Digestive Strategies Research Articles

Mirror cutting-assisted orthogonal digestion enabling large-scale and accurate protein complex characterization

蛋白质主要以复合物的形式参与各项生命活动。化学交联质谱(CXMS)技术作为近年来新兴的蛋白质复合物解析技术,不仅可实现蛋白质复合物规模化解析,而且普遍适用于任意相对分子质量和纯度的蛋白质复合物样品,因此已成为X-射线晶体衍射技术、冷冻电镜技术等蛋白质复合物解析经典技术的重要补充。目前,CXMS主要采用胰蛋白酶将交联后的蛋白质复合物进行酶切,并进行质谱鉴定。然而鉴定到的交联肽段谱图中b/y特征碎片离子的鉴定数目不足,且因肽段序列匹配连续性较差导致谱图可信度相对较低,影响了蛋白质复合物交联位点的鉴定精准度。该文基于镜像酶正交切割的互补特性,采用胰蛋白酶镜像酶与胰蛋白酶联合酶切的方法,对交联后的蛋白质复合物分别进行酶切,再利用液相色谱-质谱联用技术对酶解产生的交联肽段进行鉴定,进而实现蛋白质复合物的组成、相互作用和结构位点距离约束的解析。对牛血清白蛋白和大肠杆菌全蛋白的交联肽段的分析结果表明,该方法通过增加交联肽段谱图中特征碎裂离子的数目和连续性,鉴定到的大肠杆菌全蛋白的交联位点,较单一胰蛋白酶酶切,提高了16%。因此,镜像酶正交酶切策略能有效提高交联肽段的鉴定准确度和覆盖度,有望为实现规模化的蛋白质复合物精准解析提供新思路。

Metagenomic Comparisons between Soft and Hard Feces of Plateau Pikas (Ochotona curzoniae).

Simple SummaryPlateau pika produces hard and soft feces with different morphology, component and microbial structure. Hard feces had more abundant Firmicutes, while soft feces had more abundant Akkermansia. The differences of microbial communities between hard and soft feces were mainly driven by core microbomes. Soft feces had a comprehensive advances in predict functional pathways compared to hard feces, these strengthened functional pathways were closely associated with metabolism of energy, vitamins, and amino acid. Our study preliminarily explored the differences in microbial structure and function between hard and soft feces, provided a foundation for future systematic explorations of the cecotrophy. The division of hard and soft feces is an effective digestion strategy in the order Lagomorpha. Although previous studies have reported that hard and soft feces differ in morphology and component, the discrepancy in the microbiome remains unclear. This study explored the microbiomes of hard and soft feces in plateau pikas by sequencing the V3 and V4 regions of 16S rDNA. We found that hard feces harbored higher Firmicutes, while soft feces harbored higher Akkermansia. Increased rare bacterial taxa were observed in hard feces compared with soft feces. Moreover, hard and soft feces displayed a greater difference in terms of core operational taxonomy units (OTUs) compared to the total OTUs. The soft feces showed enhancements in all predicted Kyoto Encyclopedia of Genes and Genomes (KEGG) functions, indicating an advancing microbial metabolism compared to hard feces. The significantly upregulated pathways in soft feces were mainly enriched in metabolism of energy and carbohydrate, glycan biosynthesis, cofactors and vitamins, and amino acids—all of which are associated with increased contents of microbial proteins, vitamins, and short-chain fatty acids. Our study reports, for the first time, the differential microbiomes between hard and soft feces of pikas and provides direction for the future studies on cecotrophy.

