Fluorescent Protein Research Articles

Characterization of MAP c21873-1 as a new counter-selectable marker for unmarked genetic modification of Pichia pastoris

BackgroundSelection markers are useful in genetic modification of yeast Pichia pastoris. However, the leakage of the promoter caused undesired expression of selection markers especially those toxic proteins like MazF, halting the cell growth and hampering the genetic manipulation in procaryotic system. In this study, a new counter-selectable marker-based strategy has been established for seamless modification with high efficiency and low toxicity.ResultsAt first, the leaky expression of the enhanced green fluorescent protein (EGFP) as a reporter gene under the control of six inducible promoters of P. pastoris was investigated in two hosts Escherichia coli and P. pastoris, respectively. The results demonstrated that the DAS1 and FDH1 promoters (PDAS1 and PFDH1) had the highest leakage expression activities in procaryotes and eukaryotes, and the DAS2 promoter (PDAS2) was inducible with medium strength but low leakage expression activity, all of which were selected for further investigation. Next, Mirabilis antiviral proteins (MAPs) c21873-1, c21873-1T (truncated form of c21873-1) and c23467 were mined as the new counter-selectable markers, and hygromycin B (Hyg B) resistance gene was used as the positive-selectable marker, respectively. Then, modular plasmids with MAP-target gene-Hyg B cassettes were constructed and used to transform into P. pastoris cells after linearization, and the target genes were integrated into its genome at the BmT1 locus through single-crossover homologous recombination (HR). After counter-selection induced by methanol medium, the markers c21873-1 and c21873-1T were recycled efficiently. But c23467 failed to be recycled due to its toxic effect on the P. pastoris cells. At last, the counter-selectable marker c21873-1 under the tightly regulated PDAS2 enabled the encoding genes of reporter EGFP and tested proteins to be integrated into the target locus and expressed successfully.ConclusionsWe have developed MAP c21873-1 as a novel counter-selectable marker which could perform efficient gene knock-in by site-directed HR. Upon counter-selection, the marker could be recycled for repeated use, and no undesirable sequences were introduced except for the target gene. This unmarked genetic modification strategy may be extended to other genetic modification including but not limited to gene knock-out and site-directed mutagenesis in future.

Scalable spatial single-cell transcriptomics and translatomics in 3D thick tissue blocks.

Characterizing the transcriptional and translational gene expression patterns at the single-cell level within their three-dimensional (3D) tissue context is essential for revealing how genes shape tissue structure and function in health and disease. However, most existing spatial profiling techniques are limited to 5-20 μm thin tissue sections. Here, we developed Deep-STARmap and Deep-RIBOmap, which enable 3D in situ quantification of thousands of gene transcripts and their corresponding translation activities, respectively, within 200-μm thick tissue blocks. This is achieved through scalable probe synthesis, hydrogel embedding with efficient probe anchoring, and robust cDNA crosslinking. We first utilized Deep-STARmap in combination with multicolor fluorescent protein imaging for simultaneous molecular cell typing and 3D neuron morphology tracing in the mouse brain. We also demonstrate that 3D spatial profiling facilitates comprehensive and quantitative analysis of tumor-immune interactions in human skin cancer.

Development of a novel bacterial production system for recombinant bioactive proteins completely free from endotoxin contamination.

Endotoxins, or lipopolysaccharides (LPS), are potent immunostimulatory molecules of critical concern in bacterial recombinant protein expression systems. The gram-negative bacterium Acinetobacter baumannii exhibits an interesting and unique phenotype characterized by the complete loss of LPS. In this study, we developed a novel system for producing recombinant proteins completely devoid of endotoxin contamination using LPS-deficient A. baumannii. We purified endotoxin-free functional green fluorescent protein, which reduced endotoxin contamination by approximately three orders of magnitude, and also purified the functional cytokine tumor necrosis factor (TNF)-α. Additionally, utilization of the Omp38 signal peptide of A. baumannii enabled the extracellular production of variable domain of heavy chain of heavy chain (VHH) antibodies. With these advantages, mNb6-tri-20aa, a multivalent VHH that specifically binds to the spike protein of severe acute respiratory syndrome coronavirus 2, was purified from the culture supernatant, and endotoxin contamination was reduced by a factor of approximately 2 × 105 compared with that in conventional expression systems. A virus neutralization assay demonstrated the functionality of the purified antibody in suppressing viral infections. Moreover, we applied our system to produce ozoralizumab, a multispecific VHH that binds to human TNF-α and albumin and are marketed as a rheumatoid arthritis drug. We successfully purified a functional antibody from endotoxin contamination. This system establishes a new, completely endotoxin-free platform for the expression of recombinant proteins, which distinguishes it from other bacterial expression systems, and holds promise for future applications.

