Further Characterization of the Antiviral Transmembrane Protein MARCH8.

The cellular transmembrane protein MARCH8 impedes the incorporation of various viral envelope glycoproteins, such as the HIV-1 envelope glycoprotein (Env) and vesicular stomatitis virus G-glycoprotein (VSV-G), into virions by downregulating them from the surface of virus-producing cells. This downregulation significantly reduces the efficiency of virus infection. In this study, we aimed to further characterize this host protein by investigating its species specificity and the domains responsible for its antiviral activity, as well as its ability to inhibit cell-to-cell HIV-1 infection. We found that the antiviral function of MARCH8 is well conserved in the rhesus macaque, mouse, and bovine versions. The RING-CH domains of these versions are functionally important for inhibiting HIV-1 Env and VSV-G-pseudovirus infection, whereas tyrosine motifs are crucial for the former only, consistent with findings in human MARCH8. Through analysis of chimeric proteins between MARCH8 and non-antiviral MARCH3, we determined that both the N-terminal and C-terminal cytoplasmic tails, as well as presumably the N-terminal transmembrane domain, of MARCH8 are critical for its antiviral activity. Notably, we found that MARCH8 is unable to block cell-to-cell HIV-1 infection, likely due to its insufficient downregulation of Env. These findings offer further insights into understanding the biology of this antiviral transmembrane protein.

Engineering of IF1 -susceptive bacterial F1 -ATPase.

IF1 , an inhibitor protein of mitochondrial ATP synthase, suppresses ATP hydrolytic activity of F1 . One of the unique features of IF1 is the selective inhibition in mitochondrial F1 (MF1 ); it inhibits catalysis of MF1 but does not affect F1 with bacterial origin despite high sequence homology between MF1 and bacterial F1 . Here, we aimed to engineer thermophilic Bacillus F1 (TF1 ) to confer the susceptibility to IF1 for elucidating the molecular mechanism of selective inhibition of IF1 . We first examined the IF1 -susceptibility of hybrid F1 s, composed of each subunit originating from bovine MF1 (bMF1 ) or TF1 . It was clearly shown that only the hybrid with the ß subunit of mitochondrial origin has the IF1 -susceptibility. Based on structural analysis and sequence alignment of bMF1 and TF1 , the five non-conserved residues on the C-terminus of the ß subunit were identified as the candidate responsible for the IF1 -susceptibility. These residues in TF1 were substituted with the bMF1 residues. The resultant mutant TF1 showed evident IF1 -susceptibility. Reversely, we examined the bMF1 mutant with TF1 residues at the corresponding sites, which showed significant suppression of IF1 -susceptibility, confirming the critical role of these residues. We also tested additional three substitutions with bMF1 residues in α and γ subunits that further enhanced the IF1 -susceptibility, suggesting the additive role of these residues. We discuss the molecular mechanism by which IF1 specifically recognizes F1 with mitochondrial origin, based on the present result and the structure of F1 -IF1 complex. These findings would help the development of the inhibitors targeting bacterial F1 .

Cross-species amplification from non-human primate DNAs in commercial human DNA assay kits.

Species specificity of commercial human DNA quantification kits and short tandem repeat (STR) profiling kits was examined using primate DNA samples. These samples comprised 33 individuals from eight primate species, each with gender and kinship data, including human (Homo sapiens), chimpanzee (Pan troglodytes), gorilla (Gorilla gorilla), and orangutan (Pongo pygmaeus) of Hominidae family, and Japanese macaque (Macaca fuscata), long-tailed macaque (Macaca fascicularis), hamadryas baboon (Papio hamadryas), and savannah monkey (Chlorocebus sp.) of Cercopithecidae family. The findings revealed varying levels of cross-species amplifications in all non-human DNA samples that correlated with their evolutionary proximity to humans, both kit types. Moreover, cross-species amplification, including female DNA samples, was observed in a Y-chromosomal STR profiling kit. Additionally, species specificity differed among the commercial kits examined. The cross-species amplification data presented in this study offer valuable assistance in interpreting the results of individual human identification in forensic cases involving non-human primates.