Single-cell transcriptomic landscape of the sheep rumen provides insights into physiological programming development and adaptation of digestive strategies

As an important evolutionary innovation and unique organ, the rumen has played a crucial role in ruminant adaptation to complex ecological environments. However, the cellular basis of its complex morphology and function remains largely unknown. In this study, we identified eight major cell types from seven representative prenatal and postnatal rumen samples using ~56 600 single-cell transcriptomes. We captured the dynamic changes and high heterogeneity in cellular and molecular profiles before, during, and after the appearance of keratinized stratified squamous epithelium with neatly arranged papillae and functional maturity. Basal cells, keratinocytes, differentiating keratinocytes, terminally differentiated keratinocytes, and special spinous cells provided the cellular basis for rumen epithelium formation. Notably, we obtained clear evidence of two keratinization processes involved in early papillogenesis and papillae keratinization and identified TBX3 as a potential marker gene. Importantly, enriched stratum spinosum cells played crucial roles in volatile fatty acid (VFA) metabolism and immune response. Our results provide a comprehensive transcriptional landscape of rumen development at single-cell resolution, as well as valuable insight into the interactions between dietary metabolism and the rumen.

Single-cell transcriptomic landscape of the sheep rumen provides insights into physiological programming development and adaptation of digestive strategies

Single-cell transcriptomic landscape of the sheep rumen provides insights into physiological programming development and adaptation of digestive strategies

Simultaneous determination of three allergic proteins in rice and products by high performance liquid chromatography-tandem mass spectrometry combined with stable isotope-labeled peptides

基于稳定同位素标记特征肽段和液相色谱-质谱联用仪建立稻米及制品中3种过敏蛋白质的同时定量方法。稻米及制品样品经盐溶液提取,赖氨酰基内切酶(Lys-C)和胰蛋白酶依次水解,C18-SD柱净化后,采用纳升高效液相色谱-线性离子阱-静电场轨道阱(NanoLC-LTQ-Orbitrap)采集和Protein Discovery软件鉴定,NCBI和Uniprot数据库的基本局部搜索比对工具(BLAST)筛选验证,最终获得表征稻米及制品中α-淀粉酶/胰蛋白酶抑制剂类蛋白质(seed allergenic protein RAG2, RAG2)、乙二醛酶Ⅰ活性蛋白(glyoxalase Ⅰ)和α-球蛋白(19 kDa globulin)3种过敏蛋白质的特异性肽段。3个特异性肽段经液相色谱梯度洗脱,在Poroshell色谱柱上实现完全分离,由三重四极杆质谱仪分析。实验通过优化多反应监测(MRM)质谱参数,比较不同溶剂体系、水解酶种类和酶量等酶解条件,结合内标法定量,实现对稻米及制品中3种蛋白质的绝对定量。实验结果表明,当酶解溶剂中含1 g/L十二烷基硫酸钠,采用Lys-C和胰蛋白酶组合消化策略,可有效提高3种蛋白质的酶切效率至65.7%~97.3%。该方法在1~200 nmol/L范围内线性关系良好,相关系数均大于0.9972, 3种蛋白质的检出限和定量限分别为3 mg/kg和10 mg/kg。3种蛋白质在空白稻米制品基质中3个水平下的加标回收率为80.6%~103.7%,日间和日内精密度均小于11.5%。该方法稳定性好,检测灵敏度高,操作简便,在分析各类稻米及制品中3种过敏蛋白质含量具有广泛的应用前景。

Mirror-Cutting-Based Digestion Strategy Enables the In-Depth and Accuracy Characterization of N-Linked Protein Glycosylation.

N-linked glycosylation plays important roles in multiple physiological and pathological processes, while the analysis coverage is still limited due to the insufficient digestion of glycoproteins, as well as incomplete ion fragments for intact glycopeptide determination. Herein, a mirror-cutting-based digestion strategy was proposed by combining two orthogonal proteases of LysargiNase and trypsin to characterize the macro- and micro-heterogeneity of protein glycosylation. Using the above two proteases, the b- or y-ion series of peptide sequences were, respectively, enhanced in MS/MS, generating the complementary spectra for peptide sequence identification. More than 27% (489/1778) of the site-specific glycoforms identified by LysargiNase digestion were not covered by trypsin digestion, suggesting the elevated coverage of protein sequences and site-specific glycoforms by the mirror-cutting method. Totally, 10,935 site-specific glycoforms were identified from mouse brain tissues in the 18 h MS analysis, which significantly enhanced the coverage of protein glycosylation. Intriguingly, 27 mannose-6-phosphate (M6P) glycoforms were determined with core fucosylation, and 23 of them were found with the "Y-HexNAc-Fuc" ions after manual checking. This is hitherto the first report of M6P and fucosylation co-modifications of glycopeptides, in which the mechanism and function still needs further exploration. The mirror-cutting digestion strategy also has great application potential in the exploration of missing glycoproteins from other complex samples to provide rich resources for glycobiology research.