Bi-layered Adipose Mesenchymal Cell Sheets Improve Bladder Compliance in Spinal Cord-Injured Rats.

To improve bladder compliance in patients with low-compliance bladders, augmentation cystoplasty with the intestinal tract is performed. However, the use of the intestinal tract often leads to serious surgical complications. Tissue engineering technologies have the potential to improve bladder compliance without using the intestinal tract. In this study, we fabricated bi-layered adipose-derived mesenchymal cell (AMC) sheets and then determined whether the bi-layered AMC sheets could improve bladder compliance in rats with spinal cord injury (SCI). The abdominal adipose tissues of green fluorescence protein (GFP)-transfected Sprague-Dawley (SD) rats were harvested, and the attached and proliferating cells on type I collagen were used as AMCs. The AMCs were then cultured on temperature-responsive culture dishes. After reaching over-confluence, the AMCs that maintained cell-cell contacts were detached from the dishes and applied to a gelatin hydrogel sheet. Then, another detached AMC monolayer was accumulated on the AMC monolayer-applied gelatin. Prior to 4 weeks of transplantation, the levels of T8-9 in the spinal cords of recipient SD rats were partially transected. After producing the bi-layered AMC sheets and the rats with SCI, the detrusor muscles of the anterior bladder walls of the rats with SCI were incised, and the bi-layered AMC sheet was patch-transplanted onto the exposed bladder epithelium (n = 8). As a control, the sham operation was performed (n = 7). Four weeks after the transplantation, bladder capacity and bladder compliance in AMC sheet-transplanted SCI rats were significantly higher than those in sham-operated control SCI rats. The smooth muscle layers in AMC sheet-transplanted bladders were significantly larger than those in control bladders. In addition, the collagen fibers in the AMC sheet-transplanted bladders were significantly smaller than those in the control bladders. Some GFP-positive transplanted AMCs differentiated into smooth muscle actin- or desmin-positive cells. Furthermore, GFP-positive cells secreted transforming growth factor-β1 or vascular endothelial growth factor. Therefore, this study showed that bi-layered AMC sheets could improve bladder compliance and bladder tissues in SCI rats.

Oligodendroglia-to-pericyte conversion after lipopolysaccharide exposure is gender-dependent.

To investigate the sex-dependent differentiation of Sox10 cells and their response to pathological conditions such as lipopolysaccharide (LPS) exposure or ischemia, we utilized Sox10 Cre-ERT2, tdTomato mice. Tamoxifen administration induced the expression of red fluorescent protein (RFP) in these cells, facilitating their subsequent tracking and analysis after LPS injection and ischemia via immunofluorescence staining. Propidium iodide (PI) was injected to label necrotic cells following LPS administration. We found that the conversion of Sox10 cells to pericytes in female mice was significantly higher than in male mice, especially in those exposed to LPS. After LPS injection, the number of PI+ necrotic cells were significantly greater in females than in males. Moreover, RFP+ cells did not co-localize with glial fibrillary acidic protein (GFAP) or cluster of differentiation 11b (CD11b). Similarly, after brain ischemia, RFP+ cells did not express cluster of differentiation 13 (CD13), neuronal nuclei (NeuN), GFAP, or ionised calcium binding adaptor molecule 1 (Iba-1). These findings indicate that the conversion of Sox10 cells to pericytes following LPS exposure is sex-dependent, with neither male nor female groups showing differentiation into other cell types after LPS exposure or under ischemic conditions. The differences in LPS-induced necrosis of pericytes between sexes may explain the variations in the conversion of Sox10 cells to pericytes in both sexes.

