A fast, easy, cost-free method to remove excess dye or drug from small extracellular vesicle solution.

Tracking small extracellular vesicles (sEVs), such as exosomes, requires staining them with dyes that penetrate their lipid bilayer, a process that leaves excess dye that needs to be mopped up to achieve high specificity. Current methods to remove superfluous dye have limitations, among them that they are time-intensive, carry the risk of losing sample and can require specialized equipment and materials. Here we present a fast, easy-to-use, and cost-free protocol for cleaning excess dye from stained sEV samples by adding their parental cells to the mixture to absorb the extra dye much like sponges do. Since sEVs are considered a next-generation drug delivery system, we further show the success of our approach at removing excess chemotherapeutic drug, daunorubicin, from the sEV solution.

Removal of Azo Dyes from Water Using Natural Luffa cylindrica as a Non-Conventional Adsorbent.

Reducing high concentrations of pollutants such as heavy metals, pesticides, drugs, and dyes from water is an emerging necessity. We evaluated the use of Luffa cylindrica (Lc) as a natural non-conventional adsorbent to remove azo dye mixture (ADM) from water. The capacity of Lc at three different doses (2.5, 5.0, and 10.0 g/L) was evaluated using three concentrations of azo dyes (0.125, 0.250, and 0.500 g/L). The removal percent (R%), maximum adsorption capacity (Qm), isotherm and kinetics adsorption models, and pH influence were evaluated, and Fourier-transform infrared spectroscopy and scanning electron microscopy were performed. The maximum R% was 70.8% for 10.0 g L-1Lc and 0.125 g L-1 ADM. The Qm of Lc was 161.29 mg g-1. Adsorption by Lc obeys a Langmuir isotherm and occurs through the pseudo-second-order kinetic model. Statistical analysis showed that the adsorbent dose, the azo dye concentration, and contact time significantly influenced R% and the adsorption capacity. These findings indicate that Lc could be used as a natural non-conventional adsorbent to reduce ADM in water, and it has a potential application in the pretreatment of wastewaters.

Effectiveness of sentinel lymph node biopsy and bilateral pelvic nodal dissection using methylene blue dye in early-stage operable cervical cancer-A prospective study.

OBJECTIVE: To evaluate the effectiveness of methylene blue dye in detecting sentinel lymph nodes (SLNs) in women with early-stage operable (defined as FIGO I-IIA) cervical cancer. It also aims to evaluate procedural challenges and accuracy. METHOD: This prospective study, which focused on 20 women with early-stage cervical cancer, was carried out between June 2016 and December 2017. These patients had SLN mapping with methylene blue dye injections and thorough examinations, including imaging. All patients underwent radical hysterectomy and complete bilateral pelvic lymphadenectomy. No additional investigation was done on the lymph node in cases where a metastasis was found in the first H&E-stained segment of the sentinel node. RESULT: 20 patients were included in the analysis. The median age of the subjects was 53, and 95 % of them had squamous cell carcinoma. 90 % of the time, the identification of SLNs was effective, and 55 SLNs were found, of which 52.7 % were on the right side of the pelvis and 47.3 % on the left. The obturator group had the most nodes, followed by the external and internal iliac groups in descending order of occurrence. Metastasis was detected in 3 patients, resulting in a sensitivity of 100 % and a specificity of 93.75 % for SLN biopsy. Notably, no false-negative SLNs were found. Complications related to methylene blue usage included urine discoloration in 30 % of patients. CONCLUSION: This trial highlights the promising efficacy and safety of methylene blue dye alone for SLN identification in early-stage operable cervical cancer, with a notably higher success rate. Despite limitations like a small sample size, healthcare professionals and researchers can build upon the insights from this study to enhance cervical cancer management.

Bacteriogenic synthesis of morphologically diverse silver nanoparticles and their assessment for methyl orange dye removal and antimicrobial activity.

