METTL1 mediated tRNA m7G modification promotes leukaemogenesis of AML via tRNA regulated translational control.

BACKGROUND: RNA modifications have been proven to play fundamental roles in regulating cellular biology process. Recently, maladjusted N7-methylguanosine (m7G) modification and its modifiers METTL1/WDR4 have been confirmed an oncogene role in multiple cancers. However, the functions and molecular mechanisms of METTL1/WDR4 in acute myeloid leukemia (AML) remain to be determined. METHODS: METTL1/WDR4 expression levels were quantified using qRT-PCR, western blot analysis on AML clinical samples, and bioinformatics analysis on publicly available AML datasets. CCK-8 assays and cell count assays were performed to determine cell proliferation. Flow cytometry assays were conducted to assess cell cycle and apoptosis rates. Multiple techniques were used for mechanism studies in vitro assays, such as northern blotting, liquid chromatography-coupled mass spectrometry (LC-MS/MS), tRNA stability analysis, transcriptome sequencing, small non-coding RNA sequencing, quantitative proteomics, and protein synthesis measurements. RESULTS: METTL1/WDR4 are significantly elevated in AML patients and associated with poor prognosis. METTL1 knockdown resulted in reduced cell proliferation and increased apoptosis in AML cells. Mechanically, METTL1 knockdown leads to significant decrease of m7G modification abundance on tRNA, which further destabilizes tRNAs and facilitates the biogenesis of tsRNAs in AML cells. In addition, profiling of nascent proteins revealed that METTL1 knockdown and transfection of total tRNAs that were isolated from METTL1 knockdown AML cells decreased global translation efficiency in AML cells. CONCLUSIONS: Taken together, our study demonstrates the important role of METTL1/WDR4 in AML leukaemogenesis, which provides a promising target candidate for AML therapy.

Metagenomic insights into the composition and function of the gut microbiota of mice infected with Toxoplasma gondii.

Introduction: Despite Toxoplasma gondii infection leading to dysbiosis and enteritis, the function of gut microbiota in toxoplasmosis has not been explored. Methods: Here, shotgun metagenomics was employed to characterize the composition and function of mouse microbial community during acute and chronic T. gondii infection, respectively. Results: The results revealed that the diversity of gut bacteria was decreased immediately after T. gondii infection, and was increased with the duration of infection. In addition, T. gondii infection led to gut microbiota dysbiosis both in acute and chronic infection periods. Therein, several signatures, including depression of Firmicutes to Bacteroidetes ratio and infection-enriched Proteobacteria, were observed in the chronic period, which may contribute to aggravated gut inflammation and disease severity. Functional analysis showed that a large amount of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and carbohydrate-active enzymes (CAZy) family displayed distinct variation in abundance between infected and healthy mice. The lipopolysaccharide biosynthesis related pathways were activated in the chronic infection period, which might lead to immune system imbalance and involve in intestinal inflammation. Moreover, microbial and functional spectrums were more disordered in chronic than acute infection periods, thus implying gut microbiota was more likely to participate in disease process in the chronically infected mice, even exacerbated immunologic derangement and disease progression. Discussion: Our data indicate that the gut microbiota plays a potentially important role in protecting mice from T. gondii infection, and contributes to better understand the association between gut microbiota and toxoplasmosis.

Unravelling Strain-Specific Modifications of Toxoplasma gondii tRNA and sncRNA Using LC-MS/MS.

