Melatonin supplementation enhances browning suppression and improves transformation efficiency and regeneration of transgenic rough lemon plants (Citrus × jambhiri).

Enzymatic browning poses a significant challenge that limits in vitro propagation and genetic transformation of plant tissues. This research focuses on investigating how adding antioxidant substances can suppress browning, leading to improved efficiency in transforming plant tissues using Agrobacterium and subsequent plant regeneration from rough lemon (Citrus × jambhiri). When epicotyl segments of rough lemon were exposed to Agrobacterium, they displayed excessive browning and tissue decay. This was notably different from the 'Hamlin' explants, which did not exhibit the same issue. The regeneration process failed completely in rough lemon explants, and they accumulated high levels of total phenolic compounds (TPC) and polyphenol oxidase (PPO), which contribute to browning. To overcome these challenges, several antioxidant and osmoprotectant compounds, including lipoic acid, melatonin, glycine betaine, and proline were added to the tissue culture medium to reduce the oxidation of phenolic compounds and mitigate browning. Treating epicotyl segments with 100 or 200 µM melatonin led to a significant reduction in browning and phenolic compound accumulation. This resulted in enhanced shoot regeneration, increased transformation efficiency, and reduced tissue decay. Importantly, melatonin supplementation effectively lowered the levels of TPC and PPO in the cultured explants. Molecular and physiological analyses also confirmed the successful overexpression of the CcNHX1 transcription factor, which plays a key role in imparting tolerance to salinity stress. This study emphasizes the noteworthy impact of supplementing antioxidants in achieving successful genetic transformation and plant regeneration in rough lemon. These findings provide valuable insights for developing strategies to address enzymatic browning and enhance the effectiveness of plant tissue culture and genetic engineering methods with potential applications across diverse plant species.

The muscle regeneration marker FOXP3 is associated with muscle injury in Duchenne muscular dystrophy.

BACKGROUND: In Duchenne muscular dystrophy (DMD), the immune system cells (ISC) synthesize molecules to regulate inflammation, a process needed to regenerate muscle. The relationship between those molecules and the muscle injury is unknown. Monocytes belonging to ISC are regulated by omega-3 fatty acids (ω-3 LCPUFAs) in DMD, but whether those fatty acids influence other ISC like T-cells is unknown. OBJECTIVE: We analyzed the expression of the muscle regeneration markers (FOXP3 and AREG) in circulating leukocytes of DMD patients with different lower limb muscle functions and whether ω-3 LCPUFAs regulate the expression of those markers, and the populations of circulating T-cells, their intracellular cytokines, and disease progression (CD69 and CD49d) markers. METHODS: This placebo-controlled, double-blind, randomized study was conducted in DMD boys supplemented with ω-3 LCPUFAs (n = 18) or placebo (sunflower oil, n = 13) for six months. FOXP3 and AREG mRNA expression in leukocytes, immunophenotyping of T-cell populations, CD49d and CD69 markers, and intracellular cytokines in blood samples were analyzed at baseline and months 1, 2, 3, and 6 of supplementation. RESULTS: Patients with assisted ambulation expressed higher (P = 0.015) FOXP3 mRNA levels than ambulatory patients. The FOXP3 mRNA expression correlated (Rho = -0.526, P = 0.03) with the Vignos scale score at month six of supplementation with ω-3 LCPUFAs. CD49d + CD8 + T-cells population was lower (P = 0.037) in the ω -3 LCPUFAs group than placebo at month six of supplementation. CONCLUSION: FOXP3 is highly expressed in circulating leukocytes of DMD patients with the worst muscle function. Omega-3 LCPUFAs might modulate the synthesis of the adhesion marker CD49d + CD8 + T-cells, but their plausible impact on FOXP3 needs more research.

Periapical bacterial disinfection is critical for dental pulp regenerative cell therapy in apical periodontitis in dogs.