PSX-A-13 Late-Breaking: Establishing an in vitro model for studies of myogenesis in broiler chicks

Abstract The objective of this work was to establish protocols for isolating, maintaining, and differentiating SCs from broiler embryos and young chicks so that this model can be used to identify factors that affect myogenic development. Efficient production of lean and healthy muscle is the ultimate goal of the livestock industry. Skeletal muscle develops from a population of multipotent stem cells, also known as satellite cells (SCs), that have the potential to differentiate into both muscle cells and adipocytes. During embryonic and early postnatal development, nutritional and hormonal cues interact to influence the cell fate of this cell population, which ultimately affects body composition and muscle quality. In broilers, the factors that influence the early development of SCs are particularly important due to the high incidence of breast muscle myopathies in commercial flocks. Methods to isolate SCs from multiple species, including chicken, have been described in the literature. However, SCs in very young animals have different growth and differentiation properties, and the fiber properties of the muscle, which influence the digestion strategy, differ from those of mature birds. Protocols were developed using p. major muscle of 7 day-old-broiler chicks. Satellite cells were isolated from intact muscle by two-step enzymatic digestion and purified from other cell types based on differential adherence to tissue culture plastic. Enrichment for SCs in each procedure was evaluated based on the ability to undergo myogenic differentiation and form myotubes in vitro. Immunofluorescent staining for Pax7, a marker of SCs, and flow cytometry for CD56, a surface marker for SCs, were used to confirm a high purity population of SCs. In conclusion, we have established a model that will be valuable for assessing the impact of nutrients, metabolites, and other physiological cues on early life muscle growth and development in broilers.

Spatial epi-proteomics enabled by histone post-translational modification analysis from low-abundance clinical samples

BackgroundIncreasing evidence linking epigenetic mechanisms and different diseases, including cancer, has prompted in the last 15 years the investigation of histone post-translational modifications (PTMs) in clinical samples. Methods allowing the isolation of histones from patient samples followed by the accurate and comprehensive quantification of their PTMs by mass spectrometry (MS) have been developed. However, the applicability of these methods is limited by the requirement for substantial amounts of material.ResultsTo address this issue, in this study we streamlined the protein extraction procedure from low-amount clinical samples and tested and implemented different in-gel digestion strategies, obtaining a protocol that allows the MS-based analysis of the most common histone PTMs from laser microdissected tissue areas containing as low as 1000 cells, an amount approximately 500 times lower than what is required by available methods. We then applied this protocol to breast cancer patient laser microdissected tissues in two proof-of-concept experiments, identifying differences in histone marks in heterogeneous regions selected by either morphological evaluation or MALDI MS imaging.ConclusionsThese results demonstrate that analyzing histone PTMs from very small tissue areas and detecting differences from adjacent tumor regions is technically feasible. Our method opens the way for spatial epi-proteomics, namely the investigation of epigenetic features in the context of tissue and tumor heterogeneity, which will be instrumental for the identification of novel epigenetic biomarkers and aberrant epigenetic mechanisms.

Multidimensional trophic niche revealed by complementary approaches: Gut content, digestive enzymes, fatty acids and stable isotopes in Collembola.

