Chemical Identification of MHC-influenced Volatile Compounds in Mouse Urine. I: Quantitative Proportions of Major Chemosignals

The genes of the major histocompatibility complex (MHC) are highly polymorphic loci that encode cell surface proteins, class I and II molecules. They present peptide antigens to T cells and thereby control immunological self/nonself recognition. Increasing evidence indicates that MHC genes also influence odor and mating preferences; however, it is unclear how. Here we report the results of chemical analyses of male mouse urinary odors collected from a variety of mouse strains, including MHC-congenics, recombinants, mutants, and transgenics (i.e., beta2 microglobulin "knockouts," which lack class I expression, and transporters associated with antigen processing (TAP) knock-outs). After the identification of volatile odor components by gas chromatography/mass spectrometry, the odor profiles of urine samples were analyzed quantitatively by using stir bar sorptive extraction and gas chromatography/atomic emission detection. Results showed that MHC genes influenced the amounts of testosterone-mediated pheromones, sulfur-containing compounds, and several carbonyl metabolites. This is the first report to quantitatively link known mouse pheromones to classical, antigen-binding MHC loci. Surprisingly, these compounds were not influenced by TAP genes, even though these loci are MHC-linked and play a role in peptide presentation. Whereas identification of MHC-determined odorants does not reveal their metabolic origin, some constituents were also present in blood serum, and their levels were not altered by antibiotics.     

Nootkatone Is a Repellent for Formosan Subterranean Termite (Coptotermes formosanus)

We examined the behavior of Formosan subterranean termites toward one of the components of vetiver grass oil, the roots of which manufacture insect repellents. We found nootkatone, a sesquiterpene ketone, isolated from vetiver oil is a strong repellent and toxicant to Formosan subterranean termites. The lowest effective concentration tested was 10 g/g substrate. This is the first report of nootkatone being a repellent to insects.     

Lignoceric Acid and Hexacosanoic Acid: Major Components of Soldier Frontal Gland Secretions of the Formosan Subterranean Termite (Coptotermes formosanus)

Qualitative and quantitative analyses were performed on trimethylsilyl derivatives of soldier frontal gland secretions of the Formosan subterranean termite, Coptotermes formosanus Shiraki, by using gas chromatography–mass spectrometry. Ranges of 17.23–33.78 mg/g (acid/wet weight of secretion) of lignoceric acid and 5.39–7.67 mg/g of hexacosanoic acid were found in the soldier frontal gland secretion of three different colonies. This is the first report that organic acids occur in the soldier secretions of this species.     

No Major Role for Binding by Salivary Proteins as a Defense Against Dietary Tannins in Mediterranean Goats

We investigated whether Mediterranean goats use salivary tannin-binding proteins to cope with tannin-rich forages by determining the affinity of salivary or parotid gland proteins for tannic acid or quebracho tannin. Mixed saliva, sampled from the oral cavity, or parotid gland contents were compared to the intermediate affinity protein bovine serum albumin with a competitive binding assay. Goats that consume tannin-rich browse (Damascus) and goats that tend to avoid tannins (Mamber) were sequentially fed high (Pistacia lentiscus L.), low (vetch hay), or zero (wheat hay) tannin forages. Affinity of salivary proteins for tannins did not differ between goat breeds and did not respond to presence or absence of tannins in the diet. Proteins in mixed saliva had slightly higher affinity for tannins than those in parotid saliva, but neither source contained proteins with higher affinity for tannins than bovine serum albumin. Similarly, 3 months of browsing in a tannin-rich environment had little effect on the affinity of salivary proteins for tannin in adult goats of either breed. We sampled mixed saliva from young kids before they consumed forage and after 3 months of foraging in a tannin-rich environment. Before foraging, the saliva of Mamber kids had higher affinity for tannic acid (but not quebracho tannin) than the saliva of Damascus kids, but there was no difference after 3 months of exposure to tannin-rich browse, and the affinity of the proteins was always similar to the affinity of bovine serum albumin. Our results suggest there is not a major role for salivary tannin-binding proteins in goats. Different tendencies of goat breeds to consume tannin-rich browse does not appear be related to differences in salivary tannin-binding proteins.     

