Publications by authors named "Mingzhen Zhang"

108 Publications

Novel MAPK/AKT-impairing germline NRAS variant identified in a melanoma-prone family.

Fam Cancer 2021 Jul 3. Epub 2021 Jul 3.

Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, 9609 Medical Center Drive, EPS 7106, Bethesda, MD, 20892, USA.

While several high-penetrance melanoma risk genes are known, variation in these genes fail to explain melanoma susceptibility in a large proportion of high-risk families. As part of a melanoma family sequencing study, including 435 families from Mediterranean populations we identified a novel NRAS variant (c.170A > C, p.D57A) in an Italian melanoma-prone family. This variant is absent in exomes in gnomAD, ESP, UKBiobank, and the 1000 Genomes Project, as well as in 11,273 Mediterranean individuals and 109 melanoma-prone families from the US and Australia. This variant occurs in the GTP-binding pocket of NRAS. Differently from other RAS activating alterations, NRAS D57A expression is unable to activate MAPK-pathway both constitutively and after stimulation but enhances EGF-induced PI3K-pathway signaling in serum starved conditions in vitro. Consistent with in vitro data demonstrating that NRAS D57A does not enrich GTP binding, molecular modeling suggests that the D57A substitution would be expected to impair Mg2 + binding and decrease nucleotide-binding and GTPase activity of NRAS. While we cannot firmly establish NRAS c.170A > C (p.D57A) as a melanoma susceptibility variant, further investigation of NRAS as a familial melanoma gene is warranted.
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http://dx.doi.org/10.1007/s10689-021-00267-9DOI Listing
July 2021

The mechanism of activation of monomeric B-Raf V600E.

Comput Struct Biotechnol J 2021 4;19:3349-3363. Epub 2021 Jun 4.

Computational Structural Biology Section, Frederick National Laboratory for Cancer Research in the Laboratory of Cancer Immunometabolism, National Cancer Institute, Frederick, MD 21702, USA.

Oncogenic mutations in the serine/threonine kinase B-Raf, particularly the V600E mutation, are frequent in cancer, making it a major drug target. Although much is known about B-Raf's active and inactive states, questions remain about the mechanism by which the protein changes between these two states. Here, we utilize molecular dynamics to investigate both wild-type and V600E B-Raf to gain mechanistic insights into the impact of the Val to Glu mutation. The results show that the wild-type and mutant follow similar activation pathways involving an extension of the activation loop and an inward motion of the αC-helix. The V600E mutation, however, destabilizes the inactive state by disrupting hydrophobic interactions present in the wild-type structure while the active state is stabilized through the formation of a salt bridge between Glu600 and Lys507. Additionally, when the activation loop is extended, the αC-helix is able to move between an inward and outward orientation as long as the DFG motif adopts a specific orientation. In that orientation Phe595 rotates away from the αC-helix, allowing the formation of a salt bridge between Lys483 and Glu501. These mechanistic insights have implications for the development of new Raf inhibitors.
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http://dx.doi.org/10.1016/j.csbj.2021.06.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8215184PMC
June 2021

Changes of microbial community structure during the initial stage of biological clogging in horizontal subsurface flow constructed wetlands.

Bioresour Technol 2021 Jun 12;337:125405. Epub 2021 Jun 12.

State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China.

The clogging is a universal problem in constructed wetlands, where microorganisms play an essential role. However, the implication of micro-organism variation due to the clogging is not clear. Four horizontal subsurface flow constructed wetlands (HFCWs) were designed and operated to simulate the process of clogging. The wetland treatment performance and microbial community variation were investigated by regularly monitoring. Results showed the substrate filtration rate and the total phosphorous (TP) removal efficiency consistently decreased and the chemical oxygen demand (COD) and total nitrogen (TN) removal efficiency were at the range of 50%-85% and 10-20%, respectively. The sequencing results indicated that the clogging could affect the richness of bacterial community. The bacterial variation could be attributed to the dissolved oxygen decreasing and organic matter accumulation in the initial clogging period. These findings are expected to provide some theoretical reference for developing the biological methods to indicate the initial clogging in constructed wetlands.
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http://dx.doi.org/10.1016/j.biortech.2021.125405DOI Listing
June 2021

Oral Administration of Ginger-Derived Lipid Nanoparticles and Dmt1 siRNA Potentiates the Effect of Dietary Iron Restriction and Mitigates Pre-Existing Iron Overload in KO Mice.

Nutrients 2021 May 15;13(5). Epub 2021 May 15.

Food Science & Human Nutrition Department, University of Florida, Gainesville, FL 32611, USA.

Intestinal iron transport requires an iron importer (Dmt1) and an iron exporter (Fpn1). The hormone hepcidin regulates iron absorption by modulating Fpn1 protein levels on the basolateral surface of duodenal enterocytes. In the genetic, iron-loading disorder hereditary hemochromatosis (HH), hepcidin production is low and Fpn1 protein expression is elevated. High Fpn1-mediated iron export depletes intracellular iron, causing a paradoxical increase in Dmt1-mediated iron import. Increased activity of both transporters causes excessive iron absorption, thus initiating body iron loading. Logically then, silencing of intestinal Dmt1 or Fpn1 could be an effective therapeutic intervention in HH. It was previously established that Dmt1 knock down prevented iron-loading in weanling (encoding hepcidin) KO mice (modeling type 2B HH). Here, we tested the hypothesis that Dmt1 silencing combined with dietary iron restriction (which may be recommended for HH patients) will mitigate iron loading once already established. Accordingly, adult KO mice were switched to a low-iron (LFe) diet and (non-toxic) folic acid-coupled, ginger nanoparticle-derived lipid vectors (FA-GDLVs) were used to deliver negative-control (NC) or Dmt1 siRNA by oral, intragastric gavage daily for 21 days. The LFe diet reduced body iron burden, and experimental interventions potentiated iron losses. For example, Dmt1 siRNA treatment suppressed duodenal Dmt1 mRNA expression (by ~50%) and reduced serum and liver non-heme iron levels (by ~60% and >85%, respectively). Interestingly, some iron-related parameters were repressed similarly by FA-GDLVs carrying either siRNA, including Fe (as FeCl) absorption (~20% lower), pancreatic non-heme iron (reduced by ~65%), and serum ferritin (decreased 40-50%). Ginger may thus contain bioactive lipids that also influence iron homeostasis. In conclusion, the combinatorial approach of FA-GDLV and Dmt1 siRNA treatment, with dietary iron restriction, mitigated pre-existing iron overload in a murine model of HH.
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http://dx.doi.org/10.3390/nu13051686DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8157040PMC
May 2021

Numerical study on sector-vortex phased irradiation method using annular array transducer in High-Intensity Focused Ultrasound treatment.