Optical inactivation of intracellular molecules by fast-maturating photosensitizing fluorescence protein, HyperNova

Photosensitizing fluorescence protein is a promising tool for chromophore-assisted light inactivation (CALI) that enables specific oxidation and inactivation of intracellular molecules. However, a commonly used monomeric photosensitizing fluorescent protein, SuperNova, shows a low CALI efficiency due to its insufficient maturation at 37 °C, thereby limiting the application of CALI to various molecules, especially in mammalian cells. Here, we present a photosensitizing fluorescence protein, HyperNova, with markedly improved maturation at 37 °C, leading to greatly enhanced CALI efficiency. Exploiting this quality, HyperNova enables the application of CALI to variety of molecules such as a mitotic kinase and transcriptional factors that were highly challenging with conventional SuperNova. To further demonstrate the utility of HyperNova, we have also succeeded in developing novel CALI techniques for MAP kinases by HyperNova. Our findings suggest that HyperNova has the potential to expand the molecular toolbox for manipulating biological events in living cells, providing new avenues for investigating cellular signaling pathways.

Comparative analysis of new mScarlet-based red fluorescent tags in Caenorhabditis elegans.

One problem that has hampered the use of red fluorescent proteins in the fast-developing nematode Caenorhabditis elegans has been the substantial time delay in maturation of several generations of red fluorophores. The recently described mScarlet-I3 protein has properties that may overcome this limitation. We compare here the brightness and onset of expression of CRISPR/Cas9 genome-engineered mScarlet, mScarlet3, mScarlet-I3, and GFP reporter knock-ins. Comparing the onset and brightness of expression of reporter alleles of C. elegans golg-4, encoding a broadly expressed Golgi resident protein, we found that the onset of detection of mScarlet-I3 in the embryo is several hours earlier than older versions of mScarlet and comparable to GFP. These findings were further supported by comparing mScarlet-I3 and GFP reporter alleles for pks-1, a gene expressed in the CAN neuron and cells of the alimentary system, as well as reporter alleles for the pan-neuronal, nuclear marker unc-75. Hence, the relative properties of mScarlet-I3 and GFP do not depend on cellular or subcellular context. In all cases, mScarlet-I3 reporters also show improved signal-to-noise ratio compared to GFP.

Decreased activity in zebrafish larvae exposed to glyphosate-based herbicides during development-potential mediation by glucocorticoid receptor.

Glyphosate-based herbicides (GBH) are a widely used group of pesticides that have glyphosate (GLY) as main active compound and are used to control a wide range of weeds. Experimental and epidemiological studies point to neurotoxicity and endocrine disruption as main toxic effects. The aim of this study was to investigate the effects of developmental exposure to GLY and GBH on locomotor behavior, and the possible contribution of GR-mediated signaling. We used zebrafish (Danio rerio) larvae in a continuous exposure regimen to GLY or GBH in the rearing medium. Alongside TL wildtype, we used a mutant line carrying a mutation in the GR which prevents the GR from binding to DNA (grs357), as well as a transgenic strain expressing a variant of enhanced green fluorescent protein (d4eGFP) controlled by a promoter carrying multiple GR response elements (SR4G). We found that acute exposure to GBH, but not GLY, activates GR-mediated signaling. Using a continuous developmental exposure regime, we show that wildtype larvae exposed to GBH display decreased spontaneous activity and attenuated response to environmental stimuli, a pattern of alteration similar to the one observed in grs357 mutant larvae. In addition, developmental exposure to GBH has virtually no effects on the behavior of grs357 mutant larvae. Taken together, our data indicate that developmental exposure to GBH has more pronounced effects than GLY on behavior at 5 dpf, and that interference with GR-mediated signaling may have a relevant contribution.