Stimulation of Estrus in Female Mice by Male Urinary Proteins

Stimulation of estrus in adult female mice was obtained with major urinary proteins (MUPs) with the natural volatile ligands bound. The MUP threshold concentration for this effect was about 1.8 mg/ml. MUPs without the ligands bound, as purified by organic extraction of hydrophobic compounds, stimulated estrus in mice only when dissolved in carrier urine of juvenile or castrated adult male mouse or ovariectomized female mouse. They did not stimulate estrus when dissolved in water. Mice that had the vomeronasal organ removed were insensitive to MUPs. It is concluded that MUPs are an integral part of the mouse male pheromones that stimulate hormonal activity in females and that the vomeronasal system is involved in the estrus-stimulating effect of the major urinary proteins.     

One Solution for All: Searching for Universal Aptamers for Constantly Mutating Spike Proteins of SARS-CoV-2

Aptamers that can recognize the spike (S) protein of SARS-CoV-2 with high affinity and specificity are useful molecules towards the development of diagnostics and therapeutics to fight COVID-19. However, this S protein is constantly mutating, producing variants of concern (VoCs) that can significantly weaken the binding by aptamers initially engineered to recognize the S protein of the wildtype virus or a specific VoC. One strategy to overcome this problem is to develop universal aptamers that are insensitive to all or most of the naturally emerging mutations in the protein. We have recently demonstrated this concept by subjecting a pool of S protein-binding DNA aptamers for one-round parallel-SELEX experiments targeting 5 different S protein variants for binding-based sequence enrichment, followed by bioinformatic analysis of the enriched pools. This effort has led to the identification of a universal aptamer that recognizes 8 different variants of the spike protein with equally excellent affinity.     

An Improved Protocol for Agrobacterium-Mediated Transformation in Subterranean Clover (Trifolium subterraneum L.)

Subterranean clover (Trifolium subterraneum) is the most widely grown annual pasture legume in southern Australia. With the advent of advanced sequencing and genome editing technologies, a simple and efficient gene transfer protocol mediated by Agrobacterium tumefaciens was developed to overcome the hurdle of genetic manipulation in subterranean clover. In vitro tissue culture and Agrobacterium transformation play a central role in testing the link between specific genes and agronomic traits. In this paper, we investigate a variety of factors affecting the transformation in subterranean clover to increase the transformation efficiency. In vitro culture was optimised by including cefotaxime during seed sterilisation and testing the best antibiotic concentration to select recombinant explants. The concentrations for the combination of antibiotics obtained were as follows: 40 mg L⁻¹ hygromycin, 100 mg L⁻¹ kanamycin and 200 mg L⁻¹ cefotaxime. Additionally, 200 mg L⁻¹ cefotaxime increased shoot regeneration by two-fold. Different plant hormone combinations were tested to analyse the best rooting media. Roots were obtained in a medium supplemented with 1.2 µM IAA. Plasmid pH35 containing a hygromycin-resistant gene and GUS gene was inoculated into the explants with Agrobacterium tumefaciens strain AGL0 for transformation. Overall, the transformation efficiency was improved from the 1% previously reported to 5.2%, tested at explant level with Cefotaxime showing a positive effect on shooting regeneration. Other variables in addition to antibiotic and hormone combinations such as bacterial OD, time of infection and incubation temperature may be further tested to enhance the transformation even more. This improved transformation study presents an opportunity to increase the feeding value, persistence, and nutritive value of the key Australian pasture.     

