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Inducible Cre-dependent systems are frequently used to produce both conditional knockouts and transgenic mice with regulated expression of the gene of interest. Induction can be achieved by doxycycline-dependent transcription of the wild type gene or OH-tamoxifen-dependent nuclear translocation of the chimeric Cre/ER T2 protein. However, both of these activation strategies have some limitations. We analyzed the efficiency of knockout in different tissues and found out that it correlates with the concentration of the hydroxytamoxifen and endoxifen-the active metabolites of tamoxifen-measured by LC-MS in these tissues. We also describe two cases of Cdk8 floxed/floxed /Rosa-Cre-ER T2 mice tamoxifen-induced knockout limitations. In the first case, the standard scheme of tamoxifen administration does not lead to complete knockout formation in the brain or in the uterus. Tamoxifen metabolite measurements in multiple tissues were performed and it has been shown that low recombinase activity in the brain is due to the low levels of tamoxifen active metabolites. Increase of tamoxifen dosage (1.5 fold) and duration of activation (from 5 to 7 days) allowed us to significantly improve the knockout rate in the brain, but not in the uterus. In the second case, knockout induction during embryonic development was impossible due to the negative effect of tamoxifen on gestation. Although DNA editing in the embryos was achieved in some cases, the treatment led to different complications of the pregnancy in wild-type female mice. We propose to use doxycycline-induced Cre systems in such models.

The circadian rhythm is a biological oscillation of physiological activities with a period of approximately 24 h, that is driven by a cell-autonomous oscillator called the circadian clock. The current model of the mammalian circadian clock is based on a transcriptional-translational negative feedback loop in which the protein products of clock genes accumulate in a circadian manner and repress their own transcription. However, several studies have revealed that constitutively expressed clock genes can maintain circadian oscillations. To understand the underlying mechanism, we expressed Bmal1 in Bmal1-disrupted cells using a doxycycline-inducible promoter and monitored Bmal1 and Per2 promoter activity using luciferase reporters. Although the levels of BMAL1 and other clock proteins, REV-ERBα and CLOCK, showed no obvious rhythmicity, robust circadian oscillation in Bmal1 and Per2 promoter activities with the correct phase relationship was observed, which proceeded in a doxycycline-concentration-dependent manner. We applied transient response analysis to the Bmal1 promoter activity in the presence of various doxycycline concentrations. Based on the obtained transfer functions, we suggest that, at least in our experimental system, BMAL1 is not directly involved in the oscillatory process, but modulates the oscillation robustness by regulating basal clock gene promoter activity., (© 2022. The Author(s).)

Objective: As a highly malignant tumour, malignant rhabdoid tumours of the kidney (MRTK) are prone to metastasis and invasion, while tumour metastasis and invasion are inseparable from matrix metalloproteinases (MMPs) and epithelial-mesenchymal transformation (EMT). Moreover, the key to EMT is remodelling of the cytoskeleton. Therefore, our study is aimed at investigating whether doxycycline hydrochloride (DCH), an inhibitor of MMPs, could reverse EMT in MRTK to exert an antitumour effect by regulating MMPs and the cytoskeleton., Methods: The existence of EMT in MRTK cells was verified by bioinformatics analysis, immunofluorescence, and western blotting (WB). In vitro , the proliferation, migration, and invasion abilities of G401 cells were examined by Cell Counting Kit-8 (CCK-8), scratch, and Transwell assays, respectively. The effect of DCH on tumour growth in tumour-bearing mice was explored in in vivo experiments, and the expression of MMP2 and MMP9 and EMT correlation indexes was measured by immunofluorescence and WB, and the changes in cytoskeletal F-actin and β -tubulin were measured by fluorescence., Results: The altered extracellular matrix (ECM) composition, EMT, and high expression of MMP2 and MMP9 existed in MRTK. DCH inhibited the proliferation, migration, and invasion of G401 cells in vitro. In vivo , DCH inhibited tumour growth in mice, downregulated the expression of MMP2 and MMP9, and partially reversed EMT. Alternatively, DCH resulted in cytoskeletal rearrangements of G401 cells., Conclusions: DCH, as an MMP inhibitor, is used for the first time in MRTK research, showing good antitumour effects by reversing EMT and potentially providing new therapeutic measures for MRTK treatment., Competing Interests: The authors declare that they have no competing interests., (Copyright © 2022 Chenghao Zhanghuang et al.)

Most of the studies on doxycycline (DOX) and florfenicol (FF) remain focused on the improvement of antimicrobial activity and antimicrobial spectrum, and there is no relevant report on whether there is interaction between the two drugs after the combination. This research study evaluated the effect of DOX on FF metabolism in vitro and its mechanisms. The findings of this study showed that DOX inhibits FF metabolism in two ways. Firstly, DOX significantly inhibits the expression of CYP3A29, leading to the slower metabolism of FF; secondly, DOX affects the binding of FF to R106 and R372 by competing for the R372 and/or by a "steric-like effect", thus slowing down FF metabolism, which may increase the residual concentration of FF in edible tissues of food producing animals., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Introduction: Collagen degradation can lead to early postoperative weakness in colorectal anastomosis. Matrix metalloproteinase inhibitors (MMPIs) are shown to decrease collagen breakdown and enhance healing in anastomosis in animal models. Here, we evaluated the effectiveness of a novel anastomotic augmentation ring (AAR) that releases doxycycline, an MMPI, from a poly(lactic-co-glycolic) acid ring in porcine anastomoses., Methods: Two end-to-end stapled colorectal anastomoses were performed in 20 Yorkshire-Hampshire pigs. AAR was randomly incorporated into either the proximal or distal anastomosis as treatment, while nonaugmented anastomosis served as a control. Animals were then euthanized on days 3, 4, and 5 before anastomosis explantation and burst pressure measurement. Each anastomosis site was also collected for histology, hydroxyproline content, and gene expression microarray analyses., Results: No abscess or anastomotic leak was detected. Average burst pressures were not significantly different at any time point. There is no statistical difference in collagen content between the treatment group and controls. Gene expression analysis revealed no statistically significant in differentially expressed genes. However, genes related to inflammation, such as C-C motif chemokine ligand 11 (CCL11), CD70, and C-X-C motif chemokine ligand 10 (CXCL10), were upregulated (not statistically significant) in AAR compared to non-AAR anastomosis sites on days 3 and 4., Conclusions: This pilot study shows that doxycycline-release AAR is feasible and safe. While burst pressure and collagen content did not change significantly with doxycycline treatment, upregulating genes related to the inflammatory process for pathogen and debris clearance in AAR may improve the early stage of colorectal anastomotic healing., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Exposure of humans to aflatoxin B1 (AFB1) via ingestion of contaminated agricultural products is a major concern for human health throughout the world because epoxidized AFB1, biotransformed from AFB1 by hepatic CYP3A4, is strongly hepatotoxic and hepatocarcinogenic. Intestinal epithelial cells serve as a physical and physiological barrier against xenobiotics via their intercellular tight junction (TJ) seals and the metabolizing enzyme CYP3A4. However, the effect of AFB1 on the intestinal barrier remains unclear. Here, we investigated the influence of AFB1 on these physical and physiological intestinal barriers by means of an in vitro human intestinal model utilizing doxycycline-inducible CYP3A4-expressing Caco-2 cells, in which CYP3A4 activity is comparable to that in the adult human intestine. Cellular toxicity of AFB1 in induced Caco-2 cells (i.e., cells in which expression of CYP3A4 is induced by doxycycline) was approximately 5 times that of uninduced Caco-2 cells. Exposure to 16 µM AFB1 did not decrease the transepithelial electric resistance (TEER; a measure of TJ barrier integrity) in monolayers of uninduced Caco-2 cells to 95.8 % of that in vehicle-treated cells; in contrast, in induced Caco-2 cells, TEER was reduced to 28.8 %. Exposure to 16 µM AFB1 increased paracellular permeation of 4- and 20-kDa dextrans (paracellular permeation markers) through monolayers of induced Caco-2 cells to 5.4 and 5.2 times that through uninduced Caco-2 cells. These results together show that ingested AFB1 can modulate the intestinal barrier, and that inducible CYP3A4-expressing Caco-2 cells are a promising tool for evaluating the safety of food contaminants in the human intestine., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The presence of deoxynivalenol (DON) in feed may increase intestinal barrier permeability. Disturbance of the intestinal barrier integrity may affect the absorption of antibiotics used in animals. Since the bioavailability of orally administered antibiotics significantly affects their efficacy and safety, it was decided to evaluate how DON influences the absorption of the most commonly used antibiotics in pigs, i.e., amoxicillin (AMX) and doxycycline (DOX). The studies were conducted using jejunal explants from adult pigs. Explants were incubated in Ussing chambers, in which a buffer containing DON (30 µg/mL), AMX (50 µg/mL), DOX (30 µg/mL), a combination of AMX + DON, or a combination of DOX + DON was used. Changes in transepithelial electrical resistance (TEER), the flux of transcellular and intracellular transport markers, and the flux of antibiotics across explants were measured. DON increased the permeability of small intestine explants, expressed by a reduction in TEER and an intensification of transcellular marker transport. DON did not affect AMX transport, but it accelerated DOX transport by approximately five times. The results suggest that DON inhibits the efflux transport of DOX to the intestinal lumen, and thus significantly changes its absorption from the gastrointestinal tract.