BACKGROUND: Application of pulp regenerative cell therapy for mature teeth with periapical lesions is a critical clinical challenge. The bacterial infection in inaccessible location within the root canal system and in the periapical lesions could cause resistance and impediment, leading to limitations in successful therapy. Thus, the aim of this study was to examine the effect of residual bacteria on the outcome of pulp regeneration in mature teeth with apical periodontitis in dogs. METHODS: Periapical lesions were induced in 32 root canals of 4 dogs in two different models in severities, model A and model B. Model A (moderate infection): the canal exposed to the oral cavity for 2 weeks and then closed for 2 weeks. Model B (severe infection): the canal exposed to the oral cavity for 2 months and then closed for 5 months. All root canals were irrigated with 6% sodium hypochlorite, and 3% EDTA and further with 0.015% levofloxacin-containing nanobubbles, which was also used as an intracanal medicament. The aseptic conditions were examined by bacterial anaerobic culture and/or PCR analyses. The root canal treatment was repeated several times, and allogeneic dental pulp stem cells were transplanted into the root canals. The radiographic evaluation of periapical lesions was performed by cone-beam computed tomography before the first treatment, just after cell transplantation, and after 2 months and 6 months in both model A, model B, respectively. The animals were then sacrificed and the jaw blocks were harvested for histological and histobacteriological evaluations of pulp regeneration and periapical tissue healing. Furthermore, the DiI-labelled DPSCs were transplanted into the root canals after complete disinfection (n = 4) or without root canal treatment (n = 4) in the apical periodontitis model (model A) in one dog, and cell localization was compared 72 h after transplantation. RESULTS: In 8 out of 12 canals from model A, and 10 out of 15 canals from model B, pulp regeneration with good vascularization, innervation, and a significant reduction in the radiolucent area of the periapical lesions were observed. However, in the other 4 canals and 5 canals from model A and model B, respectively, no pulp tissue was regenerated, and inflammation in the periapical tissue, and external resorption or healed external resorption were detected. The presence of residual bacteria in the periapical tissues and severe inflammation were significantly associated with inhibition of regenerated pulp tissue in these 9 unsuccessful canals (P < 0.05, each) (OR = 0.075, each) analyzed by multiple logistic regression analysis. For cellular kinetics, transplanted cells remained in the disinfected root canals, while they were not detected in the infected root canals, suggesting their migration through the apical foramen under the influence of inflammation. CONCLUSIONS: A true pulp-dentin complex was regenerated in the root canal by the pulp regenerative therapy in mature teeth with apical lesions. The successful pulp regeneration was negatively associated both with residual bacteria and inflammation in the periapical tissue.

Designing bioinspired multifunctional nanoplatforms to support wound healing and skin regeneration: Mg-hydroxyapatite meets melanins.

Melanin is a multifunctional biological pigment that recently emerged as endowed with anti-inflammatory, antioxidant, and antimicrobial properties and with high potentialities in skin protection and regenerative medicine. Here, a biomimetic magnesium-doped nano-hydroxyapatite (MgHA) was synthesized and decorated with melanin molecules starting from two different monomeric precursors, i.e. 5,6-dihydroxyindole-2-carboxylic acid (DHICA) and dopamine (DA), demonstrating to be able to polymerize on the surface of MgHA nanostructures, thus leading to a melanin coating. This functionalization was realized by a simple and green preparation method requiring mild conditions in an aqueous medium and room temperature. Complementary spectroscopy and electron imaging analyses were carried out to define the effective formation of a stable coating, the percentage of the organic compounds, and the structural properties of resulting melanin-coated nanostructures, which showed good antioxidant activity. The in vitro interaction with a cell model, i.e. mouse fibroblasts, was investigated. The excellent biocompatibility of all bioinspired nanostructures was confirmed from a suitable cell proliferation. Finally, the enhanced biological performances of the nanostructures coated with melanin from DHICA were confirmed by scratch assays. Jointly our findings indicated that low crystalline MgHA and melanin pigments can be efficiently combined, and the resulting nanostructures are promising candidates as multifunctional platforms for a more efficient approach for skin regeneration and protection.

Effect of Naoluoxintong formula and its split prescriptions on cerebral vascular regeneration in rats with the cerebral ischemia-reperfusion.

OBJECTIVE: To observe regulatory effect of Naoluoxintong formula (, NLXT) and its split prescriptions on vascular regeneration of rats suffering from cerebral ischemia-reperfusion (IR) syndrome of Qi deficiency with blood stasis (QDBS). METHODS: NLXT is the representative prescription of Yiqi Huoxue Tongluo decoction, and NLXT is divided into Yiqi herbs and Huoxue Tongluo herbs according to their efficacies. One hundred and eight specific-pathogen-free, clean-grade, Sprague-Dawley male rats were selected to prepare the classical rat model with QDBS due to middle artery ischemia-reperfusion using the multi-factor compound simulation approach. The animals were classified into sham operation (S), model (M), Nimodping (NMDP), NLXT, YQ and HXTL groups, each having 18 rats. Cerebral ischemia was reperfused after 2 h, and 24 h later, they were administered traditional Chinese medicine treatment for 14 d twice a day. Angiogenesis changes after NLXT administration to middle cerebral artery occlusion-reperfusion (MCAO/R) rats with QDBS were analyzed using the neurological deficit score and hematoxylin-eosin staining. Cerebral infarct area by 2,3,5-Triphenyltetrazolium chloride was detected, and the ultrastructure of the blood vessel in the ischemic frontoparietal cortex was observed by transmission electron microscopy. Angiopoietin 1 (Ang1), angiopoietin 2 (Ang2), vascular endothelial growth factor A (VEGFA), vascular endothelial growth factor receptor 2 (VEGFR2), platelet endothelial cell adhesion molecule-1 (CD31), angiopoietin receptor 2 (Tie2), and P38 mitogen-activated protein kinase (MAPK) protein levels in the frontal and parietal cortex were quantified by immunofluorescence, reverse transcription-polymerase chain reaction, and Western blotting assays. RESULTS: Relative to the S group, VEGFA and VEGFR2 levels in the frontal and parietal cortex of group M were increased, and Ang1, Ang2, Tie2, CD31, and p38 MAPK levels remarkably increased (P < 0.05); cerebral infarct area was significant and pathological morphology and ultrastructure damage was obvious. Relative to the group M, VEGFA, VEGFR2, CD31, Ang1, Ang2, and Tie2 expression of group NLXT and NMDP remarkably elevated (P < 0.05) and infarct focus, pathological morphology and ultrastructure were significantly improved; VEGFA and VEGFR2 levels in the groups YQ and HXTL increased, and Ang1, Ang2, CD31, and Tie2 levels remarkably increased (P < 0.05); p38 MAPK levels in the three treatment groups decreased (P < 0.05). Relative to the group NLXT, the expression levels of p38 MAPK in group YQ and group HXTL were significantly increased, and the expression levels of other indicators were significantly decreased (P < 0.05). CONCLUSION: NLXT can promote the angiogenesis of the rat model of MCAO/R with QDBS by activating VEGFA and inhibiting P38 MAPK, and the effect is better than that of split prescription groups.

