Mutual promotion of co-condensation of KRAS G-quadruplex and a well-folded protein HMGB1.

Liquid-liquid phase separation (LLPS) of G-quadruplex (GQ) is involved in many crucial cellular processes, while the quadruplex-folding and their functions are typically modulated by specific DNA-binding proteins. However, the regulatory mechanism of binding proteins, particularly the well-folded proteins, on the LLPS of GQs is largely unknown. Here, we investigated the effect of HMGB1 on the condensation of a G-quadruplex of KRAS promoter (GQKRAS). The results show that these two rigid macro-biomolecules undergo co-condensation through a mutual promotion manner, while neither of them can form LLPS alone. Fluidity measurements confirm that the liquid-like droplets are highly dynamic. HMGB1 facilitates and stabilizes the quadruplex folding of GQKRAS, and this process enhances their co-condensation. The KRAS promoter DNA retains quadruplex folding in the droplets; interference with the GQ-folding disrupts the co-condensation of GQKRAS/HMGB1. Mechanistic studies reveal that electrostatic interaction is a key driving force of the interaction and co-condensation of GQKRAS/HMGB1; meanwhile, the recognition of two macro-biomolecules plays a crucial role in this process. This result indicates that the phase separation of GQs can be modulated by DNA binding proteins, and this process could also be an efficient way to recruit specific DNA binding proteins.

Reactions of Cisplatin with Thioredoxin-1 Regulate Intracellular Redox Homeostasis.

Cisplatin is a widely used anticancer drug. In addition to inducing DNA damage, increased levels of reactive oxygen species (ROS) play a significant role in cisplatin-induced cell death. Thioredoxin-1 (Trx1), a redox regulatory protein that can scavenge ROS, has been found to eliminate cisplatin-induced ROS, while elevated Trx1 levels are associated with cisplatin resistance. However, it is unknown whether the effect of Trx1 on the cellular response to cisplatin is due to its direct reaction and how this reaction influences the activity of Trx1. In this work, we performed detailed studies of the reaction between Trx1 and cisplatin. Trx1 is highly reactive to cisplatin, and the catalytic motif of Trx1 (CGPC) is the primary binding site of cisplatin. Trx1 can bind up to 6 platinum moieties, resulting in the structural alteration and oligomerization of Trx1 depending on the degree of platination. Platination of Trx1 inhibits its interaction with ASK1, a Trx1-binding protein that regulates cell apoptosis. Furthermore, the reaction with cisplatin suppresses drug-induced ROS generation, which could be associated with drug resistance. This study provides more insight into the mechanism of action of cisplatin.

Inter-Domain Repulsion of Dumbbell-Shaped Calmodulin during Electrospray Ionization Revealed by Molecular Dynamics Simulations.

The mechanisms whereby protein ions are released from nanodroplets at the liquid-gas interface have continued to be controversial since electrospray ionization (ESI) mass spectrometry was widely applied in biomolecular structure analysis in solution. Several viable pathways have been proposed and verified for single-domain proteins. However, the ESI mechanism of multi-domain proteins with more complicated and flexible structures remains unclear. Herein, dumbbell-shaped calmodulin was chosen as a multi-domain protein model to perform molecular dynamics simulations to investigate the structural evolution during the ESI process. For [Ca4CAM], the protein followed the classical charge residue model. As the inter-domain electrostatic repulsion increased, the droplet was found to split into two sub-droplets, while stronger-repulsive apo-calmodulin unfolded during the early evaporation stage. We designated this novel ESI mechanism as the domain repulsion model, which provides new mechanistic insights into further exploration of proteins containing more domains. Our results suggest that greater attention should be paid to the effect of domain-domain interactions on structure retention during liquid-gas interface transfer when mass spectrometry is used as the developing technique in gas phase structural biology.

α-Synuclein as a Target for Metallo-Anti-Neurodegenerative Agents.

