Human Epithelial Keratinocytes Research Articles

P19 Defining the mechanism of skin barrier restoration by Lactobacillus rhamnosus

Abstract Introduction and aims We aim to investigate the lysate of Lactobacillus rhamnosus GG (LGG), a bacterium previously established as a gut probiotic, and to examine its postulated ability to enhance and restore human skin barrier function. Compromised skin barriers are implicated in various skin conditions including atopic dermatitis. Furthermore, skin barrier function decreases with age leading to dryness and sensitivity. Previous research has shown promising effects of LGG lysates on keratinocytes, including increasing transepidermal electrical resistance, increasing tight junction protein expression, cell migration promotion and mitigation of Staphylococcus aureus toxicity. This study aims to take a closer look at the molecular mechanisms behind the effect of LGG lysates on the skin, including terminal differentiation, lamin dispersal and nuclear degradation in keratinocytes. Methods Normal human epithelial keratinocytes and rat epithelial keratinocytes were grown and treated with the LGG lysate. Western blot analysis was used to determine the relative concentrations of tight junction proteins and other differentiation markers. A phosphoproteomic study was performed to identify key pathways activated by the LGG lysate. A Staphylococcus epidermidis lysate control was used to filter out any nonspecific bacterial effects. Immunostaining of nuclear proteins was used to visualize the effects of LGG lysate on lamin dispersal and nuclear degradation. short hairpin RNA filaggrin knockdown normal human keratinocytes were developed to create a ‘barrier impaired’ model to assess ability of the lysate to ‘rescue’ the skin barrier. Results The LGG lysate was shown to increase tight junction protein expression in normal human keratinocytes and increase keratinocyte terminal differentiation markers. We present Results that show decreased tight junction protein expression in filaggrin knockdown cells. Conclusions These Results show the potential for LGG lysate to enhance and restore skin barrier function and begin to suggest potential mechanisms for this effect.

Manuka honey activates the aryl hydrocarbon receptor: Implications for skin inflammation

Manuka honey (MH) is a complex nutritional material with antimicrobial, antioxidant and anti-inflammatory activity. We have previously shown that MH down regulates IL-4-induced CCL26 expression in immortalized keratinocytes. As MH contains potential ligands of the Aryl Hydrocarbon Receptor (AHR), a key regulator of skin homeostasis, we hypothesize that this effect is mediated via AHR activation. Here, we treated HaCaT cell lines, either stable transfected with an empty vector (EV-HaCaT) or in which AHR had been stable silenced (AHR-silenced HaCaT); or primary normal human epithelial keratinocytes (NHEK) with 2% MH for 24 h. This induced a 15.4-fold upregulation of CYP1A1 in EV-HaCaTs, which was significantly reduced in AHR-silenced cells. Pre-treatment with the AHR antagonist CH223191 completely abrogated this effect. Similar findings were observed in NHEK. In vivo treatment of the Cyp1a1Cre x R26ReYFP reporter mice strain’s skin with pure MH significantly induced CYP1A1 expression compared with Vaseline. Treatment of HaCaT with 2% MH significantly decreased baseline CYP1 enzymatic activity at 3 and 6 h but increased it after 12 h, suggesting that MH may activate the AHR both through direct and indirect means. Importantly, MH downregulation of IL-4-induced CCL26 mRNA and protein was abrogated in AHR-silenced HaCaTs and by pre-treatment with CH223191. Finally, MH significantly upregulated FLG expression in NHEK in an AHR-dependent manner. In conclusion, MH activates AHR, both in vitro and in vivo, thereby providing a mechanism of its IL4-induced CCL26 downregulation and upregulation of FLG expression. These results have potential clinical implications for atopic diseases and beyond.

Screening for Lactic Acid Bacterial Strains as Probiotics Exhibiting Anti-inflammatory and Antioxidative Characteristic Via Immune Modulation in HaCaT Cell.

