Application of Deuteporfin in the Metastatic Lymph Node Mapping of Pancreatic Cancer: An in vivo Study

For most cancer patients, the presence of metastatic lymph nodes usually indicates regional recurrence and poor prognosis. Therefore, lymph node mapping is a requisite for disease staging, prognosis prediction and decision making in the treatment of cancer. Deuteporfin, a second‐generation photosensitizer, has a maximum excitation wavelength that can reach the near infrared (NIR) region (650–700 nm). We aimed to take advantage of these aspects of deuteporfin and use it as a fluorescent probe for metastatic lymph node mapping in vivo using NIR fluorescent imaging. In our study, we further investigated whether a photosensitizer could be used as a tracer for metastatic lymph node mapping of pancreatic cancer based on previous reports. Compared to normal tissues, tumor tissues including primary tumors and metastatic lymph nodes had a higher uptake ability of deuteporfin (P < 0.05). Our research confirmed this targeting property of deuteporfin using in vivo fluorescent imaging. Consistent with observations from in vivo imaging experiments, frozen sections of metastatic lymph nodes intuitively displayed significantly higher and wider distributions of deuteporfin than normal sections.     

Photocrosslinking of G‐Quadruplex‐Forming Sequences found in Human Promoters

While is it well known that human telomeric DNA sequences can adopt G‐quadruplex structures, some promoters sequences have also been found to form G‐quadruplexes, and over 40% of promoters contain putative G‐quadruplex‐forming sequences. Because UV light has been shown to crosslink human telomeric G‐quadruplexes by cyclobutane pyrimidine dimer (CPD) formation between T's on adjacent loops, UV light might also be able to photocrosslink G‐quadruplexes in promoters. To investigate this possibility, 15 potentially UV‐crosslinkable G‐quadruplex‐forming sequences found in a search of human DNA promoters were UVB irradiated in vitro, and three were confirmed to have formed nonadjacent CPDs by mass spectrometry. In addition to nonadjacent T=T CPDs found in human telomeric DNA, a nonadjacent T=U CPD was discovered that presumably arose from deamination of a nonadjacent T=C CPD. Analysis of the three sequences by circular dichroism, melting temperature analysis and chemical footprinting confirmed the presence of G‐quadruplexes that could explain the formation of the nonadjacent CPDs. The formation of nonadjacent CPDs from the sequences in vitro suggests that they might be useful probes for the presence of non‐B DNA structures, such as G‐quadruplexes, in vivo, and if they were to form in vivo, might also have significant biological consequences.     

Shining Light on the Secreted Luciferases of Marine Copepods: Current Knowledge and Applications

Copepod luciferases—a family of small secretory proteins of 18.4–24.3 kDa, including a signal peptide—are responsible for bright secreted bioluminescence of some marine copepods. The copepod luciferases use coelenterazine as a substrate to produce blue light in a simple oxidation reaction without any additional cofactors. They do not share sequence or structural similarity with other identified bioluminescent proteins including coelenterazine‐dependent Renilla and Oplophorus luciferases. The small size, strong luminescence activity and high stability, including thermostability, make secreted copepod luciferases very attractive candidates as reporter proteins which are particularly useful for nondisruptive reporter assays and for high‐throughput format. The most known and extensively investigated representatives of this family are the first cloned GpLuc and MLuc luciferases from copepods Gaussia princeps and Metridia longa, respectively. Immediately after cloning, these homologous luciferases were successfully applied as bioluminescent reporters in vivo and in vitro, and since then, the scope of their applications continues to grow. This review is an attempt to systemize and critically evaluate the data scattered through numerous articles regarding the main structural features of copepod luciferases, their luminescent and physicochemical properties. We also review the main trends of their application as bioluminescent reporters in cell and molecular biology.     

