Discovery of Species-Specific Proteotypic Peptides To Establish a Spectral Library Platform for Identification of Nontuberculosis Mycobacteria from Mass Spectrometry-Based Proteomics.

Nontuberculous mycobacteria are opportunistic bacteria pulmonary and extra-pulmonary infections in humans that closely resemble Mycobacterium tuberculosis. Although genome sequencing strategies helped determine NTMs, a common assay for the detection of coinfection by multiple NTMs with M. tuberculosis in the primary attempt of diagnosis is still elusive. Such a lack of efficiency leads to delayed therapy, an inappropriate choice of drugs, drug resistance, disease complications, morbidity, and mortality. Although a high-resolution LC-MS/MS-based multiprotein panel assay can be developed due to its specificity and sensitivity, it needs a library of species-specific peptides as a platform. Toward this, we performed an analysis of proteomes of 9 NTM species with more than 20 million peptide spectrum matches gathered from 26 proteome data sets. Our metaproteomic analyses determined 48,172 species-specific proteotypic peptides across 9 NTMs. Notably, M. smegmatis (26,008), M. abscessus (12,442), M. vaccae (6487), M. fortuitum (1623), M. avium subsp. paratuberculosis (844), M. avium subsp. hominissuis (580), and M. marinum (112) displayed >100 species-specific proteotypic peptides. Finally, these peptides and corresponding spectra have been compiled into a spectral library, FASTA, and JSON formats for future reference and validation in clinical cohorts by the biomedical community for further translation.

A single polymorphic residue in humans underlies species-specific restriction of HSV-1 by the antiviral protein MxB.

IMPORTANCE: Herpesviruses present a major global disease burden. Understanding the host cell mechanisms that block viral infections, as well as how viruses can evolve to counteract these host defenses, is critically important for understanding viral disease pathogenesis. This study reveals that the major human variant of the antiviral protein myxovirus resistance protein B (MxB) inhibits the human pathogen herpes simplex virus (HSV-1), whereas a minor human variant and orthologous MxB genes from even closely related primates do not. Thus, in contrast to the many antagonistic virus-host interactions in which the virus is successful in thwarting the host's defense systems, here the human gene appears to be at least temporarily winning at this interface of the primate-herpesvirus evolutionary arms race. Our findings further show that a polymorphism at amino acid 83 in a small fraction of the human population is sufficient to abrogate MxB's ability to inhibit HSV-1, which could have important implications for human susceptibility to HSV-1 pathogenesis.

Extreme olfactory sensitivity of silver and bighead carp to overlapping suites of 21-carbon steroids suggests that these species, and likely all other Cyprinoidei, employ them as pheromones.

Although well established that several fishes including goldfish in the suborder Cypinoidei within the family Cypriniformes use the maturation-inducing steroid 17,20ß-dihydroxy-pregn-4-ene-3-one (17,20ßP) and its metabolites as a priming pheromone which they detect with sensitivity and specificity, it is unclear whether and how other Cypriniformes might have evolved to do so. This study examined this question in the family Xenocyprididae. Using electro-olfactogram recording we tested the olfactory sensitivity of silver (Hypophthalmichthys molitrix) and bighead carp (H. nobilis) to a range of 213 steroids in 21 mixtures at 10-9M. While silver carp detected 6 of 21 mixtures, bighead carp detected 5 (p< 0.05). Silver carp were sensitive to 13 21-carbon steroids in these mixtures including 17,20ßP while bighead carp detected 9, including 8 detected by silver carp. This assortment of steroids overlapped that detected by goldfish (family Cyprinidae) but no non-Cyprinoid, suggesting common evolutionary origin and function with differences characteristic of species-specificity.

Simian Immunodeficiency Virus SIVgsn-99CM71 Vpu Employs Different Amino Acids To Antagonize Human and Greater Spot-Nosed Monkey BST-2.

