Search Results (11)

Background: Determining the optimal resection line for an organ that cannot be palpated is crucial, but challenging, in minimally invasive gastrointestinal (GI) surgery. Therefore, there is an urgent need to establish the most effective method for tumor localization. We hypothesize that our novel near-infrared (NIR) fluorescence detection system will enable the highly accurate detection of fluorescent clips marking GI cancer. Methods: Twenty-five patients with gastric cancer, esophagogastric junctional cancer, or esophageal cancer will be enrolled. NIR fluorescent clips will be placed endoscopically around the tumor on the day before surgery. Patients in whom clip dislodgement is confirmed by preoperative abdominal radiography will be excluded. The clips will be placed before the transection of the organ, and those on the surgical specimen will be observed after transection using both the novel NIR fluorescence detection system and an existing NIR fluorescence imaging system. The detection rate and time, the fluorescence intensity, surgical margins, and adverse events will be evaluated. This study has been registered in the Japan Registry of Clinical Trials, with the code jRCTs012240043. (Expected) Results: As the novel fluorescence detection system allows for higher-sensitivity detection by analyzing the spectral characteristics of fluorescence and measuring the peak values, we anticipate that this new system will detect the fluorescent clips with high accuracy. Conclusions: This study aims to establish a novel tumor-marking method using fluorescent clips and a new detection system that can be easily applied in various medical facilities. Full article

Ovarian cancer (OC) is one of the most lethal gynecologic cancers that is typically diagnosed at the very late stage of disease progression. Thus, there is an unmet need to develop diagnostic probes for early detection of OC. One approach may rely on RNA as a molecular biomarker. In this regard, FLJ22447 lncRNA is an RNA biomarker that is over-expressed in ovarian cancer (OC) and in cancer-associated fibroblasts (CAFs). CAFs appear early on in OC as they provide a metastatic niche for OC progression. FIT-PNAs (forced intercalation-peptide nucleic acids) are DNA analogs that are designed to fluoresce upon hybridization to their complementary RNA target sequence. In recent studies, we have shown that the introduction of cyclopentane PNAs into FIT-PNAs (cpFIT-PNA) results in superior RNA sensors. Herein, we report the design and synthesis of cpFIT-PNAs for the detection of this RNA biomarker in living OC cells (OVCAR8) and in CAFs. cpFIT-PNA was compared to FIT-PNA and the cell-penetrating peptide (CPP) of choice was either a simple one (four L-lysines) or a CPP with enhanced cellular uptake (CLIP6). The combination of CLIP6 with cpFIT-PNA resulted in a superior sensing of FLJ22447 lncRNA in OVCAR8 cells as well as in CAFs. Moreover, incubation of CLIP6-cpFIT-PNA in OVCAR8 cells leads to a significant decrease (ca. 60%) in FLJ22447 lncRNA levels and in cell viability, highlighting the potential theranostic use of such molecules. Full article

Unlocking cell secretion capacity is of paramount interest for the pharmaceutical industry focused on biologics. Here, we leveraged retention using a selective hook (RUSH) system for the identification of human osteosarcoma U2OS cell secretion modulators, through automated, high-throughput screening of small compound libraries. We created a U2OS cell line which co-expresses a variant of streptavidin addressed to the lumen-facing membrane of the endoplasmic reticulum (ER) and a recombinant anti-PD-L1 antibody. The heavy chain of the antibody was modified at its C-terminus, to which a furin cleavage site, a green fluorescent protein (GFP), and a streptavidin binding peptide (SBP) were added. We show that the U2OS cell line stably expresses the streptavidin hook and the recombinant antibody bait, which is retained in the ER through the streptavidin–SBP interaction. We further document that the addition of biotin to the culture medium triggers the antibody release from the ER, its trafficking through the Golgi where the GFP-SBP moiety is clipped off, and eventually its release in the extra cellular space, with specific antigen-binding properties. The use of this clone in screening campaigns led to the identification of lycorine as a secretion enhancer, and nigericin and tyrphostin AG-879 as secretion inhibitors. Altogether, our data support the utility of this approach for the identification of agents that could be used to improve recombinant production yields and also for a better understanding of the regulatory mechanism at work in the conventional secretion pathway. Full article