Nanotechnology and nanoparticles have gained massive attention in the scientific community in recent years due to their valuable properties. Among various AgNPs synthesis methods, microbial approaches offer distinct advantages in terms of cost-effectiveness, biocompatibility, and eco-friendliness. In the present research work, investigators have synthesized three different types of silver nanoparticles (AgNPs), namely AgNPs-K, AgNPs-M, and AgNPs-E, by using Klebsiella pneumoniae (MBC34), Micrococcus luteus (MBC23), and Enterobacter aerogenes (MBX6), respectively. The morphological, chemical, and elemental features of the synthesized AgNPs were analyzed by using UV-Vis spectroscopy (UV-Vis), Fourier transform-infrared spectroscopy (FTIR), X-ray diffraction (XRD), field emission scanning electron microscope (FESEM) and energy-dispersive spectroscopy (EDX). UV-Vis absorbance peaks were obtained at 475, 428, and 503 nm for AgNPs-K, AgNPs-M, and AgNPs-E, respectively. The XRD analysis confirmed the crystalline nature of the synthesized AgNPs, having peaks at 26.2°, 32.1°, and 47.2°. At the same time, the FTIR showed bands at 599, 963, 1,693, 2,299, 2,891, and 3,780 cm-1 for all the types of AgNPs indicating the presence of bacterial biomolecules with the developed AgNPs. The size and morphology of the AgNPs varied from 10 nm to several microns and exhibited spherical to porous sheets-like structures. The percentage of Ag varied from 37.8% (wt.%) to 61.6%, i.e., highest in AgNPs-K and lowest in AgNPs-M. Furthermore, the synthesized AgNPs exhibited potential for environmental remediation, with AgNPs-M exhibiting the highest removal efficiency (19.24% at 120 min) for methyl orange dye in simulated wastewater. Further, all three types of AgNPs were evaluated for the removal of methyl orange dye from the simulated wastewater, where the highest dye removal percentage was 19.24% at 120 min by AgNPs-M. Antibacterial potential of the synthesized AgNPs assessment against both Gram-positive (GPB) Bacillus subtilis (MBC23), B. cereus (MBC24), and Gram-negative bacteria Enterococcus faecalis (MBP13) revealed promising results, with AgNPs-M, exhibiting the largest zone of inhibition (12 mm) against GPB B. megaterium. Such investigation exhibits the potential of the bacteria for the synthesis of AgNPs with diverse morphology and potential applications in environmental remediation and antibacterial therapy-based synthesis of AgNPs.

Perfusion outcomes with near-infrared indocyanine green imaging system in laparoscopic total mesorectal excision for mid-rectal or low-rectal cancer (POSTER): a study protocol.

INTRODUCTION: Anastomotic leakage (AL) is defined as the failure of complete healing or disruption of the anastomosis subsequent to rectal cancer surgery, resulting in the extravasation of intestinal contents into the intra-abdominal or pelvic cavity. It is a serious complication of rectal cancer surgery, accounting for a considerable increase in morbidity and mortality. The use of fluorescence imaging technology in surgery allows surgeons to better evaluate blood perfusion. However, the conclusions of some existing studies are not consistent, so a consensus on whether the near-infrared indocyanine green (NIR-ICG) imaging system can reduce the incidence of AL is needed. METHODS: This POSTER trial is designed as a multicentre, prospective, randomised controlled clinical study adhering to the "population, interventions, comparisons, outcomes (PICO)" principles. It is scheduled to take place from August 2019 to December 2024 across eight esteemed hospitals in China. The target population consists of patients diagnosed with rectal cancer through pathological confirmation, with tumours located≤10 cm from the anal verge, eligible for laparoscopic surgery. Enrolled patients will be randomly assigned to either the intervention group or the control group. The intervention group will receive intravenous injections of ICG twice, with intraoperative assessment of anastomotic blood flow using the near-infrared NIR-ICG system during total mesorectal excision (TME) surgery. Conversely, the control group will undergo conventional TME surgery without the use of the NIR-ICG system. A 30-day follow-up period postoperation will be conducted to monitor and evaluate occurrences of AL. The primary endpoint of this study is the incidence of AL within 30 days postsurgery in both groups. The primary outcome investigators will be blinded to the application of ICG angiography. Based on prior literature, we hypothesise an AL rate of 10.3% in the control group and 3% in the experimental group for this study. With a planned ratio of 2:1 between the number of cases in the experimental and control groups, and an expected 20% lost-to-follow-up rate, the initial estimated sample size for this study is 712, comprising 474 in the experimental group and 238 in the control group. ETHICS AND DISSEMINATION: This study has been approved by Ethics committee of Beijing Friendship Hospital, Capital Medical University (approval number: 2019-P2-055-02). The results will be disseminated in major international conferences and peer-reviewed journals. TRIAL REGISTRATION NUMBER: NCT04012645.

