Species-specific Determinants Research Articles

Proteus species bloodstream infections: Comparative epidemiology of three species

BackgroundAlthough Proteus species are occasional causes of serious infections, their epidemiology has not been well defined. The objective was to describe the overall and species-specific occurrence and determinants of Proteus species bloodstream infection (BSI) in a large Australian population. MethodsAll Queensland residents with Proteus species BSI identified within the publicly funded healthcare system between 2000 and 2019 were included. ResultsA total of 2,143 incident episodes of Proteus species BSI were identified among 2,079 Queensland residents. The prevalence of comorbid illness differed with higher Charlson comorbidity scores observed with P. penneri and P. vulgaris, and higher prevalence of liver disease with P. penneri, higher comorbid cancer with P. vulgaris, and lower diabetes and renal disease prevalence with P. mirabilis BSIs. ConclusionThis study provides novel information on the epidemiology of Proteus species BSI.

Molecular Determinants of Species Specificity of α-Conotoxin TxIB towards Rat and Human α6/α3β4 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/α3β2β3 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/α3β2β3 nAChR but also human α6/α3β4 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 β4 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/α3β4 nAChRs and their mutants were evaluated through electrophysiological experiments. The results showed that the IC50 of TxIB against h[α6V32L, K61R/α3]β4L107V, V115I was 22.5 μM, a 42-fold decrease in potency compared to the native hα6/α3β4 nAChR. Val-32 and Lys-61 in the human α6/α3 subunit and Leu-107 and Val-115 in the human β4 subunit, together, were found to determine the species differences in the α6/α3β4 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.

Determination of methylmercury and inorganic mercury in human hair samples of individuals from Colombian gold mining regions by double spiking isotope dilution and GC-ICP-MS

With the aim to distinguish between routes of exposition to mercury (Hg) in artisanal and small-scale gold mining (ASGM) communities and to distinguish between Hg contamination sources, Hg species composition should be performed in human biomarkers. In this work, Hg species-specific determination were determined in human hair samples (N = 96), mostly non-directly occupied in ASGM tasks, from the six most relevant gold mining Colombian regions. Therefore, MeHg, Hg(II) and THg concentrations were simultaneously determined by double spiking species-specific isotope dilution mass spectrometry (IDMS) and GC-ICP-MS. Only 16.67% of participants were involved at some point in AGSM works and fish consumption ranged from 3 to 7 times/week, which is between medium and high intake levels. The median concentration of THg obtained from all samples is higher than the reference dose weekly acceptable of MeHg intake established by the EPA (1 ppm), whereas a 25% were more than 4 times higher than the WHO level (2.2 μg Hg g−1). Median THg value of individuals consuming fish 5–7 times per week was significantly higher (p < 0.05) than those of the other consuming groups (12.5 μg Hg g−1). Most of the samples presented a % of MeHg relative to THg higher than 80%. The average % of Hg(II)/THg was 11% and only 10 individuals presented a Hg(II) content over 30%. No significant differences (p > 0.05) were found when the amount of Hg(II) was compared between people involved in AGSM task and people not involved. Interestingly, significant differences among the evaluated groups where found when the percentage of the Hg(II)/THg ratio of these groups were compared. In fact, people involved in AGSM tasks showed 1.7 times higher Hg(II)/THg vs. inhabitants uninvolved. This suggest that Hg(II) determination by IDMS-GC-ICP-MS could be a good proxy for evaluating Hg(II) adsorption by direct exposure to mercury vapors onto hair.

The need for fast and accurate detection of dermatomycosis.

Dermatomycosis of the hair, skin or nails are one of the most common fungal infection worldwide. Beyond permanent damage to the affected area, the risk of severe dermatomycosis in immunocompromised people can be life-threatening. The potential risk of delayed or improper treatment highlights the need for a rapid and accurate diagnosis. However, with traditional methods of fungal diagnostics such as culture, a diagnosis can take several weeks. Alternative diagnostic technologies have been developed which allow for an appropriate and timely selection of an antifungal treatment, preventing nonspecific over-the-counter self-medication. Such techniques include molecular methods such as PCR, real-time PCR, DNA microarray, next-generation sequencing, in addition to matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. Molecular methods can help close the 'diagnostic gap' observed with traditional cultures and microscopy and allow for a rapid detection of dermatomycosis with increased sensitivity and specificity.

Species-specific self-DNA detection mechanisms by mammalian cyclic GMP-AMP synthases.

