Young osteocyte-derived extracellular vesicles facilitate osteogenesis by transferring tropomyosin-1.

BACKGROUND: Bone marrow mesenchymal stem cells (BMSCs) can undergo inadequate osteogenesis or excessive adipogenesis as they age due to changes in the bone microenvironment, ultimately resulting in decreased bone density and elevated risk of fractures in senile osteoporosis. This study aims to investigate the effects of osteocyte senescence on the bone microenvironment and its influence on BMSCs during aging. RESULTS: Primary osteocytes were isolated from 2-month-old and 16-month-old mice to obtain young osteocyte-derived extracellular vesicles (YO-EVs) and senescent osteocyte-derived EVs (SO-EVs), respectively. YO-EVs were found to significantly increase alkaline phosphatase activity, mineralization deposition, and the expression of osteogenesis-related genes in BMSCs, while SO-EVs promoted BMSC adipogenesis. Neither YO-EVs nor SO-EVs exerted an effect on the osteoclastogenesis of primary macrophages/monocytes. Our constructed transgenic mice, designed to trace osteocyte-derived EV distribution, revealed abundant osteocyte-derived EVs embedded in the bone matrix. Moreover, mature osteoclasts were found to release osteocyte-derived EVs from bone slices, playing a pivotal role in regulating the functions of the surrounding culture medium. Following intravenous injection into young and elderly mouse models, YO-EVs demonstrated a significant enhancement of bone mass and biomechanical strength compared to SO-EVs. Immunostaining of bone sections revealed that YO-EV treatment augmented the number of osteoblasts on the bone surface, while SO-EV treatment promoted adipocyte formation in the bone marrow. Proteomics analysis of YO-EVs and SO-EVs showed that tropomyosin-1 (TPM1) was enriched in YO-EVs, which increased the matrix stiffness of BMSCs, consequently promoting osteogenesis. Specifically, the siRNA-mediated depletion of Tpm1 eliminated pro-osteogenic activity of YO-EVs both in vitro and in vivo. CONCLUSIONS: Our findings suggested that YO-EVs played a crucial role in maintaining the balance between bone resorption and formation, and their pro-osteogenic activity declining with aging. Therefore, YO-EVs and the delivered TPM1 hold potential as therapeutic targets for senile osteoporosis.

Omentin-1 inhibits the development of benign prostatic hyperplasia by attenuating local inflammation.

BACKGROUND: Benign prostatic hyperplasia (BPH) is a prevalent disease affecting elderly men, with chronic inflammation being a critical factor in its development. Omentin-1, also known as intelectin-1 (ITLN-1), is an anti-inflammatory protein primarily found in the epithelial cells of the small intestine. This study aimed to investigate the potential of ITLN-1 in mitigating BPH by modulating local inflammation in the prostate gland. METHODS: Our investigation involved two in vivo experimental models. Firstly, ITLN-1 knockout mice (Itln-1-/-) were used to study the absence of ITLN-1 in BPH development. Secondly, a testosterone propionate (TP)-induced BPH mouse model was treated with an ITLN-1 overexpressing adenovirus. We assessed BPH severity using prostate weight index and histological analysis, including H&E staining, immunohistochemistry, and enzyme-linked immunosorbent assay. In vitro, the impact of ITLN-1 on BPH-1 cell proliferation and inflammatory response was evaluated using cell proliferation assays and enzyme-linked immunosorbent assay. RESULTS: In vivo, Itln-1-/- mice exhibited elevated prostate weight index, enlarged lumen area, and higher TNF-α levels compared to wild-type littermates. In contrast, ITLN-1 overexpression in TP-induced BPH mice resulted in reduced prostate weight index, lumen area, and TNF-α levels. In vitro studies indicated that ITLN-1 suppressed the proliferation of prostate epithelial cells and reduced TNF-α production in macrophages, suggesting a mechanism involving the inhibition of macrophage-mediated inflammation. CONCLUSION: The study demonstrates that ITLN-1 plays a significant role in inhibiting the development of BPH by reducing local inflammation in the prostate gland. These findings highlight the potential of ITLN-1 as a therapeutic target in the management of BPH.

Aptamer-functionalized hydrogels promote bone healing by selectively recruiting endogenous bone marrow mesenchymal stem cells.

