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.

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.

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.

UBXN1 promotes liver tumorigenesis by regulating mitochondrial homeostasis.

BACKGROUND: The maintenance of mitochondrial homeostasis is critical for tumor initiation and malignant progression because it increases tumor cell survival and growth. The molecular events controlling mitochondrial integrity that facilitate the development of hepatocellular carcinoma (HCC) remain unclear. Here, we report that UBX domain-containing protein 1 (UBXN1) hyperactivation is essential for mitochondrial homeostasis and liver tumorigenesis. METHODS: Oncogene-induced mouse liver tumor models were generated with the Sleeping Beauty (SB) transposon delivery system. Assessment of HCC cell growth in vivo and in vitro, including tumour formation, colony formation, TUNEL and FACS assays, was conducted to determine the effects of UBXN1 on HCC cells, as well as the involvement of the UBXN1-prohibitin (PHB) interaction in mitochondrial function. Coimmunoprecipitation (Co-IP) was used to assess the interaction between UBXN1 and PHB. Liver hepatocellular carcinoma (LIHC) datasets and HCC patient samples were used to assess the expression of UBXN1. RESULTS: UBXN1 expression is commonly upregulated in human HCCs and mouse liver tumors and is associated with poor overall survival in HCC patients. UBXN1 facilitates the growth of human HCC cells and promotes mouse liver tumorigenesis driven by the NRas/c-Myc or c-Myc/shp53 combination. UBXN1 interacts with the inner mitochondrial membrane protein PHB and sustains PHB expression. UBXN1 inhibition triggers mitochondrial damage and liver tumor cell apoptosis. CONCLUSIONS: UBXN1 interacts with PHB and promotes mitochondrial homeostasis during liver tumorigenesis.

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.

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.

PTPLAD1 Regulates PHB-Raf Interaction to Orchestrate Epithelial-Mesenchymal and Mitofusion-Fission Transitions in Colorectal Cancer.

Colorectal cancer (CRC) remains one of the leading causes of cancer-related death worldwide. The poor prognosis of this malignancy is attributed mainly to the persistent activation of cancer signaling for metastasis. Here, we showed that protein tyrosine phosphatase-like A domain containing 1 (PTPLAD1) is down-regulated in highly metastatic CRC cells and negatively associated with poor survival of CRC patients. Systematic analysis reveals that epithelial-to-mesenchymal transition (EMT) and mitochondrial fusion-to-fission (MFT) transition are two critical features for CRC patients with low expression of PTPLAD1. PTPLAD1 overexpression suppresses the metastasis of CRC in vivo and in vitro by inhibiting the Raf/ERK signaling-mediated EMT and mitofission. Mechanically, PTPLAD1 binds with PHB via its middle fragment (141-178 amino acids) and induces dephosphorylation of PHB-Y259 to disrupt the interaction of PHB-Raf, resulting in the inactivation of Raf/ERK signaling. Our results unveil a novel mechanism in which Raf/ERK signaling activated in metastatic CRC induces EMT and mitochondrial fission simultaneously, which can be suppressed by PTPLAD1. This finding may provide a new paradigm for developing more effective treatment strategies for CRC.

[Faecal microbiota study reveals specific dysbiosis in spondyloarthritis according to subtype, disease activity and treatment]. / Estudio de microbiota fecal revela disbiosis específica en espondiloartritis, según subtipo, actividad de la enfermedad y tratamiento.

