Search

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.

Objective: To investigate the expression of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) in multiple myeloma (MM) patients, and analyze the effect of doxycycline (DOX) on the expression of MMP-2 and MMP-9 in MM cells., Methods: The peripheral blood and bone marrow samples of MM patients were collected, and the patients were divided into three groups: newly diagnosed group, remission group and relapsed/refractory group, while the peripheral blood samples of 34 health people and the bone marrow samples of 17 IDA patients were selected as normal control and control group. The levels of MMP-2 and MMP-9 were detected by ELISA. The protein levels of MMP-2 and MMP-9 in H929 cells treated by different concentrations of DOX were analyzed by Western blot. After H929 cells was treated by Akt inhibitor MK-2206 2HCl in combination with DOX, Western blot was used to detect the levels of MMP-2 and MMP-9., Results: The levels of MMP-2 and MMP-9 in newly diagnosed MM patients were higher than those in control (P<0.05), while for the patients in the remission group were decreased, but still higher than those in control. The levels of MMP-2 and MMP-9 were increased again for the patients in relapsed/refractory group, and showed no significant difference as compared with those in newly diagnosed group. The levels of MMP-2 and MMP-9 could be inhibited by 10 mg/L and 15 mg/L DOX treated by H929 cell. The protein levels of MMP-2 and MMP-9 showed no altered in H929 cells treated by 5 nmol/L MK-2206 2HCl alone. DOX exerted more profound inhibitory effect to MMP-2 and MMP-9 expression in H929 cells when Akt inhibitor MK-2206 2HCl was combined with DOX., Conclusion: The levels of MMP-2 and MMP-9 are increased in MM patients and related to the disease status of MM. DOX can inhibit the expression of MMP-2 and MMP-9 in MM cells, and antagonizing its activation of Akt signaling pathway can further enhance the inhibitory effect.

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.)

Objective: To investigate the mechanism of the in vitro toxicity of doxycycline to myeloma cell line H929 and also the possible pathway involved its toxicity., Methods: Myeloma cell line H929 was treated with DOX, MEK inhibitor U0126 or RAS agonist ML-098, either alone or in combination. Then, the expression of p-MEK, caspase-3, caspase-9 and c-Jun in H929 were used to detected by Western blot; the cells proliferation and apoptosis were detected by CCK-8 assay and flow cytometry, respectively., Results: DOX significantly increased the levels of cleaved caspase-3 and caspase-9, and down-regulated the level of p-MEK in H929 (P<0.05). MEK antagonist U0126 significantly increased the levels of cleaved caspase-3 and caspase-9, and down-regulated the level of p-MEK (P<0.05). After Dox combined with ML-098 treatment of H929 cells, the apoptosis rate of H929 cells was lower than that of DOX alone treatment group(P<0.05). Compared with DOX alone treatment group, the expressions of p-MEK and p-ERK1/2 in DOX+ML-098 combined treatment group were increased, and the levels of cleaved caspase-3,9 in H929 cells were decreased (P<0.05). The levels of c-Jun mRNA and protein increased in H929 when treated by DOX alone (P<0.05)., Conclusion: DOX can induce apoptosis of H929 via intrinsic apoptosis pathway, and MEK/ERK pathway and c-Jun possibly play a role in this process.

The main objective of this study was to determine the cellular and molecular effects of doxycycline on the blood-brain barrier (BBB) and protection against secondary injuries following traumatic brain injury (TBI). Microvascular hyperpermeability and cerebral edema resulting from BBB dysfunction after TBI leads to elevation of intracranial pressure, secondary brain ischemia, herniation, and brain death. There are currently no effective therapies to modulate the underlying pathophysiology responsible for TBI-induced BBB dysfunction and hyperpermeability. The loss of BBB integrity by the proteolytic enzyme matrix metalloproteinase-9 (MMP-9) is critical to TBI-induced BBB hyperpermeability, and doxycycline possesses anti-MMP-9 effect. In this study, the effect of doxycycline on BBB hyperpermeability was studied utilizing molecular modeling (using Glide) in silico, cell culture-based models in vitro, and a mouse model of TBI in vivo. Brain microvascular endothelial cell assays of tight junction protein immunofluorescence and barrier permeability were performed. Adult C57BL/6 mice were subjected to sham versus TBI with or without doxycycline treatment and immediate intravital microscopic analysis for evaluating BBB integrity. Postmortem mouse brain tissue was collected to measure MMP-9 enzyme activity. It was found that doxycycline binding to the MMP-9 active sites have binding affinity of -7.07 kcal/mol. Doxycycline treated cell monolayers were protected from microvascular hyperpermeability and retained tight junction integrity (p < 0.05). Doxycycline treatment decreased BBB hyperpermeability following TBI in mice by 25% (p < 0.05). MMP-9 enzyme activity in brain tissue decreased with doxycycline treatment following TBI (p < 0.05). Doxycycline preserves BBB tight junction integrity following TBI via inhibiting MMP-9 activity. When established in human subjects, doxycycline, may provide readily accessible medical treatment after TBI to attenuate secondary injury., (© 2022. The Author(s).)

