Translocation Pathway Research Articles

A proton-coupled folate transporter mutation causing hereditary folate malabsorption locks the protein in an inward-open conformation

The proton-coupled folate transporter (PCFT, SLC46A1) is required for folate intestinal absorption and transport across the choroid plexus. Recent work has identified a F392V mutation causing hereditary folate malabsorption. However, the residue properties responsible for this loss of function remains unknown. Using site-directed mutagenesis, we observed complete loss of function with charged (Lys, Asp, and Glu) and polar (Thr, Ser, and Gln) Phe-392 substitutions and minimal function with some neutral substitutions; however, F392M retained full function. Using the substituted-cysteine accessibility method (with N-biotinyl aminoethyl methanethiosulfonate labeling), Phe-392 mutations causing loss of function, although preserving membrane expression and trafficking, also resulted in loss of accessibility of the substituted cysteine in P314C-PCFT located within the aqueous translocation pathway. F392V function and accessibility of the P314C cysteine were restored by insertion of a G305L (suppressor) mutation. A S196L mutation localized in proximity to Gly-305 by homology modeling was inactive. However, when inserted into the inactive F392V scaffold, function was restored (mutually compensatory mutations), as was accessibility of the P314C cysteine residue. Reduced function, documented with F392H PCFT, was due to a 15-fold decrease in methotrexate influx Vmax, accompanied by a decreased influx Kt (4.5-fold) and Ki (3-fold). The data indicate that Phe-392 is required for rapid oscillation of the carrier among its conformational states and suggest that this is achieved by dampening affinity of the protein for its folate substrates. F392V and other inactivating Phe-392 PCFT mutations lock the protein in its inward-open conformation. Reach (length) and hydrophobicity of Phe-392 appear to be features required for full activity.

The critical role of piperamide derivative D4 in the regulation of inflammatory response by the microglia and astrocytic glial cells

Acute and chronic inflammation in the central nervous system plays a critical role in the development of neurodegenerative disorders. Various pro-inflammatory cytokines, chemokines, and enzymes such as TNF-α, IL1-β, IL-6, COX-1, COX-2, iNOS, IKK, and inducible nitric oxide are expressed in several signalling pathways, and mediate the neuroinflammatory process. ROS and NF-kB nuclear translocation are the two fundamental pathways involved in neuroinflammatory pathogenesis in neuronal and glial cells. In recent years several compoundswere designed to affect the neuroinflammation and suppress neurodegenerative process. Derivatives of natural products (NPs) attract the most attention of drug developers and industries due to their safety and lesser side effects in comparison with generic drugs. One of the most well-known NP is piperine, which is a yellow crystalline alkaloid extracted from black and white pepper. Recently, we developed a novel piperine derivative (((2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)-N-(4-(hydroxymethyl)phenyl)penta-2,4-dienamide, D4) to enhance the specificity and efficacy of the base molecule. Next, we evaluated the potential anti-inflammatory properities of D4 in CHME3 and SVG cell-lines corresponding to human microglia and astrocytes, respectively. Our results indicated that D4 inhibited NF-kB translocation pathway, and significantly reduced transcript and protein levels of pro-inflammatory cytokines in comparison with Aspirin, as a well-known non-selective NSAID. Furthermore, in silico study showed excellent D4 bioavailability in oral administration. The results of the present study suggest a novel molecule with high anti-neuroinflammatory potency for further pre-clinical tests and pharmacological drug investigation.

Structural and functional diversity calls for a new classification of ABC transporters.

Members of the ATP-binding cassette (ABC) transporter superfamily translocate a broad spectrum of chemically diverse substrates. While their eponymous ATP-binding cassette in the nucleotide-binding domains (NBDs) is highly conserved, their transmembrane domains (TMDs) forming the translocation pathway exhibit distinct folds and topologies, suggesting that during evolution the ancient motor domains were combined with different transmembrane mechanical systems to orchestrate a variety of cellular processes. In recent years, it has become increasingly evident that the distinct TMD folds are best suited to categorize the multitude of ABC transporters. We therefore propose a new ABC transporter classification that is based on structural homology in the TMDs.