Bone regeneration heavily relies on bone marrow mesenchymal stem cells (BMSCs). However, recruiting endogenous BMSCs for in situ bone regeneration remains challenging. In this study, we developed a novel BMSC-aptamer (BMSC-apt) functionalized hydrogel (BMSC-aptgel) and evaluated its functions in recruiting BMSCs and promoting bone regeneration. The functional hydrogels were synthesized between maleimide-terminated 4-arm polyethylene glycols (PEG) and thiol-flanked PEG crosslinker, allowing rapid in situ gel formation. The aldehyde group-modified BMSC-apt was covalently bonded to a thiol-flanked PEG crosslinker to produce high-density aptamer coverage on the hydrogel surface. In vitro and in vivo studies demonstrated that the BMSC-aptgel significantly increased BMSC recruitment, migration, osteogenic differentiation, and biocompatibility. In vivo fluorescence tomography imaging demonstrated that functionalized hydrogels effectively recruited DiR-labeled BMSCs at the fracture site. Consequently, a mouse femur fracture model significantly enhanced new bone formation and mineralization. The aggregated BMSCs stimulated bone regeneration by balancing osteogenic and osteoclastic activities and reduced the local inflammatory response via paracrine effects. This study's findings suggest that the BMSC-aptgel can be a promising and effective strategy for promoting in situ bone regeneration.

Metformin accelerates bone fracture healing by promoting type H vessel formation through inhibition of YAP1/TAZ expression.

Due to increasing morbidity worldwide, fractures are becoming an emerging public health concern. This study aimed to investigate the effect of metformin on the healing of osteoporotic as well as normal fractures. Type H vessels have recently been identified as a bone-specific vascular subtype that supports osteogenesis. Here, we show that metformin accelerated fracture healing in both osteoporotic and normal mice. Moreover, metformin promoted angiogenesis in vitro under hypoxia as well as type H vessel formation throughout fracture healing. Mechanistically, metformin increased the expression of HIF-1α, an important positive regulator of type H vessel formation, by inhibiting the expression of YAP1/TAZ in calluses and hypoxia-cultured human microvascular endothelial cells (HMECs). The results of HIF-1α or YAP1/TAZ interference in hypoxia-cultured HMECs using siRNA further suggested that the enhancement of HIF-1α and its target genes by metformin is primarily through YAP1/TAZ inhibition. Finally, overexpression of YAP1/TAZ partially counteracted the effect of metformin in promoting type H vessel-induced angiogenesis-osteogenesis coupling during fracture repair. In summary, our findings suggest that metformin has the potential to be a therapeutic agent for fractures by promoting type H vessel formation through YAP1/TAZ inhibition.

Acquisition, loss, and replication of functional modules promote the genetic diversity of Salmonella bacteriophages.

Owing to the threats that Salmonella poses to public health and the abuse of antimicrobials, bacteriophage therapy against Salmonella is experiencing a resurgence. Although several phages have been reported as safe and efficient for controlling Salmonella, the genetic diversity and relatedness among Salmonella phages remain poorly understood. In this study, whole-genome sequences of 91 Salmonella bacteriophages were obtained from the National Center for Biological Information genome database. Phylogenetic analysis, mosaic structure comparisons, gene content analysis, and orthologue group clustering were performed. Phylogenetic analysis revealed four singletons and two major lineages (I-II), including five subdividing clades, of which Salmonella phages belonging to morphologically distinct families were clustered in the same clade. Chimeric structures (n = 31), holin genes (n = 18), lysin genes (n = 66), DNA packaging genes (n = 55), and DNA metabolism genes (n = 24) were present in these phages. Moreover, phages from different subdivided clusters harboured distinct genes associated with host cell lysis, DNA packaging, and DNA metabolism. Notably, phages belonging to morphologically distinct families shared common orthologue groups. Although several functional modules of phages SS1 and SE16 shared > 99% nucleotide sequence identity with phages SI2 and SI23, the major differences between these phages were the absence and replication of functional modules. The data obtained herein revealed the genetic diversity of Salmonella phages at genomic, structural, and gene content levels. The genetic diversity of Salmonella phages is likely owing to the acquisition, loss, and replication of functional modules.

WGS analysis of two Staphylococcus aureus bacteriophages from sewage in China provides insights into the genetic feature of highly efficient lytic phages.

