Establishment and application of TaqMan real-time PCR method for detection of Theileria annulata resistant to buparvaquone.

Tropical theileriosis is a tick-borne disease that caused by Theileria annulata, and leads to substantial economic impact in endemic area. Distinguishes to other piroplasms, Theileria is the only eukaryotic parasite could transform mammalian leukocytes. At present, buparvaquone is the most effective drug used for treatment of Theileria infection. However, frequently reported of failure treatment with buparvaquone for some T. annulata isolates. Mutation of TaPIN1 was reported to be the direct reason for failure of buparvaquone treatment. Through in vitro culture, a T. annulata isolate with a TaPIN1 mutation that is similar to the reported strain was recently identified in China. In order to understand the distribution of Theileria with mutation of TaPIN1 in China, here we developed a TaqMan probe-based real-time PCR technology to detect the mutated TaPIN1 gene. The specificity, sensitivity and reproducibility of the established TaqMan Real-time PCR method were evaluated, and field cattle blood samples collected from Xinjiang Uyghur Autonomous Region were used to test its application. Among 1683 samples, 335 samples were confirmed positive for T. annulata by traditional PCR method and 34 samples were positive for buparvaquone-resistant. The TaPIN1 gene of those 34 samples was sequenced and analyzed with the published gene sequences from NCBI database. The results showed that the sequence obtained from the present study has good consistency with those published sequences. In conclusion, the TaqMan probe-based real-time PCR targeting T. annulata mutated TaPIN1 gene was successfully established and can be used to detect clinical samples to investigation of buparvaquone-resistant parasites in Xinjiang region quickly and accurately, which will be useful for guiding clinical medicine application.

Trx4, a novel thioredoxin protein, is important for Toxoplasma gondii fitness.

BACKGROUND: To successfully replicate within the host cell, Toxoplasma gondii employs several mechanisms to overcome the host cell defenses and mitigate the harmful effects of the free radicals resulting from its own metabolic processes using effectors such as thioredoxin proteins. In this study, we characterize the location and functions of a newly identified thioredoxin in T. gondii, which was named Trx4. METHODS: We characterized the functional role of Trx4 in T. gondii Type I RH and Type II Pru strains by gene knockout and studied its subcellular localization by endogenous protein HA tagging using CRISPR-Cas9 gene editing. The enzyme-catalyzed proximity labeling technique, the TurboID system, was employed to identify the proteins in proximity to Trx4. RESULTS: Trx4 was identified as a dense granule protein of T. gondii predominantly expressed in the parasitophorous vacuole (PV) and was partially co-localized with GRA1 and GRA5. Functional analysis showed that deletion of trx4 markedly influenced the parasite lytic cycle, resulting in impaired host cell invasion capacity in both RH and Pru strains. Mutation of Trx domains in Trx4 in RH strain revealed that two Trx domains were important for the parasite invasion. By utilizing the TurboID system to biotinylate proteins in proximity to Trx4, we identified a substantial number of proteins, some of which are novel, and others are previously characterized, predominantly distributed in the dense granules. In addition, we uncovered three novel proteins co-localized with Trx4. Intriguingly, deletion of trx4 did not affect the localization of these three proteins. Finally, a virulence assay demonstrated that knockout of trx4 resulted in a significant attenuation of virulence and a significant reduction in brain cyst loads in mice. CONCLUSIONS: Trx4 plays an important role in T. gondii invasion and virulence in Type I RH strain and Type II Pru strain. Combining the TurboID system with CRISPR-Cas9 technique revealed many PV-localized proximity proteins associated with Trx4. These findings suggest a versatile role of Trx4 in mediating the processes that occur in this distinctive intracellular membrane-bound vacuolar compartment.

Role of Recognition MicroRNAs in Hemaphysalis longicornis and Theileria orientalis Interactions.

