Antimicrobial peptide thanatin fused endolysin PA90 (Tha-PA90) for the control of Acinetobacter baumannii infection in mouse model.

BACKGROUND: This study addresses the urgent need for infection control agents driven by the rise of drug-resistant pathogens such as Acinetobacter baumannii. Our primary aim was to develop and assess a novel endolysin, Tha-PA90, designed to combat these challenges. METHODS: Tha-PA90 incorporates an antimicrobial peptide (AMP) called thanatin at its N-terminus, enhancing bacterial outer membrane permeability and reducing host immune responses. PA90 was selected as the endolysin component. The antibacterial activity of the purified Tha-PA90 was evaluated using an in vitro colony-forming unit (CFU) reduction assay and a membrane permeability test. A549 cells were utilized to measure the penetration into the cytosol and the cytotoxicity of Tha-PA90. Finally, infection control was monitored in A. baumannii infected mice following the intraperitoneal administration of Tha-PA90. RESULTS: Tha-PA90 demonstrated remarkable in vitro efficacy, completely eradicating A. baumannii strains, even drug-resistant variants, at a low concentration of 0.5 µM. Notably, it outperformed thanatin, achieving only a < 3-log reduction at 4 µM. Tha-PA90 exhibited 2-3 times higher membrane permeability than a PA90 and thanatin mixture or PA90 alone. Tha-PA90 was found within A549 cells' cytosol with no discernible cytotoxic effects. Furthermore, Tha-PA90 administration extended the lifespan of A. baumannii-infected mice, reducing bacterial loads in major organs by up to 3 logs. Additionally, it decreased proinflammatory cytokine levels (TNF-α and IL-6), reducing the risk of sepsis from rapid bacterial lysis. Our findings indicate that Tha-PA90 is a promising solution for combating drug-resistant A. baumannii. Its enhanced efficacy, low cytotoxicity, and reduction of proinflammatory responses render it a potential candidate for infection control. CONCLUSIONS: This study underscores the significance of engineered endolysins in addressing the pressing challenge of drug-resistant pathogens and offers insights into improved infection management strategies.

Bacterial cancer therapy using the attenuated fowl-adapted Salmonella enterica serovar Gallinarum.

We report here a novel anti-cancer therapy based on an avian-host-specific serotype Salmonella enterica serovar Gallinarum (S. Gallinarum) deficient in ppGpp synthesis. To monitor the tumor targeting, a bioluminescent ΔppGpp S. Gallinarum was constructed and injected intravenously into mice bearing syngeneic and human xenograft tumors. Strong bioluminescent signals were detected specifically in all grafted tumors at 2 days post-injection (dpi). The bacterial counts in normal and tumor tissue at 1 dpi revealed that ΔppGpp S. Gallinarum reached >108 CFU/g in tumor tissue and 106-107 CFU/g in endothelial organs; counts were much lower in other organs. At 16 dpi, ΔppGpp S. Gallinarum counts in tumor tissue decreased to ∼106 CFU/g, while those in the other organs became undetectable. A strong anti-cancer effect was observed after the injection of ΔppGpp S. Gallinarum into BALB/c mice grafted with CT26 colon cancer cells. This could be attributed to reduced virulence, which allowed the administration of at least a 10-fold greater dose (108 CFU) of ΔppGpp S. Gallinarum than other attenuated strains of S. enterica serovar Typhimurium (≤107 CFU). An advantage of the avian-specific S. Gallinarum as a cancer therapeutic should be a reduced capacity to cause infections or harm in humans.

Constitutive Expression of a Cytotoxic Anticancer Protein in Tumor-Colonizing Bacteria.

Bacterial cancer therapy is a promising next-generation modality to treat cancer that often uses tumor-colonizing bacteria to deliver cytotoxic anticancer proteins. However, the expression of cytotoxic anticancer proteins in bacteria that accumulate in the nontumoral reticuloendothelial system (RES), mainly the liver and spleen, is considered detrimental. This study examined the fate of the Escherichia coli strain MG1655 and an attenuated strain of Salmonella enterica serovar Gallinarum (S. Gallinarum) with defective ppGpp synthesis after intravenous injection into tumor-bearing mice (~108 colony forming units/animal). Approximately 10% of the injected bacteria were detected initially in the RES, whereas approximately 0.01% were in tumor tissues. The bacteria in the tumor tissue proliferated vigorously to up to 109 colony forming units/g tissue, whereas those in the RES died off. RNA analysis revealed that tumor-associated E. coli activated rrnB operon genes encoding the rRNA building block of ribosome needed most during the exponential stage of growth, whereas those in the RES expressed substantially decreased levels of this gene and were cleared soon presumably by innate immune systems. Based on this finding, we engineered ΔppGpp S. Gallinarum to express constitutively a recombinant immunotoxin comprising TGFα and the Pseudomonas exotoxin A (PE38) using a constitutive exponential phase promoter, the ribosomal RNA promoter rrnB P1. The construct exerted anticancer effects on mice grafted with mouse colon (CT26) or breast (4T1) tumor cells without any notable adverse effects, suggesting that constitutive expression of cytotoxic anticancer protein from rrnB P1 occurred only in tumor tissue.