The mechanisms by which innate immune receptors mediate self-nonself discrimination are unclear. In this study, we found species-specific molecular determinants of self-DNA reactivity by cyclic guanosine monophosphate-adenosine monophosphate (GMP-AMP) synthase (cGAS). Human cGAS contained a catalytic domain that was intrinsically self-DNA reactive and stimulated interferon responses in diverse cell types. This reactivity was prevented by an upstream amino (N)-terminal domain. The cGAS proteins from several nonhuman primate species exhibited a similar pattern of self-DNA reactivity in cells, but chimpanzee cGAS was inactive even when its amino-terminal domain was deleted. In contrast, the N terminus of mouse cGAS promoted self-DNA reactivity. When expressed within tumors, only self-DNA-reactive cGAS proteins protected mice from tumor-induced lethality. In vitro studies of DNA- or chromatin-induced cGAS activation did not reveal species-specific activities that correlate with self-DNA reactivity observed in macrophages. Cell biological analysis revealed that self-DNA reactivity by human cGAS, but not mouse cGAS, correlated with localization to mitochondria. We found that epitope tag positions affected self-DNA reactivity in cells and that DNA present in cell lysates undermines the reliability of cGAS biochemical fractionations. These studies reveal species-specific diversity of cGAS functions, even within the primate lineage, and highlight experimental considerations for the study of this innate immune receptor.

A Guide to Sexing Salamanders in Central Appalachia, United States

Abstract Documenting the sex of individuals encountered during wildlife research and monitoring activities is important for understanding and tracking changes in populations. However, sexing salamanders can be particularly difficult because secondary sex characters are often subtle or only visible during the breeding season, and guidance on species-specific sex determination is lacking from most field guides. The purpose of this guide is to provide a reference to assist biologists in the Central Appalachian region with identifying sex of live adult salamanders. In the main text we provide summary tables and figures to serve as concise references in the field. We provide individual species accounts that contain concise yet comprehensive information for each species based on the published literature, as well as many images depicting sexually dimorphic characters. Our focal region encompasses partial or entire distributions for 56 species of salamanders in 5 families (Ambystomidae, Cryptobranchidae, Plethodontidae, Proteidae, and Salamandridae). We identified seven morphological characters that are strongly sexually dimorphic and useful for sexing live, nonanesthetized, adult salamanders in the field, with males of individual species exhibiting one to five of the characters. We identified more than 20 additional characters that are weakly sexually dimorphic, difficult to distinguish in the field, or species-specific. Our guide serves as a synthesis of sexually dimorphic characters for salamanders in Central Appalachia, and we anticipate it will have broad value for researchers, monitoring programs, and salamander enthusiasts in eastern and central North America.

Comparative analysis reveals the species-specific genetic determinants of ACE2 required for SARS-CoV-2 entry.

Coronavirus interaction with its viral receptor is a primary genetic determinant of host range and tissue tropism. SARS-CoV-2 utilizes ACE2 as the receptor to enter host cell in a species-specific manner. We and others have previously shown that ACE2 orthologs from New World monkey, koala and mouse cannot interact with SARS-CoV-2 to mediate viral entry, and this defect can be restored by humanization of the restrictive residues in New World monkey ACE2. To better understand the genetic determinants behind the ability of ACE2 orthologs to support viral entry, we compared koala and mouse ACE2 sequences with that of human and identified the key residues in koala and mouse ACE2 that restrict viral receptor activity. Humanization of these critical residues rendered both koala and mouse ACE2 capable of binding the spike protein and facilitating viral entry. Our study shed more lights into the genetic determinants of ACE2 as the functional receptor of SARS-CoV-2, which facilitates our understanding of viral entry.

COVID-19 vs influenza viruses: A cockroach optimized deep neural network classification approach.

Among Coronavirus, as with many other viruses, receptor interactions are an essential determinant of species specificity, virulence, and pathogenesis. The pathogenesis of the COVID‐19 depends on the virus's ability to attach to and enter into a suitable human host cell. This paper presents a cockroach optimized deep neural network to detect COVID‐19 and differentiate between COVID‐19 and influenza types A, B, and C. The deep network architecture is inspired using a cockroach optimization algorithm to optimize the deep neural network hyper‐parameters. COVID‐19 sequences are obtained from repository 2019 Novel Coronavirus Resource, and influenza A, B, and C sub‐dataset are obtained from other repositories. Five hundred ninety‐four unique genomes sequences are used in the training and testing process with 99% overall accuracy for the classification model.

