Fibroblast Activation Protein-Targeting Minibody-IRDye700DX for Ablation of the Cancer-Associated Fibroblast with Photodynamic Therapy.

Fibroblast activation protein (FAP), expressed on cancer-associated fibroblasts, is a target for diagnosis and therapy in multiple tumour types. Strategies to systemically deplete FAP-expressing cells show efficacy; however, these induce toxicities, as FAP-expressing cells are found in normal tissues. FAP-targeted photodynamic therapy offers a solution, as it acts only locally and upon activation. Here, a FAP-binding minibody was conjugated to the chelator diethylenetriaminepentaacetic acid (DTPA) and the photosensitizer IRDye700DX (DTPA-700DX-MB). DTPA-700DX-MB showed efficient binding to FAP-overexpressing 3T3 murine fibroblasts (3T3-FAP) and induced the protein's dose-dependent cytotoxicity upon light exposure. Biodistribution of DTPA-700DX-MB in mice carrying either subcutaneous or orthotopic tumours of murine pancreatic ductal adenocarcinoma cells (PDAC299) showed maximal tumour uptake of 111In-labelled DTPA-700DX-MB at 24 h post injection. Co-injection with an excess DTPA-700DX-MB reduced uptake, and autoradiography correlated with FAP expression in the stromal tumour region. Finally, in vivo therapeutic efficacy was determined in two simultaneous subcutaneous PDAC299 tumours; only one was treated with 690 nm light. Upregulation of an apoptosis marker was only observed in the treated tumours. In conclusion, DTPA-700DX-MB binds to FAP-expressing cells and targets PDAC299 tumours in mice with good signal-to-background ratios. Furthermore, the induced apoptosis indicates the feasibility of targeted depletion of FAP-expressing cells with photodynamic therapy.

Biochar inhibited hydrogen radical-induced Cd bioavailability in a paddy soil.

Herein, hydrogen (H·) radical was observed as a new pathway to produce hydroxyl (OH·) radicals that promoted cadmium sulfide (CdS) dissolution and thus Cd solubility in paddy soils. In soil incubation experiments, the bioavailable Cd concentrations in flooded paddy soils were increased by 8.44 % as the soil was aerated for 3d. For the first time, the H· radical was observed in aerated soil sludge. The association of CdS dissolution with free radicals was thereafter confirmed in an electrolysis experiment. Both H· and OH· radicals in electrolyzed water were confirmed by the electron paramagnetic resonance analysis. In the system with CdS, water electrolysis increased soluble Cd2+ concentration by 60.92 times, which was compromised by 43.2 % when the radical scavenger was introduced. This confirmed the free radicals can lead to oxidative dissolution of CdS. The H· radical was generated in systems with fulvic acid or catechol irradiated by ultraviolet lights, indicating soil organic carbon could be an important precursor for H· and OH· radicals. Biochar application decreased soil DTPA-Cd by 22-56 % invoking mechanisms besides adsorption. First, biochar quenched radicals and reduced CdS dissolution by 23.6 % in electrolyzed water in which -C-OH of biochar was oxidized to CO. Second, biochar boosted Fe/S-reducing bacteria and thus compromised CdS dissolution, as affirmed by a reversal correlation between soil available Fe2+ and DTPA-Cd concentrations. A similar phenomenon occurred in Shewanella oneidensis MR-1-inoculated soils. This study provided new insights into the bioavailability of Cd and offered feasible measures to remediate Cd-contaminated paddy soils with biochars.

Effects of mercapto-palygorskite application on cadmium accumulation of soil aggregates at different depths in Cd-contaminated alkaline farmland.