Size‐dependent Tumor Response to Photodynamic Therapy and Irinotecan Monotherapies Revealed by Longitudinal Ultrasound Monitoring in an Orthotopic Pancreatic Cancer Model

Longitudinal monitoring of tumor size in vivo can provide important biological information about disease progression and treatment efficacy that is not captured by other modes of quantification. Ultrasound enables high‐throughput evaluation of orthotopic mouse models via fast acquisition of three‐dimensional tumor images and calculation of volume with a reasonable degree of accuracy. Herein, we compare orthotopic pancreatic tumor volume measurements determined by ultrasound with volume measured by calipers and tumor weight, and found strong correlations between the three modalities over a large range of tumor sizes, suggesting ultrasound can accurately quantify tumor volumes in this model. Furthermore, we demonstrate the unique ability of longitudinal treatment monitoring to reveal a tumor size‐dependent response to Benzoporphyrin Derivative photodynamic therapy (BPD‐PDT) and irinotecan. Small tumors (5–35 mm³) were found to respond well to a single round of PDT, while large tumors (35–65 mm³) showed no response to the same treatment. These results highlight the role that tumor size can play in preclinical interpretation of treatment response and more generally suggest that careful evaluation of subtle biological features such as this must be carefully considered in order to grant a more comprehensive understanding of disease biology in vivo.     

Highly sensitive and selective detect of p‐arsanilic acid with a new water‐stable europium metal–organic framework

The p‐arsanilic acid (p‐ASA), as an aromatic organoarsenic compounds, had received considerable concerns for their potential toxicity and carcinogenic properties. It was essential to detect p‐ASA with a facile method. In this paper, an europium based fluorescent metal–organic framework (MOF) [Eu₂(clhex)·2H₂O)]·H₂O ( BUC‐69 ) was successfully prepared under hydrothermal conditions with 1,2,3,4,5,6‐cyclohexanehexacarboxylic acid (H₆clhex) as organic linker. BUC‐69 displayed superior fluorescence capability to achieve selective and sensitive detection toward p‐ASA in water, which presented the first example of a MOF‐based sensor to detect p‐ASA. BUC‐69 showed excellent chemical stability in solutions under pH ranging from 4 to 12, which makes it be a potential sensor both in acidity and alkalinity condition. Significantly, BUC‐69 performed well in fluorescent sensing of p‐ASA at a low concentration (10^?6 M) in the simulated wastewater prepared with real lake water, and the results were comparable to the values detected by Inductively Coupled Plasma Optical Emission Spectrometer (ICP‐OES). The corresponding mechanism of fluorescent sensing toward p‐ASA with BUC‐69 was proposed and affirmed.     

Redshifted and Near‐infrared Active Analog Pigments Based upon Archaerhodopsin‐3

Archaerhodopsin‐3 (AR3) is a member of the microbial rhodopsin family of hepta‐helical transmembrane proteins, containing a covalently bound molecule of all‐trans retinal as a chromophore. It displays an absorbance band in the visible region of the solar spectrum (λmax 556 nm) and functions as a light‐driven proton pump in the archaeon Halorubrum sodomense. AR3 and its mutants are widely used in neuroscience as optogenetic neural silencers and in particular as fluorescent indicators of transmembrane potential. In this study, we investigated the effect of analogs of the native ligand all‐trans retinal A1 on the spectral properties and proton‐pumping activity of AR3 and its single mutant AR3 (F229S). While, surprisingly, the 3‐methoxyretinal A2 analog did not redshift the absorbance maximum of AR3, the analogs retinal A2 and 3‐methylamino‐16‐nor‐1,2,3,4‐didehydroretinal (MMAR) did generate active redshifted AR3 pigments. The MMAR analog pigments could even be activated by near‐infrared light. Furthermore, the MMAR pigments showed strongly enhanced fluorescence with an emission band in the near‐infrared peaking around 815 nm. We anticipate that the AR3 pigments generated in this study have widespread potential for near‐infrared exploitation as fluorescent voltage‐gated sensors in optogenetics and artificial leafs and as proton pumps in bioenergy‐based applications.     

