Thymus linearis Extracts Ameliorate Indices of Metabolic Syndrome in Sprague Dawley Rats.

Materials and Methods: The extract library (n-hexane (NH), ethyl acetate (EA), methanol (M), distilled water (DW), and combined extract (CE)) was standardized using in vitro phytochemical, antioxidant, and α-amylase inhibition assays, after which the protective effect of selected "hit," i.e., CE against metabolic syndrome, was determined in vivo, using rats fed a high-fat diet supplemented with additional cholesterol administration. CE was administered to Sprague Dawley rats in high dose as 100 mg/kg in carboxymethyl cellulose (CMC) (1 ml; 0.75% in DW) and low-dose group as 50 mg/kg in CMC (0.5 ml; 0.75% in DW). After 10 weeks, the effects of CE on insulin resistance, lipid metabolism, nonalcoholic fatty liver disease (NAFLD), oxidative stress, and genotoxicity were assessed through histological, biochemical, and hematological investigations. Results: Phytochemical analysis including RP-HPLC analysis of the extracts showed that flavonoids and phenolics (myricetin, kaempferol, and apigenin), previously known to be effective against obesity and diabetes, are present in the extracts. Antioxidant studies revealed that the plant possesses a highly significant (p < 0.05) concentration of antioxidants. Satisfactory α-amylase inhibitory activity was also observed in in vitro experiments. In vivo studies showed that CE-administered animals had significantly (p < 0.05) lower weight gain and smaller adipocytes than the control group. Moreover, CE resisted any significant (p < 0.05) change in the organ weights. Analogous to findings from its traditional use, the plant extract had a positive modulatory effect on insulin resistance and hyperglycemia. The study also indicated that CE resisted high-fat diet-induced disturbance in lipid profile and countered any pathological changes in liver enzymes caused by fat-infused diet. Furthermore, a study on endogenous antioxidant levels indicated that CE was effective in maintaining catalase and peroxidase levels within the normal range and resisted the effects of lipid peroxidation of thiobarbituric acid reactive substances. Conclusion: In principle, the current study's findings scientifically validate the implication of T. linearis in metabolic syndrome and recommend further studies on molecular insights of the observed therapeutic activity.

Comparative appraisal of in vitro biological profile and in vivo wound healing attributes of bergenin and Bergenia ciliata (Haw.) Sternb.

ETHNOPHARMACOLOGICAL RELEVANCE: People of all ages experience injuries, whether mild or severe. The most available option to treat wounds as an alternative to allopathic care in both urban and rural populations is traditional medicine, which is mostly target inflammation. Bergenia ciliata (Haw.) Sternb rhizome and leaf powder are used in Ayurveda and local communities for various ailments including healing of wounds and burns. Owing to this property it is traditionally known as "Zakham-e-hayat" (wound healer). AIM OF THE STUDY: In the present study, we compared biological activity and wound healing potential of B. ciliata rhizome (R) extract and bergenin, a glycoside isolated from B. ciliata. MATERIALS AND METHODS: Reverse-phase high performance liquid chromatography (RP-HPLC) was performed to analyze polyphenols and bergenin in B. ciliata R extract. Samples were subjected to in vitro antioxidant assays including free radical scavenging, ferric chloride reducing power and total antioxidant capacity. Micro-broth dilution method, brine shrimp lethality assay and isolated RBC hemolysis assay were conducted to assess in vitro antibacterial and cytotoxic activities. Moreover, in vivo wound healing potential was determined by an excision wound model in mice. RESULTS: RP-HPLC showed significant content of polyphenols and bergenin (6.05 ± 0.12 µg/mg) in B. ciliata R extract. Crude extract possesses higher overall antioxidant and antibacterial capacities than bergenin due to presence of multiple phytoconstituents in extract. Both samples showed low hemolytic activity indicating their safe profile. Furthermore, mice treated with B. ciliata R extract depicted substantial decrease in wound area (99.3%; p < 0.05) as compared to bergenin, which showed 88.8% of wound closure after 12 days of treatment. Additionally, both treatments reduced epithelization duration by 1.6- and 1.4-fold in B. ciliata R extract (12.0 ± 0.6 days) and bergenin (14.2 ± 0.8 days) treated mice, respectively. This was supported by histopathological examination that showed greater epithelization, fibroblast proliferation, collagen synthesis, and revascularization in mice treated with B. ciliata R. CONCLUSION: Concisely, it is evident that B. ciliata R contains phytoconstituents in addition to bergenin, which potentiated wound healing activity of the extract. Hence, B. ciliata R is good source of compounds for treating wounds.