Background: Antibiotics are used to treat bacterial infections but also impact immunity. This is usually attributed to antibiotic-induced dysbiosis of the microbiota, but antibiotics may have a direct effect on immune cells and immunity-associated receptors, such as Toll-like receptors (TLRs)., Objectives: To investigate whether antibiotics alter TLR2/1, TLR2/6 and TLR4 activity in immune cells., Methods: We evaluated the effects of amoxicillin, ciprofloxacin, doxycycline and erythromycin on TLR2/1-, TLR2/6- and TLR4-induced NF-κB activation in THP1-XBlue™-MD2-CD14 cells. Furthermore, we studied TNF-α and IL-6 levels in THP-1-derived macrophages after exposure to these antibiotics and TLR ligands., Results: Amoxicillin had no effect on any of the TLRs studied. However, ciprofloxacin reduced TLR2/1, TLR2/6 and TLR4 activity in THP1-XBlue™-MD2-CD14 cells and decreased TLR2/1-induced TNF-α and IL-6 in macrophages. Doxycycline reduced TLR2/6 and TLR4 activity in THP1-XBlue™-MD2-CD14 cells and TNF-α and IL-6 levels in response to TLR2/6 stimulation in macrophages. Erythromycin decreased TLR2/1 and TLR4 activity in THP1-XBlue™-MD2-CD14 cells without changes in TNF-α and IL-6 levels in macrophages. In addition, ciprofloxacin decreased the expression of TLR2 mRNA., Conclusions: These results suggest that some antibiotics may attenuate TLR-dependent monocyte/macrophage responses and likely reduce bacterial clearance. The latter is particularly important in infections with AMR bacteria, where misprescribed antibiotics not only fail in control of AMR infections but might also weaken host defence mechanisms by limiting innate immune responses. Our data suggest that efforts should be made to prevent the deterioration of the immune response during and after antibiotic treatment., (© The Author(s) 2022. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy.)

The experimental need to engineer the genome both in time and space, has led to the development of several photoactivatable Cre recombinase systems. However, the combination of inefficient and non-intentional background recombination has prevented thus far the wide application of these systems in biological and biomedical research. Here, we engineer an optimized photoactivatable Cre recombinase system that we refer to as doxycycline- and light-inducible Cre recombinase (DiLiCre). Following extensive characterization in cancer cell and organoid systems, we generate a DiLiCre mouse line, and illustrated the biological applicability of DiLiCre for light-induced mutagenesis in vivo and positional cell-tracing by intravital microscopy. These experiments illustrate how newly formed HrasV12 mutant cells follow an unnatural movement towards the interfollicular dermis. Together, we develop an efficient photoactivatable Cre recombinase mouse model and illustrate how this model is a powerful genome-editing tool for biological and biomedical research., (© 2022. The Author(s).)

Patients with diabetes suffer from a variety of complications and easily develop diabetic chronic wounds. The microenvironment of diabetic wounds is characterized by an excessive amount of reactive oxygen species (ROS) and an imbalance of proinflammatory and anti-inflammatory cells/factors, which hinder the regeneration of chronic wounds. In the present study, a wound dressing with immunomodulation and electroconductivity properties was prepared and assayed in vitro and in vivo. [2-(acryloyloxy) ethyl] Trimethylammonium chloride (Bio-IL) and gelatin methacrylate (GelMA) were 3D printed onto a doxycycline hydrochloride (DOXH)-loaded and ROS-degradable polyurethane (PFKU) nanofibrous membrane, followed by UV irradiation to obtain conductive hydrogel strips. DOXH was released more rapidly under a high ROS environment. The dressing promoted migration of endothelial cells and polarization of macrophages to the anti-inflammatory phenotype (M2) in vitro. In a diabetic rat wound healing test, the combination of conductivity and DOXH was most effective in accelerating wound healing, collagen deposition, revascularization, and re-epithelialization by downregulating ROS and inflammatory factor levels as well as by upregulating the M2 macrophage ratio. STATEMENT OF SIGNIFICANCE: The microenvironment of diabetic wounds is characterized by an excessive amount of reactive oxygen species (ROS) and an imbalance of proinflammatory and anti-inflammatory cells/factors, which hinder the regeneration of chronic wounds. Herein, a wound dressing composed of a DOXH-loaded ROS-responsive polyurethane membrane and 3D-printed conductive hydrogel strips was prepared, which effectively accelerated skin regeneration in diabetic wounds in vivo with better epithelialization, angiogenesis, and collagen deposition. DOXH regulated the dysfunctional wound microenvironment by ROS scavenging and polarizing macrophages to M2 phenotype, thereby playing a dominant role in diabetic wound regeneration. This design may have great potential for preparing other similar materials for the therapy of other diseases with excessive inflammation or damage to electrophysiological organs, such as nerve defect and myocardial infarction., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Human embryonic stem cells (hESCs) have unique characteristics, such as self-renewal and pluripotency, which are distinct from those of other cell types. These characteristics of hESCs are tightly regulated by complex signaling mechanisms. In this study, we demonstrate that yes-associated protein (YAP) functions in an hESC-specific manner to maintain self-renewal and survival in hESCs. hESCs were highly sensitive to YAP downregulation to promote cell survival. Interestingly, hESCs displayed dynamic changes in YAP expression in response to YAP downregulation. YAP was critical for the maintenance of self-renewal. Additionally, the function of YAP in maintenance of self-renewal and cell survival was hESC-specific. Doxycycline upregulated YAP in hESCs and attenuated the decreased cell survival induced by YAP downregulation. However, decreased expression of self-renewal markers triggered by YAP downregulation and neural/cardiac differentiation were affected by doxycycline treatment. Collectively, the results reveal the mechanism underlying the role of YAP and the novel function of doxycycline in hESCs., (© 2022. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Enterococci are important pathogens of nosocomial infections and are increasingly difficult to treat due to their intrinsic and acquired resistance to a range of antibiotics. Therefore, there is an urgent need to develop novel antibacterial agents, while drug repurposing is a promising approach to address this issue. Our study aimed to determine the antimicrobial efficacy of halicin against enterococci and found that the minimum inhibitory concentrations (MIC) of halicin against different strains of Enterococcus faecalis and Enterococcus faecium ranged from 4 to 8 μg/ml. In addition, the synergistic antibacterial effect between halicin and doxycycline (DOX) against Enterococcus was observed through the checkerboard method, and it was observed that halicin and DOX could significantly synergistically inhibit biofilm formation and eradicate preformed biofilms at sub-MICs. Moreover, the electron microscope results revealed that halicin could also disrupt the bacterial cell membrane at high concentrations. Furthermore, it is also confirmed that the combination of halicin and DOX has no significant cytotoxic effect on erythrocytes and other human-derived cells. In addition, the mouse subcutaneous model and H&E staining showed that the combination of halicin and DOX could effectively reduce the bacterial load and inflammatory infiltration without obvious side effects. In nutshell, these results demonstrate the potential of halicin in combination with DOX as a novel therapy against infections by Enterococcus., (© The Author(s) 2022. Published by Oxford University Press on behalf of FEMS.)

Tetracycline antibiotic residues have attracted worldwide attention due to the serious damage to human health and the environment. However, most of the reported fluorescent probes were based on a single fluorescence channel-based response, which often suffered from signal fluctuation-induced poor reproducibility. Herein, by taking advantage of the unique properties of zeolitic imidazolate frameworks (ZIFs), a novel europium-doped ZIF nanocomposite (ZIF-Eu) was reported for the detection of doxycycline (DOX) in a ratiometric fluorescence manner. The synthesized probe only showed blue fluorescence at 420 nm since the fluorescence of Eu was quenched by the coordinated water molecules. However, due to the strong coordination ability of DOX to Eu atoms, the probe solution demonstrated an obvious fluorescence enhancement at 615 nm in the presence of DOX, while the blue fluorescence signal remained unchanged, realizing a ratiometric fluorescence response to DOX with a good linear range from 1 to 9 μM and a detection limit of 49 nM. Interestingly, it is found that DOX could be discriminated from other tetracycline antibiotics using this ratiometric fluorescent probe. In addition, direct detection of DOX in soil samples, the DOX removal efficiency and the reusability of the proposed ZIF-Eu nanocomposite were demonstrated to evaluate its potential in environmental remediation application.

