Comparative analysis of antimicrobial resistance phenotype and genotype of Riemerella anatipestifer.

Riemerella anatipestifer is one of the important bacterial pathogens that threaten the waterfowl farming industry. In this study, 157 suspected R. anatipestifer strains were isolated from diseased ducks and geese from seven regions of China during 2019-2020, and identified using multiple polymerase chain reaction (PCR). Antimicrobial susceptibility tests and whole-genome sequence (WGS) analysis were then performed for comparative analysis of antimicrobial resistance phenotypes and genotypes. The results showed that these strains were susceptible to florfenicol, ceftriaxone, spectinomycin, sulfafurazole and cefepime, but resistant to kanamycin, amikacin, gentamicin, and streptomycin, exhibiting multiple antimicrobial resistance phenotypes. WGS analysis revealed a wide distribution of genotypes among the 157 strains with no apparent regional pattern. Through next-generation sequencing analysis of antimicrobial resistance genes, a total of 88 resistance genes were identified. Of them, 19 tetracycline resistance genes were most commonly found, followed by 15 efflux pump resistance genes, 11 glycopeptide resistance genes and seven macrolide resistance genes. The 157 R. anatipestifer strains contained 42-55 resistance genes each, with the strains carrying 47 different resistance genes being the most abundant. By comparing the antimicrobial resistance phenotype and genotype, it was observed that a high correlation between them for most antimicrobial resistance properties was detected, except for a difference in aminoglycoside resistance phenotype and genotype. In conclusion, 157 R. anatipestifer strains exhibited severe multiple antimicrobial resistance phenotypes and genotypes, emphasizing the need for improved antimicrobial usage guidelines. The wide distribution and diverse types of resistance genes among these strains provide a foundation for studying novel mechanisms of antimicrobial resistance.

Self-heating mitochondrion-induced free radical blast for immunogenic cell death stimulation and HCC immunotherapy.

Hepatocellular carcinoma (HCC) is an immunosuppressive tumor associated with high mortality. Photothermal and photodynamic therapies have been applied to induce immunogenic cell death (ICD) in HCC, successfully eliciting immune responses but facing limitations in penetration depth in clinical trials. Here, intrinsic mitochondrial hyperthermia was used to trigger thermosensitive drug release. The mitochondria were further self-heated through 2,4-dinitrophenol uncoupling, dramatically promoting free radical initiation and inducing tumor ICD. The synthesized mitochondrial-targeting TPP-HA-TDV nanoparticles specifically generated free radicals in the mitochondria without external stimulation, and obviously enhanced the release of ICD markers, subsequently evoking immune responses. The results showed that mitochondrial hyperthermia could be an endogenous target for thermosensitive drug release. Furthermore, self-heating mitochondria-induced free radical blast could be an efficient therapeutic for deep-seated tumor therapy.

Pathogenicity of a goose astrovirus 2 strain causing fatal gout in goslings.

Gosling gout has posed a serious threat to the development of the China's goose industry since the outbreak in mainland China in 2016; goose astrovirus (GAstV) was identified as the culprit pathogen. Two genotypes of this virus have been identified: GAstV-1 and GAstV-2, of which GAstV-2 is the main epidemic strain. Our current understanding of the pathogenic mechanisms of GAstV-2 remains limited. To assess pathogenicity, 1-day-old goslings were inoculated with the GAstV-2 YC20 strain via the subcutaneous, intranasal, and oral infection routes. All the goslings showed typical gout symptoms, with those in the oral infection group exhibiting earlier and more severe clinical symptoms, the highest mortality rate, and greatest weight loss. The blood biochemical indicators, viral loads in cloacal swabs and all representative tissues, and serum antibody titers of all infection groups increased significantly, and no significant differences in these parameters were observed among the three infection groups. Histopathological studies showed that the livers, kidneys, and spleens were the main damaged organs, and the pathological changes in the oral group were more severe than those in the other groups. Further analysis revealed that hepatic sinuses narrowed or became occluded as early as 1 day post-inoculation; urate deposition occurred in the renal tubules at 2 days post-inoculation (dpi), followed by necrosis of renal tubular epithelial cells; and lymphocytic infiltration appeared in the splenic tissue at 5 dpi. These results further our understanding of the pathogenic mechanisms of GAstV-2 and provide a reference for future studies.