Aggregation-enhanced photothermal therapy of organic dyes.

Photothermal therapy (PTT) represents a groundbreaking approach to targeted disease treatment by harnessing the conversion of light into heat. The efficacy of PTT heavily relies on the capabilities of photothermal agents (PTAs). Among PTAs, those based on organic dyes exhibit notable characteristics such as adjustable light absorption wavelengths, high extinction coefficients, and high compatibility in biological systems. However, a challenge associated with organic dye-based PTAs lies in their efficiency in converting light into heat while maintaining stability. Manipulating dye aggregation is a key aspect in modulating non-radiative decay pathways, aiming to augment heat generation. This review delves into various strategies aimed at improving photothermal performance through constructing aggregation. These strategies including protecting dyes from photodegradation, inhibiting non-photothermal pathways, maintaining space within molecular aggregates, and introducing intermolecular photophysical processes. Overall, this review highlights the precision-driven assembly of organic dyes as a promising frontier in enhancing PTT-related applications. This article is categorized under: Therapeutic Approaches and Drug Discovery > Emerging Technologies Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease Diagnostic Tools > In Vivo Nanodiagnostics and Imaging.

An indocyanine green-based liquid biopsy test for circulating tumor cells for pediatric liver cancer.

BACKGROUND: Hepatoblastoma and HCC are the most common malignant hepatocellular tumors seen in children. The aim of this study was to develop a liquid biopsy test for circulating tumor cells (CTCs) for these tumors that would be less invasive and provide real-time information about tumor response to therapy. METHODS: For this test, we utilized indocyanine green (ICG), a far-red fluorescent dye used clinically to identify malignant liver cells during surgery. We assessed ICG accumulation in cell lines using fluorescence microscopy and flow cytometry. For our CTC test, we developed a panel of liver tumor-specific markers, including ICG, Glypican-3, and DAPI, and tested it with cell lines and noncancer control blood samples. We then used this panel to analyze whole-blood samples for CTC burden with a cohort of 15 patients with hepatoblastoma and HCC and correlated with patient characteristics and outcomes. RESULTS: We showed that ICG accumulation is specific to liver cancer cells, compared to nonmalignant liver cells, non-liver solid tumor cells, and other nonmalignant cells, and can be used to identify liver tumor cells in a mixed population of cells. Experiments with the ICG/Glypican-3/DAPI panel showed that it specifically tagged malignant liver cells. Using patient samples, we found that CTC burden from sequential blood samples from the same patients mirrored the patients' responses to therapy. CONCLUSIONS: Our novel ICG-based liquid biopsy test for CTCs can be used to specifically detect and quantify CTCs in the blood of pediatric patients with liver cancer.

The use of indocyanine green for lateral lymph node dissection in rectal cancer-preliminary data from an emerging procedure: a systematic review of the literature.

INTRODUCTION: Lateral lymph node dissection (LLND) for rectal cancer is still not a widely established technique owing to the existing controversy between Eastern and Western countries and the lack of well-designed studies. The risk of complications and the paucity of long-term oncological results are significant drawbacks for further applying this technique. The use of indocyanine green (ICG) near-infrared (NIR) fluorescence for LLND appears as a promising technique for enhancing postoperative and oncological outcomes. This review aims to evaluate the emerging role of ICG during LLND and present the benefits of its application. MATERIALS AND METHODS: Systematic electronic research was conducted in PubMed and Google Scholar using a combination of medical subject headings (MeSH). Studies presenting the use of ICG during LLND, especially in terms of harvested lymph nodes, were included and reviewed. Studies comparing LLND with ICG (LLND + ICG) or without ICG (LLND-alone) were further analyzed for the number of lymph nodes and postoperative outcomes. RESULTS: In total, 13 studies were found eligible and analyzed for different parameters. LLND + ICG is associated with significantly increased number of harvested lateral lymph nodes (p < 0.05), minor blood loss, decreased operative time, and probably decreased urinary retention postoperatively compared with LLND-alone. CONCLUSIONS: The use of ICG fluorescence during LLND is a safe and feasible technique for balancing postoperative outcomes and the number of harvested lymph nodes. Well-designed studies with long-term results are required to elucidate the oncological benefits and establish this promising technique.