Viral protein U (Vpu) is an accessory protein encoded by human immunodeficiency virus type 1 (HIV-1) and certain simian immunodeficiency virus (SIV) strains. Some of these viruses were reported to use Vpu to overcome restriction by BST-2 of their natural hosts. Our own recent report revealed that Vpu of SIVgsn-99CM71 (SIVgsn71) antagonizes human BST-2 through two AxxxxxxxW motifs (A22W30 and A25W33), whereas antagonizing BST-2 of its natural host, greater spot-nosed monkey (GSN), involved only the A22W30 motif. Here, we show that residues A22, A25, W30, and W33 of SIVgsn71 Vpu are all essential to antagonize human BST-2, whereas a single mutation of either A22 or W30 did not affect the ability to antagonize GSN BST-2. Similar to A18, which is located in the middle of the A14xxxxxxxW22 motif in HIV-1 NL4-3 Vpu and is essential to antagonize human BST-2, A29, located in the middle of the A25W33 motif of SIVgsn71 Vpu was found to be necessary for antagonizing human but not GSN BST-2. Further mutational analyses revealed that residues L21 and K32 of SIVgsn71 Vpu were also essential for antagonizing human BST-2. On the other hand, the ability of SIVgsn71 Vpu to target GSN BST-2 was unaffected by single amino acid substitutions but required multiple mutations to render SIVgsn71 Vpu inactive against GSN BST-2. These results suggest additional requirements for SIVgsn71 Vpu antagonizing human BST-2, implying evolution of the bst-2 gene under strong selective pressure. IMPORTANCE Genes related to survival against life-threating pathogens are important determinants of natural selection in animal evolution. For instance, BST-2, a protein showing broad-spectrum antiviral activity, shows polymorphisms entailing different phenotypes even among primate species, suggesting that the bst-2 gene of primates has been subject to strong selective pressure during evolution. At the same time, viruses readily adapt to these evolutionary changes. Thus, we found that the Vpu of an SIVgsn isolate (SIVgsn-99CM71) can target BST-2 from humans as well as from its natural host, thus potentially facilitating zoonosis. Here, we mapped residues in SIVgsn71 Vpu potentially contributing to cross-species transmission. We found that the requirements for targeting human BST-2 are distinct from and more complex than those for targeting GSN BST-2. Our results suggest that the human bst-2 gene might have evolved to acquire more restrictive phenotype than GSN bst-2 against viral proteins after being derived from their common ancestor.

Virome comparison (VC): A novel approach to comparing viromes based on virus species specificity and virome specificity diversity.

The human virome, or the viral communities distributed on or in our body, is estimated to contain about 380 trillion of viruses (individuals), which has far reaching influences on our health and diseases. Obviously, the sheer numbers of viruses alone make the comparisons of two or multiple viromes extremely challenging. In fact, the theory of computation in computer science for so-termed NP-hard problems stipulates that the problem is unsolvable when the size of virome is sufficiently large even with fastest supercomputers. Practically, one has to develop heuristic and approximate algorithms to obtain practically satisfactory solutions for NP-hard problems. Here, we extend the species-specificity and specificity-diversity framework to develop a method for virome comparison (VC). The VC method consists of a pair of metrics: virus species specificity (VS) and virome specificity diversity (VSD) and corresponding pair of random search algorithms. Specifically, the VS and VS permutation (VSP) test can detect unique virus species (US) or enriched virus species (ES) in each virome (treatment), and the VSD and VSD permutation (VSDP) test can further determine holistic differences between two viromes or their subsets (assemblages of viruses). The test with four virome data sets demonstrated that the VC method is effective, efficient, and robust.

Integrating invasive species risk assessment into environmental DNA metabarcoding reference libraries.