Mercapto-palygorskite (MP) is a novel immobilization material for cadmium (Cd) pollution, but the immobilization mechanism on alkaline Cd contaminated soil is not completely clear. In this paper, field experiment was carried out to study the effect of MP on the transfer of Cd in aggregates at different depth, the contribution of soil aggregates to the reduction of Cd in bulk soil and the immobilization mechanism of MP. The results showed that MP had no significant influence on the total Cd content, soil aggregates distribution, pH value, CEC value and enzyme activities no matter at any depth. At the depth of 0-20 cm, MP significantly reduced the DTPA-Cd in bulk soil by 60.7%, and increased the GWD and R0.25 value. Similarly, the content of DTPA-Cd in the soil aggregates was deceased by 40.2-63.6%, the OM, DOC, available Fe, Mn and S in soil aggregates were significantly increased by 15.0-19.1%, 19.2-41.7%, 24.7-41.2% and 12.5-35.1% respectively. The Cd fraction of aggregates, especially exchangeable Cd (EXE-Cd) and bound to Fe/Mn oxide Cd (OX-Cd), was reduced by 5.4-28.1% and increased by 22.3-50.4%. In addition, MP had different effects on the GSF value of soil aggregates, but there was a downward trend for AFX value at 0-20 cm soil depth. MP almost had no significant influence on the above indexes at the depth of 20-40 cm and 40-60 cm, but except the Cd fraction, the GSF and AFX value in individual aggregates. Small aggregates (<1 mm) and large aggregates (>1 mm) contributed 59.1% and 22% to the reduction of Cd in bulk soil. Partial Least Structural Equation Model (PL-SEM) revealed that S promoted the production of available Fe, Mn, OM and DOC, while the content of DOC inhibited the formation of EXE-Cd and the available Fe and Mn boosted the production of OX-Cd.

Natural field freeze-thaw process leads to different performances of soil amendments towards Cd immobilization and enrichment.

Cadmium (Cd) soil pollution is a global issue affecting crop production and food safety. Remediation methods involving in-situ Cd immobilization have been developed, but their effectiveness can diminish under seasonal freeze-thaw aging processes. In this study, we assessed the field performance of four soil treatments at a seasonally frozen rice paddy. Amendments were applied at 2 wt%, including: (i) sepiolite (a 2:1 clay mineral), (ii) superphosphate, (iii) biochar (produced by rice husk at 500 °C for 2 h), and (iv) joint application of biochar & superphosphate (1:1 mixture by weight). Immobilization performance was determined as DTPA extractable Cd and plant uptake in various organs. Overall, the four treatments significantly reduced Cd bioavailability during the plant growth period, with average DTPA-extractable concentrations decreasing by 43%, 34%, 39% and 45% for the four treatments, respectively, relative to untreated soil (control). Rice grain yields from the superphosphate and the joint application treatments increased by 8.0% and 11.8%, respectively, and Cd accumulation within those grains reduced by 14.3% and 48.9%, respectively. During the winter non-growth period, freeze-thaw aging facilitated Cd mobilization, with DTPA-extractable Cd increasing by 16.9% in the control soil, relative to the initial period. However, this reduced to 10.9%, 14.4%, 7.6% and 5.0%, for the sepiolite, superphosphate, biochar and joint application treatments, respectively. Overall, the joint application of biochar and superphosphate provided the best performance in terms of both long-term Cd immobilization and rice production enhancement, offering a green remediation option for risk management at Cd contaminated rice paddies in seasonally frozen regions.

Bacterial hydrophilins promote pathogen desiccation tolerance.

Acinetobacter baumannii is a leading cause of hospital-acquired infections, where outbreaks are driven by its ability to persist on surfaces in a desiccated state. Here, we show that A. baumannii causes more virulent pneumonia following desiccation and profile the genetic requirements for desiccation. We find that desiccation tolerance is enhanced upon the disruption of Lon protease, which targets unfolded and aggregated proteins for degradation. Notably, two bacterial hydrophilins, DtpA and DtpB, are transcriptionally upregulated in Δlon via the two-component regulator, BfmR. These proteins, both hydrophilic and intrinsically disordered, promote desiccation tolerance in A. baumannii. Additionally, recombinant DtpA protects purified enzymes from inactivation and improves the desiccation tolerance of a probiotic bacterium when heterologously expressed. These results demonstrate a connection between environmental persistence and pathogenicity in A. baumannii, provide insight into the mechanisms of extreme desiccation tolerance, and reveal potential applications for bacterial hydrophilins in the preservation of protein- and live bacteria-based pharmaceuticals.