Trophic niche differentiation may explain coexistence and shape functional roles of species. In complex natural food webs, however, trophic niche parameters depicted by single and isolated methods may simplify the multidimensional nature of consumer trophic niches, which includes feeding processes such as food choice, ingestion, digestion, assimilation and retention.Here we explore the correlation and complementarity of trophic niche parameters tackled by four complementary methodological approaches, that is, visual gut content, digestive enzyme, fatty acid and stable isotope analyses—each assessing one or few feeding processes, and demonstrate the power of method combination.Focusing on soil ecosystems, where many omnivore species with cryptic feeding habits coexist, we chose Collembola as an example. We compiled 15 key trophic niche parameters for 125 species from 40 studies. We assessed correlations among trophic niche parameters and described variation of these parameters in different Collembola species, families and across life‐forms, which represent microhabitat specialisation.Correlation between trophic niche parameters was weak in 45 out of 64 pairwise comparisons, pointing at complementarity of the four methods. Jointly, the results indicated that fungal‐ and plant‐feeding Collembola assimilate storage, rather than structural polysaccharides, and suggested bacterial feeding as a potential alternative feeding strategy. Gut content and fatty acid analyses suggested alignment between ingestion and assimilation/retention processes in fungal‐ and plant‐feeding Collembola. From the 15 trophic niche parameters, six were related to Collembola family identity, suggesting that not all trophic niche dimensions are phylogenetically structured. Only three parameters were related to the life‐forms, suggesting that species use various feeding strategies when living in the same microenvironments.Consumers can meet their nutritional needs by varying their food choices, ingestion and digestion strategies, with the connection among different feeding processes being dependent on the consumed resource and consumer adaptations. Multiple methods reveal different dimensions, together drawing a comprehensive picture of the trophic niche. Future studies applying the multidimensional trophic niche approach will allow us to trace trophic complexity and reveal niche partitioning of omnivorous species and their functional roles, especially in cryptic environments such as soils, caves, deep ocean or benthic ecosystems.

CH4/CO2 Ratios and Carbon Isotope Enrichment Between Diet and Breath in Herbivorous Mammals

Breath and diet samples were collected from 29 taxa of animals at the Zurich and Basel Zoos to characterize the carbon isotope enrichment between breath and diet. Diet samples were measured for δ13C and breath samples for CH4/CO2 ratios and for the respired component of δ13C using the Keeling plot approach. Different digestive physiologies included coprophagous and non-coprophagous hindgut fermenters, and non-ruminant and ruminant foregut fermenters. Isotope enrichments from diet to breath were 0.8 ± 0.9‰, 3.5 ± 0.8‰, 2.3 ± 0.4‰, and 4.1 ± 1.0‰, respectively. CH4/CO2 ratios were strongly correlated with isotope enrichments for both hindgut and foregut digestive strategies, although CH4 production was not the sole reason for isotope enrichment. Average CH4/CO2 ratios per taxon ranged over several orders of magnitude from 10–5 to 10–1. The isotope enrichment values for diet-breath can be used to further estimate the isotope enrichment from diet-enamel because Passey et al. (2005b) found a nearly constant isotope enrichment for breath-enamel for diverse mammalian taxa. The understanding of isotope enrichment factors from diet to breath and diet to enamel will have important applications in the field of animal physiology, and possibly also for wildlife ecology and paleontology.

Preselector.uni-jena.de: optimize your cloning-a resource for identifying restriction enzymes for preselection reactions.

Preselection digests are a common strategy to reduce the background in the ligation step of molecular cloning. However, choosing fitting restriction enzymes by hand is not trivial and may lead to errors, potentially costing a lot of time and work. We therefore created preselector.uni-jena.de (https://preselector.uni-jena.de/), a free online tool to find such restriction enzymes. The tool uses regular expressions to find recognition sites of restriction enzymes in the DNA sequences provided by the user. This new tool compares the sets of restriction sites and reports the enzymes that cut one sequence but not the other sequences to the user. These enzymes are then the ones suitable for a preselection digest. Thus, preselector.uni-jena.de is a fast, reliable, and free-to-use tool to help researchers designing preselection digestion strategies for their cloning.