Environmental DNA (eDNA) metabarcoding has shown promise as a tool for estimating biodiversity and early detection of invasive species. In aquatic systems, advantages of this method include the ability to concurrently monitor biodiversity and detect incipient invasions simply through the collection and analysis of water samples. However, depending on the molecular markers chosen for a given study, reference libraries containing target sequences from present species may limit the usefulness of eDNA metabarcoding. To explore the extent of this issue and how it may be resolved to aid biodiversity and invasive species early detection goals, we focus on fishes in the well-studied Laurentian Great Lakes region. First, we provide a synthesis of species currently known from the region and of non-indigenous species identified as threats by international, national, regional, and introduction pathway-specific fish risk assessments. With these species lists, we then evaluate 23 primer pairs commonly used in fish eDNA metabarcoding with available databases of sequence coverage and species specificity. Finally, we identify established and potentially invasive non-indigenous fish that should be prioritized for genetic sequencing to ensure robust eDNA metabarcoding for the region. Our results should increase confidence in using eDNA metabarcoding for fisheries conservation and management in the Great Lakes region and help prioritize reference sequencing efforts. The ultimate utility of eDNA metabarcoding approaches will come when conservation management of existing fish communities is integrated with early detection efforts for invasive species surveillance to assess total fish biodiversity.

Development of Specific Monoclonal Antibodies against Porcine RIG-I-like Receptors Revealed the Species Specificity.

The RIG-I-like receptors (RLRs) play critical roles in sensing and combating viral infections, particularly RNA virus infections. However, there is a dearth of research on livestock RLRs due to a lack of specific antibodies. In this study, we purified porcine RLR proteins and developed monoclonal antibodies (mAbs) against porcine RLR members RIG-I, MDA5 and LGP2, for which one, one and two hybridomas were obtained, respectively. The porcine RIG-I and MDA5 mAbs each targeted the regions beyond the N-terminal CARDs domains, whereas the two LGP2 mAbs were both directed to the N-terminal helicase ATP binding domain in the Western blotting. In addition, all of the porcine RLR mAbs recognized the corresponding cytoplasmic RLR proteins in the immunofluorescence and immunochemistry assays. Importantly, both RIG-I and MDA5 mAbs are porcine specific, without demonstrating any cross-reactions with the human counterparts. As for the two LGP2 mAbs, one is porcine specific, whereas another one reacts with both porcine and human LGP2. Thus, our study not only provides useful tools for porcine RLR antiviral signaling research, but also reveals the porcine species specificity, giving significant insights into porcine innate immunity and immune biology.

Molecular Determinants of Species Specificity of α-Conotoxin TxIB towards Rat and Human α6/α3ß4 Nicotinic Acetylcholine Receptors.

Conotoxins are widely distributed and important for studying ligand-gated ion channels. TxIB, a conotoxin consisting of 16 amino acids derived from Conus textile, is a unique selective ligand that blocks rat α6/α3ß2ß3 nAChR (IC50 = 28 nM) without affecting other rat subtypes. However, when the activity of TxIB against human nAChRs was examined, it was unexpectedly found that TxIB had a significant blocking effect on not only human α6/α3ß2ß3 nAChR but also human α6/α3ß4 nAChR, with an IC50 of 537 nM. To investigate the molecular mechanism of this species specificity and to establish a theoretical basis for drug development studies of TxIB and its analogs, different amino acid residues between human and rat α6/α3 and ß4 nAChR subunits were identified. Each residue of the human species was then substituted with the corresponding residue of the rat species via PCR-directed mutagenesis. The potencies of TxIB towards the native α6/α3ß4 nAChRs and their mutants were evaluated through electrophysiological experiments. The results showed that the IC50 of TxIB against h[α6V32L, K61R/α3]ß4L107V, V115I was 22.5 µM, a 42-fold decrease in potency compared to the native hα6/α3ß4 nAChR. Val-32 and Lys-61 in the human α6/α3 subunit and Leu-107 and Val-115 in the human ß4 subunit, together, were found to determine the species differences in the α6/α3ß4 nAChR. These results also demonstrate that the effects of species differences between humans and rats should be fully considered when evaluating the efficacy of drug candidates targeting nAChRs in rodent models.