A Potent Postentry Restriction to Primate Lentiviruses in a Yinpterochiropteran Bat.

Bats are primary reservoirs for multiple lethal human viruses, such as Ebola, Nipah, Hendra, rabies, severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome-related coronavirus (MERS-CoV), and, most recently, SARS-CoV-2. The innate immune systems of these immensely abundant, anciently diverged mammals remain insufficiently characterized. While bat genomes contain many endogenous retroviral elements indicative of past exogenous infections, little is known about restrictions to extant retroviruses. Here, we describe a major postentry restriction in cells of the yinpterochiropteran bat Pteropus alecto Primate lentiviruses (HIV-1, SIVmac) were potently blocked at early life cycle steps, with up to 1,000-fold decreases in infectivity. The block was specific, because nonprimate lentiviruses such as equine infectious anemia virus and feline immunodeficiency virus were unimpaired, as were foamy retroviruses. Interspecies heterokaryons demonstrated a dominant block consistent with restriction of incoming viruses. Several features suggested potential TRIM5 (tripartite motif 5) or myxovirus resistance protein 2 (MX2) protein restriction, including postentry action, cyclosporine sensitivity, and reversal by capsid cyclophilin A (CypA) binding loop mutations. Viral nuclear import was significantly reduced, and this deficit was substantially rescued by cyclosporine treatment. However, saturation with HIV-1 virus-like particles did not relieve the restriction at all. P. alecto TRIM5 was inactive against HIV-1 although it blocked the gammaretrovirus N-tropic murine leukemia virus. Despite major divergence in a critical N-terminal motif required for human MX2 activity, P. alecto MX2 had anti-HIV activity. However, this did not quantitatively account for the restriction and was independent of and synergistic with an additional CypA-dependent restriction. These results reveal a novel, specific restriction to primate lentiviruses in the Pteropodidae and advance understanding of bat innate immunity.IMPORTANCE The COVID-19 pandemic suggests that bat innate immune systems are insufficiently characterized relative to the medical importance of these animals. Retroviruses, e.g., HIV-1, can be severe pathogens when they cross species barriers, and bat restrictions corresponding to retroviruses are comparatively unstudied. Here, we compared the abilities of retroviruses from three genera (Lentivirus, Gammaretrovirus, and Spumavirus) to infect cells of the large fruit-eating bat P. alecto and other mammals. We identified a major, specific postentry restriction to primate lentiviruses. HIV-1 and SIVmac are potently blocked at early life cycle steps, but nonprimate lentiviruses and foamy retroviruses are entirely unrestricted. Despite acting postentry and in a CypA-dependent manner with features reminiscent of antiretroviral factors from other mammals, this restriction was not saturable with virus-like particles and was independent of P. alecto TRIM5, TRIM21, TRIM22, TRIM34, and MX2. These results identify a novel restriction and highlight cyclophilin-capsid interactions as ancient species-specific determinants of retroviral infection.

Large-Scale Comparative Analyses of Tick Genomes Elucidate Their Genetic Diversity and Vector Capacities

Among arthropod vectors, ticks transmit the most diverse human and animal pathogens, leading to an increasing number of new challenges worldwide. Here we sequenced and assembled high-quality genomes of six ixodid tick species and further resequenced 678 tick specimens to understand three key aspects of ticks: genetic diversity, population structure, and pathogen distribution. We explored the genetic basis common to ticks, including heme and hemoglobin digestion, iron metabolism, and reactive oxygen species, and unveiled for the first time that genetic structure and pathogen composition in different tick species are mainly shaped by ecological and geographic factors. We further identified species-specific determinants associated with different host ranges, life cycles, and distributions. The findings of this study are an invaluable resource for research and control of ticks and tick-borne diseases.

The Versatility of Opportunistic Infections Caused by Gemella Isolates Is Supported by the Carriage of Virulence Factors From Multiple Origins.

