Irritant-evoked activation and calcium modulation of the TRPA1 receptor.

The transient receptor potential ion channel TRPA1 is expressed by primary afferent nerve fibres, in which it functions as a low-threshold sensor for structurally diverse electrophilic irritants, including small volatile environmental toxicants and endogenous algogenic lipids1. TRPA1 is also a 'receptor-operated' channel whose activation downstream of metabotropic receptors elicits inflammatory pain or itch, making it an attractive target for novel analgesic therapies2. However, the mechanisms by which TRPA1 recognizes and responds to electrophiles or cytoplasmic second messengers remain unknown. Here we use strutural studies and electrophysiology to show that electrophiles act through a two-step process in which modification of a highly reactive cysteine residue (C621) promotes reorientation of a cytoplasmic loop to enhance nucleophilicity and modification of a nearby cysteine (C665), thereby stabilizing the loop in an activating configuration. These actions modulate two restrictions controlling ion permeation, including widening of the selectivity filter to enhance calcium permeability and opening of a canonical gate at the cytoplasmic end of the pore. We propose a model to explain functional coupling between electrophile action and these control points. We also characterize a calcium-binding pocket that is highly conserved across TRP channel subtypes and accounts for all aspects of calcium-dependent TRPA1 regulation, including potentiation, desensitization and activation by metabotropic receptors. These findings provide a structural framework for understanding how a broad-spectrum irritant receptor is controlled by endogenous and exogenous agents that elicit or exacerbate pain and itch.

Analgesic Effects of Vagus Nerve Stimulation on Visceral Hypersensitivity: A Direct Comparison Between Invasive and Noninvasive Methods in Rats.

OBJECTIVES: The aims of this study were to investigate analgesic effects of vagus nerve stimulation (VNS) on visceral hypersensitivity (VH) in a rodent model of functional dyspepsia (FD) and to compare invasive VNS with noninvasive auricular VNS (aVNS). MATERIALS AND METHODS: Eighteen ten-day-old male rats were gavaged with 0.1% iodoacetamide (IA) or 2% sucrose solution for six days. After eight weeks, IA-treated rats were implanted with electrodes for VNS or aVNS (n = 6 per group). Different parameters, varying in frequency and stimulation duty cycle, were tested to find the best parameter based on the improvement of VH assessed by electromyogram (EMG) during gastric distension. RESULTS: Compared with sucrose-treated rats, visceral sensitivity was increased significantly in IA-treated "FD" rats and ameliorated remarkably by VNS (at 40, 60, and 80 mm Hg; p ≤ 0.02, respectively) and aVNS (at 60 and 80 mm Hg; p ≤ 0.05, respectively) with the parameter of 100 Hz and 20% duty cycle. There was no significant difference in area under the curve of EMG responses between VNS and aVNS (at 60 and 80 mm Hg, both p > 0.05). Spectral analysis of heart rate variability revealed a significant enhancement in vagal efferent activity while applying VNS/aVNS compared with sham stimulation (p < 0.01). In the presence of atropine, no significant differences were noted in EMG after VNS/aVNS. Naloxone blocked the analgesic effects of VNS/aVNS. CONCLUSIONS: VNS/aVNS with optimized parameter elicits ameliorative effects on VH, mediated by autonomic and opioid mechanisms. aVNS is as effective as direct VNS and has great potential for treating visceral pain in patients with FD.

A Mass Spectrometry Strategy for Protein Quantification Based on the Differential Alkylation of Cysteines Using Iodoacetamide and Acrylamide.