Ultrasonics 2021 Aug 11;115:106464. Epub 2021 May 11.

The University of Tokyo, Dept. of Mechanical Engineering, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.

Sector-vortex phased irradiation from annular array transducer was numerically studied with breast model constructed from MRI data of real patient. Phase compensation (PC) based on time reversal pre-computation was applied in order to handle phase delay caused by heterogeneity of breast tissues, and results showed great effectiveness on single-focus case, insignificant effectiveness on multi-focus cases with 4 and 8 phase-sectors, but ineffectiveness on multi-focus case with 12 phase-sectors, where enormous undesired outer ablation occurred. For single-focus case, phase compensation not only produced real focus very close to targeted site (0.1 mm deviation), but also decreased thermal peak ratio (outer/focal) largely by 30%. However, phase compensation did not increase total ablated size. For multi-focus cases with 4 and 8 phase-sectors, deformed focal shapes by tissue heterogeneity were restored by phase compensation, but the 4-phase-sector case had higher thermal peak ratio and smaller ablation than 8-phase-sector case for strong cancelling effect between phase-sector borders. Ineffectiveness of phase compensation on multi-focus case with 12 phase-sectors had three considerable reasons. 1st, inequality of piezo-element number between sectors; 2nd, heterogeneous attenuation of breast model; 3rd, insufficient number of piezo-elements per sector; where the 2nd reason originated from breast model, and other two reasons were related to array transducer. This research gave several preliminary indications. 1st, ineffectiveness of phase compensation occurs on case with large phase-sector number when using annular array transducer; 2nd, with same input energy and same irradiation time, sector-vortex phased irradiation creates smaller focal ablation, but withstands longer than single-focus irradiation free of outer ablation; 3rd, phase-difference π between neighboring phase-sectors is disadvantageous because of energy loss; 4th, phase compensation is effective on single-focus for improving pinpoint ablation but not for increasing total ablated size.
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http://dx.doi.org/10.1016/j.ultras.2021.106464DOI Listing
August 2021

Drugging multiple same-allele driver mutations in cancer.

Expert Opin Drug Discov 2021 Aug 26;16(8):823-828. Epub 2021 Mar 26.

Computational Structural Biology Section, Frederick National Laboratory for Cancer Research in the Laboratory of Cancer ImmunoMetabolism, National Cancer Institute, Frederick U.S.A.

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http://dx.doi.org/10.1080/17460441.2021.1905628DOI Listing
August 2021

B-Raf autoinhibition in the presence and absence of 14-3-3.

Structure 2021 Jul 11;29(7):768-777.e2. Epub 2021 Mar 11.

Computational Structural Biology Section, Frederick National Laboratory for Cancer Research in the Laboratory of Cancer ImmunoMetabolism, National Cancer Institute, Frederick, MD 21702, USA; Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel. Electronic address:

Raf-activating mutations are frequent in cancer. In the basal state, B-Raf is autoinhibited by its upstream Ras-binding domain (RBD) and cysteine-rich domain (RBD-CRD) interacting with its kinase domain (KD) and the 14-3-3 dimer. Our comprehensive molecular dynamics simulations explore two autoinhibition scenarios in the presence and absence of the 14-3-3 dimer. When present, the 14-3-3 interaction with B-Raf stabilizes the RBD-CRD-KD interaction, interfering with the KD dimerization. Raf's pSer365 removal fails to induce large disruption. RBD-CRD release promotes KD fluctuations and reorientation for dimerization, consistent with experimental data. In the absence of 14-3-3, our sampled B-Raf conformations suggest that RBD-CRD can block the KD dimerization surface. Our results suggest a B-Raf activation mechanism, whereby one KD monomer is donated by 14-3-3-free B-Raf KD and the other by 14-3-3-bound KD. This mechanism can lead to homo- and heterodimers. These autoinhibition scenarios can transform autoinhibited B-Raf monomers into active B-Raf dimers.
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http://dx.doi.org/10.1016/j.str.2021.02.005DOI Listing
July 2021

Dual amyloid cross-seeding reveals steric zipper-facilitated fibrillization and pathological links between protein misfolding diseases.

J Mater Chem B 2021 04 2;9(15):3300-3316. Epub 2021 Mar 2.

Department of Chemical, Biomolecular, and Corrosion Engineering The University of Akron, Ohio, USA.

Amyloid cross-seeding, as a result of direct interaction and co-aggregation between different disease-causative peptides, is considered as a main mechanism for the spread of the overlapping pathology across different cells and tissues between different protein-misfolding diseases (PMDs). Despite the biomedical significance of amyloid cross-seeding in amyloidogenesis, it remains a great challenge to discover amyloid cross-seeding systems and reveal their cross-seeding structures and mechanisms. Herein, we are the first to report that GNNQQNY - a short fragment from yeast prion protein Sup35 - can cross-seed with both amyloid-β (Aβ, associated with Alzheimer's disease) and human islet amyloid polypeptide (hIAPP, associated with type II diabetes) to form β-structure-rich assemblies and to accelerate amyloid fibrillization. Dry, steric β-zippers, formed by the two β-sheets of different amyloid peptides, provide generally interactive and structural motifs to facilitate amyloid cross-seeding. The presence of different steric β-zippers in a variety of GNNQQNY-Aβ and GNNQQNY-hIAPP assemblies also explains amyloid polymorphism. In addition, alteration of steric zipper formation by single-point mutations of GNNQQNY and interactions of GNNQQNY with different Aβ and hIAPP seeds leads to different amyloid cross-seeding efficiencies, further confirming the existence of cross-seeding barriers. This work offers a better structural-based understanding of amyloid cross-seeding mechanisms linked to different PMDs.
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http://dx.doi.org/10.1039/d0tb02958kDOI Listing
April 2021

Application of Radiosensitizers in Cancer Radiotherapy.