Optimum Olsen Phosphorus/ZincDTPA ratio for the initial growth of maize in agricultural soils of the Mediterranean region.

BACKGROUND: Zinc (Zn) deficiency in crops is commonly aggravated by high levels of phosphorus (P) in soil. In this work, the initial performance of pot-growing maize in response to the available P and Zn in soils with low available Zn and to the application of P and Zn fertilizers was investigated. RESULTS: The soils (six non-calcareous and 14 calcareous) ranged widely in available P (Olsen P: 5.5-37.9 mg kg-1 ), were poor in available Zn [diethylenetriaminepentaacetic acid-extractable Zn (ZnDTPA ): 0.20-0.84 mg kg-1 ] and had an Olsen P/ZnDTPA ratio of 13 to 111 mg mg-1 . Soil P application generally increased aerial dry matter (ADM) yield; Zn increased ADM yield mostly when applied in combination with P; and the sole application of Zn increased yield only in a soil with a high (28 mg kg-1 ) Olsen P and a low (0.36 mg kg-1 ) ZnDTPA . The increase in ADM yield resulting from optimal application of P and/or Zn to the soil was modest in soils where the Olsen P/ZnDTPA ratio was 30-60 and Olsen P was >14 mg kg-1 . Zinc uptake by the control plants was correlated with the ZnDTPA of the soil. For a certain ZnDTPA value, the level of plant available Zn was higher in non-calcareous than in calcareous soils. CONCLUSION: Soil application of fertilizer P and Zn, in soils with low levels of available Zn, should not only aim at increasing the available P and Zn levels but also balancing them at the appropriate Olsen P/ZnDTPA ratio, which was found to lie in the 30-60 range in the present study. © 2020 Society of Chemical Industry.

Measuring the Pancreatic ß Cell Mass in Vivo with Exendin SPECT during Hyperglycemia and Severe Insulitis.

OBJECTIVE: Targeting the glucagon-like peptide-1 receptor with radiolabeled exendin is a very promising method to noninvasively determine the ß cell mass in the pancreas, which is needed to unravel the pathophysiology of type 1 and type 2 diabetes. The present study aimed to explore the effects of both hyperglycemia and insulitis on the uptake of exendin in a spontaneous type 1 diabetes mouse model, nonobese diabetic (NOD) mice. METHODS: NOD mice (n = 75, 7-21 weeks old) were injected intravenously with [111In]In-DTPA-exendin-3, and single-photon emission computed tomography (SPECT) images were acquired 1 h pi. The pancreatic accumulation of [111In]In-DTPA-exendin-3 was quantified in vivo using SPECT and by ex vivo counting and correlated to the ß cell mass (BCM). The influence of insulitis and hyperglycemia on the exendin uptake was assessed. RESULTS: The pancreas could be visualized longitudinally using SPECT. A linear correlation was found between the BCM (%) and pancreatic uptake (%ID/g) as measured by ex vivo counting (Pearson r = 0.64, p < 0.001), which was not affected by either insulitis (Pearson r = 0.66, p = 0.83) or hyperglycemia (Pearson r = 0.57, p = 0.51). Biodistribution and ex vivo autoradiography revealed remaining [111In]In-DTPA-exendin-3 uptake in the pancreas despite total ablation of BCM. CONCLUSIONS: Despite hyperglycemia and severe insulitis, we have found a good correlation between BCM and pancreatic exendin uptake, even in a suboptimal model with relatively high background activity.

Equilibrium, affinity, dissociation constants, IC5O: Facts and fantasies.

