En Route to Targeted Ribosome Editing to Replenish Skin Anchor Protein LAMB3 in Junctional Epidermolysis Bullosa.

Severe junctional epidermolysis bullosa is a rare genetic, postpartum lethal skin disease, predominantly caused by nonsense/premature termination codon (PTC) sequence variants in LAMB3 gene. LAMB3 encodes LAMB3, the ß subunit of epidermal-dermal skin anchor laminin 332. Most translational reads of a PTC mRNA deliver truncated, nonfunctional proteins, whereas an endogenous PTC readthrough mechanism produces full-length protein at minimal and insufficient levels. Conventional translational readthrough-inducing drugs amplify endogenous PTC readthrough; however, translational readthrough-inducing drugs are either proteotoxic or nonselective. Ribosome editing is a more selective and less toxic strategy. This technique identified ribosomal protein L35/uL29 (ie, RpL35) and RpL35-ligands repurposable drugs artesunate and atazanavir as molecular tools to increase production levels of full-length LAMB3. To evaluate ligand activity in living cells, we monitored artesunate and atazanavir treatment by dual luciferase reporter assays. Production levels of full-length LAMB3 increased up to 200% upon artesunate treatment, up to 150% upon atazanavir treatment, and up to 170% upon combinatorial treatment of RpL35 ligands at reduced drug dosage, with an unrelated PTC reporter being nonresponsive. Proof of bioactivity of RpL35 ligands in selective increase of full-length LAMB3 provides the basis for an alternative, targeted therapeutic route to replenish LAMB3 in severe junctional epidermolysis bullosa.

Characterization of lipoprotein supplement and influence of its oxidized lipid content on cell culture performance and monoclonal antibody production by a SP2/0 hybridoma cell line.

A challenging aspect with the use of the Sp2/0 hybridoma cell line in commercial manufacturing processes of recombinant therapeutic proteins is their exogenous lipids requirement for cell proliferation and optimal protein secretion. Lipids are commonly provided to the culture using serum or serum-derivatives, such as lipoprotein supplement. The batch-to-batch variability of these non-chemically defined raw-materials is known to impact cell culture process performance. Lipoprotein supplement variability and its impact on fed-batch production of a recombinant monoclonal antibody (mAb) expressed in Sp2/0 cells were studied using 36 batches from the same vendor. Several batches were associated with early viability drops leading to low process performance during fed-batch production. Increased caspase-3 activity (an indicator of apoptosis) was correlated to viability drops when low-performing batches were used. Addition of an antioxidant to the culture limited the increase in caspase-3 activity. Physicochemical characterization of batches confirmed that lipoproteins are mainly composed of lipids and proteins; no clear correlation between low-performing batches and lipoprotein supplement composition was observed. Controlled lipoprotein oxidation leads to lipoprotein solution browning, increasing absorbance at 276 nm and results in poor process performance. Because low-performing batches absorb more at 276 nm than other batches, oxidized lipids were suspected to be the root cause of low-performing batches. This study increased the understanding of lipoprotein supplement composition, its sensitivity to oxidation and its impact on process performance.

Comparison Between Franz Diffusion Cell and a novel Micro-physiological System for In Vitro Penetration Assay Using Different Skin Models.

In vitro diffusive models are an important tool to screen the penetration ability of active ingredients in various formulations. A reliable assessment of skin penetration enhancing properties, mechanism of action of carrier systems, and an estimation of a bioavailability are essential for transdermal delivery. Given the importance of testing the penetration kinetics of different compounds across the skin barrier, several in vitro models have been developedThe aim of this study was to compare the Franz Diffusion Cell (FDC) with a novel fluid-dynamic platform (MIVO) by evaluating penetration ability of caffeine, a widely used reference substance, and LIP1, a testing molecule having the same molecular weight but a different lipophilicity in the two diffusion chamber systems. A 0.7% caffeine or LIP1 formulation in either water or propylene glycol (PG) containing oleic acid (OA) was topically applied on the Strat-M® membrane or pig ear skin, according to the infinite-dose experimental condition (780 ul/cm2). The profile of the penetration kinetics was determined by quantify the amount of molecule absorbed at different time-points (1, 2, 4, 6, 8 hours), by means of HPLC analysis. Both diffusive systems show a similar trend for caffeine and LIP1 penetration kinetics. The Strat-M® skin model shows a lower barrier function than the pig skin biopsies, whereby the PGOA vehicle exhibits a higher penetration, enhancing the effect for both diffusive chambers and skin surrogates. Most interestingly, MIVO diffusive system better predicts the lipophilic molecules (i.e. LIP1) permeation through highly physiological fluid flows resembled below the skin models.

