Phosphoglycerate mutase 5 exacerbates alcoholic cardiomyopathy in male mice by inducing prohibitin-2 dephosphorylation and impairing mitochondrial quality control.

BACKGROUND: The induction of mitochondrial quality control (MQC) mechanisms is essential for the re-establishment of mitochondrial homeostasis and cellular bioenergetics during periods of stress. Although MQC activation has cardioprotective effects in various cardiovascular diseases, its precise role and regulatory mechanisms in alcoholic cardiomyopathy (ACM) remain incompletely understood. METHODS: We explored whether two mitochondria-related proteins, phosphoglycerate mutase 5 (Pgam5) and prohibitin 2 (Phb2), influence MQC in male mice during ACM. RESULTS: Myocardial Pgam5 expression was upregulated in a male mouse model of ACM. Notably, following ACM induction, heart dysfunction was markedly reversed in male cardiomyocyte-specific Pgam5 knockout (Pgam5cKO) mice. Meanwhile, in alcohol-treated male mouse-derived neonatal cardiomyocytes, Pgam5 depletion preserved cell survival and restored mitochondrial dynamics, mitophagy, mitochondrial biogenesis and the mitochondrial unfolded protein response (mtUPR). We further found that in alcohol-treated cardiomyocyte, Pgam5 binds Phb2 and induces its dephosphorylation at Ser91. Alternative transduction of phospho-mimetic (Phb2S91D) and phospho-defective (Phb2S9A) Phb2 mutants attenuated and enhanced, respectively, alcohol-related mitochondrial dysfunction in cardiomyocytes. Moreover, transgenic male mice expressing Phb2S91D were resistant to alcohol-induced heart dysfunction. CONCLUSIONS: We conclude that ACM-induced Pgam5 upregulation results in Pgam5-dependent Phb2S91 dephosphorylation, leading to MQC destabilisation and mitochondrial dysfunction in heart. Therefore, modulating the Pgam5/Phb2 interaction could potentially offer a novel therapeutic strategy for ACM in male mice. HIGHLIGHTS: Pgam5 knockout attenuates alcohol-induced cardiac histopathology and heart dysfunction in male mice. Pgam5 KO reduces alcohol-induced myocardial inflammation, lipid peroxidation and metabolic dysfunction in male mice. Pgam5 depletion protects mitochondrial function in alcohol-exposed male mouse cardiomyocytes. Pgam5 depletion normalises MQC in ACM. EtOH impairs MQC through inducing Phb2 dephosphorylation at Ser91. Pgam5 interacts with Phb2 and induces Phb2 dephosphorylation. Transgenic mice expressing a Ser91 phospho-mimetic Phb2 mutant are resistant to ACM.

NIR Light and GSH Dual-Responsive Upconversion Nanoparticles Loaded with Multifunctional Platinum(IV) Prodrug and RGD Peptide for Precise Cancer Therapy.

Platinum(II) drugs as a first-line anticancer reagent are limited by side effects and drug resistance. Stimuli-responsive nanosystems hold promise for precise spatiotemporal manipulation of drug delivery, with the aim to promote bioavailability and minimize side effects. Herein, a multitargeting octahedral platinum(IV) prodrug with octadecyl aliphatic chain and histone deacetylase inhibitor (phenylbutyric acid, PHB) at axial positions to improve the therapeutic effect of cisplatin was loaded on the upconversion nanoparticles (UCNPs) through hydrophobic interaction. Followed attachment of DSPE-PEG2000 and arginine-glycine-aspartic (RGD) peptide endowed the nanovehicles with high biocompatibility and tumor specificity. The fabricated nanoparticles (UCNP/Pt(IV)-RGD) can be triggered by upconversion luminescence (UCL) irradiation and glutathione (GSH) reduction to controllably release Pt(II) species and PHB, inducing profound cytotoxicity. Both in vitro and in vivo experiments demonstrated that UCNP/Pt(IV)-RGD exhibited remarkable antitumor efficiency, high tumor-targeting specificity, and real-time UCL imaging capacity, presenting an intelligent platinum(IV) prodrug-loaded nanovehicle for UCL-guided dual-stimuli-responsive combination therapy.

Epidemiology of Clostridioides difficile infection at one hospital 10 years after an outbreak of the epidemic C. difficile strain BI/027: changing strain prevalence, antimicrobial susceptibilities, and patient antibiotic exposures.