Many RNA modifications have been detected in rRNA, tRNA and small noncoding RNA (sncRNA) as well as in low-abundance RNA species such mRNA. Although RNA modifications play roles in many cellular and biological processes in various domains of life, knowledge about the diversity and role of RNA modifications in Toxoplasma gondii is limited. In this study, RNA modifications in three T. gondii strains (RH type I, PRU type II, and VEG type III) with distinct virulence abilities were determined by liquid chromatography-tandem mass spectrometry. We compared the levels of modifications of four nucleotides in tRNA and sncRNA, characterized RNA modification patterns of different T. gondii strains, and determined the diversity of RNA modifications. We detected and quantified 22 modified nucleosides in both tRNA and sncRNA. Significant differences in the diversity of the modified nucleosides were found between the three T. gondii strains. RNA modifications were correlated with the expression of many T. gondii virulence proteins. Some of the identified modifications (e.g., 2'-O-methylinosine, pseudouridine) play a role in mediating the host-parasite interaction. These results provide novel insight into the global modifications in tRNA and sncRNA, and the diversity of RNA modifications between T. gondii strains with different virulence backgrounds. IMPORTANCE Although RNA modifications play roles in many cellular and developmental processes in various domains of life, knowledge about the patterns and functions of RNA modifications in T. gondii is limited. Here, a quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach was used to study global RNA modifications in T. gondii strains of distinct virulence backgrounds. We quantified 22 modified nucleosides in both tRNA and sncRNA. Significant T. gondii strain-specific differences in RNA modifications were detected. More tRNA modifications correlated with T. gondii virulence proteins than sncRNA modifications. RNA modifications were significantly correlated with virulence proteins. Our data provide the first comprehensive profiling of the modifications tRNA and sncRNA in T. gondii, expanding the diversity of RNA modifications in this parasite and suggesting new regulators for modulating its virulence.

Human circulating small non-coding RNA signature as a non-invasive biomarker in clinical diagnosis of acute myeloid leukaemia.

Background: Acute myeloid leukaemia (AML) is the most common acute leukaemia in adults; AML is highly heterogeneous and involves abnormalities at multiple omics levels. Small non-coding RNAs (sncRNAs) present in body fluids are important regulatory molecules and considered promising non-invasive clinical diagnostic biomarkers for disease. However, the signature of sncRNA profile alteration in AML patient serum and bone marrow supernatant is still under exploration. Methods: We examined data for blood and bone marrow samples from 80 consecutive, newly-diagnosed patients with AML and 12 healthy controls for high throughput small RNA-sequencing. Differentially expressed sncRNAs were analysed to reveal distinct patterns between AML patients and controls. Machine learning methods were used to evaluate the efficiency of specific sncRNAs in discriminating individuals with AML from controls. The altered expression level of individual sncRNAs was evaluated by RT-PCR, Q-PCR, and northern blot. Correlation analysis was employed to assess sncRNA patterns between serum and bone marrow supernatant. Results: We identified over 20 types of sncRNA categories beyond miRNAs in both serum and bone marrow supernatant, with highly coordinated expression patterns between them. Non-classical sncRNAs, including rsRNA (62.86%), ysRNA (14.97%), and tsRNA (4.22%), dominated among serum sncRNAs and showed sensitive alteration patterns in AML patients. According to machine learning-based algorithms, the tsRNA-based signature robustly discriminated subjects with AML from controls and was more reliable than that comprising miRNAs. Our data also showed that serum tsRNAs to be closely associated with AML prognosis, suggesting the potential application of serum tsRNAs as biomarkers to assist in AML diagnosis. Conclusions: We comprehensively characterized the expression pattern of circulating sncRNAs in blood and bone marrow and their alteration signature between healthy controls and AML patients. This study enriches research of sncRNAs in the regulation of AML, and provides insights into the role of sncRNAs in AML.

Hypoxia induces alterations in tRNA modifications involved in translational control.