In this study, the basic probiotic characteristics and functional properties of lactic acid bacteria (LAB) were investigated using two in vitro models of inflammation induced by lipopolysaccharide (LPS) and H2O2. Fifteen strains were prescreened out of 60 LAB candidates based on their radical scavenging activity to determine the antioxidant capacity of the strains. The top 15 candidates were further investigated to evaluate their survival rate under low pH and bile salt conditions that mimic the intestinal environment. Three strains, Levilactobacillus brevis D70 (Levilact), Lactiplantibacillus pentosus S16 (Lactipla), and Limosilactobacillus fermentum MF10 (Limosilact), were capable of scavenging free radicals and survived under artificial intestinal conditions. Therefore, Levilact. brevis D70, Lactipla. pentosus S16, and Limosilact. fermentum MF10 were selected for further antioxidant, anti-inflammation, and mitochondrial activity examinations via cell models of inflammation and oxidative stress. Among the three strains, Limosilact. fermentum MF10 showed the highest anti-inflammatory activities by significantly downregulating the relative mRNA expression levels of inflammatory biomarkers such as interleukin 8 (IL-8) and interferon-gamma (IFN-γ) induced by LPS (P < 0.05). Moreover, Limosilact. fermentum MF10 was also capable of upregulating the gene expression levels of antioxidative mediator glutathione peroxidase 4 (GPX4) induced by reactive oxygen species (ROS) in both human HT-29 epithelial cells and human HaCaT keratinocytes. Limosilact. fermentum MF10 was also capable of regulating mitochondrial membrane potential (MMP), which plays a key role in the mitochondrial activity of HaCaT cells. As a result, Limosilact. fermentum MF10 showed the highest potential for probiotic properties and impacts the immune-related gut-skin axis by altering proinflammatory cytokines, antioxidative biomarkers, and MMP.

Basic amino acid-mediated cationic amphiphilic surfaces for antimicrobial pH monitoring sensor with wound healing effects

BackgroundThe wound healing process is a complex cascade of physiological events, which are vulnerable to both our body status and external factors and whose impairment could lead to chronic wounds or wound healing impediments. Conventional wound healing materials are widely used in clinical management, however, they do not usually prevent wounds from being infected by bacteria or viruses. Therefore, simultaneous wound status monitoring and prevention of microbial infection are required to promote healing in clinical wound management.MethodsBasic amino acid-modified surfaces were fabricated in a water-based process via a peptide coupling reaction. Specimens were analyzed and characterized by X-ray photoelectron spectroscopy, Kelvin probe force microscopy, atomic force microscopy, contact angle, and molecular electrostatic potential via Gaussian 09. Antimicrobial and biofilm inhibition tests were conducted on Escherichia coli and Staphylococcus epidermidis. Biocompatibility was determined through cytotoxicity tests on human epithelial keratinocytes and human dermal fibroblasts. Wound healing efficacy was confirmed by mouse wound healing and cell staining tests. Workability of the pH sensor on basic amino acid-modified surfaces was evaluated on normal human skin and Staphylococcus epidermidis suspension, and in vivo conditions.ResultsBasic amino acids (lysine and arginine) have pH-dependent zwitterionic functional groups. The basic amino acid-modified surfaces had antifouling and antimicrobial properties similar to those of cationic antimicrobial peptides because zwitterionic functional groups have intrinsic cationic amphiphilic characteristics. Compared with untreated polyimide and modified anionic acid (leucine), basic amino acid-modified polyimide surfaces displayed excellent bactericidal, antifouling (reduction ~ 99.6%) and biofilm inhibition performance. The basic amino acid-modified polyimide surfaces also exhibited wound healing efficacy and excellent biocompatibility, confirmed by cytotoxicity and ICR mouse wound healing tests. The basic amino acid-modified surface-based pH monitoring sensor was workable (sensitivity 20 mV pH−1) under various pH and bacterial contamination conditions.ConclusionHere, we developed a biocompatible and pH-monitorable wound healing dressing with antimicrobial activity via basic amino acid-mediated surface modification, creating cationic amphiphilic surfaces. Basic amino acid-modified polyimide is promising for monitoring wounds, protecting them from microbial infection, and promoting their healing. Our findings are expected to contribute to wound management and could be expanded to various wearable healthcare devices for clinical, biomedical, and healthcare applications.