In contrast to the epidemiology 10 years earlier at our hospital when the epidemic restriction endonuclease analysis (REA) group strain BI accounted for 72% of Clostridioides difficile isolates recovered from first-episode C. difficile infection (CDI) cases, BI represented 19% of first-episode CDI isolates in 2013-2015. Two additional REA group strains accounted for 31% of isolates (Y, 16%; DH, 12%). High-level resistance to fluoroquinolones and azithromycin was more common among BI isolates than among DH, Y, and non-BI/DH/Y isolates. Multivariable analysis revealed that BI cases were 2.47 times more likely to be associated with fluoroquinolone exposure compared to non-BI cases (95% confidence interval [CI]: 1.12-5.46). In addition, the odds of developing a CDI after third- or fourth-generation cephalosporin exposure was 2.83 times for DH cases than for non-DH cases (95% CI: 1.06-7.54). Fluoroquinolone use in the hospital decreased from 2005 to 2015 from a peak of 113 to a low of 56 antimicrobial days/1,000 patient days. In contrast, cephalosporin use increased from 42 to 81 antimicrobial days/1,000 patient days. These changes correlated with a decrease in geometric mean MIC for ciprofloxacin (61.03 to 42.65 mg/L, P = 0.02) and an increase in geometric mean MIC for ceftriaxone (40.87 to 86.14 mg/L, P < 0.01) among BI isolates. The BI strain remained resistant to fluoroquinolones, but an overall decrease in fluoroquinolone use and increase in cephalosporin use were associated with a decrease in the prevalence of BI, an increased diversity of C. difficile strain types, and the emergence of strains DH and Y.

Design and characterization of electroactive gelatin methacrylate hydrogel incorporated with gold nanoparticles empowered with parahydroxybenzaldehyde and curcumin for advanced tissue engineering applications.

Electroconductive polymers are the materials of interest for the fabrication of electro-conductive tissues. Metal ions through the redox systems offer polymers with electrical conductivity. In this study, we processed a gelatin methacrylate (GelMA) network with gold nanoparticles (GNPs) through a redox system with parahydroxybenzaldehyde (PHB) or curcumin to enhance its electrical conductivity. Induction of the redox system with both PHB and curcumin into the GelMA, introduced some new functional groups into the polymeric network, as it has been confirmed by H-NMR and FTIR. These new bonds resulted in higher electro-conductivity when GNPs were added to the polymer. Higher electroactivity was achieved by PHB compared to the curcumin-induced redox system, and the addition of GNPs without redox system induction showed the lowest electroactivity. MTT was used to evaluate the biocompatibility of the resultant polymers, and the PHB-treated hydrogels showed higher proliferative effects on the cells. The findings of this study suggest that the introduction of a redox system by PHB in the GelMA network along with GNPs can contribute to the electrochemical properties of the material. This electroactivity can be advantageous for tissue engineering of electro-conductive tissues like cardiac and nervous tissues.

Suppression of a Spodoptera frugiperda (Sf9) cellular microRNA following Baculovirus infection and its role in the insect cell - virus interactions.

Baculoviruses have been extensively studied for their potential in microbial pest control, but the mechanisms behind their mode of action still need to be addressed. Here we report differential expression of a cellular miRNA, Sfr-miR-184, from Sf9 cells in response to Autographa californica multicapsid Nucleopolyhedrovirus (AcMNPV) infection. Our results showed that Sfr-miR-184 is down-regulated in AcMNPV-infected cells but not with UV-inactivated virus. Prohibitin gene was determined as a target of the miRNA, which was up-regulated following AcMNPV infection. Using synthetic miRNA mimic, we found that oversupply of the miRNA resulted in decreased transcript levels of the target gene. Results suggest that Sfr-miR-184 negatively regulate prohibitin transcripts in the host cells. Antibody-mediated inhibition and silencing of the prohibitin gene revealed significant reductions in virus DNA replication suggesting a possible role for prohibitin in the virus-host interaction. These findings highlight another molecular mechanism used by baculovirus to manipulate host cells for its replication.

Evaluating the osteogenic properties of polyhydroxybutyrate-zein/multiwalled carbon nanotubes (MWCNTs) electrospun composite scaffold for bone tissue engineering applications.

