Native collagen sheet mask improves skin health and appearance: A comprehensive clinical evaluation.

BACKGROUND: Collagen, a critical structural protein found abundantly in animal skin and bones, has become increasingly recognized for its potential therapeutic role in skincare. Despite growing interest, the scientific evidence for the efficacy of collagen sheet masks remains limited. The principal objective of our study was to provide insights into the multifaceted role of collagen in skin health, with a specific focus on its application in collagen sheet masks. METHODS: The effects of a collagen sheet mask consisting of >92% native bovine collagen were investigated. The soluble protein components of the collagen matrix were analyzed and the influence of soluble collagen components on fibroblast regulation was examined. Scanning Electron Microscope (SEM) analysis was performed for structural analysis and effect on irritated skin. Five different clinical studies were conducted, including a comparison of the diversity of the skin microbiome, the tolerance and local irritating reactions in atopic dermatitis, an evaluation of skin redness after UV radiation, wrinkle reduction, and hydration and skin roughness of the collagen mask in comparison to a pre-soaked cellulose sheet mask. RESULTS: The collagen mask contains soluble protein components, including small collagen peptides. The mask showed potential for promoting fibroblast activity. SEM analysis showed a native collagen structure similar to human dermis. The mask maintained the skin microbiome diversity and decreased skin pH levels. It demonstrated good tolerability on both intact and lesional skin and had a significant effect in reducing erythema caused by UV radiation compared to other skincare products. It showed significant improvements in skin hydration and the volume of eye wrinkles and was more effective than pre-soaked cellulose sheet masks. CONCLUSION: Collagen sheet masks have the potential to positively impact skin health and appearance by increasing hydration, reducing erythema, minimizing wrinkles, and maintaining a healthy skin microbiome and skin barrier.

The glycyl-radical enzyme 2-ketobutyrate formate-lyase, TdcE, interacts specifically with the formate-translocating FNT-channel protein FocA.

Formate is a major product of mixed-acid fermentation in Escherichia coli. Because formate can act as an uncoupler at high concentration it must be excreted from the cell. The FNT (formate-nitrite transporter) membrane channel FocA ensures formate is translocated across the cytoplasmic membrane. Two glycyl-radical enzymes (GREs), pyruvate formate-lyase (PflB) and 2-ketobutyrate formate-lyase (TdcE), generate formate as a product of catalysis during anaerobic growth of Escherichia coli. We demonstrate in this study that TdcE, like PflB, interacts specifically with FocA. His-tagged variants of two other predicted GREs encoded in the genome of E. coli were over-produced and purified and were shown not to interact with FocA, indicating that interaction with FocA is not a general property of GREs per se. Together, these data show that only the GREs TdcE and PflB interact with the FNT channel protein and suggest that, like PflB, TdcE can control formate translocation by FocA.

Pyruvate formate-lyase interacts directly with the formate channel FocA to regulate formate translocation.

The FNT (formate-nitrite transporters) form a superfamily of pentameric membrane channels that translocate monovalent anions across biological membranes. FocA (formate channel A) translocates formate bidirectionally but the mechanism underlying how translocation of formate is controlled and what governs substrate specificity remains unclear. Here we demonstrate that the normally soluble dimeric enzyme pyruvate formate-lyase (PflB), which is responsible for intracellular formate generation in enterobacteria and other microbes, interacts specifically with FocA. Association of PflB with the cytoplasmic membrane was shown to be FocA dependent and purified, Strep-tagged FocA specifically retrieved PflB from Escherichia coli crude extracts. Using a bacterial two-hybrid system, it could be shown that the N-terminus of FocA and the central domain of PflB were involved in the interaction. This finding was confirmed by chemical cross-linking experiments. Using constraints imposed by the amino acid residues identified in the cross-linking study, we provide for the first time a model for the FocA-PflB complex. The model suggests that the N-terminus of FocA is important for interaction with PflB. An in vivo assay developed to monitor changes in formate levels in the cytoplasm revealed the importance of the interaction with PflB for optimal translocation of formate by FocA. This system represents a paradigm for the control of activity of FNT channel proteins.

Identification of key residues in the formate channel FocA that control import and export of formate.