The unique thermodynamic and kinetic coordination chemistry of ruthenium allows it to modulate key adverse aggregation and membrane interactions of α-synuclein (α-syn) associated with Parkinson's disease. We show that the low-toxic RuIII complex trans-[ImH][RuCl4 (Me2 SO)(Im)] (NAMI-A) has dual inhibitory effects on both aggregation and membrane interactions of α-syn with submicromolar affinity, and disassembles pre-formed fibrils. NAMI-A abolishes the cytotoxicity of α-syn towards neuronal cells and mitigates neurodegeneration and motor impairments in a rat model of Parkinson's. Multinuclear NMR and MS analyses show that NAMI-A binds to residues involved in protein aggregation and membrane binding. NMR studies reveal the key steps in pro-drug activation and the effect of activated NAMI-A species on protein folding. Our findings provide a new basis for designing ruthenium complexes which could mitigate α-syn-induced Parkinson's pathology differently from organic agents.

Native Mass Spectrometry for Peptide-Metal Interaction in Picoliter Cell Lysate.

Native mass spectrometry, which takes a high concentration of ammonium acetate (NH4OAc) for ionization, coupled with tedious and solvent-consuming purification, which separates proteins from complicated environments, has shown great potential for proteins and their complexes. A high level of nonvolatile salts in the endogenous intracellular environment results in serious ion suppression and has been one of the bottlenecks for native mass spectrometry, especially for protein complexes. Herein, an integrated protocol utilizing the inner surface of a micropipette for rapid purification, desorption, and ionization of peptide-metal interaction at subfemtomole level in cell lysate was demonstrated for native mass spectrometry. The methods showed robust and reproducibility in protein measurement within 1 min from various buffers. The E. coli cells expressing with various proteins were lysed and used to test our method. The specific interaction between the peptide-metal complex in cell lysates could be reserved and distinguished by mass spectrometry.

Treatment Outcomes and Effects of Ethanol Sclerotherapy on Systemic Coagulation Profile of Patients with Venous Malformation.

BACKGROUND: Venous malformations (VMs) and sclerotherapy may disrupt the normal systemic coagulation profile in individuals. This study investigated a correlation between the clinical efficacy of sclerotherapy in the treatment of VMs and the changes in coagulation indexes to provide data that will inform the future application of this therapy. METHODS: From September 2019 to September 2020, 61 patients were enrolled in this study to receive sclerotherapy with absolute alcohol. The clinical outcomes and the coagulation profile were assessed. RESULTS: Sclerotherapy induced increasing fibrin (original) degradation products (FDP) and D-dimer (D-D) levels. The changes in FDP and D-D level pretreatment and posttreatment were positively correlated with treatment outcomes. Moreover, a repeated treatment with absolute alcohol may restore normal levels of FDP and D-D. CONCLUSIONS: Upregulation of FDP and D-D levels after sclerotherapy results in good therapeutic outcomes. Therefore, monitoring changes in FDP and D-D levels in patients with VMs undergoing sclerotherapy may reflect the effects of sclerotherapy.

19 F NMR Allows the Investigation of the Fate of Platinum(IV) Prodrugs in Physiological Conditions.

PtIV prodrugs can overcome resistance and side effects of conventional PtII anticancer therapies. By 19 F-labeling of a PtIV prodrug (Pt-FBA, FBA=p-fluorobenzoate), the activation under physiological conditions could be investigated. Unlike single-electron reductants, multi-electron agents can efficiently promote the two electrons reduction of PtIV to PtII . The activation of Pt-FBA in cell lysate is highly dependent upon the type of cancer cells. When administered to E. coli, Pt-FBA is reduced intracellularly and free FBA can shuttle out of the cell. The reduction rate greatly increases by inducing metallothionein overexpression and is lowered by addition of ZnII ions. When injected into mice, Pt-FBA undergoes fast reduction in the bloodstream accompanied by metabolic degradation of FBA; nevertheless, unreduced Pt-FBA can accumulate to detectable levels in liver and kidneys. The 19 F NMR approach has the advantage of avoiding the interference of all background signals.

Application of Digital Subtraction Angiography in Reconstruction of Plantar Forefoot With Reverse Medial Plantar Flap.