SCARA5 inhibits oral squamous cell carcinoma via inactivating the STAT3 and PI3K/AKT signaling pathways.

Oral squamous cell carcinoma (OSCC) is a common tumor in the world. Despite the rapid development of medical care, OSCC is also accompanied by high incidence and mortality every year. Therefore, it is still necessary to continuously develop new methods or find new targets to treat OSCC. Previous research showed that scavenger receptor class A member 5 (SCARA5) was one of the potential biomarkers of OSCC, and its expression is significantly low in OSCC. This study aimed to explore the role and related molecular mechanisms of SCARA5 in OSCC. In this study, we found that the SCARA5 expression was lower in CAL-27 and SCC-9 cells than that in human normal oral epithelial keratinocytes. SCARA5 overexpression significantly inhibited cell proliferation and induced apoptosis of CAL-27 and SCC-9 cells. In addition, SCARA5 repressed OSCC cell epithelial-mesenchymal transformation (EMT), evidenced by increased E-cadherin expression and reduced N-cadherin expression. Finally, we found that SCARA5 could suppress STAT3, PI3K, and AKT phosphorylation. Therefore, SCARA5 was related to STAT3 and PI3K/AKT signaling pathways in OSCC. In conclusion, SCARA5 inhibited the proliferation and EMT and induced the apoptosis of OSCC cells through the inhibition of STAT3 and PI3K/AKT signaling pathways, thereby exerting a tumor suppressor effect.

Synthesis, characterization, solution chemistry and anticancer activity of [NiCl2(Ph2P–N(R)–PPh2)] (R = 2-CH2Py, CH2Ph and p-tol) complexes

In this work three Ni2+ complexes with general formula [NiCl2(Ph2P–N(R)–PPh2)], R = 2-CH2Py (Py = pyridine) – 1, CH2Ph (Ph = phenyl) – 2 and p-tol (p-tol = p-tolyl) – 3, were synthesized and characterized. These complexes were obtained in high yield by the reaction of NiCl2.6H2O and the corresponding diphenylphosphinoamine ligand (Ph2P–N(R)–PPh2) in CH2Cl2/MeOH (1:1) solution, at room temperature (∼25 °C), and characterized by 1H and 31P {1H} NMR, vibrational spectroscopy in the infrared region, electronic spectroscopy in the UV–Vis regions, elemental analysis (%C, %H, %N) and single-crystal X-ray diffraction. The solution chemistry was studied in CDCl3/dmso-d6 (dimethylsulfoxide) or neat dmso-d6 using complex 2 as a model. The complexes were evaluated as cytotoxic agents against two cancer cells lines, A549 (lung cancer cells), B16F10 (melanoma cells) and the health cells HaCaT (human epithelial keratinocytes).

TEAD1 Silencing Regulates Cell Proliferation and Resistance to 5-Fluorouracil in Cutaneous Squamous Cell Carcinoma.

Cutaneous squamous cell carcinoma (cSCC) is a skin malignant tumor account for approximately one-third of all nonmelanoma skin cancers. Studies have shown that TEA domain transcription factor 1 (TEAD1) is discovered to be involved in the pathogenesis of some human cancers, but to our knowledge its role in cSCC has not been reported. Samples from 16 cSCC patients and 27 healthy individuals were obtained for immunohistochemical staining of TEAD1. The expressions of TEAD1 in SCL-1, HSC-1 cells compared with the primary neonatal human epithelial keratinocytes were detected by Western blot and RT-qPCR. Proliferation and cell cycle of TEAD1 knockdown in cSCC cell lines were examined by MTT and flow cytometry analysis. Annexin V/PI and JC-1 staining were used to determine the cell apoptosis. The expression of TEAD1 decreased significantly in cSCC compared to its expression in normal skin tissues and cell lines. Down-regulation of TEAD1 in cSCC cell lines promoted cell growth via regulation of the G2/M progression. Additionally, silence of TEAD1 also protected cells against 5-Fluorouracil-induced apoptosis and decreased the expression of apoptosis-related protein (p53). Our results suggested that TEAD1 expression is down-regulated and functioned as a tumor suppressor in cSCC and that it may serve as a biomarker or therapeutic target of cSCC.