Nuclear factor-kappa B (NF-κB) signaling is an intracellular signaling pathway involved in inflammatory responses and the pathogenesis of various cancers, including ependymoma, which is a rare and chemotherapy-resistant glioma. Several isoforms of fusion proteins that consist of a nuclear protein, zinc finger translocation associated (ZFTA), and RELA (ZFTA-RELA), an NF-κB-signaling effector transcription factor, cause excessive activation of the NF-κB signaling pathway and result in supratentorial ependymomas (ST-EPN-RELA). As inhibitors of NF-κB activity induced by ZFTA-RELA are expected to be therapeutic agents for ST-EPN-RELA, we established an NF-κB responsive luciferase reporter cell line that expresses the most common isoform of ZFTA-RELA in a doxycycline-dependent manner. Using this reporter cell line, we screened fungus extracts for compounds that inhibit the NF-κB activity induced by ZFTA-RELA expression and identified aszonalenin, an alkaloid from Aspergillus novofumigatus. We also purified analogs of aszonalenin, namely acetylaszonalenin and epi-aszonalenin B and C. In a luciferase assay using cells constitutively expressing luciferase (counter assay), acetylaszonalenin and epi-aszonalenin C showed non-specific inhibition of the luciferase activity. Aszonalenin and epi-aszonalenin B inhibited the NF-κB responsive luciferase activity by expressing ZFTA-RELA more strongly than the luciferase activity in the counter assay. The upregulation of endogenous NF-κB responsive genes, such as CCND1, ICAM1, and L1CAM, by ZFTA-RELA expression was inhibited by epi-aszonalenin B, but not by aszonalenin. This study suggests that epi-aszonalenin B may be a lead compound for the therapeutic development of ST-EPN-RELA., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Purpose: Stenotrophomonas maltophilia has emerged as a critical opportunistic pathogen associated with significant morbidity and mortality. Tetracycline derivatives have been recognized as alternative treatment options, but they have varied pharmacokinetic properties. An integrated approach to different tetracycline derivatives for formulary decisions is reported., Methods: The minimum inhibitory concentration (MIC) data from clonally diverse bloodstream S. maltophilia isolates were examined, along with the pharmacokinetic profiles of 4 tetracycline derivatives, to predict achievable pharmacodynamic exposures with standard intravenous dosing regimens. Antimicrobial therapy was assessed using the ratio of daily drug acquisition cost relative to the ratio of the free-drug area under the time-concentration curve (fAUC) to minimum inhibitory concentration (MIC) for 90% of isolates (fAUC/MIC90)., Results: In our analysis, minocycline had the greatest fAUC/MIC90. Doxycycline was the most financially preferred agent, as calculated using 2020 average wholesale price for base-case estimates of drug acquisition cost., Conclusion: An integrated evaluation for antimicrobial formulary decision-making addressed local susceptibility data, pharmacokinetics, pharmacodynamics, dosing regimens, and drug acquisition costs. This comprehensive method is more objective than the conventional approach and warrants validation., (© American Society of Health-System Pharmacists 2021. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Previous studies had reported on the cytotoxic activities of generic antibiotics such as doxycycline (DOXY) and azithromycin (AZI) in multiple types of human cancers. Given that resistance to standard anti-glioblastoma multiforme (GBM) drug [temozolomide (TMZ)] is common and inevitable, alternative candidates are greatly needed., Purpose and Method: The present study was undertaken to explore the cytotoxicity and anticancer effects of DOXY and AZI on human GBM U87 cells via 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), Hoechst, Annexin V-FITC/PI, and clonogenic assays. CompuSyn software was used to determine the combination index (CI) for DOXY+AZI., Result: Individual treatment with DOXY and AZI decreased U87 cell viability in dose- and time-dependent, and quantitatively comparable to TMZ. Nevertheless, combinations of both antibiotics evidenced antagonistic behaviour in U87 cells. Increased apoptotic event was also observed with the individual treatment of DOXY and AZI. Furthermore, the proliferative and clonogenic capability of 21-day survived U87 cells was completely terminated by DOXY and AZI, but not TMZ., Conclusion: The antiproliferative and apoptosis-inducing activity exhibited by both antibiotics against U87 cells demonstrates their potential as a likely alternative to combat GBM. It would be interesting to find out more about their molecular players and cytotoxic effects in different types of GBM cells, including glioma stem cells (GSCs).

Atherogenic events promote changes in vessel walls, with alteration of the redox state, and increased activity of matrix metalloproteinases (MMPs). Thus, this study aims to evaluate aortic remodeling, MMP activity, and reactive oxygen species (ROS) levels after treatment with doxycycline in ApoE-⁄- and ovariectomized mice (OVX). Female ApoE-⁄--knockout mice (5 weeks) were submitted to ovariectomy surgery to induce experimental menopause. They then received chow enriched with 1% cholesterol to induce hypercholesterolemia. The animals were divided into two experimental groups: ApoE-⁄-/OVX vehicle and ApoE-⁄-/OVX doxycycline (30 mg/kg) administered by gavage once a day for 28 days (15th to the 18th week of life). Blood samples were collected to measure total cholesterol and fractions. The aorta was used for morphometry and to measure the activity and expression of MMP-2 and ROS levels. The ApoE-⁄-/OVX doxycycline group showed no change in total and fraction cholesterol levels. However, there was a reduction in ROS levels, MMP-2 expression, and activity that correlated with a decrease in atherosclerotic lesions relative to the ApoE-⁄-/OVX vehicle (p > 0.05). Therefore, we conclude that doxycycline in ApoE-⁄-/OVX animals promotes a reduction in atherosclerotic lesions by reducing ROS and MMP-2 activity and expression.