In vivo imaging of invasive aspergillosis with 18F-fluorodeoxysorbitol positron emission tomography.

Invasive aspergillosis is a critical complication in immunocompromised patients with hematologic malignancies or with viral pneumonia caused by influenza virus or SARS­CoV­2. Although early and accurate diagnosis of invasive aspergillosis can maximize clinical outcomes, current diagnostic methods are time-consuming and poorly sensitive. Here, we assess the ability of 2-deoxy-2-18F-fluorosorbitol (18F-FDS) positron emission tomography (PET) to specifically and noninvasively detect Aspergillus infections. We show that 18F-FDS PET can be used to visualize Aspergillus fumigatus infection of the lungs, brain, and muscles in mouse models. In particular, 18F-FDS can distinguish pulmonary aspergillosis from Staphylococcus aureus infection, both of which induce pulmonary infiltrates in immunocompromised patients. Thus, our results indicate that the combination of 18F-FDS PET and appropriate clinical information may be useful in the differential diagnosis and localization of invasive aspergillosis.

Lipocalin2 as a potential antibacterial drug against Acinetobacter baumannii infection.

Available antibiotics to treat Acinetobacter baumannii infection is limited due to increasing resistance and the emergence of multiple drug-resistant strains. Hence, discovering effective agents against A. baumannii to reduce the number of infection-related deaths is imperative. In search of novel and alternative antibiotics, the antibacterial function of lipocalin2 (Lcn2) was investigated to treat systemic infections of A. baumannii using a mouse neutropenia model. We observed a significant increase in serum Lcn2 levels upon bacterial injection into the mouse, and the administration of recombinant Lcn2 (rmLcn2) extended their survival. Such protective effects were also observed in rmLcn2-pretreated macrophages, where rmLcn2 reduced the survival of the pathogen inside the macrophages. The underlying molecular mechanism of Lcn2 protection was also investigated. We observed that pretreatment of the Raw-264.7 macrophages with rmLcn2 markedly altered the expression of tonB3, which encodes a component of the transporter for ferrisiderophores in A. baumannii. However, the expression of katG, the gene encoding catalase, remained unaffected. These indicate that Lcn2-mediated defense against the pathogen is related to nutritional immunity rather than reactive oxygen species (ROS) production. Furthermore, the addition of rmLcn2 in infected mice diminished bacterial burden in multiple organs and enhanced the expression of tonB3 in the liver, spleen, and lungs of the infected mice. Increased survival rate due to rmLcn2 treatment declined when the infection model was established using lcn2-defective (lcn2-/-) mice, which indicated the necessity of endogenous Lcn2. Therefore, the antibacterial function of Lcn2 can be exploited to develop an alternative therapeutic agent against A. baumannii.

ppGpp signaling plays a critical role in virulence of Acinetobacter baumannii.

Acinetobacter baumannii, a major nosocomial pathogen, survives in diverse hospital environments, and its multidrug resistance is a major concern. The ppGpp-dependent stringent response mediates the reprogramming of genes with diverse functions in several bacteria. We investigated whether ppGpp is involved in A. baumannii's pathogenesis by examining biofilm formation, surface motility, adhesion, invasion, and mouse infection studies. Transcriptome analysis of early stationary phase cultures revealed 498 differentially-expressed genes (≥ 2-fold change) in a ppGpp-deficient A. baumannii strain; 220 and 278 genes were up and downregulated, respectively. Csu operon expression, important in pilus biosynthesis during early biofilm formation, was significantly reduced in the ppGpp-deficient strain. Our findings suggest that ppGpp signaling influences A. baumannii biofilm formation, surface motility, adherence, and virulence. We showed the association between ppGpp and pathogenicity in A. baumannii for the first time; ppGpp may be a novel antivirulence target in A. baumannii.

Lipocalin 2 regulates iron homeostasis, neuroinflammation, and insulin resistance in the brains of patients with dementia: Evidence from the current literature.

Dementia accompanied by memory loss is considered one of the most common neurodegenerative diseases worldwide, and its prevalence is gradually increasing. Known risk factors for dementia include genetic background, certain lifestyle and dietary patterns, smoking, iron overload, insulin resistance, and impaired glucose metabolism in the brain. Here, we review recent evidence on the regulatory role of lipocalin 2 (LCN2) in dementia from various perspectives. LCN2 is a neutrophil gelatinase-associated protein that influences diverse cellular processes, including the immune system, iron homeostasis, lipid metabolism, and inflammatory responses. Although its functions within the peripheral system are most widely recognized, recent findings have revealed links between LCN2 and central nervous system diseases, as well as novel roles for LCN2 in neurons and glia. Furthermore, LCN2 may modulate diverse pathological mechanisms involved in dementia. Taken together, LCN2 is a promising therapeutic target with which to address the neuropathology of dementia.