OBJECTIVE: To compare the diversity and composition of the gastrointestinal microbiome of patients with SpA. METHODS: MiSeq sequencing of the V3-V4 region of the 16S ribosomal RNA gene was performed on DNA isolated from stool. Patients with concurrent SpA and IBD were excluded. Differences were assessed for richness and diversity indices by QIIME 2™. Differences between means >0,2% with a p-value<0,05 were assumed significant. Institutional Ethics Committee endorsement. RESULTS: 69 individuals included, 49 with SpA (ankylosing spondylitis-AS 72,9%, psoriatic arthritis-PsA 18,8%, reactive arthritis-ReA 8,3%) 5 positive controls-dysbiosis and 15 controls-eubiosis. Conventional treatment in 42,9%, anti-IL-17 16,3% and anti-TNF 40,8%. By subtype, statistically significant differences in favour of AS were found for the diversity indices. AS vs PsA there was a difference in favour of AS for Clostridium clostridioforme (p=0,002), Gemmiger formicilis (p=0,009), Roseburia inulivorans (p=0,008) and Lachnospira pectinoschiza. AS vs ReA there was a difference in favour of AS for L. pectinoschiza (p=0,009), Ruminococcus callidus (p=0.006), Clostridium ruminantium (p=0.031); G. formicilis (p=0,034). Diversity and richness showed differences in patients with high activity for Simpson's and Pielou's indices. In high activity, lower enrichment of Bacteroides eggerthii (p= 0,0003), C. ruminantium (p= 0,026) and Alistipes putredinis (p=0,035) was found. The number of ASV was higher in the anti-IL-17 vs conventional group (p=0.025) and a trend between anti-IL-17 vs anti-TNF (p=0.09). In anti-TNF there was a lower proportion for C. clostridioforme (p=0.023), G. formicilis (p=0.030) and R. callidus (p= 0.003). In anti IL-17, Alistipes indistinctus (p= 0.012) was decreased. CONCLUSIONS: There are differences in microbial diversity for SpA subtypes. The level of disease activity is plausible to influence the composition of the faecal microbiota. Anti-TNFα treatment may influence the microbiome environment favouring restoration of the gut microbiota, while anti-IL-17 may maintain an inflammatory environment.

OBJETIVO: Comparar la diversidad y composición del microbioma gastrointestinal de pacientes con EspA. MÉTODOS: La secuenciación MiSeq de la región V3-V4 del gen ARN ribosomal 16, se realizó en ADN aislado de heces. Se excluyeron pacientes con EspA y EII simultánea. Se evaluaron diferencias para los índices de riqueza y diversidad por medio de QIIME 2™. Las diferencias entre medias> 0,2%, con un valor de p< 0,05, se asumieron significativas. Aval del Comité de Ética Institucional. RESULTADOS: 69 individuos incluidos, 49 con EspA (espondilitis anquilosante-EA 72,9%, artritis psoriásica-APs 18,8%, artritis reactiva-ARe 8,3%), cinco controles positivos-disbiosis y 15 controles-eubiosis. El tratamiento convencional en 42,9%, anti-IL-17 16,3%, y anti-TNF 40,8%. Por subtipo-EasP, se encontraron diferencias estadísticamente significativas a favor de EA para los índices de diversidad. Entre EA vs APs, hubo diferencia a favor de EA para Clostridium clostridioforme (p=0,002), Gemmiger formicilis (p=0,009), Roseburia inulivorans (p=0,008) y Lachnospira pectinoschiza. Entre EA vs ARe hubo diferencia a favor de EA para L. pectinoschiza (p=0,009), Ruminococcus callidus (p = 0,006), Clostridium ruminantium (p=0,031); G. formicilis (p=0,034). La diversidad y riqueza mostraron diferencias en pacientes con alta actividad para los índices de Simpson y Pielou. En alta actividad, se encontró menor enriquecimiento de Bacteroides eggerthii (p=0,0003), C. ruminantium (p= 0,026) y Alistipes putredinis (p= 0,035). El número de ASV fue superior en el grupo de anti IL-17 vs convencional (p=0.025), y una tendencia entre anti IL-17 vs anti-TNF (p=0,09). En anti TNF hubo menor proporción para C. clostridioforme (p=0,023), G. formicilis (p=0,030) y R. callidus (p= 0,003). Y en anti IL-17, Alistipes indistinctus (p= 0,012), estuvo disminuida. CONCLUSIONES: Existen diferencias en la diversidad microbiana para los subtipos de EspA. El nivel de actividad de la enfermedad es plausible para influir en la composición de microbiota fecal. El tratamiento con anti-TNFα, puede influenciar el ambiente del microbioma favoreciendo la restauración de la microbiota intestinal, mientras los anti IL-17 podrían mantener un ambiente inflamatorio.