Laser speckle flowgraphy has comparable accuracy to indocyanine green fluorescence angiography in assessing bowel blood perfusion.

PURPOSE: To investigate the accuracy of laser speckle flowgraphy (LSFG), a noninvasive method for the quantitative evaluation of blood flow using mean blur rate (MBR) as a blood flow parameter in the assessment of bowel blood perfusion compared to indocyanine green fluorescence angiography (ICG-FA). METHODS: We enrolled 46 patients who underwent left-sided colorectal surgery. LSFG and ICG-FA were applied to assess blood bowel perfusion, with MBR and luminance as parameters, respectively. In both measurement methods, the position where the parameter suddenly decreased was defined as the blood flow boundary line. Subsequently, the blood flow boundaries created after processing the blood vessels flowing into the intestinal tract were determined using LSFG and ICG-FA, and concordance between the two was examined. Blood flow boundaries were visually identified using color tone changes on a color map created based on MBR in LSFG and using differences in luminance in ICG-FA. The distances between the transection line and blood flow boundaries determined using each method were compared. RESULTS: The location of blood flow boundaries matched in 65% (30/46) of cases. Although locations differed in the remaining 35% (16/46), all were located on the anal side near the transection line, and the difference was not clinically significant. The average distances between the transection line and blood flow boundary were 2.76 (SD = 3.25) and 3.71 (SD = 4.26) mm, respectively. There was no statistically significant difference between the two groups (p = 0.38). CONCLUSION: LSFG was shown to have comparable accuracy to ICG-FA, and may be useful for evaluating bowel perfusion.

Photo-catalytic dye degradation potentials of Ag-Pd bimetallic nanoparticles synthesized from Vitex negundo.

Plant extracts are a great alternative to synthesizing nanoparticles of different metals and metal oxides. This green synthesis method has opened up numerous possibilities in various scientific domains. In present study, Leaf extract from Vitex negundo is a non-deciduous, long-lasting shrub from the Verbenaceae family is used as capping and reducing agents for the synthesis of silver and palladium nanoparticles. The characterization study UV-vis spectrophotometer analysis showed absorbance value around 320 nm which confirming that Ag-Pd nanoparticles have been successfully obtained. Further, SEM is used to investigate the morphology of Ag-Pd NPs, which revealing their spherical and rod-like configuration, aggregation, and the size of the particles are obtained between 50 and 100 nm. The successful synthesis of Ag-Pd NPs was further confirmed by the EDAX chart, which displayed the peak of Ag and Pd at bending energies between 0.5 and 1.5 keV. According to the quantitative study, Ag and Pd ions found about 5.24 and 13.28%, respectively. In addition, surface studies with TEM confirming that synthesized Ag-Pd NPs are predominates with spheres structure morphologies, with sizes averaging 11.20 nm and ranging from 10 to 20 nm. Further, Ag-Pd nanoparticles was applied as potential photocatalyst materials to degrade methylene blue dye and found about 85% of the degradation efficiency within 150 min of the sunlight exposure thus could be used as catalyst to removal of hazardous organic dye molecules.

Harnessing solar power for enhanced photocatalytic degradation of coloured pollutants using novel Mg-doped-ZnFe2O4/S@g-C3N4 heterojunction: A facile hydrothermal synthesis approach.