The interaction between ligands and receptors is often described in terms of 50% inhibitory concentrations (IC50). However, IC50 values do not accurately reflect the dissociation constants (Kd), and the domain of application and precision of proposed approximations for Kd estimation are unclear. The effect of affinity and of experimental conditions on the differences between IC50 and Kd has been assessed from exact mass action law calculations and from computer simulations. Competitions between [111 In]DTPA-indium and a few metal-DTPA complexes for binding to a specific antibody are discussed as a practical example. Exact calculations of competition assays have been implemented in Microsoft Excel and performed for a variety of concentrations of receptor, tracer, and competitor. The results are identical to those of software packages. IC50 is found larger than Kd by less than 20% only when tracer concentration is small compared with Kd and to the receptor concentration and when this receptor concentration is small compared with Kd. Otherwise, Kd and IC50 may be very different and approximations proposed in the literature to obtain Kd values from graphically derived IC50 are not acceptable as soon as the concentrations of tracer or of receptor approach Kd. Under most experimental conditions, IC50 values do not reflect Kd values. Using available software packages to determine and report Kd values would allow for more meaningful comparisons of results obtained under different experimental conditions.

Delivery of DTPA through Liposomes as a Good Strategy for Enhancing Plutonium Decorporation Regardless of Treatment Regimen.

In this study, we assessed the efficacy of unilamellar 110-nm liposomes encapsulating the chelating agent diethylenetriaminepentaacetic acid (DTPA) in plutonium-exposed rats. Rats were contaminated by intravenous administration of the soluble citrate form of plutonium. The comparative effects of liposomal and free DTPA at similar doses were examined in terms of limitation of alpha activity burden in rats receiving various treatment regimens. Liposomal DTPA given at 1 h after contamination more significantly prevented the accumulation of plutonium in tissues than did free DTPA. Also, when compared to free DTPA, liposome-entrapped DTPA was more efficient when given at late times for mobilization of deposited plutonium. In addition, repeated injections of liposomal DTPA further improved the removal of plutonium compared to single injection. Various possible mechanisms of action for DTPA delivered through liposomes are discussed. The advantage of liposomal DTPA over free DTPA was undoubtedly directly and indirectly due to the better cell penetration of DTPA when loaded within liposomes, mainly in the tissues of the mononuclear phagocytic system. The decorporation induced by liposomal DTPA may result first from intracellular chelation of plutonium deposited in soft tissues, predominantly in the liver. Afterwards, the slow release of free DTPA molecules from these same tissues may enable a sustained action of DTPA, probably mainly by extracellular chelation of plutonium available on bone surfaces. In conclusion, decorporation of plutonium can be significantly improved by liposomal encapsulation of DTPA regardless of the treatment regimen applied.

Zinc ions regulate opening of tight junction favouring efflux of macromolecules via the GSK3ß/snail-mediated pathway.

Zinc is an essential trace element presenting in particularly high concentration in the brain. In some regions, e.g. lateral amygdala, subiculum and hippocampus, rapidly-exchangeable zinc may transiently reach even up to 600 µM. To explore the possible roles of high-concentration Zn2+ in regulating the blood-brain barrier (BBB), we investigated the effects of Zn2+ on the functions and structures of the tight junction (TJ) with an in vitro model of a Madin-Darby canine kidney (MDCK) cell monolayer. The experimental results indicated that high concentrations (>200 µM) of Zn2+ can affect the TJ integrity in a polarized manner. Basolateral addition of Zn2+ led to reversible TJ opening with pore paths of r ∼ 2 nm or more depending on Zn2+ concentration. The efflux/influx ratios of different sized probes were found to be ∼4.6 for FD4 (MW 4000) and ∼1.8 for Eu-DTPA (MW 560), suggesting that the Zn2+-induced paracelluar channels favour efflux especially for macromolecules. Further mechanistic studies revealed that the elevated intracellular Zn2+ taken from the basolateral side can increase phosphorylation of glycogen synthase kinase (GSK) 3ß, primarily due to the inhibition of calcineurin (CaN), thus resulting in the elevation of the snail transcriptional repressors. Subsequently, Zn2+ can cause the down-regulation of claudin-1, breakage of occludin and ZO-1 rings, and collapse of basolateral F-actin structures. These overall factors result in the formation of a trumpet-like paracellular channel, which allows asymmetric solute permeation. The ERK1/2 and JNK1/2 pathways may also be involved in the Zn2+-induced TJ opening process, while the activation of matrix metalloproteinase was not observed. Our results may suggest a potential role of zinc in regulation of BBB permeability associated with brain clearance of metabolites through the glymphatic system.