Nanodrug rescues liver fibrosis via synergistic therapy with H2O2 depletion and Saikosaponin b1 sustained release.

Hypoxia and hydrogen peroxide (H2O2) accumulation form the profibrogenic liver environment, which involves fibrogenesis and chronic stimulation of hepatic stellate cells (HSCs). Catalase (CAT) is the major antioxidant enzyme that catalyzes H2O2 into oxygen and water, which loses its activity in different liver diseases, especially in liver fibrosis. Clinical specimens of cirrhosis patients and liver fibrotic mice are collected in this work, and results show that CAT decrease is closely correlated with hypoxia-induced transforminmg growth factor ß1 (TGF-ß1). A multifunctional nanosystem combining CAT-like MnO2 and anti-fibrosis Saikosaponin b1 (Ssb1) is subsequently constructed for antifibrotic therapy. MnO2 catalyzes the accumulated H2O2 into oxygen, thereby ameliorating the hypoxic and oxidative stress to prevent activation of HSCs, and assists to enhance the antifibrotic pharmaceutical effect of Ssb1. This work suggests that TGF-ß1 is responsible for the diminished CAT in liver fibrosis, and our designed MnO2@PLGA/Ssb1 nanosystem displays enhanced antifibrotic efficiency through removing excess H2O2 and hypoxic stress, which may be a promising therapeutic approach for liver fibrosis treatment.

From liver fibrosis to hepatocarcinogenesis: Role of excessive liver H2O2 and targeting nanotherapeutics.

Liver fibrosis and hepatocellular carcinoma (HCC) have been worldwide threats nowadays. Liver fibrosis is reversible in early stages but will develop precancerosis of HCC in cirrhotic stage. In pathological liver, excessive H2O2 is generated and accumulated, which impacts the functionality of hepatocytes, Kupffer cells (KCs) and hepatic stellate cells (HSCs), leading to genesis of fibrosis and HCC. H2O2 accumulation is associated with overproduction of superoxide anion (O2 •-) and abolished antioxidant enzyme systems. Plenty of therapeutics focused on H2O2 have shown satisfactory effects against liver fibrosis or HCC in different ways. This review summarized the reasons of liver H2O2 accumulation, and the role of H2O2 in genesis of liver fibrosis and HCC. Additionally, nanotherapeutics targeting H2O2 were summarized for further consideration of antifibrotic or antitumor therapy.

Hemolymph Node - An Immunomorphlogical Organ: Modeling the Hemolymph Node by Allografting Renal Tissue in the Rat.

There is no authoritative characterization of the attributes of the hemolymph node (HLN) since Gibbes' first description in 1884. Early reports showed that HLN are found near the kidney in human and animals with the feature of numerous erythrocytes in sinuses. Subsequent studies mainly focused on anatomy and histology, such as the source, distribution, and quantity of erythrocytes in sinuses. Recent articles mentioned that the emergence of HLN was related to immunity, but there was no strong evidence to support this hypothesis. Therefore, it is still uncertain whether the HLN is an organ of anatomy, histology, or immunology. It has been found that the development of HLN could be elicited in the parathymic area by stimuli such as Escherichia coli, allogeneic breast cancer cells, and renal tissue that were injected/transplanted into the tail of rats in our pilot studies. In this study, the model of the HLN was established by transferring allogeneic renal tissue in the rat. Intrasinusoidal erythrocytes of the node were the component for producing a red macroscopic appearance, while macrophage-erythrocyte-lymphocyte rosettes were the major immunomorphological changes, reflecting the immune activity against the invasion of the allogeneic tissue within the node. Therefore, the HLN is an immunomorphological organ.

Molecular Characteristics and Antibiotic Resistance of Staphylococcus aureus Isolated from Patient and Food Samples in Shijiazhuang, China.