BACKGROUND: Adaptation to high-altitude hypobaric hypoxia has been shown to require a set of physiological traits enabled by an associated set of genetic modifications, as well as transcriptome regulation. These lead to both lifetime adaptation of individuals to hypoxia at high altitudes and generational evolution of populations as seen for instance in those of Tibet. Additionally, RNA modifications, which are sensitive to environmental exposure, have been shown to play pivotal biological roles in maintaining the physiological functions of organs. However, the dynamic RNA modification landscape and related molecular mechanisms in mouse tissues under hypobaric hypoxia exposure remain to be fully understood. Here, we explore the tissue-specific distribution pattern of multiple RNA modifications across mouse tissues. RESULTS: By applying an LC-MS/MS-dependent RNA modification detection platform, we identified the distribution of multiple RNA modifications in total RNA, tRNA-enriched fragments, and 17-50-nt sncRNAs across mouse tissues; these patterns were associated with the expression levels of RNA modification modifiers in different tissues. Moreover, the tissue-specific abundance of RNA modifications was sensitively altered across different RNA groups in a simulated high-altitude (over 5500 m) hypobaric hypoxia mouse model with the activation of the hypoxia response in mouse peripheral blood and multiple tissues. RNase digestion experiments revealed that the alteration of RNA modification abundance under hypoxia exposure impacted the molecular stability of tissue total tRNA-enriched fragments and isolated individual tRNAs, such as tRNAAla, tRNAval, tRNAGlu, and tRNALeu. In vitro transfection experiments showed that the transfection of testis total tRNA-enriched fragments from the hypoxia group into GC-2spd cells attenuated the cell proliferation rate and led to a reduction in overall nascent protein synthesis in cells. CONCLUSIONS: Our results reveal that the abundance of RNA modifications for different classes of RNAs under physiological conditions is tissue-specific and responds to hypobaric hypoxia exposure in a tissue-specific manner. Mechanistically, the dysregulation of tRNA modifications under hypobaric hypoxia attenuated the cell proliferation rate, facilitated the sensitivity of tRNA to RNases, and led to a reduction in overall nascent protein synthesis, suggesting an active role of tRNA epitranscriptome alteration in the adaptive response to environmental hypoxia exposure.

Alteration of RNA modification signature in human sperm correlates with sperm motility.

RNA modifications, which are introduced post-transcriptionally, have recently been assigned pivotal roles in the regulation of spermatogenesis and embryonic development. However, the RNA modification landscape in human sperm is poorly characterized, hampering our understanding about the potential role played by RNA modification in sperm. Through our recently developed high-throughput RNA modification detection platform based on liquid chromatography with tandem mass spectroscopy, we are the first to have characterized the RNA modification signature in human sperm. The RNA modification signature was generated on the basis of 49 samples from participants, including 13 healthy controls, 21 patients with asthenozoospermia (AZS) and 15 patients with teratozoospermia (TZS). In total, we identified 13 types of RNA modification marks on the total RNA in sperm, and 16 types of RNA modification marks on sperm RNA fragments of different sizes. The levels of these RNA modifications on the RNA of patients with AZS or TZS were altered, compared to controls, especially on sperm RNA fragments > 80 nt. A few types of RNA modifications, such as m1G, m5C, m2G and m1A, showed clear co-expression patterns as well as high linear correlations with clinical sperm motility. In conclusion, we characterized the RNA modification signature of human sperm and identified its correlation with sperm motility, providing promising candidates for use in clinical sperm quality assessment and new research insights for exploring the underlying pathological mechanisms in human male infertility syndromes.

Alterations of RNA Modification in Mouse Germ Cell-2 Spermatids Under Hypoxic Stress.

Hypoxia is a known stress factor in mammals and has been shown to potentially impair male fertility, which manifests as spermatogenic dysfunction and decreased semen quality. Studies have shown that RNA modifications, the novel post-transcriptional regulators, are involved in spermatogenesis, and hypoxia-induced alterations in RNA modification in testes and sperm cells may be associated with impaired spermatogenesis in mice. However, the molecular mechanisms via which RNA modifications influence spermatogenesis under hypoxic stress conditions are unclear. In this study, we generated a mouse Germ Cell-2 spermatid (GC-2spd) hypoxia model by culturing cells in a 1% O2 incubator for 48 h or treating them with CoCl2 for 24 h. The hypoxia treatment significantly inhibited proliferation and induced apoptosis in GC-2spd cells. The RNA modification signatures of total RNAs (2 types) and differentially sized RNA fragments (7 types of approximately 80 nt-sized tRNAs; 9 types of 17-50 nt-sized sncRNAs) were altered, and tRNA stability was partially affected. Moreover, the expression profiles of sncRNAs, such as microRNAs, tsRNAs, rsRNAs, and ysRNAs, were significantly regulated, and this might be related to the alterations in RNA modification and subsequent transcriptomic changes. We comprehensively analyzed alterations in RNA modification signatures in total RNAs, tRNAs (approximately 80 nt), and small RNAs (17-50 nt) as well as the expression profiles of sncRNAs and transcriptomes in hypoxia-treated GC-2spd cells; our data suggested that RNA modifications may be involved in cellular responses under hypoxic stress conditions and could provide a basis for a better understanding of the molecular mechanisms underlying male infertility.