The tetracycline repressor (tetR)-regulated system is a widely used tool to specifically control gene expression in mammalian cells. Based on this system, we generated a human osteosarcoma cell line, which allows for the inducible expression of an EGFP fusion of the TAR DNA-binding protein 43 (TDP-43), which has been linked to neurodegenerative diseases. Consistent with previous findings, TDP-43 overexpression led to the accumulation of aggregates and limited the viability of U2OS. Using this inducible system, we conducted a chemical screen with a library that included FDA-approved drugs. While the primary screen identified several compounds that prevented TDP-43 toxicity, further experiments revealed that these chemicals abrogated the doxycycline-dependent TDP-43 expression. This antagonistic effect was observed with both doxycycline and tetracycline, and in several Tet-On cell lines expressing different genes, confirming the general effect of these compounds as inhibitors of the tetR system. Using the same cell line, a genome-wide CRISPR/Cas9 screen identified epigenetic regulators such as the G9a methyltransferase and TRIM28 as potential modifiers of TDP-43 toxicity. Yet again, further experiments revealed that G9a inhibition or TRIM28 loss prevented doxycycline-dependent expression of TDP-43. In summary, we have identified new chemical and genetic regulators of the tetR system, thereby raising awareness of the limitations of this approach to conduct chemical or genetic screening in mammalian cells., (© 2022 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Purpose: To explore the effect of doxycycline on vasculogenic mimicry (VM) formation and the potential mechanism in human pterygium fibroblasts in order to find novel targets for pterygium therapy., Methods: First, we demonstrate the existence of VM in 73 pterygium specimens by CD31 and periodic acid Schiff (PAS) dual staining. Then we used cell counting kit-8, clone formation assay and flow cytometry to prove the inhibitory effect of doxycycline on cell proliferation and apoptosis. The VM formation was evaluated through wound healing assay, cell transwell assay and three-dimensional cell culture combined with PAS staining. Finally, we used Western blot to testify the correlation of the VM and the factors in protein level preliminarily., Results: Our results showed that VM existed in human pterygium specimens exactly. Otherwise, in human pterygium fibroblasts, doxycycline induced a dose-dependent inhibitory effect on cell proliferation and apoptosis induction. Besides, doxycycline significantly suppressed vasculogenic mimicry tube formation, cell migration and invasion. Furthermore, doxycycline impaired the expression of MMP-9, MMP-2 and VEGF which may related to pterygium VM formation., Conclusions: Doxycycline decelerated pterygium progression might be through inhibiting VM formation according to the downregulation of MMP-9, MMP-2 and VEGF, which may provide the basis of further studies involving doxycycline for pterygium treatment.

Some species of bacteria respond to antibiotic stresses by altering their transcription profiles, in order to produce proteins that provide protection against the antibiotic. Understanding these compensatory mechanisms allows for informed treatment strategies, and could lead to the development of improved therapeutics. To this end, studies were performed to determine whether Borrelia burgdorferi, the spirochetal agent of Lyme disease, also exhibits genetically-encoded responses to the commonly prescribed antibiotics doxycycline and amoxicillin. After culturing for 24 h in a sublethal concentration of doxycycline, there were significant increases in a substantial number of transcripts for proteins that are involved with translation. In contrast, incubation with a sublethal concentration of amoxicillin did not lead to significant changes in levels of any bacterial transcript. We conclude that B. burgdorferi has a mechanism(s) that detects translational inhibition by doxycycline, and increases production of mRNAs for proteins involved with translation machinery in an attempt to compensate for that stress., Competing Interests: The authors have declared that no competing interests exist.

As a neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP)-preferring receptor, PAC1-R mediates effective neuroprotective activity. Based on the finding that the antibiotic doxycycline (DOX) with clinical neuroprotective activity functions as a positive allosteric modulator (PAM) of neuropeptide PACAP receptor 1 (PAC1-R), we use virtual and laboratory screening to search for novel small molecule PAMs of PAC1-R. Virtual screening is carried out using a small-molecule library TargetMol. After two-level precision screening with Glide, the top five compounds with the best predicted affinities for PAC1-R are selected and named small positive allosteric modulator 1‒5 (SPAM1‒5). Our results show that only 4-{[4-(4-Oxo-3,4-2-yl)butanamido]methyl}benzoic acid (SPAM1) has stronger neuroprotective activity than DOX in the MPP+ PD cell model and MPTP PD mouse model. SPAM1 has a higher affinity for PAC1-R than DOX, but has no antibiotic activity. Moreover, both SPAM1 and DOX block the decrease of PAC1-R level in mouse brain tissues induced by MPTP. The successful screening of SPAM1 offers a novel drug for the treatment of neurodegenerative disease targeting the PAC1-R.

A clear relationship between the tau assemblies and toxicity has still to be established. To correlate the tau conformation with its proteotoxic effect in vivo, we developed an innovative cell-worm-based approach. HEK293 cells expressing tau P301L under a tetracycline-inducible system (HEK T-Rex) were employed to produce different tau assemblies whose proteotoxic potential was evaluated using C. elegans . Lysates from cells induced for five days significantly reduced the worm's locomotor activity. This toxic effect was not related to the total amount of tau produced by cells or to its phosphorylation state but was related to the formation of multimeric tau assemblies, particularly tetrameric ones. We investigated the applicability of this approach for testing compounds acting against oligomeric tau toxicity, using doxycycline (Doxy) as a prototype drug. Doxy affected tau solubility and promoted the disassembly of already formed toxic aggregates in lysates of cells induced for five days. These effects translated into a dose-dependent protective action in C. elegans . These findings confirm the validity of the combined HEK T-Rex cells and the C. elegans -based approach as a platform for pharmacological screening.

Candida glabrata is an important pathogen causing superficial to invasive disease in human. Conditional expression systems are helpful in addressing the function of genes and especially when they can be applied to in vivo studies. Tetracycline-dependent regulation systems have been used in diverse fungi to turn-on (Tet-on) or turn-off (Tet-off) gene expression either in vitro but also in vivo in animal models. Up to now, only a Tet-off expression has been constructed for gene expression in C. glabrata. Here, we report a Tet-on gene expression system which can be used in vitro and in vivo in any C. glabrata genetic background. This system was used in a mice model of systemic infection to demonstrate that the general amino acid permease Gap1 is important for C. glabrata virulence., (© The Author(s) 2022. Published by Oxford University Press on behalf of FEMS.)

Nontuberculous mycobacteria (NTM) infections are increasing worldwide. Mycobacterium avium complex (MAC) and the M. abscessus species are the most commonly cultured NTM and treatment options are limited, especially for the M. abscessus species. In this study, the in vitro activity of eravacycline, a new tetracycline derivative, was tested against 110 clinical isolates of NTM. MIC testing was performed as recommended by the Clinical and Laboratory Standards Institute against 60 isolates of rapidly growing mycobacteria (RGM), of which ~70% were tetracycline resistant. These included M. abscessus subsp. abscessus (8 isolates), M. abscessus subsp. massiliense (5), M. chelonae (10), M. immunogenum (3), M. fortuitum group (20) including 12 doxycycline-resistant isolates, and M. mucogenicum group (10) including three doxycycline-resistant isolates. Due to trailing, eravacycline MICs were read at 80% and 100% inhibition. Eravacycline was active against all RGM species, with MIC 50 ranges of ≤0.015 to 0.5 and ≤0.015 to 0.12 μg/mL for 100% and 80% inhibition, respectively. For M. abscessus subsp. abscessus , MIC 50 values were 0.12 and 0.03 μg/mL with 100% and 80% inhibition, respectively. MICs for tigecycline were generally within 1 to 2 dilutions of the 100%-inhibition eravacycline MIC values. Fifty isolates of slowly growing mycobacteria (SGM) species, including 16 isolates of MAC, were also tested. While there was no trailing observed in most SGM, the eravacycline MICs were higher (MIC range of >8 μg/mL), except for M. kansasii and M. marinum which had MIC 50 values of 1 μg/mL. This study supports further evaluation of eravacycline, including clinical trials for the development of RGM treatment regimens, especially for M. abscessus.