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.

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.

Species-specificity of the secondary biosynthetic potential in Bacillus.

Introduction: Although Bacillus species have produced a wide variety of structurally diverse and biologically active natural products, the secondary biosynthetic potential of Bacillus species is widely underestimated due to the limited number of biosynthetic gene clusters (BGCs) in this genus. The significant variation in the diversity and novelty of BGCs across different species within the Bacillus genus presents a major obstacle to the efficient discovery of novel natural products from Bacillus. Methods: In this study, the number of each class of BGCs in all 6,378 high-quality Bacillus genomes was predicted using antiSMASH, the species-specificity of BGC distribution in Bacillus was investigated by Principal component analysis. Then the structural diversity and novelty of the predicted secondary metabolites in Bacillus species with specific BGC distributions were analyzed using molecular networking. Results: Our results revealed a certain degree of species-specificity in the distribution of BGCs in Bacillus, which was mainly contributed by siderophore, type III polyketide synthase (T3PKS), and transAT-PKS BGCs. B. wiedmannii, B. thuringiensis, and B. cereus are rich in RiPP-like and siderophore BGCs, but lack T3PKS BGCs, while B. amyloliquefaciens and B. velezensis are abundant in transAT-PKS BGCs. These Bacillus species collectively encode 77,541 BGCs, with NRPS and RiPPs being the two most dominant types, which are further categorized into 4,291 GCFs. Remarkably, approximately 54.5% of GCFs and 93.8% of the predicted metabolite scaffolds are found exclusively in a single Bacillus species. Notably, B. cereus, B. thuringiensis, and B. velezensis exhibit the highest potential for producing species-specific NRPS and PKS bioinformatic natural products. Taking two species-specific NRPS gene clusters as examples, the potential of Bacillus to synthesize novel species-specific natural products is illustrated. Conclusion: This study highlights the species-specificity of the secondary biosynthetic potential in Bacillus and provides valuable insights for the targeted discovery of novel natural products from this genus.

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.

Broad spectrum and species specificity of plant allelochemicals 1,2-benzenediol and 3-indoleacrylic acid against marine and freshwater harmful algae.

Allelochemicals derived from plants have shown great potential in mitigating harmful algal blooms (HABs), although different algal species can respond differently to these chemicals. Therefore, we first investigated the allelopathic effects of two newly identified plant-derived allelochemicals, 1,2-benzenediol (1,2-BD) and 3-indoleacrylic acid (3-IDC), on six algal species. Then we further evaluated the allelopathic responses of two bloom-forming species, Microcystis aeruginosa FACHB-905 and Heterosigma akashiwo to 1,2-BD. Results showed that 1,2-BD had a broader antialgal spectrum than 3-IDC. Allelopathic response analysis indicated that 1,2-BD consistently and stably inhibit the growth of M. aeruginosa FACHB-905, with inhibitory mechanism being disruption of photosynthetic activity, overwhelming of the antioxidant system and activation of programmed cell death (PCD). H. akashiwo displayed resistance to 1,2-BD during exposure, and the growth inhibition was mainly attributed to PCD. Therefore, the species-specific allelopathic responses provide new insights for controlling HABs using 1,2-BD and 3-IDC.

Species-specific 'specialized' genomic region provides the new insights into the functional genomics characterizing metabolic polymorphisms in plants.

Functional genomics approaches with comparative omics analyses of wild-accessions and cultivars/wild species, as well as comparative genomic analyses in plant species focusing on gene clusters, have successfully detected key metabolic polymorphisms in plant specialized metabolism. In recent decades, (i) intra-species specific metabolic polymorphisms, (ii) new functionalization of tandem duplicated genes, and (iii) metabolic gene clusters were found as the main factors creating metabolic diversity of specialized metabolites in plants. However, given findings aware us that the identification of genes in plant specialized metabolism requires strategic approaches depending on the target metabolic pathways. The increasing availability of plant genome sequences and transcriptome data has facilitated inter-specific comparative analyses, including genomic analysis and gene co-expression network analysis. Here, we introduce functional genomics approaches with the integration of inter-/intra-species comparative metabolomics, their key roles in providing genomic signatures of metabolic evolution, and discuss future prospects of functional genomics on plant specialized metabolism.