Methoxy-Monobenzoylmethane Protects Skin from UV-Induced Damages in a Randomized, Placebo Controlled, Double-Blinded Human In Vivo Study and Prevents Signs of Inflammation While Improving the Skin Barrier.

INTRODUCTION: Sun protection is important in skin care and requires special attention as inefficient protection might trigger skin pathologies including polymorphic light eruption (PLE). The reduce-improve-protect (RIP) concept to avoid the onset of ultraviolet (UV) irradiation-induced diseases or damage to human skin is important. Methoxy-monobenzoylmethane (MeO-MBM), which is neither a UVB nor a UVA filter, converts to the UV filter avobenzone under UV irradiation and further acts as a photoantioxidant during its conversion process and initially as an antioxidant material. The aim of this study was to understand the mechanisms by which MeO-MBM improves the condition of UV-stressed skin through its photoantioxidant properties. The improvement of the skin condition by the activity of MeO-MBM as active ingredient was also investigated. METHODS: Potential molecular targets were identified by in silico docking to numerous cellular membrane receptors on the cell surface or nuclear membrane, followed by microarray analysis of 164 genes after MeO-MBM treatment of normal human epidermal keratinocytes (NHEK). We conducted randomized, double-blinded, intra-individual comparison vs. placebo studies on ten volunteers, aged between 34 and 65 years, to assess the effect of MeO-MBM in vivo. The effect after UV-induced inflammation was assessed in a protective and curative set-up with 2% MeO-MBM vs. 1% hydrocortisone and placebo based on the change in blood flow. The barrier function of the skin was assessed by the change in transepidermal water loss (TEWL), skin scaling and skin thickness after the treatment with MeO-MBM. Additionally, the effect of MeO-MBM after UV-induced stress on the activation of ferritin in human explants was determined ex vivo. RESULTS: A docking simulation of MeO-MBM showed a potential interaction with the retinoic acid receptor gamma and further revealed downregulation of proteins related to inflammation. In the protective treatment set-up, after 24 h MeO-MBM significantly reduced the delta blood flow compared to placebo, while this reduction was more prominent with hydrocortisone. In the curative treatment set-up, a greater reduction in delta blood flow was also observed with MeO-MBM compared to placebo and similar to hydrocortisone. Treatment with MeO-MBM revealed an improvement in skin barrier function as a result of decreased TEWL, reduced skin scaling and increased skin thickness. Immunohistochemistry staining of ferritin on human skin explants further showed that the treatment with MeO-MBM reduced the ferritin expression. CONCLUSION: Based on these results, MeO-MBM is capable of exerting an anti-aging activity via the retinoic acid receptor gamma. Its anti-inflammatory and anti-oxidative activity manifested via the downregulation of multiple anti-inflammatory genes as well as the reduction of ferritin in skin tissue. This study shows that the multidimensional functionality of MeO-MBM offers an effective approach to combat acute and chronic deleterious effects of oxidative UV damage while simultaneously enhancing the skin barrier function.

Activity-Guided Characterization of COX-2 Inhibitory Compounds in Waltheria indica L. Extracts.