In this investigation, the electrospun nanocomposite scaffolds were developed utilizing poly-3-hydroxybutyrate (PHB), zein, and multiwalled carbon nanotubes (MWCNTs) at varying concentrations of MWCNTs including 0.5 and 1 wt%. Based on the SEM evaluations, the scaffold containing 1 wt% MWCNTs (PZ-1C) exhibited the lowest fiber diameter (384 ± 99 nm) alongside a suitable porosity percentage. The presence of zein and MWCNT in the chemical structure of the scaffold was evaluated by FTIR. Furthermore, TEM images revealed the alignment of MWCNTs with the fibers. Adding 1 % MWCNTs to the PHB-zein scaffold significantly enhanced tensile strength by about 69 % and reduced elongation by about 31 %. Hydrophilicity, surface roughness, crystallinity, and biomineralization were increased by incorporating 1 wt% MWCNTs, while weight loss after in vitro degradation was decreased. The MG-63 cells exhibited enhanced attachment, viability, ALP secretion, calcium deposition, and gene expression (COLI, RUNX2, and OCN) when cultivated on the scaffold containing MWCNTs compared to the scaffolds lacking MWCNTs. Moreover, the study found that MWCNTs significantly reduced platelet adhesion and hemolysis rates below 4 %, indicating their favorable anti-hemolysis properties. Regarding the aforementioned results, the PZ-1C electrospun composite scaffold is a promising scaffold with osteogenic properties for bone tissue engineering applications.

Molecular characterization, expression profiling, and functional analysis of prohibitin 1 in red seabream, Pagrus major.

Prohibitin 1 (PHB1) is ubiquitously expressed in multiple compartments within cells and is involved in the cell cycle, cell signaling, apoptosis, transcriptional regulation, and mitochondrial biogenesis at the cellular level and in the inflammation-associated and immunological functions of B and T lymphocytes. PHB1 is an important protein that performs antioxidant regulation and immune functions inside and outside cells but has not been sufficiently studied in teleost fish. Our study aimed to elucidate the functional properties and gain new insights into the biological processes and immune system of red seabream (Pagrus major), a commercially important fish cultured in South Korea and East Asia. PHB1 mRNA was most abundantly expressed in the head kidney of healthy red seabream, and significant changes in its expression were observed after artificial infection with bacteria and viruses. On analysis, reporter gene was also significantly upregulated by polyinosinic-polycytidylic acid, lipopolysaccharides, and hydrogen peroxide. Consequent to the functional characterization of PHB1 in cells via recombinant protein preparation, the activity of leukocytes was enhanced and the reactive oxygen species-induced stress in red blood cells was reduced. The results reveal the functional characteristics of PHB1 and provide new insights into the biological processes and immune system of P. major, with beneficial implications in the study of stress responses.

Trafficking of mitochondrial double-stranded RNA from mitochondria to the cytosol.

In addition to mitochondrial DNA, mitochondrial double-stranded RNA (mtdsRNA) is exported from mitochondria. However, specific channels for RNA transport have not been demonstrated. Here, we begin to characterize channel candidates for mtdsRNA export from the mitochondrial matrix to the cytosol. Down-regulation of SUV3 resulted in the accumulation of mtdsRNAs in the matrix, whereas down-regulation of PNPase resulted in the export of mtdsRNAs to the cytosol. Targeting experiments show that PNPase functions in both the intermembrane space and matrix. Strand-specific sequencing of the double-stranded RNA confirms the mitochondrial origin. Inhibiting or down-regulating outer membrane proteins VDAC1/2 and BAK/BAX or inner membrane proteins PHB1/2 strongly attenuated the export of mtdsRNAs to the cytosol. The cytosolic mtdsRNAs subsequently localized to large granules containing the stress protein TIA-1 and activated the type 1 interferon stress response pathway. Abundant mtdsRNAs were detected in a subset of non-small-cell lung cancer cell lines that were glycolytic, indicating relevance in cancer biology. Thus, we propose that mtdsRNA is a new damage-associated molecular pattern that is exported from mitochondria in a regulated manner.

Efficacy of second-line anticonvulsant agents with adult status epilepticus: A systematic review and network meta-analysis.

BACKGROUND: Status epilepticus (SE) is potentially life-threatening, however, it is unclear which antiepileptic drugs (AEDs) should be used as second-line AEDs. OBJECTIVE: We conducted a network meta-analysis (NMA) of randomized controlled trials (RCTs) comparing multiple second-line AEDs for SE to investigate the efficacy of AEDs. METHODS: We searched MEDLINE, CENTRAL, ClinicalTrials.gov, and World Health Organization International Clinical Trials Platform Search Portal and included RCTs for patients aged ≥15 years with SE on December 31, 2023. We compared multiple second-line AEDs for SE including fosphenytoin (fPHT), lacosamide (LCM), levetiracetam (LEV), phenytoin (PHT), phenobarbital (PHB), and valproate (VPA). The primary and secondly outcomes were termination of seizures integrating the absence of seizure recurrence at 30 min and 60 min, and adverse events associated with AEDs, respectively, with expressing as relative risk (RR) with a 95% confidence interval (CI). We conducted a NMA using frequentist-based approach with multivariate random effects, and assessed the certainty based on the Grading of Recommendations, Assessment, Development, and Evaluations framework. RESULTS: Seven RCTs (n = 780) were included, and statistically significant difference was detected between VPA vs. PHB (RR, 0.67; 95% CI, 0.53-0.85; very low certainty), fPHT vs. PHB (RR, 0.66; 95% CI, 0.48-0.90; very low certainty), LCM vs. PHB (RR, 0.62; 95% CI, 0.41-0.93; very low certainty), and LEV vs. PHB (RR, 0.69; 95% CI, 0.51-0.94; very low certainty). Moreover, PHB was the highest in the ranking for termination of seizures. For adverse events, no significant reduction was observed owing to the selection of AEDs, although the ranking of PHB was the lowest. CONCLUSIONS: PHB may have been the most effective for seizure termination as second-line AEDs in adult patients with SE. However, the certainty of almost all comparisons was "very low", and careful interpretation is essential.

