Estimation of Linear Functionals in High-Dimensional Linear Models: From Sparsity to Nonsparsity.

High dimensional linear models are commonly used in practice. In many applications, one is interested in linear transformations ß âŠ¤ x of regression coefficients ß ∈ R p , where x is a specific point and is not required to be identically distributed as the training data. One common approach is the plug-in technique which first estimates ß , then plugs the estimator in the linear transformation for prediction. Despite its popularity, estimation of ß can be difficult for high dimensional problems. Commonly used assumptions in the literature include that the signal of coefficients ß is sparse and predictors are weakly correlated. These assumptions, however, may not be easily verified, and can be violated in practice. When ß is non-sparse or predictors are strongly correlated, estimation of ß can be very difficult. In this paper, we propose a novel pointwise estimator for linear transformations of ß . This new estimator greatly relaxes the common assumptions for high dimensional problems, and is adaptive to the degree of sparsity of ß and strength of correlations among the predictors. In particular, ß can be sparse or non-sparse and predictors can be strongly or weakly correlated. The proposed method is simple for implementation. Numerical and theoretical results demonstrate the competitive advantages of the proposed method for a wide range of problems.

Drug resistance and genetic characteristics of one Eimeria tenella isolate from Xiantao, Hubei Province, China.

Chicken coccidiosis causes retarded growth and low production performance in poultry, resulting in huge economic losses to the poultry industry. In order to prevent and control chicken coccidiosis, great efforts have been made to develop new drugs and vaccines, which require pure isolates of Eimeria spp. In this study, we obtained the Eimeira tenella Xiantao isolate by single oocyst isolation technology and compared its genome with the reference genome GCF_000499545.2_ETH001 of the Houghton strain. The results of the comparative genomic analysis indicated that the genome of this isolate contained 46,888 single nucleotide polymorphisms (SNPs). There were 15,107 small insertion and deletion variations (indels), 1693 structural variations (SV), and 3578 copy number variations (CNV). In addition, 64 broilers were used to determine the resistance profile of Xiantao strain. Drug susceptibility testing revealed that this isolate was completely resistant to monensin, diclazuril, halofuginone, sulfachlorpyrazine sodium, and toltrazuril, but sensitive to decoquinate. These data improve our understanding of drug resistance in avian coccidia.

Identification and molecular characterization of a novel Babesia orientalis rhoptry neck protein 4 (BoRON4).

Babesia orientalis, a protozoan parasite transmitted by the tick Rhipicephalus haemaphysaloides, holds significant economic importance along the Yangtze River. Key factors in the host invasion process include rhoptry neck proteins (RON2, RON4, and RON5) and apical membrane antigen 1 (AMA1). However, the intricacies of the interaction between AMA1 and RONs remain incompletely elucidated in B. orientalis. To better understand these crucial invasion components, the RON4 gene of B. orientalis (BoRON4) was cloned and sequenced. RON4 is 3468 base pairs long, encodes 1155 amino acids, and has a predicted molecular weight of 130 kDa. Bioinformatics analysis revealed a unique region (amino acid residues 109-452) in BoRON4, which demonstrates higher sensitivity to epitope activity. The BoRON4 gene was strategically truncated, amplified, and cloned into the pGEX-6p-1 vector for fusion expression. We successfully used the mouse polyclonal antibody to identify native BoRON4 in B. orientalis lysates. Furthermore, the corresponding BoRON4 protein band was detected in the water buffalo serum infected with B. orientalis, while no such band was observed in the control. Additionally, I-TASSER and Discovery Studio software were used to predict the tertiary structures of BoRON4 and its ligands, CH-PKA and CH-complex. These ligands can serve as lead compounds for the development of anti-babesiosis drugs. In conclusion, BoRON4 emerges as a promising candidate antigen for distinguishing water buffalo infected with B. orientalis from their normal counterparts. This study positions BoRON4 as a potential diagnostic antigen for babesiosis in water buffalo, contributing valuable insights to the field of parasitology.

Anticoccidial activity of a botanical natural product based on eucalyptus, apigenin and eugenol against Eimeria tenella in broiler chickens.