Orphan nuclear receptor ERR-γ regulates hepatic FGF23 production in acute kidney injury.

Fibroblast growth factor 23 (FGF23), a hormone generally derived from bone, is important in phosphate and vitamin D homeostasis. In acute kidney injury (AKI) patients, high-circulating FGF23 levels are associated with disease progression and mortality. However, the organ and cell type of FGF23 production in AKI and the molecular mechanism of its excessive production are still unidentified. For insight, we investigated folic acid (FA)-induced AKI in mice. Interestingly, simultaneous with FGF23, orphan nuclear receptor ERR-γ expression is increased in the liver of FA-treated mice, and ectopic overexpression of ERR-γ was sufficient to induce hepatic FGF23 production. In patients and in mice, AKI is accompanied by up-regulated systemic IL-6, which was previously identified as an upstream regulator of ERR-γ expression in the liver. Administration of IL-6 neutralizing antibody to FA-treated mice or of recombinant IL-6 to healthy mice confirms IL-6 as an upstream regulator of hepatic ERR-γ-mediated FGF23 production. A significant (P < 0.001) interconnection between high IL-6 and FGF23 levels as a predictor of AKI in patients that underwent cardiac surgery was also found, suggesting the clinical relevance of the finding. Finally, liver-specific depletion of ERR-γ or treatment with an inverse ERR-γ agonist decreased hepatic FGF23 expression and plasma FGF23 levels in mice with FA-induced AKI. Thus, inverse agonist of ERR-γ may represent a therapeutic strategy to reduce adverse plasma FGF23 levels in AKI.

The influence of moderate or deep neuromuscular block status on anesthetic depth monitoring system during total intravenous anesthesia using propofol and remifentanil: A randomized trial.

The neuromuscular block state may affect the electroencephalogram-derived index representing the anesthetic depth. We applied an Anesthetic Depth Monitoring for Sedation (ADMS) to patients undergoing laparoscopic cholecystectomy under total intravenous anesthesia, and evaluated the requirement of propofol according to the different neuromuscular block state. Adult patients scheduled to undergo laparoscopic cholecystectomy were enrolled and randomly assigned to either the moderate (MB) or deep neuromuscular block (DB) group. The UniCon sensor of ADMS was applied to monitor anesthetic depth and the unicon value was maintained between 40 and 50 during the operation. According to the group assignment, intraoperative rocuronium was administered to maintain proper neuromuscular block state, moderate or deep block state. The unicon value, electromyography (EMG) index, and total dose of propofol and rocuronium were analyzed. At similar anesthetic depth, less propofol was used in the DB group compared to the MB group (6.19 ± 1.36 in the MB mg/kg/h group vs 4.93 ± 3.02 mg/kg/h in the DM group, p = 0.042). As expected, more rocuronium were used in the DB group than in the MB group (0.8 ± 0.2 mg/kg in the MB group vs 1.2 ± 0.2 mg/kg in the DB group, p = 0.023) and the EMG indices were lower in the DB group than in the MB group, at several time points as follows: at starting operation (p < 0.001); at 15 (p = 0.019), 45 (p = 0.011), and 60 min (p < 0.001) after the initiation of the operation; at the end of operation (p = 0.003); and at 5 min after the administration of sugammadex (p < 0.001). At similar anesthetic depth, patients under the deep neuromuscular block state required less propofol with lower intraoperative EMG indices compared to those under the moderate neuromuscular block state during general anesthesia.

Label-Free White Blood Cell Classification Using Refractive Index Tomography and Deep Learning.

Objective and Impact Statement. We propose a rapid and accurate blood cell identification method exploiting deep learning and label-free refractive index (RI) tomography. Our computational approach that fully utilizes tomographic information of bone marrow (BM) white blood cell (WBC) enables us to not only classify the blood cells with deep learning but also quantitatively study their morphological and biochemical properties for hematology research. Introduction. Conventional methods for examining blood cells, such as blood smear analysis by medical professionals and fluorescence-activated cell sorting, require significant time, costs, and domain knowledge that could affect test results. While label-free imaging techniques that use a specimen's intrinsic contrast (e.g., multiphoton and Raman microscopy) have been used to characterize blood cells, their imaging procedures and instrumentations are relatively time-consuming and complex. Methods. The RI tomograms of the BM WBCs are acquired via Mach-Zehnder interferometer-based tomographic microscope and classified by a 3D convolutional neural network. We test our deep learning classifier for the four types of bone marrow WBC collected from healthy donors (n=10): monocyte, myelocyte, B lymphocyte, and T lymphocyte. The quantitative parameters of WBC are directly obtained from the tomograms. Results. Our results show >99% accuracy for the binary classification of myeloids and lymphoids and >96% accuracy for the four-type classification of B and T lymphocytes, monocyte, and myelocytes. The feature learning capability of our approach is visualized via an unsupervised dimension reduction technique. Conclusion. We envision that the proposed cell classification framework can be easily integrated into existing blood cell investigation workflows, providing cost-effective and rapid diagnosis for hematologic malignancy.