GOLPH3 inhibits erastin-induced ferroptosis in colorectal cancer cells.

Ferroptosis is a novel form of programmed cell death and is considered to be a druggable target for colorectal cancer (CRC) therapy. However, the role of ferroptosis in CRC and its underlying mechanism are not fully understood. In the present study we found that a protein enriched in the Golgi apparatus, Golgi phosphoprotein 3 (GOLPH3), was overexpressed in human CRC tissue and in several CRC cell lines. The expression of GOLPH3 was significantly correlated with the expression of ferroptosis-related genes in CRC. The overexpression of GOLPH3 in Erastin-induced Caco-2 CRC cells reduced ferroptotic phenotypes, whereas the knockdown of GOLPH3 potentiated ferroptosis in HT-29 CRC cells. GOLPH3 induced the expression of prohibitin-1 (PHB1) and prohibitin-2 (PHB2), which also inhibited ferroptosis in Erastin-treated CRC cells. Moreover, GOLPH3 interacted with PHB2 and nuclear factor erythroid 2-related factor 2 (NRF2) in Caco-2 cells. These observations indicate that GOLPH3 is a negative regulator of ferroptosis in CRC cells. GOLPH3 protects these cells from ferroptosis by inducing the expression of PHB1 and PHB2, and by interacting with PHB2 and NRF2.

Prx1/PHB2 axis mediates mitophagy in oral leukoplakia cellular senescence.

BACKGROUND: Oral leukoplakia (OLK) is the most common oral potentially malignant disorder (OPMD), which can be malignantly transformed into oral squamous cell carcinoma (OSCC). Peroxiredoxin1(Prx1) has been predicted to bind to Prohibitin2 (PHB2), which confers to affect OLK progression; however, the mechanism of Prx1/PHB2 mediated mitophagy involved in OLK remains unclear. METHODS: This study aimed to explore the mechanism of the Prx1/PHB2 axis on senescence in OLK through mediating mitophagy. The positive rate of Ki67 and the expression of p21, p16, PHB2, and LC3 in human normal, OLK, and OSCC tissues were detected by immunohistochemical staining. The mitophagy and mitochondrial function changes were then analyzed in Prx1 knockdown and Prx1C52S mutations in dysplastic oral keratinocyte (DOK) cells treated with H2O2. In situ Proximity Ligation Assay combined with co-immunoprecipitation was used to detect the interaction between Prx1 and PHB2. RESULTS: Clinically, the positive rate of Ki67 progressively increased from normal to OLK, OLK with dysplasia, and OSCC. Higher p21, p16, PHB2, and LC3 expression levels were observed in OLK with dysplasia than in normal and OSCC tissues. In vitro, PHB2 and LC3II expression gradually increased with the degree of DOK cell senescence. Prx1/PHB2 regulated mitophagy and affected senescence in H2O2-induced DOK cells. Furthermore, Prx1C52S mutation specifically reduced interaction between Prx1 and PHB2. Prx1Cys52 is associated with mitochondrial reactive oxygen species (ROS) accumulated and cell cycle arrest. CONCLUSION: Prx1Cys52 functions as a redox sensor that binds to PHB2 and regulates mitophagy in the senescence of OLK, suggesting its potential as a clinical target.

Pyruvate kinase M2 sustains cardiac mitochondrial quality surveillance in septic cardiomyopathy by regulating prohibitin 2 abundance via S91 phosphorylation.