BACKGROUND: Chicken coccidiosis is an intracellular parasitic disease that presents major challenges to the development of the commercial poultry industry. Perennial drug selective pressure has led to the multi-drug resistance of chicken coccidia, which makes the prevention and control of chicken coccidiosis extremely difficult. In recent years, natural plant products have attracted the attention of researchers due to their inherent advantages, such as the absence of veterinary drug residues. The development of these natural products provides a new direction for the prevention and treatment of chicken coccidiosis. METHODS: The anticoccidial effect of a natural plant product combination formulation (eucalyptus oil + apigenin + eugenol essential oil) was tested against Eimeria tenella in broilers. To search for the optimal concentration of the combination formulation, we screened 120 broilers in a chicken cage trial in which 100 broilers were infected with 5 × 104 sporulated Eimeria tenella oocysts; broilers receiving a decoquinate solution was set up as a chemical control. The optimal anticoccidial concentration was determined by calculating the anticoccidial index (ACI), and the suitable concentration was used as the recommended dose for a series of safety dose assessment tests, such as feed conversion ratio (FCR), hematological indices and serum biochemical indices, as well as liver and kidney sections, at onefold (low dose), threefold (medium dose) and sixfold (high dose) the recommended dose (RD). RESULTS: The results showed that this combination formulation of three plant natural products had a better anticoccidial effect than formulations containing two plant natural products or a single one, with an ACI of 169.3. The dose gradient anticoccidial test revealed that the high-dose formulation group had a better anticoccidial effect (ACI = 169.2) than the medium- and low-dose groups. The safety evaluation test showed that concentrations of the formulation at one-, three- and sixfold the RD were non-toxic to Arbor Acres broilers, indicating the high safety of the combination formulation. CONCLUSIONS: The combination formulation showed not only a moderate anticoccidial effect but also had a high safety profile for broilers. The results of this study indicate a new alternative for the prevention and control of coccidiosis in broilers.

Transplantation of miR-145a-5p modified M2 type microglia promotes the tissue repair of spinal cord injury in mice.

BACKGROUND: The traumatic spinal cord injury (SCI) can cause immediate multi-faceted function loss or paralysis. Microglia, as one of tissue resident macrophages, has been reported to play a critical role in regulating inflammation response during SCI processes. And transplantation with M2 microglia into SCI mice promotes recovery of motor function. However, the M2 microglia can be easily re-educated and changed their phenotype due to the stimuli of tissue microenvironment. This study aimed to find a way to maintain the function of M2 microglia, which could exert an anti-inflammatory and pro-repair role, and further promote the repair of spinal cord injury. METHODS: To establish a standard murine spinal cord clip compression model using Dumont tying forceps. Using FACS, to sort microglia from C57BL/6 mice or CX3CR1GFP mice, and further culture them in vitro with different macrophage polarized medium. Also, to isolate primary microglia using density gradient centrifugation with the neonatal mice. To transfect miR-145a-5p into M2 microglia by Lipofectamine2000, and inject miR-145a-5p modified M2 microglia into the lesion sites of spinal cord for cell transplanted therapy. To evaluate the recovery of motor function in SCI mice through behavior analysis, immunofluorescence or histochemistry staining, Western blot and qRT-PCR detection. Application of reporter assay and molecular biology experiments to reveal the mechanism of miR-145a-5p modified M2 microglia therapy on SCI mice. RESULTS: With in vitro experiments, we found that miR-145a-5p was highly expressed in M2 microglia, and miR-145a-5p overexpression could suppress M1 while promote M2 microglia polarization. And then delivery of miR-145a-5p overexpressed M2 microglia into the injured spinal cord area significantly accelerated locomotive recovery as well as prevented glia scar formation and neuron damage in mice, which was even better than M2 microglia transplantation. Further mechanisms showed that overexpressed miR-145a-5p in microglia inhibited the inflammatory response and maintained M2 macrophage phenotype by targeting TLR4/NF-κB signaling. CONCLUSIONS: These findings indicate that transplantation of miR-145a-5p modified M2 microglia has more therapeutic potential for SCI than M2 microglia transplantation from epigenetic perspective.

Glioma-Stem-Cell-Derived Exosomes Remodeled Glioma-Associated Macrophage via NEAT1/miR-125a/STAT3 Pathway.