Cannabidiol interactions with voltage-gated sodium channels.

Voltage-gated sodium channels are targets for a range of pharmaceutical drugs developed for the treatment of neurological diseases. Cannabidiol (CBD), the non-psychoactive compound isolated from cannabis plants, was recently approved for treatment of two types of epilepsy associated with sodium channel mutations. This study used high-resolution X-ray crystallography to demonstrate the detailed nature of the interactions between CBD and the NavMs voltage-gated sodium channel, and electrophysiology to show the functional effects of binding CBD to these channels. CBD binds at a novel site at the interface of the fenestrations and the central hydrophobic cavity of the channel. Binding at this site blocks the transmembrane-spanning sodium ion translocation pathway, providing a molecular mechanism for channel inhibition. Modelling studies suggest why the closely-related psychoactive compound tetrahydrocannabinol may not have the same effects on these channels. Finally, comparisons are made with the TRPV2 channel, also recently proposed as a target site for CBD. In summary, this study provides novel insight into a possible mechanism for CBD interactions with sodium channels.

The Twin-Arginine Translocation System Is Important for Stress Resistance and Virulence of Brucella melitensis.

Brucella, the causative agent of brucellosis, is a stealthy intracellular pathogen that is highly pathogenic to a range of mammals, including humans. The twin-arginine translocation (Tat) pathway transports folded proteins across the cytoplasmic membrane and has been implicated in virulence in many bacterial pathogens. However, the roles of the Tat system and related substrates in Brucella remain unclear. We report here that disruption of Tat increases the sensitivity of Brucella melitensis M28 to the membrane stressor sodium dodecyl sulfate (SDS), indicating cell envelope defects, as well as to EDTA. In addition, mutating Tat renders M28 bacteria more sensitive to oxidative stress caused by H2O2 Further, loss of Tat significantly attenuates B. melitensis infection in murine macrophages ex vivo Using a mouse model for persistent infection, we demonstrate that Tat is required for full virulence of B. melitensis M28. Genome-wide in silico prediction combined with an in vivo amidase reporter assay indicates that at least 23 proteins are authentic Tat substrates, and they are functionally categorized into solute-binding proteins, oxidoreductases, cell envelope biosynthesis enzymes, and others. A comprehensive deletion study revealed that 6 substrates contribute significantly to Brucella virulence, including an l,d-transpeptidase, an ABC transporter solute-binding protein, and a methionine sulfoxide reductase. Collectively, our work establishes that the Tat pathway plays a critical role in Brucella virulence.

Genomic insights into cyanobacterial protein translocation systems.

Cyanobacteria are ubiquitous oxygenic photosynthetic bacteria with a versatile metabolism that is highly dependent on effective protein targeting. Protein sorting in diderm bacteria is not trivial and, in cyanobacteria, even less so due to the presence of a complex membrane system: the outer membrane, the plasma membrane and the thylakoid membrane. In cyanobacteria, protein import into the thylakoids is essential for photosynthesis, export to the periplasm fulfills a multifunctional role in maintaining cell homeostasis, and secretion mediates motility, DNA uptake and environmental interactions. Intriguingly, only one set of genes for the general secretory and the twin-arginine translocation pathways seem to be present. However, these systems have to operate in both plasma and thylakoid membranes. This raises the question of how substrates are recognized and targeted to their correct, final destination. Additional complexities arise when a protein has to be secreted across the outer membrane, where very little is known regarding the mechanisms involved. Given their ecological importance and biotechnological interest, a better understanding of protein targeting in cyanobacteria is of great value. This review will provide insights into the known knowns of protein targeting, propose hypotheses based on available genomic sequences and discuss future directions.