Species-Specific Valid Ternary Interactions of HIV-1 Env-gp120, CD4, and CCR5 as Revealed by an Adaptive Single-Amino Acid Substitution at the V3 Loop Tip.

Molecular interactions of the variable envelope gp120 subunit of HIV-1 with two cellular receptors are the first step of viral infection, thereby playing pivotal roles in determining viral infectivity and cell tropism. However, the underlying regulatory mechanisms for interactions under gp120 spontaneous variations largely remain unknown. Here, we show an allosteric mechanism in which a single gp120 mutation remotely controls the ternary interactions between gp120 and its receptors for the switch of viral cell tropism. Virological analyses showed that a G310R substitution at the tip of the gp120 V3 loop selectively abolished the viral replication ability in human cells, despite evoking enhancement of viral replication in macaque cells. Molecular dynamics (MD) simulations predicted that the G310R substitution at a site away from the CD4 interaction site selectively impeded the binding ability of gp120 to human CD4. Consistently, virions with the G310R substitution exhibited a reduced binding ability to human lymphocyte cells. Furthermore, the G310R substitution influenced the gp120-CCR5 interaction in a CCR5-type dependent manner as assessed by MD simulations and an infectivity assay using exogenously expressed CCR5s. Interestingly, an I198M mutation in human CCR5 restored the infectivity of the G310R virus in human cells. Finally, MD simulation predicted amino acid interplays that physically connect the V3 loop and gp120 elements for the CD4 and CCR5 interactions. Collectively, these results suggest that the V3 loop tip is a cis-allosteric regulator that remotely controls intra- and intermolecular interactions of HIV-1 gp120 for balancing ternary interactions with CD4 and CCR5. IMPORTANCE Understanding the molecular bases for viral entry into cells will lead to the elucidation of one of the major viral survival strategies, and thus to the development of new effective antiviral measures. As shown recently, HIV-1 is highly mutable and adaptable in growth-restrictive cells, such as those of macaque origin. HIV-1 initiates its infection by sequential interactions of Env-gp120 with two cell surface receptors, CD4 and CCR5. A recent epoch-making structural study has disclosed that CD4-induced conformation of gp120 is stabilized upon binding of CCR5 to the CD4-gp120 complex, whereas the biological significance of this remains totally unknown. Here, from a series of mutations found in our extensive studies, we identified a single-amino acid adaptive mutation at the V3 loop tip of Env-gp120 critical for its interaction with both CD4 and CCR5 in a host cell species-specific way. This remarkable finding could certainly provoke and accelerate studies to precisely clarify the HIV-1 entry mechanism.

A novel role for ATP2B in ascidians: Ascidian-specific mutations in ATP2B contribute to sperm chemotaxis.

Sperm chemotaxis, in which sperms are attracted to conspecific eggs via species-specific attractants, plays an important role in fertilization. This phenomenon has been observed in various animals and species-specific sperm attractants have been reported in some species. However, the mechanisms involved in the reception and recognition of the species-specific attractant by the sperms is poorly studied. Previously, we found that the plasma membrane-type Ca2+ /ATPase (PMCA) is the receptor for the sperm-activating and -attracting factor (SAAF) in the ascidian Ciona intestinalis. To determine the role of PMCA in species-specific sperm chemotaxis, we identified the amino acid sequences of PMCAs derived from six Phlebobranchia species. The testis-specific splice variant of PMCA was found to be present in all the species investigated and the ascidian-specific sequence was detected near the 3'-terminus. Moreover, dN/dS analysis revealed that the extracellular loops 1, 2, and 4 in ascidian PMCA underwent a positive selection. These findings suggest that PMCA recognizes the species-specific structure of SAAF at the extracellular loops 1, 2, and 4, and its testis-specific C-terminal region is involved in the activation and chemotaxis of ascidian sperms.