Moringa oleifera Use in Maintaining Oral Health and Its Potential Use in Regenerative Dentistry.

Phytomedicine refers to the use of naturally derived products to cure and mitigate human conditions. Natural products have the advantages of causing minimum side effects, being biocompatible, available, and economical, with a wide array of biological activities. Reports have described the use of natural products with antimicrobial and anti-inflammatory properties to treat oral conditions and promote wound healing. Moringa oleifera, known as the "drumstick" or "horseradish" tree, is believed to have medicinal properties regarding a range of medical conditions, though there is limited information on its use in oral medicine. This narrative review focuses on the use of Moringa extracts in the management of oral conditions, including oral infections, inflammatory conditions, the remineralization of hard tissues, oral wound healing, and tissue regeneration, drawing from both in vitro and in vivo studies which indicate that the potential of Moringa extracts in supporting dentin-pulp regeneration after caries or trauma is worthy of more careful consideration.

The Role of Vitamin D in Skeletal Muscle Repair and Regeneration in Animal Models and Humans: A Systematic Review.

Vitamin D deficiency, prevalent worldwide, is linked to muscle weakness, sarcopenia, and falls. Muscle regeneration is a vital process that allows for skeletal muscle tissue maintenance and repair after injury. PubMed and Web of Science were used to search for studies published prior to May 2023. We assessed eligible studies that discussed the relationship between vitamin D, muscle regeneration in this review. Overall, the literature reports strong associations between vitamin D and skeletal myocyte size, and muscle regeneration. In vitro studies in skeletal muscle cells derived from mice and humans showed vitamin D played a role in regulating myoblast growth, size, and gene expression. Animal studies, primarily in mice, demonstrate vitamin D's positive effects on skeletal muscle function, such as improved grip strength and endurance. These studies encompass vitamin D diet research, genetically modified models, and disease-related mouse models. Relatively few studies looked at muscle function after injury, but these also support a role for vitamin D in muscle recovery. The human studies have also reported that vitamin D deficiency decreases muscle grip strength and gait speed, especially in the elderly population. Finally, human studies reported the benefits of vitamin D supplementation and achieving optimal serum vitamin D levels in muscle recovery after eccentric exercise and surgery. However, there were no benefits in rotator cuff injury studies, suggesting that repair mechanisms for muscle/ligament tears may be less reliant on vitamin D. In summary, vitamin D plays a crucial role in skeletal muscle function, structural integrity, and regeneration, potentially offering therapeutic benefits to patients with musculoskeletal diseases and in post-operative recovery.

Promoting FADH2 Regeneration of Hydroxylation for High-Level Production of Hydroxytyrosol from Glycerol in Escherichia coli.

Hydroxytyrosol is a natural polyphenolic compound widely used in the food and drug industries. The current commercial production of hydroxytyrosol relies mainly on plant extracts, which involve long extraction cycles and various raw materials. Microbial fermentation has potential value as an environmentally friendly and low-cost method. Here, a de novo biosynthetic pathway of hydroxytyrosol has been designed and constructed in an Escherichia coli strain with released tyrosine feedback inhibition. By introduction of hpaBC from E. coli and ARO10 and ADH6 from Saccharomyces cerevisiae, the de novo biosynthesis of hydroxytyrosol was achieved. An important finding in cofactor engineering is that the introduction of L-amino acid deaminase (LAAD) promotes not only cofactor regeneration but also metabolic flow redistribution. To further enhance the hydroxylation process, different 4-hydroxyphenylacetate 3-monooxygenase (hpaB) mutants and HpaBC proteins from different sources were screened. Finally, after optimization of the carbon source, pH, and seed medium, the optimum engineered strain produced 9.87 g/L hydroxytyrosol in a 5 L bioreactor. This represents the highest titer reported to date for de novo biosynthesis of hydroxytyrosol in microorganisms.