ABSTRACT: The reverse medial plantar flap (RMPF) raised from the nonweight-bearing region of the plantar foot represents a viable option for the soft tissue defect in planter forefoot. The anatomical basis of RMPF is the complex anastomotic branches between medial plantar artery (MPA) and deep plantar arch. Those anastomotic branches have high variation rate and may be damaged by trauma such as electric injury. Therefore, it is very important to know whether those anastomotic branches are present and intact before harvesting RMPF. Five patients with soft tissue defect in planter forefoot were enrolled into the study. The digital subtraction angiography (DSA) was performed to evaluate the plantar hemodynamics in the ipsilateral foot. The RMPF was harvested only after the anastomotic connections between MPA and deep plantar arch was confirmed. Anastomosis between superficial branch of MPA and deep plantar arch was observed in all DSA examinations. All 5 patients received the repair of soft tissue defect in plantar forefoot with RMPF. All flaps survived completely. The DSA can effectively evaluate the blood supply basis of RMPF and provide imaging evidence for the design and harvest of the flap. The main anatomical basis of RMPF is the anastomotic connections between superficial branch of MPA and deep plantar arch.

Clinical Staging, Angioarchitecture Type, and Surgical Treatment of Arteriovenous Malformations in the Head and Neck: A Single-Center Retrospective Analysis.

OBJECTIVE: Head and neck are the predilection sites of arteriovenous malformations (AVMs). Although embolization is the first-line treatment for AVMs, complete surgical removal of the lesion still has its value due to the best outcome with low recurrence rate. Here, the authors made a retrospective analysis on the surgical treatment of AVMs in the head and neck. METHODS: From January 2006 to December 2019, a total of 18 patients with AVMs in the head and neck were enrolled in this study, including 10 males. The Schobinger clinical staging, Yakes' angioarchitecture type, and surgical treatment were analyzed. The follow-up data were collected. Then, individual treatment strategies were summarized. RESULTS: According to Schobinger clinical classification system, 6 patients were at stage I, 7 patients at stage II, and 5 patients at stage III. According to Yakes' AVM classification system, 3 Type I, 4 Type II, 5 Type III, and 3 Type IV were confirmed. 3 patients cannot be confirmed due to lacking of arteriographic data. Surgical treatments included simple surgical excision (8 patients), dilator therapy (6 patients), and skin grafting after surgical excision (4 patients). In the follow-up period, 2 patients had recurrence and accepted operation again. All patients were satisfied with the appearance. CONCLUSIONS: Individual surgical treatment based on the clinical stage and angioarchitecture type can achieve satisfactory results in AVMs in the head and neck.

Repair of Defects in the Scalp and Face With Expanded Superficial Temporal Artery Flaps: A Single-Center Retrospective Study.

BACKGROUND AND OBJECTIVE: Defects resulted from the removal of large scars, benign tumors, severe pigmentation abnormalities, and vascular malformations, etc., in the scalp and face need to be repaired to restore the appearance. Here, the authors introduced the application of various expanded superficial temporal artery (STA) flaps in the repair of above defects. METHODS: From Jan. 2015 to Dec. 2018, 19 patients with craniofacial secondary defects received the repair with expanded STA flaps in our clinic. The defects were resulted from the removal of scalp scar (n = 6), neurofibroma (n = 4), sebaceous nevus (n = 3), arteriovenous malformation (n = 2), facial scar (n = 2), and port-wine stain (n = 2). The expanded STA flaps included 14 cases of flaps pedicled by parietal branch of STA, 2 cases of flaps pedicled by parietal branch of STA combined with laser hair removal, 1 case of flaps pedicled by frontal branch of STA, and 2 cases of prefabricated expanded skin flap with the superficial temporal fascia in the neck. RESULTS: The two-stage operation and water-filling expansion were accomplished in all patients. All flaps survived well, except one flap with venous congestion, which resolved after blood-letting and application of drugs promoting venous draining. In the three to six months follow-up, the flaps' color, texture, and thickness were satisfying. CONCLUSIONS: Individual application of different types of expanded STA flaps could achieve ideal results in repairing craniofacial secondary defects.

Imaging assessment and treatment of soft-tissue venous malformations: Retrospective case series study of 126 cases.

