Macrophage RAGE activation is proinflammatory in NASH.

Intrahepatic macrophages in nonalcoholic steatohepatitis (NASH) are heterogenous and include proinflammatory recruited monocyte-derived macrophages. The receptor for advanced glycation endproducts (RAGE) is expressed on macrophages and can be activated by damage associated molecular patterns (DAMPs) upregulated in NASH, yet the role of macrophage-specific RAGE signaling in NASH is unclear. Therefore, we hypothesized that RAGE-expressing macrophages are proinflammatory and mediate liver inflammation in NASH. Compared with healthy controls, RAGE expression was increased in liver biopsies from patients with NASH. In a high-fat, -fructose, and -cholesterol-induced (FFC)-induced murine model of NASH, RAGE expression was increased, specifically on recruited macrophages. FFC mice that received a pharmacological inhibitor of RAGE (TTP488), and myeloid-specific RAGE KO mice (RAGE-MKO) had attenuated liver injury associated with a reduced accumulation of RAGE+ recruited macrophages. Transcriptomics analysis suggested that pathways of macrophage and T cell activation were upregulated by FFC diet, inhibited by TTP488 treatment, and reduced in RAGE-MKO mice. Correspondingly, the secretome of ligand-stimulated BM-derived macrophages from RAGE-MKO mice had an attenuated capacity to activate CD8+ T cells. Our data implicate RAGE as what we propose to be a novel and potentially targetable mediator of the proinflammatory signaling of recruited macrophages in NASH.

Visualization of Necroptotic Cell Death through Transmission Electron Microscopy.

Transmission electron microscopy (TEM) is an all-in-one tool to visualize the complex systems of any specimen that is 1 nm in size or smaller. The current chapter provides detailed guidelines for imaging morphological changes during programmed cell necrosis using TEM as a single-step methodology. In this protocol, a novel aldehyde dehydrogenase inhibitor is used to induce cell programmed necrosis in ovarian cancer cell lines (A2780 and SKOV3). This process is followed by gradient dehydration with ethanol, chemical fixation, sampled grid preparation, and staining with 0.75% uranyl formate. Following fixation and grid preparation, cells are imaged using TEM. The resulting images reveal morphological changes consistent with necrotic morphology, including swelling of cells and organelles, appearance of vacuoles, and plasma membrane rupture followed by leakage of cellular contents. The current approach allows a single-step methodology for characterization of cell-programmed necrosis in cells based on morphology.

Buffalo liver transcriptome analysis suggests immune tolerance as its key adaptive mechanism during early postpartum negative energy balance.

Females undergo negative energy balance (NEB) during the early postpartum period to meet the lactation demands. The liver, being the key metabolic organ, plays a major role in handling NEB. Dairy animals handling high lactation demands are better models to understand the liver adaptive mechanisms during this phase. Therefore, we analyzed the liver transcriptome of dairy buffaloes during early postpartum. Liver biopsies were performed on three lactating buffaloes on the 15th and the 30th days of early postpartum and three heifers (controls) at the diestrous stage. Paired-end Next Generation Sequencing (NGS) identified 509 significantly differentially expressed genes (SDE) in the liver among the three groups. The SDE with log2 fold change > 3 and the unique SDE revealed the promotion of immune suppression (e.g., TCR), apoptosis (e.g., CCDC103), PGF2α synthesis, fat accumulation (e.g., BGLAP) and liver regeneration (e.g., FGF10) pathways, and the downregulation of antigen presentation (e.g., BOLA-DQA) on the 15th day of lactation. Consistently upregulated genes on the 15th and 30th days of early postpartum indicated the promotion of immune tolerance (e.g., IFITM3), medium and long-chain fatty acids' oxidation (e.g., ACSM3), and lipid accumulation (e.g., INSIG1). However, consistently downregulated genes during early postpartum showed immunosuppression, the downregulation of gluconeogenesis from amino acids (e.g., DDO), and the biosynthesis of taurine (e.g., CSAD) and unsaturated fatty acids (e.g., SCD). Functional annotation and network analyses also indicated the promotion of immune tolerance, fat accumulation and decreased gluconeogenesis from amino acids, and estrogen metabolism on the 15th day of lactation. Overall, the liver showed immune tolerance as an adaptive mechanism during early postpartum of buffaloes.

An efficient method for extracting next-generation sequencing quality RNA from liver tissue of recalcitrant animal species.