Circulating miRNAs Act as Diagnostic Biomarkers for Bladder Cancer in Urine

MicroRNAs (miRNAs) can be secreted into body fluids and have thus been reported as a new type of cancer biomarker. This study aimed to determine whether urinary miRNAs act as noninvasive biomarkers for diagnosing bladder cancer. Small RNA profiles from urine were generated for 10 patients with bladder cancer and 10 healthy controls by using next-generation sequencing. We identified 50 urinary miRNAs that were differentially expressed in bladder cancer compared with controls, comprising 44 upregulated and six downregulated miRNAs. Pathway enrichment analysis revealed that the biological role of these differentially expressed miRNAs might be involved in cancer-associated signaling pathways. Further analysis of the public database revealed that let-7b-5p, miR-149-5p, miR-146a-5p, miR-193a-5p, and miR-423-5p were significantly increased in bladder cancer compared with corresponding adjacent normal tissues. Furthermore, high miR-149-5p and miR-193a-5p expression was significantly correlated with poor overall survival in patients with bladder cancer. The qRT-PCR approach revealed that the expression levels of let-7b-5p, miR-149-5p, miR-146a-5p and miR-423-5p were significantly increased in the urine of patients with bladder cancer compared with those of controls. Although our results indicated that urinary miRNAs are promising biomarkers for diagnosing bladder cancer, this must be validated in larger cohorts in the future.     

The Level of Major Urinary Proteins is Socially Regulated in Wild <Emphasis Type="Italic">Mus musculus musculus</Emphasis>

Major urinary proteins (MUPs) are highly polymorphic proteins that have been shown to perform several important functions in the chemical communication of the house mouse, Mus musculus. Production of these proteins in C57Bl/6 females is cyclic, reaching the maximum just before the beginning of estrus. Social environment is an important factor that increases MUP production in both sexes. We examined responsiveness of MUP production to social stimuli in wild mice, Mus musculus musculus. The direction of change of MUP production in males depended on the sex of the stimulus animal. Males up-regulated MUP production when caged with a female, but down-regulated MUP production when caged with a male. Down-regulation was more pronounced in males that were defeated in a male–male encounter. Females responded to a male’s presence with a decrease in MUP production. We conclude that social modulation of MUP production is specific and, in coordination with other mechanisms, facilitates adjustment of the animal’s odor profile to different social contexts. Our results also suggest that in males, MUPs may play an important role in advertizing the male’s quality to females. Furthermore, we highlight the importance of analyzing data corrected with creatinine, which show MUP production on the (post)translational level as well as raw data (non-corrected with creatinine), which represent actual concentrations of MUPs in the urine.     

Searching for ways to create energetic materials based on polynitrogen compounds (review)

Polynitrogen compounds (containing only nitrogen atoms) are promising candidates as energetic materials for rocket engineering. The high energy content of these compounds is due to the significant difference in bond energy between nitrogen atoms. In particular, molecular nitrogen (N₂) is characterized by a uniquely strong triple bond — 229 kcal/mole, whereas the single-bond energy is only 38.4 kcal/mole. From theoretical estimates, use of polynitrogen compounds can provide a specific impulse of 350–500 sec with material density in a range of 2.0–3.9 g/cm³. This paper gives a brief review of the current status of experimental and theoretical studies in the chemistry of polynitrogen compounds.     

Identification of trail pheromone precursors from Subterranean termite,Coptotermes formosanus Shiraki (Isoptera: Rhinotermitidae)

Whole-body extracts of the termite,Coptotermes formosanus Shiraki served for examining the presence of trail pheromone precursor(s). Three trail pheromone precursor candidates, identified as dodecatrienyl stearate, dodecatrienyl oleate, and dodecatrienyl linoleate, were isolated using various chromatographic methods in conjunction with bioassay and by capillary GC-MS analyses.     