Glioblastoma (GBM), as the most common primary brain tumor, usually results in an extremely poor prognosis, in which glioma stem cells (GSCs) and their immunosuppressive microenvironment prominently intervene in the resistance to radiotherapy and chemotherapy that directly leads to tumor recurrence and shortened survival time. The specific mechanism through which exosomes generated from GSCs support the creation of an immunosuppressive microenvironment remains unknown, while it is acknowledged to be engaged in intercellular communication and the regulation of the glioma immunosuppressive microenvironment. The elevated expression of LncRNA-NEAT1 was found in glioma cells after radiotherapy, chemotherapy, and DNA damage stimulation, and NEAT1 could promote the malignant biological activities of GSCs. Emerging evidence suggests that lncRNAs may reply to external stimuli or DNA damage by playing a role in modulating different aspects of tumor biology. Our study demonstrated a promotive role of the carried NEAT1 by GSC-derived exosomes in the polarization of M2-like macrophages. Further experiments demonstrated the mediative role of miR-125a and its target gene STAT3 in NEAT1-induced polarization of M2-like macrophages that promote glioma progression. Our findings elucidate the mechanism by which GSCs influence the polarization of M2-like macrophages through exosomes, which may contribute to the formation of immunosuppressive microenvironments. Taken together, our study reveals the miR-125a-STAT3 pathway through which exosomal NEAT1 from treatment-resistant GSCs contributes to M2-like macrophage polarization, indicating the potential of exosomal NEAT1 for treating glioma.

Transfection of Babesia duncani: A Genetic Toolbox of this Pathogen to Advance Babesia Biology.

Human babesiosis is a tick-borne disease caused by Babesia pathogens. The disease, which presents with malaria-like symptoms, can be life-threatening, especially in individuals with weakened immune systems and the elderly. The worldwide prevalence of human babesiosis has been gradually rising, prompting alarm among public health experts. In other pathogens, genetic techniques have proven to be valuable tools for conducting functional studies to understand the importance of specific genes in development and pathogenesis as well as to validate novel cellular targets for drug discovery. Genetic manipulation methods have been established for several non-human Babesia and Theileria species and, more recently, have begun to be developed for human Babesia parasites. We have previously reported the development of a method for genetic manipulation of the human pathogen Babesia duncani. This method is based on positive selection using the hDHFR gene as a selectable marker, whose expression is regulated by the ef-1aB promoter, along with homology regions that facilitate integration into the gene of interest through homologous recombination. Herein, we provide a detailed description of the steps needed to implement this strategy in B. duncani to study gene function. It is anticipated that the implementation of this method will significantly improve our understanding of babesiosis and facilitate the development of novel and more effective therapeutic strategies for the treatment of human babesiosis. Key features This protocol provides an effective means of transfection of B. duncani, enabling genetic manipulation and editing to gain further insights into its biology and pathogenesis. The protocol outlined here for the electroporation of B. duncani represents an advancement over previous methods used for B. bovis [1]. Improvements include higher volume of culture used during the electroporation step and an enhancement in the number of electroporation pulses. These modifications likely enhance the efficiency of gene editing in B. duncani, allowing for quicker and more effective selection of transgenic parasites.

Babesia duncani Pyruvate Kinase Inhibitor Screening and Identification of Key Active Amino Acid Residues.

Babesia duncani (B. duncani), a protozoan parasite prevalent in North America, is a significant threat for human health. Given the regulatory role of pyruvate kinase I (PyK I) in glycolytic metabolism flux and ATP generation, PyK I has been considered the target for drug intervention for a long time. In this study, B. duncani PyK I (BdPyK I) was successfully cloned, expressed, and purified. Polyclonal antibodies were confirmed to recognize the native BdPyK I protein (56 kDa) using Western blotting. AlphaFold software predicted the three-dimensional structure of BdPyK I, and molecular docking with small molecules was conducted to identify potential binding sites of inhibitor on BdPyK I. Moreover, inhibitory effects of six inhibitors (tannic acid, apigenin, shikonin, PKM2 inhibitor, rosiglitazone, and pioglitazone) on BdPyK I were examined under the optimal enzymatic conditions of 3 mM PEP and 3 mM ADP, and significant activity reduction was found. Enzyme kinetics and growth inhibition assays further confirmed the reliability of these inhibitors, with PKM2 inhibitor, tannic acid, and apigenin exhibiting the highest selectivity index as specific inhibitors for B. duncani. Subsequently, key amino acid residues were mutated in both BdPyK I and Homo sapiens pyruvate kinase I (HPyK I), and two differential amino acid residues (isoleucine and phenylalanine) were identified between HPyK I and BdPyK I through PyK activity detection experiments. These findings lay foundation for understanding the role of PyK I in the growth and development of B. duncani, providing insights for babesiosis prevention and drug development.