The precise developmental dynamics of the pancreatic islet endocrine cell types, and their interrelation, are unknown. Some authors claim the persistence of islet cell differentiation from precursor cells after birth ("neogenesis"). Here, using four conditional cell lineage tracing ("pulse-and-chase") murine models, we describe the natural history of pancreatic islet cells, once they express a hormone gene, until late in life. Concerning the contribution of early-appearing embryonic hormone-expressing cells to the formation of islets, we report that adult islet cells emerge from embryonic hormone-expressing cells arising at different time points during development, without any evidence of postnatal neogenesis. We observe specific patterns of hormone gene activation and switching during islet morphogenesis, revealing that, within each cell type, cells have heterogeneous developmental trajectories. This likely applies to most maturating cells in the body, and explains the observed phenotypic variability within differentiated cell types. Such knowledge should help devising novel regenerative therapies., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Colorectal cancer (CRC) is considered the second most frequent cancer globally and one of the deadliest malignancies in humans. On the other hand, over time and facing the challenges of cancer treatment, several therapeutic approaches, including surgery, radiotherapy, chemotherapy, and immunotherapy, are being developed. Evidence showed that combination therapies had given relatively satisfactory clinical outcomes in inhibiting tumor progression and increasing patient survival compared with monotherapy. Among the available compounds and drugs used in chemotherapy, doxycycline, an antimicrobial drug, has been suitable for treating several malignancies such as CRC. It has been revealed that doxycycline has anti-tumor properties and can help control tumor growth in various mechanisms, such as inhibiting anti-apoptotic and angiogenic proteins. In addition, studies have shown that combination therapy with doxycycline and other anti-tumor drugs, such as doxorubicin, anti-angiogenic factors, and anti-check-point blockers, can inhibit tumor progression. Therefore, this review summarized the anti-tumor mechanisms of doxycycline in CRC treatment and related combination therapies., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Livestock manure is a main source of heavy metals, antibiotics and antibiotic resistance genes (ARGs) in agricultural soils. The co-existence of heavy metals and ARGs needs to be systematically studied, since manure application is greatly encouraged. In this study, we examined soils for alterations in antibiotic resistance where doxycycline, Cu, and Zn were added equivalent to those found in typical pig manure applications. The results indicated that high levels of Cu inhibited soil respiration and urease for the first 10 days. Metagenomic analysis demonstrated that Cu and Zn additions caused profound alterations in bacterial community, metal resistance genes (MRGs) and mobile genetic elements. Among the differential ARGs, efflux pump genes took a significantly high ratio compared with control for the first 5 days, emphasizing their important roles in the profile of antibiotic resistance. Moreover, the number of differential MRGs was < 30 for doxycycline treatment, but 66-87 for Cu and Zn treatments. The number of differential integrative and conjugative elements was 3 for doxycycline treatment, and 6-13 for Cu and Zn treatments. Overall, high Cu and Zn levels caused a greater influence than did doxycycline on bacterial communities and transfer of antibiotic resistance in soil., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Purpose: In the programming of tumor-targeting bacteria, various therapeutic or reporter genes are expressed by different gene-triggering strategies. Previously, we engineered pJL87 plasmid with an inducible bacterial drug delivery system that simultaneously co-expressed two genes for therapy and imaging by a bidirectional tet promoter system only in response to the administration of exogenous doxycycline (Doxy). In this multi-cassette expression approach, tetA promoter (P tetA ) was 100-fold higher in expression strength than tetR promoter (P tetR ). In the present study, we developed pJH18 plasmid with novel Doxy-inducible gene expression system based on a tet promoter., Procedures: In this system, Tet repressor (TetR) expressed by a weak constitutive promoter binds to tetO operator, resulting in the tight repression of gene expressions by P tetA and P tetR , and Doxy releases TetR from tetO to de-repress P tetA and P tetR ., Results: In Salmonella transformed with pJH18, the expression balance of bidirectional tet promoters in pJH18 was remarkably improved (P tetA :P tetR = 4~6:1) compared with that of pJL87 (P tetA :P tetR = 100:1) in the presence of Doxy. Also, the expression level by novel tet system was much higher in Salmonella transformed with pJH18 than in those with pJL87 (80-fold in rluc8 and 5-fold in clyA). Interestingly, pJH18 of the transformed Salmonella was much more stably maintained than pJL87 in antibiotic-free tumor-bearing mice (about 41-fold), because only pJH18 carries bom sequence with an essential role in preventing the plasmid-free population of programmed Salmonella from undergoing cell division., Conclusions: Overall, doxycycline-induced co-expression of two proteins at similar expression levels, we exploited bioluminescence reporter proteins with preclinical but no clinical utility. Future validation with clinically compatible reporter systems, for example, suitable for radionuclide imaging, is necessary to develop this system further towards potential clinical application., (© 2021. The Author(s).)

The aim of this study was to analyze the single and combined effects of N -acetyl cysteine (NAC) and doxycycline (Dox) on the inflammatory and angiogenic factors in the rat model of alkali-burned cornea. Rats were treated with a single and combined 0.5% NAC and 12.5 μg/mL Dox eye drops and evaluated on days 3, 7, and 28. In the corneas of various groups, the activity of Catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) enzymes was assessed. The expression of inflammatory factors ( TNF-α , Rel-a , and CXCL-1 ) and angiogenic factors ( VEGF-a, MMP2 , and MMP9 ) was measured using real-time polymerase chain reaction. The antioxidant enzyme activities decreased substantially 3 days after injury with sodium hydroxide (NaOH). NAC and combined NAC+ Dox topical treatments increased the SOD enzyme activity on day 28 ( P  < 0.05). The expression of TNF-α and Rel-a genes following single and combined treatment of NAC and Dox decreased significantly on days 7 and 28 ( P  < 0.05). The mRNA level of angiogenic factors and corneal neovascularization (CNV) level declined in NaOH-injured rats treated with Dox ( P  < 0.05). The topical treatment of Dox could attenuate inflammation and CNV complications. However, NAC treatment may not reduce the expression of angiogenic genes.

Mitochondrial dysfunction is implicated in skeletal muscle insulin resistance. Syntaxin 4 (STX4) levels are reduced in human diabetic skeletal muscle, and global transgenic enrichment of STX4 expression improves insulin sensitivity in mice. Here, we show that transgenic skeletal muscle-specific STX4 enrichment (skmSTX4tg) in mice reverses established insulin resistance and improves mitochondrial function in the context of diabetogenic stress. Specifically, skmSTX4tg reversed insulin resistance caused by high-fat diet (HFD) without altering body weight or food consumption. Electron microscopy of wild-type mouse muscle revealed STX4 localisation at or proximal to the mitochondrial membrane. STX4 enrichment prevented HFD-induced mitochondrial fragmentation and dysfunction through a mechanism involving STX4-Drp1 interaction and elevated AMPK-mediated phosphorylation at Drp1 S637, which favors fusion. Our findings challenge the dogma that STX4 acts solely at the plasma membrane, revealing that STX4 localises at/proximal to and regulates the function of mitochondria in muscle. These results establish skeletal muscle STX4 enrichment as a candidate therapeutic strategy to reverse peripheral insulin resistance., (© 2022. The Author(s).)