The endogenous mitochondrial quality control (MQC) system serves to protect mitochondria against cellular stressors. Although mitochondrial dysfunction contributes to cardiac damage during many pathological conditions, the regulatory signals influencing MQC disruption during septic cardiomyopathy (SC) remain unclear. This study aimed to investigate the involvement of pyruvate kinase M2 (PKM2) and prohibitin 2 (PHB2) interaction followed by MQC impairment in the pathogenesis of SC. We utilized LPS-induced SC models in PKM2 transgenic (PKM2TG) mice, PHB2S91D-knockin mice, and PKM2-overexpressing HL-1 cardiomyocytes. After LPS-induced SC, cardiac PKM2 expression was significantly downregulated in wild-type mice, whereas PKM2 overexpression in vivo sustained heart function, suppressed myocardial inflammation, and attenuated cardiomyocyte death. PKM2 overexpression relieved sepsis-related mitochondrial damage via MQC normalization, evidenced by balanced mitochondrial fission/fusion, activated mitophagy, restored mitochondrial biogenesis, and inhibited mitochondrial unfolded protein response. Docking simulations, co-IP, and domain deletion mutant protein transfection experiments showed that PKM2 phosphorylates PHB2 at Ser91, preventing LPS-mediated PHB2 degradation. Additionally, the A domain of PKM2 and the PHB domain of PHB2 are required for PKM2-PHB2 binding and PHB2 phosphorylation. After LPS exposure, expression of a phosphorylation-defective PHB2S91A mutant negated the protective effects of PKM2 overexpression. Moreover, knockin mice expressing a phosphorylation-mimetic PHB2S91D mutant showed improved heart function, reduced inflammation, and preserved mitochondrial function following sepsis induction. Abundant PKM2 expression is a prerequisite to sustain PKM2-PHB2 interaction which is a key element for preservation of PHB2 phosphorylation and MQC, presenting novel interventive targets for the treatment of septic cardiomyopathy.

Pyruvate kinase M2 sustains cardiac mitochondrial integrity in septic cardiomyopathy by regulating PHB2-dependent mitochondrial biogenesis.

Previous studies have highlighted the protective effects of pyruvate kinase M2 (PKM2) overexpression in septic cardiomyopathy. In our study, we utilized cardiomyocyte-specific PKM2 knockout mice to further investigate the role of PKM2 in attenuating LPS-induced myocardial dysfunction, focusing on mitochondrial biogenesis and prohibitin 2 (PHB2). Our findings confirmed that the deletion of PKM2 in cardiomyocytes significantly exacerbated LPS-induced myocardial dysfunction, as evidenced by impaired contractile function and relaxation. Additionally, the deletion of PKM2 intensified LPS-induced myocardial inflammation. At the molecular level, LPS triggered mitochondrial dysfunction, characterized by reduced ATP production, compromised mitochondrial respiratory complex I/III activities, and increased ROS production. Intriguingly, the absence of PKM2 further worsened LPS-induced mitochondrial damage. Our molecular investigations revealed that LPS disrupted mitochondrial biogenesis in cardiomyocytes, a disruption that was exacerbated by the absence of PKM2. Given that PHB2 is known as a downstream effector of PKM2, we employed PHB2 adenovirus to restore PHB2 levels. The overexpression of PHB2 normalized mitochondrial biogenesis, restored mitochondrial integrity, and promoted mitochondrial function. Overall, our results underscore the critical role of PKM2 in regulating the progression of septic cardiomyopathy. PKM2 deficiency impeded mitochondrial biogenesis, leading to compromised mitochondrial integrity, increased myocardial inflammation, and impaired cardiac function. The overexpression of PHB2 mitigated the deleterious effects of PKM2 deletion. This discovery offers a novel insight into the molecular mechanisms underlying septic cardiomyopathy and suggests potential therapeutic targets for intervention.

Synergistic effects of sulopenem in combination with cefuroxime or durlobactam against Mycobacterium abscessus.