Second-order Kinetics of DTPA and Plutonium in Rat Plasma.

In 2008, Serandour et al. reported on their in vitro experiment involving rat plasma samples obtained after an intravenous intake of plutonium citrate. Different amounts of DTPA were added to the plasma samples and the percentage of low-molecular-weight plutonium measured. Only when the DTPA dosage was three orders of magnitude greater than the recommended 30 µmol/kg was 100% of the plutonium apparently in the form of chelate. These data were modeled assuming three competing chemical reactions with other molecules that bind with plutonium. Here, time-dependent second-order kinetics of these reactions are calculated, intended eventually to become part of a complete biokinetic model of DTPA action on actinides in laboratory animals or humans. The probability distribution of the ratio of stability constants for the reactants was calculated using Markov Chain Monte Carlo. These calculations substantiate that the inclusion of more reactions is needed in order to be in agreement with known stability constants.

False-Positive Diagnosis of Brain Death Following the Pediatric Guidelines: Case Report and Discussion.

A 2-year-old boy with severe head trauma was diagnosed brain dead according to the 2011 Pediatric Guidelines. Computed tomographic (CT) scan showed massive cerebral edema with herniation. Intracranial pressures were extremely high, with cerebral perfusion pressures around 0 for several hours. An apnea test was initially contraindicated; later, one had to be terminated due to oxygen desaturation when the Pco2 had risen to 57.9 mm Hg. An electroencephalogram (EEG) was probably isoelectric but formally interpreted as equivocal. Tc-99m diethylene-triamine-pentaacetate (DTPA) scintigraphy showed no intracranial blood flow, so brain death was declared. Parents declined organ donation. A few minutes after withdrawal of support, the boy began to breathe spontaneously, so the ventilator was immediately reconnected and the death declaration rescinded. Two hours later, life support was again removed, this time for prognostic reasons; he did not breathe, and death was declared on circulatory-respiratory grounds. Implications regarding the specificity of the guidelines are discussed.

Decorporation Approach after Rat Lung Contamination with Plutonium: Evaluation of the Key Parameters Influencing the Efficacy of a Protracted Chelation Treatment.

While the efficacy of a protracted zinc (Zn)- or calcium (Ca)-diethylenetriaminepentaacetic acid (DTPA) treatment in reducing transuranic body burden has already been demonstrated, questions about therapeutic variables remain. In response to this, we designed animal experiments primarily to assess both the effect of fractionation of a given dose and the effect of the frequency of dose fraction, with the same total dose. In our study, rats were contaminated intravenously with plutonium (Pu) then treated several days later with Ca-DTPA given at once or in various split-dose regimens cumulating to the same total dose and spread over several days. Similar efficacies were induced by the injection of the total dose or by splitting the dose in several smaller doses, independent of the number of doses and the dose level per injection. In a second study, rats were pulmonary contaminated, and three weeks later they received a Ca-DTPA dose 11-fold higher than the maximal daily recommended dose, administered either as a single bolus or as numerous multiple injections cumulating to the same dose, based on different injection frequency schedules. Independent of frequency schedule, the various split-dose regimens spread over weeks/months were as efficient as single delivery of the total dose in mobilizing lung plutonium, and had a therapeutic advantage for removal of retained hepatic and bone plutonium burdens. We concluded that cumulative dose level was a therapeutic variable of greater importance than the distribution of split doses for the success of a repeated treatment regimen on retained tissue plutonium. In addition, pulmonary administration of clodronate, which aims at killing alveolar macrophages and subsequently releasing their plutonium content, and which is associated with a continuous Ca-DTPA infusion regimen, suggested that the efficacy of injected Ca-DTPA in decorporating lung deposit is limited, due to its restricted penetration into alveolar macrophages and not because plutonium, as a physicochemical form, is unavailable for chelation.

