Multiomics and bioinformatics identify differentially expressed effectors in the brain of Toxoplasma gondii infected masked palm civet.

Introduction: The masked palm civet (Paguma larvata) serves as a reservoir in transmitting pathogens, such as Toxoplasma gondii, to humans. However, the pathogenesis of T. gondii infection in masked palm civets has not been explored. We studied the molecular changes in the brain tissue of masked palm civets chronically infected with T. gondii ME49. Methods: The differentially expressed proteins in the brain tissue were investigated using iTRAQ and bioinformatics. Results: A total of 268 differential proteins were identified, of which 111 were upregulated and 157 were downregulated. KEGG analysis identified pathways including PI3K-Akt signaling pathway, proteoglycans in cancer, carbon metabolism, T-cell receptor signaling pathway. Combing transcriptomic and proteomics data, we identified 24 genes that were differentially expressed on both mRNA and protein levels. The top four upregulated proteins were REEP3, REEP4, TEP1, and EEPD1, which was confirmed by western blot and immunohistochemistry. KEGG analysis of these 24 genes identified signaling cascades that were associated with small cell lung cancer, breast cancer, Toll-like receptor signaling pathway, Wnt signaling pathways among others. To understand the mechanism of the observed alteration, we conducted immune infiltration analysis using TIMER databases which identified immune cells that are associated with the upregulation of these proteins. Protein network analysis identified 44 proteins that were in close relation to all four proteins. These proteins were significantly enriched in immunoregulation and cancer pathways including PI3K-Akt signaling pathway, Notch signaling pathway, chemokine signaling pathway, cell cycle, breast cancer, and prostate cancer. Bioinformatics utilizing two cancer databases (TCGA and GEPIA) revealed that the four genes were upregulated in many cancer types including glioblastoma (GBM). In addition, higher expression of REEP3 and EEPD1 was associated with better prognosis, while higher expression of REEP4 and TEP1 was associated with poor prognosis in GBM patients. Discussion: We identified the differentially expressed genes in the brain of T. gondii infected masked palm civets. These genes were associated with various cellular signaling pathways including those that are immune- and cancer-related.

GC/MS-based metabolic profiling for the evaluation of solid state fermentation to improve quality of Arabica coffee beans.

INTRODUCTION: Coffee fermentation has been reported as one key process in aroma and flavor development of coffee. However, natural fermentation often results in inconsistent quality of coffee. In this study, second fermentation using isolates from feces of civet (Luwak) and Cilembu sweet potato were used to improve the quality of Arabica green coffee beans. OBJECTIVES: The aim of this research was to improve the quality of various Arabica coffee from different origins in Indonesia by controlled-second fermentation. METHOD: The Arabica coffee beans used in this study were from three origins in Indonesia: Kintamani-Bali (I), Aceh-Gayo (II) and Nagarawangi-Sumedang (III). The second fermentation was done using three bacterial isolates coded as BF5C(2); UciSp14; and AF7E which belong to Bacillus genus. Quality assessment of fermented coffee was performed by cupping test following Specialty Coffee Association of America (SCAA) protocol by licensed Q graders, GC/MS metabolite profiling, and total polyphenol content measurement. RESULTS: The controlled-second fermentation for 4-8 h was successful to increase total polyphenol content well as to improve the complexity of coffee taste and coffee quality (cupping score > 84). Comparative GC/MS analysis showed that fermentation of coffee beans resulted in alteration of metabolite profiles of coffee beans compared with control, while still maintaining the characteristics of coffee based on each origin. CONCLUSION: The controlled-second fermentation was effective to increase the quality of coffee and alter metabolite composition of coffee that were associated with changes in taste profile of coffee. This report may serve as basis for producing coffee with better taste quality with higher polyphenols content through standardized fermentation production in industrial scale.

Quantification of coffee blends for authentication of Asian palm civet coffee (Kopi Luwak) via metabolomics: A proof of concept.

Asian palm civet coffee (Kopi Luwak), an animal-digested coffee with an exotic feature, carries a notorious reputation of being the rarest and most expensive coffee beverage in the world. Considering that illegal mixture of cheap coffee into civet coffee is a growing concern among consumers, we evaluated the use of metabolomics approach and orthogonal projection to latent structures (OPLS) prediction technique to quantify the degree of coffee adulteration. Two prediction sets, consisting of certified and commercial coffee, were made from a blend of civet and regular coffee with eleven mixing percentages. The prediction model exhibited accurate estimation of coffee blend percentage thus, successfully validating the prediction and quantification of the mixing composition of known-unknown samples. This work highlighted proof of concept of metabolomics application to predict degree of coffee adulteration by determining the civet coffee fraction in blends.