Int J Nanomedicine 2021 12;16:1083-1102. Epub 2021 Feb 12.

Department of Oncology, The First Affiliated Hospital, Xi'an Jiaotong University, Xi'an, Shaanxi, 710061, People's Republic of China.

Radiotherapy (RT) is a cancer treatment that uses high doses of radiation to kill cancer cells and shrink tumors. Although great success has been achieved on radiotherapy, there is still an intractable challenge to enhance radiation damage to tumor tissue and reduce side effects to healthy tissue. Radiosensitizers are chemicals or pharmaceutical agents that can enhance the killing effect on tumor cells by accelerating DNA damage and producing free radicals indirectly. In most cases, radiosensitizers have less effect on normal tissues. In recent years, several strategies have been exploited to develop radiosensitizers that are highly effective and have low toxicity. In this review, we first summarized the applications of radiosensitizers including small molecules, macromolecules, and nanomaterials, especially those that have been used in clinical trials. Second, the development states of radiosensitizers and the possible mechanisms to improve radiosensitizers sensibility are reviewed. Third, the challenges and prospects for clinical translation of radiosensitizers in oncotherapy are presented.
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http://dx.doi.org/10.2147/IJN.S290438DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7886779PMC
March 2021

A Mini Review on pH-Sensitive Photoluminescence in Carbon Nanodots.

Front Chem 2020 22;8:605028. Epub 2021 Jan 22.

Department of Biophysics, School of Basic Medical Sciences, Institute of Medical Engineering, Xi'an Jiaotong University Health Science Center, Xi'an, China.

Carbon nanodots (C-dots) with sp/sp framework and diameter of <10 nm contain abundant functional groups or polymers on their surface. C-dots have attracted immense attention because of their unique optical properties, excellent biocompatibility, facile preparation, and low cost. With these merits, C-dots have been used in a wide range of applications including sensing, bioimaging, catalysis, and light-emitting devices. C-dots exhibit good optical properties, such as tunable emission wavelength, good photostability, nonblinking, up-conversion emission, etc. Of note, C-dots show intrinsic pH-sensitive photoluminescence (PL), indicating their great potential for pH sensing, especially in biotic pH sensing. In this review, we systematically summarize the pH-sensitive PL properties and the pH-sensitive PL mechanism, as well as recent research progress of C-dots in pH sensing. The current challenges of pH-sensitive C-dots and their future research focus are also proposed here. We anticipate this review might be of great significance for understanding the characteristics of pH-sensitive C-dots and the development of photoluminescent nanomaterials with pH-sensitive properties.
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http://dx.doi.org/10.3389/fchem.2020.605028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7862559PMC
January 2021

Optimal acupoint and session of acupuncture for patients with chronic prostatitis/chronic pelvic pain syndrome: a meta-analysis.

Transl Androl Urol 2021 Jan;10(1):143-153

Department of Urology, Changhai Hospital, Naval Medical University, Shanghai, China.

Background: The study aims to perform a meta-analysis of published trials and evaluate the efficacy of acupuncture on chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) by symptom score reduction, optimal acupuncture session, and most frequently used acupoints.

Methods: A literature search was performed for randomized controlled trials (RCTs) comparing efficacy of acupuncture with sham acupuncture or standard medication on CP/CPPS. The primary outcome was the reduction of National Institute of Health-Chronic Prostatitis Index (NIH-CPSI) total score and its subscales. The optimal acupuncture session to reach its clinical efficacy and most common compatibility rule of acupoints were also evaluated.

Results: Ten trials involving 770 participants were included. Meta-analysis showed compared with sham acupuncture, acupuncture yielded significant reduction in NIH-CPSI total score [weighted mean difference (WMD): 7.28, 95% confidence interval (95% CI): 5.69-8.86), and provided better pain relief (WMD: 3.57, 95% CI: 2.07-5.08), urinary symptoms improvement (WMD: 1.68, 95% CI: 1.13-2.22), and quality of life (QOL) (WMD: 2.38, 95% CI: 1.41-3.36). Compared with standard medication, acupuncture were more efficacious in reducing NIH-CPSI total score (WMD: 3.36, 95% CI: 1.27-5.45), also showed significant greater pain relief (WMD: 2.36, 95% CI: 1.67-3.06), marginal advantage in improving QOL (WMD: 0.98, 95% CI: 0.12-1.83) but no difference in reducing urinary symptom (WMD: -0.03, 95% CI: -1.30 to 1.24). Four acupuncture sessions were the minimum "dose" to reach clinical efficacy, and prolonged acupuncture sessions continuously improved urinary symptoms and QOL. The majority of acupoint selection strategies were based on the combination of any three acupoints from CV3, CV4, BL32, SP6, and SP9.

Conclusions: Acupuncture has promising efficacy for patients with CP/CPPS, especially category IIIB, in aspects of relieving pain and urinary symptoms and improving the QOL. Acupuncture may serve as a standard treatment option when available, and a tailored comprehensive treatment strategy for CP/CPPS is the future trend.
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http://dx.doi.org/10.21037/tau-20-913DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7844493PMC
January 2021

Human disturbance on phosphorus sources, processes and riverine export in a subtropical watershed.

Sci Total Environ 2021 May 19;769:144658. Epub 2021 Jan 19.

Fujian Provincial Key Laboratory for Coastal Ecology and Environmental Studies, College of the Environment and Ecology, Xiamen University, Xiamen 361102, China; Key Laboratory of the Coastal and Wetland Ecosystems, College of the Environment and Ecology, Xiamen University, Xiamen 361102, China. Electronic address:

Phosphorus (P) is a key nutrient in freshwater systems, often acting as the limiting nutrient. The dominant sources of P in the Jiulong River watershed (S.E. China) are anthropogenic. Dissolved and particulate P species were measured in the West (WJR) and North (NJR) rivers during the wet and dry seasons of 2018 and at their river outlets during a storm (June 2019). Sources of P pollution were characterized from mainly single source subcatchments (dry season). The Agriculture source (WJR) had a total P of 114.7 ± 13.1 μg P L, which was mainly dissolved inorganic P (DIP) from excess fertilizer washed from the fields. By contrast, the West Urban source (sewage effluent) was mainly particulate (POP) and dissolved organic P (DOP). The effect of reservoirs in the main NJR was to decrease total particulate P (TPP) and DIP and increase POP, due to increased sedimentation of particles and biological uptake. An increase in all P species was observed at the beginning of the storm, followed by a decrease on the rising hydrograph due to dilution. The final concentration of all P species was higher than baseflow, confirming that storms increase the P flux out of the watershed. P was initially washed off the fields during the storm, and during the falling hydrograph P increased due to interflow and other longer-term sources. The high DIN:DIP ratio confirmed the key importance of P inputs from human activities in substantially altering P sources and cycling, and hence the importance of science-based management to alleviate the eutrophication problem.
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http://dx.doi.org/10.1016/j.scitotenv.2020.144658DOI Listing
May 2021

Novel multi-stimuli responsive functionalized PEG-based co-delivery nanovehicles toward sustainable treatments of multidrug resistant tumor.