The ability of heterogeneous photocatalysis to effectively remove organic pollutants from wastewater has shown great promise as a tool for environmental remediation. Pure zinc ferrites (ZnFe2O4) and magnesium-doped zinc ferrites (Mg@ZnFe2O4) with variable percentages of Mg (0.5, 1, 3, 5, 7, and 9 mol%) were synthesized via hydrothermal route and their photocatalytic activity was checked against methylene blue (MB) taken as a model dye. FTIR, XPS, BET, PL, XRD, TEM, and UV-Vis spectroscopy were used for the identification and morphological characterization of the prepared nanoparticles (NPs) and nanocomposites (NCs). The 7% Mg@ZnFe2O4 NPs demonstrated excellent degradation against MB under sunlight. The 7% Mg@ZnFe2O4 NPs were integrated with diverse contents (10, 50, 30, and 70 wt.%) of S@g-C3N4 to develop NCs with better activity. When the NCs were tested to degrade MB dye, it was revealed that the 7%Mg@ZnFe2O4/S@g-C3N4 NCs were more effective at utilizing solar energy than the other NPs and NCs. The synergistic effect of the interface formed between Mg@ZnFe2O4 and S@g-C3N4 was primarily responsible for the boosted photocatalytic capability of the NCs. The fabricated NCs may function as an effective new photocatalyst to remove organic dyes from wastewater.

Highly effective ultrafiltration membranes based on plastic waste for dye removal from water.

Applicable and low-cost ultrafiltration membranes based on waste polystyrene (WPS) blend and poly vinylidene fluoride (PVDF) were effectively cast on nonwoven support using phase inversion method. Analysis was done into how the WPS ratio affected the morphology and antifouling performance of the fabricated membranes. Cross flow filtration of pure water and various types of polluted aqueous solutions as the feed was used to assess the performance of the membranes. The morphology analysis shows that the WPS/PVDF membrane layer has completely changed from a spongy structure to a finger-like structure. In addition, the modified membrane with 50% WPS demonstrated that the trade-off between selectivity and permeability is met by a significant improvement in the rejection of the membrane with a reduction in permeate flux due to the addition of PVDF. With a water permeability of 50 LMH and 44 LMH, respectively, the optimized WPS-PVDF membrane with 50% WPS could reject 81% and 74% of Congo red dye (CR) and methylene blue dye (MB), respectively. The flux recovery ratio (FRR) reached to 88.2% by increasing PVDF concentration with 50% wt. Also, this membrane has the lowest irreversible fouling (Rir) value of 11.7% and lowest reversible fouling (Rr) value of 27.9%. The percent of cleaning efficiency reach to 71%, 90%, and 85% after eight cycles of humic acid (HA), CR, and MB filtration, respectively, for the modified PS-PVDF (50%-50%). However, higher PVDF values cause the membrane's pores to become clogged, increase the irreversible fouling, and decrease the cleaning efficiency. In addition to providing promising filtration results, the modified membrane is inexpensive because it was made from waste polystyrene, and as a result, it could be scaled up to treat colored wastewater produced by textile industries. PRACTITIONER POINTS: Recycling of plastic waste as an UF membrane for water/wastewater treatment was successfully prepared and investigated. Mechanical properties showed reasonable response with adding PVDF. The modified membrane with 50% PS demonstrated that the trade-off between selectivity and permeability is met by a significant improvement in the rejection.

Minimizing Structural Heterogeneity in DNA Self-Assembled Dye Templating via DNA Origami-Tuned Conformations.

With recent advances in DNA-templated dye aggregation for leveraging and engineering molecular excitons, a need exists for minimizing structural heterogeneity. Holliday Junction complexes (HJ) are commonly used to covalently template dye aggregates on their core; however, the global conformation of HJ is detrimentally dynamic. Here, the global conformation of the HJ is selectively tuned by restricting its position and orientation by using a sheet-like DNA origami construct (DOC) physisorbed on glass. The HJ arms are fixed with four different designed interduplex angles (IDAs). Atomic force microscopy confirmed that the HJs are bound to the surface of DOC with tuned IDAs. Dye orientation distributions were determined by combining dipole imaging and super-resolution microscopy. All IDAs led to dye orientations having dispersed distributions along planes perpendicular to the HJ plane, suggesting that stacking occurred between the dye and the neighboring DNA bases. The dye-base stacking interpretation was supported by increasing the size of the core cavity. The narrowest IDA minimizes structural heterogeneity and suggests dye intercalation. A strong correlation is found between the IDA and the orientation of the dye along the HJ plane. These results show that the HJ imposes restrictions on the dye and that the dye-DNA interactions are always present regardless of global conformation. The implications of our results are discussed for the scalability of dye aggregates using DNA self-assembly. Our methodology provides an avenue for the solid-supported single-molecule characterization of molecular assemblies templated on biomolecules─such as DNA and protein templates involved in light-harvesting and catalysis─with tuned conformations and restricted in position and orientation.