Mass spectrometry has become the most prominent yet evolving technology in quantitative proteomics. Today, a number of label-free and label-based approaches are available for the relative and absolute quantification of proteins and peptides. However, the label-based methods rely solely on the employment of stable isotopes, which are expensive and often limited in availability. Here we propose a label-based quantification strategy, where the mass difference is identified by the differential alkylation of cysteines using iodoacetamide and acrylamide. The alkylation reactions were performed under identical experimental conditions; therefore, the method can be easily integrated into standard proteomic workflows. Using high-resolution mass spectrometry, the feasibility of this approach was assessed with a set of tryptic peptides of human serum albumin. Several critical questions, such as the efficiency of labeling and the effect of the differential alkylation on the peptide retention and fragmentation, were addressed. The concentration of the quality control samples calculated against the calibration curves were within the ±20% acceptance range. It was also demonstrated that heavy labeled peptides exhibit a similar extraction recovery and matrix effect to light ones. Consequently, the approach presented here may be a viable and cost-effective alternative of stable isotope labeling strategies for the quantification of cysteine-containing proteins.

A conserved, buried cysteine near the P-site is accessible to cysteine modifications and increases ROS stability in the P-type plasma membrane H+-ATPase.

Sulfur-containing amino acid residues function in antioxidative responses, which can be induced by the reactive oxygen species generated by excessive copper and hydrogen peroxide. In all Na+/K+, Ca2+, and H+ pumping P-type ATPases, a cysteine residue is present two residues upstream of the essential aspartate residue, which is obligatorily phosphorylated in each catalytic cycle. Despite its conservation, the function of this cysteine residue was hitherto unknown. In this study, we analyzed the function of the corresponding cysteine residue (Cys-327) in the autoinhibited plasma membrane H+-ATPase isoform 2 (AHA2) from Arabidopsis thaliana by mutagenesis and heterologous expression in a yeast host. Enzyme kinetics of alanine, serine, and leucine substitutions were identical with those of the wild-type pump but the sensitivity of the mutant pumps was increased towards copper and hydrogen peroxide. Peptide identification and sequencing by mass spectrometry demonstrated that Cys-327 was prone to oxidation. These data suggest that Cys-327 functions as a protective residue in the plasma membrane H+-ATPase, and possibly in other P-type ATPases as well.

Insights on Aggregation of Hen Egg-White Lysozyme from Raman Spectroscopy and MD Simulations.

Protein misfolding and aggregation play a significant role in several neurodegenerative diseases. In the present work, the spontaneous aggregation of hen egg-white lysozyme (HEWL) in an alkaline pH 12.2 at an ambient temperature was studied to obtain molecular insights. The time-dependent changes in spectral peaks indicated the formation of ß sheets and their effects on the backbone and amino acids during the aggregation process. Introducing iodoacetamide revealed the crucial role of intermolecular disulphide bonds amidst monomers in the aggregation process. These findings were corroborated by Molecular Dynamics (MD) simulations and protein-docking studies. MD simulations helped establish and visualize the unfolding of the proteins when exposed to an alkaline pH. Protein docking revealed a preferential dimer formation between the HEWL monomers at pH 12.2 compared with the neutral pH. The combination of Raman spectroscopy and MD simulations is a powerful tool to study protein aggregation mechanisms.

Commonly Used Alkylating Agents Limit Persulfide Detection by Converting Protein Persulfides into Thioethers.

Protein persulfides (R-S-SH) have emerged as a common post-translational modification. Detection and quantitation of protein persulfides requires trapping with alkylating agents. Here we show that alkylating agents differ dramatically in their ability to conserve the persulfide's sulfur-sulfur bond for subsequent detection by mass spectrometry. The two alkylating agents most commonly used in cell biology and biochemistry, N-ethylmaleimide and iodoacetamide, are found to be unsuitable for the purpose of conserving persulfides under biologically relevant conditions. The resulting persulfide adducts (R-S-S-Alk) rapidly convert into the corresponding thioethers (R-S-Alk) by donating sulfur to ambient nucleophilic acceptors. In contrast, certain other alkylating agents, in particular monobromobimane and N-t-butyl-iodoacetamide, generate stable alkylated persulfides. We propose that the nature of the alkylating agent determines the ability of the disulfide bond (R-S-S-Alk) to tautomerize into the thiosulfoxide (R-(S=S)-Alk), and/or the ability of nucleophiles to remove the sulfane sulfur atom from the thiosulfoxide.