Dual Methylation-Sensitive Restriction Endonucleases Coupling with an RPA-Assisted CRISPR/Cas13a System (DESCS) for Highly Sensitive Analysis of DNA Methylation and Its Application for Point-of-Care Detection.

High-performance detection of DNA methylation possesses great significance for the diagnosis and therapy of cancer. Herein, for the first time, we present a digestion strategy based on dual methylation-sensitive restriction endonucleases coupling with a recombinase polymerase amplification (RPA)-assisted CRISPR/Cas13a system (DESCS) for accurate and sensitive determination of site-specific DNA methylation. This dual methylation-sensitive restriction endonuclease system selectively digests the unmethylated target but exhibits no response to methylated DNA. Therefore, the intact methylated DNA target triggers the RPA reaction for rapid signal amplification. In contrast, the digested unmethylated target initiates no RPA reaction. RPA products with a T7 promoter can execute the T7 transcription in the presence of T7 RNA polymerase to generate a large number of single-stranded RNA (ssRNA). This ssRNA can be recognized by CRISPR/Cas13a to induce the ssRNase activity of Cas13a, showing the indiscriminate cleavage of the collateral FQ reporter to release the fluorescence signal. With such a design, by combining the unique features of dual methylation-sensitive restriction endonucleases with RPA-assisted CRISPR/Cas13a, the DESCS system not only presents the rapid and powerful signal amplification for the determination of methylated DNA with ultrahigh sensitivity but also effectively eliminates the false positive influences from incomplete digestion of the unmethylated target. More importantly, 0.01% methylation level can be effectively distinguished with the existence of excess unmethylated DNA. In addition, the DESCS assay is integrated into the lateral flow biosensor (LFB) for the point-of-care determination of DNA methylation. In view of the superiorities in high sensitivity, outstanding selectivity, and ease of operation, the DESCS system will provide a reliable assay for site-specific analysis of methylation.

Enhanced biomethane production from corn straw by a novel anaerobic digestion strategy with mechanochemical pretreatment

The biomethane production from lignocellulose via anaerobic digestion has always suffered from low carbon conversion rate, due to the stubborn structure of lignocellulose and the inefficiency of the current anaerobic digestion systems. The integrated two-phase anaerobic reactor can form a highly efficient anaerobic digestion system with an upper acidogenic phase and a lower methanogenic phase. However, the acidogenic phase can be overaccelerated, leading to acidification failure of the whole digestion system. In this study, the acidogenesis liquid digestate (ALD) generated from the acidogenic phase was applied to impregnate corn straw coupled with disc refining (DR) to enhance biomethane production by forming a thorough and stable integrated two-phase anaerobic system. The concentration of volatile fatty acids in the acidogenic phase is adjusted by this pretreatment technology to approximately match the efficiency of methanogenic bacteria in the methanogenic phase. The acetic acid in ALD was proved to be an effective acid to swell lignocellulose and improved the performance of the acid impregnation coupled with disc refining (AIDR) pretreatment. A 47.13% higher biomethane yield was obtained from corn straw after AIDR pretreatment. Moreover, energy conversion efficiency and economic analysis indicated this anaerobic digestion process with AIDR pretreatment had a $ 21.06 ton−1 higher net profit than that without any pretreatment. This novel anaerobic digestion process creates a synergistic processing technology that integrates biomass pretreatment with a two-phase anaerobic digestion system, resulting in destroying the compact structure of lignocellulose, enhancing biomethane production efficiency, and improving the economic competitiveness of biomass conversion to biomethane.