Ticks are an important type of pathogen transmission vector, and pathogens not only cause serious harm to livestock but can also infect humans. Because of the roles that ticks play in disease transmission, reducing tick pathogen infectivity has become increasingly important and requires the identification and characterization of these pathogens and their interaction mechanisms. In this study, we determined the miRNA expression profile of Hemaphysalis longicornis infected with Theileria orientalis, predicted the target genes of miRNAs involved in this infection process, and investigated the role of miRNA target recognition during host-pathogen interactions. The results showed that longipain is a target gene of miR-5309, which was differentially expressed at different developmental stages and in various tissues in the control group. However, the miR-5309 level was reduced in the infection group. Analysis of the interaction between miRNA and the target gene showed that miR-5309 negatively regulated the expression of the longipain protein during the infection of H. longicornis with T. orientalis. To verify this inference, we compared longipain with the blocking agent orientalis. In this study, the expression of longipain was upregulated by the inhibition of miR-5309 in ticks, and the ability of the antibody produced by the tick-derived protein to attenuate T. orientalis infection was verified through animal immunity and antigen-antibody binding tests. The results showed that expression of the longipain + GST fusion protein caused the cattle to produce antibodies that could be successfully captured by ticks, and cellular immunity was subsequently activated in the ticks, resulting in a subtractive effect on T. orientalis infection. This research provides ideas for the control of ticks and tickborne diseases and a research basis for studying the mechanism underlying the interaction between ticks and pathogens.

Probiotic Bacillus licheniformis ZW3 Alleviates DSS-Induced Colitis and Enhances Gut Homeostasis.

Despite Bacillus species having been extensively utilized in the food industry and biocontrol as part of probiotic preparations, limited knowledge exists regarding their impact on intestinal disorders. In this study, we investigated the effect of Bacillus licheniformis ZW3 (ZW3), a potential probiotic isolated from camel feces, on dextran sulfate sodium (DSS)-induced colitis. The results showed ZW3 partially mitigated body weight loss, disease activity index (DAI), colon shortening, and suppressed immune response in colitis mice, as evidenced by the reduction in the levels of the inflammatory markers IL-1ß, TNF-α, and IL-6 (p < 0.05). ZW3 was found to ameliorate DSS-induced dysfunction of the colonic barrier by enhancing mucin 2 (MUC2), zonula occluden-1 (ZO-1), and occludin. Furthermore, enriched beneficial bacteria Lachnospiraceae_NK4A136_group and decreased harmful bacteria Escherichia-Shigella revealed that ZW3 improved the imbalanced gut microbiota. Abnormally elevated uric acid levels in colitis were further normalized upon ZW3 supplementation. Overall, this study emphasized the protective effects of ZW3 in colitis mice as well as some potential applications in the management of inflammation-related diseases.

The Microbial Tryptophan Metabolite Contributes to the Remission of Salmonella typhimurium Infection in Mice.

Salmonella enterica serovar Typhimurium (S. Tm) causes severe foodborne diseases. Interestingly, gut microbial tryptophan (Trp) metabolism plays a pivotal role in such infections by a yet unknown mechanism. This study aimed to explore the impact of Trp metabolism on S. Tm infection and the possible mechanisms involved. S. Tm-infected C57BL6/J mice were used to demonstrate the therapeutic benefits of the Bacillus velezensis JT3-1 (B. velezensis/JT3-1) strain or its cell-free supernatant in enhancing Trp metabolism. Targeted Trp metabolomic analyses indicated the predominance of indole-3-lactic acid (ILA), an indole derivative and ligand for aryl hydrocarbon receptor (AHR). Based on the 16S amplicon sequencing and correlation analysis of metabolites, we found that B. velezensis supported the relative abundance of Lactobacillus and Ligilactobacillus in mouse gut and showed positive correlations with ILA levels. Moreover, AHR and its downstream genes (especially IL-22) significantly increased in mouse colons after B. velezensis or cell-free supernatant treatment, suggesting the importance of AHR pathway activation. In addition, ILA was found to stimulate primary mouse macrophages to secrete IL-22, which was antagonized by CH-223191. Furthermore, ILA could protect mice from S. Tm infection by increasing IL-22 in Ahr+/- mice, but not in Ahr-/- mice. Finally, Trp-rich feeding showed amelioration of S. Tm infection in mice, and the effect depended on gut microbiota. Taken together, these results suggest that B. velezensis-associated ILA contributes to protecting mice against S. Tm infection by activating the AHR/IL-22 pathway. This study provides insights into the involvement of microbiota-derived Trp catabolites in protecting against Salmonella infection.