Modified Polyethersulfone (PES) Ultrafiltration Membranes for Enhanced Filtration of Whey Proteins

Abstract

Application of membrane technology to whey protein separation is an interesting development that has seen growth in recent years. In particular, modification of existing membranes to impart charge properties on the membrane surface or in the pores has been shown to improve membrane selectivity, product purity, and throughput of protein solutions. This paper focuses on exploring the effects of membrane charge and solution pH on filtration of the major whey proteins α‐lactalbumin (14.1 kDa) and β‐lactoglobulin (18.4 kDa) using functionalized PES membranes. The membranes have an open pore structure containing charged sulfonated grafted polymer chains that allows for greater protein retention. The modified membranes were synthesized by polymerization of styrene in the membrane pores followed by sulfuric acid treatment of the resulting polystyrene grafts. The charged membrane gave a calculated selectivity of five times better than the raw membrane at pH 7.2 based on data from single protein transmission experiments. The enhanced selectivity of the tailor‐made membrane was due to increased retention of β‐lactoglobulin due to a reduction in molecular sieving combined with electrostatic repulsion between negatively charged β‐lactoglobulin and the negatively charged membrane.     

Use of Chemical Communication by the Subterranean Rodent Ctenomys talarum (Tuco-Tuco) During the Breeding Season

Solitary subterranean rodents with a low frequency of direct contact between conspecifics are expected to use chemical communication to coordinate social and reproductive behavior. We examined whether reproductive tuco-tucos (Ctenomys talarum) were able to discriminate the reproductive condition, sex, and source population of conspecifics by means of chemical cues contained in urine, feces, soiled shavings, or anogenital secretions. During preference tests in which animals had direct contact with these chemical cues, tuco-tucos were able to determine the reproductive condition of opposite sex conspecifics independent of the source of odor. When only olfactory cues were available, both sexes discriminated reproductive condition of opposite sex individuals using urine. Females were also able to discriminate the reproductive condition of males using soiled shavings. Females spent more time investigating male odors than female odors; except in the case of feces, breeding males spent similar amounts of time investigating male and female odors. No preferences were detected for opposite sex urine from members of an animal's own versus another population. The role of chemical cues in territory defense and breeding performance by this highly territorial subterranean rodent is discussed.     

Isoelectric Split-Flow Thin (SPLITT) Fractionation of Proteins

Abstract

Electric split-flow thin (SPLITT) fractionation permits the continuous separation of charged species, particularly proteins, at gram and subgram levels. We characterized this system and separated protein mixtures based on the difference between protein isoelectric points (pI). For characterization, we examined seven variables. Buffer stability was determined by measuring pH changes per hour and the UV spectrum before and after an electrical potential of 50 V was applied. The electrical field across the channel was determined by measuring the buffer conductivity and the current passed through it. The experimental and theoretical (calculated) fractional retrieval of proteins was determined by the relative magnitude of field-induced and outlet flow rates. The protein response at various electrical fields (0, 10, 20, 30 V) and solution pHs (4.85, 5.60, 6.87, and 7.80) was examined, as were the effects of the ionic strength of the buffer, protein recovery, and protein separation with pulsed sample injection. To separate protein mixtures after the system was characterized, we ran continuous SPLITT fractionation of five protein mixtures for more than 8 hours. Characterization results show that 1) buffer stability was good for acetate and phosphate buffers, 2) the electrical field across the channel was about 60% of that predicted by a geometric estimation, 3) experimental retrieval of four proteins (ferritin, BSA, hemoglobin, and cytochrome c) agreed well with calculated retrieval, 4) protein response at the four electrical fields and four solution pHs corresponded to the difference between protein pI and solution pH, 5) lower buffer ionic strength was better for protein separation, 6) protein sample recovery was reasonable from 78 to 90% (mean 85%) for six proteins, and 7) pulsed sample injection led to successful separation of five protein mixtures. In the second part of the study, three protein mixtures were successfully separated using continuous separation over 8 hours. The collected fractions showed clean separation as confirmed by flow field-flow fractionation and spectrophotometer analysis. The throughput was around 15 mg/h and the minimum difference between protein pIs that permitted separation was about two units. We conclude that isoelectric SPLITT fractionation has potential for use in protein purification.     