Characterization and localization of relaxin-like gonad-stimulating peptide in the crown-of-thorns starfish, Acanthaster cf. solaris.

In starfish, a relaxin-like gonad-stimulating peptide (RGP) is the gonadotropin responsible for final gamete maturation. RGP comprises two different peptides, A- and B-chains with two interchain and one intrachain disulfide bonds. The existence of two isomers of RGP in the crown-of-thorns starfish, Acanthaster planci, has been reported previously, but it was recently shown that A. planci represents a species complex with four different species. Here we elucidated the authentic sequence of the Pacific species, Acanthaster cf. solaris, RGP (Aso-RGP). The Aso-RGP precursor encoded by a 354 base pair open reading frame was composed of 117 amino acids (aa). The amino acid identity of Aso-RGP to Patiria pectinifera RGP (Ppe-RGP) and Asterias amurensis RGP (Aam-RGP) was 74% and 60%, respectively. Synthetic Aso-RGP induced spawning of ovarian fragments from A. cf. solaris. Ppe-RGP and Aam-RGP also induced spawning by A. cf. solaris ovaries. In contrast, Ppe-RGP and Aso-RGP induced spawning by P. pectinifera ovaries, but Aam-RGP was inactive. Notably, anti-Ppe-RGP antibodies recognized Aso-RGP as well as Ppe-RGP. Localization of Aso-RGP was observed immunohistochemically using anti-Ppe-RGP antibodies, showing that Aso-RGP was mainly present in the radial nerve cords of A. cf. solaris. Aso-RGP was distributed not only in the epithelium of the ectoneural region but also in the neuropile of the ectoneural region. These results suggest that Aso-RGP is synthesized in the epithelium of the ectoneural region, then transferred to fibers in the neuropile of the ectoneural region in radial nerve cords.

Specific Detection of Acanthamoeba species using Polyclonal Peptide Antibody Targeting the Periplasmic Binding Protein of A. castellanii.

Acanthamoeba keratitis (AK) is a rare ocular disease, but it is a painful and sight-threatening infectious disease. Early diagnosis and adequate treatment are necessary to prevent serious complications. While AK is frequently diagnosis via several PCR assays or Acanthamoeba-specific antibodies, a more specific and effective diagnostic method is required. This study described the production of a polyclonal peptide antibody against the periplasmic binding protein (PBP) of A. castellanii and investigated its diagnostic potential. Western blot analysis showed that the PBP antibody specifically reacted with the cell lysates of A. castellanii. However, the PBP antibody did not interact with human corneal epithelial (HCE) cells and the other 3 major causative agents of keratitis. Immunocytochemistry (ICC) results revealed the specific detection of A. castellanii trophozoites and cysts by PBP antibodies when A. castellanii were co-cultured with HCE cells. PBP antibody specificity was further confirmed by co-culture of A. castellanii trophozoites with F. solani, S. aureus, and P. aeruginosa via ICC. The PBP antibody specifically reacted with the trophozoites and cysts of A. polyphaga, A. hatchetti, A. culbertsoni, A. royreba, and A. healyi, thus demonstrated its genus-specific nature. These results showed that the PBP polyclonal peptide antibody of A. castellanii could specifically detect several species of Acanthamoeba, contributing to the development of an effective antibody-based AK diagnostics.

Functional interactions between gyrase subunits are optimized in a species-specific manner.