Micropropagation and genetic transformation of Byblis liniflora

AbstractByblis, a small genus of carnivorous plants predominantly found in Australia, is characterized by its passive trapping mechanism and unique floral features. The chemical composition of Byblis, including identified phenylethanoid glycosides, particularly acteoside, highlights its pharmacological potential with various biological activities. In vitro culture techniques have been established for propagation, with micropropagation protocols developed for different Byblis species. However, information on genetic transformation, vital for trait modification and enhanced pharmacological interest, remains limited. This study focuses on optimizing micropropagation, adventitious regeneration, and genetic transformation methods for Byblis liniflora. Adventitious regeneration rates were highest in medium with reduced Murashige and Skoog salts (MS/10) and sucrose (3 gL−1) concentrations. Zeatin supplementation (1 mgL−1) further improved regeneration rates and bud development with 100% of regenerated root explants and 8.8 shoots per explant. Liquid MB3 medium supplemented with indole-3-acetic acid (IAA) 5 mgL−1 facilitated efficient rooting and acclimatization. The establishment of an efficient Rhizobium-mediated genetic transformation method yielded transgenic plants expressing green fluorescent protein (GFP). Molecular analysis confirmed transgene integration, marking the first successful genetic transformation in the Byblis genus. These advancements pave the way for exploring gene function and enhancing pharmacological properties, thereby broadening our understanding and utilization of carnivorous plants like Byblis.

Exploring Green Fluorescent Protein Brownian Motion: Temperature and Concentration Dependencies Through Luminescence Thermometry

AbstractLuminescent nanothermometry emerges as a powerful tool for studying protein dynamics. This technique was employed to perform the first measurement of the temperature dependence of protein Brownian velocity, showcasing the illustrative example of enhanced green fluorescent protein (EGFP) across physiologically relevant temperatures (30−50 °C) and concentrations (40, 60, and 80 × 10−3 kg m−3). EGFP exhibited a concentration‐dependent decrease in Brownian velocity, from (1.47 ± 0.09) × 10−3 m s−1 to (0.35 ± 0.01) × 10−3 m s−1, at 30 °C, mimicking crowded cellular environments. Notably, the protein Brownian velocity increased linearly with temperature. These results demonstrate the suitability of concentrated suspensions for modeling intracellular crowding and validate luminescent nanothermometry for protein Brownian motion studies. Furthermore, the observed linear relationship between the logarithm of the protein Brownian velocity and concentration indicates that EGFP motion is not primarily driven by diffusion, but more of a ballistic transport.

PET/CT Targeted Tissue Sampling Reveals Intravenously Administered HGN194 IgG1 Affects HIV Distribution after Rectal Exposure.

Neutralizing monoclonal antibodies hold great potential for prevention of human immunodeficiency virus (HIV) acquisition. IgG is the most abundant antibody in human serum, has a long half-life, and potent effector functions, making it a prime candidate for an HIV prevention therapeutic. We combined Positron Emission Tomography imaging and fluorescent microscopy of 64Cu-labeled, photoactivatable-green fluorescent protein HIV (PA-GFP-BaL) and fluorescently labeled HGN194 IgG1 to determine whether intravenously instilled IgG influences viral interaction with mucosal barriers and viral penetration in colorectal tissue 2 h after rectal viral challenge. Our results show that IgG1 did not alter the number of virions found throughout the colon or viral penetration into the epithelium of the rectum or descending colon. A minor increase in virions was observed in the transverse colon of IgG1 treated animals. Overall, the number of viral particles found in the mesenteric lymph nodes was low. However, IgG1 administration resulted in a significant reduction of virions found in mesenteric lymph nodes. Taken together, our results show that HGN194 IgG1 does not prevent virions from penetrating into the colorectal mucosa but may perturb HIV virion access to the lymphatic system.

In vivo multiscale analyses of spring viremia of carp virus (SVCV) infection: From model organism to target species.