Malignant melanoma is still a serious medical problem. Relatively high mortality, a still-growing number of newly diagnosed cases, and insufficiently effective methods of therapy necessitate melanoma research. Tetracyclines are compounds with pleiotropic pharmacological properties. Previously published studies on melanotic melanoma cells ascertained that minocycline and doxycycline exerted an anti-melanoma effect. The purpose of the study was to assess the anti-melanoma potential and mechanisms of action of minocycline and doxycycline using A375 and C32 human amelanotic melanoma cell lines. The obtained results indicate that the tested drugs inhibited proliferation, decreased cell viability, and induced apoptosis in amelanotic melanoma cells. The treatment caused changes in the cell cycle profile and decreased the intracellular level of reduced thiols and mitochondrial membrane potential. The exposure of A375 and C32 cells to minocycline and doxycycline triggered the release of cytochrome c and activated initiator and effector caspases. The anti-melanoma effect of analyzed drugs appeared to be related to the up-regulation of ERK1/2 and MITF. Moreover, it was noticed that minocycline and doxycycline increased the level of LC3A/B, an autophagy marker, in A375 cells. In summary, the study showed the pleiotropic anti-cancer action of minocycline and doxycycline against amelanotic melanoma cells. Considering all results, it could be concluded that doxycycline was a more potent drug than minocycline.

Background: Doxycycline was demonstrated in a retrospective study to be associated with greater survival in patients with light chain amyloidosis. Therefore, we prospectively compared the efficacy of bortezomib-cyclophosphamide-dexamethasone (CyBorD) and CyBorD combined with doxycycline for cardiac light chain amyloidosis., Methods: This was a multicenter, open-label, randomized controlled trial. Patients with Mayo 2004 stage II to III light chain amyloidosis were included. Patients were randomized to doxycycline 100 mg twice daily along with 9 cycles of CyBorD (doxycycline group) or to 9 cycles of CyBorD alone (control group). The primary outcome was 2-year progression-free survival (PFS). PFS was defined as the time from randomization to death, hematologic progression, or organ progression (heart, kidney or liver). Hematologic progression was defined on the basis of a substantial increase in free light chain. An increase in either NT-proBNP (N-terminal pro B-type natriuretic peptide) or cardiac troponin was the main criterion for defining cardiac progression. Cardiac PFS, defined as the time from randomization to cardiac progression or death, was compared between groups in an exploratory analysis. The corresponding treatment hazard ratio was estimated with a Cox regression model., Results: One hundred forty patients underwent randomization, with 70 in each group. The median age was 61 years (range, 33-78 years) with a male:female ratio of 1.75:1. Stage II disease was present in 34 (48.6%) and 33 (47.1%) patients in the doxycycline and control groups, respectively. After a median follow-up duration of 24.4 months, 32 of 70 (45.7%) patients in the doxycycline group and 30 of 70 (42.9%) patients in the control group experienced progression. PFS was not significantly different between groups (hazard ratio, 0.97 [95% CI, 0.59-1.60]; P =0.91). Cardiac progression occurred in 29 of 70 (41.4%) patients in the doxycycline group and 26 of 70 (37.1%) patients in the control group. The death rates for both groups by the end of follow-up was the same, 25 of 70 (35.7%). No significant differences were observed for either cardiac PFS (hazard ratio, 0.91 [95% CI, 0.54-1.55]; P =0.74) or overall survival (hazard ratio, 1.04 [95% CI, 0.60-1.81]; P =0.89)., Conclusions: Our trial demonstrated that doxycycline combined with CyBorD failed to prolong PFS or cardiac PFS compared with CyBorD alone in cardiac light chain amyloidosis. Registration: URL: https://www.clinicaltrials.gov; Unique identifier: NCT03401372.

Intelligent stimulus-triggered release and high drug-loading capacity are crucial requirements for drug delivery systems in cancer treatment. Based on the excessive intracellular GSH expression and pH conditions in tumor cells, a novel glutathione (GSH) and pH dual-responsive hydrogel was designed and synthesized by conjugates of glutamic acid-cysteine dendrimer with alginate ( Glu-Cys-SA ) through click reaction, and then cross-linked with polyethylene glycol (PEG) through hydrogen bonds to form a 3D-net structure. The hydrogel, self-assembled by the inner disulfide bonds of the dendrimer, is designed to respond to the GSH heterogeneity in tumors, with a remarkably high drug loading capacity. The Dox-loaded Glu-Cys-SA hydrogel showed controlled drug release behavior, significantly with a release rate of over 76% in response to GSH. The cytotoxicity investigation indicated that the prepared DOX-loaded hydrogel exhibited comparable anti-tumor activity against HepG-2 cells with positive control. These biocompatible hydrogels are expected to be well-designed GSH and pH dual-sensitive conjugates or polymers for efficient anticancer drug delivery.

Induced pluripotent stem cells (iPSCs) generated from patients are a valuable tool for disease modelling, drug screening, and studying the functions of cell/tissue-specific genes. However, for this research, isogenic iPSC lines are important for comparison of phenotypes in the wild type and mutant differentiated cells generated from the iPSCs. The advent of gene editing technologies to correct or generate mutations helps in the generation of isogenic iPSC lines with the same genetic background. Due to the ease of programming, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas9-based gene editing tools have gained pace in gene manipulation studies, including investigating complex diseases like cancer. An iPSC line with drug inducible Cas9 expression from the Adeno-Associated Virus Integration Site 1 (AAVS1) safe harbor locus offers a controllable expression of Cas9 with robust gene editing. Here, we describe a stepwise protocol for the generation and characterization of such an iPSC line (AAVS1-PDi-Cas9 iPSC) with a doxycycline (dox)-inducible Cas9 expression cassette from the AAVS1 safe harbor site and efficient editing of target genes with lentiviral vectors expressing gRNAs. This approach with a tunable Cas9 expression that allows investigating gene functions in iPSCs or in the differentiated cells can serve as a versatile tool in disease modelling studies., (© 2021. Springer Science+Business Media, LLC.)

