The tumor suppressor folliculin (FLCN) enables nutrient-dependent activation of the mechanistic target of rapamycin complex 1 (mTORC1) protein kinase via its guanosine triphosphatase (GTPase) ...activating protein (GAP) activity toward the GTPase RagC. Concomitant with mTORC1 inactivation by starvation, FLCN relocalizes from the cytosol to lysosomes. To determine the lysosomal function of FLCN, we reconstituted the human lysosomal FLCN complex (LFC) containing FLCN, its partner FLCN-interacting protein 2 (FNIP2), and the RagA
:RagC
GTPases as they exist in the starved state with their lysosomal anchor Ragulator complex and determined its cryo-electron microscopy structure to 3.6 angstroms. The RagC-GAP activity of FLCN was inhibited within the LFC, owing to displacement of a catalytically required arginine in FLCN from the RagC nucleotide. Disassembly of the LFC and release of the RagC-GAP activity of FLCN enabled mTORC1-dependent regulation of the master regulator of lysosomal biogenesis, transcription factor E3, implicating the LFC as a checkpoint in mTORC1 signaling.
The lysosomal membrane is the locus for sensing cellular nutrient levels, which are transduced to mTORC1 via the Rag GTPases and the Ragulator complex. The crystal structure of the five-subunit human ...Ragulator at 1.4 Å resolution was determined. Lamtor1 wraps around the other four subunits to stabilize the assembly. The Lamtor2:Lamtor3 dimer stacks upon Lamtor4:Lamtor5 to create a platform for Rag binding. Hydrogen-deuterium exchange was used to map the Rag binding site to the outer face of the Lamtor2:Lamtor3 dimer and to the N-terminal intrinsically disordered region of Lamtor1. EM was used to reconstruct the assembly of the full-length RagAGTP:RagCGDP dimer bound to Ragulator at 16 Å resolution, revealing that the G-domains of the Rags project away from the Ragulator core. The combined structural model shows how Ragulator functions as a platform for the presentation of active Rags for mTORC1 recruitment, and might suggest an unconventional mechanism for Rag GEF activity.
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•Crystal structure of V-shaped five subunit human Ragulator complex•Binding mode of RagA/C GTPase dimer by HDX-MS and EM•GTPase domains of RagA/C point away from and do not touch Ragulator•Ragulator affects conformation of GDP binding site in RagA without direct contact
Su et al. report the crystal structure of Ragulator, a pentameric GEF for RagA, and the EM structure of its complex with the RagA/C dimer that activates mTORC1. HDX-MS shows that Ragulator modulates the RagA GTP binding site despite no contact with its GTPase domain.
In their active GTP-bound form, Rab proteins interact with proteins termed effector molecules. In this study, we have thoroughly characterized a Rab effector domain that is present in proteins of the ...Mical and EHBP families, both known to act in endosomal trafficking. Within our study, we show that these effectors display a preference for Rab8 family proteins (Rab8, 10, 13 and 15) and that some of the effector domains can bind two Rab proteins via separate binding sites. Structural analysis allowed us to explain the specificity towards Rab8 family members and the presence of two similar Rab binding sites that must have evolved via gene duplication. This study is the first to thoroughly characterize a Rab effector protein that contains two separate Rab binding sites within a single domain, allowing Micals and EHBPs to bind two Rabs simultaneously, thus suggesting previously unknown functions of these effector molecules in endosomal trafficking.
2 Due to the vascular heterogeneity, it is difficult to predict and evaluate its efficacy with naked eyes. ...a technology that is consistent, repeatable, and non-invasive will be required to provide ...accurate images and quantitative evaluation indicators. In this study, we have reviewed dermoscopy, reflectance confocal microscopy (RCM), high-frequency ultrasound (HFUS), optical coherence tomography (OCT), and laser speckle imaging (LSI), etc. in predicting efficacy, intra-operative monitoring, and objective evaluation of effectiveness of PWS treatment Figure 1. 6.Kwiek, BRozalski, MSieczych, JPaluch, LKowalewski, CAmbroziak, M. Predictive value of dermoscopy for the treatment of port-wine stains with large spot 532 nm laser. 15.Ren, JQian, HXiang, LPan, ZZhong, LYan, S. The assessment of pulsed dye laser treatment of port-wine stains with reflectance confocal microscopy.