A detailed mapping of the readily accessible disulphide bonds in the cortex of wool fibres.

Trichocyte keratin intermediate filament proteins (keratins) and keratin associated proteins (KAPs) differ from their epithelial equivalents by having significantly more cysteine residues. Interactions between these cysteine residues within a mammalian fiber, and the putative regular organization of interactions are likely important for defining fiber mechanical properties, and thus biological functionality of hairs. Here we extend a previous study of cysteine accessibility under different levels of exposure to reducing compounds to detect a greater resolution of statistically non-random interactions between individual residues from keratins and KAPs. We found that most of the cysteines with this non-random accessibility in the KAPs were close to either the N- or C- terminal domains of these proteins. The most accessible non-random cysteines in keratins were present in the head or tail domains, indicating the likely function of cysteine residues in these regions is in readily forming intermolecular bonds with KAPs. Some of the less accessible non-random cysteines in keratins were discovered either close to or within the rod region in positions previously identified in human epithelial keratins as involved in crosslinking between the heterodimers of the tetramer. Our present study therefore provides a deeper understanding of the accessibility of disulfides in both keratins and KAPs and thus proves that there is some specificity to the disulfide bond interactions leading to these inter- and intra-molecular bonds stabilizing the fiber structure. Furthermore, these suggest potential sites of interaction between keratins and KAPs as well as keratin-keratin interactions in the trichocyte intermediate filament.

SP3-FAIMS Chemoproteomics for High-Coverage Profiling of the Human Cysteinome*.

Chemoproteomics has enabled the rapid and proteome-wide discovery of functional, redox-sensitive, and ligandable cysteine residues. Despite widespread adoption and considerable advances in both sample-preparation workflows and MS instrumentation, chemoproteomics experiments still typically only identify a small fraction of all cysteines encoded by the human genome. Here, we develop an optimized sample-preparation workflow that combines enhanced peptide labeling with single-pot, solid-phase-enhanced sample-preparation (SP3) to improve the recovery of biotinylated peptides, even from small sample sizes. By combining this improved workflow with on-line high-field asymmetric waveform ion mobility spectrometry (FAIMS) separation of labeled peptides, we achieve unprecedented coverage of >14000 unique cysteines in a single-shot 70 min experiment. Showcasing the wide utility of the SP3-FAIMS chemoproteomic method, we find that it is also compatible with competitive small-molecule screening by isotopic tandem orthogonal proteolysis-activity-based protein profiling (isoTOP-ABPP). In aggregate, our analysis of 18 samples from seven cell lines identified 34225 unique cysteines using only ∼28 h of instrument time. The comprehensive spectral library and improved coverage provided by the SP3-FAIMS chemoproteomics method will provide the technical foundation for future studies aimed at deciphering the functions and druggability of the human cysteineome.

Optimization of cysteine residue alkylation using an on-line LC-MS strategy: Benefits of using a cocktail of haloacetamide reagents.

Several common reagents for the alkylation of cysteine residues of model intact proteins were evaluated for reaction speed, yield of alkylated product and degree of over-alkylation using an online LC-MS platform. The efficiency of the alkylation reaction is found to be dependent on the (1) reagent, (2) peptide/protein, (3) reagent concentration and (4) reaction time. At high reagent concentrations, iodoacetic acid was found to produce significant levels of over-alkylation products wherein methionine residues become modified. For optimal performance of the alkylation reaction, we found the use of a cocktail of chloroacetamide, bromoacetamide and iodoacetamide worked best. The alkylating efficiency of each haloacetamide is a balance between the characteristics of the halogen leaving group and the steric hindrance of the alkylation site on the peptide or protein. A key aspect of using a cocktail of haloacetamides is that they all produce the same modification (+57.0209 Da) to the cysteine residues of the protein while the alkylation efficiency of each site may differ for each of the three reagents. Over-alkylation effects appear to be lower with the cocktail due to a lower concentration of each reagent. The haloacetamide cocktail could be useful when considering complex mixtures of proteins.