Staphylococcus aureus (S. aureus) is a common opportunistic and zoonotic pathogen in the world and could easily cause human infections and food contaminations. This study investigated the sequence typing and resistance profiles of S. aureus isolates from patient and food samples in Shijiazhuang, China. A total of 101 S. aureus isolates were distributed into six clonal complexes (CCs) and 16 singletons. A total of 86 patient isolates were distributed into six clonal CCs and 12 singletons, including a new ST. CC59, CC5, CC22, and CC398 were the predominant CCs of patient isolates. A total of 15 foodborne S. aureus isolates were distributed into 3 CCs and 4 STs, and CC1 was the most prevalent CC. Moreover, 101 S. aureus isolates had high resistance to penicillin and low resistance to chloramphenicol and rifampicin. A total of 39 strains of methicillin-resistant Staphylococcus aureus (MRSA) were detected in this study, including thirty-eight strains of patient isolates (44.2%, 38/86) and one strain of food isolates (6.7%, 1/15). MRSA-ST5, MRSA-ST59, and MRSA-ST239 were the predominant MRSA isolates in hospitals. The present study explained the relationship between S. aureus isolated from patient and food samples and indicated the risks of S. aureus in infectious diseases.

Development of a duplex real-time reverse transcription-polymerase chain reaction assay for the simultaneous detection of goose astrovirus genotypes 1 and 2.

Goose astrovirus (GAstV) is a highly infectious pathogen that causes gout in goslings (<15 old) with typical symptoms of white urate disposition on the surface of the visceral organs and articular cavity, and a high mortality rate up to 50 %. To establish a real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay for the rapid detection of the two GastV genotypes(GAstV-1 and GAstV-2), two pairs of primers and a pair of matching TaqMan probes were designed based on conserved regions of the ORF1b gene. The established duplex rRT-PCR assay showed no cross-reactivity with 10 other common waterfowl pathogens. The minimum detection limit was 10 copies/reaction for both GAstV-1 and GAstV-2. To validate the assay, 36 cloacal swabs from experimentally infected goslings and 33 field clinical samples were tested. The assay results of the experimentally infected goslings matched the infection scheme. The positive rates of GAstV-1 and GAstV-2 in the field clinical samples were 36.36 % and 54.55 %, respectively, and the co-infection rate of the two viruses was 21.21 % based on the duplex rRT-PCR assay. In conclusion, the established assay represents a specific, sensitive, and convenient tool for detecting GAstV-1, GAstV-2, and their co-infections, and for conducting epidemiological surveys.

PEDF is an antifibrosis factor that inhibits the activation of fibroblasts in a bleomycin-induced pulmonary fibrosis rat model.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a highly heterogeneous and fatal lung disease. In addition to dense fibrous tissue, abnormal angiogenesis is also an important feature of IPF. Pigment epithelium-derived factor (PEDF) is an angiogenesis inhibitor and a potential anti-fibrous factor. The purpose of this experiment is to observe the effect of PEDF on bleomycin (BLM)-induced pulmonary fibrosis in rats. METHODS: In vivo, pathological examination and detection of related factors were performed on pulmonary fibrosis induced by BLM in rats, and the temporal and spatial distribution of PEDF was investigated. Furthermore, lung gene delivery (PEDF-adeno-associated virus) was performed to investigate the effect of PEDF on pulmonary fibrosis. In vitro, lentiviral vectors were used to construct PEDF over-expression or knock out primary rat lung (PRL) fibroblasts. The effect of PEDF on fibroblast activation under TGF-ß1 stimulation was evaluated, and the activation of TGF-ß1/smad pathway and PPAR-γ expression (in the presence or absence of PPAR-γ inhibitors) were analyzed. RESULTS: In vivo results showed that PEDF expression decreased during the inflammatory phase and increased during the fibrotic phase. PEDF could inhibit the progression of pulmonary fibrosis in rats. In vitro results showed that PEDF could effectively inhibit TGF-ß1-stimulated fibroblast activation and reduce the production of α-SMA and collagen-I. PEDF could inhibit the TGF-ß1/smad pathway by up-regulating the activity of PPAR-γ. CONCLUSIONS: PEDF can act as an anti-fibrotic factor, inhibit fibroblast activation by upregulating PPAR-γ activity and reduce BLM-induced pulmonary fibrosis in rats.

PEDF reduces malignant cells proliferation and inhibits the progression of myelofibrosis in myeloproliferative neoplasms.