Mycobacterium abscessus (Mab) affects patients with immunosuppression or underlying structural lung diseases such as cystic fibrosis (CF). Additionally, Mab poses clinical challenges due to its resistance to multiple antibiotics. Herein, we investigated the synergistic effect of dual ß-lactams [sulopenem and cefuroxime (CXM)] or the combination of sulopenem and CXM with ß-lactamase inhibitors [BLIs-avibactam (AVI) or durlobactam (DUR)]. The sulopenem-CXM combination yielded low minimum inhibitory concentration (MIC) values for 54 clinical Mab isolates and ATCC19977 (MIC50 and MIC90 ≤0.25 µg/mL). Similar synergistic effects were observed in time-kill studies conducted at concentrations achievable in clinical settings. Sulopenem-CXM outperformed monotherapy, yielding ~1.5 Log10 CFU/mL reduction during 10 days. Addition of BLIs enhanced this antibacterial effect, resulting in an additional reduction of CFUs (~3 Log10 for sulopenem-CXM and AVI and ~4 Log10 for sulopenem-DUR). Exploration of the potential mechanisms of the synergy focused on their interactions with L,D-transpeptidases (Ldts; LdtMab1-LdtMab4), penicillin-binding-protein B (PBP B), and D,D-carboxypeptidase (DDC). Acyl complexes, identified via mass spectrometry analysis, demonstrated the binding of sulopenem with LdtMab2-LdtMab4, DDC, and PBP B and CXM with LdtMab2 and PBP B. Molecular docking and mass spectrometry data suggest the formation of a covalent adduct between sulopenem and LdtMab2 after the nucleophilic attack of the cysteine residue at the ß-lactam carbonyl carbon, leading to the cleavage of the ß-lactam ring and the establishment of a thioester bond linking the LdtMab2 with sulopenem. In conclusion, we demonstrated the biochemical basis of the synergy of sulopenem-CXM with or without BLIs. These findings potentially broaden the selection of oral therapeutic agents to combat Mab. IMPORTANCE: Treating infections from Mycobacterium abscessus (Mab), particularly those resistant to common antibiotics like macrolides, is notoriously difficult, akin to a never-ending struggle for healthcare providers. The rate of treatment failure is even higher than that seen with multidrug-resistant tuberculosis. The role of combination ß-lactams in inhibiting L,D-transpeptidation, the major peptidoglycan crosslink reaction in Mab, is an area of intense investigation, and clinicians have utilized this approach in the treatment of macrolide-resistant Mab, with reports showing clinical success. In our study, we found that cefuroxime and sulopenem, when used together, display a significant synergistic effect. If this promising result seen in lab settings, translates well into real-world clinical effectiveness, it could revolutionize current treatment methods. This combination could either replace the need for more complex intravenous medications or serve as a "step down" to an oral medication regimen. Such a shift would be much easier for patients to manage, enhancing their comfort and likelihood of sticking to the treatment plan, which could lead to better outcomes in tackling these tough infections. Our research delved into how these drugs inhibit cell wall synthesis, examined time-kill data and binding studies, and provided a scientific basis for the observed synergy in cell-based assays.

Prohibitin 2 deficiency in photoreceptors leads to progressive retinal degeneration and facilitated Müller glia engulfing microglia debris.

Müller glia and microglia are capable of phagocytosing fragments of retinal cells in response to retinal injury or degeneration. However, the direct evidence for their mutual interactions between Müller glia and microglia in the progression of retinal degeneration (RD) remains largely unclear. This study aims to construct a progressive RD mouse model and investigate the activated pattern of Müller glia and the interplay between Müller glia and microglia in the early stage or progression of RD. A Prohibitin 2 (Phb2) photoreceptor-specific knockout (RKO) mouse model was generated by crossing Phb2flox/flox mice with Rhodopsin-Cre mice. Optical Coherence Tomography (OCT), histological staining, and Electroretinography (ERG) assessed retinal structure and function, and RKO mice exhibited progressive RD from six weeks of age. In detail, six-week-old RKO mice showed no significant retinal impairment, but severe vision dysfunction and retina thinning were shown in ten-week-old RKO mice. Furthermore, RKO mice were sensitive to Light Damage (LD) and showed severe RD at an early age after light exposure. Bulk retina RNA-seq analysis from six-week-old control (Ctrl) and RKO mice showed reactive retinal glia in RKO mice. The activated pattern of Müller glia and the interplay between Müller glia and microglia was visualized by immunohistology and 3D reconstruction. In six-week-old RKO mice or light-exposed Ctrl mice, Müller glia were initially activated at the edge of the retina. Moreover, in ten-week-old RKO mice or light-exposed six-week-old RKO mice with severe photoreceptor degeneration, abundant Müller glia were activated across the whole retinas. With the progression of RD, phagocytosis of microglia debris by activated Müller glia were remarkably increased. Altogether, our study establishes a Phb2 photoreceptor-specific knockout mouse model, which is a novel mouse model of RD and can well demonstrate the phenotype of progressive RD. We also report that Müller glia in the peripheral retina is more sensitive to the early damage of photoreceptors. Our study provides more direct evidence for Müller glia engulfing microglia debris in the progression of RD due to photoreceptor Phb2 deficiency.

Preparation, characterization and evaluation of HPßCD-PTX/PHB nanoparticles for pH-responsive, cytotoxic and apoptotic properties.