Inorganic polyphosphate and ion transport across biological membranes.

Inorganic polyphosphate (polyP) is widely recognized for playing important roles and processes involved in energy and phosphate storage, regulation of gene expression, and calcium signaling. The less well-known role of polyP is as a direct mediator of ion transport across biological membranes. Here, we will briefly summarize current knowledge of the molecular mechanisms of how polyP can be involved in membrane ion transport. We discuss three types of mechanisms that might involve polyP: (1) formation of non-protein channel complex that includes calcium, polyP, and polyhydroxybutyrate (PHB); (2) modulation of the channel activity of PHBlated protein channels; and (3) direct effects of polyP on the function of the voltage-gated ion channels in the process that do not involve PHB.

Moringin, an isothiocyanate modulates multiple cellular signalling molecules in breast cancer cells.

Prohibitin (PHB) is a pleiotropic molecule with a variety of known functions and subcellular locations. PHB's function in breast cancer is poorly understood. Herein, we report that PHB is expressed in cancer types of diverse origin including breast cancer. The cancer patients with changes in PHB were reported to have significantly reduced 'overall survival' in comparison to the cases without alterations in PHB. The expression of PHB was increased by H2O2 and also by Moringin (MG), which is an isothiocyanate derived from the seeds of Moringa oleifera. MG interacted with PHB, DRP1, and SLP2 and inhibited the growth of MCF-7 and MDAMB-231 cells. The isothiocyanate triggered apoptosis in breast cancer cells as revealed by AO/PI assay, phosphatidylserine externalization, cell cycle analysis and DAPI staining. MG induced proapoptotic proteins expression such as cytochrome c, p53, and cleaved caspase-7. Further, cell survival proteins such as survivin, Bcl-2, and Bcl-xL were suppressed. A depolarization of membrane potential suggested that the apoptosis was triggered through mitochondria. The isothiocyanate suppressed the cancer cell migration and interacted with NF-κB subunits. MG suppressed p65 nuclear translocation induced by TNF-α. The reactive oxygen species generation was also induced by the isothiocyanate in breast cancer cells. MG also modulated the expression of lncRNAs. Collectively, the functions of PHB in breast cancer growth is evident from this study. The activities of MG against breast cancer might result from its ability to modulate multiple cancer-related targets.

Impact of the environmental parameters on single cell protein production and composition by Cupriavidus necator.

Due to the rapid increase in the world's population, many developing countries are facing malnutrition problems, including famine and food insecurity. Particularly, the deficiency of protein sources becomes a serious problem for human and animal nutrition. In this context, Single Cell Proteins, could be exploited as an alternative source of unconventional proteins. The aim of the study was to investigate SCP production and composition by Cupriavidus necator under various environmental conditions, temperature and pH values. A mono-factorial approach was implemented using batch bioreactor cultures under well-controlled conditions. Results were compared in terms of bacterial growth and SCP composition (proteins, nucleic acids, amino acids and elemental formula). Complementary analyses were performed by flow cytometry to study cell morphology, membrane permeability and the presence of Poly(3-hydroxybutyrate) (PHB) production. Our data confirmed the ability of C. necator to produce high amount of proteins (69 %DW at 30 °C and pH7). The results showed that temperature and pH independently impact SCP production and composition. This impact was particularly observed at the highest temperature (40 °C) and also the lowest pH value (pH5) providing lower growth rates, cell elongation, changes in granularity and lower amounts of proteins (down to 44 %DW at pH5) and nucleic acids. These low percentages were related to the production of PHB production (up to 44 %DW at 40 °C) which is the first report of a PHB accumulation in C. necator under nutrient unlimited conditions.

Codelivery of CPT and siPHB1 with GSH/ROS Dual-Responsive Hybrid Nanoparticles Based on a [12]aneN3-Derived Lipid for Synergistic Lung Cancer Therapy.