Isolation, Identification, Genomic Diversity, and Antimicrobial Resistance Analysis of Streptococcus suis in Hubei Province of China from 2021 to 2023.

Streptococcus suis (S. suis) is a zoonotic pathogen capable of causing severe diseases in humans and pigs, including meningitis, sepsis, polyserositis, arthritis, and endocarditis. This study aimed to investigate the biological characteristics of 19 strains of S. suis isolated from diseased pigs in Hubei Province between 2021 and 2023. Through bioinformatics analysis, we investigated the serotype, MLST, pan-genome characteristics, SNP, AMR, and ICE of the 19 S. suis isolates. Among the 19 S. suis strains, ten serotypes were identified, and serotype 9 was the most prevalent (21.05%). Ten new alleles and nine new sequence types (STs) were discovered, with ST28 and ST243 emerging as the predominant STs. The results of the pan-genomic analysis of S. suis indicate that there are 943 core genes, 2259 shell genes, and 5663 cloud genes. Through SNP evolutionary analysis, we identified a strong genetic similarity between SS31 and the reference genome P1/7. The analysis of antibiotic resistance genes revealed widespread presence of erm(B) and tet(O) genes among 19 strains of S. suis. This association may be linked to the high resistance of S. suis to lincosamides, macrolides, and tetracyclines. Integrative and conjugative elements (ICEs) and integrative and mobilizable elements (IMEs) were identified in 16 strains, with a carriage rate of 84.21%, and resistance genes were identified within the ICE/IME elements of 8 strains. Antimicrobial susceptibility testing revealed that all strains showed sensitivity to vancomycin and lincomycin but resistance to tilmicosin, tiamulin, amoxicillin, and doxycycline. This study contributes to our understanding of the genomic diversity of S. suis in Hubei Province of China, providing essential data for the comprehensive prevention and control of S. suis infections in China.

A novel easy-to-desorb eluant contributes to address environmental contamination of African swine fever virus.

African swine fever virus (ASFV) is a highly pathogenic and rapidly disseminated virus with strong viability in the environment, suggesting the importance of environmental detection for prevention and control in all the pig industry. However, the detection results of environmental swabs cannot always reflect the accurate status of viral pollution, leading to persistent ASFV environmental contamination. In this study, we developed an ASFV eluant with higher environmental ASFV detection efficiency relative to 0.85% saline solution, which obtains the patent certificate issued by the China Intellectual Property Office (patent number:202010976050.9). qPCR analysis showed that in the environmental swab samples, the number of viral copies was 100 times higher for the ASFV eluant treatment than the traditional eluant treatment (0.85% saline solution). And besides, the high sensitivity of the ASFV eluant had be verified in a slaughterhouse environmental sampling detection. In soil samples, the ASFV eluent showed the same extraction effect as the TIANamp Soil DNA Kit, in contrast to no extraction effect for 0.85% saline solution. Simultaneously, this eluent could protect ASFV from degradation and allow the transportation of samples at ambient temperature without refrigeration. In clinical practice, we monitored the environmental contamination condition of the ASFV in a large-scale pig farm. The results shown that the ASFV load decreased after every disinfection in environment. This study provides an effective solution for surveilling the potential threat of ASFV in environment.

Phenotypic comparison and the potential antitumor function of immortalized bone marrow-derived macrophages (iBMDMs).