Luciferin‐Regenerating Enzyme Mediates Firefly Luciferase Activation Through Direct Effects of D‐Cysteine on Luciferase Structure and Activity

Luciferin‐regenerating enzyme (LRE) contributes to in vitro recycling of D‐luciferin. In this study, reinvestigation of the luciferase‐based LRE assay is reported. Here, using quick change site‐directed mutagenesis seven T‐LRE (Lampyris turkestanicusLRE) mutants were constructed and the most functional mutant of T‐LRE (T⁶⁹R) was selected for this research and the effects of D‐ and L‐cysteine on T⁶⁹R T‐LRE‐luciferase‐coupled assay are examined. Our results demonstrate that bioluminescent signal of T⁶⁹R T‐LRE‐luciferase‐coupled assay increases and then reach equilibrium state in the presence of 5 mm D‐cysteine. In addition, results reveal that 5 mm D‐ and L‐cysteine in the absence of T⁶⁹R T‐LRE cause a significant increase in bioluminescence intensity of luciferase over a long time as well as decrease in decay rate. Based on activity measurements, far‐UV CD analysis, ANS fluorescence and DLS (Dynamic light scattering) results, D‐cysteine increases the activity of luciferase due to weak redox potential, antiaggregatory effects, induction of changes in conformational structure and kinetics properties. In conclusion, in spite of previous reports on the effect of LRE on luciferase bioluminescent intensity, the majority of increase in luciferase light output and time‐course originate from the direct effects of D‐cysteine on structure and activity of firefly luciferase.     

Toward a Holistic Understanding and Models of Nonphotochemical Quenching Effects on In Vivo Fluorometry of Chlorophyll a in Coastal Waters

Nonphotochemical quenching (NPQ) is known to depress in vivo fluorescence (IVF) of chlorophyll a (Chla) in aquatic environments, which makes it difficult to interpret the hour-to-hour variations in Chla measured by in situ fluorometers. We hypothesized that ratios between quenched and unquenched IVF are a function of both NPQ and photochemical quenching. In this study, two diatom model species Thalassiosira pseudonana (CCMP1335) and Thalassiosira weissflogii (CCMP1047) incubated under a sinusoidal light:dark cycle were studied; IVF was recorded continuously, and Chla and photo-physiological variables were measured seven times a day. The maximal decline in Chla-specific IVF (IVF^B) attributable to quenching was 50% under the experimental settings. An NPQ and photochemical quenching-based modeling equation exhibited a better match to the measured IVF^B than equations representing the sole NPQ effect. Photochemical quenching induced by measuring light beam varied substantially during the day, and the part of the model for this process is excitation intensity-dependent (which is differed between models of in situ fluorometers, implying no straightforward method to correct Chla for all instrument models, instrument-specific parameterization is required). The forms of the IVF^B-light relationship are discussed as well. The findings foster a holistic understanding of NPQ effects on in vivo Chla fluorometry.     

LLLT Effects on Oral Keratinocytes in an Organotypic 3D Model

Several in vitro studies evaluated the cellular and molecular events related to interactions between phototherapy and target tissues, including oral keratinocytes and fibroblasts, providing elucidative data about phototherapy‐induced healing. However, these interactions were limited to the application of a bidimensional cell culture model of oral mucosal cells. Thus, thisstudy evaluated the use of an organotypic oral epithelium model to elucidate the morphological and phenotypic responses of cells subjected to low‐level laser therapy (LLLT). Oral keratinocytes were seeded in the ex vivo ‐produced oral mucosal equivalent (EVPOME ) model, with a porcine acellular dermal matrix. LLLT was applied by means of the LaserTABLE device (780 nm, 25 mW ) at 0.5, 1.5 and 3 J cm⁻². After three irradiations, morphology, proliferation and gene expression of growth factors were assessed. LLLT and control groups presented similar morphological features, characterized by the formation of a stratified, differentiated and keratinized epithelium. LLLT enhanced the cell proliferation and gene expression of keratinocytes (hKGF ) as well as epidermal (hEGF ) growth factors. In general, analysis of these data shows that the three‐dimensional cell culture model can be applied for phototherapy studies and that the positive effects of LLLT were confirmed by the use of an organotypic model.     