J Mater Chem B 2021 02;9(5):1297-1314

School of Food and Biological Engineering, Shaanxi University of Science and Technology, Xi'an 710021, China.

The efficacy of ongoing anticancer treatment is often compromised by some barriers, such as low drug content, nonspecific release of drug delivery system, and multidrug resistance (MDR) effect of tumors. Herein, in the research a novel functionalized PEG-based polymer cystine-(polyethylene glycol)2-b-(poly(2-methacryloyloxyethyl ferrocenecarboxylate)2) (Cys-(PEG45)2-b-(PMAOEFC)2) with multi-stimuli sensitive mechanism was constructed, in which doxorubicin (DOX) was chemical bonded through Schiff base structure to provide acid labile DOX prodrug (DOX)2-Cys-(PEG45)2-b-(PMAOEFC)2. Afterwards, paclitaxel (PTX) and its diselenide bond linked PTX dimer were encapsulated into the prodrug through physical loading, to achieve pH and triple redox responsive (DOX)2-Cys-(PEG45)2-b-(PMAOEFC)[email protected] and (DOX)2-Cys-(PEG45)2-b-(PMAOEFC)[email protected] dimer with ultrahigh drugs content. The obtained nanovehicles could self-assemble into globular micelles with good stability based on fluorescence spectra and TEM observation. Moreover, there was a remarkable "reassembly-disassembly" behavior caused by phase transition of micelles under the mimic cancerous physiological environment. DOX and PTX could be on-demand released in acid and redox stress mode, respectively. Meanwhile, in vivo anticancer studies revealed the significant tumor inhibition of nanoformulas. This work offered facile strategies to fabricate drug nanaovehicles with tunable drug content and types, it has a profound significance in overcoming MDR effect, which provided more options for sustainable cancer treatment according to the desired drug dosage and the stage of tumor growth.
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http://dx.doi.org/10.1039/d0tb02192jDOI Listing
February 2021

Endoglucanase activity of cellulolytic bacteria from lake sediments and its application in hydrophyte degradation.

FEMS Microbiol Lett 2020 01;367(24)

State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, No. 7 Donghu South Road, Wuchang District, Wuhan, Hubei Province, P.R. China.

Hydrophytes are plants that grow in or on water. Their overgrowth adversely affects the ecosystem because of crowding out other aquatic organisms and polluting the environment with plant residue. In principle, cellulolytic bacteria can be used to degrade hydrophyte biomass. We here isolated and characterized four cellulolytic bacterial strains from Lake Donghu sediments (Wuhan, China) that are rich in organic matter and plant residues. The isolates (WDHS-01 to 04) represent Bacillus, Micromonospora and Streptomyces genera. The bacteria exhibited pronounced endoglucanase activities (from 0.022 to 0.064 U mL-1). They did not extensively degrade the emerged plant Canna indica L. However, in an Hydrilla verticillata (submerged plant) degradation medium, strain WDHS-02 exhibited a high degradation rate (54.91%), endoglucanase activity of 0.35 U mL-1 and the conversion rate of cellulose to reducing sugars of 7.15%. Correlation analysis revealed that bacterial endoglucanase activity was significantly correlated with the degradation rate, and acid detergent lignin, ash and cellulose content of the residual H. verticillata powder. In conclusion, the identified bacteria efficiently decomposed submerged plants without the need for acid-base pretreatment. They expand the set of known cellulolytic bacteria and can be used for natural degradation of submerged plants.
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http://dx.doi.org/10.1093/femsle/fnaa200DOI Listing
January 2020

Phosphorylation and Driver Mutations in PI3Kα and PTEN Autoinhibition.

Mol Cancer Res 2021 04 7;19(4):543-548. Epub 2020 Dec 7.

Computational Structural Biology Section, Frederick National Laboratory for Cancer Research in the Laboratory of Cancer Immunometabolism, NCI, Frederick, Maryland.

PI3K and PTEN are the second and third most highly mutated proteins in cancer following only p53. Their actions oppose each other. PI3K phosphorylates signaling lipid PIP to PIP PTEN dephosphorylates it back. Driver mutations in both proteins accrue PIP PIP recruits AKT and PDK1 to the membrane, promoting cell-cycle progression. Here we review phosphorylation events and mutations in autoinhibition in PI3K and PTEN from the structural standpoint. Our purpose is to clarify how they control the autoinhibited states. In autoinhibition, a segment or a subunit of the protein occludes its functional site. Protein-protein interfaces are often only marginally stable, making them sensitive to changes in conditions in living cells. Phosphorylation can stabilize or destabilize the interfaces. Driver mutations commonly destabilize them. In analogy to "passenger mutations," we coin "passenger phosphorylation" to emphasize that the presence of a phosphorylation recognition sequence logo does not necessarily imply function. Rather, it may simply reflect a statistical occurrence. In both PI3K and PTEN, autoinhibiting phosphorylation events are observed in the occluding "piece." In PI3Kα, the "piece" is the p85α subunit. In PTEN, it is the C-terminal segment. In both enzymes the stabilized interface covers the domain that attaches to the membrane. Driver mutations that trigger rotation of the occluding piece or its deletion prompt activation. To date, both enzymes lack specific, potent drugs. We discuss the implications of detailed structural and mechanistic insight into oncogenic activation and how it can advance allosteric precision oncology.
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http://dx.doi.org/10.1158/1541-7786.MCR-20-0818DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8026595PMC
April 2021

PI3K Driver Mutations: A Biophysical Membrane-Centric Perspective.

