Frontiers and development in live-cell super-resolution fluorescence microscopy / 生物医学工程学杂志

This paper reviews the research progress on live-cell super-resolution fluorescence microscopy, discusses the current research status and hotspots in this field, and summarizes the technological application of super-resolution fluorescence microscopy for live-cell imaging. To date, this field has gained progress in numerous aspects. Specifically, the structured illumination microscopy, stimulated emission depletion microscopy, and the recently introduced minimal photon fluxes microscopy are the current research hotspots. According to the current progress in this field, future development trend is likely to be largely driven by artificial intelligence as well as advances in fluorescent probes and relevant labelling methods.

Development of flow cytometry and its application in plant research / 生物工程学报

Flow cytometry is a multi-parameter, rapid and efficient method for qualitative analysis and quantitative determination of various fluorescently labeled particles in liquid flow. Flow cytometry has been applied in multiple disciplines such as immunology, virology, molecular biology, cancer biology and infectious disease monitoring. However, the application of flow cytometry in plant research is hampered due to the special composition and structure of plant tissues and cells, such as cell walls and secondary metabolites. In this paper, the development, composition and classification of flow cytometry were introduced. Subsequently, the application, research progress and application limitations of flow cytometry in plant field were discussed. At last, the development trend of flow cytometry in plant research was prospected, which provides new perspectives for broadening the potential application scope of plant flow cytometry.

Genetic code expansion and its application in characterization and imaging of proteins / 生物工程学报

Genetic code expansion (GCE) allows the incorporation of unnatural amino acids into proteins via using stop codons. GCE may achieve site-specific labeling of proteins in combination with the click reaction. Compared with other labeling tools such as fluorescent proteins and tagged antibodies, the compound molecules used in protein labeling by GCE technology are smaller, and therefore, may less interfere the conformational structure of proteins. In addition, through click reaction, GCE allows a 1:1 stoichiometric ratio of the target protein molecule and the fluorescent dye, and the protein can be quantified based on the fluorescence intensity. Thus, GCE technology has great advantages in the researches that require the exposition of living cells under high laser power for longer time, for example, in the context of single molecule tracing and super-resolution microscopic imaging. Meanwhile, this technology lays the foundation for improving the accuracy of positioning and molecule counting in the imaging process of living cells. This review summarized the GCE technology and its recent applications in functionally characterizing, labeling and imaging of proteins.

Detection of drug resistance genes of mycobacterium tuberculosis by rolling circle amplification technique with multicolor fluorescent probes / 中华预防医学杂志

The aim of this study was to construct a simple, rapid and ultra-sensitive optical biosensing technique based on rolling circle amplification (RCA), and to apply it to multiple detection of drug-resistant genes of mycobacterium tuberculosis. The common mutation sites of isoniazid, rifampicin and streptomycin resistance genes are katG315 (AGC➝ACC), rpoB531 (CAC➝TAC) and rpsL43 (AAG➝AGG). For these three gene sites, from February 2020 to May 2021, in the Department of Laboratory Medicine of the First Affiliated Hospital of Army Military Medical University, the padlock probe (PLP), primers and capture probes were designed. And a solid-phase RCA constant temperature amplification reaction system based on magnetic beads was constructed and the experimental parameters were optimized. The RCA products were accurately captured by the multicolor fluorescent probes (Cy3/Cy5/ROX), and the single-tube multiple detection of three mutation genes was realized. The sensitivity, specificity and linear range of this method were further verified. The results showed that the response range of katG315 in the same reaction system ranged from 1.0 pmol/L to 0.1 nmol/L. The response range of rpoB531 and rpsL43 ranged from 1.0 pmol/L to 50.0 pmol/L and 1.0 pmol/L to 20.0 pmol/L, and the method had good specificity and sensitivity, and could accurately identify single base mutations in mixed targets, with the minimum detection limit as low as 1.0 pmol/L. The recoveries of simulated serum samples were 95.0%-105.2%. In conclusion, the constant temperature amplification multiple detection method constructed in this study can quickly realize the single-tube multiple detection of three drug resistance mutation sites. This technology is low-cost, simple and rapid, and does not rely on large equipment, providing a new analysis method for pathogen drug resistance gene detection.