The molecular basis of the pathogenesis of the opportunistic invasive infections caused by isolates of the Gemella genus remains largely unknown. Moreover, inconsistencies in the current species assignation were detected after genome-level comparison of 16 public Gemella isolates. A literature search detected that, between the two most pathogenic species, Gemella morbillorum causes about twice the number of cases compared to Gemella haemolysans. These two species shared their mean diseases – sepsis and endocarditis – but differed in causing other syndromes. A number of well-known virulence factors were harbored by all species, such as a manganese transport/adhesin sharing 83% identity from oral endocarditis-causing streptococci. Likewise, all Gemellae carried the genes required for incorporating phosphorylcholine into their cell walls and encoded some choline-binding proteins. In contrast, other proteins were species-specific, which may justify the known epidemiological differences. G. haemolysans, but not G. morbillorum, harbor a gene cluster potentially encoding a polysaccharidic capsule. Species-specific surface determinants also included Rib and MucBP repeats, hemoglobin-binding NEAT domains, peptidases of C5a complement factor and domains that recognize extracellular matrix molecules exposed in damaged heart valves, such as collagen and fibronectin. Surface virulence determinants were associated with several taxonomically dispersed opportunistic genera of the oral microbiota, such as Granulicatella, Parvimonas, and Streptococcus, suggesting the existence of a horizontally transferrable gene reservoir in the oral environment, likely facilitated by close proximity in biofilms and ultimately linked to endocarditis. The identification of the Gemella virulence pool should be implemented in whole genome-based protocols to rationally predict the pathogenic potential in ongoing clinical infections caused by these poorly known bacterial pathogens.

Routineausdrücke in deutschen Gesetzestexten – Versuch einer Klassifizierung

In the linguistics of language for special purposes / special text, one of the characteristics of the legal language is its formulaicity. It should be regarded as a particularly distinctive and even constitutive feature of legal texts. For many genres of texts used in legal professional communication, it is not only the compositional standardization that is characteristic, but above all the formulaicity on the level of expression, the main exhibit of which is the pattern of routine expressions repeated in copies of a given genre of text. Due to their species-specific determination, they should only be described in relation to a specific species/quality variant of text. The aim of the article is to attempt a typology of routine expressions occurring in German primary statutory texts.

Structural Basis for a Species-Specific Determinant of an SIV Vif Protein toward Hominid APOBEC3G Antagonism

Primate lentiviruses encode a Vif protein that counteracts the host antiviral APOBEC3 (A3) family members. The adaptation of Vif to species-specific A3 determinants is a critical event that allowed the spillover of a lentivirus from monkey reservoirs to chimpanzees and subsequently to humans, which gave rise to HIV-1 and the acquired immune deficiency syndrome (AIDS) pandemic. How Vif-A3 protein interactions are remodeled during evolution is unclear. Here, we report a 2.94Å crystal structure of the Vif substrate receptor complex from simian immunodeficiency virus isolated from red-capped mangabey (SIVrcm). The structure of the SIVrcm Vif complex illuminates the stage of lentiviral Vif evolution that is immediately prior to entering hominid primates. Structure-function studies reveal the adaptations that allowed SIVrcm Vif to antagonize hominid A3G. These studies show a partitioning between an evolutionarily dynamic specificity determinant and a conserved protein interacting surface onVif that enables adaptation while maintaining protein interactions required for potent A3 antagonism.

Species-specific mechanisms of cytotoxicity toward immune cells determine the successful outcome of Vibrio infections

Vibrio species cause infectious diseases in humans and animals, but they can also live as commensals within their host tissues. How Vibrio subverts the host defenses to mount a successful infection remains poorly understood, and this knowledge is critical for predicting and managing disease. Here, we have investigated the cellular and molecular mechanisms underpinning infection and colonization of 2 virulent Vibrio species in an ecologically relevant host model, oyster, to study interactions with marine Vibrio species. All Vibrio strains were recognized by the immune system, but only nonvirulent strains were controlled. We showed that virulent strains were cytotoxic to hemocytes, oyster immune cells. By analyzing host and bacterial transcriptional responses to infection, together with Vibrio gene knock-outs, we discovered that Vibrio crassostreae and Vibrio tasmaniensis use distinct mechanisms to cause hemocyte lysis. Whereas V. crassostreae cytotoxicity is dependent on a direct contact with hemocytes and requires an ancestral gene encoding a protein of unknown function, r5.7, V. tasmaniensis cytotoxicity is dependent on phagocytosis and requires intracellular secretion of T6SS effectors. We conclude that proliferation of commensal vibrios is controlled by the host immune system, preventing systemic infections in oysters, whereas the successful infection of virulent strains relies on Vibrio species-specific molecular determinants that converge to compromise host immune cell function, allowing evasion of the host immune system.