Insight into Hyalomma anatolicum biology by comparative genomics analyses.

Hyalomma anatolicum is an obligatory blood-sucking ectoparasite and contributes to the transmission of Crimean-Congo haemorrhagic fever (CCHF) virus, Theileria spp. and Babesia spp. Progress in exploring the adaptive strategy of this ectoparasite and developing tools to fight it has been hindered by the lack of a complete genome. Herein, we assembled the genome using diverse sources of data from multiple sequencing platforms and annotated the 1.96 Gb genome of Hy. anatolicum. Comparative genome analyses and the predicted protein encoding genes reveal unique facets of this genome, including gene family expansion associated with blood feeding and digestion, multi-gene families involved in detoxification, a great number of neuropeptides and corresponding receptors regulating tick growth, development, and reproduction, and glutathione S-transferase genes playing roles in insecticide resistance and detoxification of multiple xenobiotic factors. This high quality reference genome provides fundamental data for obtaining insights into a variety of aspects of tick biology and developing novel strategies to fight notorious tick vectors of human and animal pathogens.

OAS1 suppresses African swine fever virus replication by recruiting TRIM21 to degrade viral major capsid protein.

IMPORTANCE: African swine fever virus (ASFV) completes the replication process by resisting host antiviral response via inhibiting interferon (IFN) secretion and interferon-stimulated genes (ISGs) function. 2', 5'-Oligoadenylate synthetase gene 1 (OAS1) has been reported to inhibit the replication of various RNA and some DNA viruses. However, the regulatory mechanisms involved in the ASFV-induced IFN-related pathway still need to be fully elucidated. Here, we found that OAS1, as a critical host factor, inhibits ASFV replication in an RNaseL-dependent manner. Furthermore, overexpression of OAS1 can promote the activation of the JAK-STAT pathway promoting innate immune responses. In addition, OAS1 plays a new function, which could interact with ASFV P72 protein to suppress ASFV infection. Mechanistically, OAS1 enhances the proteasomal degradation of P72 by promoting TRIM21-mediated ubiquitination. Meanwhile, P72 inhibits the production of avSG and affects the interaction between OAS1 and DDX6. Our findings demonstrated OAS1 as an important target against ASFV replication and revealed the mechanisms and intrinsic regulatory relationships during ASFV infection.

Interferon-stimulated gene 15 facilitates BTV replication through interacting with the NS1 protein.

Bluetongue virus (BTV) infection effectively activates the innate immune response, followed by the expression of interferon (IFN) and multiple interferon-stimulated genes (ISGs). ISG15 is one of the most induced ISGs, and often plays a role in inhibiting virus replication. This study aims to explore the role and specific mechanisms of ovine ISG15 (oISG15) in BTV infection. We found that the transcription level of oISG15 was upregulated in a time-dependent and BTV multiplicity of infection-dependent manner. The overexpression of exogenous oISG15 enhances BTV replication, whereas the knockdown of endogenous oISG15 inhibits BTV replication. The viral protein in wild-type oISG15-overexpressed cells and ISGylation defective oISG15-overexpressed cells have no significant differences, which indicated that oISG15 promoted BTV replication in an ISGylation-independent manner. A co-immunoprecipitation assay showed that four viral BTV proteins-VP3, VP4, VP5, and NS1-interacted with oISG15. We also found that the VP4 and NS1 proteins associated with ubiquitin via co-immunoprecipitation, and that oISG15 overexpression improved the stability of both proteins. Further results showed that the degradation of NS1 was involved in lysine 63-linked polyubiquitin. This suggested that oISG15 may interfere with NS1 degradation via the autophagy pathway. This study provides new insights on the interaction between BTV and ISG15, and enriches our understanding of the regulation and biological function of ISG15 with virus replication.

Identification and Characterization of Nanobodies from a Phage Display Library and Their Application in an Immunoassay for the Sensitive Detection of African Swine Fever Virus.