Berberine inhibits erastin-induced ferroptosis of mouse hippocampal neuronal cells possibly by activating the Nrf2-HO-1/GPX4 pathway / 南方医科大学学报

OBJECTIVE@#To explore the mechanism by which berberine inhibits ferroptosis of mouse hippocampal neuronal cells (HT22).@*METHODS@#Cultured HT22 cells were pretreated with 30 or 60 μmol/L berberine for 2 h before exposure to 0.5 μmol/L erastin for 8 h, and the cell proliferation, intracellular ferric iron level, changes in intracellular reactive oxygen species (ROS) and cell apoptosis were detected using CCK-8, Fe2+ fluorescent probe, fluorescent dye (DAPI) and fluorescent probe (H2DCFH-DA). RT-qPCR and Western blotting were used to detect the mRNA and protein expressions of Nrf2, HO-1 and GPX4 in the cells. We further tested the effects of treatments with 2 μmol/L ML385 (a Nrf2 inhibitor), 60 μmol/L berberine and erastin in the cells to explore the protective mechanism of berberine against erastin-induced ferroptosis in the neuronal cells.@*RESULTS@#Treatment with 0.5 μmol/L erastin significantly lowered the viability of HT22 cells (P < 0.05) and increased the production of ROS, cell apoptosis rate and ferric iron level (P < 0.05). Pretreatment with 30 and 60 μmol/L berberine both significantly increased the vitality of erastin-exposed cells (P < 0.05) and lowered the levels of intracellular ROS and ferric iron content (P < 0.05). RT-qPCR and Western blotting showed that berberine obviously promoted the expressions of Nrf2, HO-1 and GPX4 in the cells (P < 0.05), and treatment with ML385 significantly inhibited the Nrf2-HO-1/GPX4 pathway, increased intracellular ROS and ferric iron contents and mitigated the protective effect of berberine against erastin-induced ferroptosis (P < 0.05).@*CONCLUSION@#Berberine can inhibit erastin-induced ferroptosis in HT22 cells possibly by activating the Nrf2-HO-1/ GPX4 pathway.

Carbenoxolone enhances inhibitory effect of RSL3 against cisplatin-resistant testicular cancer cells by promoting ferroptosis / 南方医科大学学报

OBJECTIVE@#To investigate the inhibitory effect of RSL3 on the proliferation, invasion and migration of cisplatinresistant testicular cancer cells (I-10/DDP) and the effect of carbenoxolone on the activity of RSL3 against testicular cancer.@*METHODS@#MTT assay was used to evaluate the survival rate of I-10/DDP cells following treatment with RSL3 (1, 2, 4, 8, 16 or 32 μmol/L) alone or in combination with carbenoxolone (100 μmol/L) or after treatment with Fer-1 (2 μmol/L), RSL3 (4 μmol/L), RSL3+Fer-1, RSL3+carbenoxolone (100 μmol/L), or RSL3+Fer-1+carbenoxolone. Colony formation assay was used to assess the proliferation ability of the treated cells; wounding-healing assay and Transwell assay were used to assess the invasion and migration ability of the cells. The expression of GPX4 was detected using Western blotting, the levels of lipid ROS were detected using C11 BODIPY 581/591 fluorescent probe, and the levels of Fe2+ were determined with FerroOrange fluorescent probe.@*RESULTS@#RSL3 dose-dependently decreased the survival rate of I-10/DDP cells, and the combined treatment with 2, 4, or 8 μmol/L RSL3 with carbenoxolone, as compared with RSL3 treatment alone, resulted in significant reduction of the cell survival rate. The combination with carbenoxolone significantly enhanced the inhibitory effect of RSL3 on colony formation, wound healing rate (P=0.005), invasion and migration of the cells (P < 0.001). Fer-1 obviously attenuated the inhibitory effects of RSL3 alone and its combination with carbenoxolone on I-10/DDP cells (P < 0.01). RSL3 treatment significantly decreased GPX4 expression (P=0.001) and increased lipid ROS level (P=0.001) and Fe2+ level in the cells, and these effects were further enhanced by the combined treatment with carbenoxolone (P < 0.01).@*CONCLUSION@#Carbenoxolone enhances the inhibitory effect of RSL3 on the proliferation, invasion and migration of cisplatin-resistant testicular cancer cells by promoting RSL3-induced ferroptosis.