In vitro anti-Toxoplasma gondii efficacy of synthesised benzyltriazole derivatives.

Toxoplasma gondii, an obligate intracellular parasite, is the aetiological agent of toxoplasmosis, a disease that affects approximately 25% - 30% of the world's population. At present, no safe and effective vaccine exists for the prevention of toxoplasmosis. Current treatment options for toxoplasmosis are active only against tachyzoites and may also cause bone marrow toxicity. To contribute to the global search for novel agents for the treatment of toxoplasmosis, we herein report the in vitro activities of previously synthesised benzyltriazole derivatives. The effects of these compounds against T. gondii in vitro were evaluated by using a expressing green fluorescent protein (GFP) type I strain parasite (RH-GFP) and a type II cyst-forming strain of parasite (PruΔku80Δhxgprt). The frontline antitubercular drug isoniazid, designated as Frans J. Smit -isoniazid (FJS-INH), was also included in the screening as a preliminary test in view of future repurposing of this agent. Of the compounds screened, FJS-302, FJS-303, FJS-403 and FJS-INH demonstrated 80% parasite growth inhibition with IC50 values of 5.6 µg/mL, 6.8 µg/µL, 7.0 µg/mL and 19.8 µg/mL, respectively. FJS-302, FJS-303 and FJS-403 inhibited parasite invasion and replication, whereas, sulphadiazine (SFZ), the positive control, was only effective against parasite replication. In addition, SFZ induced bradyzoite differentiation in vitro, whilst FJS-302, FJS-303 and FJS-403 did not increase the bradyzoite number. These results indicate that FJS-302, FJS-303 and FJS-403 have the potential to act as a viable source of antiparasitic therapeutic agents.

Hypoxia-induced alteration of RNA modifications in the mouse testis and sperm†.

Hypobaric hypoxia as an extreme environment in a plateau may have deleterious effects on human health. Studies have indicated that rush entry into a plateau may reduce male fertility and manifest in decreased sperm counts and weakened sperm motility. RNA modifications are sensitive to environmental changes and have recently emerged as novel post-transcriptional regulators in male spermatogenesis and intergenerational epigenetic inheritance. In the present study, we generated a mouse hypoxia model simulating the environment of 5500 m in altitude for 35 days, which led to compromised spermatogenesis, decreased sperm counts, and an increased sperm deformation rate. Using this hypoxia model, we further applied our recently developed high-throughput RNA modification quantification platform based on liquid chromatography with tandem mass spectrometry, which exhibited the capacity to simultaneously examine 25 types of RNA modifications. Our results revealed an altered sperm RNA modifications signature in the testis (6 types) and mature sperm (11 types) under the hypoxia model, with 4 types showing overlap (Am, Gm, m7G, and m22G). Our data first drew the signature of RNA modification profiles and comprehensively analyzed the alteration of RNA modification levels in mouse testis and sperm under a mouse hypoxia model. These data may be highly related to human conditions under a similar hypoxia environment.

PLK:Δgra9 Live Attenuated Strain Induces Protective Immunity Against Acute and Chronic Toxoplasmosis.