Spring viremia of carp virus (SVCV) has a broad fish host spectrum and is responsible for a disease that generally affects juvenile fishes with a mortality rate of up to 90%. In the absence of treatments or vaccines against SVCV, the search for prophylactic or therapeutic solutions is thus relevant, particularly to identify solutions compatible with mass vaccination. In addition to being a threat to aquaculture and ecosystems, SVCV is a unique pathogen to study virus-host interactions in the zebrafish model. Establishing the first reverse genetics system for SVCV and the design of recombinant SVCV (rSVCV) expressing fluorescent or bioluminescent proteins adds a new dimension for the study of these interactions using innovative imaging techniques. The infection by bath immersion of zebrafish larvae with rSVCV expressing mCherry allows us to define the first SVCV replication sites and the host innate immune responses using different transgenic lines of zebrafish. The fins were found as the main initial sites of infection in both zebrafish and carp, its natural host. Hence, new insights into the physiopathology of SVCV infection have been described. We report that neutrophils are recruited at the sites of infection and persist up to the death of the animal leading to an uncontrolled inflammation correlated with the expression of the pro-inflammatory cytokine IL1β. Tissue damage was observed at the site of initial replication, a likely consequence of virus-induced injury or the pro-inflammatory response. Interestingly, SVCV infection by bath immersion triggers a persistent pro-inflammatory response rather than activation of the antiviral IFN signaling pathway as observed following intravenous injection, highlighting the importance of the route of infection on the progression of pathogenicity. Thus, this model of zebrafish larvae infection by rSVCV offers new perspectives to study in detail virus-host interactions and to discover new prophylactic or therapeutic solutions.

Small GTPase ActIvitY ANalyzing (SAIYAN) system: A method to detect GTPase activation in living cells.

Small GTPases are essential in various cellular signaling pathways, and detecting their activation within living cells is crucial for understanding cellular processes. The current methods for detecting GTPase activation using fluorescent proteins rely on the interaction between the GTPase and its effector. Consequently, these methods are not applicable to factors, such as Sar1, where the effector also functions as a GTPase-activating protein. Here, we present a novel method, the Small GTPase ActIvitY ANalyzing (SAIYAN) system, for detecting the activation of endogenous small GTPases via fluorescent signals utilizing a split mNeonGreen system. We demonstrated Sar1 activation at the endoplasmic reticulum (ER) exit site and successfully detected its activation state in various cellular conditions. Utilizing the SAIYAN system in collagen-secreting cells, we discovered activated Sar1 localized both at the ER exit sites and ER-Golgi intermediate compartment (ERGIC) regions. Additionally, impaired collagen secretion confined the activated Sar1 at the ER exit sites, implying the importance of Sar1 activation through the ERGIC in collagen secretion.

Biodegradation of combined pollutants of polyethylene terephthalate and phthalate esters by esterase-integrated Pseudomonas sp. JY-Q with surface-co-displayed PETase and MHETase

The waste pollution problem caused by polyethylene terephthalate (PET) plastics poses a huge threat to the environment and human health. As plasticizers, Phthalate esters (PAEs) are widely used in PET production and become combined pollutants with PET. Synthetic biology make it possible to construct engineered cells for microbial degradation of combined pollutants of PET and PAEs. PET hydroxylase (PETase) and monohydroxyethyl terephthalate hydroxylase (MHETase) isolated from Ideonella sakaiensis 201-F6 exhibit the capability to depolymerize PET. However, PET cannot enter cells, thus enzymatic degradation or cell surface displaying technology of PET hydrolase are the potential strategies. In this study, Pseudomonas sp. JY-Q was selected as a chassis strain, which exhibits robust stress tolerance. First, a truncated endogenous outer membrane protein cOmpA and its variant Signal (OprF)-cOmpA were selected as anchor motifs for exogenous protein to display on the cell surface. These anchor motifs were fused at the N-terminal of PET hydrolase and MHETase and transformed into Pseudomonas sp. JY-Q, the mutant strains successfully display the enzymes on cell surface, after verification by green fluorescent protein labeling and indirect immunofluorescence assay. The resultant strains also showed the catalytic activity of co-displaying PETase and MHETase for PET biodegradation. Then, the cell surface displaying PET degradation module was introduced to a JY-Q strain which genome was integrated with PAEs degrading enzymes and exhibited PAEs degradation ability. The resultant strain JY-Q-R1-R4-SFM-TPH have the ability of degradation PET and PAEs simultaneously. This study provided a promising strain resource for PET and PAEs pollution control.