Pigment epithelial-derived factor (PEDF) exerts a broad spectrum of activities and has been implicated in diverse biological processes and a variety of diseases. However, the role of PEDF in myeloproliferative neoplasms (MPN) remains unknown. In this study, we found that PEDF expression was down-regulated in MPN patients and MPLW515L-transuduced mice. Exogenous PEDF inhibited the peripheral blood cell proliferation in MPLW515L-transuduced mice, reduced tumor cells in bone marrow and spleen, ameliorated hepatosplenomegaly, reduced extramedullary hemopoiesis in the spleen, and prolonged the overall survival of MPN mice. More importantly, PEDF inhibited the progression of myelofibrosis. Moreover, PEDF significantly reduced the proliferation of MPN cells in vitro, especially megakaryocyte-biased HSCs. Furthermore, PEDF induced the apoptosis of MPN cells and reduced the secretion of TGF-ß1 in cell culture supernatant. Exogenous PEDF inhibits the proliferation of MPN cells and the progression of myelofibrosis, indicating that it might play an anti-tumor and anti-fibrotic role in MPN. This study implies that PEDF might be a novel agent for the treatment of MPN.

Strategies to Attenuate Myocardial Infarction and No-Reflow Through Preservation of Vascular Integrity by Pigment Epithelium-Derived Factor.

The phenomenon of no-reflow seriously limits the therapeutic value of coronary recanalization and leads to poor prognosis. Recent studies have demonstrated the potential role of pigment epithelium-derived factor (PEDF) in stabilizing endothelial cell junction, reducing vascular permeability and maintaining a quiescent vasculature. In this study, intramyocardial gene delivery was performed 5 days before the acute myocardial infarction/recanalization experiment in male rats. Positron emission tomography perfusion imaging with 13N-NH3 indicated PEDF to promote microvascular reperfusion significantly 4 h postcoronary occlusion. PEDF was observed to maintain the stability of endothelial adherens junctions (AJs), thus preventing the occurrence of no-reflow. PEDF reduced the hypoxia-induced vascular endothelial (VE)-cadherin endocytosis through PEDF/LR/Src/VE-cadherin S665 axis in vitro, which was remarkably observed to maintain endothelial AJs. Generally, PEDF might function as a relevant target for therapeutic vasculoprotection by way of regulating the phosphorylation level of VE-cadherin according to our data, thus being crucial for preventing no-reflow.

Biological Characterization and Evolution of Bacteriophage T7-△holin During the Serial Passage Process.

Bacteriophage T7 gene 17.5 coding for the only known holin is one of the components of its lysis system, but the holin activity in T7 is more complex than a single gene, and evidence points to the existence of additional T7 genes with holin activity. In this study, a T7 phage with a gene 17.5 deletion (T7-△holin) was rescued and its biological characteristics and effect on cell lysis were determined. Furthermore, the genomic evolution of mutant phage T7-△holin during serial passage was assessed by whole-genome sequencing analysis. It was observed that deletion of gene 17.5 from phage T7 delays lysis time and enlarges the phage burst size; however, this biological characteristic recovered to normal lysis levels during serial passage. Scanning electron microscopy showed that the two opposite ends of E. coli BL21 cells swell post-T7-△holin infection rather than drilling holes on cell membrane when compared with T7 wild-type infection. No visible progeny phage particle accumulation was observed inside the E. coli BL21 cells by transmission electron microscopy. Following serial passage of T7-△holin from the 1st to 20th generations, the mRNA levels of gene 3.5 and gene 19.5 were upregulated and several mutation sites were discovered, especially two missense mutations in gene 19.5, which indicate a potential contribution to the evolution of the T7-△holin. Although the burst size of T7-△holin increased, high titer cultivation of T7-△holin was not achieved by optimizing the culture process. Accordingly, these results suggest that gene 19.5 is a potential lysis-related component that needs to be studied further and that the T7-△holin strain with its gene 17.5 deletion is not adequate to establish the high-titer phage cultivation process.

TRA2A Binds With LncRNA MALAT1 To Promote Esophageal Cancer Progression By Regulating EZH2/ß-catenin Pathway.