Engineered Bio-Heterojunction with Infection-Primed H2 S Liberation for Boosted Angiogenesis and Infectious Cutaneous Regeneration.

Photodynamic therapy (PDT) acts as a powerful weapon against infectious diseases for its enormous antimicrobial activity that quickly elicits storms of reactive oxygen species (ROS). Nevertheless, redundant ROS during treatment inevitably bring detriments in revascularization. To address this dilemma, an innovative P-N bio-heterojunction (bio-HJ) material consisting of p-type copper sulfide (p-CuS), n-type bismuth sulfide (n-Bi2 S3 ), and lactate oxidase (LOx) for effective treatment of recalcitrant infectious wounds by promoting angiogenesis is devised. LOx exhausts lactic acid accumulated in infection environment and converts it to hydrogen peroxide (H2 O2 ), which subsequently yields bactericidal hydroxyl radicals (·OH) via Fenton-like reactions. Ultimately, the P-N bio-HJs exert synergistic photothermal, photodynamic, and chemodynamic effects for rapid bacterial annihilation. Moreover, in vitro and RNA-seq analyses reveal that the crafted bio-HJs dramatically expedite the proliferation of L929 cells and promote angiogenesis by up-regulating angiogenic gene expression in hypoxia-inducible factor-1 (HIF-1) signaling pathway, which may ascribe to the evolution of H2 S in response to the infection microenvironment. Critically, results of in vivo experiments have authenticated that the bio-HJs significantly boost healing rates of full-thickness wounds by slaughtering bacteria, elevating angiogenesis, and promoting cytothesis. As envisioned, this work furnishes a novel tactic for the effective treatment of bacteria-invaded wound using H2 S-liberating P-N bio-HJs.

[Observation on the effect of electroacupuncture on the regeneration of endometrium in rats with intrauterine adhesion].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on the degree of endometrial fibrosis and inflammatory response in the rat model of intrauterine adhesion (IUA), so as to explore the possible mechanism of EA underlying improving IUA and promoting endometrium regeneration. METHODS: Forty-five female SD rats were randomly divided into blank, model and EA groups, with 15 rats in each group. The IUA model was established by mechanical scratching combined with lipopolysaccharide infection. EA was applied to bilateral "Zigong" (EX-CA1) and "Sanyinjiao" (SP6), with acupuncture applied to "Guanyuan" (CV4) for rats in the EA group, started from the 2nd day after modeling, 15 minutes every time, once a day for 2 consecutive estrous cycles. Samples from 5 rats in each group were collected during estrus period. Changes of endometrial histopathology and number of glands were observed after HE staining. The area of endometrial fibrosis was observed and calculated after Masson staining. The positive expressions of collagen type I (Col-I) and transforming growth factor ß1 (TGF-ß1) proteins in endometrial tissue were detected by immunohistochemistry method. The protein expression of integrin αγß3 in uterine tissue was detected by Western blot. The contents of interleukin (IL)-1ß and tumor necrosis factor α (TNF-α) in uterine tissue were detected by ELISA. Samples from remaining 10 rats in each group were collected on the 8th day of gestation for calculation of the embryo implantation numbers of the rats. RESULTS: HE staining showed complete uterine tissue structure of the rats in the blank group during estrus period, with clear endometrial layer, unobstructed and regular uterine cavity, and dense glands. Destroyed endometrial layer, narrowed and adhered uterine cavity, and sparse glands of the rats were seen in the model group, which was relatively milder in the EA group. Following modeling, the number of endometrial glands, the protein expression of Integrin αγß3, the number of implanted uterine embryos on the injured side of the model group were significantly decreased (P<0.01), while the area of endometrial fibrosis, the positive expressions of Col-I and TGF-ß1 proteins, and the contents of IL-1ß and TNF-α in the uterine tissue were significantly increased (P<0.01) in comparison with those in the blank group. After intervention, the number of endometrial glands, the protein expression of Integrin αγß3, the number of implanted uterine embryos on the injured side of the EA group were significantly increased (P<0.01，P<0.05), while the area of endometrial fibrosis, the positive expressions of Col-I and TGF-ß1 proteins, and the contents of IL-1ß and TNF-α in the uterine tissue were significantly decreased (P<0.01,P<0.05) compared with the model group. CONCLUSION: EA can enhance endometrial receptivity, and promote endometrial regeneration, be conducive to embryo implantation in IUA model rats, which may be related to its effect in alleviating endometrial fibrosis and reducing inflammatory response.

["Huayu Tongluo" moxibustion improves learning-memory ability and promotes myelin regeneration by regulating Sonic Hedgehog signaling pathway in vascular dementia rats].