DNA gyrase is a bacterial DNA topoisomerase that catalyzes ATP-dependent negative DNA supercoiling and DNA decatenation. The enzyme is a heterotetramer comprising two GyrA and two GyrB subunits. Its overall architecture is conserved, but species-specific elements in the two subunits are thought to optimize subunit interaction and enzyme function. Toward understanding the roles of these different elements, we compared the activities of Bacillus subtilis, Escherichia coli, and Mycobacterium tuberculosis gyrases and of heterologous enzymes reconstituted from subunits of two different species. We show that B. subtilis and E. coli gyrases are proficient DNA-stimulated ATPases and efficiently supercoil and decatenate DNA. In contrast, M. tuberculosis gyrase hydrolyzes ATP only slowly and is a poor supercoiling enzyme and decatenase. The heterologous enzymes are generally less active than their homologous counterparts. The only exception is a gyrase reconstituted from mycobacterial GyrA and B. subtilis GyrB, which exceeds the activity of M. tuberculosis gyrase and reaches the activity of the B. subtilis gyrase, indicating that the activities of enzymes containing mycobacterial GyrB are limited by ATP hydrolysis. The activity pattern of heterologous gyrases is in agreement with structural features present: B. subtilis gyrase is a minimal enzyme, and its subunits can functionally interact with subunits from other bacteria. In contrast, the specific insertions in E. coli and mycobacterial gyrase subunits appear to prevent efficient functional interactions with heterologous subunits. Understanding the molecular details of gyrase adaptations to the specific physiological requirements of the respective organism might aid in the development of species-specific gyrase inhibitors.

Viperin has species-specific roles in response to herpes simplex virus infection.

Viperin is a gene with a broad spectrum of antiviral functions and various mechanisms of action. The role of viperin in herpes simplex virus type 1 (HSV-1) infection is unclear, with conflicting data in the literature that is derived from a single human cell type. We have addressed this gap by investigating viperin during HSV-1 infection in several cell types, spanning species and including immortalized, non-immortalized and primary cells. We demonstrate that viperin upregulation by HSV-1 infection is cell-type-specific, with mouse cells typically showing greater increases compared with those of human origin. Further, overexpression and knockout of mouse, but not human viperin significantly impedes and increases HSV-1 replication, respectively. In primary mouse fibroblasts, viperin upregulation by infection requires viral gene transcription and occurs in a predominantly IFN-independent manner. Further we identify the N-terminal domain of viperin as being required for the anti-HSV-1 activity. Interestingly, this is the region of viperin that differs most between mouse and human, which may explain the apparent species-specific activity against HSV-1. Finally, we show that HSV-1 virion host shutoff (vhs) protein is a key viral factor that antagonises viperin in mouse cells. We conclude that viperin can be upregulated by HSV-1 in mouse and human cells, and that mouse viperin has anti-HSV-1 activity.

Transposable elements: a jump toward the future of expression vectors.

Expression vectors (EVs) are artificial nucleic acid molecules with a modular structure that allows for the transcription of DNA sequences of interest in either cellular or cell-free environments. These vectors have emerged as cross-disciplinary tools with multiple applications in an expanding Life Sciences market. The cis-regulatory sequences (CRSs) that control the transcription in EVs are typically sourced from either viruses or from characterized genes. However, the recent advancement in transposable elements (TEs) technology provides attractive alternatives that may enable a significant improvement in the design of EVs. Commonly known as "jumping genes," due to their ability to move between genetic loci, TEs are constitutive components of both eukaryotic and prokaryotic genomes. TEs harbor native CRSs that allow the regulated transcription of transposition-related genes. However, some TE-related CRSs display striking characteristics, which provides the opportunity to reconsider TEs as lead actors in the design of EVs. In this article, we provide a synopsis of the transcriptional control elements commonly found in EVs together with an extensive discussion of their advantages and limitations. We also highlight the latest findings that may allow for the implementation of TE-derived sequences in the EVs feasible, possibly improving existing vectors. By introducing this new concept of TEs as a source of regulatory sequences, we aim to stimulate a profitable discussion of the potential advantages and benefits of developing a new generation of EVs based on the use of TE-derived control sequences.

Two Amino Acids Proximate to the Renin Cleavage Site of Human Angiotensinogen Do Not Affect Blood Pressure and Atherosclerosis in Mice-Brief Report.