The combination of small-interfering RNA (siRNA)-mediated gene silencing and chemotherapeutic agents for lung cancer treatment has attracted widespread attention in terms of a greater therapeutic effect, minimization of systemic toxicity, and inhibition of multiple drug resistance (MDR). In this work, three amphiphiles, CBN1-CBN3, were first designed and synthesized as a camptothecin (CPT) conjugate and gene condensation agents by the combination of CPT prodrugs and di(triazole-[12]aneN3) through the ROS-responsive phenylborate ester and different lengths of alkyl chains (with 6, 9, 12 carbon chains for CBN1-CBN3, respectively). CBN1-CBN3 were able to be self-assembled into liposomes with an average diameter in the range of 320-240 nm, showing the ability to effectively condense siRNA. Among them, CBN2, with a nine-carbon alkyl chain, displayed the best anticancer efficiency in A549 cells. In order to give nanomedicines a stealth property and PEGylation/dePEGylation transition, a GSH-responsive PEGylated TPE derivative containing a disulfide linkage (TSP) was further designed and prepared. A combination of CBN2/siRNA complexes and DOPE with TSP resulted in GSH/ROS dual-responsive lipid-polymer hybrid nanoparticles (CBN2-DP/siRNA NPs). In present GSH and H2O2, CBN2-DP/siRNA NPs were decomposed, resulting in the controlled release of CPT drug and siRNA. In vitro, CBN2-DP/siPHB1 NPs showed the best anticancer activity for suppression of about 75% of A549 cell proliferation in a serum medium. The stability of CBN2-DP/siRNA NPs was significantly prolonged in blood circulation, and they showed effective accumulation in the A549 tumor site through an enhanced permeability and retention (EPR) effect. In vivo, CBN2-DP/siPHB1 NPs demonstrated enhanced synergistic cancer therapy efficacy and tumor inhibition as high as 71.2%. This work provided a strategy for preparing lipid-polymer hybrid NPs with GSH/ROS dual-responsive properties and an intriguing method for lung cancer therapy.

A stagewise response to mitochondrial dysfunction in mitochondrial DNA maintenance disorders.

Mitochondrial DNA (mtDNA) deletions which clonally expand in skeletal muscle of patients with mtDNA maintenance disorders, impair mitochondrial oxidative phosphorylation dysfunction. Previously we have shown that these mtDNA deletions arise and accumulate in perinuclear mitochondria causing localised mitochondrial dysfunction before spreading through the muscle fibre. We believe that mito-nuclear signalling is a key contributor in the accumulation and spread of mtDNA deletions, and that knowledge of how muscle fibres respond to mitochondrial dysfunction is key to our understanding of disease mechanisms. To understand the contribution of mito-nuclear signalling to the spread of mitochondrial dysfunction, we use imaging mass cytometry. We characterise the levels of mitochondrial Oxidative Phosphorylation proteins alongside a mitochondrial mass marker, in a cohort of patients with mtDNA maintenance disorders. Our expanded panel included protein markers of key signalling pathways, allowing us to investigate cellular responses to different combinations of oxidative phosphorylation dysfunction and ragged red fibres. We find combined Complex I and IV deficiency to be most common. Interestingly, in fibres deficient for one or more complexes, the remaining complexes are often upregulated beyond the increase of mitochondrial mass typically observed in ragged red fibres. We further find that oxidative phosphorylation deficient fibres exhibit an increase in the abundance of proteins involved in proteostasis, e.g. HSP60 and LONP1, and regulation of mitochondrial metabolism (including oxidative phosphorylation and proteolysis, e.g. PHB1). Our analysis suggests that the cellular response to mitochondrial dysfunction changes depending on the combination of deficient oxidative phosphorylation complexes in each fibre.

CgPHB2 involved in the haemocyte mitophagy in response to Vibrio splendidus stimulation in Pacific oyster Crassostrea gigas.

