Topical application of jojoba (Simmondsia chinensis L.) wax enhances the synthesis of pro-collagen III and hyaluronic acid and reduces inflammation in the ex-vivo human skin organ culture model.

Jojoba (Simmondsia chinensis L.) wax was previously reported to increase cutaneous wound healing, ameliorate acne and psoriasis manifestations, and reduce oxidative stress and inflammation. However, its potential cosmetic properties have not been fully investigated. Thus, the current study aimed to evaluate the anti-inflammatory activities of jojoba wax and its impact on the synthesis of extracellular components following topical application. The fatty acid and fatty alcohol profiles of two industrial and two lab-scale cold-press jojoba waxes were analyzed along with total tocopherol and phytosterol content. The dermo-cosmetic effect of all jojoba wax preparations was evaluated ex-vivo, using the human skin organ culture model, which emulates key features of intact tissue. The ability of jojoba wax to reduce secreted levels of key pro-inflammatory cytokines and the safety of the applications in the ex-vivo model were evaluated. In addition, the impact on the synthesis of pro-collagen and hyaluronic acid levels upon treatment was investigated. The results demonstrate that topically applied jojoba wax can reduce LPS-induced secretion of IL-6, IL-8, and TNFα by approx. 30% compared to untreated skin. This effect was enhanced when treatment was combined with low non-toxic levels of Triton X-100, and its efficacy was similar to the anti-inflammatory activity of dexamethasone used as a positive control. In addition, mRNA and protein levels of collagen III and synthesis of hyaluronic acid were markedly increased upon topical application of jojoba. Moreover, the enhanced content of extracellular matrix (ECM) components correlated with the enhanced expression of TGFß1. Collectively, our results further demonstrate that jojoba can reduce local skin inflammation, and this effect may be increased by emulsifier which increases its bioavailability. In addition, the finding that topical application of jojoba wax enhances the synthesis of pro-collagen and hyaluronic acid and may be beneficial in the treatment of age-related manifestations.

Skin microbiome bacteria enriched following long sun exposure can reduce oxidative damage.

Sun exposure is harmful to the skin and increases the risk of skin aging and skin cancer. Here we examined the effects of daily exposure to sun radiation on the skin microbiome in order to determine whether skim microbiome bacteria can contribute to protection from solar damage. Skin swabs were collected from ten lifeguards before and after the summer to analyse the skin microbiome. The results indicate that specific skin microbiome bacteria were enriched following the seasonal sun exposure. Especially interesting were two bacterial families - Sphingomonas and Erythrobacteraceae - which may have the ability to protect against UV radiation as they produce potentially protective compounds. We concentrated on a Sphingomonas strain and could show that it was highly resistant to UV irradiation and was able to reduce reactive oxygen species levels in human keratinocytes. These results provide a proof-of-concept for the role of the skin microbiome in protection from solar radiation.

In vitro, ex vivo, and clinical evaluation of anti-aging gel containing EPA and CBD.