Introduction: Macrophages are an important component of innate immunity and involved in the immune regulation of multiple diseases. The functional diversity and plasticity make macrophages to exhibit different polarization phenotypes after different stimuli. During tumor progression, the M2-like polarized tumor-associated macrophages (TAMs) promote tumor progression by assisting immune escape, facilitating tumor cell metastasis, and switching tumor angiogenesis. Our previous studies demonstrated that functional remodeling of TAMs through engineered-modifying or gene-editing provides the potential immunotherapy for tumor. However, lack of proliferation capacity and maintained immune memory of infused macrophages restricts the application of macrophage-based therapeutic strategies in the repressive tumor immune microenvironment (TIME). Although J2 retrovirus infection enabled immortalization of bone marrow-derived macrophages (iBMDMs) and facilitated the mechanisms exploration and application, little is known about the phenotypic and functional differences among multi kinds of macrophages. Methods: HE staining was used to detect the biosafety of iBMDMs, and real-time quantitative PCR, immunofluorescence staining, and ELISA were used to detect the polarization response and expression of chemokines in iBMDMs. Flow cytometry, scratch assay, real-time quantitative PCR, and crystal violet staining were used to analyze its phagocytic function, as well as its impact on tumor cell migration, proliferation, and apoptosis. Not only that, the inhibitory effect of iBMDMs on tumor growth was detected through subcutaneous tumor loading, while the tumor tissue was paraffin sectioned and flow cytometry was used to detect its impact on the tumor microenvironment. Results: In this study, we demonstrated iBMDMs exhibited the features of rapid proliferation and long-term survival. We also compared iBMDMs with RAW264.7 cell line and mouse primary BMDMs with in vitro and in vivo experiments, indicating that the iBMDMs could undergo the same polarization response as normal macrophages with no obvious cellular morphology changes after polarization. What's more, iBMDMs owned stronger phagocytosis and pro-apoptosis functions on tumor cells. In addition, M1-polarized iBMDMs could maintain the anti-tumor phenotypes and domesticated the recruited macrophages of receptor mice, which further improved the TIME and repressed tumor growth. Discussion: iBMDMs can serve as a good object for the function and mechanism study of macrophages and the optional source of macrophage immunotherapy.

Novel Strategy for In Vitro Validation of Babesia orientalis Heat Shock Proteins Chaperone Activity and Thermostability.

BACKGROUND: Babesia orientalis is an intra-erythrocytic protozoan parasite that causes babesiosis in water buffalo. The genome of B. orientalis has been reported and various genes have been accurately annotated, including heat shock proteins (HSP). Three B. orientalis HSPs (HSP90, HSP70 and HSP20) have been previously identified as potential antigenic targets. Here, a new validation strategy for the chaperone activities and cell protection characteristics of the three HSPs was developed in vitro. METHODS: BoHSP20, BoHSP70 and BoHSP90B were amplified from cDNA, followed by cloning them into the pEGFP-N1 vector and transfecting the vector plasmid separately into 293T and Hela mammalian cells. Their expression and localization were determined by fluorescence microscopy. The biological functions and protein stability were testified through an analysis of the fluorescence intensity duration. Their role in the protection of cell viability from heat-shock treatments was examined by MTT assay (cell proliferation assay based on thiazolyl blue tetrazolium bromide). RESULTS: Fusion proteins pEGFP-N1-BoHSP20, pEGFP-N1-BoHSP70, and pEGFP-N1-BoHSP90B (pBoHSPs: pBoHSP20; pBoHSP70 and pBoHSP90B) were identified as 47 kDa/97 kDa/118 kDa with a 27 kDa GFP tag, respectively. Prolonged fluorescent protein half-time was observed specifically in pBoHSPs under heat shock treatment at 55 °C, and BoHSP20 showed relatively better thermotolerance than BoHSP70 and BoHSP90B. Significant difference was found between pBoHSPs and controls in the cell survival curve after 2 h of 45 °C heat shock. CONCLUSION: Significant biological properties of heat stress-associated genes of B. orientalis were identified in eukaryote by a new strategy. Fusion proteins pBoHSP20, pBoHSP70 and pBoHSP90B showed good chaperone activity and thermo-stability in this study, implying that BoHSPs played a key role in protecting B. orientalis against heat-stress environment during parasite life cycle. In conclusion, the in vitro model explored in this study provides a new way to investigate the biological functions of B. orientalis proteins during the host-parasite interaction.

Cold to Hot: Tumor Immunotherapy by Promoting Vascular Normalization Based on PDGFB Nanocomposites.