Prohibitin2 (PHB2) is recently identified as a novel inner membrane mitophagy receptor to mediate mitophagy. In the present study, the function of CgPHB2 in mediating mitophagy in response to Vibrio splendidus stimulation was investigated in Crassostrea gigas. CgPHB2 protein was mainly distributed in the cytoplasm of three subpopulations of haemocytes. After V. splendidus stimulation, the expressions of CgPHB2 mRNA in haemocytes were up-regulated significantly at 6, 12 and 24 h, and the abundance of CgPHB2 protein was also enhanced at 12-24 h compared to control group. Furthermore, the green signals of CgPHB2 were colocalized respectively with the red signals of mitochondria and CgLC3 in the haemocytes at 12 h after V. splendidus stimulation, and the co-localization value of CgPHB2 and mtphagy Dye was significantly increased. The direct interaction between CgPHB2 and CgLC3 was simulated by molecular docking. In PHB2-inhibitor Fluorizoline-treated oysters, the mRNA expressions of mitophagy-related genes and the ratio of mitophagy were significantly decreased in haemocytes of oysters after V. splendidus stimulation. All the results collectively suggested that CgPHB2 participated in mediating the haemocyte mitophagy in the antibacterial immune response of oysters.

Progesterone-induced progesterone receptor membrane component 1 rise-to-decline changes are essential for decidualization.

BACKGROUND: Decidualization of endometrial cells is the prerequisite for embryo implantation and subsequent placenta formation and is induced by rising progesterone levels following ovulation. One of the hormone receptors contributing to endometrial homeostasis is Progesterone Receptor Membrane Component 1 (PGRMC1), a non-classical membrane-bound progesterone receptor with yet unclear function. In this study, we aimed to investigate how PGRMC1 contributes to human decidualization. METHODS: We first analyzed PGRMC1 expression profile during a regular menstrual cycle in RNA-sequencing datasets. To further explore the function of PGRMC1 in human decidualization, we implemented an inducible decidualization system, which is achieved by culturing two human endometrial stromal cell lines in decidualization-inducing medium containing medroxyprogesterone acetate and 8-Br-cAMP. In our system, we measured PGRMC1 expression during hormone induction as well as decidualization status upon PGRMC1 knockdown at different time points. We further conferred proximity ligation assay to identify PGRMC1 interaction partners. RESULTS: In a regular menstrual cycle, PGRMC1 mRNA expression is gradually decreased from the proliferative phase to the secretory phase. In in vitro experiments, we observed that PGRMC1 expression follows a rise-to-decline pattern, in which its expression level initially increased during the first 6 days after induction (PGRMC1 increasing phase) and decreased in the following days (PGRMC1 decreasing phase). Knockdown of PGRMC1 expression before the induction led to a failed decidualization, while its knockdown after induction did not inhibit decidualization, suggesting that the progestin-induced 'PGRMC1 increasing phase' is essential for normal decidualization. Furthermore, we found that the interactions of prohibitin 1 and prohibitin 2 with PGRMC1 were induced upon progestin treatment. Knocking down each of the prohibitins slowed down the decidualization process compared to the control, suggesting that PGRMC1 cooperates with prohibitins to regulate decidualization. CONCLUSIONS: According to our findings, PGRMC1 expression followed a progestin-induced rise-to-decline expression pattern during human endometrial decidualization process; and the correct execution of this expression program was crucial for successful decidualization. Thereby, the results of our in vitro model explained how PGRMC1 dysregulation during decidualization may present a new perspective on infertility-related diseases.

Did mitophagy follow the origin of mitochondria?