Development of a 2A peptide-based multigene expression system and its application for enhanced production of ganoderic acids in Ganoderma lucidum

Ganoderma has received much attention for its medicinal value, but the manipulation of multiple genes remains a challenge, hindering the genetic engineering of this species for the development of cell factories. Here, we first showed that the presence of an intron is necessary for the efficient expression of the endogenous cDNA of carboxin-resistant gene (cbx) in G. lucidum. Then, the self-cleaving function of 2 A peptide was investigated in G. lucidum by linking cbx cDNA to the codon-optimized hygromycin B-resistant gene (ophph) using the 2A-peptide sequence. The results showed that cbx cDNA and ophph can be successfully expressed in G. lucidum in a bicistronic manner from a single transcript. Moreover, the expression of both genes was not affected by the order within the 2 A cassette. In addition, simultaneous expression of cbx cDNA, ophph, and codon-optimized yellow fluorescent protein gene (opyfp) was conducted for the first time in G. lucidum using the 2 A peptide-based approach. The developed method was successfully applied to express both cDNA of the 3-hydroxy-3-methylglutaryl coenzyme A reductase (hmgr) and squalene epoxidase gene (se) for enhanced production of ganoderic acids (GAs) in G. lucidum. The engineered strain produced the maximum content of GA-Mk, GA-T, GA-S, and GA-Me were 26.56±3.53,39.58±3.75, 16.54±2.16, and 19.1±1.87 μg/100 mg dry weight, respectively. These values were 3.85-, 4.74-, 3.65-, and 3.23-fold higher than those produced by the control strain. The developed method will be useful for the manipulation of complex metabolic or regulatory pathways involving multiple genes in Ganoderma.

A frugal CRISPR kit for equitable and accessible education in gene editing and synthetic biology

Equitable and accessible education in life sciences, bioengineering, and synthetic biology is crucial for training the next generation of scientists, fostering transparency in public decision-making, and ensuring biotechnology can benefit a wide-ranging population. As a groundbreaking technology for genome engineering, CRISPR has transformed research and therapeutics. However, hands-on exposure to this technology in educational settings remains limited due to the extensive resources required for CRISPR experiments. Here, we develop CRISPRkit, an affordable kit designed for gene editing and regulation in high school education. CRISPRkit eliminates the need for specialized equipment, prioritizes biosafety, and utilizes cost-effective reagents. By integrating CRISPRi gene regulation, colorful chromoproteins, cell-free transcription-translation systems, smartphone-based quantification, and an in-house automated algorithm (CRISPectra), our kit offers an inexpensive (~$2) and user-friendly approach to performing and analyzing CRISPR experiments, without the need for a traditional laboratory setup. Experiments conducted by high school students in classroom settings highlight the kit’s utility for reliable CRISPRkit experiments. Furthermore, CRISPRkit provides a modular and expandable platform for genome engineering, and we demonstrate its applications for controlling fluorescent proteins and metabolic pathways such as melanin production. We envision CRISPRkit will facilitate biotechnology education for communities of diverse socioeconomic and geographic backgrounds.

Abstract Mo121: Sarcomere activation biosensor reveals key functional differences in live cell active states between cardiac and skeletal muscle