Cancer Res 2021 01 12;81(2):237-247. Epub 2020 Oct 12.

Computational Structural Biology Section, Frederick National Laboratory for Cancer Research in the Laboratory of Cancer Immunometabolism, National Cancer Institute, Frederick, Maryland.

Ras activates its effectors at the membrane. Active PI3Kα and its associated kinases/phosphatases assemble at membrane regions enriched in signaling lipids. In contrast, the Raf kinase domain extends into the cytoplasm and its assembly is away from the crowded membrane surface. Our structural membrane-centric outlook underscores the spatiotemporal principles of membrane and signaling lipids, which helps clarify PI3Kα activation. Here we focus on mechanisms of activation driven by PI3Kα driver mutations, spotlighting the PI3Kα double (multiple) activating mutations. Single mutations can be potent, but double mutations are stronger: their combination is specific, a single strong driver cannot fully activate PI3K, and two weak drivers may or may not do so. In contrast, two strong drivers may successfully activate PI3K, where one, for example, H1047R, modulates membrane interactions facilitating substrate binding at the active site () and the other, for example, E542K and E545K, reduces the transition state barrier (), releasing autoinhibition by nSH2. Although mostly unidentified, weak drivers are expected to be common, so we ask here how common double mutations are likely to be and why PI3Kα with double mutations responds effectively to inhibitors. We provide a structural view of hotspot and weak driver mutations in PI3Kα activation, explain their mechanisms, compare these with mechanisms of Raf activation, and point to targeting cell-specific, chromatin-accessible, and parallel (or redundant) pathways to thwart the expected emergence of drug resistance. Collectively, our biophysical outlook delineates activation and highlights the challenges of drug resistance.
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http://dx.doi.org/10.1158/0008-5472.CAN-20-0911DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7855922PMC
January 2021

Highly Biocompatible Functionalized Layer-by-Layer Ginger Lipid Nano Vectors Targeting P-selectin for Delivery of Doxorubicin to Treat Colon Cancer.

Adv Ther (Weinh) 2019 Dec 18;2(12). Epub 2019 Sep 18.

Institute for Biomedical Sciences, Center for Diagnostics and Therapeutics, Digestive Disease Research Group, Georgia State University, Atlanta, Georgia, 30302, United States.

A biocompatible natural nanoparticle drug delivery system that has specific cancer-targeting function holds vast promise for cancer therapy. Here, a fucoidan/poly-lysine-functionalized layer-by-layer ginger-derived lipid vector (LbL-GDLV) was designed to target P-selectin (overexpressed by endothelial cells) and deliver a loaded drug into vascularized colon cancer. , LbL-GDLVs selectively bound to P-selectin, and the degradation of the fucoidan outer layer in a milieu similar to the cancer microenvironment resulted in rapid attachment of the cancer cell and internalization of the remaining positively charged poly-lysine coated-GDLVs. Upon enzymolysis of the poly-lysine layer inside the cancer cell, the GDLV core released loaded doxorubicin (Dox) which had the expected effects. bio-distribution studies showed that intravenously injected LbL-GDLVs exhibited enhanced accumulation at the vascularized tumor site (~ 4.4-fold higher than control vesicles), presumably due to P-selectin-mediated targeting plus the enhanced permeability and retention effect (EPR). In two animal models used to screen anti-cancer efficacy (Luc-HT-29 and HCT-116 xenografts), Dox-loaded LbL-GDLVs (LbL-GDLVs/Dox) significantly inhibited tumor growth and demonstrated much better therapeutic efficiency than free Dox. More importantly, LbL-GDLVs/Dox exhibited excellent biocompatibility, and LbL-GDLVs encapsulation largely reduced the cardiotoxicity of free Dox and avoided the notorious drug resistance of colon cells against free Dox. Together, these findings demonstrate the potential of our newly designed and highly biocompatible plant-derived LbL nanoparticles and their precise colon cancer drug delivery function.
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http://dx.doi.org/10.1002/adtp.201900129DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7546358PMC
December 2019

PI3K inhibitors: review and new strategies.

Chem Sci 2020 Jun 19;11(23):5855-5865. Epub 2020 May 19.

Computational Structural Biology Section , Frederick National Laboratory for Cancer Research , National Cancer Institute at Frederick , Frederick , MD 21702 , USA . Email: ; Tel: +1-301-846-5579.

The search is on for effective specific inhibitors for PI3Kα mutants. PI3Kα, a critical lipid kinase, has two subunits, catalytic and inhibitory. PIK3CA, the gene that encodes the p110α catalytic subunit is a highly mutated protein in cancer. Dysregulation of PI3Kα signalling is commonly associated with tumorigenesis and drug resistance. Despite its vast importance, only recently the FDA approved the first drug (alpelisib by Novartis) for breast cancer. A second (GDC0077), classified as PI3Kα isoform-specific, is undergoing clinical trials. Not surprisingly, these ATP-competitive drugs commonly elicit severe concentration-dependent side effects. Here we briefly review PI3Kα mutations, focus on PI3K drug repertoire and propose new, to-date unexplored PI3Kα therapeutic strategies. These include (1) an allosteric and orthosteric inhibitor combination and (2) taking advantage of allosteric rescue mutations to guide drug discovery.
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http://dx.doi.org/10.1039/d0sc01676dDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7472334PMC
June 2020

Structural Features that Distinguish Inactive and Active PI3K Lipid Kinases.

J Mol Biol 2020 11 10;432(22):5849-5859. Epub 2020 Sep 10.

Computational Structural Biology Section, Frederick National Laboratory for Cancer Research, National Cancer Institute at Frederick, Frederick, MD 21702, USA; Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel. Electronic address:

PI3K lipid kinases signal through the PI3K/Akt pathway, regulating cell growth and proliferation. While the structural features that distinguish between the active and inactive states of protein kinases are well established, that has not been the case for lipid kinases, and neither was the structural mechanism controlling the switch between the two states. Class I PI3Ks are obligate heterodimers with catalytic and regulatory subunits. Here, we analyze PI3K crystal structures. Structures with the nSH2 (inactive state) are featured by collapsed activation loop (a-loop) and an IN kinase domain helix 11 (kα11). In the active state, the a-loop is extended and kα11 in the OUT conformation. Our analysis suggests that the nSH2 domain in the regulatory subunit regulates activation, catalysis and autoinhibition through the a-loop. Inhibition, activation and catalytic scenarios are shared by class IA PI3Ks; the activation is mimicked by oncogenic mutations and the inhibition offers an allosteric inhibitor strategy.
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http://dx.doi.org/10.1016/j.jmb.2020.09.002DOI Listing
November 2020

A novel fluorescent enhancing platform based on DNA-scaffolded silver nanoclusters for potential inflammatory bowel disease-associated microRNA detection.