The study of bacteriophages is experiencing a resurgence with the increasing development of antimicrobial resistance in Staphylococcus aureus. Nonetheless, the genetic features of highly efficient lytic S. aureus phage remain to be explored. In this study, two lytic S. aureus phages, SapYZU11 and SapYZU15, were isolated from sewage samples from Yangzhou, China. The phage morphology, one-step growth, host spectrum and lytic activity of these phages were examined, and their whole-genome sequences were analysed and compared with 280 published genomes of staphylococcal phages. The structural organisation and genetic contents of SapYZU11 and SapYZU15 were investigated. The Podoviridae phage SapYZU11 and Herelleviridae phage SapYZU15 effectively lysed all of the 53 S. aureus strains isolated from various sources. However, SapYZU15 exhibited a shorter latent period, larger burst size and stronger bactericidal ability with an anti-bacterial rate of approximately 99.9999% for 24 h. Phylogenetic analysis revealed that Herelleviridae phages formed the most ancestral clades and the S. aureus Podoviridae phages were clustered in the staphylococcal Siphoviridae phage clade. Moreover, phages in different morphology families contain distinct types of genes associated with host cell lysis, DNA packaging and lysogeny. Notably, SapYZU15 harboured 13 DNA metabolism-related genes, 5 lysin genes, 1 holin gene and 1 DNA packaging gene. The data suggest that S. aureus Podoviridae and Siphoviridae phages originated from staphylococcal Herelleviridae phages, and the module exchange of S. aureus phages occurred in the same morphology family. Moreover, the extraordinary lytic capacity of SapYZU15 was likely due to the presence of specific genes associated with DNA replication, DNA packaging and the lytic cycle.

African Swine Fever Virus Cysteine Protease pS273R Inhibits Type I Interferon Signaling by Mediating STAT2 Degradation.

African swine fever virus (ASFV) is a large DNA virus that causes African swine fever (ASF), an acute and hemorrhagic disease in pigs with lethality rates of up to 100%. To date, how ASFV efficiently suppress the innate immune response remains enigmatic. In this study, we identified ASFV cysteine protease pS273R as an antagonist of type I interferon (IFN). Overexpression of pS273R inhibited JAK-STAT signaling triggered by type I IFNs. Mechanistically, pS273R interacted with STAT2 and recruited the E3 ubiquitin ligase DCST1, resulting in K48-linked polyubiquitination at K55 of STAT2 and subsequent proteasome-dependent degradation of STAT2. Furthermore, such a function of pS273R in JAK-STAT signaling is not dependent on its protease activity. These findings suggest that ASFV pS273R is important to evade host innate immunity. IMPORTANCE ASF is an acute disease in domestic pigs caused by infection with ASFV. ASF has become a global threat with devastating economic and ecological consequences. To date, there are no commercially available, safe, and efficacious vaccines to prevent ASFV infection. ASFV has evolved a series of strategies to evade host immune responses, facilitating its replication and transmission. Therefore, understanding the immune evasion mechanism of ASFV is helpful for the development of prevention and control measures for ASF. Here, we identified ASFV cysteine protease pS273R as an antagonist of type I IFNs. ASFV pS273R interacted with STAT2 and mediated degradation of STAT2, a transcription factor downstream of type I IFNs that is responsible for induction of various IFN-stimulated genes. pS273R recruited the E3 ubiquitin ligase DCST1 to enhance K48-linked polyubiquitination of STAT2 at K55 in a manner independent of its protease activity. These findings suggest that pS273R is important for ASFV to escape host innate immunity, which sheds new light on the mechanisms of ASFV immune evasion.

Tumor Growth Rate in Spinal Giant Cell Tumors of Bone and Association With the Immune Microenvironment and Denosumab Treatment Responsiveness: A Multicenter Study.