Toxoplasmosis is a zoonotic parasitic disease caused by the obligate intracellular protozoa Toxoplasma gondii, which threatens a range of warm-blooded mammals including humans. To date, it remains a challenge to find safe and effective drug treatment or vaccine against toxoplasmosis. In this study, our results found that the development of a mutant strain based on gene disruption of dense granule protein 9 (gra9) in type II PLK strain decreased parasite replication in vivo, severely attenuated virulence in mice, and significantly reduced the formation of cysts in animals. Hence, we developed an immunization scheme to evaluate the protective immunity of the attenuated strain of Δgra9 in type II PLK parasite as a live attenuated vaccine against toxoplasmosis in the mouse model. Δgra9 vaccination-induced full immune responses characterized by significantly high levels of pro-inflammatory cytokine interferon gamma (IFN-γ) and interleukin-12 (IL-12), maintained the high T. gondii-specific immunoglobulin G (IgG) level, and mixed high IgG1/IgG2a levels. Their levels provided the complete protective immunity which is a combination of cellular and humoral immunity in mouse models against further infections of lethal doses of type I RH, type II PLK wild-type tachyzoites, or type II PLK cysts. Results showed that Δgra9 vaccination proved its immunogenicity and potency conferring 100% protection against acute and chronic T. gondii challenges. Together, Δgra9 vaccination provided safe and efficient immune protection against challenging parasites, suggesting that PLK:Δgra9 is a potentially promising live attenuated vaccine candidate.

Development and evaluation of an enzyme-linked immunosorbent assay based on recombinant TgSRS2 for serodiagnosis of Toxoplasma gondii infection in cats.

An enzyme-linked immunosorbent assay (ELISA) based on recombinant SAG1-related sequence 2 of Toxoplasma gondii (rTgSRS2) was developed to detect toxoplasmosis in cats. The specificity and sensitivity of rTgSRS2 ELISA were confirmed using a series of serum samples from T. gondii-experimentally infected mice. A total of 76 field samples from cats were examined by the developed ELISA. The rTgSRS2 ELISA showed a good diagnostic performance characterized by high concordance (88.16) and kappa value (0.76) with latex agglutination test (LAT). The sensitivity and specificity of the test were 92.68% and 82.86%, respectively. These results suggest that the ELISA based on rTgSRS2 could be a useful tool for serodiagnosis of T. gondii infection in cats.

Babesia microti Confers Macrophage-Based Cross-Protective Immunity Against Murine Malaria.

Malaria and babesiosis, the two primary intraerythrocytic protozoan diseases of humans, have been reported in multiple cases of co-infection in endemic regions. As the geographic range and incidence of arthropod-borne infectious diseases is being affected by climate change, co-infection cases with Plasmodium and Babesia are likely to increase. The two parasites have been used in experimental settings, where prior infection with Babesia microti has been shown to protect against fatal malarial infections in mice and primates. However, the immunological mechanisms behind such phenomena of cross-protection remain unknown. Here, we investigated the effect of a primary B. microti infection on the outcome of a lethal P. chabaudi challenge infection using a murine model. Simultaneous infection with both pathogens led to high mortality rates in immunocompetent BALB/c mice, similar to control mice infected with P. chabaudi alone. On the other hand, mice with various stages of B. microti primary infection were thoroughly immune to a subsequent P. chabaudi challenge. Protected mice exhibited decreased levels of serum antibodies and pro-inflammatory cytokines during early stages of challenge infection. Mice repeatedly immunized with dead B. microti quickly succumbed to P. chabaudi infection, despite induction of high antibody responses. Notably, cross-protection was observed in mice lacking functional B and T lymphocytes. When the role of other innate immune effector cells was examined, NK cell-depleted mice with chronic B. microti infection were also found to be protected against P. chabaudi. Conversely, in vivo macrophage depletion rendered the mice vulnerable to P. chabaudi. The above results show that the mechanism of cross-protection conferred by B. microti against P. chabaudi is innate immunity-based, and suggest that it relies predominantly upon the function of macrophages. Further research is needed for elucidating the malaria-suppressing effects of babesiosis, with a vision toward development of novel tools to control malaria.

Drug screening of food and drug administration-approved compounds against Babesia bovis in vitro.