OBJECTIVE: Renin cleavage of angiotensinogen has species specificity. As the residues at positions 11 and 12 are different between human angiotensinogen and mouse angiotensinogen, we determined whether these 2 residues in angiotensinogen affect renin cleavage and angiotensin II-mediated blood pressure regulation and atherosclerosis using an adenoassociated viral approach for manipulating angiotensinogen in vivo. Approach and Results: Hepatocyte-specific angiotensinogen deficient (hepAGT-/-) mice in an LDL receptor-deficient background were infected with adenoassociated virals containing a null insert, human angiotensinogen, or mouse angiotensinogen expressing the same residues of the human protein at positions 11 and 12 (mouse angiotensinogen [L11V;Y12I]). Expression of human angiotensinogen in hepAGT-/- mice led to high plasma human angiotensinogen concentrations without changes in plasma endogenous mouse angiotensinogen, plasma renin concentrations, blood pressure, or atherosclerosis. This is consistent with human angiotensinogen not being cleaved by mouse renin. To determine whether the residues at positions 11 and 12 in human angiotensinogen lead to the inability of mouse renin to cleave human angiotensinogen, hepAGT-/- mice were injected with adenoassociated viral vector encoding mouse angiotensinogen (L11V;Y12I). Expression of mouse angiotensinogen (L11V;Y12I) in hepAGT-/- mice resulted in increased plasma mouse angiotensinogen concentrations, reduced renin concentrations, and increased renal AngII concentrations that were comparable to their concentrations in hepAGT+/+ mice. This mouse angiotensinogen variant increased blood pressure and atherosclerosis in hepAGT-/- mice to the magnitude of hepAGT+/+ mice. CONCLUSIONS: Replacement of L11 and Y12 to V11 and I12, respectively, in mouse angiotensinogen does not affect renin cleavage, blood pressure, and atherosclerosis in LDL receptor-deficient mice.

Functional analysis of a species-specific inhibitor selective for human Na+-coupled citrate transporter (NaCT/SLC13A5/mINDY).

The Na+-coupled citrate transporter (NaCT/SLC13A5/mINDY) in the liver delivers citrate from the blood into hepatocytes. As citrate is a key metabolite and regulator of multiple biochemical pathways, deletion of Slc13a5 in mice protects against diet-induced obesity, diabetes, and metabolic syndrome. Silencing the transporter suppresses hepatocellular carcinoma. Therefore, selective blockers of NaCT hold the potential to treat various diseases. Here we report on the characteristics of one such inhibitor, BI01383298. It is known that BI01383298 is a high-affinity inhibitor selective for human NaCT with no effect on mouse NaCT. Here we show that this compound is an irreversible and non-competitive inhibitor of human NaCT, thus describing the first irreversible inhibitor for this transporter. The mouse NaCT is not affected by this compound. The inhibition of human NaCT by BI01383298 is evident for the constitutively expressed transporter in HepG2 cells and for the ectopically expressed human NaCT in HEK293 cells. The IC50 is ∼100 nM, representing the highest potency among the NaCT inhibitors known to date. Exposure of HepG2 cells to this inhibitor results in decreased cell proliferation. We performed molecular modeling of the 3D-structures of human and mouse NaCTs using the crystal structure of a humanized variant of VcINDY as the template, and docking studies to identify the amino acid residues involved in the binding of citrate and BI01383298. These studies provide insight into the probable bases for the differential effects of the inhibitor on human NaCT versus mouse NaCT as well as for the marked species-specific difference in citrate affinity.

Host affinity of endophytic fungi and the potential for reciprocal interactions involving host secondary chemistry.