A Selective Fluorescent Probe for the Determination and Imaging of Fluoride in Living Cells

Abstract Fluoride anions are indispensable trace elements required for sustaining life. To investigate the homeostasis and action of fluoride in the body, a new highly sensitive and selective fluorescence detection method was designed for fluoride in aqueous solutions. A fluorescent probe for fluoride (FP-F) was synthesized for imaging F- in living cells. The design strategy for the probe was based on the specific reaction between fluoride and silica to mediate deprotection of this probe to fluorescein. Upon treatment with F-, FP-F, a closed and weakly fluorescent lactone, was transformed into an open and strongly fluorescent product. Under the optimum conditions, the detection limit for fluoride was 0.526 nM. FP-F could detect micromolar changes in F- concentrations in living cells by confocal microscopy.

Application of organic fluorescent probe-assisted near infrared fluorescence imaging in cervical cancer diagnosis / 生物工程学报

Fluorescence imaging has been widely used in the fields of biomedicine and clinical diagnosis. Compared with traditional fluorescence imaging in the visible spectral region (400-760 nm), near-infrared (NIR, 700-1 700 nm) fluorescence imaging is more helpful to improve the signal-to-noise ratio and the sensitivity of imaging. Highly-sensitive fluorescent probes are required for high-quality fluorescence imaging, and the rapid development of nanotechnology has led to the emergence of organic dyes with excellent fluorescent properties. Among them, organic fluorescent probes with the advantages of high safety, good biocompatibility, and high optical stability, are more favorable than inorganic fluorescent probes. Therefore, NIR fluorescence imaging assisted with organic fluorescent probes can provide more structural and dynamic information of biological samples to the researchers, which becomes a hot spot in the interdisciplinary research field of optics, chemistry and biomedicine. This review summarizes the application of NIR organic fluorescent probes in cervical cancer imaging. Several typical organic fluorescent probes (such as indocyanine green, heptamethine cyanine dye, rhodamine and polymer fluorescent nanoparticles) assisted NIR fluorescence imaging and their applications in cervical cancer diagnosis were introduced, and the future development and application of these techniques were discussed.

PSO+: nonlinear fitting fluorescence data based on particle swarm optimizing combine with other iteration algorithm / 生物医学工程学杂志

The convective polymerase chain reaction (CCPCR) uses the principle of thermal convection to allow the reagent to flow in the test tube and achieve the purpose of amplification by the temperature difference between the upper and lower portions of the test tube. In order to detect the amplification effect in real time, we added a fluorophore to the reagent system to reflect the amplification in real time through the intensity of fluorescence. The experimental results show that the fluorescence curve conforms to the S-type trend of the amplification curve, but there is a certain jitter condition due to the instability of the thermal convection, which is not conducive to the calculation of the cycle threshold (CT value). In order to solve this problem, this paper uses the dynamic method, using the double S-type function model to fit the curve, so that the fluorescence curve is smooth and the initial concentration of the nucleic acid can be deduced better to achieve the quantitative purpose based on the curve. At the same time, the PSO+ algorithm is used to solve the double s-type function parameters, that is, particle swarm optimization (PSO) algorithm combined with Levenberg-Marquardt, Newton-CG and other algorithms for curve fitting. The proposed method effectively overcoms PSO randomness and the shortcoming of traditional algorithms such as Levenberg-Marquardt and Newton-CG which are easy to fall into the local optimal solution. The of the data fitting result can reach 0.999 8. This study is of guiding significance for the future quantitative detection of real-time fluorescent heat convection amplification.

Monitoring Glutathione Dynamics and Heterogeneity in Living Stem Cells

Glutathione (GSH) is a major antioxidant in cells, and plays vital roles in the cellular defense against oxidants and in the regulation of redox signals. In a previous report, we demonstrated that stem cell function is critically affected by heterogeneity and dynamic changes in cellular GSH concentration. Here, we present a detailed protocol for the monitoring of GSH concentration in living stem cells using FreSHtracer, a real-time GSH probe. We describe the steps involved in monitoring GSH concentration in single living stem cells using confocal microscopy and flow cytometry. These methods are simple, rapid, and quantitative, and able to demonstrate intracellular GSH concentration changes in real time. We also describe the application of FreSHtracer to the sorting of stem cells according to their GSH content using flow cytometry. Typically, microscopic or flow cytometric analyses of FreSHtracer and MitoFreSHtracer signals in living stem cells take ~2~3 h, and the fractionation of stem cells into subpopulations on the basis of cellular GSH levels takes 3~4.5 h. This method could be applied to almost every kind of mammalian cell with minor modifications to the protocol described here.