Background: Acne is the pilosebaceous units' disorder. The most important cause of acne is the colonization of bacteria in the follicles. Among antibiotics, doxycycline hyclate kills a wide range of bacteria., Objectives: The study aims to prevent oral administration's side effects, overcome the barriers of conventional topical treatment, and improve the therapeutic effectiveness; this drug was loaded into niosomal nanocarriers for topical application., Methods: Doxycycline hyclate was loaded into four niosomal formulations prepared by the thinfilm hydration method with different percentages of constituents. Drug-containing niosomal systems were evaluated for morphological properties via scanning electron microscopy, particle size, drug entrapment efficiency, zeta potential, in vitro drug release, physical stability after 60 days, in vitro drug permeation through rat skin, in vitro drug deposition in rat skin, toxicity on human dermal fibroblasts (HDF) by MTT method after 72 hours, and antibacterial properties against the main acne-causing bacteria via antibiogram test., Results: The best formulation had the appropriate particle size of 362.88 ± 13.05 nm to target follicles, entrapment efficiency of 56.3 ± 2.1%, the zeta potential of - 24.46±1.39 mV, in vitro drug release of 54.93 ± 1.99% after 32 hours, and the lowest permeation of the drug through the rat skin among all other formulations. Improved cell viability, increased antibacterial activity, and an approximately three-fold increase in drug deposition were the optimal niosomal formulation features relative to the free drug., Conclusion: This study demonstrated the ability of nano-niosomes containing doxycycline hyclate to treat skin acne compared with the free drug., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Background: The antibacterial mechanism of doxycycline is known, but its effects on the nerve-muscle system are still not unclear., Objective: The aim of the study was to combine molecular targets of the neuromuscular machinery using the in situ neuronal blocker effect of doxycycline, a semisynthetic second-generation tetracycline derivative, on mice neuromuscular preparations., Methods: The effects of doxycycline were assessed on presynaptic, synaptic cleft, and postsynaptic neurotransmission, along with the muscle fiber, using the traditional myographic technique. Precisely, the effects of doxycycline were categorized into "all" or "nothing" effects depending on the concentration of doxycycline used; "all" was obtained with 4 μM doxycycline, and "nothing" was obtained with 1-3 μM doxycycline. The rationale of this study was to apply known pharmacological tools against the blocker effect of 4 μM doxycycline, such as F55-6 (Casearia sylvestris), CaCl 2 (or Ca 2+ ), atropine, neostigmine, polyethylene glycol (PEG 400), and d-Tubocurarine. The evaluation of cholinesterase enzyme activity and the diaphragm muscle histology were performed, and protocols on the neuromuscular preparation submitted to indirect or direct stimuli were complementary., Results: Doxycycline does not affect cholinesterase activity nor causes damage to skeletal muscle diaphragm; it acts on ryanodine receptor, sarcolemmal membrane, and neuronal sodium channel with a postjunctional consequence due to the decreased availability of muscle nicotinic acetylcholine receptors., Conclusion: In conclusion, in addition to the neuronal blocker effect of doxycycline, we showed that doxycycline acts on multiple targets. It is antagonized by F55-6, a neuronal Na+-channel agonist, and Ca 2+ , but not by neostigmine., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Objective: The present study aims to assess a proposed treatment approach or therapy for periodontitis by using the in-silico technique. The proposed treatment strategy offers a singular vehicular system consisting of minocycline (antibiotic), celecoxib (selective COX-II inhibitor), doxycycline hyclate (matrix metalloproteinase inhibitor), and hydroxyapatite (osteogenic agent)., Material and Methods: Molecular docking studies of drugs were performed using Maestro version 9.4 software Schrödinger, and 3-Dimensional Crystallographic X-ray protein structures of targeted proteins were downloaded from RCSB protein data bank in .pdb file format. These agents were docked, and their affinities towards the receptors/protein/enzyme were calculated. Furthermore, their affinities were compared with the standard drug., Results: The study suggests that minocycline and metronidazole possess equal affinity towards the RGPB and Inlj protein of P.gingivalis. Celecoxib, a well-known inhibitor of the COX-II enzyme, showed very high affinity. Selective inhibitor of MMP-8 possessed higher affinity than doxycycline, whereas CMT-3 showed equal affinity as doxycycline for MMP-13. Similarly, hydroxyapatite and simvastatin also showed a comparatively similar affinity for osteopontin receptor., Conclusion: Based upon molecular docking results, it can be concluded that the proposed treatment strategy would be a suitable approach for periodontitis and all the selected therapeutic agents have potential similar to the standard drugs, thereby constituting a reliable system for periodontitis., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Introduction: Brucellosis is a zoonotic disease that is prevalent in livestock animals. The bacteria reside inside the macrophage cells of the host. The WHO has endorseda combination treatment therapy for brucellosis against the conventional monotherapy to avoid relapse and resistance. Therefore, we developed nanoparticles incorporating doxycycline and rifampicin in combination., Aim: The aim of the study is to develop polymeric nanoparticles incorporating doxycycline as well as rifampicin and investigate the antibacterial activity of nanoparticles in U937 human macrophage cells infected with B. abortus., Methods: Polymeric nanoparticles were developed by the emulsion-solvent diffusion method, and characterization was performed., Results: The nanoparticles with high entrapment efficiency of both the drugs were developed successfully. Scanning electron microscopy revealed a spherical morphology with a size ranging ~450nm, which can be easily engulfed by the macrophages. Zeta potential confirmed the colloidal stability. Differential scanning calorimetry and X-ray diffraction suggested amorphization of doxycycline and rifampicin in nanoparticles. Fourier transfer infrared spectroscopy could not confirm the interaction of drugs with AOT. In vitro haemolysis study confirmed the safety of nanoparticles (<10%) for IV administration. Further, nanoparticles revealed the sustained release of both drugs, which followed diffusion kinetics. Nanoparticles were found stable for 6 months as per WHO guidelines. The internalization study revealed nanoparticles could be easily uptaken by U-937 human macrophage cells. The efficacy study demonstrated significantly high antibacterial activity of nanoparticles as compared to free drug solution in U937 human macrophages cells infected with Brucella abortus., Conclusion: It can be concluded that the developed nanoparticles entrapping doxycycline and rifampicin combination can be considered as a promising delivery system for enhancing the antibacterial activity against Brucella abortus., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Several degenerative amyloid diseases, with no fully effective treatment, affect millions of people worldwide. These pathologies-amyloidoses-are known to be associated with the formation of ordered protein aggregates and highly stable and insoluble amyloid fibrils, which are deposited in multiple tissues and organs. The disruption of preformed amyloid aggregates and fibrils is one possible therapeutic strategy against amyloidosis; however, only a few compounds have been identified as possible fibril disruptors in vivo to date. To properly identify chemical compounds as potential fibril disruptors, a reliable, fast, and economic screening protocol must be developed. For this purpose, three amyloid fibril formation protocols using transthyretin (TTR), a plasma protein involved in several amyloidoses, were studied using thioflavin-T fluorescence assays, circular dichroism (CD), turbidity, dynamic light scattering (DLS), and transmission electron microscopy (TEM), in order to characterize and select the most appropriate fibril formation protocol. Saturation transfer difference nuclear magnetic resonance spectroscopy (STD NMR) was successfully used to study the interaction of doxycycline, a known amyloid fibril disruptor, with preformed wild-type TTR (TTRwt) aggregates and fibrils. DLS and TEM were also used to characterize the effect of doxycycline on TTRwt amyloid species disaggregation. A comparison of the TTR amyloid morphology formed in different experimental conditions is also presented.