Bioreactor-scale cell performance and protein production can be substantially increased by using a secretion signal that drives co-translational translocation in Pichia pastoris

Pichia pastoris (Komagataella spp.) has become one of the most important host organisms for production of heterologous proteins of biotechnological interest, many of them extracellular. The protein secretion pathway has been recognized as a limiting process in which many roadblocks have been pinpointed. Recently, we have identified a bottleneck at the ER translocation level. In earlier exploratory studies, this limitation could be largely overcome by using an improved chimeric secretion signal to drive proteins through the co-translational translocation pathway. Here, we have further tested at bioreactor scale the improved secretion signal consisting of the pre-Ost1 signal sequence, which drives proteins through co-translational translocation, followed by the pro region from the secretion signal of the Saccharomyces cerevisiae α-factor mating pheromone. For comparison, the commonly used full-length α-factor secretion signal, which drives proteins through post-translational translocation, was tested. These two secretion signals were fused to three different model proteins: the tetrameric red fluorescent protein E2-Crimson, which can be used to visualize roadblocks in the secretory pathway; the lipase 2 from Bacillus thermocatenulatus (BTL2); and the Rhizopus oryzae lipase (ROL). All strains were tested in batch cultivation to study the different growth parameters obtained. The strains carrying the improved secretion signal showed increased final production of the proteins of interest. Interestingly, they were able to grow at significantly higher maximum specific growth rates than their counterparts carrying the conventional secretion signal. These results were corroborated in a 5 L fed-batch cultivation, where the final product concentration and volumetric productivity were also shown to be improved.

Tripartite transporters as mechanotransmitters in periplasmic alternating-access mechanisms.

To remove xenobiotics from the periplasmic space, Gram-negative bacteria utilise unique tripartite efflux systems in which a molecular engine in the plasma membrane connects to periplasmic and outer membrane subunits. Substrates bind to periplasmic sections of the engine or sometimes to the periplasmic subunits. Then, the tripartite machines undergo conformational changes that allow the movement of the substrates down the substrate translocation pathway to the outside of the cell. The transmembrane (TM) domains of the tripartite resistance-nodulation-drug-resistance (RND) transporters drive these conformational changes by converting proton motive force into mechanical motion. Similarly, the TM domains of tripartite ATP-binding cassette (ABC) transporters transmit mechanical movement associated with nucleotide binding and hydrolysis at the nucleotide-binding domains to the relevant subunits in the periplasm. In this way, metabolic energy is coupled to periplasmic alternating-access mechanisms to achieve substrate transport across the outer membrane.

Cryo-EM structure of the entire mammalian F-type ATP synthase.

The majority of adenosine triphosphate (ATP) powering cellular processes in eukaryotes is produced by the mitochondrial F1Fo ATP synthase. Here, we present the atomic models of the membrane Fo domain and the entire mammalian (ovine) F1Fo, determined by cryo-electron microscopy. Subunits in the membrane domain are arranged in the 'proton translocation cluster' attached to the c-ring and a more distant 'hook apparatus' holding subunit e. Unexpectedly, this subunit is anchored to a lipid 'plug' capping the c-ring. We present a detailed proton translocation pathway in mammalian Fo and key inter-monomer contacts in F1Fo multimers. Cryo-EM maps of F1Fo exposed to calcium reveal a retracted subunit e and a disassembled c-ring, suggesting permeability transition pore opening. We propose a model for the permeability transition pore opening, whereby subunit e pulls the lipid plug out of the c-ring. Our structure will allow the design of drugs for many emerging applications in medicine.