Uso de marcadores fluorescentes na engenharia tecidual óssea: estudo piloto / Use of fluorescent markers in bone tissue engineering: pilot study

Introdução: a regeneração e o reparo de tecidos ósseos perdidos é objeto de estudo da Bioengenharia Tecidual. O uso de biomateriais substitutos ósseos biomiméticos visa estimular os sistemas celulares e bioquímicos para restabelecer de modo mais eficiente o tecido ósseo nos casos de sua reconstrução. Ao investigar o processo de remodelação, é vital identificar áreas de novo crescimento para avaliar a eficácia dos biomateriais implantados e respectivos regimes de tratamento. A avaliação qualitativa e quantitativa da regeneração óssea pode ser realizada através da aplicação de marcadores como o Xilenol, a Tetraciclina, a Calceína e a Alizarina. A administração desses marcadores de forma associada possibilita ainda marcar sequencialmente camadas de nova deposição e remodelação durante o reparo. Objetivo: estabelecer um protocolo para utilização dos marcadores fluorescentes de reparo ósseo xilenol, tetraciclina, calceína e alizarina, em ratos. Metodologia: foram utilizados 35 ratos da linhagem Wistar, machos adultos, com massa corpórea entre 350 e 400g, e idade aproximada de 4 a 5 meses, distribuídos randomicamente em 5 grupos experimentais, submetidos à confecção de defeito ósseo circular de 8 mm em região de calvária, e administração dos diferentes marcadores segundo os grupos; XO ­ Xilenol; Ca ­ Calceína; Al ­ Alizarina; Te ­ Tetraciclina; C ­ Controle. Após 15 dias de experimento, os animais foram eutanasiados e as calvárias processadas e analisadas por histomorfometria, microscopia de epifluorescência e microscopia de fluorescência. Resultados: todos protocolos empregados para utilização dos marcadores fluorescentes xilenol, calceína, alizarina e tetracicilina foram úteis para identificar área de deposição mineral durante o período analisado de regeneração óssea em ratos. As imagens obtidas pela microscopia de fluorescência revela a presença dos marcadores incorporados à matriz óssea neoformada, no entanto a utilização da Alizarina e Calceína dentro dos protocolos testados mostraram-se mais eficientes. Conclusão: os protocolos testados nesse estudo apresentaram-se viáveis para utilização em pesquisas envolvendo marcadores de regeneração óssea, com resultados superiores para Alizarina e Calceína

Introduction: The regeneration and repair of lost bone tissues is the subject of a study of Tissue Bioengineering. The use of biomimetic biomaterial bone substitutes aims to stimulate the cellular and biochemical systems to restore more efficiently the bone tissue in the cases of its reconstruction. When investigating the remodeling process, it is vital to identify areas of new growth to evaluate the efficacy of implanted biomaterials and their treatment regimens. The qualitative and quantitative evaluation of bone regeneration can be performed through the use of markers such as Xylenol, Tetracycline, Calcein and Alizarin. The administration of such markers in an associated manner also makes it possible to sequentially mark layers of new deposition and remodeling during the repair. Objective: to establish a protocol for the use of fluorescent xylenol, tetracycline, calcein and alizarin bone repair markers in rats. Metodology: thirtyfive male adult Wistar rats with a body mass ranging from 350 to 400 g and approximately 4 to 5 months old were randomly assigned to 5 experimental groups submitted to a circular bone defect of 8 mm in the region of calvaria, and administration of the different markers according to the groups; XO ­ Xylenol; Ca ­ Calcein; Al-Alizarin; Te ­ Tetracycline; C ­ Control. After 15 days of experiment, the animals were euthanized and the calvaria processed and analyzed by histomorphometry, epifluorescence microscopy and fluorescence microscopy. Results: all protocols used for fluorescence markers xylenol, calcein, alizarin and tetracycline were useful to identify area of mineral deposition during the analyzed period of bone regeneration in rats. The images obtained by fluorescence microscopy revealed the presence of the markers incorporated into the neoformed bone matrix, however the use of Alizarin and Calcein within the protocols tested were more efficient. Conclusion: the protocols tested in this study were feasible for use in research involving markers of bone regeneration, with superior results for Alizarin and Calcein.