Chemical Tagging of Protein Lipoylation.

Protein lipoylation is a post-translational modification of emerging importance in both prokaryotes and eukaryotes. However, labeling and large-scale profiling of protein lipoylation remain challenging. Here, we report the development of iLCL (iodoacetamide-assisted lipoate-cyclooctyne ligation), a chemoselective reaction that enables chemical tagging of protein lipoylation. We demonstrate that the cyclic disulfide of lipoamide but not linear disulfides can selectively react with iodoacetamide to produce sulfenic acid, which can be conjugated with cyclooctyne probes. iLCL enables tagging of lipoylated proteins for gel-based detection and cellular imaging. Furthermore, we apply iLCL for proteomic profiling of lipoylated proteins in both bacteria and mammalian cells. In addition to all of the eight known lipoylated proteins, we identified seven candidates for novel lipoylated proteins. The iLCL strategy should facilitate uncovering the biological function of protein lipoylation.

Design and Evaluation of Artificial Hybrid Photoredox Biocatalysts.

A pair of 9-mesityl-10-phenyl acridinium (Mes-Acr+ ) photoredox catalysts were synthesized with an iodoacetamide handle for cysteine bioconjugation. Covalently tethering of the synthetic Mes-Acr+ cofactors with a small panel of thermostable protein scaffolds resulted in 12 new artificial enzymes. The unique chemical and structural environment of the protein hosts had a measurable effect on the photophysical properties and photocatalytic activity of the cofactors. The constructed Mes-Acr+ hybrid enzymes were found to be active photoinduced electron-transfer catalysts, controllably oxidizing a variety of aryl sulfides when irradiated with visible light, and possessed activities that correlated with the photophysical characterization data. Their catalytic performance was found to depend on multiple factors including the Mes-Acr+ cofactor, the protein scaffold, the location of cofactor immobilization, and the substrate. This work provides a framework toward adapting synthetic photoredox catalysts into artificial cofactors and includes important considerations for future bioengineering efforts.

Effects of electroacupuncture on stress-induced gastric dysrhythmia and mechanisms involving autonomic and central nervous systems in functional dyspepsia.

Electroacupuncture (EA) is widely used as an effective method to treat stress-related disorders. However, its mechanisms remain largely unknown. The aim of this study was to investigate the effects and mechanisms of EA on gastric slow wave (GSW) dysrhythmia and c-Fos expression in the nucleus of the solitary tract (NTS) induced by stress in a rodent model of functional dyspepsia (FD). Rats in the neonatal stage were treated using intragastric iodoacetamide. Eight weeks later, the rats were implanted with electrodes in the stomach for the measurement of GSW and electrodes into accupoints ST36 for EA. Autonomic functions were assessed by spectral analysis of heart rate variability. Rats were placed for 30 min in a cylindrical plastic tube for acute restraint stress. The involvement of a central afferent pathway was assessed by measuring c-Fos-immunoreactive cells in the NTS. 1) EA normalized restraint stress-induced impairment of GSW in FD rats. 2) EA significantly increased vagal activity (P = 0.002) and improved sympathovagal balance (P = 0.004) under stress in FD rats. 3) In FD rats under restraint stress, plasma norepinephrine concentration was increased substantially (P < 0.01), which was suppressed with EA. 4) The EA group showed increased c-Fos-positive cell counts in the NTS compared with the sham EA group (P < 0.05) in FD rats. Acute restraint stress induces gastric dysrhythmia in a rodent model of FD. EA at ST36 improves GSW under stress in FD rats mediated via the central and autonomic pathways, involving the NTS and vagal efferent pathway.

Mechanistic Studies of Cytochrome P450 3A4 Time-Dependent Inhibition Using Two Cysteine-Targeting Electrophiles.