African swine fever (ASF) is one of the most lethal and devastating diseases of domestic and wild swine. The continual spread and frequent outbreaks of ASF have seriously threatened the pig and pig-related industries, causing great socioeconomic losses at unprecedented proportions. Although ASF has been documented for a century, no effective vaccine or antiviral treatment is currently available. Nanobodies (Nbs) derived from heavy-chain-only antibodies in camelids have been discovered to be effective as therapeutics and robust biosensors in imaging and diagnostic applications. In the present study, a high-quality phage display library containing specific Nbs raised against ASFV proteins was successfully constructed, and 19 nanobodies specific to ASFV p30 were preliminarily identified by phage display technology. After extensive evaluation, nanobodies Nb17 and Nb30 were employed as immunosensors and applied to develop a sandwich enzyme-linked immunosorbent assay (ELISA) for the detection of ASFV in clinical specimens. This immunoassay showed a detection limit of approximately 1.1 ng/mL target protein and 102.5 hemadsorption (HAD50/mL) of ASFV and exhibited high specificity with no cross-reaction with the other porcine viruses tested. The performances of the newly developed assay and a commercial kit in testing 282 clinical swine samples were very similar (93.62% agreement). However, the novel sandwich Nb-ELISA showed higher sensitivity than the commercial kit when serial dilutions of ASFV-positive samples were tested. The present study describes a valuable alternative technique for the detection and surveillance of ASF in endemic regions. Furthermore, additional nanobodies specific to ASFV may be developed using the generated VHH library and employed in different biotechnology fields.

Antioxidant effects of Bifidobacterium longum T37a in mice weight loss and aging model induced by D-galactose.

BACKGROUND: Probiotics can reduce free radical scavenging rate and oxidative damage, and improve activity of crucial antioxidative enzymes in host cells. This study aimed to isolate Bifidobacterium spp. from faeces of babies, and investigate the antioxidant effects of the Bif. longum T37a in mice weight loss and aging model induced by D-galactose. RESULTS: T37a have good antioxidant properties in the DPPH assay and anti-lipid peroxidation test. Compared with the model group, T37a low group significantly increased the thymus index and the levels of T-AOC and GSH-Px of mice. T37a high group significantly decreased the spleen and liver index of mice and the levels of MDA in liver, significantly increased in liver HDL-C levels, and decreased LDL-C in liver. CONCLUSIONS: T37a may be an anti-aging and weight-loss probiotics for its antioxidant capacity, and it is necessary to study further the molecular mechanism of T37a as antioxidant.

Comparative genomics reveals unique features of two Babesia motasi subspecies: Babesia motasi lintanensis and Babesia motasi hebeiensis.

Parasites of the Babesia genus are prevalent worldwide and infect a wide diversity of domestic animals and humans. Herein, using Oxford Nanopore Technology and Illumina sequencing technologies, we sequenced two Babesia subspecies, Babesia motasi lintanensis and Babesia motasi hebeiensis. We identified 3,815 one-to-one ortholog genes that are specific to ovine Babesia spp. Phylogenetic analysis reveals that the two B. motasi subspecies form a distinct clade from other piroplasmas. Consistent with their phylogenetic position, comparative genomic analysis reveals that these two ovine Babesia spp. share higher colinearity with Babesia bovis than with Babesia microti. Concerning the speciation date, B. m. lintanensis split from B. m. hebeiensis approximately 17 million years ago. Genes correlated to transcription, translation, protein modification and degradation, as well as differential/specialized gene family expansions in these two subspecies may favor adaptation to vertebrate and tick hosts. The close relationship between B. m. lintanensis and B. m. hebeiensis is underlined by a high degree of genomic synteny. Compositions of most invasion, virulence, development, and gene transcript regulation-related multigene families, including spherical body protein, variant erythrocyte surface antigen, glycosylphosphatidylinositol anchored proteins, and transcription factor Apetala 2 genes, is largely conserved, but in contrast to this conserved situation, we observe major differences in species-specific genes that may be involved in multiple functions in parasite biology. For the first time in Babesia spp., we find abundant fragments of long terminal repeat-retrotransposons in these two species. We provide fundamental information to characterize the genomes of B. m. lintanensis and B. m. hebeiensis, providing insights into the evolution of B. motasi group parasites.