BACKGROUND: Currently, little is known about the prognostic value of tumor growth rate (TGR) in spinal giant cell tumors of bone (GCTB). OBJECTIVE: To investigate the correlation of TGR with clinicopathological features, immune microenvironment, prognosis, and response to denosumab treatment of spinal GCTB. METHODS: A total of 128 patients with spinal GCTB treated at 5 centers from 2011 to 2021 were included. TGR was assessed by 2 independent neuroradiologists using at least 2 preoperative thin-section magnetic resonance imaging scans at a minimum interval of 2 months. Immunohistochemistry was used to assess tumor-infiltrating lymphocyte subtypes for CD3, CD4, CD8, CD20, PD-1, PD-L1, and Foxp3. Then, these parameters were analyzed for their associations with patient outcomes (progression-free survival and overall survival), clinicopathological features, and denosumab treatment responsiveness. RESULTS: High TGR predicted both poor progression-free survival and overall survival (both P < .001). In addition, TGR was associated with postoperative neurological dysfunction ( P < .001), Enneking staging ( P = .016), denosumab treatment responsiveness ( P = .035), and the number of CD3 + ( P < .001), PD-1 + ( P = .009), PD-L1 + ( P < .001), and FoxP3 + tumor-infiltrating lymphocyte ( P = .02). Importantly, TGR outperformed the traditional Enneking, Campanacci, and American Joint Committee on Cancer staging systems in predicting the clinical outcomes of spinal GCTB. CONCLUSION: These data support the use of TGR as a reliable predictive tool for clinically relevant outcomes and response to denosumab therapy of spinal GCTB, which may be helpful in guiding prognostic risk stratification and therapeutic optimization of patients.

Safety and protective effects of an avirulent Salmonella Gallinarum isolate as a vaccine candidate against Salmonella Gallinarum infections in young chickens.

Fowl typhoid is an important disease of chickens and turkeys, which is caused by Salmonella Gallinarum (S. Gallinarum). Vaccines with high levels of protective effects against fowl typhoid need to be developed for the poultry industry. In this study, a S. Gallinarum strain, named SG01, was isolated from a poultry farm in Mashan region of Wuxi City, China, and identified through biochemical tests and specific PCR amplifications. Then, safety evaluations of the SG01 strain were performed in young chickens. No clinical symptom including depression and diarrhea and gross lesion involved in the cardiac nodule, hepatic necrotic lesion and splenic necrotic lesion, was determined on fifteen-day-old chickens after immunization with 1 × 1010 CFU of the SG01 strain through the oral route. However, diarrhea symptoms and hepatic lesions were identified from chickens immunized with the commercial vaccine strain SG9R by the same dose and route. At 14 days post inoculation, SG01 strain was eliminated in the liver and spleen from SG01-immunized chickens, while the SG9R strain still could be identified from SG9R-immunized chickens. After challenge with the virulent S. Gallinarum strain, significant reduction of the morbidity rate was found in the SG01 immunized group (20 %) compared to the challenge group (100 %) according to signs scoring systems for clinical symptoms and gross lesions. Additionally, immunization with the SG01 strain could provide more than 8 weeks of protection periods against fowl typhoid. These results demonstrate the SG01 strain is avirulent to young chickens and might be safer compared to the SG9R strain. In addition, SG01 strain is a potential vaccine candidate against fowl typhoid in young chickens.

Ångstrom-scale silver particles potently combat SARS-CoV-2 infection by suppressing the ACE2 expression and inflammatory responses.

The SARS-CoV-2 pandemic has become a severe global public health event, and the development of protective and therapeutic strategies is urgently needed. Downregulation of angiotensin converting enzyme 2 (ACE2; one of the important SARS-CoV-2 entry receptors) and aberrant inflammatory responses (cytokine storm) are the main targets to inhibit and control COVID-19 invasion. Silver nanomaterials have well-known pharmaceutical properties, including antiviral, antibacterial, and anticancer properties. Here, based on a self-established metal evaporation-condensation-size graded collection system, smaller silver particles reaching the Ångstrom scale (AgÅPs) were fabricated and coated with fructose to obtain a stabilized AgÅP solution (F-AgÅPs). F-AgÅPs potently inactivated SARS-CoV-2 and prevented viral infection. Considering the application of anti-SARS-CoV-2, a sterilized F-AgÅP solution was produced via spray formulation. In our model, the F-AgÅP spray downregulated ACE2 expression and attenuated proinflammatory factors. Moreover, F-AgÅPs were found to be rapidly eliminated to avoid respiratory and systemic toxicity in this study as well as our previous studies. This work presents a safe and potent anti-SARS-CoV-2 agent using an F-AgÅP spray.