PAH bioaccumulation in two polluted sites along the eastern coast of the Red Sea, Saudi Arabia

Significant amounts of polycyclic aromatic hydrocarbons (PAHs) accumulate in specific habitats and organisms, such as mangroves and benthic organisms, due to the PAHs’ direct attachment to sediments in the environment or the feeding activity of the organisms, especially in contaminated coastal areas. The present study investigates the effect of sewage effluents on the bioaccumulation of PAHs in two polluted sites along the coastal area of Jeddah city. PAH levels in the mangrove sediments, roots, and leaves ranged between 230.09 and 17.94, 32.79 and 409.86, and 111.14 and 795.2 ng/g, respectively, following the general trend of sediments < roots < leaves. Though all mangrove roots showed bioaccumulation factors (BAFroots) higher than 1, the leaves bioaccumulation factors (BAFleaves) exceeded 26, clearly indicating PAHs accumulated not only via a translocation pathway but also from the surrounding atmosphere. These results suggest that mangroves could serve as an in situ eco-friendly resource for the dissipation of PAHs in marine sediments and the atmosphere. Although higher levels of PAHs were detected in Al-Arbaeen Lagoon sediments (ranging between 89.18 and 1056.46 ng/g), lower levels of PAHs were observed in the gastropod Littorina Littorea (range 113.21–265.96 ng/g) in this area, with biota-sediment accumulation factors (BSAF) ranging from 0.23 to 2.10. Only one Al-Arbaeen site presented sediments with a petrogenic origin, with a BSAF value higher than 1 in the gastropod. Based on sediment quality guidelines, adverse biological effects should rarely occur at the studied sites under the present PAH levels.

Plastid chaperone HSP90C guides precursor proteins to the SEC translocase for thylakoid transport.

Chloroplast stromal factors involved in regulating thylakoid protein targeting are poorly understood. We previously reported that in Arabidopsis thaliana, the stromal-localized chaperone HSP90C (plastid heat shock protein 90) interacted with the nuclear-encoded thylakoid lumen protein PsbO1 (PSII subunit O isoform 1) and suggested a role for HSP90C in aiding PsbO1 thylakoid targeting. Using in organello transport assays, particularly with model substrates naturally expressed in stroma, we showed that light, exogenous ATP, and HSP90C activity were required for Sec-dependent transport of green fluorescent protein (GFP) led by the PsbO1 thylakoid targeting sequence. Using a previously identified PsbO1T200A mutant, we provided evidence that a stronger interaction between HSP90C and PsbO1 better facilitated its stroma-thylakoid trafficking. We also demonstrated that SecY1, the channel protein of the thylakoid SEC translocase, specifically interacted with HSP90C in vivo. Inhibition of the chaperone ATPase activity suppressed the association of the PsbO1GFP-HSP90C complex with SecY1. Together with analyzing the expression and accumulation of a few other thylakoid proteins that utilize the SRP, TAT, or SEC translocation pathways, we propose a model in which HSP90C forms a guiding complex that interacts with thylakoid protein precursors and assists in their specific targeting to the thylakoid SEC translocon.

Dual targeting of TatA points to a chloroplast-like Tat pathway in plant mitochondria

The biogenesis of membrane-bound electron transport chains requires membrane translocation pathways for folded proteins carrying complex cofactors, like the Rieske Fe/S proteins. Two independent systems were developed during evolution, namely the Twin-arginine translocation (Tat) pathway, which is present in bacteria and chloroplasts, and the Bcs1 pathway found in mitochondria of yeast and mammals. Mitochondria of plants carry a Tat-like pathway which was hypothesized to operate with only two subunits, a TatB-like protein and a TatC homolog (OrfX), but lacking TatA. Here we show that the nuclearly encoded TatA from pea has dual targeting properties, i.e., it can be imported into both, chloroplasts and mitochondria. Dual targeting of TatA was observed with in organello experiments employing chloroplasts and mitochondria isolated from pea as well as after transient expression of suitable reporter constructs in leaf tissue from pea and Nicotiana benthamiana. The extent of transport of these constructs into mitochondria of transiently transformed leaf cells was relatively low, causing a demand for highly sensitive methods to be detected, like the sasplitGFP approach. Yet, the dual import of TatA into mitochondria and chloroplasts observed here points to a common mechanism of Tat transport for folded proteins within both endosymbiotic organelles in plants.

Structure and mechanism of the Mrp complex, an ancient cation/proton antiporter.