Paclitaxel (PTX) is a potent anticancer drug. However, PTX exhibits extremely poor solubility in aqueous solution along with severe side effects. Therefore, in this study, an inclusion complex was prepared between PTX and hydroxypropyl-ß-cyclodextrin (HPßCD) by solvent evaporation to enhance the drug's solubility. The HPßCD-PTX inclusion complex was then encapsulated in poly-3-hydroxybutyrate (PHB) to fabricate drug-loaded nanoparticles (HPßCD-PTX/PHB NPs) by nanoprecipitation. The HPßCD-PTX/PHB NPs depicted a higher release of PTX at pH 5.5 thus demonstrating a pH-dependent release profile. The cytotoxic properties of HPßCD-PTX/PHB NPs were tested against MCF-7, MDA-MB-231 and SW-620 cell lines. The cytotoxic potential of HPßCD-PTX/PHB NPs was 2.59-fold improved in MCF-7 cells in comparison to free PTX. Additionally, the HPßCD-PTX/PHB NPs improved the antimitotic (1.68-fold) and apoptotic (8.45-fold) effects of PTX in MCF-7 cells in comparison to PTX alone. In summary, these pH-responsive nanoparticles could be prospective carriers for enhancing the cytotoxic properties of PTX for the treatment of breast cancer.

Stability and release mechanism of double emulsification (W1/O/W2) for biodegradable pH-responsive polyhydroxybutyrate/cellulose acetate phthalate microbeads loaded with the water-soluble bioactive compound niacinamide.

Microbeads of biodegradable polyhydroxybutyrate (PHB) offer environmental benefits and economic competitiveness. The aim of this study was to encapsulate a water-soluble bioactive compound, niacinamide (NIA), in a pH-responsive natural matrix composed of PHB and cellulose acetate phthalate (CAP) by double emulsification (W1/O/W2) to improve the encapsulation efficiency (%EE) and loading capacity (%LC). PHB was produced in-house by Escherichia coli JM109 pUC19-23119phaCABA-04 without the inducing agent isopropyl ß-D-1-thiogalactopyranoside (IPTG). The influences of PHB and polyvinyl alcohol (PVA) concentrations, stirring rate, PHB/CAP ratio and initial NIA concentration on the properties of NIA-loaded pH-responsive microbeads were studied. The NIA-loaded pH-responsive PHB/CAP microbeads exhibited a spherical core-shell structure. The average size of the NIA-loaded pH-responsive microbeads was 1243.3 ± 11.5 µm. The EE and LC were 33.3 ± 0.5 % and 28.5 ± 0.4 %, respectively. The release profiles of NIA showed pH-responsive properties, as 94.2 ± 3.5 % of NIA was released at pH 5.5, whereas 99.3 ± 2.4 % of NIA was released at pH 7.0. The NIA-loaded pH-responsive PHB/CAP microbeads were stable for >90 days at 4 °C under darkness, with NIA remaining at 73.65 ± 1.86 %. A cytotoxicity assay in PSVK1 cells confirmed that the NIA-loaded pH-responsive PHB/CAP microbeads were nontoxic at concentrations lower than 31.3 µg/mL, in accordance with ISO 10993-5.

Formulation of biogenic fluorescent pigmented PHB nanoparticles from Rhodanobacter sp. for drug delivery.

Biogenic nanoparticles (NPs) have emerged as promising therapeutic formulations in effective drug delivery. Despite of various positive attributes, these NPs are often conjugated with various cytotoxic organic fluorophores for bioimaging, thereby reducing its effectiveness as a potential carrier. Herein, we aim to formulate biogenic fluorescent pigmented polyhydroxybutyrate (PHB) NPs from Rhodanobacter sp. strain KT31 (OK001852) for drug delivery. The bacterial strain produced 0.5 g L-1 of polyhydroxyalkanoates (PHAs) from 2.04 g L-1 of dry cell weight (DCW) under optimised conditions via submerged fermentation. Further, structural, thermal, and morphological charactersiation of the extracted PHAs was conducted using advance analytical technologies. IR spectra at 1719.25 cm-1 confirmed presence of C = O functional group PHB. NMR and XRD analysis validated the chemical structure and crystallinity of PHB. TG-DTA revealed Tm (168 °C), Td (292 °C), and Xc (35%) of the PHB. FE-SEM imaging indicated rough surface of the PHB film and the biodegradability was confirmed from open windro composting. WST1 assay showed no significant cell death (> 50%) from 100 to 500 µg/mL, endorsing non-cytotoxic nature of PHB. PHB NPs were uniform, smooth and spherical with size distribution and mean zeta potential 44.73 nm and 0.5 mV. IR and XRD peaks obtained at 1721.75 cm-1 and 48.42 Å denoted C = O and crystalline nature of PHB. Cell proliferation rate of PHB NPs was quite significant at 50 µg/mL, establishing the non-cytotoxic nature of NPs. Further, in vitro efficacy of the PHB NPs needs to be evaluated prior to the biomedical applications.