Venous malformations (VMs) are common slow-flow vascular malformations, which affect almost anywhere of the body. From January 2010 to October 2019, 126 patients with VMs who had complete imaging and follow-up data were enrolled into this study, including 75 males. The initial treatment age ranged from 5 to 72 years. The role of imaging results on the choice of treatment measures and the application were summarized. In this study, we retrospectively analyzed the imaging examinations, treatment measures, and follow-up results of the patients with VMs in our clinic. In this series, imaging examinations included ultrasound, magnetic resonance imaging, computed tomography (CT) scan and enhanced scan, percutaneous sinus angiography and three-dimensional CT imaging, plain film, CT venography, CT angiography, and digital subtraction angiography. Treatment measures included surgical excision (n = 20), sclerotherapy (n = 86, including absolute ethanol [n = 75], polidocanol [n = 8], and pingyangmycin [n = 3]), and combination treatment with intralesional copper wire retention and sclerotherapy(n = 20). After treatment, most of the lesions shrunk obviously or disappeared, and the symptoms were largely relieved. Comprehensive and accurate imaging assessment of VMs is necessary for selecting appropriate treatment. Individual strategy and sequential treatment can achieve effective results and avoid potential complications.

The Efficacy of Absolute Ethanol and Polidocanol in the Treatment of Venous Malformations.

INTRODUCTION: Using meta-analysis to evaluate the efficacy of absolute ethanol and polidocanol in the treatment of venous malformations. MATERIALS AND METHODS: A systematic search of the English literature was conducted in April 2019 including PubMed, Embase and Web of Science. Article selection was based on preset criteria. The included literature was scored on the MINORS scale, and the meta-analysis and the forest plot were made using the R 3.5.1 software for efficiency. RESULTS: Ten articles were included in the meta-analysis. Absolute ethanol response rate ranged between 79% and 92% with a pooled rate of 85%, and polidocanol response rate ranged between 63% and 94% with a pooled rate of 77%. DISCUSSION: Although sclerotherapy is effective in most studies, a large number of randomized controlled trials are still needed to confirm the best treatment options at different sites.

Application of Superficial Temporal Artery Flap in Wound Repairing After the Resection of Craniofacial Malignant Tumors.

BACKGROUND AND OBJECTIVE: Craniofacial malignant tumors require not only extended resection but also appropriate reconstruction to restore appearance, which remains a major challenge. Here the authors introduced the application of superficial temporal artery (STA) flap in wound repairing after the resection of craniofacial malignant tumors. METHODS: From January 2015 to December 2018, 16 patients with craniofacial malignant tumors were enrolled into the study, including squamous cell carcinoma (n = 6), basal cell carcinoma (n = 3), melanoma (n = 4), neuroendocrine carcinoma (n = 2), and dermatofibrosarcoma protuberance (n = 1). All of the tumors underwent extended resection. The defects formed were repaired by flaps pedicled with superior or frontal branch of STA. Donor sites were repaired with skin grafts. Patients were followed up for 6 months to 3 years to monitor the recurrence of tumor. RESULTS: All the flaps survived well. Venous congestion occurred in two cases but resolved after blood-letting and application of drugs promoting venous draining. During the follow-up, no recurrence of tumors was observed and the appearance of flaps was satisfying. But flap donor sites suffered from relatively poor appearance or alopecia deformity. CONCLUSIONS: The STA flap is reliable for wound repairing after resection of craniofacial malignant tumors. The STA parietal branch flap is preferred for repairing scalp defects, while the STA frontal branch flap is preferred for repairing facial defects. However, the STA flap should be used prudently due to its disadvantage of the deformity in scalp donor sites.

Ultrafast Microelectrophoresis: Behind Direct Mass Spectrometry Measurements of Proteins and Metabolites in Living Cell/Cells.

Direct chemical profiling and protein identification from living single cells using mass spectrometry (MS) have been demonstrated to further our understanding of biological variability and differential susceptibility to several diseases and treatments. Despite the great challenge from extremely complicated cytoplasm, we recently proposed a versatile MS strategy to achieve direct mass spectrometric characterization of both proteins and metabolite-like small molecules directly from living cells or single cells. Although the capability to directly handle cell cytoplasm was presumably attributed to microelectrophoresis in our previous studies, the assumption had only been partially explored by some preliminary experiments. To better understand the mechanism, herein, we systematically characterized its separation behavior with a series of model compounds covering a wide range of molecular size. With the merit of in situ separation, microelectrophoresis herein has been further demonstrated as an attractive and alternative tool, which can potentially contribute to direct MS measurements of more protein interactions or metabolic pathways in living single cells or a few cells.

Covalent versus Noncovalent Binding of Ruthenium η6 -p-Cymene Complexes to Zinc-Finger Protein NCp7.