The sarcomere is the functional unit of striated muscle contraction. Previous studies have provided valuable information about the sarcomeric proteins and modulators that are necessary for activation of contraction through inter-myofilament signaling. This work has revealed key differences in contraction kinetics and paradigms between cardiac, slow skeletal, and fast skeletal muscle. We seek here to expand these studies through the use of myofilament incorporated Förster Resonance Energy Transfer (FRET) fluorescent biosensor in live, intact muscle. This biosensor fuses fluorescent donor and acceptor proteins to the N- and C-terminals of fast skeletal and cardiac (slow) troponin C, respectively, and allows for the monitoring in real-time of the stepwise physiological mechanism of muscle sarcomere activation in an intact system which preserves significant features of the muscle, including excitation-contraction coupling and load. This biosensor has been designed and validated in both cardiac and skeletal muscle and using intact muscles from transgenic animals we have demonstrated FRET fluorescence in cardiac papillaries, extensor digitorum longus, and soleus muscles. This biosensor has shown unique sarcomere activation properties between cardiac and skeletal muscle, notably the emergence of a prolonged return to baseline of the FRET signal in loaded fast and slow skeletal muscle, which is evidence of a “primed state” of myofilament activation unique to skeletal muscles under load. Elucidation of the “primed state” of sarcomere activation is evidence of a transient memory of recent activity enabling the enhanced contraction properties of skeletal muscle. This finding advances previous understanding of skeletal muscle activation, including summation and tetanic contraction, which is not possible in cardiac muscle. The implementation of this biosensor provides a new approach to interrogate the essential physiological features of muscle sarcomere activation and contraction and adds a valuable new tool for small molecule and gene-based discoveries for muscle disease remediation.

Abstract We123: Cardiomyocyte Specific Sustained Atg7 Overexpression Activates Mitochondrial Autophagy in the Heart

Introduction: Accumulating evidence indicates that autophagy-related protein 7 (Atg7) is essential in cellular autophagy. Our earlier studies showed that Atg7 is required to promote macro-autophagy in the heart. However, it is unknown if the activation of Atg7 has a role in the heart to regulate selective mitochondrial autophagy (mitophagy). Objectives: We employed in vitro and in vivo approaches to delineate the molecular function and mechanisms of Atg7-mediated activation of mitophagy in the heart. Methods and Results: Subcellular fractionation and quantum dot-labeled electron microscopy revealed Atg7’s localization on the mitochondrial fraction in the heart. Atg7 co-localization to Mitotracker-labeled mitochondria was confirmed using isolated heart mitochondria. To quantitate the mitophagy, we overexpressed Atg7 in cultured primary cardiomyocytes, treated with cyanide 3-chlorophenylhydrazone (CCCP) to induce mitophagy, and stained with pH-sensitive fluorescent mitophagy dye to enumerate mitophagy dots by microscopy. We found significantly increased mitophagy dots in Atg7 overexpressed cardiomyocytes than in β-gal expressing control. To assess the activation of mitophagy in vivo , we treated the cardiac-specific Atg7 overexpressing transgenic (αMHC-Atg7 Tg) and their littermate non-transgenic (Ntg) mice with CCCP. We found an increased accumulation of mature autophagolysosomal marker protein LC3-II in isolated mitochondria from Atg7 Tg mice hearts post-CCCP treatment. To assess the extent of mitochondrial lysosomal delivery in Atg7 overexpressing hearts, we crossed αMHC-Atg7 Tg mice with cardiac-specific Mito-Keima transgenic (Mt-Keima Tg) mice. Mt-Keima Tg mice bear mitochondria-targeted pH-dependent fluorescent lysosomal protease-resistance Keima protein, enabling the measurement of mitophagy activation in vivo . We observed increased Mt-Keima high red-to-green areas in αMHC-Atg7xMt-Keima Tg mice hearts compared to Mt-Keima mice. Conclusions: Our biochemical and genetic approaches revealed that Atg7 localizes to the mitochondria, leading to the recruitment and initiation of mitophagy machinery. Our data further confirm that genetic activation of Atg7 leads to increased mitophagy activity in the heart.

