Although many vector-borne agents are potential zoonoses and cause substantial morbidity and mortality in dogs worldwide, there are limited data on these organisms in dogs of China. Quantitative PCRs for vector-borne agents were performed to investigate their prevalences in convenience whole blood samples obtained from 1114 dogs from 21 veterinary clinics and a commercial dog breeding facility in ten provinces of China. In addition, the PCRs were performed on 146 Rhipicephalus sanguineus senso lato and 37 Linognathus setosus collected from dogs in the commercial dog breeding facility. DNAs of Babesia gibsoni and B. vogeli (1.2 %), Ehrlichia canis (1.3 %), Hepatozoon canis (1.8 %) and Theileria orientalis (0.1 %) or a closely related organism were detected in the bloods of the dogs studied, and Babesia vogeli (3.4 %) and Ehrlichia canis (4.1 %) in R. sanguineus senso lato. The qPCRs for Anaplasma spp., Dirofilaria immitis and Leishmania spp. were negative for all blood samples, ticks and lice. At least one vector-borne agent was found in dogs from 5 of the 10 provinces investigated in this study. Overall, 4.4 % (49/1117) of the dogs studied were positive for at least one vector-borne agent with the prevalence being highest in the commercial breeding colony (24/97; 24.7 %). Our study confirms that B. vogeli, B. gibsoni, H. canis, and E. canis occur in China. Also, we present evidence that T. orientalis or a closely related organism can infect dogs.

The Ehrlichia are obligate intracellular Gram-negative tick-borne bacteria that are important human and animal pathogens. There is a need for assays to rapidly and reliably detect and differentiate the five generally recognized species into groups in a single reaction: E. canis, E. chaffeensis, E. ewingii, E. muris and E. ruminantium. We developed primers and probes against the 16S rRNA gene to enable us to reliably detect the five major Ehrlichia spp. in a single FRET-qPCR. We tested the Ehrlichia FRET-qPCR on reference strains and on DNA from the blood of domestic ruminants from five Caribbean islands. The Ehrlichia present were determined using melting point analysis and by sequencing the Ehrlichia FRET-qPCR products as well as those of a nested PCR against the citrate synthase gene (gltA). Our Ehrlichia FRET-qPCR was negative for the closely related Anaplasma marginale and A. phagocytophilum but gave positive reactions with reference strains of the most generally recognized species and with other less characterized Ehrlichia of domestic ruminants, mainly E. ovina, the Panola Mountain Ehrlichia, and Ehrlichia sp. BOV2010. Melting point analysis revealed 4 distinct groups: E. ruminantium (T m ~55.8 °C); E. chaffeensis and E. ewingii (T m ~57.7 °C); E. canis, E. muris, E. ovina and Ehrlichia sp. BOV 2010 (T m ~62.0 °C); and the Panola Mountain Ehrlichia (T m ~65.5 °C). The detection limit of the FRET-qPCR was ~ 5 gene copies in a reaction and the sequences of the FRET-qPCR products were as expected. With DNA from domestic ruminants from the Caribbean we found 12.2 % (134/1,101) positive: cattle (76/385; 19.7 %), sheep (45/340; 13.2 %) and goats (13/376; 3.5 %). Melting point analysis and sequencing of the FRET-qPCR and nested PCR gltA products showed the Ehrlichia we detected were E. canis or very closely related organisms. In a single reaction, our Ehrlichia FRET-qPCR can detect the Ehrlichia spp. we studied and differentiate them into four groups. Domestic ruminants in the Caribbean are not uncommonly exposed to Ehrlichia, possibly E. canis or very closely related organisms.

Polymerase chain reaction (PCR) is a widely used technique in gene expression analysis, diagnostics, and various molecular biology applications. However, the accuracy and sensitivity of PCR can be compromised by primer–template mismatches, potentially leading to erroneous results. In this study, we strategically designed 111 primer–template combinations with varying numbers, types, and locations of mismatches to meticulously assess their impact on qPCR performance while two distinctly different types of DNA polymerases were used. Notably, when a single-nucleotide mismatch occurred at the 3’ end of the primer, we observed significant decreases in the analytical sensitivity (0–4%) with Invitrogen™ Platinum™ Taq DNA Polymerase High Fidelity, while the analytical sensitivity remained unchanged with Takara Ex Taq Hot Start Version DNA Polymerase. Leveraging these findings, we designed a highly specific PCR to amplify Babesia while effectively avoiding the genetically close Theileria. Through elucidating the critical interplay between types of DNA polymerases and primer–template mismatches, this research provides valuable insights for improving PCR accuracy and performance. These findings have important implications for researchers aiming to achieve robust qPCR results in various molecular biology applications.