OBJECTIVE: To investigate the effect of "Huayu Tongluo" (resolving blood stagnation to dredge meridian-collaterals) moxibustion on remyelination and Sonic Hedgehog (Shh) signaling pathway in the corpus callosum of vascular dementia (VD) rats, so as to explore its mechanisms underlying improvement of VD. METHODS: Male Wistar rats were randomized into sham-operation, model, medication and moxibustion groups, with 12 rats in each group.The VD model was established by bilateral common carotid artery occlusion. Moxibustion was applied to "Shenting"(GV24), "Baihui"(GV20) and "Dazhui"(GV14) for 20 min once a day, 7 d as a treatment course, for 3 courses, with one day's rest between every two courses. Rats of the medication group were treated by gavage of 10 mg/kg of chloromastine solution once a day, and the course of treatment was the same as that of the moxibustion group. The rat's learning-memory ability was assessed by Morris water maze test (escape latency). The neurological deficits were evaluated by using Longa's scale.The mRNA and protein expressions of Shh and Gli1 in the corpus callosum were measured by quantitative real-time fluorescence PCR and Western blot, separately. The ultrastructure of the myelin sheath and myelinated axons was observed under transmission electron microscopy (TCM). RESULTS: Compared with the sham-operation group, the neurologic score and escape latency were significantly increased and prolonged (P<0.01), and the mRNA and protein expression levels of Shh and Gli1 and the number of myelinated axons were obviously decreased in the model group (P<0.01). In comparison with the model group, the escape latency was apparently shortened (P<0.05), while the mRNA and protein expression levels of Shh and Gli1 as well as the number of myelinated axons were strikingly increased in both moxibustion and medication groups (P<0.01). Results of TCM showed that in the model group, the arrangement of myelin coil structures was sparse and fuzzy, and some structures were bulged and disbanded. The oligodendrocytes were irregular, and the number of myelin sheath was rare. These situations were relatively milder in both moxibustion and medication groups. CONCLUSION: "Huayu Tongluo" moxibustion can promote the differentiation and maturation of oligodendrocyte precursor cells after cerebral ischemia by regulating the expressions of Shh and Gli1 in Shh signaling pathway, thus promoting the regeneration of cerebral white matter myelin sheaths in VD rats, which may contribute to improving learning-memory ability.

WUSCHEL controls genotype-dependent shoot regeneration capacity in potato.

Plant cells can reprogram their fate. The combinatorial actions of auxin and cytokinin dedifferentiate somatic cells to regenerate organs, which can develop into individual plants. As transgenic plants can be generated from genetically modified somatic cells through these processes, cell fate transition is an unavoidable step in crop genetic engineering. However, regeneration capacity closely depends on the genotype, and the molecular events underlying these variances remain elusive. In the present study, we demonstrated that WUSCHEL (WUS)-a homeodomain transcription factor-determines regeneration capacity in different potato (Solanum tuberosum) genotypes. Comparative analysis of shoot regeneration efficiency and expression of genes related to cell fate transition revealed that WUS expression coincided with regeneration rate in different potato genotypes. Moreover, in a high-efficiency genotype, WUS silencing suppressed shoot regeneration. Meanwhile, in a low-efficiency genotype, regeneration could be enhanced through the supplementation of a different type of cytokinin that promoted WUS expression. Computational modeling of cytokinin receptor-ligand interactions suggested that the docking pose of cytokinins mediated by hydrogen bonding with the core residues may be pivotal for WUS expression and shoot regeneration in potatoes. Furthermore, our whole-genome sequencing analysis revealed core sequence variations in the WUS promoters that differentiate low- and high-efficiency genotypes. The present study revealed that cytokinin responses, particularly WUS expression, determine shoot regeneration efficiency in different potato genotypes.

FTZ promotes islet ß-cell regeneration in T1DM mice via the regulation of nuclear proliferation factors.

ETHNOPHARMACOLOGICAL RELEVANCE: Fufang-Zhenzhu-Tiaozhi capsule (FTZ), a Traditional Chinese Medicine (TCM) patent prescription commonly used in clinical practice, has a significant curative effect on hyperglycemia and hyperlipidemia. Previous studies have shown that FTZ can treat diabetes, but the effect of FTZ on ß-cell regeneration needs to be further explored in T1DM mice. AIM OF THE STUDY: The aim is to investigate the role of FTZ in promoting ß-cell regeneration in T1DM mice, and to further explore its mechanism. MATERIALS AND METHODS: C57BL/6 mice were used as control. NOD/LtJ mice were divided into the Model group and FTZ group. Oral glucose tolerance, fasting blood glucose, and fasting insulin level were measured. Immunofluorescence staining was used to detect the level of ß-cell regeneration and the composition of α-cells and ß-cells in islets. Hematoxylin and eosin staining was used to detect the infiltration degree of inflammatory cells. The apoptosis of islet cells was detected by terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling. Western blotting was used to detect the expression levels of Pancreas/duodenum homeobox protein 1 (PDX-1), V-maf musculoaponeurotic fibrosarcoma oncogene homolog A (MAFA), and Neurogenin-3 (NGN3). RESULTS: FTZ could increase insulin levels and reduce the glucose level of T1DM mice and promote ß-cell regeneration. FTZ also inhibited the invasion of inflammatory cells and the islet cell apoptosis, and maintained the normal composition of islet cells, thus preserving the quantity and quality of ß-cells. Furthermore, FTZ promoting ß-cell regeneration was accompanied by increasing the expression of PDX-1, MAFA, and NGN3. CONCLUSION: FTZ can restore the insulin-secreting function of the impaired pancreatic islet, improve blood glucose level, possibly via the enhancing ß cell regeneration via upregulation of PDX-1, MAFA, and NGN3 in T1DM mice, and may be a potential therapeutic drug for T1DM.