Mitophagy is the process of selective autophagy that removes superfluous and dysfunctional mitochondria. Mitophagy was first characterized in mammalian cells and is now recognized to follow several pathways including basal forms in specific organs. Mitophagy pathways are regulated by multiple, often interconnected factors. The present review aims to streamline this complexity and evaluate common elements that may define the evolutionary origin of mitophagy. Key issues surrounding mitophagy signaling at the mitochondrial surface may fundamentally derive from mitochondrial membrane dynamics. Elements of such membrane dynamics likely originated during the endosymbiosis of the alphaproteobacterial ancestor of our mitochondria but underwent an evolutionary leap forward in basal metazoa that determined the currently known variations in mitophagy signaling.Abbreviations: AGPAT, 1-acylglycerol-3-phosphate O-acyltransferase; ATG, autophagy related; BCL2L13, BCL2 like 13; BNIP3, BCL2 interacting protein 3; BNIP3L, BCL2 interacting protein 3 like; CALCOCO, calcium binding and coiled-coil domain; CL, cardiolipin; ER, endoplasmic reticulum; ERMES, ER-mitochondria encounter structure; FBXL4, F-box and leucine rich repeat protein 4; FUNDC1, FUN14 domain containing 1; GABARAPL1, GABA type A receptor associated protein like 1; HIF, hypoxia inducible factor; IMM, inner mitochondrial membrane; LBPA/BMP, lysobisphosphatidic acid; LIR, LC3-interacting region; LPA, lysophosphatidic acid; MAM, mitochondria-associated membranes; MAP1LC3/LC3, microtubule associated protein 1 light chain 3; MCL, monolysocardiolipin; ML, maximum likelihood; NBR1, NBR1 autophagy cargo receptor; OMM, outer mitochondrial membrane; PA, phosphatidic acid; PACS2, phosphofurin acidic cluster sorting protein 2; PC/PLC, phosphatidylcholine; PE, phosphatidylethanolamine; PHB2, prohibitin 2; PINK1, PTEN induced kinase 1; PtdIns, phosphatidylinositol; SAR, Stramenopiles, Apicomplexa and Rhizaria; TAX1BP1, Tax1 binding protein 1; ULK1, unc-51 like autophagy activating kinase 1; VDAC/porin, voltage dependent anion channel.

Dual-specificity phosphatase 1 interacts with prohibitin 2 to improve mitochondrial quality control against type-3 cardiorenal syndrome.

Type-3 cardiorenal syndrome (CRS-3) is acute kidney injury followed by cardiac injury/dysfunction. Mitochondrial injury may impair myocardial function during CRS-3. Since dual-specificity phosphatase 1 (DUSP1) and prohibitin 2 (PHB2) both promote cardiac mitochondrial quality control, we assessed whether these proteins were dysregulated during CRS-3-related cardiac depression. We found that DUSP1 was downregulated in heart tissues from a mouse model of CRS-3. DUSP1 transgenic (DUSP1Tg) mice were protected from CRS-3-induced myocardial damage, as evidenced by their improved heart function and myocardial structure. CRS-3 induced the inflammatory response, oxidative stress and mitochondrial dysfunction in wild-type hearts, but not in DUSP1Tg hearts. DUSP1 overexpression normalized cardiac mitochondrial quality control during CRS-3 by suppressing mitochondrial fission, restoring mitochondrial fusion, re-activating mitophagy and augmenting mitochondrial biogenesis. We found that DUSP1 sustained cardiac mitochondrial quality control by binding directly to PHB2 and maintaining PHB2 phosphorylation, while CRS-3 disrupted this physiological interaction. Transgenic knock-in mice carrying the Phb2S91D variant were less susceptible to cardiac depression upon CRS-3, due to a reduced inflammatory response, suppressed oxidative stress and improved mitochondrial quality control in their heart tissues. Thus, CRS-3-induced myocardial dysfunction can be attributed to reduced DUSP1 expression and disrupted DUSP1/PHB2 binding, leading to defective cardiac mitochondrial quality control.

Oxidative stress suppresses PHB2-mediated mitophagy in ß-cells via the Nrf2/PHB2 pathway.