Cell‐specific Retention and Action of Pheophorbide‐based Photosensitizers in Human Lung Cancer Cells

This study determined in primary cultures of human lung cancer cells the cell specificity of chlorin‐based photosensitizers. Epithelial cells (ECs) preferentially retained 3‐[1‐hexyloxyethyl]‐2‐devinylpyropheophorbide‐a (HPPH) and related structural variants. Tumor‐associated fibroblasts (Fb) differ from EC by a higher efflux rate of HPPH. Immunoblot analyses indicated dimerization of STAT3 as a reliable biomarker of the photoreaction. Compared to mitochondria/ER‐localized photoreaction by HPPH, the photoreaction by lysosomally targeted HPPH‐lactose showed a trend toward lower STAT3 cross‐linking. Lethal consequence of the photoreaction differed between EC and Fb with the latter cells being more resistant. A survey of lung tumor cases indicated a large quantitative range by which EC retains HPPH. The specificity of HPPH retention defined in vitro could be confirmed in vivo in selected cases grown as xenografts. HPPH retention as a function of the tetrapyrrole structure was evaluated by altering side groups on the porphyrin macrocycle. The presence or absence of a carboxylic acid at position 17² proved to be critical. A benzyl group at position 20 enhanced retention in a subset of cancer cells with low HPPH binding. This study indicated experimental tools that are potentially effective in defining the photosensitizer preference and application for individual patient's cancer lesions.     

The Evolution and Functional Role of Flavin‐based Prokaryotic Photoreceptors

Flavin‐based photoreceptor proteins of the LOV (light, oxygen and voltage) superfamily are ubiquitous and appear to be essential blue‐light sensing systems not only in plants, algae and fungi, but also in prokaryotes, where they are represented in more than 10% of known species. Despite their broad occurrence, only in few cases LOV proteins have been correlated with important phenomena such as bacterial infectivity, selective growth patterns or/and stress responses; nevertheless these few known roles are helping us understand the multiple ways by which prokaryotes can exploit these soluble blue‐light photoreceptors. Given the large number of sequences now deposited in databases, it becomes meaningful to define a signature for bona fide LOV domains, a procedure that facilitates identification of proteins with new properties and phylogenetic analysis. The latter clearly evidences that a class of LOV proteins from alpha‐proteobacteria is the closest prokaryotic relative of eukaryotic LOV domains, whereas cyanobacterial sequences cluster with the archaeal and the other bacterial LOV domains. Distance trees built for LOV domains suggest complex evolutionary patterns, possibly involving multiple horizontal gene transfer events. Based on available data, the in vivo relevance and evolution of prokaryotic LOV is discussed.     

Hexanuclear 3d − 4f metal–organic cages assembled from a carboxylic acid‐functionalized tris‐triazamacrocycle for highly selective fluorescent sensing of picric acid

Two Ln^III ions are sandwiched by dinuclear Co^II building blocks derived from a tris‐triazamacrocyclic ligand bearing pendant carboxylic acid functionality, 1,3,5‐tris((4,7‐bis(2‐carboxyethyl)‐1,4,7‐triazacyclonon‐1‐yl)methyl)‐benzene (H₆L), giving rising to two nanoscale heterometallic metal–organic cages formulated as [Co₄Ln₂(LH_2.5)₂(H₂O)₄]·(ClO₄)₆·NO₃·nH₂O [Ln = Dy, n = 12 ( 1 ); Ln = Yb, n = 9 ( 2 )], whose internal cavity accommodates a guest NO₃^? anion. Their hexanuclear cage‐like architectures are maintained both in solution and solid states as confirmed by mass spectrum as well as X‐ray diffraction experiments. These two cages display ligand‐based fluorescence emissions and therefore both were chosen to be operated as fluorescent chemosensors for the detection of nitroaromatic compounds. Attractively, these metal–organic cages allow highly selective and sensitive detection of picric acid (PA) over other nitroaromatics in solution and suspension, and the fluorescence resonance energy transfer (FRET) between the cage probes and PA is mainly responsible for the remarkable detection efficiency.     

Luciferin‐Regenerating Enzyme Crystal Structure Is Solved but its Function Is Still Unclear

Contribution of luciferin‐regenerating enzyme (LRE) for in vitro recycling of D‐luciferin has been reported. According to crystal structure of LRE, it is a beta‐propeller protein which is a type of all β‐protein architecture. In this overview, reinvestigation of the luciferase‐based LRE assays and its function is reported. Until now, sequence of LRE genes from four different species of firefly has been reported. In spite of previous reports, T‐LRE (from Lampyris turkestanicus) was cloned and expressed in Escherichia coli as well as Pichia pastoris in a nonsoluble form as inclusion body. According to recent investigations, bioluminescent signal of soluble T‐LRE–luciferase‐coupled assay increased and then reached an equilibrium state in the presence of D‐cysteine. In addition, the results revealed that both D‐ and L‐cysteine in the absence of T‐LRE caused a significant increase in bioluminescence intensity of luciferase over a long time. Based on activity measurements and spectroscopic results, D‐cysteine increased the activity of luciferase due to its redox potential and induction of conformational changes in structure and kinetics properties. In conclusion, in spite of previous reports on the effect of LRE (at least T‐LRE) on luciferase activity, most of the increase in luciferase activity is caused by direct effect of D‐cysteine on structure and activity of firefly luciferase. Moreover, bioinformatics analysis cannot support the presence of LRE in peroxisome of photocytes in firefly lanterns.     