Growth dynamic of allogeneic and autogenous bone grafts in a vertical model

Abstract Several techniques have been proposed for vertical bone regeneration, and many of them use bone autogenous and allogeneic grafts. The purpose of this study was to compare demineralised freeze-dried bone allografts (DFDBA), fresh-frozen (FF) allografts, autogenous bone grafts to find differences between volumetric and histological quantity of bone formation and vertical bone growth dynamic. A vertical tissue regeneration bone model was performed in rabbit calvarias under general anaesthesia. Four hollow cylinders of pure titanium were screwed onto external cortical bone calvarias in eight rabbits. Each one of the cylinders was randomly filled with one intervention: DFDBA, FF, autogenous bone, or left to be filled with blood clot (BC) as control. Allogeneic grafts were obtained from a ninth animal following international standardised protocols for the harvesting, processing, and cryopreservation of allografts. Autogenous graft was obtained from the host femur scraping before adapting hollow cylinders. Animals were euthanized at 13 weeks. Vertical volume was calculated after probe device measurements of the new formed tissue inside the cylinders and after titanium cylinders were removed. Histomorphometry and fluorochrome staining were used to analyse quantity and dynamic of bone formation, respectively. Results showed that DFDBA and fresh-frozen bone improved the velocity and the quantity of bone deposition in distant portions of the basal plane of grafting. Remaining material in allograft groups was more intense than in autogenous group. Both allografts can be indicated as reliable alternatives for volume gain and vertical bone augmentation.

Resumo Várias técnicas foram propostas para regeneração óssea vertical, e muitas delas usam enxertos ósseos e alogênicos ósseos. O objetivo deste estudo foi comparar os aloenxertos ósseos congelados desmineralizados (DFDBA), os aloenxertos congelados frescos (FF) com os enxertos ósseos autógenos para encontrar diferenças entre o volume, a histologia da formação óssea e a dinâmica do crescimento ósseo vertical. Um modelo ósseo de regeneração tecidual vertical foi realizado em calvarias de coelho sob anestesia geral. Quatro cilindros ocos de titânio puro foram parafusados nas calvarias de osso cortical externo em oito coelhos. Cada um dos cilindros foi preenchido aleatoriamente com uma intervenção: DFDBA, FF, osso autógeno ou com coágulo sanguíneo (BC) como controle. Os enxertos alogênicos foram obtidos a partir de um nono animal seguindo protocolos internacionais padronizados para a coleta, processamento e criopreservação de aloenxertos. O enxerto autógeno foi obtido da raspagem do fêmur do hospedeiro antes de adaptar os cilindros ocos. Os animais foram eutanasiados após 13 semanas. O volume vertical foi calculado após a medição, por meio de sonda milimetrada, do novo tecido formado dentro dos cilindros e após a remoção dos cilindros de titânio. Histomorfometria e coloração com fluorocromios foram utilizados para analisar a quantidade e a dinâmica da formação óssea. Os resultados mostraram que DFDBA e osso fresco congelado melhoraram a velocidade e a quantidade de deposição óssea em porções distantes do plano basal de enxerto. O material remanescente nos grupos de aloenxerto foi mais intenso do que em grupo autógeno. Ambos os aloenxertos podem ser indicados como alternativas confiáveis para ganho de volume e aumento ósseo vertical.

Development of Molecular Diagnosis Using Multiplex Real-Time PCR and T4 Phage Internal Control to Simultaneously Detect Cryptosporidium parvum, Giardia lamblia, and Cyclospora cayetanensis from Human Stool Samples

This study aimed to develop a new multiplex real-time PCR detection method for 3 species of waterborne protozoan parasites (Cryptosporidium parvum, Giardia lamblia, and Cyclospora cayetanensis) identified as major causes of traveler's diarrhea. Three target genes were specifically and simultaneously detected by the TaqMan probe method for multiple parasitic infection cases, including Cryptosporidium oocyst wall protein for C. parvum, glutamate dehydrogenase for G. lamblia, and internal transcribed spacer 1 for C. cayetanensis. Gene product 21 for bacteriophage T4 was used as an internal control DNA target for monitoring human stool DNA amplification. TaqMan probes were prepared using 4 fluorescent dyes, FAM™, HEX™, Cy5™, and CAL Fluor Red® 610 on C. parvum, G. lamblia, C. cayetanensis, and bacteriophage T4, respectively. We developed a novel primer-probe set for each parasite, a primer-probe cocktail (a mixture of primers and probes for the parasites and the internal control) for multiplex real-time PCR analysis, and a protocol for this detection method. Multiplex real-time PCR with the primer-probe cocktail successfully and specifically detected the target genes of C. parvum, G. lamblia, and C. cayetanensis in the mixed spiked human stool sample. The limit of detection for our assay was 2×10 copies for C. parvum and for C. cayetanensis, while it was 2×10³ copies for G. lamblia. We propose that the multiplex real-time PCR detection method developed here is a useful method for simultaneously diagnosing the most common causative protozoa in traveler's diarrhea.