Ruthenium-arene complexes are a unique class of organometallic compounds that have been shown to have prominent therapeutic potencies. Here, we have investigated the interactions of Ru-cymene complexes with a zinc-finger protein NCp7, aiming to understand the effects of various ligands on the reaction. Five different binding modes were observed on selected Ru-complexes. Ru-cymene complex can bind to proteins through either noncovalent binding alone or through a combination of covalent and noncovalent binding modes. Moreover, the noncovalent interaction can promote the coordination of RuII to NCp7, resulting synergistic effects of the different ligands. The binding of Ru(Cym) complexes leads to dysfunction of NCp7 through zinc-ejection and structural perturbation. These results indicate that the reactivity of Ru-complexes can be modulated by ligands through different approaches, which could be closely correlated to their different therapeutic effects.

Charge-dependent modulation of specific and nonspecific protein-metal ion interactions in nanoelectrospray ionization mass spectrometry.

RATIONALE: Previous studies found that charge state could affect both specific and nonspecific binding of protein-metal ion interactions in nanoelectrospray ionization mass spectrometry (nESI-MS). However, the two kinds of interactions have been studied individually in spite of the problem that they often coexist in the same system. Thus, it is necessary to study the effects of charge state on specific and nonspecific protein-metal ion interactions in one system to reveal more accurate binding state. METHODS: The HIV-1 nucleocapsid protein (NCp7(31-55)) which can bind specifically and nonspecifically to Zn2+ served as the model to show the charge-dependent protein-metal ion interactions. Hydrogen/deuterium exchange (HDX) and photodissociation (PD) were used to demonstrate that specific binding state was correlated with protein structure. In addition to NCp7(31-55), three other model proteins were used to investigate the reason for the charge-dependent nonspecific binding. RESULTS: For specific binding, we proposed that protein ions with different charge states had different conformations. The HDX results showed that labile protons in the NCp7(31-55)-Zn complex were exchanged in a charge-state-dependent way. The PD experiments revealed differential fragment yields for different charge states. For nonspecific binding, higher charge states had more Zn2+ additions, but less SO4 2- additions. The effects of charge states on nonspecific binding levels were entirely the opposite for Zn2+ and SO4 2- . These results could reveal that the nonspecific binding was caused by electrostatic interaction. CONCLUSIONS: For specific binding, NCp7(31-55) with lower charge states have folding and undenatured structures. The binding states of lower charge states can better reflect more native binding states. For nonspecific binding, when multiple metal ions adduct to proteins, the proteins have more net positive charges, which tend to generate higher charge ions during electrospray.

The facile and visualizable identification of broad-spectrum inhibitors of MDM2/p53 using co-expressed protein complexes.

MDM2 is a well-known oncoprotein overexpressed in a variety of cancers, and the identification of inhibitors that disrupt the MDM2/p53 interaction is of great interest in anticancer drug development. Here we designed a platform for the facile and visualizable identification of inhibitors of MDM2 using co-expressed protein complexes of MDM2/p53. A hexahistidine-tag on MDM2 allows the binding of the protein complex to the Ni-NTA affinity resin, while the fluorescent protein fused to p53 enables the direct visualization of the interaction of p53 with MDM2. Hence, the inhibition of the MDM2/p53 interaction can be observed with the naked eye. The assay can be set up by directly loading cell lysate to the Ni-NTA affinity resin, and no chemical modification of proteins is needed. In addition to the qualitative analyses, the binding affinity of inhibitors to the MDM2 protein can be quantified by fluorescence titration. The applications of this system have been verified using small molecules and peptide inhibitors. As a proof of concept, we screened a small library using this platform. Interestingly, two types of novel inhibitors of MDM2, including cyclohexyl-triphenylamine derivatives and platinum complexes, were identified and their binding affinities were obtained. Quantitative measurements show that these new types of inhibitors demonstrate a high binding affinity (up to Kd = 51.9 nM) to MDM2.

Reaction of Histone H1 with trans-Platinum Complexes and the Effect on DNA Platination.