Particulate matter-induced hypomethylation of Alu and LINE1 in normal human bronchial epithelial cells and epidermal keratinocytes

BackgroundAirborne particulate matter (PM), a complex mixture of organic and inorganic compounds, is a major public health concern due to its adverse health effects. Understanding the biological action of PM is of particular importance in the improvement of public health. Differential methylation of repetitive elements (RE) by PM might have severe consequences for the structural integrity of the genome and on transcriptional activity, thereby affecting human health. This study aimed to evaluate the effect of inhaled and non-inhaled PM (PM2.5, PM10, and PM10-PAH) exposure on DNA methylation. We quantitatively measured the methylation content of Alu and LINE1 in PM-treated normal human bronchial epithelial cells (NHBE) and normal human epidermal keratinocytes (NHEK) by using whole-genome bisulfite sequencing and pyrosequencing.ResultsAll PMs exposure significantly lowered Alu and LINE1 methylation in both cells than in mock-treated controls. Hypomethylation was more prominent in PM10-PAH exposed-NHBE and PM10 exposed-NHEK. Alu and LINE1 methylation change exhibited different sensitivity according to the subfamily evolutionary ages, with stronger effects on the oldest L1-M and Alu J in NHBE, and oldest L1-M and youngest Alu S in NHEK.ConclusionsThese results demonstrate that the differential susceptibility of PM-induced hypomethylation of Alu and LINE1 depends upon RE evolutionary age and PM type.

An In vitro evaluation of effect of implant abutment on human gingival epithelial keratinocytes.

Abutment surfaces are being designed to promote gingival soft tissue attachment and integration. This confirms implant survival for long term by forming a seal around the prosthetics. This study was done to compare the biocompatibility of three implant abutments: titanium uncoated, Ti-nitride coated, and modified polyetheretherketone (PEEK) with human gingival keratinocytes. The titanium-uncoated, titanium-nitride-coated, and modified PEEK discs (13 mm × 3 mm) were fabricated and compared with uncoated polyester cell culture discs, which were used as controls. These three implant abutments were evaluated for biocompatibility with respect to human gingival keratinocytes for viability, morphology, proliferation, and migration by scanning electron microscopy imaging and scratch wound healing assays. Measurements of roughness show changes between the investigated surfaces. Keratinocytes cultured on all examined surfaces indicated adhesion and attachment. An assay of cell viability showed no substantial variances among the groups. The modified PEEK surface showed greater cell proliferation and migration among the three abutment materials. All three abutment material surface types showed similar epithelial biological responses. However, modified PEEK material showed the highest biocompatibility.

Transcriptomic and Functional Evidence Show Similarities between Human Amniotic Epithelial Stem Cells and Keratinocytes.

Amniotic epithelial stem cells (AESCs) are considered as potential alternatives to keratinocytes (KCs) in tissue-engineered skin substitutes used for treating skin damage. However, their clinical application is limited since similarities and distinctions between AESCs and KCs remain unclear. Herein, a transcriptomics analysis and functional evaluation were used to understand the commonalities and differences between AESCs and KCs. RNA-sequencing revealed that AESCs are involved in multiple epidermis-associated biological processes shared by KCs and show more similarity to early stage immature KCs than to adult KCs. However, AESCs were observed to be heterogeneous, and some possessed hybrid mesenchymal and epithelial features distinct from KCs. A functional evaluation revealed that AESCs can phagocytose melanosomes transported by melanocytes in both 2D and 3D co-culture systems similar to KCs, which may help reconstitute pigmented skin. The overexpression of TP63 and activation of NOTCH signaling could promote AESC stemness and improve their differentiation features, respectively, bridging the gap between AESCs and KCs. These changes induced the convergence of AESC cell fate with KCs. In future, modified reprogramming strategies, such as the use of small molecules, may facilitate the further modulation human AESCs for use in skin regeneration.