The Inhibitory Effect of Polyphenon 60 from Green Tea on Melanin and Tyrosinase in Zebrafish and A375 Human Melanoma Cells.

Polyphenon 60 (PP60) from green tea has long been used as an antioxidant, anticancer, antimicrobial, and antimutagenic. Aim of the Study. To investigate tyrosinase inhibition-related kinetic mechanism and antimelanogenesis potential of PP60. Materials and Methods. The effect of PP60 on melanin and tyrosinase was evaluated in A375 melanoma cells and zebrafish embryos. The melanoma cells were treated with 20, 40, and 60 µg/mL of PP60, and tyrosinase expression was induced by using L-DOPA. The western blot method was used for the evaluation of tyrosinase expression. Cell lysates were prepared from treated and untreated cells for cellular tyrosinase and melanin quantification. Furthermore, zebrafish embryos were treated with 20, 40, and 60 µg/mL of PP60 and reference drug kojic acid for determination of depigmentation and melanin quantification. In vitro assays were also performed to examine the impact of PP60 on mushroom tyrosinase activity. To determine cytotoxicity, MTT was used against melanoma cell line A375. Results. PP60 showed good tyrosinase inhibitory activity with an IC50 value of 0.697 ± 0.021 µg/mL as compared to kojic acid a reference drug with an IC50 value of 2.486 ± 0.085 µg/mL. Kinetic analysis revealed its mixed type of inhibition against mushroom tyrosinase. In addition, western blot analysis showed that at 60 µg/mL dose of PP60 significantly reduced L-DOPA-induced tyrosinase expression in melanoma cells. PP60 significantly inhibits the cellular tyrosinase (p < 0.05) and reduces the melanin (p < 0.05) contents of melanoma cells. Furthermore, PP60 was found to be very potent in significantly reducing the zebrafish embryos' pigmentation (p < 0.05) and melanin (p < 0.05) content at the dose of 60 µg/mL. Conclusions. Our results demonstrate that PP60 has a strong potency to reduce pigmentation. It may be useful for the cosmetic industries to develop skin whitening agents with minimal toxic effects.

Green nanoparticles as multifunctional nanomedicines: Insights into anti-inflammatory effects, growth signaling and apoptosis mechanism in cancer.

Recently, green nanotechnology got great attention due to their reliable, sustainable, and eco-friendly synthesis protocols. The green nanoparticles (GNPs) are preferred over chemically synthesized nanoparticles owing to less destructive effects associated with the synthesis procedures as well as therapeutic involvement. In this review, we have discussed the applications of GNPs in inflammation-mediated disorders, with special emphasis on cancer, initiated due to oxidative stress and inflammatory cascade. Real-time mechanism based studies on GNPs have suggested their anticancer effects through inducing apoptosis, inhibiting angiogenesis, tissue invasion metastasis, reduced replicative capabilities in addition to target specific different signaling molecules and cascades involved in the development or progression of cancer. Moreover, the association of GNPs with the inhibition or induction of autophagy for the management of cancer has also been discussed. A large number of studies showed the GNPs have multifunctional biomedical properties of theranostic prominence. Therefore, the development of GNPs with naturally established systems could upsurge their definite applications as biomedicines including target specific destruction of the cancerous cells.

Cordyceps militaris as a Bio Functional Food Source: Pharmacological Potential, Anti-Inflammatory Actions and Related Molecular Mechanisms.

Cordyceps militaris (C. militaris) is a medicinal mushroom possessing a variety of biofunctionalities. It has several biologically important components such as polysaccharides and others. The diverse pharmacological potential of C. militaris has generated interest in reviewing the current scientific literature, with a particular focus on prevention and associated molecular mechanisms in inflammatory diseases. Due to rising global demand, research on C. militaris has continued to increase in recent years. C. militaris has shown the potential for inhibiting inflammation-related events, both in in vivo and in vitro experiments. Inflammation is a multifaceted biological process that contributes to the development and severity of diseases, including cancer, colitis, and allergies. These functions make C. militaris a suitable functional food for inhibiting inflammatory responses such as the regulation of proinflammatory cytokines. Therefore, on the basis of existing information, the current study provides insights towards the understanding of anti-inflammatory activity-related mechanisms. This article presents a foundation for clinical use, and analyzes the roadmap for future studies concerning the medical use of C. militaris and its constituents in the next generation of anti-inflammatory drugs.