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.

Human and mouse cortical astrocytes: a comparative view from development to morphological and functional characterization.

The vision of astroglia as a bare scaffold to neuronal circuitry has been largely overturned. Astrocytes exert a neurotrophic function, but also take active part in supporting synaptic transmission and in calibrating blood circulation. Many aspects of their functioning have been unveiled from studies conducted in murine models, however evidence is showing many differences between mouse and human astrocytes starting from their development and encompassing morphological, transcriptomic and physiological variations when they achieve complete maturation. The evolutionary race toward superior cognitive abilities unique to humans has drastically impacted neocortex structure and, together with neuronal circuitry, astrocytes have also been affected with the acquisition of species-specific properties. In this review, we summarize diversities between murine and human astroglia, with a specific focus on neocortex, in a panoramic view that starts with their developmental origin to include all structural and molecular differences that mark the uniqueness of human astrocytes.

Intestinal microbiota analyses of five economic fishery resources in the South China Sea.

The investigation of intestinal microbiota can provide evidence for revealing the growth and development regulation, feeding habits, environmental adaptability and pollutant indication of marine organisms. To data, the intestinal microbiota of marine organisms in the South China Sea is relatively lacking. To supplement these information, we sequenced intestinal microbiota from five fishery resources (including Auxis rochei, A. thazard, Symplectoteuthis oualaniensis, Thunnus albacores, and Coryphaena equiselis) in the South China Sea using high-throughput Illumina sequencing technology. After filtering, a total of 18,706,729 reads were finally produced and then clustered into OTUs. The mean number of OTUs detected in A. rochei, A. thazard, C. equiselis, S. oualaniensis, and T. albacores was 127, 137, 52, 136, and 142, respectively. Although the Actinobacteria, Bacteroidetes, Cyanobacteria, Deferribacteres, Firmicutes, Proteobacteria, Spirochaetes, Tenericutes, [Thermi], and unclassified_Bacteria were the most abundant in the five species, Photobacterium is the most abundant microbiota. Meanwhile, intestinal microbiota showed species- and sampling sites- specificity, thus only 84 microbiota species were common to all species. Additionally, the potential functions of OTUs in the five species is mainly involved in the synthesis and metabolism of carbohydrate, amino acid, fatty acid and vitamin. This study can provide basic data for clarifying the diversity and species- specificity of intestinal microbiota of five species in the South China Sea, and help to improve the intestinal microbiota database of marine organisms.

Association of nine pathobionts with periodontitis in four South American and European countries.

Aim: Our aim was to compare the prevalence and load of nine pathobionts in subgingival samples of healthy individuals and periodontitis patients from four different countries. Methods: Five hundred and seven subgingival biofilm samples were collected from healthy subjects and periodontitis patients in Belgium, Chile, Peru and Spain. The prevalence and load of Eubacterium brachy, Filifactor alocis, Fretibacterium fastidiosum, Porphyromonas endodontalis, Porphyromonas gingivalis, Selenomonas sputigena, Treponema denticola, Tannerella forsythia and Treponema socranskii were measured by quantitative PCR. Results: The association with periodontitis of all species, except for T. socranskii, was confirmed in all countries but Peru, where only P. endodontalis, P. gingivalis and T. denticola were found to be significantly associated. Moreover, most species showed higher loads at greater CAL and PPD, but not where there was BOP. Through Principal Component Analysis, samples showed clearly different distributions by diagnosis, despite observing a smaller separation in Peruvian samples. Conclusions: Unlike prevalence, relative load was found to be a reliable variable to discriminate the association of the species with periodontitis. Based on this, F. alocis, P. endodontalis, P. gingivalis, T. denticola and T. forsythia may be biomarkers of disease in Belgium, Chile and Spain, due to their significantly higher abundance in periodontitis patients.

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.