Babesia (B.) bovis is one of the main etiological agents of bovine babesiosis, causes serious economic losses to the cattle industry. Control of bovine babesiosis has been hindered by the limited treatment selection for B. bovis, thus, new options are urgently needed. We explored the drug library and unbiasedly screened 640 food and drug administration (FDA) approved drug compounds for their inhibitory activities against B. bovis in vitro. The initial screening identified 13 potentially effective compounds. Four potent compounds, namely mycophenolic acid (MPA), pentamidine (PTD), doxorubicin hydrochloride (DBH) and vorinostat (SAHA) exhibited the lowest IC50 and then selected for further evaluation of their in vitro efficacies using viability, combination inhibitory and cytotoxicity assays. The half-maximal inhibitory concentration (IC50) values of MPA, PTD, DBH, SAHA were 11.38 ± 1.66, 13.12 ± 4.29, 1.79 ± 0.15 and 45.18 ± 7.37 µM, respectively. Of note, DBH exhibited IC50 lower than that calculated for the commonly used antibabesial drug, diminazene aceturate (DA). The viability result revealed the ability of MPA, PTD, DBH, SAHA to prevent the regrowth of treated parasite at 4 × and 2 × of IC50. Antagonistic interactions against B. bovis were observed after treatment with either MPA, PTD, DBH or SAHA in combination with DA. Our findings indicate the richness of FDA approved compounds by novel potent antibabesial candidates and the identified potent compounds especially DBH might be used for the treatment of animal babesiosis caused by B. bovis.

Hydroxylamine and Carboxymethoxylamine Can Inhibit Toxoplasma gondii Growth through an Aspartate Aminotransferase-Independent Pathway.

Toxoplasma gondii is an obligate intracellular protozoan parasite and a successful parasitic pathogen in diverse organisms and host cell types. Hydroxylamine (HYD) and carboxymethoxylamine (CAR) have been reported as inhibitors of aspartate aminotransferases (AATs) and interfere with the proliferation in Plasmodium falciparum Therefore, AATs are suggested as drug targets against Plasmodium The T. gondii genome encodes only one predicted AAT in both T. gondii type I strain RH and type II strain PLK. However, the effects of HYD and CAR, as well as their relationship with AAT, on T. gondii remain unclear. In this study, we found that HYD and CAR impaired the lytic cycle of T. gondiiin vitro, including the inhibition of invasion or reinvasion, intracellular replication, and egress. Importantly, HYD and CAR could control acute toxoplasmosis in vivo Further studies showed that HYD and CAR could inhibit the transamination activity of rTgAAT in vitro However, our results confirmed that deficiency of AAT in both RH and PLK did not reduce the virulence in mice, although the growth ability of the parasites was affected in vitro HYD and CAR could still inhibit the growth of AAT-deficient parasites. These findings indicated that HYD and CAR inhibition of T. gondii growth and control of toxoplasmosis can occur in an AAT-independent pathway. Overall, further studies focusing on the elucidation of the mechanism of inhibition are warranted. Our study hints at new substrates of HYD and CAR as potential drug targets to inhibit T. gondii growth.

Molecular investigation of tick-borne infections in cattle from Xinjiang Uygur Autonomous Region, China.

Tick-borne diseases cause significant losses to livestock production in tropical and subtropical regions. However, information about the tick-borne infections in cattle in Xinjiang Uygur Autonomous Region (XUAR), northwestern China, is scarce. In this study, nested polymerase chain reaction (PCR) assays and gene sequencing were used to detect and analyze epidemiological features of Babesia bovis, B. bigemina, Coxiella burnetii and Anaplasma bovis infections in XUAR. Out of 195 samples tested, 24 (12.3%), 67 (34.4%), 40 (20.5%) and 10 (5.1%) were positive for B. bovis, B. bigemina, C. burnetii and A. bovis, respectively. Sequencing analysis indicated that B. bovis SBP-4, B. bigemina Rap1a, C. burnetii htpB and A. bovis 16S rRNA genes from XUAR showed 99%-100% identity with documented isolates from other countries. Phylogenetic analyses revealed that B. bovis SBP-4, B. bigemina Rap1a, C. burnetii htpB and A. bovis 16S rRNA gene sequences clustered in the same clade with isolates from other countries. To the best of our knowledge, this is the first report of C. burnetii infection of cattle in XUAR. Furthermore, this study provides important data for understanding the distribution of tick-borne pathogens, and is expected to improve the approach for prevention and control of tick-borne diseases in China.