Activation of telomerase activity and telomere elongation of host cells by Theileria annulata infection.

Theileria annulata-transformed cells share many phenotypes with cancer cells, including uncontrolled proliferation, immortalization, and dissemination. Telomeres are DNA-protein complex at the end of eukaryotic chromosomes that function to maintain genome stability and cell replicative capacity. Telomere length maintenance is primarily dependent on telomerase activity. In up to 90% of human cancer cells, telomerase is reactivated through expression of its catalytic subunit TERT. However, the effect of T. annulata infection on telomere and telomerase activity in bovine cells has not yet been described. In the present study, we confirmed that telomere length and telomerase activity are upregulated after T. annulata infection in three types of cell lines. This change depends on the presence of parasites. After eliminating Theileria from cells with antitheilerial drug buparvaquone, telomerase activity and the expression level of bTERT were decreased. In addition, inhibition of bHSP90 by novobiocin led to decreased AKT phosphorylation levels and telomerase activity, indicating that the bHSP90-AKT complex is a potent factor modulates telomerase activity in T. annulata-infected cells.

Joint toxicity of insecticides against Hyalomma asiaticum.

Hyalomma asiaticum, a hematophagous ectoparasite, causes severe economic losses. We studied the acute toxicity of five pesticides (three single-agent and two combination preparations) to this organism. Engorged larval ticks were immersed in ten serial concentrations of each insecticide and observed for 20 days. The LC50 values of the five insecticides and the cotoxicity coefficients (CTCs) of the two mixtures were estimated for H. asiaticum. The CTCs of lambda-cyhalothrin + etoxazole and lambda-cyhalothrin + fipronil were 128.83 and 331.58, respectively, each demonstrating synergism. The results indicated that these two mixtures were more effective than individual insecticides, and this study suggests a substitutional approach to the control of ticks.

OGG1 inhibition suppresses African swine fever virus replication.

African swine fever virus (ASFV) is an important pathogen that causes a highly contagious and lethal disease in swine, for which neither a vaccine nor treatment is available. The DNA repair enzyme 8-oxoguanine DNA glycosylase 1 (OGG1), which excises the oxidative base lesion 8-oxo-7,8-dihydroguanine (8-oxoG), has been linked to the pathogenesis of different diseases associated with viral infections. However, the role of OGG1-base excision repair (BER) in ASFV infection has been poorly investigated. Our study aimed to characterize the alteration of host reactive oxygen species (ROS) and OGG1 and to analyse the role of OGG1 in ASFV infection. We found that ASFV infection induced high levels and dynamic changes in ROS and 8-oxoG and consistently increased the expression of OGG1. Viral yield, transcription level, and protein synthesis were reduced in ASFV-infected primary alveolar macrophages (PAMs) treated by TH5487 or SU0268 inhibiting OGG1. The expression of BER pathway associated proteins of ASFV was also suppressed in OGG1-inhibited PAMs. Furthermore, OGG1 was found to negatively regulate interferon ß (IFN-ß) production during ASFV infection and IFN-ß could be activated by OGG1 inhibition with TH5487 and SU0268, which blocked OGG1 binding to 8-oxoG. Additionally, the interaction of OGG1 with viral MGF360-14-L protein could disturb IFN-ß production to further affect ASFV replication. These results suggest that OGG1 plays the crucial role in successful viral infection and OGG1 inhibitors SU0268 or TH5487 could be used as antiviral agents for ASFV infection.

Changes in TFG gene expression in bovine leucocytes transformed by Theileria annulata.