PCR-RFLP at 18S rRNA gene for identification of Sarcocystis species and their prevalence in cattle of Andhra Pradesh

In the present study, the species specific determination of Sarcocystis cysts isolated from 137 naturally infected and slaughtered cattle from different regions of Chittoor district, Andhra Pradesh, India was achieved by PCR-RFLP. Diagnosis of sarcocysts in cattle was based on macroscopic examination and pepsin digestion method. Genomic DNA was extracted individually from bradyzoites of all 137 sarcocystosis positive cattle and all the isolates were characterized at 18S rRNA. Digestion of PCR amplicons (900 bp) with restriction endonuclease revealed 3 different electromorphs which were referred to Sarcocystis cruzi (513 and 343 bp), S. hirsuta (525, 241 and 141 bp) and S. fusiformis (532 and 335 bp). Sarcocystis cruzi (93.43%) was significantly more prevalent in comparison with the S. hirsuta (4.38%) and S. fusiformis (2.19%). Infection of cattle with S. hominis was not observed in the study area. Occurrence of S. fusiformis in cattle supports that Sarcocystis species of buffaloes are not strictly intermediate host specific however can infect cattle.

Cytological characterization of three closely related pines - Pinus mugo, P. uliginosa and P. \xd7 rhaetica from the Pinus mugo complex (Pinaceae)

The Pinus mugo complex comprises closely related pines in different taxonomic ranks, which commonly occur in major European mountain chains. This large and polymorphic complex has been, and still is, a considerable challenge for taxonomists due to highly variable phenotypical characteristics within particular taxa, ongoing hybridization in sympatric populations, lack of distinct morphological species-specific determinants, and the absence of distinct genetic differentiation among them. Moreover, cytogenetic data on pines from this complex are very scarce. Therefore, in this study, we used C-banding methods and flow cytometric analysis in order to provide new data on karyotypical features and DNA content of three closely-related pines from the Pinus mugo complex, i.e., Pinus mugo (Turra), Pinus uliginosa (Neumann), and Pinus × rhaetica (Brügger) and to gain more insight into the taxonomic relationships between these pines. Our results confirmed that the basic number of chromosome is the same amongst the analyzed taxa (2n = 2x = 24), and that the karyotypes have a similar morphology. The number of secondary constrictions (SCs) and C-banding patterns differed slightly between P. mugo, P. uliginosa and P. × rhaetica. The nuclear DNA content (2C/pg) among analyzed taxa is from 46.41 pg for P. mugo and P. × rhaetica to 46.48 pg for P. uliginosa. For each of these pines, certain taxa-specific cytogenetic determinants were identified. Our results indicate that they may represent two different taxa, and so their names should not be used interchangeably in scientific literature.

Evolution of Hepatitis B Virus Receptor NTCP Reveals Differential Pathogenicities and Species Specificities of Hepadnaviruses in Primates, Rodents, and Bats.

Human hepatitis B virus (HBV) is a global health problem, affecting more than 250 million people worldwide. HBV-like viruses, named orthohepadnaviruses, also naturally infect nonhuman primates, rodents, and bats, but their pathogenicity and evolutionary history are unclear. Here, we determined the evolutionary history of the HBV receptors NTCP and GPC5 over millions of years of primate, rodent, and bat evolution. We use this as a proxy to understand the pathogenicity of orthohepadnaviruses in mammalian hosts and to determine the implications for species specificity. We found that NTCP, but not GPC5, has evolved under positive selection in primates (27 species), rodents (18 species), and bats (21 species) although at distinct residues. Notably, the positively selected codons map to the HBV-binding sites in primate NTCP, suggesting past genetic "arms races" with pathogenic orthohepadnaviruses. In rodents, the positively selected codons fall outside and within the presumed HBV-binding sites, which may contribute to the restricted circulation of rodent orthohepadnaviruses. In contrast, the presumed HBV-binding motifs in bat NTCP are conserved, and none of the positively selected codons map to this region. This suggests that orthohepadnaviruses may bind to different surfaces in bat NTCP. Alternatively, the patterns may reflect adaptive changes associated with metabolism rather than pathogens. Overall, our findings further point to NTCP as a naturally occurring genetic barrier for cross-species transmissions in primates, which may contribute to the narrow host range of HBV. In contrast, this constraint seems less important in bats, which may correspond to greater orthohepadnavirus circulation and diversity.IMPORTANCE Chronic infection with hepatitis B virus (HBV) is a major cause of liver disease and cancer in humans. Mammalian HBV-like viruses are also found in nonhuman primates, rodents, and bats. As for most viruses, HBV requires a successful interaction with a host receptor for replication. Cellular receptors are thus key determinants of host susceptibility as well as specificity. One hallmark of pathogenic virus-host relationships is the reciprocal evolution of host receptor and viral envelope proteins, as a result of their antagonistic interaction over time. The dynamics of these so-called "evolutionary arms races" can leave signatures of adaptive selection, which in turn reveal the evolutionary history of the virus-host interaction as well as viral pathogenicity and the genetic determinants of species specificity. Here, we show how HBV-like viruses have shaped the evolutionary history of their mammalian host receptor, as a result of their ancient pathogenicity, and decipher the genetic determinants of cross-species transmissions.