SALBUTAMOL AMELIORATES THE PHENOTYPE OF THE SKIN INFLAMMATORY DISEASE PSORIASIS ACCORDING TO SKIN SPHEROID MODELS

Psoriasis is a multifactorial chronic inflammatory disorder resulting by the interplay of genetics, the immune system and the environment. It is characterized by the hyperproliferation of epithelial cells, generating red, itchy psoriatic plaques which have no cure but have great negative impact in patients’ life. Although corticosteroids or vitamin D analogs might help recovery to some extent, there is yet no total cure for the disease. In this study, we sought to generate three-dimensional (3D) stress-related psoriatic skin spheroids with the screening of the potential efficacy of a β2-adrenergic receptor agonist, salbutamol. 3D Culture spheroids with human dermal fibroblasts (HDF), human epithelial keratinocytes (HEK) and human monocytic cell line (THP-1) were generated as a representative model of skin and the protocol of stress-related modelling was conducted. The efficacy of the drug salbutamol was evaluated by the changes in mRNA and protein expression levels of selected genes, as well as by several metabolic assays. We developed a method for culturing spherical organoid models of psoriasis in vitro. We tested the potential theurapetic effects of salbutamol on psoriasis spheroids. Spheroids treated with salbutamol indicated the effictiveness of the treatment. 3D spheroid system was found partially efficient for mimicking the physiological features of psoriasis in vitro. This present work may be a starting point for future investigation as it is the first to generate a stress-related psoriatic model and first to try a β2 agonist as a potential treatment option. Considering the effects and suitability of topical application of salbutamol, its efficacy should not be underestimated and should be investigated further for translating this knowledge into clinics.

Biochemical impact of solar radiation exposure on human keratinocytes monitored by Raman spectroscopy; effects of cell culture environment.

Understanding and amelioration of the effects of solar radiation exposure are critical in preventing the occurrence of skin cancer. Towards this end, many studies have been conducted in 2D cell culture models under simplified and unrealistic conditions. 3D culture models better capture the complexity of in vivo physiology, although the effects of the 3D extracellular matrix have not been well studied. Monitoring the instantaneous and resultant cellular responses to exposure, and the influence of the 3D environment, could provide an enhanced understanding of the fundamental processes of photocarcinogenesis. This work presents an analysis of the biochemical impacts of simulated solar radiation (SSR) occurring in immortalised human epithelial keratinocytes (HaCaT), in a 3D skin model, compared to 2D culture. Cell viability was monitored using the Alamar Blue colorimetric assay (AB), and the impact of the radiation exposure, at the level of the biomolecular constituents (nucleic acids and proteins), were evaluated through the combination of Raman microspectroscopy and multivariate statistical analysis. The results suggest that SSR exposure induces alterations of the conformational structure of DNA as an immediate impact, whereas changes in the protein signature are primarily seen as a subsequent response.

Ultraviolet B irradiation decreases CXCL10 expression in keratinocytes through endoplasmic reticulum stress.

Ultraviolet radiation is one of the standard treatment selections for psoriasis. interferon (IFN)-γ and IFN-γ-induced CXCL10, which are highly expressed by keratinocytes in psoriasis lesion, are therapeutic targets for psoriasis. In this study, we found that ultraviolet B (UVB) irradiation inhibited IFN-γ signaling events, including STAT1 phosphorylation and induction of CXCL10 messenger RNA (mRNA) expression in keratinocytes. IFN-γ-induced expression of CXCL10 mRNA in HaCaT cells, a human keratinocyte cell line, and human epithelial keratinocytes were also inhibited by H2 O2 or endoplasmic reticulum (ER) stress inducers. Conversely, a mixture of antioxidants, Trolox and ascorbic acid, and the ER stress inhibitor salubrinal partially counteracted the inhibitory effect of UVB on IFN-γ-induced CXCL10 mRNA expression in HaCaT cells. We also found that UVB and ER stress reduced IFN-γ receptor 1 protein levels in the plasma membrane fraction of keratinocytes. These observations suggested that ER stress and the generation of reactive oxygen species are essential for the inhibitory effect of UVB on IFN-γ-induced CXCL10 mRNA in keratinocytes.