Pgam5 aggravates hyperglycemia-induced myocardial dysfunction through disrupting Phb2-dependent mitochondrial dynamics.

This study aims to elucidate the roles of Phosphoglycerate Mutase Family Member 5 (Pgam5) and Prohibitin 2 (Phb2) in the context of hyperglycemia-induced myocardial dysfunction, a critical aspect of diabetic cardiomyopathy. The research employed primary cardiomyocytes, which were then subjected to hyperglycemia treatment to mimic diabetic conditions. We used siRNA transfection to knock down Pgam5 and overexpressed Phb2 using adenovirus transfection to assess their individual and combined effects on cardiomyocyte health. Mitochondrial function was evaluated through measurements of mitochondrial membrane potential using the JC-1 probe, and levels of mitochondrial reactive oxygen species (ROS) were assessed. Additionally, the study involved qPCR analysis to quantify the transcriptional changes in genes related to mitochondrial fission and mitophagy. Our findings indicate that hyperglycemia significantly reduces cardiomyocyte viability and impairs mitochondrial function, as evidenced by decreased mitochondrial membrane potential and increased ROS levels. Pgam5 knockdown was observed to mitigate these adverse effects, preserving mitochondrial function and cardiomyocyte viability. On the molecular level, Pgam5 was found to regulate genes associated with mitochondrial fission (such as Drp1, Mff, and Fis1) and mitophagy (including Parkin, Bnip3, and Fundc1). Furthermore, overexpression of Phb2 countered the hyperglycemia-induced mitochondrial dysfunction and normalized the levels of key mitochondrial antioxidant enzymes. The combined data suggest a protective role for both Pgam5 knockdown and Phb2 overexpression against hyperglycemia-induced cellular and mitochondrial damage. The study elucidates the critical roles of Pgam5 and Phb2 in regulating mitochondrial dynamics in the setting of hyperglycemia-induced myocardial dysfunction. By modulating mitochondrial fission and mitophagy, Pgam5 and Phb2 emerge as key players in preserving mitochondrial integrity and cardiomyocyte health under diabetic conditions. These findings contribute significantly to our understanding of the molecular mechanisms underlying diabetic cardiomyopathy and suggest potential therapeutic targets for mitigating myocardial dysfunction in diabetes.

A new strategy for the treatment of middle ear infection using ciprofloxacin/amoxicillin-loaded ethyl cellulose/polyhydroxybutyrate nanofibers.

A middle ear infection occurs due to the presence of several microorganisms behind the eardrum (tympanic membrane) and is very challenging to treat due to its unique location and requires a well-designed treatment. If not treated properly, the infection can result in severe symptoms and unavoidable side effects. In this study, excellent biocompatible ethyl cellulose (EC) and biodegradable polyhydroxybutyrate (PHB) biopolymer were used to fabricate drug-loaded nanofiber scaffolds using an electrospinning technique to overcome antibiotic overdose and insufficient efficacy of drug release during treatment. PHB polymer was produced from Halomonas sp., and the purity of PHB was found to around be 90 %. Additionally, ciprofloxacin (CIP) and amoxicillin (AMX) are highly preferable since both drugs are highly effective against gram-negative and gram-positive bacteria to treat several infections. Obtained smooth nanofibers were between 116.24 and 171.82 nm in diameter and the addition of PHB polymer and antibiotics improved the morphology of the nanofiber scaffolds. Thermal properties of the nanofiber scaffolds were tested and the highest Tg temperature resulted at 229 °C. The mechanical properties of the scaffolds were tested, and the highest tensile strength resulted in 4.65 ± 6.33 MPa. Also, drug-loaded scaffolds were treated against the most common microorganisms that cause the infection, such as S.aureus, E.coli, and P.aeruginosa, and resulted in inhibition zones between 10 and 21 mm. MTT assay was performed by culturing human adipose-derived mesenchymal stem cells (hAD MSCs) on the scaffolds. The morphology of the hAD MSCs' attachment was tested with SEM analysis and hAD MSCs were able to attach, spread, and live on each scaffold even on the day of 7. The cumulative drug release kinetics of CIP and AMX from drug-loaded scaffolds were analysed in phosphate-buffered saline (pH: 7.4) within different time intervals of up to 14 days using a UV spectrophotometer. Furthermore, the drug release showed that the First-Order and Korsmeyer-Peppas models were the most suitable kinetic models. Animal testing was performed on SD rats, matrix and collagen deposition occurred on days 5 and 10, which were observed using Hematoxylin-eosin and Masson's trichrome staining. At the highest drug concentration, a better repair effect was observed. Results were promising and showed potential for novel treatment.