Highly toxic type Ⅱ ribosome-inactivating proteins ricin and abrin and their detection methods: a review

Ⅱ型核糖体失活蛋白(RIPs)是一类重要的蛋白毒素,该类毒素大都具有一对二硫键连接的A-B链结构特征,B链具有半乳糖结合特性,能够与真核细胞膜表面受体特异性结合,将具有N-糖苷酶活性的A链导入细胞,与核糖体特定位点发生脱嘌呤作用使核糖体失活,最终通过抑制蛋白质合成而展现出细胞毒性。Ⅱ型RIPs毒素毒性极强,来源于植物的蓖麻毒素(ricin)和相思子毒素(abrin)的毒性分别是神经性毒剂维埃克斯(Vx)的385倍和2885倍。同时,该类毒素来源广泛、易于制备、稳定性好,成为一类潜在化生恐怖战剂,受到国内外广泛关注,其中蓖麻毒素作为唯一的蛋白毒素被收录于禁止化学武器公约禁控清单。近年来发生的多次蓖麻毒素邮件恐怖事件,进一步促进了有关Ⅱ型RIPs毒素的准确、灵敏、快速的检测鉴定技术的发展。剧毒性Ⅱ型RIPs毒素的检测鉴定方法主要涉及免疫分析法为代表的特异性识别和生物质谱分析为主的定性定量检测方法,以及基于脱嘌呤反应活性和细胞毒性的毒素活性检测方法。基于抗原-抗体作用的免疫检测法及基于寡核苷酸适配体的特异性识别检测法具有速度快、灵敏度高的优势,但对于复杂样品中高度同源蛋白的检测,易产生假阳性结果。随着生物质谱技术的快速发展,电喷雾离化(ESI)或基质辅助激光解吸离化(MALDI)等技术广泛应用于蛋白质的准确鉴定,不仅能够提供蛋白毒素的准确分子量和结构序列信息,而且能够实现准确定量。酶解质谱法是应用最为广泛的检测鉴定方法,通过酶解肽指纹谱分析,实现蛋白毒素的准确鉴定;对于复杂样品中蛋白毒素的分析,通过多种蛋白酶酶解策略获得丰富的特异性肽段标志物,然后进行肽段标志物的靶向质谱分析从而获得准确的定性及定量信息,方法有效提升了Ⅱ型RIPs毒素鉴定的准确度和灵敏度。免疫分析法和生物质谱法能够准确鉴定Ⅱ型RIPs毒素,但无法识别毒素是否还保持毒性。对于Ⅱ型RIPs毒素的活性分析,主要包括基于N-糖苷酶活性的脱嘌呤反应测定法和细胞毒性测定法,两种方法均可实现毒素毒性的简便、快速、灵敏的分析检测,是Ⅱ型RIPs毒素检测方法的有效补充。由于该类毒素的高度敏感性,国际禁止化学武器组织(OPCW)对相关样品中Ⅱ型RIPs毒素的分析提出了唯一性鉴定的技术要求。该文引用了Ⅱ型RIPs毒素及其检测方法相关的70篇文献,综述了以上Ⅱ型RIPs毒素的结构性质、中毒机理及典型剧毒性Ⅱ型RIPs毒素检测方法的研究进展,对不同检测方法的特点和应用潜力进行了总结,并结合OPCW对Ⅱ型RIPs毒素唯一性鉴定的技术需求,展望了未来Ⅱ型RIPs毒素检测技术研究的发展趋势。

Multiple-parallel-protease digestion coupled with high-resolution mass spectrometry: An approach towards comprehensive peptide mapping of therapeutic mAbs