Protein–induced aggregation of fluorescent conjugated polyelectrolytes with sulfonate groups: Synthesis and its sensing application

Anionically charged fluorescent conjugated polyelectrolytes of poly{[4,7‐(2,1,3‐benzothiadiazole)‐alt‐1,4‐phenylene]‐co‐[2,5‐bis(4‐sulfonatobutoxy)‐alt‐1,4‐phenylene]} ( P1 ) and poly{[4,7‐(bis(thiophen‐2‐yl)benzo‐2,1,3‐thiadiazole)‐alt‐1,4‐phenylene]‐co‐[2,5‐bis(4‐sulfonatobutoxy)‐alt‐1,4‐phenylene]} ( P2 ) were synthesized by Suzuki crosscoupling polymerization in the presence of a palladium catalyst. The conjugated polyelectrolytes with sulfonate groups, as efficient signal amplifying reporters, were carefully designed to be soluble in water over the entire pH range examined and interact with proteins through intermolecular forces. The polymers exhibited blue emission in aqueous solutions but green or red emission in solid form depending on the conjugation length due to intermolecular exciton migration. The anionic conjugated polymers exhibited blue‐to‐green or blue‐to‐red changes in fluorescence upon exposure to charged proteins, indicating that the polymers have potential applications in fluorescent array systems for protein. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2010     

Disinfect Porphyromonas gingivalis Biofilm on Titanium Surface with Combined Application of Chlorhexidine and Antimicrobial Photodynamic Therapy

Various antimicrobial modalities have been proposed to treat peri‐implantitis but resulted in limited outcomes. The aim of this in vitro study was to evaluate the disinfection efficacy of combined application of chlorhexidine digluconate (CHX) and antimicrobial photodynamic therapy (aPDT) of titanium surfaces previously contaminated with Porphyromonas gingivalis biofilm. P. gingivalis biofilms were grown on 32 polished and 32 sandblasted large‐grit acid‐etched (SLA) titanium surfaces. Titanium disks were allocated into four groups as follows: (1) immersed in phosphate‐buffered saline (PBS), (2) immersed in 0.2% CHX, (3) application of aPDT and (4) immersed in 0.2% CHX and subsequent aPDT. Residual bacteria were determined by microbial culture analysis and by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) imaging. Combination protocol (CHX+ aPDT) was the most effective in eradicating P. gingivalis (P < 0.05) on both polished and SLA surfaces. There was no significant difference in the number of remaining P. gingivalis between polished titanium disks and the SLA ones in four groups (P > 0.05). Under the limitation of this study, combined technique of preceding application of CHX and subsequent aPDT was shown to be an efficient method in reducing P. gingivalis numbers in both polished and SLA titanium surfaces.     

Dissection of Environmental Changes at the Cytoplasmic Surface of Light‐activated Bacteriorhodopsin and Visual Rhodopsin: Sequence of Spectrally Silent Steps†