Contact lenses coated with hybrid multifunctional ternary nanocoatings (Phytomolecule-coated ZnO nanoparticles:Gallic Acid:Tobramycin) for the treatment of bacterial and fungal keratitis

Contact lenses are widely used for visual corrections. However, while wearing contact lenses, eyes typically face discomforts (itching, irritation, burning, etc.) due to foreign object sensation, lack of oxygen permeability, and tear film disruption as opposed to a lack of wetting agents. Eyes are also prone to ocular infections such as bacterial keratitis (BK) and fungal keratitis (FK) and inflammatory events such as contact lens-related acute red eye (CLARE), contact lens peripheral ulcer (CLPU), and infiltrative keratitis (IK) caused by pathogenic bacterial and fungal strains that contaminate contact lenses. Therefore, a good design of contact lenses should adequately address the need for wetting, the supply of antioxidants, and antifouling and antimicrobial efficacy. Here, we developed multifunctional gallic acid (GA), phytomolecules-coated zinc oxide nanoparticles (ZN), and phytomolecules-coated zinc oxide nanoparticles + gallic acid + tobramycin (ZGT)-coated contact lenses using a sonochemical technique. The coated contact lenses exhibited significant antibacterial (>log10 5.60), antifungal, and antibiofilm performance against BK-causing multidrug resistant bacteria (Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia. coli) and FK-related pathogenic fungal strains (Candida albicans, Aspergillus fumigatus, and Fusarium solani). The gallic acid, tobramycin, and phytomolecules-coated zinc oxide nanoparticles have different functionalities (-OH, -NH2, -COOH, -COH, etc.) that enhanced wettability of the coated contact lenses as compared to that of uncoated ones and further enabled them to exhibit remarkable antifouling property by prohibiting adhesion of platelets and proteins. The coated contact lenses also showed significant antioxidant activity by scavenging DPPH and good cytocompatibility to human corneal epithelial cells and keratinocytes cell lines. Statement of significance• Multifunctional coated lenses were developed with an efficient sonochemical approach.• Lens surface was modified with nanocoatings of ZnO nanoparticles, gallic acid, and tobramycin.• This synergistic combination endowed the lenses with remarkable antimicrobial activity.• Coated lenses also showed noteworthy antifouling and biofilm inhibition activities.• Coated lenses showed good antioxidant, biocompatibility, and wettability characteristics.

Bardoxolone-Methyl (CDDO-Me) Impairs Tumor Growth and Induces Radiosensitization of Oral Squamous Cell Carcinoma Cells.

Radiotherapy represents a common treatment strategy for patients suffering from oral squamous cell carcinoma (OSCC). However, application of radiotherapy is immanently limited by radio-sensitivity of normal tissue surrounding the tumor sites. In this study, we used normal human epithelial keratinocytes (NHEK) and OSCC cells (Cal-27) as models to investigate radio-modulating and anti-tumor effects of the synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9,-dien-28-oic acid methyl ester (CDDO-Me). Nanomolar CDDO-Me significantly reduced OSCC tumor xenograft-growth in-ovo applying the chick chorioallantoic membrane (CAM) assay. In the presence of CDDO-Me reactive oxygen species (ROS) were found to be reduced in NHEK when applying radiation doses of 8 Gy, whereas ROS levels in OSCC cells rose significantly even without radiation. In parallel, CDDO-Me was shown to enhance metabolic activity in malignant cells only as indicated by significant accumulation of reducing equivalents NADPH/NADH. Furthermore, antioxidative heme oxygenase-1 (HO-1) levels were only enhanced in NHEK and not in the OSCC cell line, as shown by immunoblotting. Clonogenic survival was left unchanged by CDDO-Me treatment in NHEK but revealed to be abolished almost completely in OSCC cells. Our results indicate anti-cancer and radio-sensitizing effects of CDDO-Me treatment in OSCC cells, whereas nanomolar CDDO-Me failed to provoke clear detrimental consequences in non-malignant keratinocytes. We conclude, that the observed differential aftermath of CDDO-Me treatment in malignant OSCC and non-malignant skin cells may be utilized to broaden the therapeutic range of clinical radiotherapy.