First description of Coxiella burnetii and Rickettsia spp. infection and molecular detection of piroplasma co-infecting horses in Xinjiang Uygur Autonomous Region, China.

Q fever, spotted fever rickettsioses and equine piroplasmosis, are some of the most serious equine tick-borne diseases caused by Coxiella burnetii, Rickettsia spp., Babesia caballi and/or Theileria equi. This study surveyed and molecularly characterized these pathogens infecting horses in ten ranches from XUAR, China using molecular technology. Among 200 horse blood samples, 163 (81.5%) were infected with at least one of the pathogens. Rickettsia spp. was the most prevalent pathogen (n = 114, 57.0%), followed by C. burnetii (n = 79, 39.5%), T. equi (n = 79, 39.5%) and B. caballi (n = 49, 24.5%). Co-infections were observed in 61.3% of positive samples in this study. Statistically significant differences were observed between the sampling regions for C. burnetii, B. caballi and T. equi, and also in different age group for C. burnetii and T. equi. The genotype analysis indicated that C. burnetii htpB, Rickettsia spp. ompA, B. caballi rap-1, B. caballi 18S rRNA, T. equi EMA-1 and T. equi 18S rRNA gene sequences from horses in XUAR were variable. To the best of our knowledge, this study is the first report of C. burnetii and Rickettsia spp. infection and co-infected with piroplasma in horses in China. Overall, this study revealed the high infection rate of the pathogens in horses in XUAR, China. The current findings are expected to provide a basis for better tick-borne disease control in the region.

Effects of KIF2A on the prognosis of nasopharyngeal carcinoma and nasopharyngeal carcinoma cells.

Nasopharyngeal carcinoma (NPC) is a common tumor in south China. Kinesin family member 2A (KIF2A) belongs to the kinesin-13 family and is associated with the growth and invasion of a number of different types of human cancer, including ovarian, breast and prostate cancer. The aim of the present study was to evaluate the expression of KIF2A in NPC and explore the relationship between KIF2A and the basic characteristics of 5-8F cells. Immunohistochemistry was performed on tissues from 97 patients with NPC to assess KIF2A protein expression. KIF2A was knocked down by a specific short interfering (si)RNA in 5-8F cell lines. Cell proliferation, apoptosis and cycle were analyzed by MTT assay and flow cytometry. The invasive ability and angiogenesis were evaluated by Matrigel assay and reverse transcription-quantitative PCR. The level of KIF2A was associated with the growth and migration of primary tumor, nodal status and tumor stage. The viability of KIF2A-knockdown cells was decreased compared with that of the control cells. The number of apoptotic cells, as well as the percentage of cells in the G0/G1 phase, was higher in the KIF2A siRNA group compared with the control group. The invasive and angiogenetic ability of 5-8F cells in the KIF2A siRNA group was decreased compared with the control group. In conclusion, the expression of KIF2A correlated with the poor clinicopathological features in NPC. Therefore, KIF2A may serve an important role in the progression of NPC and proliferation of 5-8F cells, which might present a potential therapeutic target for patients with NPC.

Molecular detection and genetic characterisation of pathogenic Theileria, Anaplasma and Ehrlichia species among apparently healthy sheep in central and western Kenya.