Human non-typhoidal salmonellosis is among the leading cause of morbidity and mortality worldwide, resulting in huge economic losses and threatening the public health systems. To date, epidemiological characteristics of non-typhoidal Salmonella (NTS) implicated in human salmonellosis in China are still obscure. Herein, we investigate the antimicrobial resistance and genomic features of NTS isolated from outpatients in Shaoxing city in 2020. Eighty-seven Salmonella isolates were recovered and tested against 28 different antimicrobial agents, representing 12 categories. The results showed high resistance to cefazolin (86.21%), streptomycin (81.61%), ampicillin (77.01%), ampicillin-sulbactam (74.71%), doxycycline (72.41%), tetracycline (71.26%), and levofloxacin (70.11%). Moreover, 83.91% of isolates were resistant to ≥3 categories, which were considered multi-drug resistant (MDR). Whole-genome sequencing (WGS) combined with bioinformatic analysis was used to predict serovars, MLST types, plasmid replicons, antimicrobial resistance genes, and virulence genes, in addition to the construction of phylogenomic to determine the epidemiological relatedness between isolates. Fifteen serovars and 16 STs were identified, with the dominance of S . I 4, [5], 12:i:- ST34 (25.29%), S . Enteritidis ST11 (22.99%), and S . Typhimurium ST19. Additionally, 50 resistance genes representing ten categories were detected with a high prevalence of aac(6')-Iaa (100%), bla TEM-1B (65.52%), and tet(A) (52.87%), encoding resistance to aminoglycosides, β-lactams, and tetracyclines, respectively; in addition to chromosomic mutations affecting gyrA gene. Moreover, we showed the detection of 18 different plasmids with the dominance of IncFIB(S) and IncFII(S) (39.08%). Interestingly, all isolates harbor the typical virulence genes implicated in the virulence mechanisms of Salmonella , while one isolate of S . Jangwani contains the cdtB gene encoding typhoid toxin production. Furthermore, the phylogenomic analysis showed that all isolates of the same serovar are very close to each other and clustered together in the same clade. Together, we showed a high incidence of MDR among the studied isolates which is alarming for public health services and is a major threat to the currently available treatments to deal with human salmonellosis; hence, efforts should be gathered to further introduce WGS in routinely monitoring of AMR Salmonella in the medical field in order to enhance the effectiveness of surveillance systems and to limit the spread of MDR clones., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Chen, Ed-Dra, Zhou, Wu, Zhang and Yue.)

The adsorption of ceftriaxone (CET) and doxycycline (DOX) from aqueous solution using ferrihydrite/plant-based composites (silica rice husk) to reduce their negative impact on the ecosystem was adequately studied. On the other hand, phosphate and humic acid are often found in water and soil; in view of this, their effects on the adsorption of CET and DOX were investigated. The results showed that the removal of ceftriaxone decreased with an increase in pH, while that of doxycycline did not. Ferrihydrite with 10% silica rice husk (Fh-10%SRH) has the highest maximum adsorption capacity of 139 and 178 mg g -1 for CET and DOX, respectively, at room temperature based on Liu's adsorption isotherm. This implies that the presence of silica rice husk increases CET and DOX uptake due to an increase in the pore volume of FH-10%SRH. The results showed that phosphate had a significant inhibition role on CET adsorption and minor on DOX, whereas humic acid salt affected neither case. Increase in temperature up to 333 K favored the adsorption of both contaminants. The proposed adsorption mechanisms of ceftriaxone are electrostatic interaction, n-π interaction, and hydrogen bond, while that of DOX entails n-π interaction and hydrogen bond., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Antibiotics predispose travellers to acquire multidrug-resistant bacteria, such as extended-spectrum beta-lactamase-producing Enterobacterales (ESBL-PE). Although widely used in antimalarial prophylaxis, doxycycline has scarcely been studied in this respect., Methods: We explored the impact of doxycycline on rates of traveller's diarrhoea (TD), ESBL-PE acquisition and, particularly, doxycycline co-resistance among travel-acquired ESBL-PE in a sample of 412 visitors to low- and middle-income countries. We reviewed the literature on traveller studies of doxycycline/tetracycline resistance among stool pathogens and the impact of doxycycline on TD rates, ESBL-PE acquisition, and doxycycline/tetracycline resistance., Results: The TD rates were similar for doxycycline users (32/46; 69.6%) and non-users (256/366; 69.9%). Of the 90 travel-acquired ESBL-PE isolates, 84.4% were co-resistant to doxycycline: 100% (11/11) among users and 82.3% (65/79) among non-users. The literature on doxycycline's effect on TD was not conclusive nor did it support a recent decline in doxycycline resistance. Although doxycycline did not increase ESBL-PE acquisition, doxycycline-resistance among stool pathogens proved more frequent for users than non-users., Conclusions: Our prospective data and the literature review together suggest the following: 1) doxycycline does not prevent TD; 2) doxycycline use favours acquisition of doxy/tetracycline-co-resistant intestinal bacteria; 3) although doxycycline does not predispose to travel-related ESBL-PE acquisition per se, it selects ESBL-PE strains co-resistant to doxycycline; 4) doxycycline resistance rates are high among stool bacteria in general with no evidence of any tendency to decrease., Competing Interests: Declaration of competing interest AK has received investigator-initiated grants from Valneva and Pfizer, neither of which are relevant for the current manuscript. SM and TL declare no conflicts of interest., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Gene therapy is a powerful approach to promote spinal cord regeneration. For a clinical application it is important to restrict therapeutic gene expression to the appropriate time window to limit unwanted side effects. The doxycycline (dox)-inducible system is a widely used regulatable gene expression platform, however, this system depends on a bacterial-derived immunogenic transactivator. The foreign origin of this transactivator prevents reliable regulation of therapeutic gene expression and currently limits clinical translation. The glycine-alanine repeat (GAR) of Epstein-Barr virus nuclear antigen-1 protein inhibits its presentation to cytotoxic T cells, allowing virus-infected cells to evade the host immune system. We developed a chimeric transactivator (GARrtTA) and show that GARrtTA has an immune-evading advantage over "classical" rtTA in vivo. Direct comparison of lentiviral vectors expressing rtTA and GARrtTA in the rat spinal cord shows that the GARrtTA system is inducible for 6 doxycycline-cycles over a 47 week period, whereas with the rtTA-based system luciferase reporter expression declines during the 3rd cycle and is no longer re-inducible, indicating that GARrtTA provides an immune-advantage over rtTA. Immunohistochemistry revealed that GARrtTA expressing cells in the spinal cord appear healthier and survive better than rtTA expressing cells. Characterization of the immune response shows that expression of GARrtTA, in contrast to rtTA, does not recruit cytotoxic T-cells to the transduced spinal cord. This study demonstrates that fusion of the GAR domain to rtTA results in a functional doxycycline-inducible transactivator with a clear immune-advantage over the classical rtTA in vivo., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Mitohormesis defines the increase in fitness mediated by adaptive responses to mild mitochondrial stress. Tetracyclines inhibit not only bacterial but also mitochondrial translation, thus imposing a low level of mitochondrial stress on eukaryotic cells. We demonstrate in cell and germ-free mouse models that tetracyclines induce a mild adaptive mitochondrial stress response (MSR), involving both the ATF4-mediated integrative stress response and type I interferon (IFN) signaling. To overcome the interferences of tetracyclines with the host microbiome, we identify tetracycline derivatives that have minimal antimicrobial activity, yet retain full capacity to induce the MSR, such as the lead compound, 9-tert-butyl doxycycline (9-TB). The MSR induced by doxycycline (Dox) and 9-TB improves survival and disease tolerance against lethal influenza virus (IFV) infection when given preventively. 9-TB, unlike Dox, did not affect the gut microbiome and also showed encouraging results against IFV when given in a therapeutic setting. Tolerance to IFV infection is associated with the induction of genes involved in lung epithelial cell and cilia function, and with downregulation of inflammatory and immune gene sets in lungs, liver, and kidneys. Mitohormesis induced by non-antimicrobial tetracyclines and the ensuing IFN response may dampen excessive inflammation and tissue damage during viral infections, opening innovative therapeutic avenues.