Talanta 2020 Oct 4;218:121122. Epub 2020 May 4.

Institute of Medical Engineering, Department of Biophysics, School of Basic Medical Sciences, Xi'an Jiaotong University Health Science Center, Xi'an, Shaanxi, 710061, China. Electronic address:

DNA-scaffolded silver nanoclusters (DNA/AgNC) probes are widely used to detect microRNAs (miRNAs) for diagnosing diseases. However, current available DNA/AgNC probes, which primarily based on fluorescence quenching (turn-off) method, suffer from low detection accuracy caused by bio-matrix interferences. Herein, we designed a new DNA/AgNC-cDNA probe to detect miRNA based on a fluorescence enhancing (turn-on) strategy. Using miR-223, a potential biomarker of inflammatory bowel diseases (IBD), as the target miRNA, we devised the partially hybridized DNA/AgNC-cDNA fluorescent probe. The cDNA was the sequence that completely paired against miR-223 and served as a quencher to the fluorescent DNA/AgNC moiety. Upon the presence of miR-223, which could competitively bind the cDNA, then the DNA/AgNC was set free from the DNA/AgNC-cDNA complex accompanied by an increase in the fluorescence of the DNA/AgNC. Further, by fluorescence decay and polyacrylamide gel electrophoresis (PAGE) experiments, we tentatively addressed the probe working mechanism: the restriction of photo-induced electron-transfer from complementary nucleobases to DNA/AgNC. Compared with the traditional fluorescence turn-off approach, our newly designed probe significantly improved the sensitivity (10 times) and demonstrated excellent specificity. This rapid, label-free, and low-cost fluorescence enhancing method can potentially be applied in the diagnosis of miR-223 associated disease, such as IBD.
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http://dx.doi.org/10.1016/j.talanta.2020.121122DOI Listing
October 2020

Effects of rhaponticin on retinal oxidative stress and inflammation in diabetes through NRF2/HO-1/NF-κB signalling.

J Biochem Mol Toxicol 2020 Nov 14;34(11):e22568. Epub 2020 Jul 14.

Department of Biophysics, School of Basic Medical Sciences, Institute of Medical Engineering, Xi'an Jiaotong University Health Science Center, Xi'an, Shaanxi, China.

Oxidative stress and inflammation have long been considered to be responsible for the development and progression of diabetic retinopathy. On the other hand, rhaponticin (RN) has received scientific attention due to its various pharmacological properties. Keeping all these in view, the present study was performed to investigate the potential protective effects of RN on the retina in diabetic rats. Rats were randomly divided into three groups: control group rats, diabetic group rats, diabetic + RN (20 mg/kg body weight for 28 days through oral route) group rats. RN supplementation to diabetic rats significantly prevent the reduction of final body weight loss, reduced weekly fasting blood glucose levels and HbA1c levels with a significant increase in serum insulin levels. quantitative polymerase chain reaction and immunohistochemical analysis found upregulation of Nrf2, NQO-1, HO-1 and upregulation of Keap1 genes and protein distribution along with significantly reduced levels of malondialdehyde and increased activity of superoxide dismutase, catalase and glutathione peroxidase in RN-treated diabetic rats as compared to diabetic rats. Furthermore, treatment of diabetic rats with RN showed downregulated expression of tumour necrosis factor-α, matrix metalloproteinase-2 and upregulated expression of interleukin-10 (IL-10) and TIMP-1 in the retina. RN treatment decreased nuclear factor kappa-light-chain-enhancer of activated B cells distribution and increased IL-10 protein distribution in the retinae of diabetic rats. In addition, RN treatment ameliorated morphological changes observed in retinae of diabetic rats. Altogether, these results provided clear evidence that treatment of diabetic rats with RN attenuated diabetic retinal changes through its hypoglycaemic, antioxidant and anti-inflammatory effects.
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http://dx.doi.org/10.1002/jbt.22568DOI Listing
November 2020

Dynamic Subcellular Localization, Accumulation, and Interactions of Proteins From and Its Associated Betasatellite.

Front Plant Sci 2020 16;11:840. Epub 2020 Jun 16.

State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China.

Geminiviruses contain the largest number of species of plant viruses, and cause devastating crop diseases worldwide. The development of resistance to these viruses will require a clear understanding of viral protein function and interactions. Tomato yellow leaf curl China virus (TYLCCNV) is a typical monopartite geminivirus, which is associated with a tomato yellow leaf curl China betasatellite (TYLCCNB) in the field; the complex infection of TYLCCNV/TYLCCNB leads to serious economic losses in solanaceous plants. The functions of each protein encoded by the TYLCCNV/TYLCCNB complex have not yet been examined in a targeted manner. Here, we show the dynamic subcellular localization and accumulation of six viral proteins encoded by TYLCCNV and the βC1 protein encoded by TYLCCNB in plants over time, and analyzed the effect of TYLCCNV or TYLCCNV/TYLCCNB infection on these parameters. The interaction among the seven viral proteins was also tested in this study: C2 acts as a central player in the viral protein interaction network, since it interacts with C3, C4, V2, and βC1. Self-interactions were also found for C1, C2, and V2. Together, the data presented here provide a template for investigating the function of viral proteins with or without viral infection over time, and points at C2 as a pivotal protein potentially playing a central role in the coordination of the viral life cycle.
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http://dx.doi.org/10.3389/fpls.2020.00840DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7308551PMC
June 2020

Isolation and Characterization of Exosomes from Mouse Feces.

Bio Protoc 2020 Apr;10(8)

Institute for Biomedical Sciences, Center for Diagnostics and Therapeutics, Digestive Disease Research Group, Georgia State University, Atlanta, Georgia, 30302, United States.