The next-generation RNA sequencing technologies expedite the discovery of a large number of novel transcripts and genes associated with various pathophysiological conditions. These technologies involve poly(A) enrichment, which in turn requires micrograms of high-quality total RNA. Unfortunately, the available RNA isolation approaches produce poor quality total RNA from difficult-to-isolate animal tissues, such as the liver with high glycogen content. Moreover, the extraction efficiencies of these approaches vary significantly depending on the animal species. To address this challenge, we optimized a three-step protocol for the extraction of high-yield and high-quality total RNA from the liver tissue (LT). The procedure effectively resolved the problem of glycogen coprecipitation by its stepwise removal. No signs of RNA degradation on gel electrophoresis analysis and RNA integrity number values ≥8.5 indicated that the extracted RNA is suitable for downstream processing, such as poly(A) enrichment and transcriptome profiling. To demonstrate the robustness of the novel protocol, a comparison was made with other currently available RNA extraction approaches from diverse resources. Whereas other protocols yielded partially degraded bands with either decreased or reversed ribosomal RNA (rRNA) ratio, our protocol yielded intact rRNA with a ratio of 2.0 ± 0.1. This optimized protocol was also successfully followed for other animal tissues, such as the bone and muscles. In conclusion, the study has described a highly efficient method for the next-generation sequencing quality RNA isolation from LT across a broad range of animal species, with extended applicability to other difficult-to-isolate tissues.

High serum free fatty acids and low leptin levels: Plausible metabolic indicators of negative energy balance in early lactating Murrah buffaloes.

Lactation is a highly demanding event in mammals, including buffaloes. It modulates the partitioning of nutrients, energy utilization, and food intake of the mother to meet her own and infant's energy needs. Failure to satisfy these energy needs leads to Negative Energy Balance (NEB). Currently, the only available indirect NEB indicator is Body Condition Score (BCS). However, direct dependency of the BCS on the peak depletion of body fat causes its inefficient use in a dairy farm. Thus, to establish objective NEB indicators in buffaloes, the serum levels of biochemical (serum ß-hydroxybutyrate [BHBA] and free fatty acids [FFAs]), and endocrine (Growth Hormone [GH], insulin-like growth factor1 [IGF1], Insulin, and leptin) parameters were estimated in buffaloes. Our results revealed that serum FFA levels were significantly (p < 0.05) higher in high milk yielders (HMY) than low milk yielders (LMY) and heifers (H) during the 3rd and the 4th weeks of postpartum. The serum FFA levels were also significantly (p < 0.001) higher in the postpartum buffaloes with BCS < 3 in the field conditions. Further, serum leptin levels were significantly (p < 0.05) lower in HMY than LMY during the 3rd week of postpartum. However, the BHBA, GH, IGF1, and insulin levels were not significantly different between lactating buffaloes and H. These observations indicated that the NEB condition is probably restricted to the first month of early lactation in buffaloes. In conclusion, the simultaneous higher FFA and lower leptin levels could act as direct plausible metabolic indicators of NEB in buffaloes.

A highly efficient method for extracting next-generation sequencing quality RNA from adipose tissue of recalcitrant animal species.

The next-generation sequencing (NGS) based RNA sequencing (RNA-Seq) and transcriptome profiling offers an opportunity to unveil complex biological processes. Successful RNA-Seq and transcriptome profiling requires a large amount of high-quality RNA. However, NGS-quality RNA isolation is extremely difficult from recalcitrant adipose tissue (AT) with high lipid content and low cell numbers. Further, the amount and biochemical composition of AT lipid varies depending upon the animal species which can pose different degree of resistance to RNA extraction. Currently available approaches may work effectively in one species but can be almost unproductive in another species. Herein, we report a two step protocol for the extraction of NGS quality RNA from AT across a broad range of animal species.

Salivary miR-16, miR-191 and miR-223: intuitive indicators of dominant ovarian follicles in buffaloes.

Estrus or sexual receptivity determination is utmost important for efficient breeding programs for female buffaloes. Prominent estrus behavioral symptoms are the result of several molecular and neuroendocrine events involving the ovary and the brain. Expression of estrus behavior is poor in buffaloes during the summer season. Hence, the discovery of biomarkers specific to the estrus stage or its related ovarian events, like the presence of dominant ovarian follicle, is helpful for developing an easy estrus determination method. MicroRNA are small non-coding RNA with a potential to be biomarkers. Therefore, the present study targeted to investigate the potential of estrogen responsive miRNAs (miR-24, miR-200c, miR-16, miR-191, miR-223 and miR-203) as estrus biomarkers in buffalo saliva, a non-invasive fluid representing animals' pathophysiology. There was a significant (P < 0.05) increase in the salivary presence of the miR-16, miR-191 and miR-223 at 6th and 18th-19th days than the 0 day (estrus), 10th day and the following consecutive estrus day. These observations may indicate an association between the representative lower presence of these miRNA in saliva and the presence of dominant ovarian follicles. To test this association, pathway analysis, target gene identification, functional annotation and protein-protein interaction networks (PPI) were performed for miR-16, miR-191 and miR-223 by different bioinformatics tools. Interestingly, the top pathways (fatty acid biosynthesis and oocyte meiosis), target genes (FGF, BDNF and IGF1) and PPI hub genes (KRAS, BCL2 and IGF1) of these miRNAs were found essential for ovarian follicular dominance. In conclusion, the miR-16, miR-191 and miR-223 may not be the perfect estrus stage-specific biomarkers. However, their lower presence in saliva at estrus and 9th-10th day of estrous cycles, when the ovary usually has a dominant follicle in buffaloes, may intuitively indicate the follicular dominance. Further studies are needed to prove this association in a large population.