Microarray‐Based Identification of Lectins for the Purification of Human Urinary Extracellular Vesicles Directly from Urine Samples

As cellular‐derived vesicles largely maintain the biomolecule composition of their original tissue, exosomes, which are found in nearly all body fluids, have enormous potential as clinical disease markers. A major bottleneck in the development of exosome‐based diagnostic assays is the challenging purification of these vesicles; this requires time‐consuming and instrument‐based procedures. We employed lectin arrays to identify potential lectins as probes for affinity‐based isolation of exosomes from the urinary matrix. We found three lectins that showed specific interactions to vesicles and no (or only residual) interaction with matrix proteins. Based on these findings a bead‐based method for lectin‐based isolation of exosomes from urine was developed as a sample preparation step for exosome‐based biomarker research.     

DNA Origami as Scaffolds for Self-Assembly of Lipids and Proteins

Since first being reported in 2006, the DNA origami approach has attracted increasing attention due to programmable shapes, structural stability, biocompatibility, and fantastic addressability. Herein, we provide an account of recent developments of DNA origami as scaffolds for templating the selfassembly of distinct biocomponents, essentially proteins and lipids, into a diverse spectrum of integrated supramolecular architectures. First, the historical development of the DNA origami concept is briefly reviewed. Next, various applications of DNA origami constructs in controllable directed assembly of soluble proteins are discussed. The manipulation and self-assembly of lipid membranes and membrane proteins by using DNA origami as scaffolds are also addressed. Furthermore, recent progress in applying DNA origami in cryoelectron microscopy analysis is discussed. These advances collectively emphasize that the DNA origami approach is a highly versatile, fast evolving tool that may be integrated with lipids and proteins in a way that meets future challenges in molecular biology and nanomedicine.     

Novel Strategies for Drug Discovery Based on Intrinsically Disordered Proteins (IDPs)

Intrinsically disordered proteins (IDPs) are proteins that usually do not adopt well-defined native structures when isolated in solution under physiological conditions. Numerous IDPs have close relationships with human diseases such as tumor, Parkinson disease, Alzheimer disease, diabetes, and so on. These disease-associated IDPs commonly play principal roles in the disease-associated protein-protein interaction networks. Most of them in the disease datasets have more interactants and hence the size of the disease-associated IDPs interaction network is simultaneously increased. For example, the tumor suppressor protein p53 is an intrinsically disordered protein and also a hub protein in the p53 interaction network; α-synuclein, an intrinsically disordered protein involved in Parkinson diseases, is also a hub of the protein network. The disease-associated IDPs may provide potential targets for drugs modulating protein-protein interaction networks. Therefore, novel strategies for drug discovery based on IDPs are in the ascendant. It is dependent on the features of IDPs to develop the novel strategies. It is found out that IDPs have unique structural features such as high flexibility and random coil-like conformations which enable them to participate in both the "one to many" and "many to one" interaction. Accordingly, in order to promote novel strategies for drug discovery, it is essential that more and more features of IDPs are revealed by experimental and computing methods.     

Loops In Proteins (LIP)--a comprehensive loop database for homology modelling

One of the most important and challenging tasks in protein modellingis the prediction of loops, as can be seen in the large varietyof existing approaches. Loops In Proteins (LIP) is a databasethat includes all protein segments of a length up to 15 residuescontained in the Protein Data Bank (PDB). In this study, theapplicability of LIP to loop prediction in the framework ofhomology modelling is investigated. Searching the database forloop candidates takes less than 1 s on a desktop PC, and rankingthem takes a few minutes. This is an order of magnitude fasterthan most existing procedures. The measure of accuracy is theroot mean square deviation (RMSD) with respect to the main-chainatoms after local superposition of target loop and predictedloop. Loops of up to nine residues length were modelled witha local RMSD <1 Å and those of length up to 14residues with an accuracy better than 2 Å. The resultswere compared in detail with a thoroughly evaluated and testedab initio method published recently and additionally with twofurther methods for a small loop test set. The LIP method producedvery good predictions. In particular for longer loops it outperformedother methods. Received May 12, 2003; revised October 17, 2003; accepted October 21, 2003