Therapeutic monoclonal antibodies (mAbs) are structurally large and complex molecules. To be safe and efficacious, a biosimilar mAb must show high similarity to its reference product in Critical Quality Attributes (CQA). mAbs are highly sensitive to protein expression, production, manufacturing, supply chain, and storage conditions. All these factors make biosimilar mAbs intrinsically susceptible for variability during production. Accordingly, several lots of references and tests are required to establish the biosimilarity of a test mAb. The primary structure is a CQA of a mAb affecting its safety and efficacy. Here, we apply peptide mapping as an analytical method to decipher the primary structure and associated modifications for a quick quality assessment of TrastuzumAb and RituximAb innovator and biosimilar. A multiple-parallel-protease digestion strategy followed by high-resolution mass spectrometric analysis consistently achieved 100% sequence coverage along with reliable detection of post-translational modifications. Additionally, the use of supporting methods such as intact mass analysis and circular dichroism helped us to decipher the primary and higher order structures of these mAbs. We identify discernible variations in the profile of the innovator and biosimilar mAbs and validate the method for quick yet deep comparability analysis of the primary structure of biosimilar mAbs sold in the market. SignificancePeptide mapping using bottom-up approach is one of the most common methods for the characterization of therapeutic monoclonal antibodies. Herein, we describe a multi-parallel-protease digestion strategy using a combination of five different proteases followed by high-resolution mass spectrometric analysis with TrastuzumAb and RituximAb as an example. This resulted in a comprehensive identification of peptides with increased reliability and identification of different PTMs. Additional supporting orthogonal methods like intact mass and higher-order structure analysis helped evaluate broader conformational properties.

Chemical Digestion of the -Asp-Cys- Sequence for Preparation of Post-translationally Modified Proteins.

Numerous studies of native proteins have been reported on protein folding in this half century. Recently, post-translationally modified proteins are also focused on protein folding. However, it is still difficult to prepare such types of proteins because it requires not only the chemical but also the recombinant techniques. Native chemical ligation (NCL) is a powerful technique for producing target proteins when combined with recombinant techniques, such as expressed protein ligation (EPL). NCL basically requires an N-terminal peptide with a thioester and a C-terminal peptide which should possess a Cys residue at the N-terminus. Numerous efforts have been made to prepare N-terminal peptides carrying a thioester or a derivative thereof. However, a method for preparing C-terminal Cys-peptides with post-translational modifications has not been well developed, making it difficult to prepare such C-terminal Cys-peptides, except for chemical syntheses or enzymatic digestion. We report here on the development of a convenient technique that involves acid hydrolysis at the -Asp-Cys- sequence, to effectively obtain a C-terminal peptide fragment that can be used for any protein synthesis when combined with EPL, even under denatured conditions. Thus, this chemical digestion strategy permits the NCL strategy to be dramatically accelerated for protein syntheses in which post-translational modifications, such as glycosylation, phosphorylation, etc. are involved. In addition, this method should be useful to prepare the post-translationally modified proteins for protein folding.

Low activities of digestive enzymes in the guts of herbivorous grouse (Aves: Tetraoninae)

Avian herbivores face the exceptional challenge of digesting recalcitrant plant material while under the selective pressure to reduce gut mass as an adaptation for flight. One mechanism by which avian herbivores may overcome this challenge is to maintain high activities of intestinal enzymes that facilitate the digestion and absorption of nutrients. However, previous studies in herbivorous animals provide equivocal evidence as to how activities of digestive enzymes may be adapted to herbivorous diets. For example, “rate-maximizing” herbivores generally exhibit rapid digesta transit times and high activities of digestive enzymes. Conversely, “yield-maximizing” herbivores utilize long gut retention times and express lower activities of digestive enzymes. Here, we investigated the activities of digestive enzymes (maltase, sucrase, aminopeptidase-N) in the guts of herbivorous grouse (Aves: Tetraoninae) and compared them to activities measured in several other avian species. We found that several grouse species exhibit activities of enzymes that are dramatically lower than those measured in other birds. We propose that grouse may use a “yield-maximizing” strategy of digestion, which is characterized by relatively long gut retention times and generally lower enzyme activities. These low activities of intestinal digestive enzyme could have ecological and evolutionary consequences, as grouse regularly consume plants with compounds known to inhibit digestive enzymes. However, more comprehensive studies on passage rates, digestibility, and microbial contributions will be necessary to understand the full process of digestion in herbivorous birds.