Experiments designed to identify the mechanism of cytochrome P450 inactivation are critical to drug discovery. Small molecules irreversibly inhibit P450 enzymatic activity via two primary mechanisms: apoprotein adduct formation or heme modification. Understanding the interplay between chemical structures of reactive electrophiles and the impact on CYP3A4 structure and function can ultimately provide insights into drug design to minimize P450 inactivation. In a previous study, raloxifene and N-(1-pyrene) iodoacetamide (PIA) alkylated CYP3A4 in vitro; however, only raloxifene influenced enzyme activity. Here, two alkylating agents with cysteine selectivity, PIA and pyrene maleimide (PM), were used to investigate this apparent compound-dependent disconnect between CYP3A4 protein alkylation and activity loss. The compound's effect on 1) enzymatic activity, 2) carbon monoxide (CO) binding capacity, 3) intact heme content, and 4) protein conformation were measured. Results showed that PM had a large time-dependent loss of enzyme activity, whereas PIA did not. The differential effect on enzymatic activity between PM and PIA was mirrored in the CO binding data. Despite disruption of CO binding, neither compound affected the heme concentrations, inferring there was no destruction or alkylation of the heme. Lastly, differential scanning fluorescence showed PM-treated CYP3A4 caused a shift in the onset temperature required to induce protein aggregation, which was not observed for CYP3A4 treated with PIA. In conclusion, alkylation of CYP3A4 apoprotein can have a variable impact on catalytic activity, CO binding, and protein conformation that may be compound-dependent. These results highlight the need for careful interpretation of experimental results aimed at characterizing the nature of P450 enzyme inactivation. SIGNIFICANCE STATEMENT: Understanding the mechanism of CYP3A4 time-dependent inhibition is critical to drug discovery. In this study, we use two cysteine-targeting electrophiles to probe how subtle variation in inhibitor structure may impact the mechanism of CYP3A4 time-dependent inhibition and confound interpretation of traditional diagnostic experiments. Ultimately, this simplified system was used to reveal insights into CYP3A4 biochemical behavior. The insights may have implications that aid in understanding the susceptibility of CYP enzymes to the effects of electrophilic intermediates generated via bioactivation.

A lanthipeptide library used to identify a protein-protein interaction inhibitor.

In this article we describe the production and screening of a genetically encoded library of 106 lanthipeptides in Escherichia coli using the substrate-tolerant lanthipeptide synthetase ProcM. This plasmid-encoded library was combined with a bacterial reverse two-hybrid system for the interaction of the HIV p6 protein with the UEV domain of the human TSG101 protein, which is a critical protein-protein interaction for HIV budding from infected cells. Using this approach, we identified an inhibitor of this interaction from the lanthipeptide library, whose activity was verified in vitro and in cell-based virus-like particle-budding assays. Given the variety of lanthipeptide backbone scaffolds that may be produced with ProcM, this method may be used for the generation of genetically encoded libraries of natural product-like lanthipeptides containing substantial structural diversity. Such libraries may be combined with any cell-based assay to identify lanthipeptides with new biological activities.

The synthesis of non-structural carbohydrates under the network security model.

In order to explore the pathway of non-structural carbohydrate synthesis, an analysis of the pathway of non-structural carbohydrate synthesis under the network security model was proposed. Taking non-structural carbohydrates as the research object, the experimental materials and equipment were selected under the network security model. Through the establishment of detection methods, the preparation of freeze-dried carbohydrates, the influence of synthesis pathway-specific inhibitors on the synthesis of non-structural carbohydrates, the influence of precursors and intermediates in the pathway on the synthesis of non-structural carbohydrates, the effect of multiple factors on the synthesis of non-structural carbohydrates, the influence of polysaccharide synthesis, the treatment of reaction solution for detection, the preparation of detection sample, the detection conditions of a liquid phase, the detection conditions of LC-MS and the determination of carbohydrate biomass were studied. The results showed that the synthesis of nonstructural carbohydrates requires the participation of the glycolysis, shikimic acid and phenylpropane pathways, but not the polyketone pathway.