Lung fluid clearance in chronic heart failure patients.

Chronic elevation of pulmonary microvascular pressure (Pmv) consistently leads to alveolocapillary barrier thickening and reduction in the filtration coefficient. In animal models of chronic heart failure (CHF) the lung remains dry despite hydrostatic forces. As fluid flux is bi-directional, it has been postulated that an increase in alveolar fluid clearance may facilitate the dry lung when Pmv is chronically elevated. In this study we aimed to examine alveolar fluid clearance in ambulatory patients with CHF secondary to left ventricular (LV) systolic dysfunction compared against non-CHF controls. Lung clearance following aerosol delivery of 99mtechnetium (Tc)-diethyl triaminepentaacetic acid (DTPA) was measured non-invasively by scintigraphy and half time of 99mTc-DTPA clearance (T (1/2)) was calculated by mono-exponential curve fit. Alveolar fluid clearance measured as half time DTPA clearance was significantly faster in CHF patients than controls (P=0.001). This was further defined by NYHA classification. No correlation was found between DTPA clearance and plasma epinephrine, norepinephrine or aldosterone hormone (P>0.05). Our results support an association between increasing alveolar fluid clearance and disease severity in CHF, and the concept of controlled bi-directional fluid flux in CHF associated with increasing Pmv, and represents another defence mechanism of the lung against pulmonary oedema.

Decorporation of Pu/Am Actinides by Chelation Therapy: New Arguments in Favor of an Intracellular Component of DTPA Action.

Diethylenetriaminepentaacetic acid (DTPA) is currently still the only known chelating drug that can be used for decorporation of internalized plutonium (Pu) and americium (Am). It is generally assumed that chelation occurs only in biological fluids, thus preventing Pu/Am deposition in target tissues. We postulate that actinide chelation may also occur inside cells by a mechanism called "intracellular chelation". To test this hypothesis, rats were given DTPA either prior to (termed "prophylactic" treatment) or belatedly after (termed "delayed" treatment) Pu/Am injection. DTPA decorporation efficacy was systematically tested for both plutonium and americium. Both prophylactic and delayed DTPA elicited marked decreases in liver Pu/Am. These results can be explained by chelation within subcellular compartments where DTPA efficacy increased as a function of a favorable intracellular DTPA-to-actinide molar ratio. The efficacy of intracellular chelation of liver actinides decreased with the delay of treatment. This is probably explained by progressive actinide binding to the high-affinity ligand ferritin followed by migration to lysosomes. Intracellular chelation was reduced as the gap between prophylactic treatment and contamination increased. This may be explained by the reduction of the intracellular DTPA pool, which declined exponentially with time. Skeletal Pu/Am was also reduced by prophylactic and delayed DTPA treatments. This decorporation of bone actinides may mainly result from extracellular chelation on bone surfaces. This work provides converging evidence for the involvement of an intracellular component of DTPA action in the decorporation process. These results may help to improve the interpretation of biological data from DTPA-treated contamination cases and could be useful to model DTPA therapy regimens.

Biotransformation Capacity of Carboxylesterase in Skin and Keratinocytes for the Penta-Ethyl Ester Prodrug of DTPA.