AIMS/INTRODUCTION: Mitochondrial damage caused by oxidative stress is a main driver of pancreatic ß-cell dysfunction in the pathogenesis of type 2 diabetes mellitus. Prohibitin2 (PHB2) is a vital inner mitochondrial membrane protein that participates in mitophagy to remove the damaged mitochondria. This study aimed to investigate the role and mechanisms of PHB2-mediated mitophagy in oxidative stress-induced pancreatic ß-cell dysfunction. MATERIALS AND METHODS: PHB2 and mitophagy-related protein expression were analyzed by real-time polymerase chain reaction and western blotting in RINm5F cells treated with H2O2 and islets of diabetic rats. Mitophagy was observed by mitochondrial and lysosome colocalization. RINm5F cells were transfected by phb2 siRNA or overexpression plasmid to explore the role of PHB2 in mitophagy of RINm5F cells. The mechanism of Nrf2 regulating PHB2 was explored by Nrf2 inhibitor and agonist. RESULTS: The expression of PHB2, mitophagy related protein PINK1, and Parkin were decreased in RINm5F cells incubated with H2O2 and in islets of diabetic rats. Overexpression of PHB2 protected ß-cells from oxidative stress by promoting mitophagy and inhibiting cell apoptosis, whereas transfection with PHB2 siRNA suppressed mitophagy. Furthermore, PHB2-mediated mitophagy induced by oxidative stress was through the Nrf2/PHB2 pathway in ß-cells. Antioxidant NAC alleviated oxidative stress injury by promoting PHB2-mediated mitophagy. CONCLUSION: Our study suggested that PHB2-mediated mitophagy can protect ß-cells from apoptosis via the Nrf2/PHB2 pathway under oxidative stress. Antioxidants may protect ß-cell from oxidative stress by prompting PHB2-mediated mitophagy. PHB2-mediated mitophagy as a potential mechanism takes part in the oxidative stress induced ß-cell injury.

Selection and identification of a prohibitin 2-binding DNA aptamer for tumor tissue imaging and targeted chemotherapy.

Tumor cell-targeting molecules play a vital role in cancer diagnosis, targeted therapy, and biomarker discovery. Aptamers are emerging as novel targeting molecules with unique advantages in cancer research. In this work, we have developed several DNA aptamers through cell-based systematic evolution of ligands by exponential enrichment (Cell-SELEX). The selected SYL-6 aptamer can bind to a variety of cancer cells with high signal. Tumor tissue imaging demonstrated that SYL-6-Cy5 fluorescent probe was able to recognize multiple clinical tumor tissues but not the normal tissues, which indicates great potential of SYL-6 for clinical tumor diagnosis. Meanwhile, we identified prohibitin 2 (PHB2) as the molecular target of SYL-6 using mass spectrometry, pull-down and RNA interference assays. Moreover, SYL-6 can be used as a delivery vehicle to carry with doxorubicin (Dox) chemotherapeutic agents for antitumor targeted chemotherapy. The constructed SYL-6-Dox can not only selectively kill tumor cells in vitro, but also inhibit tumor growth with reduced side effects in vivo. This work may provide a general tumor cell-targeting molecule and a potential biomarker for cancer diagnosis and targeted therapy.

Bakuchiol targets mitochondrial proteins, prohibitins and voltage-dependent anion channels: New insights into developing antiviral agents.

We previously reported that bakuchiol, a phenolic isoprenoid anticancer compound, and its analogs exert anti-influenza activity. However, the proteins targeted by bakuchiol remain unclear. Here, we investigated the chemical structures responsible for the anti-influenza activity of bakuchiol and found that all functional groups and C6 chirality of bakuchiol were required for its anti-influenza activity. Based on these results, we synthesized a molecular probe containing a biotin tag bound to the C1 position of bakuchiol. With this probe, we performed a pulldown assay for Madin-Darby canine kidney cell lysates and purified the specific bakuchiol-binding proteins with SDS-PAGE. Using nanoLC-MS/MS analysis, we identified prohibitin (PHB) 2, voltage-dependent anion channel (VDAC) 1, and VDAC2 as binding proteins of bakuchiol. We confirmed the binding of bakuchiol to PHB1, PHB2, and VDAC2 in vitro using Western blot analysis. Immunofluorescence analysis showed that bakuchiol was bound to PHBs and VDAC2 in cells and colocalized in the mitochondria. The knockdown of PHBs or VDAC2 by transfection with specific siRNAs, along with bakuchiol cotreatment, led to significantly reduced influenza nucleoprotein expression levels and viral titers in the conditioned medium of virus-infected Madin-Darby canine kidney cells, compared to the levels observed with transfection or treatment alone. These findings indicate that reducing PHBs or VDAC2 protein, combined with bakuchiol treatment, additively suppressed the growth of influenza virus. Our findings indicate that bakuchiol exerts anti-influenza activity via a novel mechanism involving these mitochondrial proteins, providing new insight for developing anti-influenza agents.