Electroacupuncture Ameliorates Gastric Hypersensitivity via Adrenergic Pathway in a Rat Model of Functional Dyspepsia.

OBJECTIVE: Electroacupuncture (EA) is effective in treating visceral pain associated with functional dyspepsia (FD). The aim of this study was to explore the effect of chronic EA (CEA) on gastric hypersensitivity and the involvement of sympathetic nervous system in a rodent model of FD. MATERIALS AND METHODS: Gastric hypersensitivity in adulthood was induced by iodoacetamide (IA) in neonatal rats. The IA-treated rats were randomized to receive no treatment (control), sham-CEA, CEA, or adrenergic antagonists, for one week. Gastric sensitivity to graded gastric distensions was then assessed by electromyogram (EMG) analysis. Autonomic functions were assessed from the spectral analysis of heart rate variability (HRV) to derive the low-frequency (LF, sympathetic activity) and high-frequency (HF, mainly vagal activity) components expressed as percentage of total spectral power. Blood was collected for the measurement of corticosterone (CORT) and norepinephrine (NE). RESULTS: 1) CEA, but not sham-CEA, reduced the EMG response to graded gastric distension in IA-treated control rats at 40 mmHg (128 ± 6% vs. 171 ± 15%, p = 0.009), 60 mmHg (204 ± 14% vs. 271 ± 24%, p = 0.010) and 80 mmHg (269 ± 19% vs. 364 ± 33%, p = 0.025), respectively. 2) CEA, but not sham CEA, increased HF component (0.61 ± 0.02 vs. 0.46 ± 0.04 in IA-treated rats, p = 0.003) and decreased LF component (0.39 ± 0.02 vs. 0.54 ± 0.04, p = 0.003). 3) Adrenergic antagonists reduced the EMG response to graded gastric distension. 4) CEA significantly reduced plasma CORT and NE in IA-treated rats. CONCLUSIONS: EA ameliorates gastric hypersensitivity in IA-treated rats and the effect may be related to the improved sympathovagal balance and the decrease of stress hormones.

Upregulation of the TRPA1 Ion Channel in the Gastric Mucosa after Iodoacetamide-Induced Gastritis in Rats: A Potential New Therapeutic Target.

Acute gastritis is often untreatable by acid secretion-inhibiting drugs. Understanding the protective mechanisms including the role of Transient Receptor Potential Ankyrin1 (TRPA1) and Vanilloid1 (TRPV1) channels localized on capsaicin-sensitive afferents and non-neuronal structures might identify novel therapeutic approaches. Therefore, we characterized a translational gastritis model using iodoacetamide (IAA) and investigated TRPA1/V1 expressions. Wistar rats and CD1, C57Bl/6J mice were exposed to IAA-containing (0.05, 0.1, 0.2, 0.3, 0.5%) drinking water for 7 or 14 days. Body weight and water consumption were recorded daily. Macroscopic lesions were scored, qualitative histopathologic investigation was performed, TRPA1/V1 immunopositivity and mRNA expressions were measured. IAA induced a concentration-dependent weight loss and reduced water intake in both species. Hyperemia, submucosal edema, inflammatory infiltration and hemorrhagic erosions developed after 7 days, while ulcers after 14 days in rats. Trpa1 mRNA/protein expressions were upregulated at both timepoints. Meanwhile, TRPV1 immunopositivity was upregulated in the gastric corpus after 0.05% IAA ingestion, but downregulated after 0.2%, whereas Trpv1 mRNA did not change. Interestingly, no macroscopic/microscopic changes were observed in mice. These are the first data for the concentration- and duration-dependent changes in the IAA-induced gastritis in rats accompanied by TRPA1 upregulation, therefore, its therapeutic potential in gastritis should further be investigated.