Combining topical dermal infused exosomes with injected calcium hydroxylapatite for enhanced tissue biostimulation.

BACKGROUND: Exosome research continues to flourish. Subsequent knowledge surrounding indications, dose-response, safety, efficacy, and the ability to combine exosome treatment as a "skin primer"-for biostimulation modalities such as calcium hydroxylapatite (CaHA), platelet-rich plasma (PRP), and platelet-rich fibrin matrix (PRFM) is growing rapidly. The objective of this study was to develop safe, reproducible methods of improving topical exosome absorption to enhance the quality of skin either by themselves, or in combination with injectable CaHA. METHODS: Under IRB Approval (International Cell Surgical Society: ICSS-2022-007), 40 patients were enrolled in this study. Twenty patients underwent facial biostimulatory dermal infusion alone, to determine if this method allowed adequate exosome absorption. Five patients underwent facial biostimulatory infusion followed immediately by Dilute CaHA injection (1:1 dilution) to the face. Five patients underwent exosome biostimulatory dermal infusion followed immediately by hyperdilute CaHA (dilution 1:4) injection to the neck. Five patients underwent Facial Dilute CaHA injection (1:1 dilution) alone, without dermal infusion. Five patients underwent neck hyperdilute CaHA injection (1:4 dilution) alone, without dermal infusion. All patients had pretreatment Quantificare 3-D photo-documentation and skin analysis (Quantificare, France). In all patients, the skin was first cleansed with a gentle glycolic acid facial wash (Gregory MD). To induce a "homing inflammatory environment" for the exosomes, sea salt exfoliation was performed (SaltFacial®, SaltMed, Cardiff, CA). A nitric oxide-generating serum (N101 Pneuma Nitric Oxide, Austin, TX) was then applied to act as an enhanced vehicle for absorption. A 3 MHz ultrasound (SaltFacial®, SaltMed, Cardiff, CA) was then utilized to further deepen the absorption of the nitric oxide serum. A topical emulsion containing equal volumes (1.0 cc containing 1 million) of exosomes (Kimera Labs, Miramar, FL), 25 units of botulinum toxin (Xeomin, Merz Aesthetics, Raleigh, NC) and hyaluronic acid (Belatero, Merz Aesthetics, Raleigh, NC) was mixed via back-and-forth propulsion in a 3-cc syringe. When adequately mixed, the emulsion was then applied to the treatment areas. The cavitating ultrasound was then used to aid in the absorption of the emulsion. The patients were then treated with high-intensity LED therapy (SaltFacial®, SaltMed, Cardiff, CA), utilizing the collagen restoration preset program of combination red (660 nm) near-infrared (930 nm) wavelength for 20 min. Post-treatment Quantificare analysis was performed at 15 and 30 days after treatment. RESULTS: Without exception, all dermal infusion alone and CaHA injection alone patients showed an improvement in the tone, quality, and texture of their skin. Quantificare results showed consistent improvement in wrinkles, pores, skin evenness, improved vascularity, and a reduction in oiliness and unwanted pigment. When employed as a skin primer prior to injections (CaHA), enhanced and more rapid results were seen. CONCLUSIONS: Biostimulatory dermal infusion can be achieved utilizing topical placental mesenchymal stem cell-derived exosomes. These exosomes can be used alone, or mixed with ancillary ingredients such as botulinum toxin, hyaluronic acid dermal filler, and CaHA to customize and personalize treatments based upon individual patient needs. Topical absorption is enhanced with sea salt exfoliation, a topical nitric oxide-generating serum, and 3 MHz cavitating ultrasound. Post-absorption activity is enhanced with high-intensity LED treatment. The addition of CaHA injections after the topical exosome "priming of the skin" yielded enhanced skin quality faster than exosomes or CaHA alone.

Engineering Streptomyces albulus to enhance ε-poly-L-lysine production by introducing a polyphosphate kinase-mediated ATP regeneration system.