PREMISE: Interactions between fungal endophytes and their host plants present useful systems for identifying important factors affecting assembly of host-associated microbiomes. Here we investigated the role of secondary chemistry in mediating host affinity of asymptomatic foliar endophytic fungi using Psychotria spp. and Theobroma cacao (cacao) as hosts. METHODS: First, we surveyed endophytic communities in Psychotria species in a natural common garden using culture-based methods. Then we compared differences in endophytic community composition with differences in foliar secondary chemistry in the same host species, determined by liquid chromatography-tandem mass spectrometry. Finally, we tested how inoculation with live and heat-killed endophytes affected the cacao chemical profile. RESULTS: Despite sharing a common environment and source pool for endophyte spores, different Psychotria host species harbored strikingly different endophytic communities that reflected intrinsic differences in their leaf chemical profiles. In T. cacao, inoculation with live and heat-killed endophytes produced distinct cacao chemical profiles not found in uninoculated plants or pure fungal cultures, suggesting that endophytes, like pathogens, induce changes in secondary chemical profiles of their host plant. CONCLUSIONS: Collectively our results suggest at least two potential processes: (1) Plant secondary chemistry influences assembly and composition of fungal endophytic communities, and (2) host colonization by endophytes subsequently induces changes in the host chemical landscape. We propose a series of testable predictions based on the possibility that reciprocal chemical interactions are a general property of plant-endophyte interactions.

Evolutionary diversification of Japanese Stomaphis aphids (Aphididae, Lachninae) in relation to their host plant use and ant association.

Phytophagous insects are among the most diverse of the earth's organisms, and their diversification patterns and the driving forces behind these have attracted considerable research interest. Host shifting to closely related plant species is thought to play an important role in phytophagous insect diversification, but the extent to which other interactions such as mutualistic associations affect diversification is not yet known. In this study, we reconstructed the molecular phylogeny of Japanese Stomaphis aphids and determined whether host shifting or mutualistic association with different ant species could explain diversification in this aphid genus. We analyzed 12 species of Stomaphis and grouped them into ten well-supported DNA lineages. Species in each lineage used a single or a few host plant species, but were mutualistically associated with many ant species of the genus Lasius. This result suggests that Stomaphis evolutionarily diversified primarily through host plant shifts. Interestingly, the reconstructed phylogeny suggests that Stomaphis host shifts occasionally occurred between very distantly related host plant taxa (spanning up to five plant orders). The dependence of Stomaphis on long-lasting Lasius ant colonies situated in temperate deciduous forests where Lasius is the dominant ant genus may have led the aphids to shift to distantly related but spatially adjacent host tree species.

Effect of chimeric relaxin-like gonad-stimulating peptides on oocyte maturation and ovulation in the starfish Asterias rubens and Aphelasterias japonica.

A relaxin-like gonad-stimulating peptide (RGP), comprising two peptide chains (A- and B-chains) linked by two interchain bonds and one intrachain disulfide bond, acts as a gonadotropin in starfish. RGP orthologs have been identified in several starfish species, including Patiria pectinifera (PpeRGP), Asterias rubens (AruRGP) and Aphelasterias japonica (AjaRGP). To analyze species-specificity, this study examined the effects on oocyte maturation and ovulation in ovaries of A. rubens and A. japonica of nine RGP derivatives comprising different combinations of A- and B-chains from the three species. All nine RGP derivatives induced spawning in A. rubens and A. japonica ovaries. However, AruRGP, AjaRGP and their chimeric derivatives were more potent than peptides containing the A- or B-chain of PpeRGP. Three-dimensional models of the structures of the RGP derivatives revealed that residues in the B-chains, such as AspB6, MetB10 and PheB13 in PpeRGP and GluB7, MetB11, and TyrB14 in AruRGP and AjaRGP, respectively, are likely to be involved in receptor binding. Conversely, it is likely that ArgA18 in the A-chain of AruRGP and AjaRGP impairs binding of these peptides to the PpeRGP receptor in P. pectinifera. In conclusion, this study provides new insights into the structural basis of RGP bioactivity and RGP receptor activation in starfish.