Multiple resistance and pH adaptation (Mrp) antiporters are multi-subunit Na+ (or K+)/H+ exchangers representing an ancestor of many essential redox-driven proton pumps, such as respiratory complex I. The mechanism of coupling between ion or electron transfer and proton translocation in this large protein family is unknown. Here, we present the structure of the Mrp complex from Anoxybacillus flavithermus solved by cryo-EM at 3.0 Å resolution. It is a dimer of seven-subunit protomers with 50 trans-membrane helices each. Surface charge distribution within each monomer is remarkably asymmetric, revealing probable proton and sodium translocation pathways. On the basis of the structure we propose a mechanism where the coupling between sodium and proton translocation is facilitated by a series of electrostatic interactions between a cation and key charged residues. This mechanism is likely to be applicable to the entire family of redox proton pumps, where electron transfer to substrates replaces cation movements.

Partitioning, Translocation Pathways and Environmental Risk Evaluation of Selected Polychlorinated Biphenyls and Pesticides

Degradation-resistant chemical contaminants of health concern such as dichlorodiphenyltrichloroethane (DDT) pesticides and polychlorinated biphenyls (PCBs) in the environment are redistributed between different environmental compartments, where they partition between biotic heterotrophic routes and abiotic features (water and immobile soil components). Their fate and the potential risk they pose is a function of translocation, interaction, environmental behavior, and bio-translation/activities. In this study, the partitioning, translocation, bio-translation pathways of 3-DDT and 6-PCB congeners in dosed soils cultured root and leaf vegetables were investigated to predict their soil–vegetable mobility, depuration, and exposure risk. Results showed that PCB_110 and PCB_180 were the least and highest PCBs concentrated in both the leaf and root vegetables. The variations in the 3-DDT and 6-PCB concentrations in the leaf and root vegetables may be attributed to differences in their solubility and partitioning. Total residual mass fraction 3-DDT taken up by the leaf and root vegetables indicated time-dependent preferentiality in pesticide-type vascular tissue translocation to the vegetables. Mann–Whitney non-parametric test showed evidence of spatial variations in levels of the 3-DDT and 6-PCBs across the farmland; however, the variations in the distribution were not significant (PFML1–FML6 <<Pstat). Risk factors for 3-PCBs and 3-DDT tend to 1(f < 1.0), while that for PCBs_149, 153, and _180 were far less than 0.4 (<1.0). It is therefore uncertain that any immediate health risks could arise from exposure to vegetables containing such congener levels. Continuous exposure to an extrapolated estimate of 25% vegetable population has a 20 ± 10% probability of leading to undesirable chronic effects.

Tidal Freshwater Zones as Hotspots for Biogeochemical Cycling: Sediment Organic Matter Decomposition in the Lower Reaches of Two South Texas Rivers

While organic and inorganic nutrient inputs from land are recognized as a major driver of primary production in estuaries, remarkably little is known about how processes within the tidal freshwater zones (TFZs) of rivers modify these inputs. This study quantifies organic matter (OM) decomposition rates in surface sediment layers in the lower reaches of two south Texas river channels and identifies key parameters that influence sediment decomposition rates. Sediment cores were collected from non-tidal and tidal freshwater sites in the Mission and Aransas rivers during two summers (June 2015 and June 2016) and two winters (February 2016, January 2017). We measured oxygen consumption rates, organic carbon and nitrogen content, stable isotope ratios (δ13C and δ15N of OM), and sediment porosity. O2 consumption rates in TFZ sediments were 385 ± 88 μmol O2 m−2 h−1 (summer) and 349 ± 87 μmol O2 m−2 h−1 (winter) in the Aransas River and 767 ± 153 μmol O2 m−2 h−1 (summer) and 691 ± 95 μmol O2 m−2 h−1 (winter) in the Mission River. These rates in TFZs were similar to rates in estuaries and higher than rates at non-tidal riverine sites. Rates of sediment O2 consumption were primarily controlled by OM content and temperature. Sediment OM was dominated by algal biomass from in situ production in both TFZs. We hypothesize that algal production and sinking within TFZs is a major pathway for translocation of watershed-derived nutrients from the water column to the sediments within TFZs. Further work is needed to quantify linkages between decomposition, nutrient remineralization, and potential removal through processes such as denitrification.