Antibiotic treatment in early life influences gastrointestinal (GI) microbial composition and function. In humans, the resultant intestinal dysbiosis is associated with an increased risk for certain diseases later in life. The objective of this study was to determine the temporal effects of antibiotic treatment on the GI microbiome of young cats. Fecal samples were collected from cats randomly allocated to receive either amoxicillin/clavulanic acid (20 mg/kg q12h) for 20 days (AMC group; 15 cats) or doxycycline (10 mg/kg q24h) for 28 days (DOX group;15 cats) as part of the standard treatment of upper respiratory tract infection. In addition, feces were collected from healthy control cats (CON group;15 cats). All cats were approximately two months of age at enrolment. Samples were collected on days 0 (baseline), 20 or 28 (AMC and DOX, respectively; last day of treatment), 60, 120, and 300. DNA was extracted and sequencing of the 16S rRNA gene and qPCR assays were performed. Fecal microbial composition was different on the last day of treatment for AMC cats, and 1 month after the end of antibiotic treatment for DOX cats, compared to CON cats. Species richness was significantly greater in DOX cats compared to CON cats on the last day of treatment. Abundance of Enterobacteriales was increased, and that of Erysipelotrichi was decreased in cats of the AMC group on the last day of treatment compared to CON cats. The abundance of the phylum Proteobacteria was increased in cats of the DOX group on days 60 and 120 compared to cats of the CON group. Only minor differences in abundances between the treatment groups and the control group were present on day 300. Both antibiotics appear to delay the developmental progression of the microbiome, and this effect is more profound during treatment with amoxicillin/clavulanic acid and one month after treatment with doxycycline. Future studies are required to determine if these changes influence microbiome function and whether they have possible effects on disease susceptibility in cats., Competing Interests: The authors have declared that no competing interests exist.

Obesity is closely linked to metabolic diseases, particularly non-alcoholic steatohepatitis (NASH) or non-alcoholic fatty liver disease (NAFLD), ultimately leading to hepatocellular carcinoma (HCC). However, the molecular mechanisms of NASH-associated HCC (NAHCC) remain elusive. To explore the impact of Max dimerization protein 3 (MXD3), a transcription factor that regulates several cellular functions in disorders associated with metabolic diseases, we conditionally expressed Mxd3 proteins using Tet-on mxd3 transgenic zebrafish (MXs) with doxycycline (MXs + Dox) or without doxycycline (MXs - Dox) treatment. Overexpression of global MXD3 (gMX) or hepatic Mxd3 (hMX) was associated with obesity-related NAFLD pathophysiology in gMX + Dox, and liver fibrosis and HCC in hMX + Dox. Oil Red O (ORO)-stained signals were seen in intravascular blood vessels and liver buds of larval gMX + Dox, indicating that Mxd3 functionally promotes lipogenesis. The gMX + Dox-treated young adults exhibited an increase in body weight and visceral fat accumulation. The hMX + Dox-treated young adults showed normal body characteristics but exhibited liver steatosis and NASH-like phenotypes. Subsequently, steatohepatitis, liver fibrosis, and NAHCC were found in 6-month-old gMX + Dox adults compared with gMX - Dox adults at the same stage. Overexpression of Mxd3 also enhanced AR expression accompanied by the increase of AR-signaling pathways resulting in hepatocarcinogenesis in males. Our results demonstrate that global actions of Mxd3 are central to the initiation of obesity in the gMX zebrafish through their effects on adipogenesis and that MXD3 could serve as a therapeutic target for obesity-associated liver diseases.

Background: Increasing resistance of microorganisms to antimicrobial agents is a growing concern and there is a lack of novel agents. This has stimulated the exploration of novel strategies for treatment of infection., Objective: To investigate synergistic interactions between five tetracyclines and tobramycin with an iron chelator (CP762) against two reference strains and nine clinical isolates of Pseudomonas aeruginosa from cystic fibrosis patients., Method: Microdilution assays for minimal inhibitory concentration determination and checkerboard assays were used to assess synergy between antibiotics and CP762. Given the iron-binding capacity of tetracyclines, the binding of iron with doxycycline was investigated using Job's plot methodology. Synergy between the iron-bound form of doxycycline and CP762 was compared with that of unbound doxycycline and CP762. Enhancement of doxycycline anti-biofilm activity was also assessed., Results: There was synergy between CP762 and all tetracyclines, except minocycline, against the reference strains but that against clinical isolates was variable. Synergy was not demonstrated for tobramycin against any of the strains tested. This led to the hypothesis that iron chelation preserves the binding of tetracyclines to the bacterial ribosome. Susceptibility to iron-bound doxycycline was decreased by two- to four-fold and synergistic interactions with the iron chelator were consistently more intense with iron-bound doxycycline than with doxycycline alone. The doxycycline-iron chelator combination also significantly reduced cell viability in established biofilms., Conclusion: The data in this study provide evidence that iron chelation enhances the anti-pseudomonal activity of tetracyclines, specifically doxycycline., Competing Interests: Declarations of Competing Interests The authors have no conflict of interests to declare, (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Hypermobile Ehlers-Danlos syndrome (hEDS) is the most frequent type of EDS and is characterized by generalized joint hypermobility and musculoskeletal manifestations which are associated with chronic pain, and mild skin involvement along with the presence of more than a few comorbid conditions. Despite numerous research efforts, no causative gene(s) or validated biomarkers have been identified and insights into the disease-causing mechanisms remain scarce. Variability in the spectrum and severity of symptoms and progression of hEDS patients' phenotype likely depend on a combination of age, gender, lifestyle, and the probable multitude of genes involved in hEDS. However, considering the clinical overlap with other EDS forms, which lead to abnormalities in extracellular matrix (ECM), it is plausible that the mechanisms underlying hEDS pathogenesis also affect the ECM to a certain extent. Herein, we performed a series of in vitro studies on the secretome of hEDS dermal fibroblasts that revealed a matrix metalloproteinases (MMPs) dysfunction as one of the major disease drivers by causing a detrimental feedback loop of excessive ECM degradation coupled with myofibroblast differentiation. We demonstrated that doxycycline-mediated inhibition of MMPs rescues in hEDS cells a control-like ECM organization and induces a partial reversal of their myofibroblast-like features, thus offering encouraging clues for translational studies confirming MMPs as a potential therapeutic target in hEDS with the expectation to improve patients' quality of life and alleviate their disabilities.