Theileria annulata schizont-infected host cells in culture in vitro show unlimited proliferation similar to tumor cells; thus far, T. annulata and T. parva are the only eukaryotes that have been found to transform mammalian cells (immortalized). The transformation of these cells is reversible; when the parasite is eliminated in transformed cells by buparvaquone (BW720c), the host cells show normal growth and apoptosis. TFG is a tropomyosin-receptor kinase fused gene that is conserved among many species and is an important proto-oncogene. In this study, the bovine TFG gene was amplified by PCR from the cDNA of T. annulata schizont-transformed cells, cloned into the pGEX-4T-1 vector and expressed in Escherichia coli BL21 (DE3). After purification, the fusion protein was injected into rabbits to produce polyclonal antibodies. Using T. annulata-transformed cells together with BW720c treatment to kill the parasite, we aimed to identify changes in TFG gene expression by real-time PCR and Western blotting. The results showed that the bovine TFG gene was ~582 bp in size; SDS-PAGE analysis showed that the fusion protein was expressed in BL21 (DE3) cells with a molecular mass of 48 kD, and Western blotting indicated that the polyclonal antibodies could react with bovine TFG proteins from T. annulata-transformed cells and showed high specificity. Compared with that in the control group, the transcription level of the host TFG gene decreased significantly in the BW720c test group, and the expression of host tumor-related TFG protein decreased sharply after 72 h of drug treatment, suggesting that the TFG protein expression in transformed cells was directly related to T. annulata. This finding laid a foundation for further study on the interaction between T. annulata and host cells.

Adaptation to low nitrogen and salt stresses in the desert poplar by effective regulation of nitrogen assimilation and ion balance.

As a main desert plant from arid regions of Central Asia, Populus euphratica always encounters with nitrogen shortage in its long life, apart from salt or drought stress. However, it remains unknown how this species responds to low nitrogen and combined stresses of low nitrogen and salinity. Thus, saplings of P. euphratica with uniform size were exposed to normal or low nitrogen condition (150 and 15 ppm ammonium nitrate separately) individually or in combination with salinity. Under low nitrogen conditions we found a positive effect on P. euphratica root growth, which could be associated to high level of nitrogen allocation to support root growth and effective regulation of nitrogen assimilation in comparison with the other poplar species reported before. Under salt stress the root growth of P. euphratica was significantly inhibited, with the side effects of oxidative stress, as saplings stored higher Na+ and Cl- contents in roots. Under the combined stressors of both salinity and low nitrogen, P. euphratica undergo a risky strategy, as stimulated root growth is accompanied by further oxidative stress.The concentrations of root K+ and whole plant NO3- were increased to support the tolerance of the combined stressors in P. euphratica, showing same characteristics with halophytes. Overall, our results provide evidence that the desert poplar can adapt to the salt stress/low nitrogen bundle, by effective regulation of nitrogen assimilation and ion homoeostasis.

Identification and characterization of nanobodies specifically against African swine fever virus major capsid protein p72.

African swine fever virus (ASFV) is a large and very complex DNA virus. The major capsid protein p72 is the most predominant structural protein and constitutes the outmost icosahedral capsid of the virion. In the present study, the nanobodies against ASFV p72 protein were screened from a camelid immune VHH library by phage display technique. Nine distinct nanobodies were identified according to the amino acid sequences of the complementary determining regions (CDRs), and contain typical amino acid substitutions in the framework region 2 (FR2). Six nanobodies were successfully expressed in E. coli, and their specificity and affinity to p72 protein were further evaluated. The results showed that nanobodies Nb25 had the best affinity to both recombinant and native p72 protein of ASFV. The Nb25 possesses an extremely long CDR3 with 23 amino acids compared with other nanobodies, which may allow this nanobody to access the hidden epitopes of target antigen. Furthermore, the Nb25 can specifically recognize the virus particles captured by polyclonal antibody against ASFV in a sandwich immunoassay, and its application as a biosensor to target virus in PAM cells was verified by an immunofluorescence assay. Nanobodies have been proven to possess many favorable properties with small size, high affinity and specificity, easier to produce, low costs and deep tissue penetration that make them suitable for various biotechnological applications. These findings suggest that nanobody Nb25 identified herein could be a valuable alternative tool and has potential applications in diagnostic and basic research on ASFV.

Theileria annulata SVSP455 interacts with host HSP60.