Due to the mechanistic coupling between solar-induced chlorophyll fluorescence (SIF) and photosynthesis, SIF has an advantage over greenness-based vegetation indices in detecting drought. Since photosystem I (PSI) contributes very little to red SIF, red SIF is assumed to be more responsive to environmental stress than far-red SIF. However, in addition to affecting photosynthesis, drought also has an impact on vegetation chlorophyll concentration and thus affects the reabsorption process of red SIF. When these responses are entangled, the sensitivity of SIF in the red and far-red regions in response to drought is not yet clear. In this study, we conducted a water stress experiment on maize in the field and measured the upward and downward leaf SIF spectra by a spectrometer assembled with a leaf clip. Simultaneously, leaf-level active fluorescence was measured with a pulse-amplified modulation (PAM) fluorometer. We found that SIF, after normalization by photosynthetically active radiation (PAR) and dark-adapted minimal fluorescence ($F_o$), is a better estimation of SIF yield. By comparing the wavelength-dependent link between SIF yield and nonphotochemical quenching (NPQ) across the range of 660 to 800 nm, the results show that red SIF and far-red SIF have different sensitivities in response to drought. SIF yield in the far-red region has a strong and stable correlation with NPQ. Drought not only reduces red SIF due to photosynthetic regulation, but it also increases red SIF by reducing chlorophyll content (weakening the reabsorption effect). The co-existence of these two contradictory effects makes the red SIF of leaf level unable to reliably indicate NPQ. In addition, the red:far-red ratio of downward SIF and the ratio between the downward SIF and upward SIF at the red peak can be good indicators of chlorophyll content. These findings can help to interpret SIF variations in remote sensing techniques and fully exploit SIF information in red and far-red regions when monitoring plant water stress. Full article

Two heteroctanuclear Au₄Ag₄ cluster complexes of 4,5-diethynylacridin-9-one (H₂L) were prepared through the self-assembly reactions of [Au(tht)₂](CF₃SO₃), Ag(tht)(CF₃SO₃), H₂L and PPh₃ or PPh₂Py (2-(diphenylphosphino)pyridine). The Au₄Ag₄ cluster consists of a [Au₄L₄]⁴⁻ and four [Ag(PPh₃)]⁺ or [Ag(PPh₂Py)]⁺ units with Au₄L₄ framework exhibiting a twisted paper clip structure. In CH₂Cl₂ solutions at ambient temperature, both compounds show ligand fluorescence at ca. 463 nm as well as phosphorescence at 650 nm for 1 and 630 nm for 2 resulting from admixture of ³IL (intraligand) of L ligand, ³LMCT (from L ligand to Au₄Ag₄) and ³MC (metal-cluster) triplet states. Crystals or crystalline powders manifest bright yellow-green phosphorescence with vibronic-structured emission bands at 530 (568sh) nm for complex 1 and 536 (576sh) nm for complex 2. Upon mechanical grinding, yellow-green emission in the crystalline state is dramatically converted to red luminescence centered at ca. 610 nm with a drastic redshift of the emission after crystal packing is destroyed. Full article

Cervical spinal cord injury (SCI) remains a devastating event without adequate treatment options despite decades of research. In this context, the usefulness of common preclinical SCI models has been criticized. We, therefore, aimed to use a clinically relevant animal model of severe cervical SCI to assess the long-term effects of neural precursor cell (NPC) transplantation on secondary injury processes and functional recovery. To this end, we performed a clip contusion-compression injury at the C6 level in 40 female Wistar rats and a sham surgery in 10 female Wistar rats. NPCs, isolated from the subventricular zone of green fluorescent protein (GFP) expressing transgenic rat embryos, were transplanted ten days after the injury. Functional recovery was assessed weekly, and FluoroGold (FG) retrograde fiber-labeling, as well as manganese-enhanced magnetic resonance imaging (MEMRI), were performed prior to the sacrifice of the animals eight weeks after SCI. After cryosectioning of the spinal cords, immunofluorescence staining was conducted. Results were compared between the treatment groups (NPC, Vehicle, Sham) and statistically analyzed (p < 0.05 was considered significant). Despite the severity of the injury, leading to substantial morbidity and mortality during the experiment, long-term survival of the engrafted NPCs with a predominant differentiation into oligodendrocytes could be observed after eight weeks. While myelination of the injured spinal cord was not significantly improved, NPC treated animals showed a significant increase of intact perilesional motor neurons and preserved spinal tracts compared to untreated Vehicle animals. These findings were associated with enhanced preservation of intact spinal cord tissue. However, reactive astrogliosis and inflammation where not significantly reduced by the NPC-treatment. While differences in the Basso–Beattie–Bresnahan (BBB) score and the Gridwalk test remained insignificant, animals in the NPC group performed significantly better in the more objective CatWalk XT gait analysis, suggesting some beneficial effects of the engrafted NPCs on the functional recovery after severe cervical SCI. Full article

The correct fertilization of vegetable crops is commonly determined on the basis of soil and plant costly destructive analyses, demanding more sustainable non-invasive optical detection. Here, we tested the ability of the combined transmittance/fluorescence leaf clip Dualex device for determining the nitrogen (N) status of cabbage plants. Fully developed leaves from plants grown under different N rates of 0; 100; 200; 300 kg N ha⁻¹ in 2018 and 2019 were measured in the field by the Dualex sensor twice a year in July and October. The chlorophyll (Chl) and nitrogen (nitrogen balance index, NBI) indices and the flavonols (Flav) index of the sensor were positively and negatively correlated to leaf nitrogen, respectively. Merging the two-years data, the NBI versus leaf N correlation was less point dispersed in October than July (R² = 0.76 and 0.64, respectively). NBI was also correlated to cabbage yield, better in July than October. Our results showed that the multiparametric Dualex device can be used as precision agriculture tool for the early prediction of plant N and cabbage yield with economic advantage for the growers and reduced environmental contamination due to nitrate leaching. Full article