BACKGROUND: Skin aging manifestation, such as coarse wrinkles, loss of elasticity, pigmentation, and rough-textured appearance, is a multifactorial process that can be exacerbated by air pollution, smoking, poor nutrition, and sun exposure. Exposure to UV radiation is considered the primary cause of extrinsic skin aging and accounts for about 80% of facial aging. Extrinsic skin aging signs can be reduced with demo-cosmetic formulations. Both cannabidiol (CBD) and eicosapentaenoic acid (EPA) have been previously suggested as potent active dermatological ingredients. AIMS: The objective of the current research was to evaluate the compatibility of both agents in the prevention and treatment of skin aging. First, the impact of both agents was assessed using standard photoaging models of UV-induced damage, both in vitro (HaCaT cells) and ex vivo (human skin organ culture). Then, a clinical validation study (n = 33) was performed using an optimized topical cream formulation tested at different time points (up to Day 56). RESULTS: EPA was found to potentiate the protective effects of CBD by reducing the secretion of prostaglandin E2 (PGE2 ) and interleukin-8 (IL-8), two primary inflammatory agents associated with photoaging. In addition, a qualitative histological examination signaled that applying the cream may result in an increase in extracellular matrix (ECM) remodeling following UV radiation. This was also evidenced clinically by a reduction of crow's feet wrinkle area and volume, as well as a reduction of fine line wrinkle volume as measured by the AEVA system. The well-established age-dependent subepidermal low-echogenic band (SLEB) was also reduced by 8.8%. Additional clinical results showed significantly reduced red spots area and count, and an increase in skin hydration and elasticity by 31.2% and 25.6% following 56 days of cream application, respectively. These impressive clinical results correlated with high satisfaction ratings by the study participants. DISCUSSION AND CONCLUSIONS: Collectively, the results show a profound anti-aging impact of the developed formulation and strengthen the beneficial derm-cosmetic properties of CBD-based products.

Oligochitosan and oxidized nucleoside-based bioderived hydrogels for wound healing.

Herein, we report biocompatible hydrogel for wound healing that was prepared using nature-sourced building blocks. For the first time, OCS was employed as a building macromolecule to form bulk hydrogels along with the nature-sourced nucleoside derivative (inosine dialdehyde, IdA) as the cross-linker. A strong correlation was obtained between the mechanical properties and stability of the prepared hydrogels with a cross-linker concentration. The Cryo-SEM images of IdA/OCS hydrogels showed an interconnected spongy-like porous structure. Alexa 555 labeled bovine serum albumin was incorporated into the hydrogels matrix. The release kinetics studies under physiological conditions indicated that cross-linker concentration could also control the release rate. The potential of hydrogels in wound healing applications was tested in vitro and ex vivo on human skin. Topical application of the hydrogel was excellently tolerated by the skin with no impairment of epidermal viability or irritation, determined by MTT and IL-1α assays, respectively. The hydrogels were used to load and deliver epidermal growth factor (EGF), showing an increase in its ameliorating action, effectively enhancing wound closure inflicted by punch biopsy. Furthermore, BrdU incorporation assay performed in both fibroblast and keratinocyte cells revealed an increased proliferation in hydrogel-treated cells and an enhancement of EGF impact in keratinocytes.

COP I and II dependent trafficking controls ER-associated degradation in mammalian cells.

Misfolded proteins and components of the endoplasmic reticulum (ER) quality control and ER associated degradation (ERAD) machineries concentrate in mammalian cells in the pericentriolar ER-derived quality control compartment (ERQC), suggesting it as a staging ground for ERAD. By tracking the chaperone calreticulin and an ERAD substrate, we have now determined that the trafficking to the ERQC is reversible and recycling back to the ER is slower than the movement in the ER periphery. The dynamics suggest vesicular trafficking rather than diffusion. Indeed, using dominant negative mutants of ARF1 and Sar1 or the drugs Brefeldin A and H89, we observed that COPI inhibition causes accumulation in the ERQC and increases ERAD, whereas COPII inhibition has the opposite effect. Our results suggest that targeting of misfolded proteins to ERAD involves COPII-dependent transport to the ERQC and that they can be retrieved to the peripheral ER in a COPI-dependent manner.

Anti-Herpes Simplex 1 Activity of Simmondsia chinensis (Jojoba) Wax.