BACKGROUND: ε-Poly-L-lysine (ε-PL) is a natural and safe food preservative that is mainly produced by filamentous and aerobic bacteria Streptomyces albulus. During ε-PL biosynthesis, a large amount of ATP is used for the polymerization of L-lysine. A shortage of intracellular ATP is one of the major factors limiting the increase in ε-PL production. In previous studies, researchers have mainly tried to increase the oxygen supply to enhance intracellular ATP levels to improve ε-PL production, which can be achieved through the use of two-stage dissolved oxygen control, oxygen carriers, heterologous expression of hemoglobin, and supplementation with auxiliary energy substrates. However, the enhancement of the intracellular ATP supply by constructing an ATP regeneration system has not yet been considered. RESULTS: In this study, a polyphosphate kinase (PPK)-mediated ATP regeneration system was developed and introduced into S. albulus to successfully improve ε-PL production. First, polyP:AMP phosphotransferase (PAP) from Acinetobacter johnsonii was selected for catalyzing the conversion of AMP into ADP through an in vivo test. Moreover, three PPKs from different microbes were compared by in vitro and in vivo studies with respect to catalytic activity and polyphosphate (polyP) preference, and PPK2Bcg from Corynebacterium glutamicum was used for catalyzing the conversion of ADP into ATP. As a result, a recombinant strain PL05 carrying coexpressed pap and ppk2Bcg for catalyzing the conversion of AMP into ATP was constructed. ε-PL production of 2.34 g/L was achieved in shake-flask fermentation, which was an increase of 21.24% compared with S. albulus WG608; intracellular ATP was also increased by 71.56%. In addition, we attempted to develop a dynamic ATP regulation route, but the result was not as expected. Finally, the conditions of polyP6 addition were optimized in batch and fed-batch fermentations, and the maximum ε-PL production of strain PL05 in a 5-L fermenter was 59.25 g/L by fed-batch fermentation, which is the highest ε-PL production reported in genetically engineered strains. CONCLUSIONS: In this study, we proposed and developed a PPK-mediated ATP regeneration system in S. albulus for the first time and significantly enhanced ε-PL production. The study provides an efficient approach to improve the production of not only ε-PL but also other ATP-driven metabolites.

Multifaceted applications of ulvan polysaccharides: Insights on biopharmaceutical avenues.

Ulvans are water-soluble sulfated polysaccharides predominantly found in the cell wall of green algae. They hold unique characteristics that are attributed to their 3D conformation, functional groups along with the presence of saccharides and sulfate ions. Traditionally, ulvans are widely used as food supplements and probiotics owing to the high content of carbohydrates. Despite their widespread usage in food industry, an in-depth understanding is required for extrapolating their potential application as a nutraceutical and medicinal agent which could be beneficial in promoting human health and well-being. This review emphasizes novel therapeutic avenues where ulvan polysaccharides can be used beyond their nutritional applications. A collection of literature points towards multifarious applications of ulvan in various biomedical fields. Structural aspects along with extraction and purification methods have been discussed. The underlying molecular mechanisms associated with its biomedical potential in different therapeutic fields like oncology, infectious diseases, inflammation, neuroprotection and tissue engineering, etc. have been unravelled. Challenges associated with clinical translation and future perspectives have been deliberated.

Amelioration of Lung Fibrosis by Total Flavonoids of Astragalus via Inflammatory Modulation and Epithelium Regeneration.

Idiopathic Pulmonary Fibrosis (IPF) is identifiable by the excessive increase of mesenchyme paired with the loss of epithelium. Total flavonoids of Astragalus (TFA), the main biologically active ingredient of the traditional Chinese medicine, Astragalus membranaceus (Huangqi), shows outstanding effects on treating pulmonary disorders, including COVID-19-associated pulmonary dysfunctions. This study was designed to evaluate the efficacy of TFA on treating pulmonary fibrosis and the possible mechanisms behind these effects. A549 cells were treated with TGF-[Formula: see text]1 and TFA to observe the potential effects of TFA on regulating alveolar epithelial cell proliferation, TGF-[Formula: see text]1-induced EMT, and the underlying mechanisms in vitro. Then, mouse pulmonary fibrosis was induced with a single intra-tracheal injection of bleomycin, and TFA was administrated by i.p. injection. Lung fibrosis was evaluated through histological and molecular analyses, and the possible mechanisms were explored using immunological methods. The results demonstrated that TFA could promote cell proliferation but inhibit TGF-[Formula: see text]1-induced EMT on A549 cells. TFA attenuated BLM-induced pulmonary fibrosis in mice by modulating inflammatory infiltration and M2 macrophage polarization; it furthermore modulated EMT through regulating the TGF-[Formula: see text]1/Smad pathway. In addition, TFA augmented the expression of the Wnt7b protein, which plays an important role in alveolar epithelium reparation. In conclusion, TFA alleviated bleomycin-induced mouse lung fibrosis by preventing the fibrotic response and increasing epithelium regeneration.

Repairing and Regenerating Injured Endometrium Methods.