Exosomes secreted by colonic epithelial cells are present in feces and contain valuable epigenetic information, such as miRNAs, proteins, and metabolites. An in-depth study of this information is conducive to the diagnosis or treatment of relevant diseases. A crucial prerequisite of such a study is to establish an efficient isolation method, through which we can obtain a relatively more significant amount of exosomes from feces. This protocol is designed to effectively isolate a large number of exosomes from contaminants and other particles in feces by a combined method with fast filtration and sucrose density gradient ultracentrifugation. Exosomes generated by this method are suitable for further RNA, protein, and lipid analysis.
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http://dx.doi.org/10.21769/bioprotoc.3584DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7241525PMC
April 2020

Design principles and fundamental understanding of biosensors for amyloid-β detection.

J Mater Chem B 2020 07;8(29):6179-6196

Department of Chemical, Biomolecular, and Corrosion Engineering, The University of Akron, Ohio, USA.

Alzheimer's disease (AD), as an age-related, progressive neurodegenerative disease, poses substantial challenges and burdens on public health and disease research. While significant research, investment, and progress have been made for the better understanding of pathological mechanisms and risk factors of AD, all clinical trials for AD treatment and diagnostics have failed so far. Since early and accurate diagnostics of AD is key to AD prevention and treatment, the development of probes for AD-related biomarkers is highly important but challenging for AD diagnosis. In this review, emerging evidence highlights the importance of the Aβ cascade hypothesis and indicates a significant role of Aβ and its aggregates as biomarkers in the pathogenesis of AD; we present an up-to-date summary on Aβ-based biosensor systems. Four typical biosensor systems for Aβ detection and representative examples from each type of biosensor are carefully selected and discussed in terms of their sensing strategies, materials, and mechanisms. Finally, we address the remaining challenges and opportunities for the development of future sensing platforms for Aβ detection and Aβ-based diagnostics of AD.
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http://dx.doi.org/10.1039/d0tb00344aDOI Listing
July 2020

Natural-lipid nanoparticle-based therapeutic approach to deliver 6-shogaol and its metabolites M2 and M13 to the colon to treat ulcerative colitis.

J Control Release 2020 07 23;323:293-310. Epub 2020 Apr 23.

Institute for Biomedical Sciences, Digestive Disease Research Group, Georgia State University, Atlanta, GA 30303, USA; Atlanta Veterans Affairs Medical Center, Decatur, GA 30302, USA.

The anti-inflammatory drug candidate, 6-shogaol, has demonstrated excellent efficacies in various in vitro studies. However, its rapid metabolism after oral administration results in poor bioavailability and undetectable in vivo pharmacokinetics. Here, we constructed a natural-lipid (NL) nanoparticle drug delivery system (NP-DDS) to encapsulate 6-shogaol and undertake its controlled release to the proposed drug target (colon). Our in vitro drug-release assay revealed that NL-encapsulated 6-shogaol (6-S-NL) exhibits a delayed drug-release profile compared to free 6-shogaol (free-6-S). Consistent with our expectations, orally administrated 6-S-NL exhibits a superior anti-inflammatory efficacy likely due to the controlled release compared to free 6-S in a dextran sulfate sodium (DSS)-induced mouse model of colitis. Although 6-S-NL treatment yields an enhanced concentration of 6-shogaol at the target site (colon), this concentration is still far below the effective level. We hypothesize that the released 6-shogaol undergoes rapid metabolism and that the metabolites of 6-shogaol may contribute to the anti-inflammatory efficacy of 6-S-NL. We thus examined the in vitro anti-inflammatory efficacies of two highly abundant colonic metabolites, M2 (a cysteine-conjugated metabolite) and M13 (a glutathione-conjugated metabolite), against macrophage cells. Reverse transcription-polymerase chain reaction (RT-PCR) data showed that both M2 and M13 (at 1.0 μg/mL) could down-regulate pro-inflammatory factors (TNF-α, IL-1β, and IL-6) and up-regulate an anti-inflammatory factor (IL-10) in inflamed Raw 264.7 cells. Subsequent in vitro wound-healing assays also confirmed that M2 and M13 accelerate the wound recovery process of Caco-2 cells at the concentrations seen in the colon (1.0 μg/mL). Further, in the DSS-induced mouse model of colitis, oral administration of M2- or M13-loaded NL nanoparticles (M2-NL, M13-NL) demonstrated excellent in vivo wound-healing effects, and these activities were better than those observed for 6-S-NL. Combined with the 6-S-NL's bio-distribution assay, our data show that: the 6-shogaol metabolites, M2 and M13, are more potent anti-inflammatory compounds than 6-shogaol itself; NL nanoparticles can effectively deliver 6-shogaol to the colon, with little accumulation seen in the kidney or liver; and the actions of M2 and M13 mostly confer the anti-inflammatory effect of 6-S-NL. Our results explained the discrepancy between the low tissue concentrations of NL delivered 6-shogaol and its effectiveness against ulcerative colitis (UC) in a mouse model. This study paved the way for further developing the NL-loaded active metabolites, M2 or M13, as novel targeted therapeutic approaches for curing UC.
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http://dx.doi.org/10.1016/j.jconrel.2020.04.032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7299768PMC
July 2020

The quaternary assembly of KRas4B with Raf-1 at the membrane.

Comput Struct Biotechnol J 2020 25;18:737-748. Epub 2020 Mar 25.

Computational Structural Biology Section, Basic Science Program, Frederick National Laboratory for Cancer Research, Frederick, MD 21702, USA.

Proximally located in the membrane, oncogenic Ras dimers (or nanoclusters) can recruit and promote Raf dimerization and MAPK (Raf/MEK/ERK) signaling. Among Ras isoforms, KRas4B is the most frequently mutated. Recent data on the binary KRas4B-Raf-1 complex suggested that Raf-1 CRD not only executes membrane anchorage, but also supports the high-affinity interaction of Raf-1 RBD with KRas4B catalytic domain. For a detailed mechanistic picture of Raf activation at the membrane, we employ explicit MD simulations of the quaternary KRas4B-Raf-1 complex. The complex contains two active GTP-bound KRas4B proteins forming a dimer through the allosteric lobe interface and two tandem RBD-CRD segments of Raf-1 interacting with the effector lobes at both ends of the KRas4B dimer. We show that Raf-1 RBD-CRD supports stable KRas4B dimer at preferred interface and orientation at the membrane, thereby cooperatively enhancing the affinity of the KRas4B-Raf-1 interaction. We propose that a Ras dimer at the membrane can increase the population of proximal Raf kinase domains, promoting kinase domain dimerization in the cytoplasm. Collectively, the dynamic Ras-Raf assembly promotes Raf activation not by allostery; instead, Ras activates Raf by shifting its ensemble toward kinase domain-accessible states through enhanced affinity at the membrane.
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http://dx.doi.org/10.1016/j.csbj.2020.03.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7125320PMC
March 2020

The Mystery of Rap1 Suppression of Oncogenic Ras.