Leaf anatomical traits determine the 18 O enrichment of leaf water in coastal halophytes.

Anatomical adaptations to high-salinity environments in mangrove leaves may be recorded in leaf water isotopes. Recent studies observed lower 18 O enrichment (ΔL ) of leaf water with respect to source water in three mangrove species relative to adjacent freshwater trees, but the factors that govern this phenomenon remain unclear. To resolve this issue, we investigated leaf traits and ΔL in 15 species of true mangrove plants, 14 species of adjacent freshwater trees, and 4 species of semi-mangrove plants at five study sites along south-eastern coast of China. Our results confirm that ΔL was generally 3-4‰ lower for mangrove species than for adjacent freshwater or semi-mangrove species. We hypothesized that higher leaf water content (LWC) and lower leaf stomatal density (LS) both played important roles in reducing ΔL in mangroves relative to nearby freshwater plants. Both differences acted to elongate effective leaf mixing length (L) in mangroves by about 200% and lower stomatal conductance by about 30%. Péclet models based on both LWC and LS could accurately predict ΔL . Our findings highlight the potential species-specific anatomical determinants of ΔL (or L), which has important implications for the interpretation of environmental information from metabolites produced by leaf water isotopes in palaeoclimate research.

Asynchronous CDX2 expression and polarization of porcine trophoblast cells reflects a species-specific trophoderm lineage determination progress model.

Upregulation of Cdx2 expression in outer cells is a key event responsible for cell lineage segregation between the inner cell mass and the trophoderm (TE) in mouse morula-stage embryos. In TE cells, polarization can regulate Hippo and Rho-associated kinase (Rho-ROCK) signaling to induce the nuclear location of YAP, which has been demonstrated to further induce the expression of Cdx2. However, we found that CDX2 expression could not be detected in the outer cells of porcine morula-stage embryos butonly in some TE cells at the early blastocyst stage. The biological significance and the regulation mechanism of this species-specific CDX2 expression pattern have still not been determined. We show here that an asynchronous CDX2 expression pattern exists in porcine TE cells during the development of the blastocyst. We demonstrate that CDX2 expression in porcine TE cells depends on the nuclear localization of YAP and polarization of the embryo through Y27632 treatment. We found that the polarization process in the morula to the late blastocyst stage porcine embryos was asynchronous, which was revealed by the apical localization of phosphorylated EZRIN staining. Artificially enhancing the number of polarized blastomeres by culturing the separated blastomeres of four-cell stage porcine embryos resulted in increased CDX2-positive cell numbers. These results indicate that the mechanism of CDX2 expression regulation is conserved, butthe polarization progress is not conserved between the pig and themouse, and results in a species-specific trophoblast determination progress model.

Reverse transcription loop-mediated isothermal amplification for species-specific detection of tomato chlorotic spot orthotospovirus

Tomato chlorotic spot orthotospovirus (TCSV) is an emerging orthotospovirus that can cause severe disease on tomato plants. There are at least four orthotospoviruses infecting tomato, and mixed infection of two or more orthotospoviruses in a single tomato plant is quite common in the field. With similarity in the symptomatology and cross serological reactivity among tomato-infecting orthotospoviruses, especially between TCSV and groundnut ringspot orthotospovirus (GRSV), the current serological tests could not achieve definite and accurate species-specific determination in disease diagnosis. Here, a one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for TCSV. Under optimum conditions, the virus was detected in as little as 0.2 ng of total RNA or in 1:10,000 dilution of a simple diluted tissue extract, which was ten times more sensitive than a conventional RT-PCR assay. The RT-LAMP assay was highly specific for TCSV, with no cross reaction with the other two orthotospoviruses: GRSV and tomato spotted wilt orthotospovirus (TSWV). These results demonstrate that this simple and sensitive RT-LAMP could be used to achieve species-specific detection for TCSV under field conditions.