Increasing prevalence of hypervirulent ST5 methicillin susceptible Staphylococcus aureus subtype poses a serious clinical threat

ABSTRACT Staphylococcus aureus (S. aureus) is a clinical pathogen of great significance causing metastatic or complicated infections. ST5 clonotype isolates have dominated S. aureus infections for more than 10 years in Shanghai, China, and the proportion of methicillin-susceptible S. aureus (MSSA) has remarkably increased in the past decades. By whole-genome sequencing (WGS) 121 ST5 clonotype S. aureus isolates using next-generation sequencing (NGS) platforms and characterizing the evolutionary dynamics of ST5 linages, we found that MSSA evolved independently, making it a subtype differed from other MRSA clones. Drug resistance gene analysis by using the NGS data demonstrated that ST5 clonotype MRSA might be more tolerant under the threat of antimicrobials, which was confirmed in further in vitro susceptibility tests. However, MSSA subtype isolates exhibited relatively high virulence upon the analysis of virulence factors. Furthermore, MSSA subtype isolates displayed higher hemolysis capacity and higher ability to adhere to epithelial cells including A549 human alveolar epithelial cells and HaCaT human skin keratinocytes, caused more severe infections in murine abscess model. With its high virulence and enhanced magnitude in the past decades, the ST5 MSSA subtype poses a serious clinical threat hence more attention should be paid to the prevention and control.

Monitoring the biochemical changes occurring to human keratinocytes exposed to solar radiation by Raman spectroscopy.

Solar radiation exposure is recognised to be a significant contributor to the development of skin cancer. Monitoring the simultaneous and consecutive mechanisms of interaction could provide a greater understanding of the process of photocarcinogenesis. This work presents an analysis of the biochemical and morphological changes occurring to immortalised human epithelial keratinocyte (HaCaT) cell cultures exposed to simulated solar radiation (SSR). Cell viability was monitored with the aid of the Alamar Blue assay, morphological examination was done with haematoxylin and eosin staining (H&E) and changes to the biochemical constituents (nucleic acids and proteins) as a result of the radiation insult were demonstrated through a combination of Raman microspectroscopy and multivariate analysis of spectral patterns. The spectral results suggest that SSR induces changes to the conformational structure of DNA as an immediate result of the radiation, whereas alteration in the protein signature is mostly seen as a later response.

Development of Moringa oleifera Standardized Leaf Extract Nanofibers Impregnated onto Hydrocolloid Film as A Potential Chronic Wound Dressing

This research focuses on the development of a hybrid dressing, i.e. Moringa oleifera (MOL) leave extract nanofibers impregnated onto an alginate-pectin hydrocolloid film for chronic wound healing. The MOL nanofibers impregnated hydrocolloid films were characterised and optimised by physicomechanical properties, moisture transmission, swelling ratio, microscopy analysis, Franz diffusion drug release and cell viability assays. From the preformulation studies, PEO 6 % solution with viscosity above 1.3 Pa·s was found suitable to be electrospun into uniform nanofibers (diameter 322.7±78.02 nm). The moisture transmission rate and swelling ratio studies revealed that the MOL nanofiber-film was useful for the application of light to medium suppurating wounds. The Franz diffusion cell study showed that the electrospun duration could influence the amount of MOL released from the nanofibers-film. Finally, cell viability assays revealed that the MOL nanofibers-films hybrid dressing did not cause cytotoxicity to human dermal fibroblast and human epithelial keratinocytes cell lines. In conclusion, a hybrid MOL nanofibers impregnated hydrocolloid films dressing was successfully formulated and this study has implied that MOL nanofibers-film could be an alternative treatment option for treating chronic wounds.