We evaluated antibiotic activity against the intracellular bacterium Coxiella burnetii using an activated THP-1 cell model of infection. At clinically relevant concentrations, the intracellular bacterial load was reduced 300-fold by levofloxacin and finafloxacin, 40-fold by doxycycline, and 4-fold by ciprofloxacin and was unaffected by azithromycin. Acidification of the culture medium reduced antibiotic activity, with the exceptions of doxycycline (no change) and finafloxacin (slight improvement). This model may be used to select antibiotics to be evaluated in vivo .

P21-activated kinase 3 (PAK3) gene mutations are linked to several neurodevelopmental disorders, but the underlying mechanisms remain unclear. In this study, we used a tetracycline-inducible system to control the expression of a mutant PAK3 (mPAK3) protein in immediate early gene, namely cFos, positive cells to disrupt PAK signaling, specifically in cells activated by social interaction in transgenic mice. We show that the expression of mPAK3-GFP proteins was in cFos-expressing excitatory and inhibitory neurons in various brain regions, such as the cortex and hippocampus, commonly activated during learning and memory. Basal expression of mPAK3-GFP proteins in cFos-positive cells resulted in social recognition memory deficits in the three-chamber social interaction test, without affecting locomotor activity or other forms of memory. The social memory deficit was rescued by doxycycline to halt the mPAK3-GFP transgene expression. In addition, we show that the expression of mPAK3-GFP proteins in a subset of cFos-positive cells, induced by an antecedent short social interaction, termed social pairing, was sufficient to impair social recognition memory. These results indicate that normal PAK signaling in cFos-positive cells activated during social interaction is critical for social memory.

Purpose: Cardiac transition from concentric (C-LVH) to eccentric left ventricle hypertrophy (E-LVH) is a maladaptive response of hypertension. Matrix metalloproteinases (MMPs), in particular MMP-2, may contribute to tissue remodeling by proteolyzing extra- and intracellular proteins. Troponin I and dystrophin are two potential targets of MMP-2 examined in this study and their proteolysis would impair cardiac contractile function. We hypothesized that MMP-2 contributes to the decrease in troponin I and dystrophin in the hypertensive heart and thereby controls the transition from C-LVH to E-LVH and cardiac dysfunction., Methods: Male Wistar rats were divided into sham or two kidney-1 clip (2K-1C) hypertensive groups and treated with water (vehicle) or doxycycline (MMP inhibitor, 15 mg/kg/day) by gavage from the tenth to the sixteenth week post-surgery. Tail-cuff plethysmography, echocardiography, gelatin zymography, confocal microscopy, western blot, mass spectrometry, in silico protein analysis and immunofluorescence were performed., Results: 6 out of 23 2K-1C rats (26%) had E-LVH followed by reduced ejection fraction. The remaining had C-LVH with preserved cardiac function. Doxycycline prevented the transition from C-LVH to E-LVH. MMP activity is increased in C-LVH and E-LVH hearts which was inhibited by doxycycline. This effect was associated with an increase in troponin I cleavage products and a decline in dystrophin in the left ventricle of E-LVH rats, which was prevented by doxycycline., Conclusion: Hypertension causes increased cardiac MMP-2 activity which proteolyzes troponin I and dystrophin, contributing to the transition from C-LVH to E-LVH and cardiac dysfunction., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Lung cancer constitutes a threat to human health. BHLHE41 plays important roles in circadian rhythm and cell differentiation as a negative regulatory transcription factor. This study investigates the role of BHLHE41 in lung cancer progression. We analyzed BHLHE41 function via in silico and immunohistochemical studies of 177 surgically resected non-small cell lung cancer (NSCLC) samples and 18 early lung squamous cell carcinoma (LUSC) cases. We also examined doxycycline (DOX)-inducible BHLHE41-expressing A549 and H2030 adenocarcinoma cells. BHLHE41 expression was higher in normal lung than in lung adenocarcinoma (LUAD) tissues and was associated with better prognosis for the overall survival (OS) of patients. In total, 15 of 132 LUAD tissues expressed BHLHE41 in normal lung epithelial cells. Staining was mainly observed in adenocarcinoma in situ and the lepidic growth part of invasive cancer tissue. BHLHE41 expression constituted a favorable prognostic factor for OS ( p = 0.049) and cause-specific survival ( p = 0.042) in patients with LUAD. During early LUSC, 7 of 18 cases expressed BHLHE41, and this expression was inversely correlated with the depth of invasion. DOX suppressed cell proliferation and increased the autophagy protein LC3, while chloroquine enhanced LC3 accumulation and suppressed cell death. In a xenograft model, DOX suppressed tumor growth. Our results indicate that BHLHE41 expression prevents early lung tumor malignant progression by inducing autophagic cell death in NSCLC.