Immunotherapy is a promising cancer therapeutic strategy. However, the "cold" tumor immune microenvironment (TIME), characterized by insufficient immune cell infiltration and immunosuppressive status, limits the efficacy of immunotherapy. Tumor vascular abnormalities due to defective pericyte coverage are gradually recognized as a profound determinant in "cold" TIME establishment by hindering immune cell trafficking. Recently, several vascular normalization strategies by improving pericyte coverage have been reported, whereas have unsatisfactory efficacy and high rates of resistance. Herein, a combinatorial strategy to induce tumor vasculature-targeted pericyte recruitment and zinc ion-mediated immune activation with a platelet-derived growth factor B (PDGFB)-loaded, cyclo (Arg-Gly-Asp-D-Phe-Lys)-modified zeolitic imidazolate framework 8 (PDGFB@ZIF8-RGD) nanoplatform is proposed. PDGFB@ZIF8-RGD effectively induced tumor vascular normalization, which facilitated trafficking and infiltration of immune effector cells, including natural killer (NK) cells, M1-like macrophages and CD8+ T cells, into tumor microenvironment. Simultaneously, vascular normalization promoted the accumulation of zinc ions inside tumors to trigger effector cell immune activation and effector molecule production. The synergy between these two effects endowed PDGFB@ZIF8-RGD with superior capabilities in reprogramming the "cold" TIME to a "hot" TIME, thereby initiating robust antitumor immunity and suppressing tumor growth. This combinatorial strategy for improving immune effector cell infiltration and activation is a promising paradigm for solid tumor immunotherapy.

A Subunit Vaccine Based on the VP2 Protein of Porcine Parvovirus 1 Induces a Strong Protective Effect in Pregnant Gilts.

Porcine parvovirus 1 (PPV1) is one of the most prevalent pathogens that can cause reproductive disorder in sows. The VP2 protein of PPV1 is the most important immunogenic protein that induces neutralizing antibodies and protective immunity. Thus, VP2 is considered an ideal target antigen for the development of a genetically engineered PPV1 vaccine. In this study, the baculovirus transfer vector carrying the HR5-P10-VP2 expression cassette was successfully constructed with the aim of increasing the expression levels of the VP2 protein. The VP2 protein was confirmed using SDS‒PAGE and Western blot analyses. Electronic microscope analysis showed that the recombinant VP2 proteins were capable of self-assembling into VLPs with a diameter of approximately 25 nm. The immunogenicity of the VP2 subunit vaccine was evaluated in pigs. The results showed that VP2 protein emulsified with ISA 201VG adjuvant induced higher levels of HI antibodies and neutralizing antibodies than VP2 protein emulsified with IMS 1313VG adjuvant. Furthermore, the gilts immunized with the ISA 201VG 20 µg subunit vaccine acquired complete protection against PPV1 HN2019 infection. In contrast, the commercial inactivated vaccine provided incomplete protection in gilts. Therefore, the VP2 subunit vaccine is a promising genetically engineered vaccine for the prevention and control of PPV1.

Bioinspired porous three-coordinated single-atom Fe nanozyme with oxidase-like activity for tumor visual identification via glutathione.

Inspired by structures of natural metalloenzymes, a biomimetic synthetic strategy is developed for scalable synthesis of porous Fe-N3 single atom nanozymes (pFeSAN) using hemoglobin as Fe-source and template. pFeSAN delivers 3.3- and 8791-fold higher oxidase-like activity than Fe-N4 and Fe3O4 nanozymes. The high catalytic performance is attributed to (1) the suppressed aggregation of atomically dispersed Fe; (2) facilitated mass transfer and maximized exposure of active sites for the created mesopores by thermal removal of hemoglobin (2 ~ 3 nm); and (3) unique electronic configuration of Fe-N3 for the oxygen-to-water oxidation pathway (analogy with natural cytochrome c oxidase). The pFeSAN is successfully demonstrated for the rapid colorimetric detection of glutathione with a low limit of detection (2.4 nM) and wide range (50 nM-1 mM), and further developed as a real-time, facile, rapid (~6 min) and precise visualization analysis methodology of tumors via glutathione level, showing its potentials for diagnostic and clinic applications.

Myeloid-specific blockade of notch signaling alleviates dopaminergic neurodegeneration in Parkinson's disease by dominantly regulating resident microglia activation through NF-κB signaling.