This study aimed to evaluate the effects of untreated pig manure from diets incorporating growth-promoting supplements (antibiotics and Zn oxide) on the survival and reproduction of Eisenia andrei earthworms. The tested manures were obtained from four different groups of pigs fed with four different diets: CS, a diet based on corn and soymeal; TR, a diet based on corn, soymeal, and ground wheat (15%); CSa, a diet based on corn and soymeal + 100 ppm of doxycycline + 50 ppm of colistin + 2500 ppm of Zn oxide; and TRa, a diet based on corn, soymeal, and ground wheat (15%) + 100 ppm of doxycycline + 50 ppm of colistin + 2500 ppm of Zn oxide. The study used two soils representative of the Southern region of Brazil (Oxisol and Entisol). In general, there were no significant differences between the different manures tested in each soil. However, there were differences in the toxicity manure on E. andrei between the soils, and the magnitude of this effect was dependent on the applied dose. In Oxisol, LC 50 values were higher than 80 m 3  ha -1 , and EC 50 varied from 9 to 27 m 3  ha -1 . In Entisol, the LC 50 values were below the lowest dose tested (< 25 m 3  ha -1 ), and EC 50 remained around 5 m 3  ha -1 . It may be possible that the effects observed were attributed to an excess of nitrogen, copper, and zinc, promoted by the addition of the untreated manure and how these factors interacted with soil type., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Introduction: The present study aims to determine the effect of different post-space final irrigants ethylenediaminetetraacetic acid (EDTA), a mixture of Doxycycline citric acid and detergent (MTAD), Riboflavin (RF) and Pineapple peel extract (PPE) on the bond integrity of GFPs bonded to radicular dentin., Materials and Methods: Crowns of forty human mandibular premolars teeth were sectioned up to cementoenamel junction CEJ. Root canal treatment was performed. Gates Glidden drills were used to retrieve gutta-percha from the canal. All the specimens were randomly divided into four groups based on the final irrigant used. Group 1 (NaOCl with EDTA), group 2 NaOCl+ MTAD, group 3 NaOCl+RF and group 4 NaOCl+PPE. Cementation of glass fiber posts (GFPs) was performed using self-etch dual-cure resin cement. Sectioning was performed using a slow-speed saw from the coronal, middle, and apical third under constant water irrigation. A universal testing machine was used to assess push-out bond strength (PBS) and failure mode was evaluated using a Stereomicroscope at 40x magnification. One-way analysis of variance (ANOVA) and Tukey multiple comparison tests were used for statistical analysis (p = 0.05)., Results: Group 4 (2.5% NaOCl+ PPE) displayed the highest PBS at all three root levels (Cervical: 11.22 ± 0.74 MPa, Middle: 8.99 ± 0.97 MPa, and Apical: 6.00 ± 0.88 MPa). However, Group 1 (2.5% NaOCl+17% EDTA) demonstrated the lowest EBS (Cervical: 9.25 ± 0.17 MPa, Middle: 7.01 ± 1.84 MPa, and Apical: 4.41 ± 0.17 MPa). Inter-group comparison displayed that PBS to be comparable between group 1 and group 3 (2.5% NaOCl+ Riboflavin) (Cervical: 9.74 ± 0.41 MPa, Middle: 7.44 ± 1.18 MPa, and Apical: 4.21 ± 0.05 MPa) (p > 0.05). Whereas, group 2 (1.3% NaOCl+ MTAD) (Cervical: 9.32 ± 1.36 MPa, Middle: 7.17 ± 1.75 MPa, and Apical: 4.85 ± 0.19 MPa) and group 4 demonstrated comparable values of PBS (p > 0.05)., Conclusion: Pineapple peel extract and a mixture of Doxycycline citric acid and a detergent when used as a final irrigant demonstrated better bond integrity of GFPs to radicular dentin at all three levels coronal, middle and apical., (Copyright © 2022. Published by Elsevier B.V.)

Mycoplasma hyorhinis is an emerging swine pathogen bacterium causing polyserositis and polyarthritis in weaners and finishers. The pathogen is distributed world-wide, generating significant economic losses. No commercially available vaccine is available in Europe. Therefore, besides improving the housing conditions for prevention, antimicrobial therapy of the diseased animals is the only option to control the infection. Our aim was to determine the minimal inhibitory concentrations (MIC) of ten antimicrobials potentially used against M. hyorhinis infection. The antibiotic susceptibility of 76 M. hyorhinis isolates from Belgium, Germany, Hungary, Italy and Poland collected between 2019 and 2021 was determined by broth micro-dilution method and mismatch amplification mutation assay (MAMA). Low concentrations of tiamulin (MIC90 0.312 μg/ml), doxycycline (MIC90 0.078 μg/ml), oxytetracycline (MIC90 0.25 μg/ml), florfenicol (MIC90 2 μg/ml) and moderate concentrations of enrofloxacin (MIC90 1.25 μg/ml) inhibited the growth of the isolates. For the tested macrolides and lincomycin, a bimodal MIC pattern was observed (MIC90 >64 μg/ml for lincomycin, tulathromycin, tylosin and tilmicosin and 5 μg/ml for tylvalosin). The results of the MAMA assay were in line with the conventional method with three exceptions. Based on our statistical analyses, significant differences in MIC values of tiamulin and doxycycline were observed between certain countries. Our results show various levels of antimicrobial susceptibility among M. hyorhinis isolates to the tested antibiotics. The data underline the importance of susceptibility monitoring on pan-European level and provides essential information for proper antibiotic choice in therapy., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: U. Klein and W. Depondt are the employees of Huvepharma® NV, the producer of various veterinary antibiotic products. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Bacteria of the genus Wolbachia are endosymbionts of parasitic filarial nematodes, including Dirofilaria immitis, and are a target for the treatment of canine heartworm disease. In the present study, 53 naturally-infected dogs were divided in three groups, based on their positivity to D. immitis by antigen and Knott tests, to assess the efficacy of doxycycline treatment in eliminating Wolbachia from circulating blood. At T0, dogs that scored positive to both tests (G1) or to antigen only (G2) were submitted to doxycycline (10 mg/kg BID PO) treatment and to 10% Imidacloprid + 2.5% Moxidectin (Advocate®), while those negative to both tests (G3) received only 10% Imidacloprid + 2.5% Moxidectin (Advocate®). All dogs were followed-up for one year, monthly treated with Advocate® and regularly monitored by antigen and Knott tests. During the whole period, all blood samples were screened for Wolbachia-D. immitis DNA load by quantitative real-time PCR (qPCR). At T0, 88.2% of the microfilariemic dogs were positive for Wolbachia DNA, while none of the dogs from G2 or G3 were positive. Wolbachia DNA was no longer detectable in dogs from G1 following 1 month of doxycycline treatment and microfilariae (mfs) were cleared at T2. All dogs from the G1 and G2 were negative for D. immitis antigen at 12 months. Results of this study suggest that successful elimination of mfs by doxycycline is associated with complete clearance of Wolbachia DNA in D. immitis-naturally infected dogs., (Copyright © 2022. Published by Elsevier B.V.)

Neurodegenerative diseases are still a challenge for effective treatments. The high cost of approved drugs, severity of side effects, injection site pain, and restrictions on drug delivery to the Central Nervous System (CNS) can overshadow the management of these diseases. Due to the chronic and progressive evolution of neurodegenerative disorders and since there is still no cure for them, new therapeutic strategies such as the combination of several drugs or the use of existing drugs with new therapeutic applications are valuable strategies. Tetracyclines are traditionally classified as antibiotics. However, in this class of drugs, doxycycline and minocycline exhibit also anti-inflammatory effects by inhibiting microglia/macrophages. Hence, they have been studied as potential agents for the treatment of neurodegenerative diseases. The results of in vitro and in vivo studies confirm the effective role of these two drugs as anti-inflammatory agents in experimentally induced models of neurodegenerative diseases. In clinical studies, satisfactory results have been obtained in Multiple sclerosis (MS) but not yet in other disorders such as Alzheimer's disease (AD), Parkinson's disease (PD), or Amyotrophic lateral sclerosis (ALS). In recent years, researchers have developed and evaluated nanoparticulate drug delivery systems to improve the clinical efficacy of these two tetracyclines for their potential application in neurodegenerative diseases. This study reviews the neuroprotective roles of minocycline and doxycycline in four of the main neurodegenerative disorders: AD, PD, ALS and MS. Moreover, the potential applications of nanoparticulate delivery systems developed for both tetracyclines are also reviewed., (Copyright © 2022. Published by Elsevier B.V.)

Background: The collagenase of the bacterium Clostridium histolyticum (CCH) is already an established treatment for fibroproliferative diseases like M. Dupuytren and M. Peyronie Although results are comparable to surgical intervention, skin laceration is a severe and relevant side effect. Doxycycline (DOX) recently rose interest as an inhibitor of matrix-metalloproteinases alongside its capabilities of skin accumulation. It therefore might be a potential skin protective agent in the use of CCH., Methods: For simulation of a fibroproliferative disease adjacent to the skin, we utilized a rodent model of capsular fibrosis involving silicone implants and subsequent fibrotic capsule formation. For in-vitro studies, fibrotic capsules were excised and incubated with 0.9 mg/ml CCH and four different doses of DOX. For in-vivo experiments, animals received 0.0, 0.3 or 0.9 mg/ml CCH injections into the fibrotic capsules with or without prior oral DOX administration. Outcome analysis included histology, immunohistochemistry, gene expression analysis, chemical collagen and DOX concentration measurements as well as μCT imaging., Results: In-vitro, DOX showed a dose-dependent inhibition of CCH activity associated with increasing capsule thickness and collagen density and content. In-vivo, oral DOX administration did neither interfere with capsule formation nor in effectiveness of CCH dissolving fibrotic capsule tissue. However, skin thickness and especially collagen density was significantly higher compared to control groups. This led to a reduced rate of clinical skin lacerations after DOX administration., Conclusion: DOX inhibits CCH and accumulates in the skin. Thereby, DOX can effectively reduce skin laceration after CCH treatment., Competing Interests: The authors have declared that no competing interests exist.