Isolation and characterization of a bat SARS-like coronavirus that uses the ACE2 receptor.

The 2002-3 pandemic caused by severe acute respiratory syndrome coronavirus (SARS-CoV) was one of the most significant public health events in recent history. An ongoing outbreak of Middle East respiratory syndrome coronavirus suggests that this group of viruses remains a key threat and that their distribution is wider than previously recognized. Although bats have been suggested to be the natural reservoirs of both viruses, attempts to isolate the progenitor virus of SARS-CoV from bats have been unsuccessful. Diverse SARS-like coronaviruses (SL-CoVs) have now been reported from bats in China, Europe and Africa, but none is considered a direct progenitor of SARS-CoV because of their phylogenetic disparity from this virus and the inability of their spike proteins to use the SARS-CoV cellular receptor molecule, the human angiotensin converting enzyme II (ACE2). Here we report whole-genome sequences of two novel bat coronaviruses from Chinese horseshoe bats (family: Rhinolophidae) in Yunnan, China: RsSHC014 and Rs3367. These viruses are far more closely related to SARS-CoV than any previously identified bat coronaviruses, particularly in the receptor binding domain of the spike protein. Most importantly, we report the first recorded isolation of a live SL-CoV (bat SL-CoV-WIV1) from bat faecal samples in Vero E6 cells, which has typical coronavirus morphology, 99.9% sequence identity to Rs3367 and uses ACE2 from humans, civets and Chinese horseshoe bats for cell entry. Preliminary in vitro testing indicates that WIV1 also has a broad species tropism. Our results provide the strongest evidence to date that Chinese horseshoe bats are natural reservoirs of SARS-CoV, and that intermediate hosts may not be necessary for direct human infection by some bat SL-CoVs. They also highlight the importance of pathogen-discovery programs targeting high-risk wildlife groups in emerging disease hotspots as a strategy for pandemic preparedness.

Selection of discriminant markers for authentication of Asian palm civet coffee (Kopi Luwak): a metabolomics approach.

Kopi Luwak, an exotic Indonesian coffee, is made from coffee berries that have been eaten by the Asian palm civet ( Paradoxurus hermaphroditus ). Despite being known as the world's most expensive coffee, there is no reliable, standardized method for determining its authenticity. GC-MS-based multimarker profiling was employed to explore significant metabolites as discriminant markers for authentication. Extracts of 21 coffee beans ( Coffea arabica and Coffea canephora ) from three cultivation areas were analyzed and subjected to multivariate analyses, principal component analysis, and orthogonal projection to latent structures discriminant analysis. Citric acid, malic acid, and the inositol/pyroglutamic acid ratio were selected for further verification by evaluating their differentiating abilities against various commercial coffee products. The markers demonstrated potential application in the differentiation of original, fake Kopi Luwak, regular coffee, and coffee blend samples with 50 wt % Kopi Luwak content. This is the first report to address the selection and successful validation of discriminant markers for the authentication of Kopi Luwak.

Mechanisms of host receptor adaptation by severe acute respiratory syndrome coronavirus.

The severe acute respiratory syndrome coronavirus (SARS-CoV) from palm civets has twice evolved the capacity to infect humans by gaining binding affinity for human receptor angiotensin-converting enzyme 2 (ACE2). Numerous mutations have been identified in the receptor-binding domain (RBD) of different SARS-CoV strains isolated from humans or civets. Why these mutations were naturally selected or how SARS-CoV evolved to adapt to different host receptors has been poorly understood, presenting evolutionary and epidemic conundrums. In this study, we investigated the impact of these mutations on receptor recognition, an important determinant of SARS-CoV infection and pathogenesis. Using a combination of biochemical, functional, and crystallographic approaches, we elucidated the molecular and structural mechanisms of each of these naturally selected RBD mutations. These mutations either strengthen favorable interactions or reduce unfavorable interactions with two virus-binding hot spots on ACE2, and by doing so, they enhance viral interactions with either human (hACE2) or civet (cACE2) ACE2. Therefore, these mutations were viral adaptations to either hACE2 or cACE2. To corroborate the above analysis, we designed and characterized two optimized RBDs. The human-optimized RBD contains all of the hACE2-adapted residues (Phe-442, Phe-472, Asn-479, Asp-480, and Thr-487) and possesses exceptionally high affinity for hACE2 but relative low affinity for cACE2. The civet-optimized RBD contains all of the cACE2-adapted residues (Tyr-442, Pro-472, Arg-479, Gly-480, and Thr-487) and possesses exceptionally high affinity for cACE2 and also substantial affinity for hACE2. These results not only illustrate the detailed mechanisms of host receptor adaptation by SARS-CoV but also provide a molecular and structural basis for tracking future SARS-CoV evolution in animals.