Photodynamic therapy (PDT) is an emerging technique for treating tumors. Especially, topical administration of photosensitizers (PSs) is more favorable for superficial tumor treatments with low ...systematic phototoxicity. Yet, ineffective migration of PSs to targeted tumor tissues and rapid consumption of O2 during PDT greatly limit their effects. Herein, PS‐loaded microneedle (MN) patches with O2 propellant for a deeper and faster transdermal delivery of PS and improved PDT by embedding sodium percarbonate (SPC) into dissolving poly(vinyl pyrrolidone) MNs are presented. It is shown that SPC in the MNs can react with surrounding fluid to generate gaseous oxygen bubbles, forming vigorous fluid flows and thus greatly enhancing PS of chlorin e6 (Ce6) penetration in both hydrogel models and skin tissues. Reactive oxygen species (ROS) in hypoxic breast cancer cells (4T1 cells) are greatly increased by rapid penetration of PS and relief of hypoxia in vitro, and Ce6‐loaded SPC MNs show an excellent cell‐killing effect. Moreover, lower tumor growth rate and tumor mass after a 20‐d treatment in tumor‐bearing mice model verify the improved PDT in gaseous oxygen‐droved delivery of PS. This study demonstrates a facile yet effective route of MN delivery of PSs for improved PDT in hypoxic tumor treatment.
Photosensitizer‐loaded microneedles with O2 propellant that can actively deliver photosensitizer to the tumor by generating gaseous oxygen bubbles are developed, greatly enhancing the penetration of photosensitizer (PS) and relieving the hypoxia for improved photodynamic therapy.
Background
This study aimed to assess the independent prognostic value of tumor size compared with other clinical and pathologic features of primary invasive cutaneous melanoma (CM).
Methods
This ...study included 28,593 patients with primary invasive CM in Surveillance, Epidemiology, and End Results Program database diagnosed from 2004 through 2016. Tumor size was divided into five subgroups (≤6, 7‐12, 13‐30, 31‐42, and >42 mm). The primary endpoint was melanoma‐specific survival (MSS).
Results
The relationship between tumor size and survival was piecewise. After adjusting for age, sex, primary site, histopathologic cell type, Breslow thickness, ulceration, mitotic rate, regional metastasis, and distant metastasis, the hazard ratio (HR) of MSS increased with increasing tumor size until a peak at 31‐42 mm (HRs, 1.33, 1.59, 2.41, respectively; all P < .0001), and then decreased when tumor size was larger than 42 mm using tumor size ≤ 6 mm as the reference (HR, 2.11; 95% confidence interval CI, 1.84 −2.42; P < .0001). This pattern mostly remained after stratification by T subcategories from T1 to T4 in localized primary CM except that tumor size >42 mm subgroup had the shortest MSS in T4. In addition, tumor size with a cutoff value of 12 mm showed stronger prognostic value for MSS (HR, 2.32; 95% CI, 1.80‐2.98; P < .0001) than Breslow thickness and mitotic rate in primary CM with T1N0M0.
Conclusions
Tumor size was an important independent prognostic factor for MSS in patients with primary invasive CM. Tumor size larger than 30 mm would provide additional and important prognostic information in each T subcategory of localized CM. Furthermore, tumor size with a cutoff value of 12 mm has great potential in improving the accuracy of melanoma T1 substaging.
Tumor size was an important independent prognostic factor for MSS in patients with primary invasive CM. Tumor size larger than 30 mm would provide additional important prognostic information in each T subcategory of localized CM.Tumor size with a cutoff value of 12 mm has great potential in improving the accuracy of melanoma T1 substaging.
Dissolving microneedle (DMN) patches are emerging as a minimally invasive and efficient transdermal drug delivery platform. Generally, noncrystalline, water‐soluble, and high‐molecular‐weight ...polymers are employed in DMNs because their sufficient intermolecular interactions can endow the DMNs with necessary mechanical strength and toughness. However, high viscosity and heavy chain entanglement of polymer solutions greatly hinder processing and dissolution of polymeric DMNs. Here, a strong and tough supramolecular DMN patch made of highly water‐soluble cyclodextrin (CD) derivatives is described. Due to the synergy of multiple supramolecular interactions, the CD DMN patch exhibits robust mechanical strength outperforming the state‐of‐the‐art polymeric DMNs. The CD DMN displays ultrafast dissolution (<30 s) in skin models by virtue of the dynamic and weak noncovalent bonds, which also enables the CD DMN and its payloads to diffuse rapidly into the deep skin layer. Moreover, the unique supramolecular structure of CD allows the CD DMNs to load not only hydrophilic drugs (e.g., rhodamine B as a model) but also hydrophobic model drugs (e.g., ibuprofen). As a proof‐of‐concept, CD DMNs loading ibuprofen show a rapid onset of therapeutic action in a xylene‐induced acute inflammation model in mice. This work opens a new avenue for the development of mechanically robust supramolecular DMNs and broadens the applications of supramolecular materials.