Lactic Acid Bacteria Fermented Cordyceps militaris (GRC-SC11) Suppresses IgE Mediated Mast Cell Activation and Type I Hypersensitive Allergic Murine Model.

Cordyceps militaris (C. militaris) has various biomedical applications in traditional oriental medicine for different diseases including inflammatory and immune-dysregulated diseases. It is a reservoir of nutritional components such as cordycepin, polysaccharides, and antioxidants. To improve its bioactivity, we fermented C. militaris with a Pediococcus pentosaceus strain isolated from a salted small octopus (SC11). The current study aimed to evaluate whether P. pentosaceus (SC11) fermentation could enhance the anti-allergic potential of C. militaris cultured on germinated Rhynchosia nulubilis (GRC) against a type I hypersensitive reaction in in vitro and in vivo studies. Total antioxidant capacity and cordycepin content were significantly increased in GRC after SC11 fermentation. GRC-SC11 showed significantly enhanced anti-allergic responses by inhibiting immunoglobulin E (IgE)/antigen-induced degranulation in RBL-2H3 cells, compared to GRC. The results demonstrated the significant inhibition of phosphorylated spleen tyrosine kinase (Syk)/ p38/GRB2-associated binding protein 2 (Gab2)/c-jun in IgE/Ag-triggered RBL-2H3 cells. Furthermore, suppressed mRNA levels of interleukin-4 (IL-4) and tumor necrosis factor-α (TNF-α) in IgE/Ag-activated RBL-2H3 cells were observed. GRC-SC11 significantly ameliorated IgE-induced allergic reactions by suppressing the ear swelling, vascular permeability, and inflammatory cell infiltration in passive cutaneous anaphylaxis (PCA) BALB/c mice. In conclusion, GRC fermented with P.pentosaceus exerted enhanced anti-allergic effects, and increased the cordycepin content and antioxidants potential compared to GRC. It can be used as bio-functional food in the prevention and management of type I allergic diseases.

Synthesis of Silver Nanoparticles using Euphorbia wallichii Extract and Assessment of their Bio-functionalities.

BACKGROUND: Silver nanoparticles synthesized by the bio-green method have been applied to various biomedical applications. These procedures are simple, eco-friendly and serve as an alternative to complex chemical methods for the preparation of nanomaterials. OBJECTIVE: In the present study, phytosynthesis of silver nanoparticles, to examine their antioxidant potential, toxic effects towards bacterial-, fungal-strains, brine shrimp nauplii and cancer cells was focused. METHODS: Methanolic extract of Euphorbia wallichii roots was used for the synthesis of silver nanoparticles. The synthesis was monitored and confirmed by UV-visible spectroscopy, Fourier Transform Infra-Red (FTIR) spectrometric analysis, Field Emission Scanning Electron Microscope (FESEM), Energy Dispersive X-ray (EDX) and X-Ray Powder Diffraction (XRD). RESULTS: The synthesized particles were average 63±8 nm in size. Involvement of phenolic (46.7±2.4 µg GAE/mg) and flavonoid (11.7±1.2 µg QE/mg) compounds as capping agents was also measured. Nanoparticles showed antioxidant properties in terms of free radical scavenging potential (59.63±1.0 %), reducing power (44.52±1.34 µg AAE/mg) and total antioxidant capacity (60.48±2.2 µg AAE/mg). The nanoparticles showed potent cytotoxic effects against brine shrimp nauplii (LD50 66.83 µg/ml), proliferation and cell death of HeLa cells as determined by MTT (LD50 0.3923 µg/ml) and TUNEL assays, respectively. Antimicrobial results revealed that silver nanoparticles were found to be more potent against pathogenic fungal (maximum active against A. fumigatus, MIC 15 µg/disc) and bacterial strains (maximum active against S. aureus, MIC 3.33 µg/disc) than the E. wallichii extract alone. CONCLUSION: These results support the advantages of using an eco-friendly and cost-effective method for synthesis of nanoparticles with antioxidant, cytotoxic and antimicrobial potential.