Indocyanine Green-Fluorescent Pancreatic Perfusion-Guided Resection of Distal Pancreas in Solid Pseudopapillary Neoplasm: Usefulness and Feasibility During Pancreaticobiliary Surgery

ICG is widely applied in real-time imaging during abdominal surgery, plastic surgery, as well as oncologic staging and treatment. A twenty-eight year-old female patient was found to have a 4.5 cm solid pseudopapillary neoplasm in the tail of the pancreas. Under ICG-fluorescent pancreatic perfusion-guidance, we easily defined the margin of the pancreatic tumor and secured the resection margin when performing laparoscopic distal pancreatosplenectomy in the patient. No clinically relevant complications, including postoperative pancreatic fistula, were noted. Intravenous ICG can be very easily and quickly detected in the pancreas under near infrared light. This enhanced vision gives strong contrast to the organ compared to a necrotic tumor with poor blood perfusion, such as solid pseudopapillary neoplasm. Based on our current experience, ICG pancreatic perfusion-guided determination of appropriate resection margin is useful and feasible during pancreaticoduodenectomy.

Daily melatonin administration improves osseointegration in pinealectomized rats

Abstract The hypothesis of this study was that the peri-implant bone healing of the group of pinealectomized rats would differ from the control group. The samples were subjected to immunohistochemical, microtomographic (total porosity and connectivity density), and fluorochrome (mineralized surface) analyses. Objectives The goal of this study was to investigate the cellular changes and bone remodeling dynamics along the bone/implant interface in pinealectomized rats. Material and Methods The total of 18 adult male rats (Rattus norvegicus albinus, Wistar) was divided into three groups (n=6): control (CO), pinealectomized without melatonin (PNX) and pinealectomized with melatonin (PNXm). All animals were submitted to the first surgery (pinealectomy), except the CO group. Thirty days after the pinealectomy without melatonin, the second surgery was conducted, in which all animals received an implant in each tibia (36 titanium implants with surface treatment were installed - Implalife® São Paulo, SP, Brazil). By gavage, the rats of the PNX group received the vehicle solution, and the procedure. Results Immunohistochemical analysis for runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), osteopontin (OP) and osteocalcin (OC) showed that the bone repair process in the PNXm group was similar to that of the CO group, whereas the PNX group showed a delay. The microtomographic parameters of total porosity [Po(tot)] and bone surface (BS) showed no statistically significant differences, whereas for the connective density (Conn.Dn) a statistical difference was found between the CO and PNXm groups. Fluorochrome analysis of the active mineralized surface showed statistically significant difference between the CO and PNX and between the CO and PNXm groups. Conclusion The absence of the pineal gland impaired the bone repair process during osseointegration, however the daily melatonin replacement was able to restore this response.

Fluorescent test accurately predicts leak of ischemic colon anastomosis in rats

Abstract Purpose: To investigate if fluorescein fluorescent test can predict dehiscence in a model of ischemic colonic anastomosis in rats. Methods: This experimental controlled trial randomly assigned 55 rats to four groups. Anastomoses were performed in non-ischemic colon segments (control group) and in ischemic colon segments measuring 1, 2 or 3 cm long (groups 1, 2 and 3, respectively). Fluorescein was injected and the tissues were examined under ultraviolet light. Seven days later, a second-look surgery was performed to check for the presence or absence of anastomosis dehiscence. Results: Twenty-four rats presented anastomotic dehiscence during the second-look surgery. Reticular and nonfluorescent patterns were significantly associated with the occurrence of anastomotic dehiscence. Fluorescein fluorescence had a sensitivity of 95.8%, specificity of 89.2%, positive predictive value of 88.4%, negative predictive value of 96.2%, and accuracy of 92.3% to predict anastomotic dehiscence. Conclusion: Fluorescein fluorescent test can accurately predict leak in a model of ischemic colonic anastomosis in rats.