Supramolecular dissolving microneedle (DMN) patches composed of highly water‐soluble cyclodextrin (CD) derivatives demonstrate strong and tough mechanical properties by virtue of multiple supramolecular interactions, surpassing that of most state‐of‐the‐art polymeric DMN patches. CD DMN can load hydrophilic and hydrophobic drugs, and dissolve extremely fast to achieve a rapid onset of its payloads, which is unachievable with conventional polymeric DMN patches.
Glucocorticoid-based creams are commonly used for treatments of psoriatic skin lesions while showing poor permeation because the thickened stratum corneum severely limits drug absorption. Although ...dissolving microneedle (DMN) patches have been employed in treating skin disease by virtue of their direct target to the lesion site, conventional DMN patches are generally fabricated from the water-soluble matrix, making them difficult to efficiently encapsulate hydrophobic glucocorticoids. Here, we develop a mechanically robust supramolecular DMN composed of hydroxypropyl β-cyclodextrin (HPCD) to effectively and uniformly load triamcinolone acetonide (TA). The TA-loaded HPCD DMN (TAMN) exhibits excellent mechanical performance that can easily pierce the thickened psoriasis lesions and deliver TA efficiently. Owing to the increased water solubility and bioavailability of TA after inclusion into HPCD, TAMN shows a superior in vitro inhibitory effect on immortalized human keratinocyte (HaCaT) cells. Importantly, the administration of TAMN twice a week effectively alleviates psoriatic signs and reduces the expression of Ki67, IL-23, and IL-17 in the ear lesions of imiquimod-induced psoriasis-like mice. This supramolecular DMN provides a promising strategy for the efficient treatment of psoriasis and other skin diseases, greatly broadens the applications of supramolecular materials in transdermal drug delivery, and widens the range of drugs in DMNs.
Both clinical and animal studies showed that the impaired functions of the orbitofrontal cortex (OFC) underlie the compulsive drug-seeking behavior of drug addiction. However, the functional changes ...of the microcircuit in the OFC and the underlying molecular mechanisms in drug addiction remain elusive, and little is known for whether microcircuits in the OFC contributed to drug addiction-related behaviors. Utilizing the cocaine-induced conditioned-place preference model, we found that the malfunction of the microcircuit led to disinhibition in the OFC after cocaine withdrawal. We further showed that enhanced Somatostatin-Parvalbumin (SST-PV) inhibitory synapse strength changed microcircuit function, and SST and PV inhibitory neurons showed opposite contributions to the drug addiction-related behavior of mice. Brevican of the perineuronal nets of PV neurons regulated SST-PV synapse strength, and the knockdown of Brevican alleviated cocaine preference. These results reveal a novel molecular mechanism of the regulation of microcircuit function and a novel circuit mechanism of the OFC in gating cocaine preference.
Bacterial biofilms pose severe threats to public health worldwide and are intractable by conventional antibiotic treatment. Antimicrobial photodynamic therapy (PDT) is emerging as a promising ...strategy for eradicating biofilms by virtue of low invasiveness, broad-spectrum antibacterial activity, and nondrug resistance. However, its practical efficacy is impeded by the low water solubility, severe aggregation, and poor penetration of photosensitizers (PSs) into the dense extracellular polymeric substances (EPS) of biofilms. Herein, we develop a dissolving microneedle (DMN) patch composed of a sulfobutylether-β-cyclodextrin (SCD)/tetra(4-pyridyl)-porphine (TPyP) supramolecular PS for enhanced biofilm penetration and eradication. The inclusion of TPyP into the SCD cavity can drastically inhibit the aggregation of TPyP, thereby allowing for nearly tenfold reactive oxygen species production and high photodynamic antibacterial efficacy. Moreover, the TPyP/SCD-based DMN (TSMN) possesses excellent mechanical performance that can easily pierce the EPS of biofilm with a penetration depth of ∼350 μm, enabling sufficient contact of TPyP with bacteria and optimal photodynamic elimination of bacterial biofilms. Furthermore, TSMN could efficiently eradicate Staphylococcus aureus biofilm infection in vivo with good biosafety. This study offers a promising platform for supramolecular DMN for efficient biofilm elimination and other PDTs.
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