Synthesis, Photophysical Properties and Application of New Porphyrin Derivatives for Use in Photodynamic Therapy and Cell Imaging.

New derivatives of tetrakis(4-carboxyphenyl) porphyrin were designed, synthesized and characterized by IR, proton NMR and mass spectroscopy. The ground and excited state nature of new derivatives were examined using UV-Vis. absorption and fluorescence spectroscopy, fluorescence quantum yield and fluorescence lifetime studies. The singlet oxygen quantum yield of each synthesized derivative of porphyrin was estimated for their further efficacy as potential photosensitizer in biological studies. The significant photophysical data of all synthesized derivatives was supplementary accessed to examine the cell imaging and cytotoxicity against two cancer cell lines viz. MBA-MD-231 and A375. The fluorescence lifetime, fluorescence quantum yield and efficiency of singlet oxygen generation suggests alkyl amine and alkyl hydrazide linked new porphyrin photosensitizers can be useful for PDT agent in cancer treatment.

Undaria pinnatifida a Rich Marine Reservoir of Nutritional and Pharmacological Potential: Insights into Growth Signaling and Apoptosis Mechanisms in Cancer.

Seaweeds are an important part of diet consumed in a different part of the world such as New Zealand, Ireland, Wales, and Asian countries including Korea, China, and Japan. In addition, seaweed is nutritious sources possessing health improving effects and therapeutic potential. Recently, one of the widely eaten seaweed species Undaria pinnatifida (U. pinnatifida) has got much attention because of its pharmacological properties for the prevention of various ailments, including cancer, inflammation, and other diseases. It is rich in all essential amino acids, physiologically significant fatty acids, vitamins, minerals, and has a variety of bioactive constituents which include fucoidan, carotenoids, and fucoxanthin. The present study reviews the nutritional aspects, key bioactivities specifically focusing on anticancer potential along with apoptosis and growth signaling mechanisms of U. pinnatifida or its constituents. It exhibited anticancer effects both in vitro and in vivo studies in a variety of experimental models. Due to a variety of pharmacological properties of U. pinnatifida can not only fulfilling nutritional necessities, but it can be used for treating, curing and preventing cancer.

Oxidative stress, consequences and ROS mediated cellular signaling in rheumatoid arthritis.

There are numerous extra- and intra-cellular processes involved in the production of reactive oxygen species (ROS). Augmented ROS generation can cause the damage of biomolecules such as proteins, nucleic acid and lipids. ROS act as an intracellular signaling component and is associated with various inflammatory responses, chronic arthropathies, including rheumatoid arthritis (RA). It is well documented that ROS can activate different signaling pathways having a vital importance in the patho-physiology of RA. Hence, understanding of the molecular pathways and their interaction might be advantageous in the development of novel therapeutic approaches for RA.

In Vitro, In Silico Elucidation of Antiurease Activity, Kinetic Mechanism and COX-2 Inhibitory Efficacy of Coagulansin A of Withania coagulans.

Urease enzyme plays a crucial role in the survival of Helicobacter pylori that contributes to different diseases, including peptic ulcer (gastric and duodenal ulcers). Coagulansin A is the steroidal lactone (withanolide) found in plants of solanaceae family such Withania coagulans. The current study was carried out to examine the in vitro urease, COX-2 inhibitory activity and effect on type II collagen expression of coagulansin A. Moreover, we investigated cytotoxic effects on rabbit articular chondrocytes through MTT assay. COX-2 and type II collagen expressions were determined through a Western blot method. Molecular docking and simulation studies of urease (PDBID 4H9M) and COX-2 (PDBID 5F1A) proteins were also performed as an in silico approach. Results showed that COX-2 expression was decreased dose dependably, significantly higher expression of type II collagen was observed at higher doses. In the current study, coagulansin A was found as non-toxic, and showed notable urease inhibitory activity in non-competitive manner with IC50 23.14 µm in comparison to reference drug thiourea 17.81 µm. Significant decrease in COX-2 expression (40%) and increase in type II collagen (20%) were observed as compared to control. In silico results unveiled the strong binding affinities of coagulansin A with both of these urease and COX-2 proteins. Therefore, herein we proposed the significant antiurease potential of this compound that could be used in treating different diseases such as ulcers. Moreover, detailed in vivo studies and molecular mechanism based studies are suggested.