Inflammation is the body's response to infection or tissue injury in order to restore and maintain homeostasis. Prostaglandin E2 (PGE-2) derived from arachidonic acid (AA), via up-regulation of cyclooxygenase-2 (COX-2), is a key mediator of inflammation and can also be induced by several other factors including stress, chromosomal aberration, or environmental factors. Targeting prostaglandin production by inhibiting COX-2 is hence relevant for the successful resolution of inflammation. Waltheria indica L. is a traditional medicinal plant whose extracts have demonstrated COX-2 inhibitory properties. However, the compounds responsible for the activity remained unknown. For the preparation of extracts with effective anti-inflammatory properties, characterization of these substances is vital. In this work, we aimed to address this issue by characterizing the substances responsible for the COX-2 inhibitory activity in the extracts and generating prediction models to quantify the COX-2 inhibitory activity without biological testing. For this purpose, an extract was separated into fractions by means of centrifugal partition chromatography (CPC). The inhibitory potential of the fractions and extracts against the COX-2 enzyme was determined using a fluorometric COX-2 inhibition assay. The characterizations of compounds in the fractions with the highest COX-2 inhibitory activity were conducted by high resolution mass spectrometry (HPLC-MS/MS). It was found that these fractions contain alpha-linolenic acid, linoleic acid and oleic acid, identified and reported for the first time in Waltheria indica leaf extracts. After analyzing their contents in different Waltheria indica extracts, it could be demonstrated that these fatty acids are responsible for up to 41% of the COX-2 inhibition observed with Waltheria indica extract. Additional quantification of secondary metabolites in the extract fractions revealed that substances from the group of steroidal saponins and triterpenoid saponins also contribute to the COX-2 inhibitory activity. Based on the content of compounds contributing to COX-2 inhibition, two mathematical models were successfully developed, both of which had a root mean square error (RMSE) = 1.6% COX-2 inhibitory activity, demonstrating a high correspondence between predicted versus observed values. The results of the predictive models further suggested that the compounds contribute to COX-2 inhibition in the order linoleic acid > alpha linolenic acid > steroidal saponins > triterpenoid saponins. The characterization of substances contributing to COX-2 inhibition in this study enables a more targeted development of extraction processes to obtain Waltheria indica extracts with superior anti-inflammatory properties.

Methoxy-Monobenzoylmethane Protects Human Skin against UV-Induced Damage by Conversion to Avobenzone and Radical Scavenging.

Avobenzone, one of the most commonly used UV filters in topical sunscreens, is susceptible to photodegradation with a consequential reduction of its UV absorbing properties. This loss of function may lead to skin irritation, photodermatosis, and photoallergic reactions caused by photodegradation byproducts. In this work, we aim to address this issue with a substance named methoxy-monobenzoylmethane (MeO-MBM), which is neither a UVB nor a UVA filter, but which converts to avobenzone, a known and approved UVA filter, under mainly UVB light irradiation. The antioxidant and intracellular radical formation properties of MeO-MBM were compared to the ones of avobenzone. The UV irradiation of MeO-MBM led to an increase in UV absorption primarily in the UVA range after conversion, both in vitro and in vivo. HPTLC and UHPLC studies illustrate the conversion of MeO-MBM to avobenzone in vitro after irradiation at 250 kJ/m2, reaching a conversion rate of 48.8%. A stable molecular antioxidant activity was observed, since 100-µM MeO-MBM was measured to be 11.2% in the DPPH assay, with a decrease to 9.7% after irradiation. In comparison, the molecular antioxidant activity of 100-µM avobenzone was determined to be 0.8%. In keratinocytes, MeO-MBM reduces the intracellular ROS by 90% and avobenzone by 75% with tBHP as the inducer and by 53% and 57%, respectively, when induced by pyocyanin, indicating the redox scavenging capacity of both these molecules. These results indicate that MeO-MBM functions initially as an antioxidant material and as a photoantioxidant during its conversion process to avobenzone. This research provides insight into the development of active ingredients for topical applications with dynamic functionalities. Using this approach, we demonstrate the possibility to extend the UV protection offered to skin cells while combating cellular stress in parallel.