Biogenesis of Mitochondrial Metabolite Carriers.

Metabolite carriers of the mitochondrial inner membrane are crucial for cellular physiology since mitochondria contribute essential metabolic reactions and synthesize the majority of the cellular ATP. Like almost all mitochondrial proteins, carriers have to be imported into mitochondria from the cytosol. Carrier precursors utilize a specialized translocation pathway dedicated to the biogenesis of carriers and related proteins, the carrier translocase of the inner membrane (TIM22) pathway. After recognition and import through the mitochondrial outer membrane via the translocase of the outer membrane (TOM) complex, carrier precursors are ushered through the intermembrane space by hexameric TIM chaperones and ultimately integrated into the inner membrane by the TIM22 carrier translocase. Recent advances have shed light on the mechanisms of TOM translocase and TIM chaperone function, uncovered an unexpected versatility of the machineries, and revealed novel components and functional crosstalk of the human TIM22 translocase.

Transcriptional Modulation of Resistance against Xanthomonas oryzae pv. oryzae Korean Race K2 in japonica Rice

Bacterial blight is a common disease found in the rice-growing regions in the Korean peninsula. Identification of the gene network involved against Xanthomonas oryzae pv. oryzae Korean race K2 in popular japonica cultivars is essential in underpinning the molecular mechanism of resistance. A microarray of two popular Korean japonica rice cultivars, a bacterial blight susceptible Dongjin and resistant Jinbaek, was performed to investigate the transcripts of inducible genes at 48 h post-inoculation. A total of 771 differentially expressed genes were identified in Jinbaek, whereas 298 were found in Dongjin. The resistance observed in Jinbaek is likely participated by genes with predicted functions in transmembrane perception, intracellular signal transduction, and transcription activity. Moreover, the remarkable involvement of numerous WRKY proteins signifies orchestration of defense signals via robust cellular reprogramming, which leads to resistance. To discover genes essential to bacterial blight resistance in Jinbaek, 13 highly expressed genes encoding different protein classes were cloned and overexpressed in rice. Although none of the overexpression plants exhibited resistance comparable to Jinbaek, four candidate genes, including one twin-arginine translocation pathway signal (LOC_Os01g45640.1), one cytochrome p450 (LOC_Os09g10340.1), and two uncharacterized expressed protein (LOC_Os08g26230.4, LOC_Os09g04310.1) conferred partial resistance. However, of these four genes, only p450s have been reported to play an important role in the synthesis of plant defense compounds. These findings revealed the complexity of key immune signaling conduits critical to mounting a full defense against Xanthomonas. oryzae pv. oryzae race K2 in japonica rice.

Jejunal villus absorption and paracellular tight junction permeability are major routes for early intestinal uptake of food-grade TiO2 particles: an in vivo and ex vivo study in mice