Major cause of antibody artifact bands on non-reducing SDS-PAGE and methods for minimizing artifacts.

When monoclonal antibodies (mAbs) are analysed by non-reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), method-induced artifacts are a frequent phenomenon. Previous studies suggested that incomplete denaturation and disulfide-bond scrambling are two main causes of artifact bands. Thus, in practice samples are normally heated and treated with alkylating agent iodoacetamide (IAM) before loading to promote denaturation and block free sulfhydryl groups, respectively. In this work, we further studied the major cause of artifact bands on non-reducing SDS-PAGE and ways of eliminating artifacts with two purified mAbs. In both cases, it was found that artifact bands on non-gradient Tris-glycine gels are mainly caused by incomplete denaturation under typical gel conditions. In general, heating minimizes artifact bands due to incomplete denaturation but it also generates some extra bands. Combining heating with IAM treatment achieved slightly better results than heating alone. As an alternative to heating, treating the samples with 8 M urea also allows close to complete denaturation of samples and thus minimizes artifact bands. In addition, we learned that untreated samples (samples that are not heated or treated with urea) may look different on Bis-Tris gel depending on gel composition (non-gradient vs. gradient) and the buffer used (MES vs. MOPS). In certain case, the apparent lack of artifact bands on gradient Bis-Tris gel may be in fact due to insufficient resolution. In conclusion, this study further confirmed that full-denaturation of sample is critical for minimizing/avoiding artifact bands on non-reducing SDS-PAGE.

Specific inhibitors of lysozyme and peptidases inhibit the growth of the rumen protozoan Entodinium caudatum without decreasing feed digestion or fermentation in vitro.

AIMS: Experiments were designed to determine the effects of different chemical inhibitors of lysozyme and peptidases on rumen protozoa and the associated prokaryotes, and in vitro fermentation using Entodinium caudatum as a model protozoan species. METHODS AND RESULTS: Imidazole (a lysozyme inhibitor), phenylmethylsulphonyl fluoride (PMSF, a serine peptidase inhibitor) and iodoacetamide (IOD, a cysteine peptidase inhibitor) were evaluated in vitro both individually and in two- and three-way combinations using E. caudatum monocultures with respect to their ability to inhibit the protozoan and their effect on feed digestion, fermentation and the microbiota. All the three inhibitors, both individually and in combination, decreased E. caudatum counts (P < 0·001), and IOD and its combinations with the other inhibitors significantly (P < 0·01) decreased ammonia concentration, with the two- and three-way combinations showing additive effective. Feed digestion was not affected, but fermentation and microbial diversity were affected mostly by PMSF, IOD and their combinatorial treatments potentially due to the overgrowth of Streptococcus luteciae accompanying with the disappearance of host ciliates. CONCLUSIONS: Entodinium caudatum depends on lysozyme and peptidase for digestion and utilization of the engulfed microbes and specific inhibition of these enzymes can inhibition E. caudatum without adversely affecting feed digestion or fermentation even though they changed the microbiota composition in the cultures. SIGNIFICANCE AND IMPACT OF THE STUDY: The peptidase inhibitors may have the potential to be used in controlling rumen protozoa to improve ruminal nitrogen utilization efficiency.

Cysteine-Selective Phosphonamidate Electrophiles for Modular Protein Bioconjugations.

We describe a new technique in protein synthesis that extends the existing repertoire of methods for protein modification: A chemoselective reaction that induces reactivity for a subsequent bioconjugation. An azide-modified building block reacts first with an ethynylphosphonite through a Staudinger-phosphonite reaction (SPhR) to give an ethynylphosphonamidate. The resulting electron-deficient triple bond subsequently undergoes a cysteine-selective reaction with proteins or antibodies. We demonstrate that ethynylphosphonamidates display excellent cysteine-selective reactivity combined with superior stability of the thiol adducts, when compared to classical maleimide linkages. This turns our technique into a versatile and powerful tool for the facile construction of stable functional protein conjugates.