The RNA binding protein TRA2A, a member of the transformer 2 homolog family, plays a crucial role in the alternative splicing of pre-mRNA. However, it remains unclear whether TRA2A is involved in non-coding RNA regulation and, if so, what are the functional consequences. By analyzing expression profiling data, we found that TRA2A is highly expressed in esophageal cancer and is associated with disease-free survival and overall survival time. Subsequent gain- and loss-of-function studies demonstrated that TRA2A promotes proliferation and migration of esophageal squamous cell carcinoma and adenocarcinoma cells. RNA immunoprecipitation and RNA pull-down assay indicated that TRA2A can directly bind specific sites on MALAT1 in cells. In addition, ectopic expression or depletion of TRA2A leads to MALAT expression changes accordingly, thus modulates EZH2/ß-catenin pathway. Together, these findings elucidated that TRA2A triggers carcinogenesis via MALAT1 mediated EZH2/ß-catenin axis in esophageal cancer cells.

Molecular Characteristics of Staphylococcus aureus From Food Samples and Food Poisoning Outbreaks in Shijiazhuang, China.

As an opportunistic pathogen worldwide, Staphylococcus aureus can cause food poisoning and human infections. This study investigated the sequence typing, the penicillin (blaZ) and methicillin (mec) resistance profiles of S. aureus from food samples and food poisoning outbreaks in Shijiazhuang City, and the staphylococcal enterotoxin (SE) types of the S. aureus isolates from food poisoning. A total of 138 foodborne S. aureus isolates were distributed into 8 clonal complexes (CCs) and 12 singletons. CC1, CC5, CC8, CC15, CC97, CC59, CC398, CC88, and CC7 were the predominant CCs of foodborne S. aureus isolates. Moreover, CC59, CC15, and CC5 were the most prevalent CCs in food poisoning outbreaks. SEE was the most commonly detected SE in food poisoning isolates. One hundred thirty-three S. aureus isolates harbored the penicillin-resistant gene blaZ, and nine isolates carried the mec gene. The present study further explained the relationship between S. aureus and foods and food poisoning and indicated the potential risk of S. aureus infection.

A Riemerella anatipestifer Metallophosphoesterase That Displays Phosphatase Activity and Is Associated with Virulence.

Riemerella anatipestifer is an important pathogen of waterfowl, causing septicemic and exudative diseases. In our previous study, we demonstrated that bacterial virulence and secretion proteins of the type IX secretion system (T9SS) mutant strains Yb2ΔgldK and Yb2ΔgldM were significantly reduced, in comparison to those of wild-type strain Yb2. In this study, the T9SS secretion protein AS87_RS00980, which is absent from the secretion proteins of Yb2ΔgldK and Yb2ΔgldM, was investigated by construction of gene mutation and complementation strains. The virulence assessment showed >1,000-fold attenuated virulence and significantly reduced bacterial loads in the blood of ducks infected with Yb2Δ00980, the AS87_RS00980 gene deletion mutant strain. Bacterial virulence was recovered in complementation strain cYb2Δ00980 Further study indicated that the T9SS secretion protein AS87_RS00980 is a metallophosphoesterase (MPPE), which displayed phosphatase activity and was cytomembrane localized. Moreover, the optimal reactive pH and temperature were determined to be 7.0 and 60°C, respectively, and the Km and Vmax were determined to be 3.53 mM and 198.1 U/mg. The rMPPE activity was activated by Zn2+ and Cu2+ but inhibited by Fe3+, Fe2+, and EDTA. There are five conserved sites, namely, N267, H268 H351, H389, and H391, in the metallophosphatase domain. Mutant proteins Y267-rMPPE and Y268-rMPPE retained 29.30% and 19.81% relative activity, respectively, and mutant proteins Y351-rMPPE, Y389-rMPPE, and Y391-rMPPE lost almost all MPPE activity. Taken together, these results indicate that the R. anatipestiferAS87_RS00980 gene encodes an MPPE that is a secretion protein of T9SS that plays an important role in bacterial virulence.IMPORTANCERiemerella anatipestifer T9SS was recently discovered to be associated with bacterial gliding motility and secretion of virulence factors. Several T9SS genes have been identified, but no effector has been reported in R. anatipestifer to date. In this study, we identified the T9SS secretion protein AS87_RS00980 as an MPPE that displays phosphatase activity and is associated with bacterial virulence. The enzymatic activity of the rMPPE was determined, and the Km and Vmax were 3.53 mM and 198.1 U/mg, respectively. Five conserved sites were also identified. The AS87_RS00980 gene deletion mutant strain was attenuated >1,000-fold, indicating that MPPE is an important virulence factor. In summary, we identified that the R. anatipestiferAS87_RS00980 gene encodes an important T9SS effector, MPPE, which plays an important role in bacterial virulence.