Green Synthesis of Silver Nanoparticles using Bidens Frondosa Extract and their Tyrosinase Activity.

Herbal nanoparticles gain lot of attention because of their pharmaceutical importance. The present study reports the eco-friendly synthesis, characterization and their tyrosinase activity of silver nanoparticles (AgNPs) using aqueous extract of Bidens frondosa. The appearance of brown color indicated the formation of B. frondosa AgNPs. The Formation of AgNPs was confirmed by UV-Vis spectroscopy, FTIR, FESEM and EDS analysis. The formation of herbal AgNPs of size ranging 20-70 nm further was assured by energy dispersive X-ray spectroscopy (EDS) and field emission scanning electron microscopy (FESEM). The mushroom tyrosinase inhibitory activity of synthesized AgNPs was evaluated. Nanoparticles were found to have significant higher tyrosinase inhibitory activity compared to control. The IC50 values of crude extract, AgNP and Kojic acid were found to be 9, 15, and 2.37 µg/mL, respectively. AgNPs of B. frondosa may be considered as potential candidate for the production of medical and cosmetic products.

Acetazolamide Inhibits the Level of Tyrosinase and Melanin: An Enzyme Kinetic, In Vitro, In Vivo, and In Silico Studies.

Melanin is the major factor that determines skin color and protects from ultraviolet radiation. In present study we evaluated the anti-melanogenesis effect of acetazolamide (ACZ) using four different approaches: enzyme kinetic, in vitro, in vivo and in silico. ACZ demonstrated significant inhibitory activity (IC50 7.895 ± 0.24 µm) against tyrosinase as compared to the standard drug kojic acid (IC50 16.84 ± 0.64 µm) and kinetic analyses showed that ACZ is a non-competitive inhibitor without cytotoxic effect. In in vitro experiments, A375 human melanoma cells were treated with 20 or 40 µm of ACZ with or without 50 µm of l-DOPA. Western blot results showed that ACZ significantly (P < 0.05) decreased the expression level of tyrosinase at 40 µm. Zebrafish embryos were treated with 10, 20 or 40 µm of ACZ and of positive control kojic acid. At 72 h of treatment with ACZ and kojic acid, ACZ significantly (P < 0.001) decreased the embryos pigmentation to 40.8% of untreated embryos at the dose of 40 µm of ACZ while kojic acid decreased only 25.0% of pigmentation at the same dose of kojic acid. In silico docking were performed against tyrosinase using PyRx tool. Docking studies suggested that His244, Asn260 and His85 are the major interacting residues in the binding site of the protein. In conclusion, our results suggest that ACZ is a good candidate for the inhibition of melanin and it could be used as a lead for developing the drugs for hyperpigmentary disorders and skin whitening.

In vitro and in vivo evaluation of anti-arthritic, antioxidant efficacy of fucoidan from Undaria pinnatifida (Harvey) Suringar.

Seaweed and their constituents have been traditionally employed for the management of various human pathologic conditions such as edema, urinary disorders and inflammatory anomalies. The current study was performed to investigate the antioxidant and anti-arthritic effects of fucoidan from Undaria pinnatifida. A noteworthy in vitro antioxidant potential at 500µg/ml in 2, 2-diphenyl-1-picrylhydrazyl scavenging assay (80% inhibition), nitrogen oxide inhibition assay (71.83%), hydroxyl scavenging assay (71.92%), iron chelating assay (73.55%) and a substantial ascorbic acid equivalent reducing power (399.35µg/mg ascorbic acid equivalent) and total antioxidant capacity (402.29µg/mg AAE) suggested fucoidan a good antioxidant agent. Down regulation of COX-2 expression in rabbit articular chondrocytes in a dose (0-100µg) and time (0-48h) dependent manner, unveiled its in vitro anti-inflammatory significance. In vivo carrageenan induced inflammatory rat model demonstrated a 68.19% inhibition of inflammation whereas an inflammation inhibition potential of 79.38% was recorded in anti-arthritic complete Freund's adjuvant-induced arthritic rat model. A substantial ameliorating effect on altered hematological and biochemical parameters in arthritic rats was also observed. Therefore, findings of the present study prospects fucoidan as a potential antioxidant that can effectively abrogate oxidative stress, edema and arthritis-mediated inflammation and mechanistic studies are recommended for observed activities.