The impact of subdermal adipose derived stem cell injections and early excision on systemic oxidative stress and wound healing in rats with severe scald burns

AimThis study aims to develop an experimental treatment model effective against oxidative stress in the acute period of severe burns and to analyze the mechanisms of healing large wound defects. MethodsFive rats, including 2 females and 3 males, were used as donors to obtain adipose-derived stem cells (ADSC) from the inguinal fat pad. The stem cells were labeled with green fluorescent protein. The study included four groups of 17 rats, each with grade 3 scalding burns on 30 % of their body surface, and a control group of 10 rats with an equal number of males and females. After early excision, 106 ADSC-derived stem cells were administered subdermally to the burned wound and autografted to the stem cell group (n = 17). The early excision group (n = 17) received early excision and autograft, with 2 ml of normal saline injected subdermally into the burn wound edge. The PLM group (n = 17) was treated with a polylactic membrane (PLM) dressing after the burn. No treatment was given to the burn group (n = 17). Ten rats from all groups were sacrificed on the 4th day post-burn for oxidative stress evaluation. The control group (n = 10) was sacrificed on day 4. Blood and tissue samples were collected post-sacrifice. Oxidative stress and inflammation in the blood, as well as cell damage in the skin, liver, kidneys, and lungs, were investigated histopathologically and biochemically on the 4th day post-burn. On the 70th day after burn, wound healing was examined macroscopically and histopathologically. ResultsOn the 4th day, oxidative stress results showed that the levels of Total Oxidative Capacity (TOC) in the blood were lowest in the stem cell (7.4 [6–8.8]), control (6.7 [5.9–7.6]), and early excision (7.5 [6.6–8.5]) groups, with no significant difference between them. The burn group (14.7 [12.5–16.9]) had the highest TOC levels. The PLM group (9.7 [8.6–10.7]) had lower TOC levels than the burn group but higher levels than the other groups.Histopathological examination on the 4th day revealed low liver caspase-3 immunoreactivity in the stem cell and early excision groups among the burn groups. Caspase-3 immunoreactivity levels were as follows: stem cell group (20 [10–30]), early excision group (25 [15–50]), PLM group (70 [50–100]), control group (0), and burn group (80 [60–120]). Other oxidative stress and end-organ damage outcomes were consistent with these results.All rats in the stem cell group had burn wounds that healed completely by the 70th day. Examination of the skin and its appendages from the stem cell group with an immunofluorescence microscope demonstrated green coloration, indicating incorporation of stem cells. ConclusionStem cells may have the potential to form new skin and its appendages, providing better healing for large skin defects. Early excision treatment, by removing local necrotic tissues after extensive and deep burns, can prevent end-organ damage due to systemic oxidative stress and inflammation. We also believe that when these two treatments are used together, they can achieve the best results.

Anoectochilus roxburghii Extract Extends the Lifespan of Caenorhabditis elegans through Activating the daf-16/FoxO Pathway.

As a significant global issue, aging is prompting people's interest in the potential anti-aging properties of Anoectochilus roxburghii (A. roxburghii), a plant traditionally utilized in various Asian countries for its purported benefits in treating diabetes and combating aging. However, the specific anti-aging components and mechanisms of A. roxburghii remain unclear. This study aims to investigate the anti-aging effects and mechanisms of A. roxburghii extract E (ARE). Caenorhabditis elegans (C. elegans) were exposed to media containing different concentrations of ARE whose superior in vitro radical scavenging capacity was thus identified. Lifespan assays, stress resistance tests, and RT-qPCR analyses were conducted to evaluate anti-aging efficacy, reactive oxygen species (ROS) levels, antioxidant enzyme activity, and daf-16, sod-3, and gst-4 levels. Additionally, transcriptomic and metabolomic analyses were performed to elucidate the potential anti-aging mechanisms of ARE. Fluorescence protein assays and gene knockout experiments were employed to validate the impacts of ARE on anti-aging mechanisms. Our results revealed that ARE not only prolonged the lifespan of C. elegans but also mitigated ROS and lipofuscin accumulation, and boosted resistance to UV and heat stress. Furthermore, ARE modulated the expression of pivotal anti-aging genes including daf-16, sod-3, and gst-4, facilitating the nuclear translocation of DAF-16. Significantly, ARE failed to extend the lifespan of daf-16-deficient C. elegans (CF1038), indicating its dependency on the daf-16/FoxO signaling pathway. These results underscored the effectiveness of ARE as a natural agent for enhancing longevity and stress resilience to C. elegans, potentially to human.