Transplatin is an inactive platinum drug; however, a number of analogues, such as trans-EE and trans-PtTz, demonstrate promising antitumor activity in vitro and in vivo. Although the ultimate target is nuclear DNA, increasing evidence indicate that proteins also play important roles in the display of antitumor activity. The linker histone H1 is situated by the portal between the unwrapped DNA and the nucleosome core. Our recent study revealed that H1 can readily react with cisplatin, and the adducts tend to form ternary complexes with DNA. In this work, we have investigated the reaction of histone H1 with two antitumor-active trans-oriented complexes, trans-EE and trans-PtTz, and the effect of H1 upon the platination of DNA. The results show that trans-platinum drugs are much more reactive than cisplatin toward H1. Interestingly, in addition to the expected bidentate adducts (by displacement of the two labile chlorido ligands), also a tridentate adduct can be formed by displacement of one nonlabile carrier ligand of trans-EE or trans-PtTz. The trans-Pt/H1 adducts can then react with DNA and generate protein-Pt-DNA ternary complexes. Additionally, platinum can be transferred from trans-Pt/H1 adducts to DNA, generating binary trans-Pt/DNA complexes. Such a transfer of the platinum agent to DNA was not observed in the reaction of cisplatin. Furthermore, the detailed investigation carried out on a model peptide indicates that H1 promotes the DNA platination by trans- EE, while it reduces that of trans-PtTz and cisplatin. These results suggest that H1 can play a key role in the DNA platination and modulate the efficacy of different platinum agents.

Treatment of Large Scars in Children Using Artificial Dermis and Scalp Skin Grafting.

BACKGROUND AND OBJECTIVES: Large scars formed after burns injury can seriously hamper appearance and function in children. Surgical resection of scars and secondary skin or flap grafting often brings severe damages to donor sites, which may lead to physiological and psychological development disorders in children. Here, we introduce the use of artificial dermis and skin grafts from scalps to treat large scars in children to minimize the donor site morbidity. METHODS: A retrospective char review was performed including 7 children with large scars between January 2016 and December 2017. First, the scars were resected, and artificial dermis was applied to the secondary wounds. Twelve days later, outer silicone membrane was removed. Another 2 days later, scalp skin grafts of 0.3 mm were transplanted to the wounds. Manchester Scar Scale and Visual Analog Scale were used to evaluate scar appearance before and after the treatment respectively. One special patient with extensive scars was treated twice at an interval of 1 year. The first therapy was performed with both conventional method of resection and skin grafting and the new method described above. In the second therapy, 4 samples were taken from 4 different sites-the normal skin, scars, the skin where artificial dermis and scalp skin grafting were performed, and the skin where only scalp skin grafting was performed. H-E staining, Masson staining, Aldehyde fuchsin staining, and scanning electron microscopy were used for histological observation. RESULTS: All skin grafts survived well. The Manchester Scar Scale score of the graft area was significantly reduced (P < 0.01) after the treatment. Histological examination showed obviously better dermis arrangement where artificial dermis and scalp grafting was performed. CONCLUSION: The therapy achieves better appearances and minimizes donor site morbidity. It is beneficial to physical and psychological development of children and provides an alternative to treat children with large scars.

In Situ Living Cell Protein Analysis by Single-Step Mass Spectrometry.

In situ living cell protein analysis would enable the structural identification and functional interrogation of intracellular proteins in native cellular environments. Previously, we have presented an in situ mass spectrometry (MS) strategy to identify protein and protein/metal ion complex with relatively small molecular weight ( Anal. Chem. 2016, 88, 10860-10866). However, it is still challenging to directly identify larger proteins and protein/ligand complexes in cell, due to numerous nonspecific bindings of ligands, solvents, and other cellular constituents. Here we present a versatile single-step mass spectrometric strategy, "in-cell" mass spectrometry ("in-cell" MS), for in situ protein identification and dynamic protein-ligand interaction monitoring directly from living cells. "In-cell" MS combined all-ion-fragmentation mode with our previous method; thus, on a high-resolution MS instrument, we can greatly improve the signal/noise ratio of the larger proteins and protein/ligand complexes. Meanwhile, we also achieved a much wider mass range for protein complex and detection of 17 proteins with molecular weight ranging from 4 to 44 kDa. In addition, "in-cell" MS could also monitor dynamic protein interactions in living cells. Calcium-regulated calmodulin-melittin interaction was tested to demonstrate the proof of concept. "In-cell" MS provides an alternative for in situ analysis of living cells, which might contribute to rapid protein analysis and quality control in biochemistry laboratories, protein engineering, and even protein industry.