Isolation, characterization, and in silico, in vitro and in vivo antiulcer studies of isoimperatorin crystallized from Ostericum koreanum.

CONTEXT: Ostericum koreanum (Maxim.) Kitagawa (Apiaceae) roots are traditionally used as an analgesic and antiulcer agent. However, the antiulcer potential of isoimperatorin isolated from O. koreanum has not yet been explored. AIM: To evaluate the antiulcer activity of isoimperatorin isolated from the roots of O. koreanum. MATERIALS AND METHODS: Isoimperatorin was isolated as cubic crystals by repeated column chromatography of the ethyl acetate fraction and structure was verified with 1H NMR, 13C NMR and high-resolution mass spectrometry (HRMS-FAB). The crystals obtained were analyzed with the single crystal X-ray method. The MTT assay was used to determine its cytotoxicity against chondrocytes at different concentrations (0.0-737.74 µM, 24 h). The in vivo antiulcer activity of isoimperatorin (40 mg/kg) was determined against ethanol-, indomethacin- and pyloric ligation-induced ulcers in Sprague-Dawley rats. Furthermore, the effect of isoimperatorin (0.0-737.74 µM, 24 h) on the expression of type II collagen in chondrocytes was determined using western blot method. The in vitro urease inhibitory activity of isoimperatorin (0-80 µM) and molecular docking was also performed against urease. RESULTS AND DISCUSSION: Isoimperatorin demonstrated significant inhibitory activity (IC50 36.43 µM) against urease as compared to the standard drug thiourea (IC50 33.57 µM) without cytotoxic effects. It provided 70.9%, 67.65% and 54.25% protection in ulcer models induced by ethanol, indomethacin and pyloric ligation, respectively. Isoimperatorin showed the highest expression level of type II collagen at 368.87 µM. The docking results confirmed strong binding affinity with the target protein. CONCLUSION: Isoimperatorin may be used to develop antiulcer drugs with decreased side effects.

Applications of Chondrocyte-Based Cartilage Engineering: An Overview.

Chondrocytes are the exclusive cells residing in cartilage and maintain the functionality of cartilage tissue. Series of biocomponents such as different growth factors, cytokines, and transcriptional factors regulate the mesenchymal stem cells (MSCs) differentiation to chondrocytes. The number of chondrocytes and dedifferentiation are the key limitations in subsequent clinical application of the chondrocytes. Different culture methods are being developed to overcome such issues. Using tissue engineering and cell based approaches, chondrocytes offer prominent therapeutic option specifically in orthopedics for cartilage repair and to treat ailments such as tracheal defects, facial reconstruction, and urinary incontinence. Matrix-assisted autologous chondrocyte transplantation/implantation is an improved version of traditional autologous chondrocyte transplantation (ACT) method. An increasing number of studies show the clinical significance of this technique for the chondral lesions treatment. Literature survey was carried out to address clinical and functional findings by using various ACT procedures. The current study was conducted to study the pharmacological significance and biomedical application of chondrocytes. Furthermore, it is inferred from the present study that long term follow-up studies are required to evaluate the potential of these methods and specific positive outcomes.

Synthesis, characterization, applications, and challenges of iron oxide nanoparticles.

Recently, iron oxide nanoparticles (NPs) have attracted much consideration due to their unique properties, such as superparamagnetism, surface-to-volume ratio, greater surface area, and easy separation methodology. Various physical, chemical, and biological methods have been adopted to synthesize magnetic NPs with suitable surface chemistry. This review summarizes the methods for the preparation of iron oxide NPs, size and morphology control, and magnetic properties with recent bioengineering, commercial, and industrial applications. Iron oxides exhibit great potential in the fields of life sciences such as biomedicine, agriculture, and environment. Nontoxic conduct and biocompatible applications of magnetic NPs can be enriched further by special surface coating with organic or inorganic molecules, including surfactants, drugs, proteins, starches, enzymes, antibodies, nucleotides, nonionic detergents, and polyelectrolytes. Magnetic NPs can also be directed to an organ, tissue, or tumor using an external magnetic field for hyperthermic treatment of patients. Keeping in mind the current interest in iron NPs, this review is designed to report recent information from synthesis to characterization, and applications of iron NPs.