Chemotherapy remains the standard treatment for triple‐negative breast cancer (TNBC); however, chemoresistance compromises its efficacy. The RNA‐binding protein Hu antigen R (HuR) could be a potential therapeutic target to enhance the chemotherapy efficacy. HuR is known to mainly stabilize its target mRNAs, and/or promote the translation of encoded proteins, which are implicated in multiple cancer hallmarks, including chemoresistance. In this study, a docetaxel‐resistant cell subline (231‐TR) was established from the human TNBC cell line MDA‐MB‐231. Both the parental and resistant cell lines exhibited similar sensitivity to the small molecule functional inhibitor of HuR, KH‐3. Docetaxel and KH‐3 combination therapy synergistically inhibited cell proliferation in TNBC cells and tumor growth in three animal models. KH‐3 downregulated the expression levels of HuR targets (e.g., β‐Catenin and BCL2) in a time‐ and dose‐dependent manner. Moreover, KH‐3 restored docetaxel's effects on activating Caspase‐3 and cleaving PARP in 231‐TR cells, induced apoptotic cell death, and caused S‐phase cell cycle arrest. Together, our findings suggest that HuR is a critical mediator of docetaxel resistance and provide a rationale for combining HuR inhibitors and chemotherapeutic agents to enhance chemotherapy efficacy.

Triple-negative breast cancer (TNBC) presents therapeutic challenges due to limited targeted treatment options and resistance to chemotherapy drugs, such as doxorubicin. This study investigated doxorubicin resistance mechanisms and a strategy to overcome it. A doxorubicin-resistant cell subline (231-DR) was developed from MDA-MB-231 TNBC cells, and enhanced expression of cellular FLICE-inhibitory protein (cFLIP) in 231-DR cells was identified as a potential driver of the resistance. Overexpression of cFLIP conferred resistance to doxorubicin-induced apoptosis, whereas siRNA-mediated cFLIP depletion induced apoptosis, particularly in 231-DR cells. Furthermore, the RNA-binding protein Hu antigen R (HuR) was found to regulate cFLIP expression. HuR Inhibition with KH-3 or RNA interference reduced cFLIP levels. Importantly, KH-3 sensitized TNBC cells to doxorubicin-induced apoptosis. In summary, this study delineates cFLIP’s role in mediating doxorubicin resistance and identifies HuR as a positive regulator of cFLIP, offering a novel therapeutic avenue against chemoresistance in TNBC by combining HuR inhibition with doxorubicin.

Ehrlichia chaffeensis causes human monocytic ehrlichiosis. Little is known about how this and other related tick-borne rickettsia pathogens maintain pH homeostasis in acidified phagosomes and the extracellular milieu. The membrane-bound sodium (cation)/proton antiporters are found in a wide range of organisms aiding pH homeostasis. We recently reported a mutation in an antiporter gene of E. chaffeensis (ECH_0379) which causes bacterial in vivo attenuation. The E. chaffeensis genome contains 10 protein coding sequences encoding for predicted antiporters. We report here that nine of these genes are transcribed during the bacterial growth in macrophages and tick cells. All E. chaffeensis antiporter genes functionally complemented antiporter deficient Escherichia coli. Antiporter activity for all predicted E. chaffeensis genes was observed at pH 5.5, while gene products of ECH_0179 and ECH_0379 were also active at pH 8.0, and ECH_0179 protein was complemented at pH 7.0. The antiporter activity was independently verified for the ECH_0379 protein by proteoliposome diffusion analysis. This is the first description of antiporters in E. chaffeensis and demonstrates that the pathogen contains multiple antiporters with varying biological functions, which are likely important for the pH homeostasis of the pathogen’s replicating and infectious forms.