The physico‐chemical properties as well as the conformation of the cytoplasmic surface of the 7‐helix retinal proteins bacteriorhodopsin (bR) and visual rhodopsin change upon light activation. A recent study found evidence for a transient softening of bR in its key intermediate M [Pieper et al. (2008) Phys. Rev. Lett. 100 , 228103] as a direct proof for the functional significance of protein flexibility. In this report we compare environmental and flexibility changes at the cytoplasmic surface of light‐activated bR and rhodopsin detected by time‐resolved fluorescence spectroscopy. The changes in fluorescence of covalently bound fluorescent probes and protein real‐time dynamics were investigated. We found that in fluorescently labeled bR and rhodopsin the intensity of fluorescein and Atto647 increased upon formation of the key intermediates M and metarhodopsin‐II, respectively, suggesting different surface properties compared to the dark state. Furthermore, time‐resolved fluorescence anisotropy experiments reveal an increase in steric restriction of loop flexibility because of changes in the surrounding protein environment in both the M‐intermediate as well as the active metarhodopsin‐II state. The kinetics of the fluorescence changes at the rhodopsin surface uncover multiple transitions, suggesting metarhodopsin‐II substates with different surface properties. Proton uptake from the aqueous bulk phase correlates with the first transition, while late proton release seems to parallel the second transition. The last transition between states of different surface properties correlates with metarhodopsin‐II decay.     

A Fluorescent Blue Phosphazene Dye: Synthesis,Structure and Optical Properties of 1,6‐Bis(Dimethylamino)‐2,5,7,10‐Tetraazo‐1,6λ5‐Diphosphapyrene

Herein we report on the synthesis, structure, and optical properties of the fluorescent blue phosphazene dye 1,6‐bis(dimethylamino)‐2,5,7,10‐tetraazo‐1,6λ⁵‐diphosphapyrene, which was isolated as the unexpected product of the reaction between 1,4,5,8‐(tetraamino)naphthalene and [P(NMe₂)₃Br]Br. This dye, which turned out to be soluble in water and a range of organic solvents (including hexane, tetrahydrofuran / petroleum ether, acetonitrile, and ethanol), was structurally characterized by XRD. Its absorption as well as emission spectra and their sensitivity to pH variations were analyzed. The experimental work is complemented by quantum chemical calculations on the possible intermediate on the way to the isolated product and on its pK_a value.     

Active‐Site‐Matched Fluorescent Probes for Rapid and Direct Detection of Vicinal‐Sulfydryl‐Containing Peptides/Proteins in Living Cells

Vicinal‐sulfydryl‐containing peptides/proteins (VSPPs) play a crucial role in human pathologies. Fluorescent probes that are capable of detecting intracellular VSPPs in vivo would be useful tools to explore the mechanisms of some diseases. In this study, by regulating the spatial separation of two maleimide groups in a fluorescent dye to match that of two active cysteine residues contained in the conserved amino acid sequence (–CGPC–) of human thioredoxin, two active‐site‐matched fluorescent probes, o‐Dm‐Ac and m‐Dm‐Ac, were developed for real‐time imaging of VSPPs in living cells. As a result, the two probes can rapidly respond to small peptide models and reduced proteins, such as WCGPCK (W‐6), WCGGPCK (W‐7), and WCGGGPCK (W‐8), reduced bovine serum albumin (rBSA), and reduced thioredoxin (rTrx). Moreover, o‐Dm‐Ac displays a higher binding sensitivity with the above‐mentioned peptides and proteins, especially with W‐7 and rTrx. Furthermore, o‐Dm‐Ac was successfully used to rapidly and directly detect VSPPs both in vitro and in living cells. Thus, a novel probe‐design strategy was proposed and the synthesized probe applied successfully in imaging of target proteins in situ.     

New ent‐Pimarane Diterpenes from the Roots of Aralia dumetorum

Four new ent‐pimarane diterpenes were isolated from the EtOH extract of Aralia dumetorum, together with three known compounds involving ent‐pimar‐8(14),15‐dien‐19‐oic acid ( 5 ), ent‐pimar‐8(14),15‐dien‐19‐ol ( 6 ), and ent‐kaur‐16‐en‐19‐oic acid ( 7 ). By detailed analyses of the MS, IR, and NMR data, the structures of four new diterpenes were characterized as (5β,9β,10α,13α)‐pimara‐6,8(14),15‐trien‐18‐oic acid ( 1 ), (5β,7β,9β,10α,13α)‐7‐methoxypimara‐8(14),15‐dien‐18‐oic acid ( 2 ), (5β,9β,10α,13α,14β)‐14‐methoxypimara‐7,15‐dien‐18‐oic acid ( 3 ), and (5β,10α,13α,14α)‐14‐hydroxypimara‐7,9(11),15‐trien‐18‐oic acid ( 4 ). The cytotoxic activities of compounds 1  –  7 were assayed in vitro through MTT method.