A Cornflower Extract Containing N-Feruloylserotonin Reduces Inflammation in Human Skin by Neutralizing CCL17 and CCL22 and Inhibiting COX-2 and 5-LOX.

Thymus and Activation-Regulated Chemokine (TARC/CCL17) and Macrophage-Derived Chemokine (MDC/CCL22) are two key chemokines exerting their biological effect via binding and activating a common receptor CCR4, expressed at the surface of type 2 helper T (Th2) cells. By recruiting Th2 cells in the dermis, CCL17 and CCL22 promote the development of inflammation in atopic skin. The aim of this research was to develop a plant extract whose biological properties, when applied topically, could be beneficial for people with atopic-prone skin. The strategy which was followed consisted in identifying ligands able to neutralize the biological activity of CCL17 and CCL22. Thus, an in silico molecular modeling and a generic screening assay were developed to screen natural molecules binding and blocking these two chemokines. N-Feruloylserotonin was identified as a neutraligand of CCL22 in these experiments. A cornflower extract containing N-feruloylserotonin was selected for further in vitro tests: the gene expression modulation of inflammation biomarkers induced by CCL17 or CCL22 in the presence or absence of this extract was assessed in the HaCaT keratinocyte cell line. Additionally, the same cornflower extract in another vehicle was evaluated in parallel with N-feruloylserotonin for cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) enzymatic cellular inhibition. The cornflower extract was shown to neutralize the two chemokines in vitro, inhibited COX-2 and 5-LOX, and demonstrated anti-inflammatory activities due mainly to the presence of N-feruloylserotonin. Although these findings would need to be confirmed in an in vivo study, the in vitro studies lay the foundation to explain the benefits of the cornflower extract when applied topically to individuals with atopic-prone skin.

Characterization of soy protein hydrolysates and influence of its iron content on monoclonal antibody production by a murine hybridoma cell line.

A challenging aspect with the use of protein hydrolysates in commercial manufacturing processes of recombinant therapeutic proteins is their impacts on the protein production due to a lack of understanding of batch-to-batch variability. Soy hydrolysates variability and its impact on fed-batch production of a recombinant monoclonal antibody (mAb) expressed in Sp2/0 cells were studied using 37 batches from the same vendor. The batch-to-batch variability of soy hydrolysates impacted cell growth, titer and product quality. Physicochemical characterization of batches confirmed that soy hydrolysates are mainly a source of amino acids and peptides containing lower amounts of other components such as carbohydrates and chemical elements in cell culture media. Soy hydrolysates composition of different batches was consistent except for trace elements. Statistical analyses identified iron as a potential marker of a poor process performance. To verify this correlation, two forms of iron, ferric ammonium citrate and ferrous sulfate, were added to a batch of soy hydrolysates associated to a low level of iron during cell culture. Both forms of iron reduced significantly cell growth, mAb titer and increased level of the acidic charge variants of the mAb. Consequently, trace element composition of soy hydrolysates or of all incoming raw materials might lead to significant impacts on process performance and product quality and therefore need to be tightly controlled.

Review: High temperature short time treatment of cell culture media and feed solutions to mitigate adventitious viral contamination in the biopharmaceutical industry.

Events of viral contaminations occurring during the production of biopharmaceuticals have been publicly reported by the biopharmaceutical industry. Upstream raw materials were often identified as the potential source of contamination. Viral contamination risk can be mitigated by inactivating or eliminating potential viruses of cell culture media and feed solutions. Different methods can be used alone or in combination on raw materials, cell culture media, or feed solutions such as viral inactivation technologies consisting mainly of high temperature short time, ultraviolet irradiation, and gamma radiation technologies or such as viral removal technology for instance nanofiltration. The aim of this review is to present the principle, the advantages, and the challenges of high temperature short time (HTST) technology. Here, we reviewed effectiveness of HTST treatment and its impact on media (filterability of media, degradation of components), on process performance (cell growth, cell metabolism, productivity), and product quality based on knowledge shared in the literature.