The penta-ethyl ester prodrug of the chelating agent diethylene triamine pentaacetic acid (DTPA), referred to as C2E5, effectively accelerated clearance of americium after transdermal delivery. Carboxylesterases (CESs) play important roles in facilitating C2E5 hydrolysis. However, whether CESs in human skin hydrolyze C2E5 remains unknown. We evaluated the gene and protein expression of CESs in distinctive human epidermal cell lines: HEKa, HEKn, HaCaT, and A431. The substrates p-nitrophenyl acetate (pNPA) and 4-nitrophenyl valerate (4-NPV) were used to access esterase and CES activity. C2E5 hydrolysis was measured by radiometric high-performance liquid chromatography after incubation of [(14)C]C2E5 with supernatant fractions after centrifugation at 9000g (S9) prepared from skin cell lines. CES-specific inhibitors were used to access metabolism in human skin S9 fractions with analysis by liquid chromatography-tandem mass spectrometry. We identified the human carboxylesterase 1 and 2 (CES1 and CES2) bands in a Western blot. The gene expression of these enzymes was supported by a real-time polymerase chain reaction (qPCR). pNPA and 4-NPV assays demonstrated esterase and CES activity in all the cell lines that were comparable to human skin S9 fractions. The prodrug C2E5 was hydrolyzed by skin S9 fractions, resulting in a primary metabolite, C2E4. In human skin S9 fractions, inhibition of C2E5 hydrolysis was greatest with a pan-CES inhibitor (benzil). CES1 inhibition (troglitazone) was greater than CES2 (loperamide), suggesting a primary metabolic role for CES1. These results indicate that human keratinocyte cell lines are useful for the evaluation of human cutaneous metabolism and absorption of ester-based prodrugs. However, keratinocytes from skin provide a small contribution to the overall metabolism of C2E5.

Development and Characterization of the Recombinant Human VEGF-EGF Dual-Targeting Fusion Protein as a Drug Delivery System.

The design, preparation, as well as structural and functional characterizations of the recombinant fusion protein hVEGF-EGF as a dual-functional agent that may target both EGFR (R: receptor) and angiogenesis are reported. hVEGF-EGF was found to bind to EGFR more strongly than did EGF, and to bind to VEGFR similarly to VEGF. Mass spectrometry measurements showed that the sites of DTPA (diethylenetriaminepentaacetic acid) conjugated hVEGF-EGF (for radiolabeling) were the same as those of its parent hEGF and hVEGF proteins. All DTPA-conjugated proteins retained similar binding capacities to their respective receptors as compared to their respective parent proteins. In vitro cell binding studies using BAEC (a bovine aortic endothelial cell) and MDA-MB-231 (a human breast cancer) cells expressing both EGFR and VEGFR confirmed similar results. Treating BAEC cells with hVEGF-EGF induced remarkable phosphorylation of EGFR, VEGFR, and their downstream targets ERK1/2. Nevertheless, the radiolabeled (111)In-DTPA-hVEGF-EGF showed cytotoxicity against MDA-MB-231 cells. Pharmacokinetic studies using (111)In-DTPA-hVEGF-EGF in BALB/c nude mice showed that appreciable tracer activities were accumulated in liver and spleen. In all, this study demonstrated that the fusion protein hVEGF-EGF maintained the biological specificity toward both EGFR and VEGFR and may be a potential candidate as a dual-targeting moiety in developing anticancer drugs.

Development of magnetic separation and quantum dots labeled immunoassay for the detection of mercury in biological samples.

A rapid and sensitive immunoassays of mercury (Hg) in biological samples was developed using quantum dots (QDs) and magnetic beads (MBs) as fluorescent and separated probes, respectively. A monoclonal antibody (mAb) that recognizes an Hg detection antigen (BSA-DTPA-Hg) complex was produced by the injection of BALB/c mice with an Hg immunizing antigen (KLH-DTPA-Hg). Then the ascites monoclonal antibodies were purified. The Hg monoclonal antibody (Hg-mAb) is conjugated with MBs to separate Hg from biological samples, and the other antibody, which is associated with QDs, is used to detect the fluorescence. The Hg in biological samples can be quantified using the relationship between the QDs fluorescence intensity and the concentration of Hg in biological samples following magnetic separation. In this method, the detection linear range is 1-1000ng/mL, and the minimum detection limit is 1ng/mL. The standard addition recovery rate was 94.70-101.18%. The relative standard deviation values were 2.76-7.56%. Furthermore, the Hg concentration can be detected in less than 30min, the significant interference of other heavy metals can be avoided, and the simultaneous testing of 96 samples can be performed. These results indicate that the method could be used for rapid monitoring Hg in the body.