BACKGROUND: Theileria annulata, a transforming parasite, invades bovine B cells, dendritic cells and macrophages, promoting the uncontrolled proliferation of these cells. This protozoan evolved intricate strategies to subvert host cell signaling pathways related to antiapoptotic signaling to enable survival and proliferation within the host cells. However, the molecular mechanisms of the cell transformation induced by T. annulata remain largely unclear. Although some studies have predicted that the subtelomere-encoded variable secreted protein (SVSP) family plays roles in host-parasite interactions, the evidence for this is limited. METHODS: In the present study, the SVSP455 (TA05545) gene, a member of the SVSP gene family, was used as the target molecule. The expression pattern of SVSP455 in different life-cycle stages of T. annulata infection was explored using a quantitative real-time PCR assay, and the subcellular distribution of SVSP455 was observed using confocal microscopy. The host cell proteins interacting with SVSP455 were screened using the Y2H system, and their interactions were verified in vivo and in vitro using both bimolecular fluorescence complementation and confocal microscopy, and co-immunoprecipitation assays. The role played by SVSP455 in cell transformation was further explored by using overexpression, RNA interference and drug treatment experiments. RESULTS: The highest level of the SVSP455 transcript was detected in the schizont stage of T. annulata, and the protein was located both on the surface of schizonts and in the host cell cytoplasm. In addition, the interaction between SVSP455 and heat shock protein 60 was shown in vitro, and their link may regulate host cell apoptosis in T. annulata-infected cells. CONCLUSION: Our findings are the first to reveal that T. annulata-secreted SVSP455 molecule directly interacts with both exogenous and endogenous bovine HSP60 protein, and that the interaction of SVSP455-HSP60 may manipulate the host cell apoptosis signaling pathway. These results provide insights into cancer-like phenotypes underlying Theilera transformation and therapeutics for protection against other pathogens.

Overexpression of PeNAC122 gene promotes wood formation and tolerance to osmotic stress in poplars.

Finding the adequate balance between wood formation and abiotic stress resistance is still an important challenge for industrial woody crops. In this study, PeNAC122, a member of the NAC transcription factor (TF) family highly expressed in xylem, was cloned from Populus euphratica. Tissue expression and ß-glucuronidase (GUS) staining showed that PeNAC122 was exclusively expressed in phloem fiber and secondary xylem of stems. Subcellular and yeast transactivation assays confirmed that PeNAC122 protein existed in the nucleus and did not have transcriptional activation and inhibitory activity. Overexpression of PeNAC122 poplar lines exhibited reduced plant height, thickened xylem, and accumulated lignin content in stems, and also upregulates the expression of secondary cell wall biosynthetic genes. Moreover, overexpression of PeNAC122 lines displayed more tolerance to PEG6000-induced osmotic stress, with stronger photosynthetic performance, higher antioxidant enzyme activity, and less accumulation of reactive oxygen species in leaves, and higher expression levels of stress response genes DREB2A, RD29, and NCED3. These results indicate that PeNAC122 plays a crucial role in wood formation and abiotic stress tolerance, which, in addition to potential use in improving wood quality, provides further insight into the role of NAC family TFs in balancing wood development and abiotic stress resistance.

Comparative genomic analysis of Babesia duncani responsible for human babesiosis.

BACKGROUND: Human babesiosis, caused by parasites of the genus Babesia, is an emerging and re-emerging tick-borne disease that is mainly transmitted by tick bites and infected blood transfusion. Babesia duncani has caused majority of human babesiosis in Canada; however, limited data are available to correlate its genomic information and biological features. RESULTS: We generated a B. duncani reference genome using Oxford Nanopore Technology (ONT) and Illumina sequencing technology and uncovered its biological features and phylogenetic relationship with other Apicomplexa parasites. Phylogenetic analyses revealed that B. duncani form a clade distinct from B. microti, Babesia spp. infective to bovine and ovine species, and Theileria spp. infective to bovines. We identified the largest species-specific gene family that could be applied as diagnostic markers for this pathogen. In addition, two gene families show signals of significant expansion and several genes that present signatures of positive selection in B. duncani, suggesting their possible roles in the capability of this parasite to infect humans or tick vectors. CONCLUSIONS: Using ONT sequencing and Illumina sequencing technologies, we provide the first B. duncani reference genome and confirm that B. duncani forms a phylogenetically distinct clade from other Piroplasm parasites. Comparative genomic analyses show that two gene families are significantly expanded in B. duncani and may play important roles in host cell invasion and virulence of B. duncani. Our study provides basic information for further exploring B. duncani features, such as host-parasite and tick-parasite interactions.