The role of LR-TIMAP/PP1c complex in the occurrence and development of no-reflow.

BACKGROUND: The presence of no-reflow can increase the risk of major adverse cardiac events and is widely regarded as an important sign of serious prognosis. Previous studies show that laminin receptor (LR) is closely related to the morphology and function of microvessels. However, whether LR is involved in the occurrence and development of no-reflow is still unknown. METHODS: In vivo, positron emission tomography (PET) perfusion imaging was performed to detect the effects of intramyocardial gene (LR-AAV and LR-siRNA-AAV) delivery treatment on the degree of no-reflow. In vitro, LC-MS/MS analysis was conducted to identify the LR phosphorylation sites of human cardiac microvascular endothelial cells (HCMECs) treated with oxygen-glucose deprivation (OGD) for 4 h. Western blot analyses were used to evaluate the phosphorylation levels of LR at residues Tyr47 (phospho-Tyr47-LR/pY47-LR) and Thr125 (phospho-Thr125-LR/pT125-LR) and their effects on the phosphorylation of VE-cadherin residue Ser665 (phospho-Ser665-VE-cad). FINDINGS: LR over-expression, LRT125A (phosphonull) and LRY47A (phosphonull) treatments were found to reduce the level of phospho-Ser665-VE-cad, and subsequently maintain adherent junctions and endothelial barrier integrity in hypoxic environments. Mechanistically, TIMAP/PP1c can combine with LR on the cell membrane to form a novel LR-TIMAP/PP1c complex. The level of pY47-LR determined the stability of LR-TIMAP/PP1c complex. The binding of TIMAP/PP1c on LR activated the protein phosphatase activity of PP1c and regulated the level of pT125-LR. INTERPRETATION: This study demonstrates that low level of phospho-LR reduces no-reflow area through stabilizing the LR-TIMAP/PP1c complex and promoting the stability of adherens junctions, and may help identify new therapeutic targets for the treatment of no-reflow.

Coronary Collateral Microcirculation Reserve Becomes Vestigial with Aging.

INTRODUCTION: Our previous study indicated that coronary collateral microcirculation reserve (CCMR), native collaterals, transports blood flow to an ischemic area to reduce ischemic tissue injury. This study aimed to observe the changes of CCMR in the hearts of different month-old rats. METHODS: We selected 2-, 8-, 16-, and 24-month-old rats as the research objects to monitor the changes of CCMR in rats with aging. After acute myocardial infarction, lectin-FITC was injected into the femoral vein vessels of rats to mark CCMR vessels in the ischemic area. RESULTS: Results of the lectin-FITC perfusion experiment indicated that the number and collagen IV coverage of CCMR vessels declined with aging. Moreover, data suggested a correlation between endothelial nitric oxide synthase and a decline in the number of CCMR vessels. CONCLUSION: Aging causes CCMR decline in rats.

Preparation and Characterization of Protein-loaded PFC Nanoemulsions for the Treatment of Heart Diseases by Pulmonary Administration.

In the treatment of heart disease, strategies for the targeted delivery of protein therapeutics to the heart by inhalation are still immature. Perfluorocarbons (PFCs) are inert chemicals with good biocompatibility, and unique physico-chemical properties that have recently led to their applications in numerous fields. In this study, we combined the advantages of protein-phospholipid complexes and PFC emulsions and then synthesized protein-loaded PFC nanoemulsions (PNEs) to test whether, after inhalation, these nanoemulsions could deliver therapeutic proteins to the heart. After preparing protein-phospholipid complexes by lyophilization, we obtained PNEs by extrusion. The particle size and surface charge of PNEs were about 140 nm and -50 mV, respectively. In vitro results showed that the PNEs had a fine particle fraction of 35% and exhibited sustained protein release. Translocation studies were done using three types of pulmonary epithelial cells, and ~7% translocation was observed in the Calu-3 cell line. Further, they were easily absorbed by cells and had therapeutic effects in culture. In vivo results showed that the PNEs successfully delivered proteins to the myocardial tissue of rats and reduced ischemic myocardial injury caused by acute myocardial infarction (AMI). This study suggests that inhalation of PNEs is a new potential strategy to deliver proteins to cardiac tissues for treating heart diseases.