Discrete element modeling of particles sphericity effect on sand direct shear performance.

Particle surface morphology is an important factor influencing sand structure and mechanical properties. In this study, the effect of sand particle sphericity on sand direct shear performance is investigated by using the discrete element method (DEM). Two ways are adapted to simulate different approaching methods from round particles to irregular sand. The macroresponse shows that irregular sand has a higher shear strength at lower normal stress than round particles. The shape of the particle has less influence on shear strength at higher normal stress. The irregular shape of sand leads to an increase in the shear band proportion. However, the shear band proportion is not related to the sphericity. Under all conditions, particles within the shear band have a larger average rotation angle than those outside the shear band. When the particle shape approaches round (regardless of the round particle proportion and particle shape), the average rotation angle of particles within and without shear bands increase, while the coordinate number and contact anisotropy decrease.

Aged bone matrix-derived extracellular vesicles as a messenger for calcification paradox.

Adipocyte differentiation of bone marrow mesenchymal stem/stromal cells (BMSCs) instead of osteoblast formation contributes to age- and menopause-related marrow adiposity and osteoporosis. Vascular calcification often occurs with osteoporosis, a contradictory association called "calcification paradox". Here we show that extracellular vesicles derived from aged bone matrix (AB-EVs) during bone resorption favor BMSC adipogenesis rather than osteogenesis and augment calcification of vascular smooth muscle cells. Intravenous or intramedullary injection of AB-EVs promotes bone-fat imbalance and exacerbates Vitamin D3 (VD3)-induced vascular calcification in young or old mice. Alendronate (ALE), a bone resorption inhibitor, down-regulates AB-EVs release and attenuates aging- and ovariectomy-induced bone-fat imbalance. In the VD3-treated aged mice, ALE suppresses the ovariectomy-induced aggravation of vascular calcification. MiR-483-5p and miR-2861 are enriched in AB-EVs and essential for the AB-EVs-induced bone-fat imbalance and exacerbation of vascular calcification. Our study uncovers the role of AB-EVs as a messenger for calcification paradox by transferring miR-483-5p and miR-2861.

Physical optics framework for electromagnetic scattering from electrically large targets coated with a uniaxial electric anisotropic medium based on point-source excitation.

Aiming to determine the scattered estimation of complex and electrically large targets coated with the uniaxial electric anisotropic medium (UEAM) from a distributed excitation source, the demanding study is simplified by constructing the physical optics (PO) architecture which consists of three aspects, the discrete facet modeling, the tangent plane approximation, and the scattering of an infinite PEC plate coated with the UEAM based on point-source excitation, including the electric and magnetic dipole. We depict the outer surface of an electrically large scatterer as the constitution of countless tiny triangular facets. From the tangent plane approximation employed in the PO method, the scattered fields of any discretized facet induced by the equivalent electromagnetic currents (EECs) can be further evaluated as the surface fields of an infinite UEAM-coated PEC slab. Therefore, the rigorous solution of the dyadic Green's function (DGFs) for an infinite anisotropic-medium-coated PEC plate under point-source incidence is computed first. Moreover, characterizing the ray propagation process of the plane wave spectrum, the asymptotic technique of the saddle point is employed to obtain the scattered ray field in the spatial domain. Finally, the total scattered fields are obtained by the field superposition of the overall illuminated facets under point-source excitation. Compared with the reference solution, the proposed method is validated, and the simulation results of the representative shapes coated with the UEAM layer from a point source are presented.

First report on the phylogenetic relationship, genetic variation of Echinococcus shiquicus isolates in Tibet Autonomous Region, China.