Chlamydia (C.) abortus is an emerging zoonotic pathogen. Since data on its antimicrobial susceptibility are lacking, we aimed to determine minimal inhibitory (MIC) and minimal bactericidal concentrations (MBC) for azithromycin and doxycycline in HeLa-cells and primary human macrophages (M1). We also examined the MDM2-p53-inhibitor nutlin-3, an anti-infective imidazoline analog. Azithromycin and doxycycline demonstrated MICs and MBCs equal or below their peak serum concentrations (PSC) after standard dosing in both cell types. While doxycycline exhibited an MIC 64-fold and an MBC 4-fold below its PSC in HeLa-cells, the MIC of azithromycin was 4-fold below, the MBC equal to the PSC. However, azithromycin revealed lower MBCs in M1. The pharmacological advantage of azithromycin accumulation in phagocytes and their role as chlamydial reservoirs remain uncertain. However, our data suggest possible therapeutic advantages of doxycycline in epithelial cells and we provide first evidence for an anti-C. abortus effect of nutlin-3 in M1., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Objective: Tamoxifen is widely used for inducible Cre-LoxP systems but has several undesirable side effects for researchers investigating metabolism or energy balance, including weight loss, lipoatrophy, and drug incorporation into lipid stores. For this reason, we sought to determine whether a doxycycline-inducible system would be more advantageous for adipocyte-specific Cre mouse models, but serendipitously discovered widespread ectopic tetracycline response element Cre (TRE-Cre) recombinase activity., Methods: Adipocyte-specific tamoxifen- and doxycycline-inducible Cre mice were crossed to fluorescent Cre reporter mice and visualized by confocal microscopy to assess efficiency and background activity. TRE-Cre mice were crossed to stop-floxed diphtheria toxin mice to selectively ablate cells with background Cre activity., Results: Tamoxifen- and doxycycline-inducible systems performed similarly in adipose tissues, but ectopic Cre recombination was evident in numerous other cell types of the latter, most notably neurons. The source of ectopic Cre activity was isolated to the TRE-Cre transgene, driven by the pTet (tetO7) tetracycline-inducible promoter. Ablation of cells with ectopic recombination in mice led to stunted growth, diminished survival, and reduced brain mass., Conclusions: These results indicate that tamoxifen- and doxycycline-inducible adipocyte-specific Cre mouse models are similarly efficient, but the TRE-Cre component of the latter is inherently leaky. TRE-Cre background activity is especially pronounced in the brain and peripheral nerve fibers, and selective ablation of these cells impairs mouse development and survival. Caution should be taken when pairing TRE-Cre with floxed alleles that have defined roles in neural function, and additional controls should be included when using this model system., (Copyright © 2022 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Human extended pluripotent stem cell (hEPS) is a novel type of pluripotent stem cell, which possesses bi-potency towards both embryonic and extraembryonic lineages. Here, we generated a hEPS cell line (hEPS1-iCas9-B) from the cell line named hEPS1, carrying a doxycycline-inducible Cas9 expression cassette along with a constitutive reverse tetracycline transactivator (M2rtTA) expression cassette at the AAVS1 locus, thus we could efficiently generate genetically modified hEPS for studies. This cell lined remained self-renewal, differentiation potential and normal karyotype. Meanwhile, it showed robust transcriptional expression of Cas9 with doxycycline induction and could target the site where the sgRNA guided., (Copyright © 2022. Published by Elsevier B.V.)

Objectives: The manufacturing of polymethylmethacrylate(PMMA) microspheres loaded with doxycycline(DOX) and/or silver sulfate(Ag 2 SO 4 ) to be incorporated into glass ionomer cement(GIC)., Methods: PMMA microspheres were manufactured with Ag 2 SO 4 (1-5%) and/or DOX(5-15%). Particle size, encapsulation efficiency and drug release were measured by light microscope, ICP, and HPLC. Microspheres were added to a dental GIC(20%w/w). Drug release and DTS were investigated. Minimum inhibitory concentration and antibacterial effects of PMMA microspheres into GIC materials were tested., Results: The median diameter of 50 µm was obtained for microspheres. DOX was encapsulated at an efficiency of 8.3% using a theoretical loading of 15%DOX + 5%Ag 2 SO 4 . The Ag 2 SO 4 encapsulation efficiency was 0.63% using a theoretical loading of 5%AgSO 4 . All groups showed burst release within the first day and continued released up to 15 days, with 60-83% of DOX and approximately 30% of silver. For GIC, approximately 15% of DOX and 0.18% of silver were released in a 7-day period. Microbiological results showed an antimicrobial effect against S. mutans when the lead formulation of microspheres was added. The DTS was reduced by the inclusion of microspheres., Significance: PMMA microspheres containing DOX and Ag 2 SO 4 offer a sustained antimicrobial activity for dental applications and promising potential for the biomedical field., (Crown Copyright © 2022. Published by Elsevier Inc. All rights reserved.)

The accurate and early diagnosis of infection is an important feature in the biomedical sciences for better treatment and to decrease the rate of morbidity associated with diseases. Doxycycline (DC) is a semisynthetic antibiotic that belongs to tetracycline family and usually prescribed to treat a variety of infections. The objective of the present research work was to develop a new radiopharmaceutical 99m Tc-Doxycycline ( 99m Tc-DC), by using SnCl 2 ·2H 2 O as a reducing agent for diagnostic applications. It was confirmed through this study that 99m Tc-DC possessed high radiolabeling yield (95%). In vitro studies were performed by incubating 99m Tc-DC in human serum at 37 °C. The in vitro binding interaction of the labeled antibiotic was analyzed with bacterial strain (live Staphylococcus aureus cells), and its stability was further determined. Moreover, for in vivo infection imaging study, the infection was induced with S. aureus (gram positive) cells intramuscularly injected in mice models followed by biodistribution studies for 99m Tc-DC that were performed. Biodistribution studies of 99m Tc-DC showed that the radiotracer was significantly accumulated at the site of infection and indicated the renal route of excretion. Scintigraphic images obtained as a result of in vivo study showed good uptake of prepared radiotracer ( 99m Tc-DC) in the infectious lesions at 1-, 4-, and 24-h post-injection. Target-to-non-target ratios for 99m Tc-DC were significantly different for the infectious lesions and non-infected tissues and remained 2.13 ± 0.3 up to 24-h post-injection of 99m Tc-DC. 99m Tc-DC showed preferential binding to living bacterial infected sites as compared to other parts of the body, and thus it can be inferred that 99m Tc-DC might be a potential candidate to diagnose the infection., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The novel antibiotic-exploiting strategy in the treatment of Alzheimer's (AD) and Parkinson's (PD) disease has emerged as a potential breakthrough in the field. The research in animal AD/PD models provided evidence on the antiamyloidogenic, anti-inflammatory, antioxidant and antiapoptotic activity of tetracyclines, associated with cognitive improvement. The neuroprotective effects of minocycline and doxycycline in animals initiated investigation of their clinical efficacy in AD and PD patients which led to inconclusive results and additionally to insufficient safety data on a long-standing doxycycline and minocycline therapy in these patient populations. The safety issues should be considered in two levels; in AD/PD patients (particularly antibiotic-induced alteration of gut microbiota and its consequences), and as a world-wide threat of development of bacterial resistance to these antibiotics posed by a fact that AD and PD are widespread incurable diseases which require daily administered long-lasting antibiotic therapy. Recently proposed subantimicrobial doxycycline doses should be thoroughly explored for their effectiveness and long-term safety especially in AD/PD populations. Keeping in mind the antibacterial activity-related far-reaching undesirable effects both for the patients and globally, further work on repurposing these drugs for a long-standing therapy of AD/PD should consider the chemically modified tetracycline compounds tailored to lack antimicrobial but retain (or introduce) other activities effective against the AD/PD pathology. This strategy might reduce the risk of long-term therapy-related adverse effects (particularly gut-related ones) and development of bacterial resistance toward the tetracycline antibiotic agents but the therapeutic potential and desirable safety profile of such compounds in AD/PD patients need to be confirmed., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

A telomerase reverse transcriptase (Tert) lineage-tracing mouse line was developed to investigate the behavior and fate of adult tissue stem cells, by crossing the 'Tet-On' system oTet-Cre mouse with a novel reverse tetracycline transactivator (rtTA) transgene linked to the Tert promoter, which we have demonstrated marks a novel population of adult brain stem cells. Here, administration of the tetracycline derivative doxycycline to mTert-rtTA::oTet-Cre mice will indelibly mark a population of cells that express a 4.4 kb fragment of the promoter region of the gene Tert. When combined the Rosa-mTmG reporter, mTert-rtTA::oTet-Cre::Rosa-mTmG mice will express membrane tdTomato (mTomato) until doxycycline treatment induces the replacement of mTomato expression with membrane EGFP (mGFP) in cells that also express Tert. Therefore, when these triple-transgenic lineage tracing mice receive doxycycline (the "pulse" period during which TERT expressing cells are marked), these cells will become indelibly marked mGFP+ cells, which can be tracked for any desirable amount of time after doxycycline removal (the "chase" period), even if Tert expression is subsequently lost. Brains are then perfusion-fixed and processed for immunofluorescence and other downstream applications in order to interpret changes to stem cell activation, proliferation, lineage commitment, migration to various brain niches, and differentiation to mature cell types. Using this system, any rtTA mouse can be mated to oTet-Cre and a Rosa reporter to conduct doxycycline-inducible "pulse-chase" lineage tracing experiments using markers of stem cells.