Jojoba (Simmondsia chinensis (Link) Schneider) wax is used for various dermatological and pharmaceutical applications. Several reports have previously shown beneficial properties of Jojoba wax and extracts, including antimicrobial activity. The current research aimed to elucidate the impact of Jojoba wax on skin residential bacterial (Staphylococcus aureus and Staphylococcus epidermidis), fungal (Malassezia furfur), and virus infection (herpes simplex 1; HSV-1). First, the capacity of four commercial wax preparations to attenuate their growth was evaluated. The results suggest that the growth of Staphylococcus aureus, Staphylococcus epidermidis, and Malassezia furfur was unaffected by Jojoba in pharmacologically relevant concentrations. However, the wax significantly attenuated HSV-1 plaque formation. Next, a complete dose-response analysis of four different Jojoba varieties (Benzioni, Shiloah, Hatzerim, and Sheva) revealed a similar anti-viral effect with high potency (EC50 of 0.96 ± 0.4 µg/mL) that blocked HSV-1 plaque formation. The antiviral activity of the wax was also confirmed by real-time PCR, as well as viral protein expression by immunohistochemical staining. Chemical characterization of the fatty acid and fatty alcohol composition was performed, showing high similarity between the wax of the investigated varieties. Lastly, our results demonstrate that the observed effects are independent of simmondsin, repeatedly associated with the medicinal impact of Jojoba wax, and that Jojoba wax presence is required to gain protection against HSV-1 infection. Collectively, our results support the use of Jojoba wax against HSV-1 skin infections.

Copper Ions Ameliorated Thermal Burn-Induced Damage in ex vivo Human Skin Organ Culture.

INTRODUCTION: The zone of stasis is formed around the coagulation zone following skin burning and is characterized by its unique potential for salvation. The cells in this zone may die or survive depending on the severity of the burn and therefore are target for the local treatments of burns. Their low survival rate is consistent with decreased tissue perfusion, hypotension, infection, and/or edema, resulting in a significant increase in the wound size following burning. Copper is an essential trace mineral needed for the normal function of almost all body tissues, including the skin. OBJECTIVE: The aim of the work was to study the effect copper ions have on skin burn pathophysiology. METHODS: Skin obtained from healthy patients undergoing abdominoplasty surgery was cut into 8 × 8 mm squares, and round 0.8-mm diameter burn wounds were inflicted on the skin explants. The burned and control intact skin samples were cultured up to 27 days after wounding. Immediately following injury and then again every 48 h, saline only or containing 0.02 or 1 µM copper ions was added onto the skin explant burn wounds. RESULTS: We found that exposing the wounded sites immediately after burn infliction to 0.02 or 1 µM copper ions reduced the deterioration of the zone of stasis and the increase in wound size. The presence of the copper ions prevented the dramatic increase of pro-inflammatory cytokines (interleukin (IL)-6 and IL-8) and transforming growth factor beta-1 that followed skin burning. We also detected re-epithelialization of the skin tissue and a greater amount of collagen fibers upon copper treatment. CONCLUSION: The deterioration of the zone of stasis and the increase in wound size following burning may be prevented or reduced by using copper ion-based therapeutic interventions.

Kinetic Cytokine Secretion Profile of LPS-Induced Inflammation in the Human Skin Organ Culture.

Several in vitro models that mimic different aspects of local skin inflammation exist. The use of ex vivo human skin organ culture (HSOC) has been reported previously. However, comprehensive evaluation of the cytokine secretory capacity of the system and its kinetics has not been performed. Objective: the aim of the current study was to investigate the levels and secretion pattern of key cytokine from human skin tissue upon lipopolysaccharide (LPS) stimulation. HSOC maintained in an air-liquid interface was used. Epidermal and tissue viability was monitored by MTT and Lactate Dehydrogenase (LDH) activity assay, respectively. Cytokine levels were examined by ELISA and multiplex array. HSOCs were treated without or with three different LPS subtypes and the impact on IL-6 and IL-8 secretion was evaluated. The compounds enhanced the secreted levels of both cytokines. However, differences were observed in their efficacy and potency. Next, a kinetic multiplex analysis was performed on LPS-stimulated explants taken from three different donors to evaluate the cytokine secretion pattern during 0-72 h post-induction. The results revealed that the pro-inflammatory cytokines IL-6, IL-8, TNFα and IL-1ß were up-regulated by LPS stimuli. IL-10, an anti-inflammatory cytokine, was also induced by LPS, but exhibited a different secretion pattern, peak time and maximal stimulation values. IL-1α and IL-15 showed donor-specific changes. Lastly, dexamethasone attenuated cytokine secretion in five independent repetitions, supporting the ability of the system to be used for drug screening. The collective results demonstrate that several cytokines can be used as valid inflammatory markers, regardless of changes in the secretion levels due to donor's specific alterations.