BACKGROUND: Cystic or alveolar echinococcosis caused by the larval stages of Echinococcus spp. is a very severe zoonotic helminth infection. Echinococcus shiquicus is a newly discovered species that has only been reported in the Qinghai and Sichuan provinces of the Qinghai-Tibet plateau, China where, to date, it has only been confirmed in Tibetan foxes and wild small mammal populations of the Tibetan plateau. Information on its genetic and evolutionary diversity is scanty. The aim of this study was to investigate the prevalence of E. shiquicus in plateau pikas (Ochotona curzoniae), a known intermediate host, and to determine the genetic variation and phylogenetic relationship of the E. shiquicus population in the Tibet region of China based on mitochondrial DNA. METHODS: Echinococcus shiquicus samples were collected from Damxung and Nyêmo counties (located in Tibet Autonomous Region, China). The mitochondrial cox1 and nad1 gene sequences were analyzed, and the genetic diversity and epidemiology of E. shiquicus in the region were discussed based on the results. RESULTS: The prevalence of E. shiquicus in pikas in Damxung and Nyêmo counties was 3.95% (6/152) and 6.98% (9/129), respectively. In combination with previous public sequence data, the haplotype analysis revealed 12 haplotypes (H) characterized by two distinct clusters (I and II), and a sequence distance of 99.1-99.9% from the reference haplotype (H1). The diversity and neutrality indices for the entire E. shiquicus populations were: haplotype diversity (Hd) ± standard deviation (SD) 0.862 ± 0.035; nucleotide diversity (Hd ± SD) 0.0056 ± 0.0003; Tajima's D 0.876 (P > 0.05); and Fu's F 6.000 (P > 0.05). CONCLUSIONS: This was the first analysis of the newly discovered E. shiquicus in plateau pikas in the Tibet Autonomous Region of China. The neutrality indices suggest a deficiency of alleles, indicative of a recent population bottleneck.

Regulation of LOX-1 on adhesion molecules and neutrophil infiltration in mouse Aspergillus fumigatus keratitis.

AIM: To determine whether lectin-like ox-LDL receptor (LOX-1) regulates adhesion molecules expression and neutrophil infiltration in Aspergillus fumigatus (A. fumigatus) keratitis of C57BL/6 mice. METHODS: C57BL/6 mice were pretreated with a neutralizing antibody to LOX-1 (5 µg/5 µL) or control nonspecific IgG (5 µg/5 µL), LOX-1 inhibitor Poly-I (2 µg/5 µL) or PBS by subconjunctival injection. Fungal keratitis (FK) mouse models of C57BL/6 mice were established by scraping corneal central epithelium, smearing A. fumigatus on the corneal surface and covering the eye with contact lenses. The corneal response to infection was assessed via clinical score. The mRNA levels of the adhesion molecules intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), P-selectin and E-selectin were tested in control and infected corneas by reverse transcription-polymerase chain reaction (RT-PCR). The protein levels of ICAM-1 were evaluated by immunofluorescence (IF) and Western blot. Neutrophils were extracted from the abdominal cavity of C57BL/6 mice followed by pretreatment using antibody to LOX-1 (10 µg/mL) or control nonspecific IgG (10 µg/mL), the Poly-I (4 µg/mL) or PBS. The cells were then stimulated with A. fumigatus and tested mRNA and protein levels of lymphocyte function-associated antigen-1 (LFA-1) using RT-PCR and Western blot. IF and myeloperoxidase (MPO) assays were used to assess neutrophil infiltration in mice corneas. RESULTS: Pretreatment of LOX-1 antibody or the Poly-I reduced the degree of inflammation of cornea and decreased the clinical FK score compared with pretreatment of IgG or PBS (both P<0.01). And these pretreatment also displayed an obvious decline in the mRNA levels of ICAM-1, VCAM-1, P-selectin, E-selectin and LFA-1 expression compared with control groups (all P<0.01). Furthermore, pretreated with LOX-1 antibody or Poly-I, the protein levels of ICAM-1 and LFA-1 also decreased compared with control groups (all P<0.05). Neutrophil infiltration in the cornea was significantly reduced after pretreatment of LOX-1 antibody or Poly-I compared with control groups by IF and MPO assays (both P<0.01). CONCLUSION: Inhibition of LOX-1 can decrease the expression of adhesion molecules and reduce neutrophil infiltration in A. fumigatus infected corneas of C57BL/6 mice.

Expression and role of aryl hydrocarbon receptor in Aspergillus fumigatus keratitis.