Trends Cancer 2020 05 2;6(5):369-379. Epub 2020 Mar 2.

VIB Center for the Biology of Disease and KU Leuven Department of Oncology, Leuven Cancer Institute, Leuven, Belgium.

Decades ago, Rap1, a small GTPase very similar to Ras, was observed to suppress oncogenic Ras phenotype, reverting its transformation. The proposed reason, persisting since, has been competition between Ras and Rap1 for a common target. Yet, none was found. There was also Rap1's puzzling suppression of Raf-1 versus activation of BRAF. Reemerging interest in Rap1 envisages capturing its Ras suppression action by inhibitors. Here, we review the literature and resolve the enigma. In vivo oncogenic Ras exists in isoform-distinct nanoclusters. The presence of Rap1 within the nanoclusters reduces the number of the clustered oncogenic Ras molecules, thus suppressing Raf-1 activation and mitogen-activated protein kinase (MAPK) signaling. Nanoclustering suggests that Rap1 suppression is Ras isoform dependent. Altogether, a potent Rap1-like inhibitor appears unlikely.
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http://dx.doi.org/10.1016/j.trecan.2020.02.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7211489PMC
May 2020

Charge-reversal nanocarriers: An emerging paradigm for smart cancer nanomedicine.

J Control Release 2020 03 14;319:46-62. Epub 2019 Dec 14.

The Second Affiliated Hospital and Yuying Children's Hospital, Wenzhou Medical University, 109 Xueyuan West Road, Wenzhou, Zhejiang 325027, PR China; WMU-Monash University BDI Alliance in Clinical & Experimental Biomedicine, Wenzhou Medical University, Wenzhou, Zhejiang 325027, PR China. Electronic address:

The surface charge of nanoparticles (NPs) plays an essential role in determining their biological properties both in vitro and in vivo. In view of the complex features associated with the biological or physiological microenvironment of solid tumors, such as electrostatic interactions between NPs and serum components, cellular membrane, or intracellular organelles, drug-loaded NPs (or nanocarriers) should intelligently accommodate such unique extra- or intracellular microenvironment in order to achieve maximum therapeutic and/or diagnostic efficacy. To that end, the surface charge of nanocarriers needs to be readily converted at the target site by means of charge reversal, i.e., conversion from anionic to cationic, or vice versa, depending on specific microenvironment. In such a manner, the payloads could be efficiently released at the desired tumor site. This review discusses 1) the physicochemical aspects related to long-circulating nanocarriers for systemic applications; 2) the recent progress in charge-reversal nanocarriers, which are loaded with drugs, nucleic acids, proteins or imaging agents and triggered by various biological signals (i.e., pH, redox, ROS or enzyme) associated with tumor microenvironment, with an emphasis on those induced by acidic tumoral pH; and 3) the perspectives of charge-reversal nanocarriers regarding thorough investigations on how the chemical structure of charge-reversal moiety temporally affects the responsiveness of the resulting nanocarriers toward the rational design of precision cancer nanomedicine.
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http://dx.doi.org/10.1016/j.jconrel.2019.12.024DOI Listing
March 2020

Isolation, Purification, and Characterization of Ginger-derived Nanoparticles (GDNPs) from Ginger, Rhizome of .

Bio Protoc 2019 Oct;9(19)

Institute for Biomedical Science, Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA, USA.

Factors implicated in the pathophysiology of intestinal inflammation include defects in intestinal epithelial barrier function, abnormal immune responses, and activities of the gut microbiota. Current agents used to treat human Inflammatory Bowels Disease (IBD), chronic inflammation of digestive tract, have serious side effects. In addition, most of these treatments target the damaging factors while not providing pro-healing factors that repair the damaged intestine. Here we provide a method to isolate, purify and characterize a specific population from ginger (ginger-derived nanoparticles: GDNPs 2) with anti-inflammatory activities. GDNPs 2 as a drug vehicle are a novel natural, nontoxic delivery system, which target the inflamed intestinal mucosa, blocks damaging factors while promoting pro-healing factors and could easily be developed for large-scale production aimed at the treatment of IBD.
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http://dx.doi.org/10.21769/BioProtoc.3390DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6857645PMC
October 2019

Ca-Dependent Switch of Calmodulin Interaction Mode with Tandem IQ Motifs in the Scaffolding Protein IQGAP1.

Biochemistry 2019 12 26;58(49):4903-4911. Epub 2019 Nov 26.

Center for Cancer Research, National Cancer Institute , National Institutes of Health , Frederick , Maryland 20892 , United States.

IQ domain GTPase-activating scaffolding protein 1 (IQGAP1) mediates cytoskeleton, cell migration, proliferation, and apoptosis events. Calmodulin (CaM) modulates IQGAP1 functions by binding to its four tandem IQ motifs. Exactly how CaM binds the IQ motifs and which functions of IQGAP1 CaM regulates and how are fundamental mechanistic questions. We combine experimental pull-down assays, mutational data, and molecular dynamics simulations to understand the IQ-CaM complexes with and without Ca at the atomic level. Apo-CaM favors the IQ3 and IQ4 motifs but not the IQ1 and IQ2 motifs that lack two hydrophobic residues for interactions with apo-CaM's hydrophobic pocket. Ca-CaM binds all four IQ motifs, with both N- and C-lobes tightly wrapped around each motif. Ca promotes IQ-CaM interactions and increases the amount of IQGAP1-loaded CaM for IQGAP1-mediated signaling. Collectively, we describe IQ-CaM binding in atomistic detail and feature the emergence of Ca as a key modulator of the CaM-IQGAP1 interactions.
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http://dx.doi.org/10.1021/acs.biochem.9b00854DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8195445PMC
December 2019