Use of fluorescent oligonucleotide probes for differentiation between Paracoccidioides brasiliensis and Paracoccidioides lutzii in yeast and mycelial phase

BACKGROUND Fluorescence in situ hybridisation (FISH) associated with Tyramide Signal Amplification (TSA) using oligonucleotides labeled with non-radioactive fluorophores is a promising technique for detection and differentiation of fungal species in environmental or clinical samples, being suitable for microorganisms which are difficult or even impossible to culture. OBJECTIVE In this study, we aimed to standardise an in situ hybridisation technique for the differentiation between the pathogenic species Paracoccidioides brasiliensis and Paracoccidioides lutzii, by using species-specific DNA probes targeting the internal transcribed spacer-1 (ITS-1) of the rRNA gene. METHODS Yeast and mycelial phase of each Paracoccidioides species, were tested by two different detection/differentiation techniques: TSA-FISH for P. brasiliensis with HRP (Horseradish Peroxidase) linked to the probe 5’ end; and FISH for P. lutzii with the fluorophore TEXAS RED-X® also linked to the probe 5’ end. After testing different protocols, the optimised procedure for both techniques was accomplished without cross-positivity with other pathogenic fungi. FINDINGS The in silico and in vitro tests show no reaction with controls, like Candida and Cryptococcus (in silico) and Histoplasma capsulatum and Aspergillus spp. (in vitro). For both phases (mycelial and yeast) the in situ hybridisation showed dots of hybridisation, with no cross-reaction between them, with a lower signal for Texas Red probe than HRP-TSA probe. The dots of hybridisation was confirmed with genetic material marked with 4’,6-diamidino-2-phenylindole (DAPI), visualised in a different filter (WU) on fluorescent microscopic. MAIN CONCLUSION Our results indicated that TSA-FISH and/or FISH are suitable for in situ detection and differentiation of Paracoccidioides species. This approach has the potential for future application in clinical samples for the improvement of paracoccidioidomycosis patients prognosis.

NIRF Heptamethine Cyanine Dye Nanocomplexes for Multi Modal Theranosis of Tumors / 전남의대학술지

Heptamethine cyanine dyes are categorized as a class of near infrared fluorescent (NIRF) dyes which have been discovered to have tumor targeting and accumulation capability. This unique feature of NIRF dye makes it a promising candidate for imaging, targeted therapy and also as a drug delivery vehicle for various types of cancers. The favored uptake of dyes only in cancer cells is facilitated by several factors which include organic anion-transporting polypeptides, high mitochondrial membrane potential and tumor hypoxia in cancer cells. Currently nanotechnology has opened possibilities for multimodal or multifunctional strategies for cancer treatment. Including heptamethine cyanine dyes in nanoparticle based delivery systems have generally improved its theranostic ability by several fold owing to the multiple functionalities and structural features of heptamethine dyes. For this reason, nanocomplexes with NIRF heptamethine cyanine dye probe are preferred over non-targeting dyes such as indo cyanine green (ICG). This review sums up current trends and progress in NIRF heptamethine cyanine dye, including dye properties, multifunctional imaging and therapeutic applications in cancer.

Structure-Inherent Targeting of Near-Infrared Fluorophores for Image-Guided Surgery / 전남의대학술지

Although various clinical imaging modalities have been developed to visualize internal body structures and detect abnormal tissues prior to surgical procedures, most medical imaging modalities do not provide disease-specific images in real-time. Optical imaging can provide the surgeon with real-time visualization of the surgical field for intraoperative image-guided surgery. Imaging in the near-infrared (NIR) window (650-900 nm), also known as the “therapeutic window” has high potential by offering low absorbance and scattering in tissues resulting in minimized background autofluorescence. Clinically, optical fluorescence imaging with the targeted contrast agents provides opportunities for significant advances in intraoperative image-guided surgery. There are only two clinically available NIR fluorophores, indocyanine green (ICG) and methylene blue (MB), that support the image-guided surgery. However, neither of them perform in vivo by providing optimum specificity and stability for targeted image guidance. Therefore, it is of paramount importance to develop targeted NIR fluorophores for unmet clinical needs. Using the right combination of an NIR fluorescence imaging system and a targeted fluorophore, the desired target tissues can be imaged to provide real-time fluorescence guidance without changing the field-of-view during surgery. Thus, in a clinical discipline, the development of NIR fluorophores for ‘structure-inherent targeting’ is an unmet need for early phase diagnostics with accurate targeting.