Homologues of the Chlamydia trachomatis and Chlamydia muridarum Inclusion Membrane Protein IncS Are Interchangeable for Early Development but Not for Inclusion Stability in the Late Developmental Cycle.

Chlamydia trachomatis is an obligate intracellular bacterium, which undergoes a biphasic developmental cycle inside a vacuole termed the inclusion. Chlamydia-specific effector proteins embedded into the inclusion membrane, the Inc proteins, facilitate inclusion interaction with cellular organelles. A subset of Inc proteins engages with specific host factors at the endoplasmic reticulum (ER)-inclusion membrane contact site (MCS), which is a discrete point of contact between the inclusion membrane and the endoplasmic reticulum (ER). Here, we report that the C. trachomatis Inc protein CTL0402/IncSCt is a novel component of the ER-inclusion MCS that specifically interacts with and recruits STIM1, a previously identified host component of the ER-inclusion MCS with an unassigned interacting partner at the inclusion membrane. In comparison, the Chlamydia muridarum IncS homologue (TC0424/IncSCm) does not interact with or recruit STIM1 to the inclusion, indicating species specificity. To further investigate IncS function and overcome the recently reported early developmental defect of the incS mutant, we achieved temporal complementation by expressing IncS exclusively during the early stages of the developmental cycle. Additionally, we used allelic exchange to replace the incSCt open reading frame with incSCm in the C. trachomatis chromosome. Inclusions harboring either of these strains progressed through the developmental cycle but were STIM1 negative and displayed increased inclusion lysis 48 h postinfection. Expression of incSCt in trans complemented these phenotypes. Altogether, our results indicate that IncS is necessary and sufficient to recruit STIM1 to C. trachomatis inclusion and that IncS plays an early developmental role conserved in C. trachomatis and C. muridarum and a late role in inclusion stability specific to C. trachomatis. IMPORTANCE Obligate intracellular pathogens strictly rely on the host for replication. Specialized pathogen-encoded effector proteins play a central role in sophisticated mechanisms of host cell manipulation. In Chlamydia, a subset of these effector proteins, the inclusion membrane proteins, are embedded in the membrane of the vacuole in which the bacteria replicate. Chlamydia encodes 50 to 100 putative Inc proteins. Many are conserved among species, including the human and mouse pathogens Chlamydia trachomatis and Chlamydia muridarum, respectively. However, whether the function(s) of Inc proteins is indeed conserved among species is poorly understood. Here, we characterized the function of the Inc protein IncS conserved in C. trachomatis and C. muridarum. Our work reveals that a single effector protein can play multiple functions at various stages of the developmental cycle. However, these functions are not necessarily conserved across species, suggesting a complex evolutionary path among Chlamydia species.

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.

The Incompetence of Mosquitoes-Can Zika Virus Be Adapted To Infect Culex tarsalis Cells?