The formate-nitrite transporter (FNT) family comprises pentameric channels that transport monovalent anions. The prototype of this family is the formate channel (FocA), which was originally identified as a formate channel in Escherichia coli. Each protomer in the channel has a pore with structural features that include periplasmic and cytoplasmic constriction sites, which are likely important for bi-directional gating of substrate passage. Highly conserved amino acid residues within FocA previously identified in structural studies are predicted to be important in the control of formate translocation. Here we present a first detailed in vivo analysis of these residues using a combined targeted amino acid exchange and formate-responsive lacZ fusion-based reporter approach. Sixteen exchanges were made and each variant was shown to be largely unaffected in its secondary and quaternary structure. The invariant H209 and T91 residues, which form part of the lower constriction site linking the Ω-loop with the pore cavity, proved to be important in governing the directionality of formate passage through the pore. A predicted salt-bridge triad of E208-K156-N213 along with the cytoplasmically-oriented N-terminal helix are also involved in pH-dependent gating of the channel. Together, our data are consistent with passive export and import of formate or formic acid through the channel.

Coordination of FocA and pyruvate formate-lyase synthesis in Escherichia coli demonstrates preferential translocation of formate over other mixed-acid fermentation products.

Enterobacteria such as Escherichia coli generate formate, lactate, acetate, and succinate as major acidic fermentation products. Accumulation of these products in the cytoplasm would lead to uncoupling of the membrane potential, and therefore they must be either metabolized rapidly or exported from the cell. E. coli has three membrane-localized formate dehydrogenases (FDHs) that oxidize formate. Two of these have their respective active sites facing the periplasm, and the other is in the cytoplasm. The bidirectional FocA channel translocates formate across the membrane delivering substrate to these FDHs. FocA synthesis is tightly coupled to synthesis of pyruvate formate-lyase (PflB), which generates formate. In this study, we analyze the consequences on the fermentation product spectrum of altering FocA levels, uncoupling FocA from PflB synthesis or blocking formate metabolism. Changing the focA translation initiation codon from GUG to AUG resulted in a 20-fold increase in FocA during fermentation and an ∼3-fold increase in PflB. Nevertheless, the fermentation product spectrum throughout the growth phase remained similar to that of the wild type. Formate, acetate, and succinate were exported, but only formate was reimported by these cells. Lactate accumulated in the growth medium only in mutants lacking FocA, despite retaining active PflB, or when formate could not be metabolized intracellularly. Together, these results indicate that FocA has a strong preference for formate as a substrate in vivo and not other acidic fermentation products. The tight coupling between FocA and PflB synthesis ensures adequate substrate delivery to the appropriate FDH.

Unexpected oligomeric structure of the FocA formate channel of Escherichia coli : a paradigm for the formate-nitrite transporter family of integral membrane proteins.

FocA is a predicted formate channel with a deduced mass of 31 kDa that catalyzes the bidirectional movement of formate across the cytoplasmic membrane of Escherichia coli and is the archetype of the formate-nitrite transporter (FNT) family. Overproduced FocA variants with either an N- or a C-terminal Strep-tag increased formate import into anaerobic E. coli cells as determined by the enhanced activity of a single-copy formate-dependent fdhF::lacZ fusion. Using anti-FocA antibodies, we could show that both FocA variants were integrated into the cytoplasmic membrane. Circular dichroism spectroscopy of purified FocA(Strep-N) revealed a high alpha-helical content of 56% consistent with the predicted six transmembrane helices present in the protein. Analysis of the oligomeric state by blue-native polyacrylamide gel electrophoresis revealed FocA to have an unexpected pentameric quaternary structure. This study reports the first isolation of an FNT family member.

Volume flow rate evaluation in patients with obstructive arteriosclerotic disease.

This study was performed to quantify the volumetric impact of extracranial arteriosclerotic lesions. We investigated patients with different degrees of carotid stenosis as defined by conventional velocity-based duplex criteria and different patterns of collateralization. We studied the volume flow rate (color duplex M-mode device) in 37 patients with symmetrical internal carotid artery (ICA) plaques (<50% stenosis) and compared these data to 43 patients with a unilateral 50-74% stenosis, 75-99% stenosis (n = 73) and occlusion of the ICA (n = 37). A 75-99% stenosis caused an ipsilateral flow reduction of 36% with a wide interindividual variability, and an occlusion of 51%. Collateralization via the ophthalmic artery only caused a nonsignificant ipsilateral volume flow increase, whereas the contralateral volume flow was significantly higher in patients with a patent anterior communicating artery. The increase was more pronounced in patients with an occluded vessel. Our data demonstrate a wide diversity of the hemodynamic impact of carotid stenosis as defined by 'classical' Doppler criteria. Collateral flow reflects a dynamic quantitative process.