Enhancers are critical cis-regulatory elements that regulate spatiotemporal gene expression and control cell fates. However, the identification of enhancers in native cellular contexts still remains a challenge. Here, we develop an inducible CRISPR activation (CRISPRa) system by transgenic expression of doxycycline (Dox)-inducible dCas9-VPR in mouse embryonic stem cells (iVPR ESC). With this line, a simple introduction of specific guide RNAs targeting promoters or enhancers allows us to realize the effect of CRISPRa in an inducible, reversible, and Dox concentration-dependent manner. Taking advantage of this system, we induce tiled CRISPRa across genomic regions (105 kilobases) surrounding T (Brachyury), one of the key mesodermal development regulator genes. Moreover, we identify several CRISPRa-responsive elements with chromatin features of putative enhancers, including a region the homologous sequence in which humans harbors a body height risk variant. Genetic deletion of this region in ESC does affect subsequent T gene activation and osteogenic differentiation. Therefore, our inducible CRISPRa ESC line provides a convenient platform for high-throughput screens of putative enhancers., Competing Interests: Conflict of interest The authors declare that they have no competing interests., (Copyright © 2021 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Ltd. All rights reserved.)

Traumatic brain injury (TBI) is one of the significant causes of death among young people worldwide. Doxycycline (DOX), an antibiotic with anti-inflammatory effects, has not been used as a therapeutic agent to modify the inflammatory response after the traumatic brain injury. In this study, intraperitoneal administration of DOX reduced significantly the acute inflammatory markers like IL-6 and CD3, microglial migration to the damaged area marked with Iba-1, and neuronal apoptosis assessed with TUNEL assay at 72 h after the trauma. The low dose, 10 mg/kg of DOX had a dominant anti-inflammatory effect; while the high dose, 100 mg/kg of DOX, was more effective in decreasing neuronal apoptosis. In early hours after the head trauma, use of a low dose (10 mg/kg) of DOX for decreasing the acute form of inflammation followed by a high dose (100 mg/kg) for the anti-apoptotic effects particularly in severe head traumas, would be a promising approach to alleviate the brain injury., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Natural products have been the main source of bioactive molecules for centuries. We tested the biological profile of two metabolites extracted from Gentiana lutea L. by means of computational techniques and in vitro assays. The two molecules (loganic acid and gentiopicroside) were tested in silico using an innovative technique, named Inverse Virtual Screening (IVS), to highlight putative partners among a panel of proteins involved in inflammation and cancer events. A positive binding with cyclooxygenase-2 (COX-2), alpha-1-antichymotrypsin, and alpha-1-acid glycoprotein emerged from the computational experiments and the outcomes from the promising interaction with COX-2 were confirmed by Western blot, highlighting the reliability of IVS in the field of the natural products.

Background and Aims: Induced pluripotent stem cells (iPSCs) provide an important tool for the generation of patient-derived cells, including hepatocyte-like cells, by developmental cues through an endoderm intermediate. However, most iPSC lines fail to differentiate into endoderm, with induction resulting in apoptosis., Approach and Results: To address this issue, we built upon published methods to develop an improved protocol. We discovered that doxycycline dramatically enhances the efficiency of iPSCs to endoderm differentiation by inhibiting apoptosis and promoting proliferation through the protein kinase B pathway. We tested this protocol in >70 iPSC lines, 90% of which consistently formed complete sheets of endoderm. Endoderm generated by our method achieves similar transcriptomic profiles, expression of endoderm protein markers, and the ability to be further differentiated to downstream lineages., Conclusions: Furthermore, this method achieves a 4-fold increase in endoderm cell number and will accelerate studies of human diseases in vitro and facilitate the expansion of iPSC-derived cells for transplantation studies., (© 2021 by the American Association for the Study of Liver Diseases.)

Objectives: The anti-cancer activity of doxycycline has been reported in many cancers but not renal cell carcinoma (RCC). This study aimed to determine the efficacy of doxycycline alone and in combination with paclitaxel and analyze the underlying mechanism in RCC., Methods: Proliferation, colony formation and apoptosis assays were performed in RCC cell lines after drug treatments. An RCC xenograft mouse model was generated, and tumor growth was monitored. Mechanistic studies focused on mitochondrial translation and functions., Results: Doxycycline at clinically achievable concentrations inhibited proliferation and colony formation and induced apoptosis in RCC cell lines. In normal kidney cells, doxycycline at the same concentrations either had no effect or was less effective. The combination index value demonstrated that doxycycline and paclitaxel were synergistic in vitro . Consistently, this combination therapy was significantly more effective than the monotherapy in RCC xenograft mice without causing significant toxicity. Mechanistic studies revealed that doxycycline acts on RCC cells via preferentially inhibiting mitochondrial DNA translation, thereby disrupting multiple mitochondrial complexes and impairing mitochondrial respiration., Conclusions: Doxycycline is a useful addition to the treatment strategy for RCC. Our work also highlights the therapeutic value of mitochondrial translation inhibition in sensitizing RCC to chemotherapy.

Through the efficient clearance of extracellular glutamate, high affinity astrocytic glutamate transporters constantly shape excitatory neurotransmission in terms of duration and spreading. Even though the glutamate transporter GLT-1 (also known as EAAT2/SLC1A2) is amongst the most abundant proteins in the mammalian brain, its density and activity are tightly regulated. In order to study the influence of changes in the expression of GLT-1 on glutamate uptake capacity, we have developed a model in HEK cells where the density of the transporter can be manipulated thanks to a tetracycline-inducible promoter. Exposing the cells to doxycycline concentration-dependently increased GLT-1 expression and substrate uptake velocity. However, beyond a certain level of induction, increasing the density of transporters at the cell surface failed to increase the maximal uptake. This suggested the progressive generation of a pool of spare transporters, a hypothesis that was further validated using the selective GLT-1 blocker WAY-213613 of which potency was influenced by the density of the transporters. The curve showing inhibition of uptake by increasing concentrations of WAY-213613 was indeed progressively rightward shifted when tested in cells where the transporter density was robustly induced. As largely documented in the context of cell-surface receptors, the existence of 'spare' glutamate transporters in the nervous tissue and particularly in astrocytes could impact on the consequences of physiological or pathological regulation of these transporters., (Copyright © 2021. Published by Elsevier Ltd.)