Lyme disease (LD) due to Borrelia burgdorferi is the most prevalent vector-borne disease in the United States. There is a poor understanding of how immunity contributes to bacterial control, pathology, or both during LD. Dogs in an area of endemicity were screened for B. burgdorferi and Anaplasma exposure and stratified according to seropositivity, presence of LD symptoms, and doxycycline treatment. Significantly elevated serum interleukin-21 (IL-21) and increased circulating CD3 + CD94 + lymphocytes with an NK-like CD8 + T cell phenotype were predominant in asymptomatic dogs exposed to B. burgdorferi. Both CD94 + T cells and CD3 - CD94 + lymphocytes, corresponding to NK cells, from symptomatic dogs expressed gamma interferon (IFN-γ) at a 3-fold-higher frequency upon stimulation with B. burgdorferi than the same subset among endemic controls. Surface expression of activating receptor NKp46 was reduced on CD94 + T cells from LD, compared to cells after doxycycline treatment. A higher frequency of NKp46-expressing CD94 + T cells correlated with significantly increased peripheral blood mononuclear cell (PBMC) cytotoxic activity via calcein release assay. PBMCs from dogs with symptomatic LD showed significantly reduced killing ability compared with endemic control PBMCs. An elevated NK-like CD8 + T cell response was associated with protection against development of clinical LD, while excess IFN-γ was associated with clinical disease.

Doxycycline targets the 16S rRNA and is widely used for the treatment of sexually transmitted infections. While it is not highly effective at eradicating Mycoplasma genitalium infections, it can reduce organism load. The aim of this study was to investigate the association between single nucleotide polymorphisms (SNPs) in the 16S rRNA gene of M. genitalium and change in organism load. M. genitalium samples were collected from 56 men prior to commencing doxycycline and at a median of 13 of 14 doses. These were sequenced for the 16S rRNA, and the association between 16S rRNA SNPs and change in organism load was determined. 16S rRNA sequences were available for 52/56 (92.9%) M. genitalium-infected men, of which 20 (38.5%) had an undetectable load, 26 (50.0%) had a decrease in M. genitalium load (median change of 105-fold), and 6 (11.5%) had an increase in load (median change of 5-fold). The most common SNPs identified were A742G (10/52 [19.2%]), GG960-961TT/C (7/52 [13.5%]), and C1435T (28/52 [53.8%]) (M. genitalium numbering). None were associated with a change in organism load ( P  = 0.76, 0.16, and 0.98, respectively). Using pooled published data from 28 isolates, no clear relationship between the SNPs and doxycycline MIC was identified. In conclusion, the low efficacy of doxycycline against M. genitalium does not appear to be due to variation in the 16S rRNA gene.

Antibiotic tolerance is typically associated with a phenotypic change within a bacterial population, resulting in a transient decrease in antibiotic susceptibility that can contribute to treatment failure and recurrent infections. Although tolerant cells may emerge prior to treatment, the stress of prolonged antibiotic exposure can also promote tolerance. Here, we sought to determine how Yersinia pseudotuberculosis responds to doxycycline exposure, to then verify if these gene expression changes could promote doxycycline tolerance in culture and in our mouse model of infection. Only four genes were differentially regulated in response to a physiologically-relevant dose of doxycycline: osmB and ompF were upregulated, tusB and cnfy were downregulated; differential expression also occurred during doxycycline treatment in the mouse. ompF, tusB and cnfy were also differentially regulated in response to chloramphenicol, indicating these could be general responses to ribosomal inhibition. cnfy has previously been associated with persistence and was not a major focus here. We found deletion of the OmpF porin resulted in increased antibiotic accumulation, suggesting expression may promote diffusion of doxycycline out of the cell, while OsmB lipoprotein had a minor impact on antibiotic permeability. Overexpression of tusB significantly impaired bacterial survival in culture and in the mouse, suggesting that tRNA modification by tusB, and the resulting impacts on translational machinery, promotes survival during treatment with an antibiotic classically viewed as bacteriostatic. We believe this may be the first observation of bactericidal activity of doxycycline under physiological conditions, which was revealed by reversing tusB downregulation., Competing Interests: The authors have declared that no competing interests exist.

Due to the pathophysiological complexity of Alzheimer's disease, multitarget approaches able to mitigate several pathogenic mechanisms are of interest. Previous studies have pointed to the neuroprotective potential of Doxycycline (Dox), a safe and inexpensive second-generation tetracycline. Dox has been particularly reported to slow down aggregation of misfolded proteins but also to mitigate neuroinflammatory processes. Here, we have evaluated the pre-clinical potential of Dox in the APP/PS1 mouse model of amyloidogenesis. Dox was provided to APP/PS1 mice from the age of 8 months, when animals already exhibit amyloid pathology and memory deficits. Spatial memory was then evaluated from 9 to 10 months of age. Our data demonstrated that Dox moderately improved the spatial memory of APP/PS1 mice without exerting major effect on amyloid lesions. While Dox did not alleviate overall glial reactivity, we could evidence that it rather enhanced the amyloid-dependent upregulation of several neuroinflammatory markers such as CCL3 and CCL4. Finally, Dox exerted differentially regulated the levels of synaptic proteins in the hippocampus and the cortex of APP/PS1 mice. Overall, these observations support that chronic Dox delivery does not provide major pathophysiological improvements in the APP/PS1 mouse model., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Doxycycline (DOX), an antibiotic commonly used in medicine and veterinary, is frequently detected in natural waterways. Exposition of bacteria to DOX residuals poses a selective pressure leading to a common occurrence of DOX-resistance genetic determinants among microorganisms, including virulent human pathogens. In view of diminishment of the available therapeutic options, we developed a continuous-flow reaction-discharge system generating pulse-modulated radio-frequency atmospheric pressure glow discharge (pm-rf-APGD) intended for DOX removal from liquid solutions. A Design of Experiment and a Response Surface Methodology were implemented in the optimisation procedure. The removal efficiency of DOX equalling 79 ± 4.5% and the resultant degradation products were identified by High-Performance Liquid Chromatography-Diode Array Detection, Liquid Chromatography Quadruple Time of Flight Mass Spectrometry, Ultraperformance Liquid Chromatography-Tandem Mass Spectrometry, total organic carbon, total nitrogen, Attenuated Total Reflectance Furrier Transform-Infrared, and UV/Vis-based methods. The pm-rf-APGD-treated DOX solution due to the generated Reactive Oxygen and Nitrogen Species either lost its antimicrobial properties towards Escherichia coli ATCC25922 or significantly decreased biocidal activities by 37% and 29% in relation to Staphylococcus haemolyticus ATCC29970 and Staphylococcus aureus ATCC25904, respectively. Future implementation of this efficient and eco-friendly antibiotic-degradation technology into wastewater purification systems is predicted., (© 2022. The Author(s).)

Bacterial and fungal coinfections have posed great clinical challenges in recent years, and combination therapy may be a useful way to treat these mixed infections. The objective of this study was to find an effective drug combination to treat dual-species cultures of fungi and bacteria. In this study, we focused on poorly investigated mixed cultures of Candida albicans and Staphylococcus epidermidis . In this research, we investigated the effects of fluconazole (FLC) and doxycycline (DOX) against dual-species cultures of C. albicans and S. epidermidis. Both the fractional inhibitory concentration index model and Δ E model revealed a synergistic antimicrobial effect between FLC and DOX against the four groups of dual-species cultures. Mechanistic studies revealed that the synergism of FLC and DOX against dual-species cultures may be associated with the inhibition of biofilms and calcium dysregulation. Fluconazole+doxycycline appears to be a potential drug combination for the treatment of bacterial and fungal coinfections. These findings are of great significance for overcoming clinical bacterial and fungal coinfections and might provide novel insights into drug discovery for combination therapy.