Increased pro-collagen 1, elastin, and TGF-ß1 expression by copper ions in an ex-vivo human skin model.

BACKGROUND: Clinical studies demonstrated that continued exposure to copper oxide-embedded textiles, such as pillowcases, significantly reduces depth of facial wrinkles and skin sagging and enhances skin elasticity. OBJECTIVE: Study the mechanisms by which the exposure to copper ions improve the well-being of the skin. METHODS: Human skin explants, cultured ex-vivo, were exposed topically to saline alone or saline containing 0.02 or 1 µmol/L copper ions. The skin explants viability, histology and secretion of elastin, pro-collagen 1, and TGF-ß1 to the culture medium were determined at various time intervals. RESULTS: Exposure to saline containing 0.02 or 1 µmol/L copper ions did not affect the viability or morphological profile of the explants as compared to control explants treated with saline only. Notably, exposure of the skin grafts to 0.02 or to 1 µmol/L of copper ions resulted in ~100% and ~20% increases in elastin and pro-collagen 1 concentrations, respectively, in the culture supernatants already after 1 day of incubation, which remained statistically significantly elevated also after 6 days on incubation, as compared to the control explants. In addition, ~2- and ~4-fold increases in TGF-ß1 levels in the culture supernatants of explants exposed to the copper ions were detected after 4 and 6 days of culture, as compared to the explants exposed to saline alone. CONCLUSION: This study substantiated the anti-aging effect that copper ions have on the skin and gave insights into the mechanisms by which exposure of the skin to copper ions improves the skin well-being.

[2-3H]Mannose-labeling and Analysis of N-linked Oligosaccharides.

Modifications of N-linked oligosaccharides of glycoproteins soon after their biosynthesis correlate to glycoprotein folding status. These alterations can be detected in a sensitive way by pulse-chase analysis of [2-3H]mannose-labeled glycoproteins, with enzymatic removal of labeled N-glycans, separation according to size by HPLC and radioactive detection in a scintillation counter.

Mannosidase IA is in Quality Control Vesicles and Participates in Glycoprotein Targeting to ERAD.

Endoplasmic reticulum-associated degradation (ERAD) of a misfolded glycoprotein in mammalian cells requires the removal of 3-4 alpha 1,2 linked mannose residues from its N-glycans. The trimming and recognition processes are ascribed to ER Mannosidase I, the ER-degradation enhancing mannosidase-like proteins (EDEMs), and the lectins OS-9 and XTP3-B, all residing in the ER, the ER-derived quality control compartment (ERQC), or quality control vesicles (QCVs). Folded glycoproteins with untrimmed glycans are transported from the ER to the Golgi complex, where they are substrates of other alpha 1,2 mannosidases, IA, IB, and IC. The apparent redundancy of these enzymes has been puzzling for many years. We have now determined that, surprisingly, mannosidase IA is not located in the Golgi but resides in QCVs. We had recently described this type of vesicles, which carry ER α1,2 mannosidase I (ERManI). We show that the overexpression of alpha class I α1,2 mannosidase IA (ManIA) significantly enhances the degradation of ERAD substrates and its knockdown stabilizes it. Our results indicate that ManIA trims mannose residues from Man9GlcNAc2 down to Man5GlcNAc2, acting in parallel with ERManI and the EDEMs, and targeting misfolded glycoproteins to ERAD.

Protein aggregation and ER stress.