Identification of plant metabolite classes from Waltheria Indica L. extracts regulating inflammatory immune responses via COX-2 inhibition.

ETHNOPHARMACOLOGICAL RELEVANCE: Waltheria Indica L. is traditionally used in Africa, South America and Hawaii to treat pain, anemia, diarrhea, epilepsy and inflammatory related diseases. AIM OF THE STUDY: This study aimed to identify extraction parameters to maximize tiliroside yield and to quantitative secondary metabolite composition of Waltheria Indica under various extraction conditions. The extracts were tested for COX-2 inhibition and their activity correlated with the type and quantity of the secondary metabolites. Insight was gained about how extraction parameters influence the extract composition and thus the COX-2 enzymatic inhibitory activity. MATERIALS AND METHODS: Powdered leaves of Waltheria Indica were extracted using water, methanol, ethyl acetate and ethanol at different temperatures. Tiliroside was identified by HPLC-HRMS n and quantified using a tiliroside standard. The compound groups of the secondary metabolites were quantified by spectrometric methods. Inhibitory potential of different Waltheria extracts against the COX-2 enzyme was determined using a fluorometric COX-2 inhibition assay. RESULTS: The molecule, tiliroside, exhibited a COX-2 inhibition of 10.4% starting at a concentration of 15 µM and increased in a dose dependent manner up to 51.2% at 150 µM. The ethanolic extract at 30 °C and the ethyl acetate extract at 90 °C inhibited COX-2 with 37.7% and 38.9%, while the methanolic and aqueous extract showed a lower inhibition of 21.9% and 9.2% respectively. The results concerning phenol, alkaloid and tiliroside concentration in the extracts showed no dependence on COX-2 inhibition. The extracts demonstrated a direct correlation of COX-2 inhibitory activity with their triterpenoid-/steroidal-saponin concentration. COX-2 inhibition increased linearly with the concentration of the saponins. CONCLUSION: The data suggest that Waltheria Indica extracts inhibit the key inflammatory enzyme, COX-2, as a function of triterpenoid- and steroidal-saponin concentration and support the known efficacy of extracted Waltheria Indica leaves as a traditional treatment against inflammation related diseases.

Improvement and simplification of fed-batch bioprocesses with a highly soluble phosphotyrosine sodium salt.

Fed-batch culture bioprocesses are currently used predominantly for the production of recombinant proteins, especially monoclonal antibodies. In these cultures, concentrated feeds are added during cultivation to prevent nutrient depletion, thus extending the cellular growth phase and increasing product concentrations. One limitation in these bioprocesses arises from the low solubility or stability of some compounds at high concentrations, in particular amino acids. This study describes the synthesis and evaluation of a phosphotyrosine disodium salt as a tyrosine source in fed-batch processes. This molecule is highly soluble in concentrated feeds at neutral pH. Mechanistic studies demonstrated that the molecule is cleaved in the cell culture supernatant after processing by released phosphatases, leading to phosphate and free L-tyrosine which can be taken up by the cells. No intact phosphotyrosine was detected intracellularly or incorporated into the sequence of the monoclonal antibody. The use of this new molecule allows the simplification of fed-batch processes in large scale manufacturing via the implementation of neutral pH, highly concentrated feeds.

Cellular fractionation--mammalian cells.

In proteomics research, one essential step among enrichment techniques is subcellular fractionation. This is of special importance for analyzing intracellular organelles and multiprotein complexes. Subcellular fractionation is a flexible and adjustable approach to reducing sample complexity and is most efficiently combined with high-resolution 2-D gel/mass spectrometry analysis as well as with gel-independent techniques.

Cellular fractionation--yeast cells.

In proteomics research, one essential step among enrichment techniques is subcellular fractionation. This is of special importance for analyzing intracellular organelles and multiprotein complexes. Subcellular fractionation is a flexible and adjustable approach to reducing sample complexity and is most efficiently combined with high-resolution 2-D gel/mass spectrometry analysis as well as with gel-independent techniques.