Good endometrium is the prerequisite and guarantee for reproduction and maternal and child health. Endometrial injury caused by operation or non-operation can lead to menstrual irregularities, amenorrhea, abortion, infertility, and other gynecological diseases to bother women. Intrauterine adhesions (IUA) and thin endometrium are common diseases caused by abnormal repair after endometrium damage. The incidence of IUA is not low after uterine operative surgery, and the recurrence is pretty high after uterine adhesiolysis. At present, there were many methods for endometrial repair in clinic or in the laboratory, but the efficacy was different from methods to methods. They are mainly including estrogen therapy, stem cell therapy, complementary medicine therapy, and some physical barrier therapy. In order to guide the effective repair and regeneration of endometrium in clinic, this paper reviews the merit and demerit of these methods for endometrium regeneration and repair that have been proved to be effective in experiments and clinical in recent years.

TiO2 nanoparticle synthesis, characterization and application to shoot regeneration of water hyssop (Bacopa monnieri L. Pennel) in vitro.

Water hyssop (Bacopa monnieri L. Pennel) is a medicinal aquatic herb used to treat diseases in South Asia. Various regeneration protocols have been developed or modified in vitro to ensure the availability of biomass and secondary metabolites of Bacopa. We applied hydrothermally treated titanium dioxide (TiO2) nanoparticles (NPs) (TiO2-NPs) at different concentrations. Three explants, distal portion of half leaf (DPHL), proximal portion of half leaf (PPHL) and full leaf (FL), were used to evaluate response to TiO2. Regeneration from the three explants in vitro was similar except for shoot length. Application of TiO2-NPs exerted significant, but variable, effects on all parameters except percentage of shoot formation, which was 100%. Interactive effects of explant and TiO2-NPs exhibited significant, but variable, effects on fresh weight and percentage of callus formation. All explants produced more shoots using TiO2-NPs compared to control treatments. DPHL explants with application of 8 mg/l TiO2 produced more shoots than controls. Similarly, FL explant treated with 2 mg/l TiO2-NPs produced more shoots/explant than controls. All concentrations of TiO2-NPs produced significantly longer shoots compared to controls. Increased elongation of shoots justifies use of TiO2-NPs for propagation of plants in vitro during acclimatization. Use of TiO2-NPs for rapid elongation of shoots ultimately fosters survival of plants.

Fufang-zhenzhu-tiaozhi formula protects islet against injury and promotes ß cell regeneration in diabetic mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Fufang-zhenzhu-tiaozhi formula (FTZ) is a patented preparation of traditional Chinese medicine that has been used to treat hyperglycemia and hyperlipidemia in the clinic for almost 10 years. Our previous study had demonstrated that FTZ can protect islet ß cell injury in vitro. However, the efficacy of FTZ on ß cell regeneration in vivo and the involved anti-diabetic mechanism remains unknown. AIM OF THE STUDY: We aim to investigate the effects of FTZ as a good remedy for islet protection and ß cell regeneration, and to reveal the underlying mechanism. MATERIALS AND METHODS: C57BL/6 mice were fed with high-fat diet for 3 weeks and then intraperitoneally injected with streptozotocin (90 mg/kg/d × 1 d) to establish type 2 diabetes (T2D) models. Mice in each group were divided into three batches that sacrificed after 3, 7 and 28 days of FTZ administration. Body weight, blood glucose, and oral glucose tolerance test were measured at indicated time points. Fasting insulin was determined by enzyme-linked immunosorbent assay (ELISA) kit. Neonatal ß cell was assessed by insulin & PCNA double immunofluorescence staining, and the underlying mechanisms related to ß cell regeneration were further performed by hematoxylin-eosin staining, insulin & glucagon double immunofluorescence staining and Western blot. RESULTS: FTZ and metformin can significantly help with the symptoms of DM, such as alleviating weight loss, reducing blood glucose, improving the level of insulin in vivo, and relieving insulin resistance, suggesting FTZ and metformin treatment maintained the normal morphological function of islet. Notably, ß cell regeneration, which is indicated by insulin and PCNA double-positive cells, was promoted by FTZ, whereas few neonatal ß cells were observed in metformin group. Hematoxylin-eosin staining, and its quantification results showed that FTZ effectively prevented the invasion of inflammatory cells into the islets in diabetic mice. Most ß cells in the islets of diabetic model mice were devoid, and the islets were almost all α cells, while the diabetic mice administered FTZ could still maintain about half of the ß cells in the islet. Furthermore, FTZ upregulated the expression of critical transcription factors during ß cell development and maturation (such as PDX-1, MAFA and NGN3) in diabetic mice. CONCLUSIONS: FTZ can alleviate diabetes symptoms and promote ß cell regeneration in diabetic mice. Moreover, FTZ promotes ß cell regeneration by preserving islet (resisting inflammatory cells invading islets), maintaining the number of ß cells in islets, and increasing the expression of PDX-1, MAFA and NGN3.