Protein aggregation is a common feature of the protein misfolding or conformational diseases, among them most of the neurodegenerative diseases. These disorders are a major scourge, with scarce if any effective therapies at present. Recent research has identified ER stress as a major mechanism implicated in cytotoxicity in these diseases. Whether amyloid-ß or tau in Alzheimer's, α-synuclein in Parkinson's, huntingtin in Huntington's disease or other aggregation-prone proteins in many other neurodegenerative diseases, there is a shared pathway of oligomerization and aggregation into amyloid fibrils. There is increasing evidence in recent years that the toxic species, and those that evoke ER stress, are the intermediate oligomeric forms and not the final amyloid aggregates. This review focuses on recent findings on the mechanisms and importance of the development of ER stress upon protein aggregation, especially in neurodegenerative diseases, and possible therapeutic approaches that are being examined. This article is part of a Special Issue entitled SI:ER stress.

Mammalian ER mannosidase I resides in quality control vesicles, where it encounters its glycoprotein substrates.

Endoplasmic reticulum α1,2 mannosidase I (ERManI), a central component of ER quality control and ER-associated degradation (ERAD), acts as a timer enzyme, modifying N-linked sugar chains of glycoproteins with time. This process halts glycoprotein folding attempts when necessary and targets terminally misfolded glycoproteins to ERAD. Despite the importance of ERManI in maintenance of glycoprotein quality control, fundamental questions regarding this enzyme remain controversial. One such question is the subcellular localization of ERManI, which has been suggested to localize to the ER membrane, the ER-derived quality control compartment (ERQC), and, surprisingly, recently to the Golgi apparatus. To try to clarify this controversy, we applied a series of approaches that indicate that ERManI is located, at the steady state, in quality control vesicles (QCVs) to which ERAD substrates are transported and in which they interact with the enzyme. Both endogenous and exogenously expressed ERManI migrate at an ER-like density on iodixanol gradients, suggesting that the QCVs are derived from the ER. The QCVs are highly mobile, displaying dynamics that are dependent on microtubules and COP-II but not on COP-I vesicle machinery. Under ER stress conditions, the QCVs converge in a juxtanuclear region, at the ERQC, as previously reported. Our results also suggest that ERManI is turned over by an active autophagic process. Of importance, we found that membrane disturbance, as is common in immunofluorescence methods, leads to an artificial appearance of ERManI in a Golgi pattern.

Glycan regulation of ER-associated degradation through compartmentalization.

The internal environment of the eukaryotic cell is divided by membranes into various organelles, containing diverse functional subcompartments, which allow complex cellular life. The quality control of newly made secretory proteins relies on the ability of the endoplasmic reticulum (ER) to segregate and compartmentalize molecules at different folding states. These folding states are communicated by N-glycans present on most secretory proteins. In ER-associated degradation (ERAD), protein molecules that have been identified as terminally misfolded are sent for degradation at the cytosolic proteasomes after being dislocated from the ER to the cytosol. This review will focus on how misfolded glycoprotein molecules are segregated from their properly folded counterparts and targeted to ERAD. The pathway involves compartmentalization, which is intimately linked to differential N-glycan processing. Recent data suggests that these processes are very dynamic, and include transient assembly of ERAD machinery complexes.

Compartmentalization of endoplasmic reticulum quality control and ER-associated degradation factors.

Recent studies are delineating a detailed picture of the architecture and function of the endoplasmic reticulum (ER) and the early secretory pathway, showing the existence of dynamic compartmentalization of ER quality control and ER-associated degradation (ERAD) factors. The compartmentalization is regulated by ER protein load and in turn regulates protein processing and cell fate. This compartmentalization is intimately linked to the protein quality control processes, protein disposal through ERAD, the unfolded protein response, and the initiation of apoptosis. It includes novel compartments, the ER-derived quality control compartment (ERQC), vesicles implicated in "ERAD-tuning," and the mitochondria-associated membranes (MAMs).