore Sequencing Technologies for Transcriptomic Landscape Identification of Penaeus monodon

With the advantages that long-read sequencing platforms such as Pacific Biosciences (Menlo Park, CA, USA) (PacBio) and Oxford Nanopore Technologies (Oxford, UK) (ONT) can offer, various research fields such as genomics and transcriptomics can exploit their benefits. Selecting an appropriate sequencing platform is undoubtedly crucial for the success of the research outcome, thus there is a need to compare these long-read sequencing platforms and evaluate them for specific research questions. This study aims to compare the performance of PacBio and ONT platforms for transcriptomic analysis by utilizing transcriptome data from three different tissues (hepatopancreas, intestine, and gonads) of the juvenile black tiger shrimp, Penaeus monodon. We compared three important features: (i) main characteristics of the sequencing libraries and their alignment with the reference genome, (ii) transcript assembly features and isoform identification, and (iii) correlation of the quantification of gene expression levels for both platforms. Our analyses suggest that read-length bias and differences in sequencing throughput are highly influential factors when using long reads in transcriptome studies. These comparisons can provide a guideline when designing a transcriptome study utilizing these two long-read sequencing technologies

Absolute expressions of hypoxia-inducible factor-1 alpha (HIF1A) transcript and the associated genes in chicken skeletal muscle with white striping and wooden breast myopathies.

Development of white striping (WS) and wooden breast (WB) in broiler breast meat have been linked to hypoxia, but their etiologies are not fully understood. This study aimed at investigating absolute expression of hypoxia-inducible factor-1 alpha subunit (HIF1A) and genes involved in stress responses and muscle repair using a droplet digital polymerase chain reaction. Total RNA was isolated from pectoralis major collected from male 6-week-old medium (carcass weight ≤ 2.5 kg) and heavy (carcass weight > 2.5 kg) broilers. Samples were classified as "non-defective" (n = 4), "medium-WS" (n = 6), "heavy-WS" (n = 7) and "heavy-WS+WB" (n = 3) based on abnormality scores. The HIF1A transcript was up-regulated in all of the abnormal groups. Transcript abundances of genes encoding 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 (PFKFB4), lactate dehydrogenase-A (LDHA), and phosphorylase kinase beta subunit (PHKB) were increased in heavy-WS but decreased in heavy-WS+WB. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was up-regulated in non-defective samples. The muscle-specific mu-2 isoform of glutathione S-transferases (GSTM2) was up-regulated in the abnormal samples, particularly in the heavy groups. The genes encoding myogenic differentiation (MYOD1) and myosin light chain kinase (MYLK) exhibited similar expression pattern, of which medium-WS and heavy-WS significantly increased compared to non-defective whereas expression in heavy-WS+WB was not different from either non-defective or WS-affected group. The greatest and the lowest levels of calpain-3 (CAPN3) and delta-sarcoglycan (SCGD) were observed in heavy-WS and heavy-WS+WB, respectively. Based on micrographs, the abnormal muscles primarily comprised fibers with cross-sectional areas ranging from 2,000 to 3,000 μm2. Despite induced glycolysis at the transcriptional level, lower stored glycogen in the abnormal muscles corresponded with the reduced lactate and higher pH within their meats. The findings support hypoxia within the abnormal breasts, potentially associated with oversized muscle fibers. Between WS and WB, divergent glucose metabolism, cellular detoxification and myoregeneration at the transcriptional level could be anticipated