The molecular evolutionary mechanisms underpinning virus-host interactions are increasingly recognized as key drivers of virus emergence, host specificity, and the likelihood that viruses can undergo a host shift that alters epidemiology and transmission biology. Zika virus (ZIKV) is mainly transmitted between humans by Aedes aegypti mosquitoes. However, the 2015 to 2017 outbreak stimulated discussion regarding the role of Culex spp. mosquitoes in transmission. Reports of ZIKV-infected Culex mosquitoes, in nature and under laboratory conditions, resulted in public and scientific confusion. We previously found that Puerto Rican ZIKV does not infect colonized Culex quinquefasciatus, Culex pipiens, or Culex tarsalis, but some studies suggest they may be competent ZIKV vectors. Therefore, we attempted to adapt ZIKV to Cx. tarsalis by serially passaging virus on cocultured Ae. aegypti (Aag2) and Cx. tarsalis (CT) cells to identify viral determinants of species specificity. Increasing fractions of CT cells resulted in decreased overall virus titer and no enhancement of Culex cell or mosquito infection. Next-generation sequencing of cocultured virus passages revealed synonymous and nonsynonymous variants throughout the genome that arose as CT cell fractions increased. We generated nine recombinant ZIKVs containing combinations of the variants of interest. None of these viruses showed increased infection of Culex cells or mosquitoes, demonstrating that variants associated with passaging were not specific to increased Culex infection. These results reveal the challenge of a virus adapting to a new host, even when pushed to adapt artificially. Importantly, they also demonstrate that while ZIKV may occasionally infect Culex mosquitoes, Aedes mosquitoes likely drive transmission and human risk. IMPORTANCE ZIKV is mainly transmitted between humans by Aedes mosquitoes. In nature, ZIKV-infected Culex mosquitoes have been found, and ZIKV infrequently infects Culex mosquitoes under laboratory conditions. Yet, most studies show that Culex mosquitoes are not competent vectors for ZIKV. We attempted to adapt ZIKV to Culex cells to identify viral determinants of species specificity. We sequenced ZIKV after it was passaged on a mixture of Aedes and Culex cells and found that it acquired many variants. We generated recombinant viruses containing combinations of the variants of interest to determine if any of these changes enhance infection in Culex cells or mosquitoes. Recombinant viruses did not show increased infection in Culex cells or mosquitoes, but some variants increased infection in Aedes cells, suggesting adaptation to those cells instead. These results reveal that arbovirus species specificity is complex, and that virus adaptation to a new genus of mosquito vectors likely requires multiple genetic changes.

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.

Species-Specific Gamete Interaction during Sea Urchin Fertilization: Roles of the Egg Jelly and Vitelline Layer.

In sea urchins, the sequence of the cellular and molecular events characterizing the fertilization process has been intensively studied. We have learned that to activate the egg, the fertilizing sperm must undergo morphological modifications (the acrosome reaction, AR) upon reaching the outer gelatinous layer enveloping the egg (egg jelly), which triggers the polymerization of F-actin on the sperm head to form the acrosomal process. The AR exposes bindin, an adhesive sperm protein essential for the species-specific interaction with the cognate receptor on the egg vitelline layer. To investigate the specific roles of the egg jelly and vitelline layer at fertilization of sea urchin eggs, Paracentrotus lividus eggs were incubated in acidic seawater, which removes the egg jelly, i.e., experimental conditions that should prevent the occurrence of the AR, and inseminated in the same medium. At variance with the prevailing view, our results have shown that these dejellied P. lividus eggs can still interact with sperm in acidic seawater, albeit with altered fertilization responses. In particular, the eggs deprived of the vitelline layer reacted with multiple sperm but with altered Ca2+ signals. The results have provided experimental evidence that the plasma membrane, and not the vitelline layer, is where the specific recognition between gametes occurs. The vitelline layer works in unfertilized eggs to prevent polyspermy.

Cross-species comparisons reveal resistance of human skeletal stem cells to inhibition by non-steroidal anti-inflammatory drugs.

Fracture healing is highly dependent on an early inflammatory response in which prostaglandin production by cyclo-oxygenases (COX) plays a crucial role. Current patient analgesia regimens favor opioids over Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) since the latter have been implicated in delayed fracture healing. While animal studies broadly support a deleterious role of NSAID treatment to bone-regenerative processes, data for human fracture healing remains contradictory. In this study, we prospectively isolated mouse and human skeletal stem cells (SSCs) from fractures and compared the effect of various NSAIDs on their function. We found that osteochondrogenic differentiation of COX2-expressing mouse SSCs was impaired by NSAID treatment. In contrast, human SSCs (hSSC) downregulated COX2 expression during differentiation and showed impaired osteogenic capacity if COX2 was lentivirally overexpressed. Accordingly, short- and long-term treatment of hSSCs with non-selective and selective COX2 inhibitors did not affect colony forming ability, chondrogenic, and osteogenic differentiation potential in vitro. When hSSCs were transplanted ectopically into NSG mice treated with Indomethacin, graft mineralization was unaltered compared to vehicle injected mice. Thus, our results might contribute to understanding species-specific differences in NSAID sensitivity during fracture healing and support emerging clinical data which conflicts with other earlier observations that NSAID administration for post-operative analgesia for treatment of bone fractures are unsafe for patients.