T4 Pili Promote Colonization and Immune Evasion Phenotypes of Nonencapsulated M4 Streptococcus pyogenes.

Streptococcus pyogenes (group A Streptococcus [GAS]) is an important human pathogen causing a broad spectrum of diseases and associated with significant global morbidity and mortality. Almost all GAS isolates express a surface hyaluronic acid capsule, a virulence determinant that facilitates host colonization and impedes phagocyte killing. However, recent epidemiologic surveillance has reported a sustained increase in both mucosal and invasive infections caused by nonencapsulated GAS, which questions the indispensable role of hyaluronic acid capsule in GAS pathogenesis. In this study, we found that pilus of M4 GAS not only significantly promotes biofilm formation, adherence, and cytotoxicity to human upper respiratory tract epithelial cells and keratinocytes, but also promotes survival in human whole blood and increased virulence in murine models of invasive infection. T4 antigen, the pilus backbone protein of M4 GAS, binds haptoglobin, an abundant human acute-phase protein upregulated upon infection and inflammation, on the bacterial surface. Haptoglobin sequestration reduces the susceptibility of nonencapsulated M4 GAS to antimicrobial peptides released from activated neutrophils and platelets. Our results reveal a previously unappreciated virulence-promoting role of M4 GAS pili, in part mediated by co-opting the biology of haptoglobin to mitigate host antimicrobial defenses.IMPORTANCE Group A Streptococcus (GAS) is a strict human pathogen causing more than 700 million infections globally each year. The majority of the disease-causing GAS are encapsulated, which greatly guarantees survival and dissemination in the host. Emergence of the capsule-negative GAS, such as M4 GAS, in recent epidemiologic surveillance alarms the necessity to elucidate the virulence determinants of these pathogens. Here, we found that M4 pili play an important role in promoting M4 GAS adherence and cytotoxicity to human pharyngeal epithelial cells and keratinocytes. The same molecule also significantly enhanced M4 GAS survival and replication in human whole blood and experimental murine infection. T4 antigen, which composes the backbone of M4 pili, was able to sequester the very abundant serum protein haptoglobin to further confer M4 GAS resistance to antibacterial substances released by neutrophils and platelets.

Expanding the Spectrum of Antibiotics Capable of Killing Multidrug-Resistant Staphylococcus aureus and Pseudomonas aeruginosa.

Infections from antibiotic-resistant Staphylococcus aureus and Pseudomonas aeruginosa are a serious threat because reduced antibiotic efficacy complicates treatment decisions and prolongs the disease state in many patients. To expand the arsenal of treatments against antimicrobial-resistant (AMR) pathogens, 600-Da branched polyethylenimine (BPEI) can overcome antibiotic resistance mechanisms and potentiate β-lactam antibiotics against Gram-positive bacteria. BPEI binds cell-wall teichoic acids and disables resistance factors from penicillin binding proteins PBP2a and PBP4. This study describes a new mechanism of action for BPEI potentiation of antibiotics generally regarded as agents effective against Gram-positive pathogens but not Gram-negative bacteria. 600-Da BPEI is able to reduce the barriers to drug influx and facilitate the uptake of a non-β-lactam co-drug, erythromycin, which targets the intracellular machinery. Also, BPEI can suppress production of the cytokine interleukin IL-8 by human epithelial keratinocytes. This enables BPEI to function as a broad-spectrum antibiotic potentiator, and expands the opportunities to improve drug design, antibiotic development, and therapeutic approaches against pathogenic bacteria, especially for wound care.