Yolk sac-derived microglia and peripheral monocyte-derived macrophages play a key role during Parkinson's disease (PD) progression. However, the regulatory mechanism of microglia/macrophage activation and function in PD pathogenesis remains unclear. Recombination signal-binding protein Jκ (RBP-J)-mediated Notch signaling regulates macrophage development and activation. In this study, with an 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) hydrochloride-induced acute murine PD model, we found that Notch signaling was activated in amoeboid microglia accompanied by a decrease in tyrosine hydroxylase (TH)-positive neurons. Furthermore, using myeloid-specific RBP-J knockout (RBP-JcKO) mice combined with a PD model, our results showed that myeloid-specific disruption of RBP-J alleviated dopaminergic neurodegeneration and improved locomotor activity. Fluorescence-activated cell sorting (FACS) analysis showed that the number of infiltrated inflammatory macrophages and activated major histocompatibility complex (MHC) II+ microglia decreased in RBP-JcKO mice compared with control mice. Moreover, to block monocyte recruitment by using chemokine (C-C motif) receptor 2 (CCR2) knockout mice, the effect of RBP-J deficiency on dopaminergic neurodegeneration was not affected, indicating that Notch signaling might regulate neuroinflammation independent of CCR2+ monocyte infiltration. Notably, when microglia were depleted with the PLX5622 formulated diet, we found that myeloid-specific RBP-J knockout resulted in more TH+ neurons and fewer activated microglia. Ex vitro experiments demonstrated that RBP-J deficiency in microglia might reduce inflammatory factor secretion, TH+ neuron apoptosis, and p65 nuclear translocation. Collectively, our study first revealed that RBP-J-mediated Notch signaling might participate in PD progression by mainly regulating microglia activation through nuclear factor kappa-B (NF-κB) signaling.

Transcriptional variation in Babesia gibsoni (Wuhan isolate) between in vivo and in vitro cultures in blood stage.

BACKGROUND: Babesia gibsoni, the causative agent of canine babesiosis, belongs to the phylum Apicomplexa. The development of in vitro culture technology has driven research progress in various kinds of omics studies, including transcriptomic analysis of Plasmodium spp. between in vitro and in vivo environments, which has prompted the observation of diagnostic antigens and vaccine development. Nevertheless, no information on Babesia spp. could be obtained in this respect, which greatly hinders the further understanding of parasite growth and development in the blood stage. METHODS: In this study, considerable changes in the morphology and infectivity of continuous in vitro cultured B. gibsoni (Wuhan isolate) were observed compared to in vivo parasites. Based on these changes, B. gibsoni (Wuhan isolate) was collected from both in vivo and in vitro cultures, followed by total RNA extraction and Illumina transcriptome sequencing. The acquired differentially expressed genes (DEGs) were validated using qRT-PCR, and then functionally annotated through several databases. The gene with the greatest upregulation after in vitro culture was cloned from the genome of B. gibsoni (Wuhan isolate) and characterized by western blotting and indirect immunofluorescence assay for detecting the native form and cellular localization. RESULTS: Through laboratory cultivation, multiple forms of parasites were observed, and the infectivity of in vitro cultured parasites in dogs was found to be lower. Based on these changes, Illumina transcriptome sequencing was conducted, showing that 377 unigenes were upregulated and 334 unigenes were downregulated. Notably, an AP2 transcription factor family, essential for all developmental stages of parasites, was screened, and the transcriptional changes in these family members were tested. Thus, the novel AP2 transcription factor gene (BgAP2-M) with the highest upregulated expression after in vitro adaptation was selected. This gene comprises an open reading frame (ORF) of 1989 base pairs encoding a full-length protein of 662 amino acids. BgAP2-M contains one AP2 domain and one ACDC conserved domain, which may be involved in the nuclear biology of parasites. The prepared polyclonal antibodies against the BgAP2-M peptides further detected a native size of ~ 73 kDa and were localized to the nuclei of B. gibsoni. CONCLUSION: This study presents a thorough transcriptome analysis of B. gibsoni in vivo and in vitro for the first time, contributing to a detailed understanding of the effects of environmental changes on the growth and development of parasites in the blood stage. Moreover, it also provides a deeper investigation for the different members of the ApiAP2 transcription factor family as various life stage regulators in Babesia spp.