Inhibitory potential of three zinc chelating agents against the proteolytic, hemorrhagic, and myotoxic activities of Echis carinatus venom.

Viperbites undeniably cause local manifestations such as hemorrhage and myotoxicity involving substantial degradation of extracellular matrix (ECM) at the site of envenomation and lead to progressive tissue damage and necrosis. The principle toxin responsible is attributed to snake venom metalloproteases (SVMPs). Treatment of such progressive tissue damage induced by SVMPs has become a challenging task for researchers and medical practitioners who are in quest of SVMPs inhibitors. In this study, we have evaluated the inhibitory potential of three specific zinc (Zn(2+)) chelating agents; N,N,N',N'-tetrakis (2-pyridylmethyl) ethane-1,2-diamine (TPEN), diethylene triamine pentaacetic acid (DTPA), tetraethyl thiuram disulfide (TTD) on Echis carinatus venom (ECV) induced hemorrhage and myotoxicity. Amongst them, TPEN has high affinity for Zn(2+) and revealed potent inhibition of ECV metalloproteases (ECVMPs) in vitro (IC50: 6.7 µM) compared to DTPA and TTD. The specificity of TPEN towards Zn(2+) was confirmed by spectral and docking studies. Further, TPEN, DTPA, and TTD completely blocked the hemorrhagic and myotoxic activities of ECV in a dose dependent manner upon co-injection; whereas, only TPEN successfully neutralized hemorrhage and myotoxicity following independent injection. Histological examinations revealed that TPEN effectively prevents degradation of dermis and basement membrane surrounding the blood vessels in mouse skin sections. TPEN also prevents muscle necrosis and accumulation of inflammatory cells at the site of ECV injections. In conclusion, a high degree of structural and functional homology between mammalian MMPs and SVMPs suggests that specific Zn(2+) chelators currently in clinical practice could be potent first aid therapeutic agents in snakebite management, particularly for local tissue damage.

A drug-repositioning screening identifies pentetic acid as a potential therapeutic agent for suppressing the elastase-mediated virulence of Pseudomonas aeruginosa.

Pseudomonas aeruginosa, a Gram-negative bacterium of clinical significance, produces elastase as a predominant exoprotease. Here, we screened a library of chemical compounds currently used for human medication and identified diethylene triamine penta-acetic acid (DTPA, pentetic acid) as an agent that suppresses the production of elastase. Elastase activity found in the prototype P. aeruginosa strain PAO1 was significantly decreased when grown with a concentration as low as 20 µM DTPA. Supplementation with Zn(2+) or Mn(2+) ions restored the suppressive effect of DTPA, suggesting that the DTPA-mediated decrease in elastase activity is associated with ion-chelating activity. In DTPA-treated PAO1 cells, transcription of the elastase-encoding lasB gene and levels of the Pseudomonas quinolone signal (PQS), a molecule that mediates P. aeruginosa quorum sensing (QS), were significantly downregulated, reflecting the potential involvement of the PQS QS system in DTPA-mediated elastase suppression. Biofilm formation was also decreased by DTPA treatment. When A549 alveolar type II-like adenocarcinoma cells were infected with PAO1 cells in the presence of DTPA, A549 cell viability was substantially increased. Furthermore, the intranasal delivery of DTPA to PAO1-infected mice alleviated the pathogenic effects of PAO1 cells in the animals. Together, our results revealed a novel function for a known molecule that may help treat P. aeruginosa airway infection.