Background: Mycobacterium chimaera is a slowly growing non-tuberculous mycobacterium associated with outbreaks of fatal infections in patients after cardiac surgery, and it is increasingly being detected in patients with chronic lung conditions. M chimaera can cause disseminated disease, osteomyelitis, and chronic skin or soft-tissue infections. We aimed to find new inhibitory compounds and drug repurposing opportunities for M chimaera, as current therapeutic options often result in poor outcomes., Methods: In an open drug discovery approach, we screened the Medicines for Malaria Venture (MMV) Pathogen Box to assess the in-vitro antimicrobial drug susceptibility of M chimaera compared with the antimicrobial drug susceptibility of the slowly growing, major human pathogen Mycobacterium tuberculosis, and the rapidly growing Mycobacterium abscessus reference strains. Compounds identified from an initial resazurin microtitre cell viability assay screen were further characterised by determining the minimum inhibitory concentration (MIC) of MMV Pathogen Box compounds against M chimaera; and the MICs of a panel of 20 drugs commonly used to treat mycobacterial infections against M tuberculosis, M abscessus, and M chimaera. We also assessed the time-kill kinetics of doxycycline, clarithromycin, ethambutol, and rifabutin against M chimaera., Findings: M chimaera was inhibited by 21 (5%) of 400 compounds in the Pathogen Box. Ten compounds were active against all three mycobacteria. MMV675968, with activity against slowly growing mycobacteria that probably targets folate metabolism, had a mean MIC of 2·22 μM (0·80 μg/mL) against M chimaera. Antimicrobial susceptibility testing showed that oxazolidinones such as linezolid (mean MIC 3·13 μg/mL) were active against M chimaera and that bedaquiline was the most potent compound (mean MIC 0·02 μg/mL). Doxycycline, a broad-spectrum antimicrobial drug with excellent tissue penetration properties, also inhibited M chimaera with a mean MIC of 6·25 μg/mL., Interpretation: Molecular diagnostics present an opportunity for more effective, targeted drug therapies-treating bacterial infections at the species level. Using an open drug discovery platform, we identified compounds that inhibit the newly recognised pathogen M chimaera. The existing evidence base is poor and the option for expensive drug discovery is improbable; therefore, we have also found options for drug repurposing. Future in-vivo efficacy studies will reveal whether these findings result in new, targeted treatment regimens for M chimaera., Funding: Wellcome Trust, National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs), and the University of Sussex Junior Research Associate scheme., Competing Interests: Declaration of interests We declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

Temporal regulation of CRISPRi activity is critical for genetic screens. Here, we present an inducible CRISPRi platform enabling selection of iPSC-derived cardiomyocytes and reversible gene knockdown. We targeted a doxycycline-inducible dCas9-KRAB-mCherry cassette into the AAVS1 locus in an MYL7-mGFP reporter iPSC line. A clone with bi-allelic integration displayed minimally leaky CRISPRi activity and strong expression upon addition of doxycycline in iPSCs, iPSC-derived cardiomyocytes, and multilineage differentiated cells. The CRISPRi activity was validated by targeting the MYOCD gene in iPSC cardiomyocytes. In summary, we developed a robust inducible CRISPRi platform to interrogate gene function in human iPSC-derived cardiomyocytes and other cells., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Due to their marked larvicidal activity, macrocyclic lactones (MLs) are used for the prevention of heartworm disease ( Dirofilaria immitis) in dogs. They have also been shown to eliminate adult parasites after long-term administration, with a so-called "slow-kill" effect. In addition, recent studies have established that a combination of doxycycline, which eliminates the endosymbiont Wolbachia, and MLs has superior adulticide effects when compared to MLs alone. It has been hypothesized that the apparent synergism between doxycycline/MLs may be due to interaction with drug efflux transport proteins. The aim of the present study was to evaluate gene expression of several transport proteins in D. immitis adults treated in vitro either with doxycycline alone, ivermectin alone, moxidectin alone, or a combination of ivermectin or moxidectin with doxycycline for 12 h. Quantitative PCR analysis showed a sex-dependent response to treatments. In female worms, Dim-pgp-10, Dim-haf-1 and Dim-haf-5 were upregulated compared to controls with doxycycline alone and when combined with ivermectin. Moxidectin did not induce any changes in gene expression. In males, moxidectin administered alone induced a slight increase in Dim-pgp-10, Dim-pgp-11and Di-avr-14, while ivermectin in combination with doxycycline produced significant upregulation of the ML receptor Di-avr-14. These results suggest possible synergism between the two drug classes and different susceptibility of males vs. females to adulticide effects., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Antibiotic resistance represents the main challenge of Helicobacter pylori infection worldwide. This study investigates the potential bactericidal effects of fosfomycin combinations with clarithromycin, metronidazole, ciprofloxacin, amoxicillin, rifampicin, and doxycycline against thirty-six H. pylori strains using the checkerboard and time-kill assay methods. The results showed that ≥ 50% of the strains were resistant to the six antibiotics. Remarkably, only six strains exerted resistance to these antibiotics, with the minimum inhibitory concentrations (MICs) ranges of (3.2-12.8 mg/l), (32-256 mg/l), (3.2-51.2 mg/l), (3.2-25.6 mg/l), (1.6-3.2 mg/l), and (25.6 > 51.2 mg/l), respectively. The seven antibiotics were evaluated through in silico studies for their permeability and ability to bind UDP-N-acetylglucosamine1-carboxyvinyltransferase (MurA) of H. pylori. The results indicated that fosfomycin exhibited the highest predicted membrane permeability (membrane ∆G insert = - 37.54 kcal/mol) and binding affinity (docking score = - 5.310 kcal/mol) for H. pylori MurA, compared to other tested antibiotics. The combinations of fosfomycin with these antibiotics exerted synergistic interactions (Fractional inhibitory concentration, FIC index < 1) against the six strains. Importantly, the combinations of fosfomycin with clarithromycin, doxycycline and rifampicin achieved bactericidal effects (reduction ≥ 3.0 Log 10 cfu/ml) against the most resistant H. pylori strain. Notably, these effects increased with presence of metronidazole, which enhanced the activity of the fosfomycin combination with amoxicillin from a weak inhibition to bactericidal effect. This study provides evidence that the combination of fosfomycin with either clarithromycin, amoxicillin, doxycycline, or rifampicin (especially with the presence of metronidazole) could be a promising option for treating MDR H. pylori infection., (© 2022. The Author(s).)

Asthma is a respiratory disease; involving millions of people worldwide. The main cause of asthma is allergy and immune response dysregulation. The effects of azithromycin and doxycycline as asthma-controlling drugs were evaluated in this study. Mice asthma model was produced and asthmatic mice were treated with azithromycin (75 mg/kg, orally) and doxycycline (20 mg/kg, orally). Eosinophils and neutrophils count, interleukin (IL)-4, IL-5, IL-12, IL-13, and total immunoglobulin E (IgE) levels were measured. Histological study and evaluating the genes expression of Muc5ac, Muc5b, IL-33, COX2, MYD88, and TRAF6 were performed. Azithromycin and doxycycline did not affect eosinophil and neutrophil percentage, IL-4, IL-5, IL-12, and total IgE levels, peribronchial and perivascular inflammation, goblet cell hyperplasia, and gene expression of MYD88, TRAF6, and COX2. Treatment with azithromycin significantly decreased IL-13 level, mucus secretion, and gene expression of IL-33, Muc5ac, and Muc5b; compared to the non-treated asthma group. Azithromycin administration controls mucus secretion and inflammation. Azithromycin therapy and not doxycycline might be an effective adjuvant option in asthma with reducing mucus in the airway.

Functionally graded membranes (FGM) with regenerative signals and nanofibrous topography mimicking the native extracellular matrix have been shown to improve the outcome of alveolar ridge regeneration (ARR). This study developed a novel FGM with doxycycline-enamel matrix derivative (EMD) nanofibrous composites deposition to coordinate anti-inflammation and differentiation signals, thus facilitating ARR. Doxycycline-loaded PDLLA nanofibers (PD), EMD-loaded chitosan nanospheres (CE), and CE-embedded PD (CE-PD) were fabricated by electrospinning, deposited on the surfaces of barrier membrane to develop a FGM, and the efficacy was validated by delivering the FGM to regenerate experimental alveolar ridge defects in rats. Results revealed that PD had potent antibacterial capability, and CE-PD allowed sustained release of EMD to promote osteogenesis in vitro. In the alveolar ridge defects, FGM with PD on the outer surface downregulated MMP-8, and wound dehiscence was further reduced with Cbfa1 upregulation in those treated by FGM with CE-PD on the inner surface at 1 week. FGM with CE-PD revealed significantly greater new bone formation and defect fill at 4 weeks. In conclusion, FGM with PD reduced early tissue breakdown and with CE-PD nanofibrous composites accelerated wound healing and facilitated osteogenesis, and thus could be an advantageous strategy for ARR., (Copyright © 2022 Elsevier B.V. All rights reserved.)