Biodecolorization and biodegradation of Reactive Green 12 textile industry dye and their post-degradation phytotoxicity-genotoxicity assessments.

The employment of versatile bacterial strains for the efficient degradation of carcinogenic textile dyes is a sustainable technology of bioremediation for a neat, clean, and evergreen globe. The present study has explored the eco-friendly degradation of complex Reactive Green 12 azo dye to its non-toxic metabolites for safe disposal in an open environment. The bacterial degradation was performed with the variable concentrations (50, 100, 200, 400, and 500 mg/L) of Reactive Green 12 dye. The degradation and toxicity of the dye were validated by high-performance liquid chromatography, Fourier infrared spectroscopy analysis, and phytotoxicity and genotoxicity assay, respectively. The highest 97.8% decolorization was achieved within 12 h. Alternations in the peaks and retentions, thus, along with modifications in the functional groups and chemical bonds, confirmed the degradation of Reactive Green 12. The disappearance of a major peak at 1450 cm-1 corresponding to the -N=N- azo link validated the breaking of azo bonds and degradation of the parent dye. The 100% germination of Triticum aestivum seed and healthy growth of plants verified the lost toxicity of degraded dye. Moreover, the chromosomal aberration of Allium cepa root cell treatment also validated the removal of toxicity through bacterial degradation. Thereafter, for efficient degradation of textile dye, the bacterium is recommended for adaptation to the sustainable degradation of dye and wastewater for further application of degraded metabolites in crop irrigation for sustainable agriculture.

Intraoperative bladder visualization by indocyanine green filling and subsequent washout in endoscopic hysterectomy: ICG-Washout method.

INTRODUCTION: Despite a potential risk of bladder injury in laparoscopic hysterectomy (LH) and robot-assisted LH (RaLH), an intraoperative method for delineating the entire bladder with indocyanine green (ICG) has not been established. METHODS: We conducted a preliminary experiment using porcine bladders to verify the appropriate amount of ICG for intraoperative bladder visualization. Afterward, intraoperative bladder visualization was tried in LH and RaLH in two patients suspected of having adhesions around the bladder after previous abdominal surgery. RESULTS: Although near-infrared (NIR) fluorescence was well observed through the wall of the porcine bladder filled with ICG solution at a concentration of 0.024 mg/mL, the subsequent replacement of the ICG solution with saline made the NIR fluorescence brighter. In both patients, the bladder was successfully delineated by NIR fluorescence after filling the bladder with ICG solution and the subsequent washout with saline. CONCLUSION: The ICG-Washout method for locating the bladder by NIR fluorescence could be useful in LH and RaLH.

Quantum dot fluorescent microsphere-based immunochromatographic strip for detecting PRRSV antibodies.