AIM: To observe the expression and role of aryl hydrocarbon receptor (AhR) in the immune response of mouse cornea infected with Aspergillus fumigatus (A. fumigatus). METHODS: Murine models of A. fumigatus keratitis were established by scraping the central epithelium of mouse cornea, daubing A. fumigatus on the cornea and covering with a contact lens. The mice were randomly divided into the control group and the A. fumigatus-infected (A.F.) group for 1, 3 and 5d respectively, which corneas were daily monitored by a slit lamp microscope and the clinical scores were also recorded timely after infection. In this study, immunofluorescence staining was used to detect the expression and localization of AhR in mouse corneas, and the mRNA and protein of AhR were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. In addition, mouse peritoneal macrophages were stimulated by A. fumigatus with or without the pretreatment of AhR antagonist CH223191 and AhR agonist FICZ, and the tumor necrosis factor alpha (TNF-α), inducible nitric oxide synthase (iNOS), interleukin-10 (IL-10) and Arg-1 mRNA were detected by RT-PCR. RESULTS: According to the results of the slit light photography, it was clearly indicated that the corneal inflammation were the most severe and the clinical score became the highest as well on the 3rd day after the infection of A. fumigatus. Contrasted with the control group, the expression of AhR in the corneal epithelial cells infected with A. fumigatus was significantly increased detected by immunofluorescence staining. AhR mainly expressed in the nucleus and cytoplasm of corneal epithelial cells. Consistent with the transcriptional level of AhR mRNA, the expression level of AhR protein reached the peak on the 3rd day after infection which was detected by Western blot. Furthermore, RT-PCR showed that CH223191 up-regulated the expression of TNF-α and iNOS and down-regulated the expression of IL-10 and Arg-1 in peritoneal macrophages; inversely, FICZ reduced the expression of TNF-α and iNOS while elevated the expression of IL-10 and Arg-1. CONCLUSION: AhR is involved in the pathogenesis of A. fumigatus keratitis and induced immune protection in anti-A. fumigatus immune response by inhibiting M1 and increasing M2 phenotype macrophage-related inflammatory factors.

Changes in corneal innervation and pain responses in fungal keratitis.

AIM: To characterize changes in the cornea nerve and pain responses in fungal keratitis (FK). METHODS: A retrospective analysis of in vivo confocal microscopy images of 11 FK corneas was performed, and the results were compared with those for 11 normal corneas. Subbasal corneal nerves were analyzed for total nerve number, main nerve trunk number, branching patterns and tortuosity. C57BL/6 mice were infected with Aspergillus fumigatus. Disease severity was determined through clinical scoring and slit lamp photography. Corneas were harvested at 1, 3, 5, and 7d post infection (p.i.) and assessed for ß III tubulin. Corneal mechanical sensitivity thresholds were detected by von Frey test. ß-endorphin (ß-EP) and µ receptor protein expression was detected through Western blotting. RESULTS: Total nerve number, main nerve trunk number, and nerve branching were significantly lower in FK patients than in controls, but tortuosity was not significantly different. In infected mice, subbasal nerve density decreased from 1d p.i., reaching a minimum at 5d p.i. Clinical scores rose at 1d p.i., peaked at 3d p.i., and decreased at 5d p.i. Mechanical sensitivity thresholds showed the same trends. ß-EP and µ receptor protein expression increased after infection. CONCLUSION: Corneal nerve density is lower in FK patients and Aspergillus fumigatus-infected mice than in controls. Pain sensitivity decreases with postinfection corneal ulcer aggravation. ß-EP and µ receptor proteins are both upregulated in infected mouse corneas.

Review of Cystic Echinococcosis in Nigeria: A Story of Neglect.

PURPOSE: Cystic echinococcosis (CE) caused by Echinococcus granulosus sensu lato is a widespread zoonotic disease of global concern. In Nigeria, the exact picture/status of CE is unclear, as most of the states are largely uninvestigated. Yet, as with every parasitic zoonosis, the first step towards planning a comprehensive management and control programme involves assessment of available national/regional prevalence data, host range, and risk factors at play in the transmission dynamics. METHODS: Published articles on echinococcosis were searched on PubMed and Africa Journal Online (AJOL) databases. Inclusion criteria were based on studies reporting prevalence of echinococcosis in animals and humans (including case reports) from 1970 to 2018. RESULTS: In this study, we evaluated and summarized cystic echinococcosis reports in Nigeria and found that post 1970-80s, studies on cystic echinococcosis have remained sparse regardless of the high prevalence recorded in the early years of CE investigation. In addition, information on the genetic population structure and the role of wildlife in CE transmission is still lacking. CONCLUSIONS: This study appraises the prevalence and distribution of CE in Nigeria and identified areas where surveillance and control efforts should be focused and intensified.