Tick-borne diseases (TBDs) caused by Theileria, Babesia, Anaplasma and Ehrlichia species are common in tropical and subtropical regions. In this study, we investigated the presence and genetic diversity of Theileria spp., Anaplasma ovis, B. ovis, E. ruminantium and Anaplasma spp. in sheep from the Machakos and Homa Bay counties of Kenya. In order to improve the diagnosis and control of ovine TBDs, a total of 76 blood samples from apparently healthy sheep were screened using a polymerase chain reaction (PCR). The assays were conducted using primers based on Theileria spp. 18S rRNA, Anaplasma ovis Major surface protein-4 (AoMSP4), B. ovis 18S rRNA, E. ruminantium pCS20 and Anaplasma spp. 16S rRNA. The overall infection rates for Theileria spp., A. ovis, E. ruminantium and Anaplasma spp. were 39/76 (51.3%), 26/76 (34.2%), 6/76 (7.9%) and 31/76 (40.8%), respectively. The overall co-infection was 47/76 (61.8%). All Theileria spp. positive samples were confirmed to be of Theileria ovis on sequencing. A phylogenetic analysis of the 18S rRNA gene sequences of T. ovis revealed that all isolates of this study clustered with T. ovis sequences extracted from the GenBank suggesting this gene is highly conserved. E. ruminantium pCS20 sequences were in the same clade on the phylogenetic tree. However, three AoMSP4 sequences from this study appeared in the same clade, while one sequence formed a separate branch revealing genetic divergence. The 16S rRNA sequencing revealed uncharacterised Anaplasma spp. and A. ovis. The phylogenetic analyses of the uncharacterised Anaplasma spp. revealed that the two sequences from this study appear in an independent clade from other sequences extracted from the GenBank. This study provides important information regarding the occurrence of tick-borne pathogens and their degree of genetic diversity among sheep in Kenya, which is useful for the diagnosis and control of TBDs.

Molecular evidence of hemotropic mycoplasmas in goats from Cebu, Philippines.

Hemotropic mycoplasma (hemoplasma), a neglected vector-borne pathogen in goats, causes extensive economic damage to farmers due to production losses. In this study, 107/295 (36.27%) goats sampled from 4 farms (Barili, Danao City, Dumanjug and Minglanilla) in Cebu, Philippines tested positive for PCR targeting the 16S rRNA gene of Mycoplasma. All hemoplasma-positive goats were from Barili and no clinical sign was observed. Sex (P=0.0005) and age (P=0.03) were found associated with hemoplasma infection. Mycoplasma ovis, Candidatus Mycoplasma haemobos, Candidatus Mycoplasma haemominutum and 3 Uncultured Mycoplasma sp. sequences were identified by sequencing analysis. This is the first report of molecular detection and genetic characterization of hemoplasmas in goats in the Philippines.

First molecular detection and characterization of tick-borne pathogens in water buffaloes in Bohol, Philippines.

The water buffalo industry is a vital part of the Philippine livestock economy and is an essential contributor to the developing local dairy industry. Although relatively less susceptible to diseases, water buffaloes can still be infected and can act as reservoirs of tick-borne pathogens (TBPs). However, limited information is available regarding the prevalence of tick-borne infections in water buffaloes in the Philippines. This study was conducted to identify TBPs harbored by water buffaloes and to characterize these pathogens molecularly. One hundred water buffalo blood samples collected from three areas in Bohol, Visayas region, Philippines were screened for various TBPs using pathogen-specific PCR assays. TBPs were detected in 46% of the samples (39% singly infected, 7% coinfected). The pathogens detected were Anaplasma marginale (29%), Babesia bovis (21%), and B. bigemina (3%). None of the blood samples were positive for Theileria annulata, T. orientalis, and B. ovata. A. marginale infection rates were significantly higher (37.5%) among water buffaloes aged ≤6 years (P = 0.046) than those >6 years old (18.2%) and was detected only in Bulgarian Murrah (36.1%) and US Murrah (25.9%) breeds. Phylogenetic analyses revealed that groEL sequences of A. marginale were 100% identical with isolates from the Philippines (Batangas and Cebu) and China. Two B. bigemina RAP-1a gene sequences were identical to each other and were homologous with previous isolates from Thailand, Indonesia, Uruguay, and the Philippines. Moreover, four B. bovis SBP-2 partial sequences obtained in this study had 92.4-99.7% identities. This study is the first molecular detection and characterization of A. marginale, B. bigemina and B. bovis in water buffaloes in the Visayas region, and the first molecular confirmation of B. bovis infection in water buffaloes in the country. The findings presented in this study may serve as baseline data for crafting effective tick-borne disease surveillance and prevention programs in Bohol and in the Philippines.