Porcine reproductive and respiratory syndrome (PRRS) is an immunosuppressive disease caused by the porcine reproductive and respiratory syndrome virus (PRRSV). Current vaccine prevention and treatment approaches for PRRS are not adequate, and commercial vaccines do not provide sufficient cross-immune protection. Therefore, establishing a precise, sensitive, simple, and rapid serological diagnostic approach for detecting PRRSV antibodies is crucial. The present study used quantum dot fluorescent microspheres (QDFM) as tracers, covalently linked to the PRRSV N protein, to develop an immunochromatography strip (ICS) for detecting PRRSV antibodies. Monoclonal antibodies against PRRSV nucleocapsid (N) and membrane (M) proteins were both coated on nitrocellulose membranes as control (C) and test (T) lines, respectively. QDFM ICS identified PRRSV antibodies under 10 min with high sensitivity and specificity. The specificity assay revealed no cross-reactivity with the other tested viruses. The sensitivity assay revealed that the minimum detection limit was 1.2 ng/mL when the maximum dilution was 1:2,048, comparable to the sensitivity of enzyme-linked immunosorbent assay (ELISA) kits. Moreover, compared to PRRSV ELISA antibody detection kits, the sensitivity, specificity, and accuracy of QDFM ICS after analyzing 189 clinical samples were 96.7%, 97.9%, and 97.4%, respectively. Notably, the test strips can be stored for up to 6 months at 4 °C and up to 4 months at room temperature (18-25 °C). In conclusion, QDFM ICS offers the advantages of rapid detection time, high specificity and sensitivity, and affordability, indicating its potential for on-site PRRS screening. KEY POINTS: • QDFM ICS is a novel method for on-site and in-lab detection of PRRSV antibodies • Its sensitivity, specificity, and accuracy are on par with commercial ELISA kits • QDFM ICS rapidly identifies PRRSV, aiding the swine industry address the evolving virus.

UFObow: A single-wavelength excitable Brainbow for simultaneous multicolor ex-vivo and in-vivo imaging of mammalian cells.

Brainbow is a genetic cell-labeling technique that allows random colorization of multiple cells and real-time visualization of cell fate within a tissue, providing valuable insights into understanding complex biological processes. However, fluorescent proteins (FPs) in Brainbow have distinct excitation spectra with peak difference greater than 35 nm, which requires sequential imaging under multiple excitations and thus leads to long acquisition times. In addition, they are not easily used together with other fluorophores due to severe spectral bleed-through. Here, we report the development of a single-wavelength excitable Brainbow, UFObow, incorporating three newly developed blue-excitable FPs. We have demonstrated that UFObow enables not only tracking the growth dynamics of tumor cells in vivo but also mapping spatial distribution of immune cells within a sub-cubic centimeter tissue, revealing cell heterogeneity. This provides a powerful means to explore complex biology in a simultaneous imaging manner at a single-cell resolution in organs or in vivo.

FAST, a method based on split-GFP for the detection in solution of proteins synthesized in cell-free expression systems.

Cell-free protein synthesis (CFPS) systems offer a versatile platform for a wide range of applications. However, the traditional methods for detecting proteins synthesized in CFPS, such as radioactive labeling, fluorescent tagging, or electrophoretic separation, may be impractical, due to environmental hazards, high costs, technical complexity, and time consuming procedures. These limitations underscore the need for new approaches that streamline the detection process, facilitating broader application of CFPS. By harnessing the reassembly capabilities of two GFP fragments-specifically, the GFP1-10 and GFP11 fragments-we have crafted a method that simplifies the detection of in vitro synthesized proteins called FAST (Fluorescent Assembly of Split-GFP for Translation Tests). FAST relies on the fusion of the small tag GFP11 to virtually any gene to be expressed in CFPS. The in vitro synthesized protein:GFP11 can be rapidly detected in solution upon interaction with an enhanced GFP1-10 fused to the Maltose Binding Protein (MBP:GFP1-10). This interaction produces a fluorescent signal detectable with standard fluorescence readers, thereby indicating successful protein synthesis. Furthermore, if required, detection can be coupled with the purification of the fluorescent complex using standardized MBP affinity chromatography. The method's versatility was demonstrated by fusing GFP11 to four distinct E. coli genes and analyzing the resulting protein synthesis in both a homemade and a commercial E. coli CFPS system. Our experiments confirmed that the FAST method offers a direct correlation between the fluorescent signal and the amount of synthesized protein:GFP11 fusion, achieving a sensitivity threshold of 8 ± 2 pmol of polypeptide, with fluorescence plateauing after 4 h. Additionally, FAST enables the investigation of translation inhibition by antibiotics in a dose-dependent manner. In conclusion, FAST is a new method that permits the rapid, efficient, and non-hazardous detection of protein synthesized within CFPS systems and, at the same time, the purification of the target protein.