Genetic variation of Echinococcus spp. in yaks and sheep in the Tibet Autonomous Region of China based on mitochondrial DNA.

BACKGROUND: Cystic echinococcosis (CE) in humans and livestock is caused by Echinococcus granulosus (sensu lato). In China where CE is endemic, a number of studies have shown that Echinococcus granulosus (sensu stricto) is majorly responsible for CE. However, E. canadensis (G6) which is the second leading cause of CE is now being detected in most parts of the country. In this study, the species diversity and genetic variation of Echinococcus granulosus (s.l.) in four counties in Tibet Autonomous Region of China were investigated. METHODS: Infection with Echinococcus granulosus (s.s.) in yaks and sheep was identified using NADH dehydrogenase subunit 1 and 5 (nad1 and nad5) mitochondrial genes while the genotype G6 of E. canadensis initially diagnosed with NADH dehydrogenase subunit 1 (nad1) was further confirmed by analysis of the complete mitochondrial genome and a phylogenetic network constructed based on the nad2 and nad5 genes. RESULTS: Out of 85 hydatid cyst samples collected from slaughtered sheep (n = 54) and yaks (n = 31), 83 were identified as E. granulosus (s.s.) G1 (n = 77), G3 (n = 6) and 2 were identified as E. canadensis G6. Analysis of the nad1/nad5 genes revealed 16/17 mutations with 9/14 parsimony informative sites resulting in 15/14 haplotypes, respectively. Haplotype diversity (Hd) and nucleotide diversity (π) of E. granulosus (s.s.) population were 0.650 and 0.00127 for nad1 and 0.782 and 0.00306 for nad5, respectively, with an overall negative Tajima's D and Fu's Fs. A low FST indicated no genetic difference between isolates from sheep and yaks. CONCLUSION: Pockets of infection with E. canadensis (G6, G7, G8 and G10) have been previously reported in sheep, goats, yaks and/or humans in different parts of China. While the G6 genotype has been previously reported in sheep in the Tibet Autonomous Region, the detection in a yak in the present study represents the first to the best of our knowledge. Therefore, we recommend future surveys and control efforts to comprehensively investigate other potential intermediate hosts for the prevalence and genetic diversity of the E. canadensis group (G6, G7, G8 and G10) across the country and their inclusion into the existing CE control programme.

First molecular description, phylogeny and genetic variation of Taenia hydatigena from Nigerian sheep and goats based on three mitochondrial genes.

BACKGROUND: Cysticercosis caused by the metacestode larval stage of Taenia hydatigena is a disease of veterinary and economic importance. A considerable level of genetic variation among isolates of different intermediate hosts and locations has been documented. Generally, data on the genetic population structure of T. hydatigena is scanty and lacking in Nigeria. Meanwhile, similar findings in other cestodes like Echinococcus spp. have been found to be of epidemiological importance. Our aim, therefore, was to characterize and compare the genetic diversity of T. hydatigena population in Nigeria based on three mitochondrial DNA markers as well as to assess the phylogenetic relationship with populations from other geographical regions. METHODS: In the present study, we described the genetic variation and diversity of T. hydatigena isolates from Nigerian sheep and goats using three full-length mitochondrial genes: the cytochrome c oxidase subunit 1 (cox1), NADH dehydrogenase subunit 1 (nad1), and NADH dehydrogenase subunit 5 (nad5). RESULTS: The median-joining network of concatenated cox1-nad1-nad5 sequences indicated that T. hydatigena metacestodes of sheep origin were genetically distinct from those obtained in goats and this was supported by high FST values of nad1, cox1, and concatenated cox1-nad1-nad5 sequences. Genetic variation was also found to be higher in isolates from goats than from sheep. CONCLUSIONS: To the best of our knowledge, the present study described the genetic variation of T. hydatigena population for the first time in Nigeria using full-length mitochondrial genes and suggests the existence of host-specific variants. The population indices of the different DNA markers suggest that analysis of long mitochondrial DNA fragments may provide more information on the molecular ecology of T. hydatigena. We recommend that future studies employ long mitochondrial DNA sequence in order to provide reliable data that would explain the extent of genetic variation in different hosts/locations and the biological and epidemiological significance.