A specious relevance between theory and practice: Insights into temperature parameter and multi-phase strategy of anaerobic digestion of straw

Anaerobic digestion (AD) of straw is a highly complex and dynamic process. The temperature range of mesophilic (30–40 °C) and thermophilic (50–65 °C) are usually recommended in textbook notion. The two-phase strategy is usually applied based on the classical theory, including acidification-phase and methanation-phase. However, both the optimized temperature parameter and the enhanced multi-phase strategy solely focus on the local AD process. A specious relevance between theory and practice during AD process of straw has always been argued. Classical AD theory was not necessarily the sufficient approach to guide the anaerobic biological transformation of straw. More profound investigations of optimum temperature are still needed, uniquely synergistic mechanisms of functional microorganisms, as well as process stability, should be taken into account. Besides, additional research should focus on the matching between the physicochemical properties and process parameters/strategies choosing. A multi-stage operation strategy based on straw material composition is a potential operation approach to improve its efficiency. Furthermore, more comprehensive attention should be paid to the collaborative response mechanism by coupling substrate, temperature, and microbial in complex AD systems for straws.

Batch anaerobic co-digestion of food waste and sludge: a multi criteria decision modelling (MCDM) approach

The present study investigates the application of Multi Criteria Decision Modelling (MCDM) on batch anaerobic co-digestion of kitchen food waste (FW) and the fresh septic tank sludge (STS) for robust output. The batch experiment was carried out at a temperature of 37 ± 2 °C. Different ratio of FW and STS (FW: STS:100:0; 75:25; 60:40; 50:50; 25:75; 0:100) were adopted based on volatile solid (VS) basis as co digestion strategy for 90 days digestion period. Results revealed that experimentally, mono-digestion of FW showed maximum biogas yield (544 ± 65 mL/g VS) followed by blending ratio of 75:25 (FW: STS). However, mono-digestion of STS showed only 150 ± 15 mL/g VS of biogas yield and also showed negative effect on co digestion with FW for every blending on VS basis as STS was high in minerals. MCDM revealed that the FW is the best possible alternative amongst the other blends with STS considering pH, chemical oxygen demand, VS reduction, alkalinity, C/N and biogas yield as output.

Direct interspecies electron transfer in anaerobic digestion: Research and technological application

Anaerobic digestion (AD), an economical and eco-friendly strategy for renewable energy recovery, has been widely investigated and applied in the stabilization of various organic wastes. However, two major factors involved in the slow fermentation rate and low methane conversion efficiency still limit its large-scale applications around the world. Methane formation via conventional methanogenic pathway in AD undergoes the three stages: Hydrolysis/acidogenesis, acetogenesis and methanogenesis. The structure of some saccharide- and protein-like organic compounds contained in complex organic waste is relatively complex and stable, limiting the rate of converting them into amino acids, fatty acids and alcohols by fermentative bacteria. Therefore, the hydrolysis/acidogenesis is widely recognized as a rate-limiting step of AD in practical applications. On the other hand, the obligate anaerobic methanogens can only utilize methanol, methylamine, formate, acetate and hydrogen/carbon dioxide as substrates. Methane formation with amino acids, fatty acids and alcohols as substrates requires the essential conversion of them into acetate and hydrogen/carbon dioxide by acetogenic bacteria, and then depends on acetate- and hydrogen-utilizing methanogens to metabolize acetate and hydrogen/carbon dioxide into methane, respectively. This working mode of syntrophic metabolism of organic compounds into methane between acetogenic bacteria and methanogens that depends on hydrogen as an interspecies electron carrier has been widely described as Interspecies Hydrogen Transfer (IHT). Formate may be also served as a substitute for hydrogen as an interspecies electron carrier (Interspecies Formate Transfer, IFT). Syntrophic metabolism of organic compounds is strictly limited by the hydrogen partial pressure, since the oxidation of organic compounds into acetate with H+ as an electron acceptor is not feasible in thermodynamics ( E H + / H 2 = −0.414 V, pH 7). Syntrophic metabolism via IHT functions well as long as the hydrogen-utilizing methanogens continuously maintain the hydrogen partial pressure low enough (