BackgroundFood-grade TiO2 (E171 in the EU) is widely used as a coloring agent in foodstuffs, including sweets. Chronic dietary exposure raises concerns for human health due to proinflammatory properties and the ability to induce and promote preneoplastic lesions in the rodent gut. Characterization of intestinal TiO2 uptake is essential for assessing the health risk in humans. We studied in vivo the gut absorption kinetics of TiO2 in fasted mice orally given a single dose (40 mg/kg) to assess the ability of intestinal apical surfaces to absorb particles when available without entrapment in the bolus. The epithelial translocation pathways were also identified ex vivo using intestinal loops in anesthetized mice.ResultsThe absorption of TiO2 particles was analyzed in gut tissues by laser-reflective confocal microscopy and ICP-MS at 4 and 8 h following oral administration. A bimodal pattern was detected in the small intestine: TiO2 absorption peaked at 4 h in jejunal and ileal villi before returning to basal levels at 8 h, while being undetectable at 4 h but significantly present at 8 h in the jejunal Peyer’s patches (PP). Lower absorption occurred in the colon, while TiO2 particles were clearly detectable by confocal microscopy in the blood at 4 and 8 h after treatment. Ex vivo, jejunal loops were exposed to the food additive in the presence and absence of pharmacological inhibitors of paracellular tight junction (TJ) permeability or of transcellular (endocytic) passage. Thirty minutes after E171 addition, TiO2 absorption by the jejunal villi was decreased by 66% (p < 0.001 vs. control) in the presence of the paracellular permeability blocker triaminopyrimidine; the other inhibitors had no significant effect. Substantial absorption through a goblet cell (GC)-associated pathway, insensitive to TJ blockade, was also detected.ConclusionsAfter a single E171 dose in mice, early intestinal uptake of TiO2 particles mainly occurred through the villi of the small intestine, which, in contrast to the PP, represent the main absorption surface in the small intestine. A GC-associated passage and passive diffusion through paracellular TJ spaces between enterocytes appeared to be major absorption routes for transepithelial uptake of dietary TiO2.

Cell Membrane Penetration without Pore Formation: Chameleonic Properties of Dendrimers in Response to Hydrophobic and Hydrophilic Environments

AbstractThe mechanism by which cell‐penetrating peptides and antimicrobial peptides cross plasma membranes is unknown, as is how cell‐penetrating peptides facilitate drug delivery, mediating the transport of small molecules. Once nondisruptive and nonendocytotic pathways are excluded, pore formation is one of the proposed mechanisms, including toroidal, barrel‐stave, or carpet models. Spontaneous pores are observed in coarse‐grained simulations and less often in molecular dynamics simulations. While pores are widely assumed and inferred, there is no unambiguous experimental evidence of the existence of pores. Some recent experimental studies contradict the mechanistic picture of pore formation, however, highlighting the possibility of a direct translocation pathway that is both nondisruptive and nonendocytotic.In this work, a model is proposed a model for peptide (linear and dendritic) translocation which does not require the presence of pores and which potentially accords with such experiments. It is suggested that a charged peptide, as it experiences an increasingly hydrophobic environment within the membrane surface, can utilize a proton chain transfer mechanism to shed its protons to counter ions or potentially phospholipid head groups in the membrane skin region, thereby becoming compatible with the hydrophobic interior of the membrane. This increases the likelihood to move into the highly hydrophobic core of the membrane and ultimately reach the opposite leaflet to re‐acquire protons again, suggesting a potential “chameleon” mechanism for non‐disruptive and non‐endocytotic membrane translocation. The molecular dynamics simulations reveal stability of peptide bridges joining two membrane leaflets and demonstrate that this can facilitate cross‐membrane transport of small drug molecules.

Slide-free clinical imaging of melanin with absolute quantities using label-free third-harmonic-generation enhancement-ratio microscopy.

The capability to image the 3D distribution of melanin in human skin in vivo with absolute quantities and microscopic details will not only enable noninvasive histopathological diagnosis of melanin-related cutaneous disorders, but also make long term treatment assessment possible. In this paper, we demonstrate clinical in vivo imaging of the melanin distribution in human skin with absolute quantities on mass density and with microscopic details by using label-free third-harmonic-generation (THG) enhancement-ratio microscopy. As the dominant absorber in skin, melanin provides the strongest THG nonlinearity in human skin due to resonance enhancement. We show that the THG-enhancement-ratio (erTHG) parameter can be calibrated in vivo and can indicate the melanin mass density. With an unprecedented clinical imaging resolution, our study revealed erTHG-microscopy's unique capability for long-term treatment assessment and direct clinical observation of melanin's micro-distribution to shed light into the unknown pathway and regulation mechanism of melanosome transfer and translocation.