Successful measurements of chlorophyll fluorescence (ChlF) spectral properties (typically in the wavelength range of 650–850 nm) across plant species, environmental conditions, and stress levels are a first step towards establishing a quantitative link between solar-induced chlorophyll fluorescence (SIF), which can only be measured at discrete ChlF spectral bands, and photosynthetic functionality. Despite its importance and significance, the various methodologies for the estimation of leaf-level ChlF spectral properties have not yet been compared, especially when applied to leaves with complex morphology, such as needles. Here we present, to the best of our knowledge, a first comparison of protocols for measuring leaf-level ChlF spectra: a custom-made system designed to measure ChlF spectra at ambient and 77 K temperatures (optical chamber, OC), the widely used FluoWat leaf clip (FW), and an integrating sphere setup (IS). We test the three methods under low-light conditions, across two broadleaf species and one needle-like species. For the conifer, we characterize the effect of needle arrangements: one needle, three needles, and needle mats with as little gap fraction as technically possible. We also introduce a simple baseline correction method to account for non-fluorescence-related contributions to spectral measurements. Baseline correction was found especially useful in recovering the spectra nearby the filter cut-off. Results show that the shape of the leaf-level ChlF spectra remained largely unaffected by the measurement methodology and geometry in OC and FW methods. Substantially smaller red/far-red ratios were observed in the IS method. The comparison of needle arrangements indicated that needle mats could be a practical solution to investigate temporal changes in ChlF spectra of needle-like leaves as they produced more reproducible results and higher signals. Full article

Laparoscopic surgery is now a standard treatment for gastric cancer. Currently, the location of the gastric cancer is identified during laparoscopic surgery via the preoperative endoscopic injection of charcoal ink around the primary tumor; however, the wide spread of injected charcoal ink can make it difficult to accurately visualize the specific site of the tumor. To precisely identify the locations of gastric tumors, we developed a fluorescent detection system comprising clips with glass phosphor (Yb³⁺, Nd³⁺ doped to Bi₂O₃-B₂O₃-based glasses, size: 2 mm × 1 mm × 3 mm) fixed in the stomach and a laparoscopic fluorescent detection system for clip-derived near-infrared (NIR) light (976 nm). We conducted two ex vivo experiments to evaluate the performance of this fluorescent detection system in an extirpated pig stomach and a freshly resected human stomach and were able to successfully detect NIR fluorescence emitted from the clip in the stomach through the stomach wall by the irradiation of excitation light (λ: 808 nm). These results suggest that the proposed combined NIR light-emitting clip and laparoscopic fluorescent detection system could be very useful in clinical practice for accurately identifying the location of a primary gastric tumor during laparoscopic surgery. Full article

Precise detection of leaf nitrogen concentration (LNC) is helpful for nutrient diagnosis and fertilization guidance in farm crops. Numerous researchers have estimated LNC with techniques based on reflectance spectra or active chlorophyll fluorescence, which have limitations of low accuracy or small scale in the field. Given the correlation between chlorophyll and nitrogen contents, the response of sun-induced chlorophyll fluorescence (SIF) to chlorophyll (Chl) content reported in a few papers suggests the feasibility of quantifying LNC using SIF. Few studies have investigated the difference and power of the upward and downward SIF components on monitoring LNC in winter wheat. We conducted two field experiments to evaluate the capacity of SIF to monitor the LNC of winter wheat during the entire growth season and compare the differences of the upward and downward SIF for LNC detection. A FluoWat leaf clip coupled with a ASD spectrometer was used to measure the upward and downward SIF under sunlight. It was found that three (↓FY687, ↑FY687/↑FY739, and ↓FY687/↓FY739) out of the six SIF yield (FY) indices examined were significantly correlated to the LNC (R² = 0.6, 0.51, 0.75, respectively). The downward SIF yield indices exhibited better performance than the upward FY indices in monitoring the LNC with the ↓FY687/↓FY739 being the best FY index. Moreover, the LNC models based on the three SIF yield indices are insensitive to the chlorophyll content and the leaf mass per area (LMA). These findings suggest the downward SIF should not be neglected for monitoring crop LNC at the leaf scale, although it is more difficult to measure with current instruments. The downward SIF could play an increasingly important role in understanding of the SIF emission for LNC detection at different scales. These results could provide a solid foundation for elucidating the mechanism of SIF for LNC estimation at the canopy scale. Full article

Communication of molecular species through dynamic association and/or dissociation at various cellular sites governs biological functions. Understanding these physiological processes require delineation of molecular events occurring at the level of individual complexes in a living cell. Among the few non-invasive approaches with nanometer resolution are methods based on Förster Resonance Energy Transfer (FRET). FRET is effective at a distance of 1–10 nm which is equivalent to the size of macromolecules, thus providing an unprecedented level of detail on molecular interactions. The emergence of fluorescent proteins and SNAP- and CLIP- tag proteins provided FRET with the capability to monitor changes in a molecular complex in real-time making it possible to establish the functional significance of the studied molecules in a native environment. Now, FRET is widely used in biological sciences, including the field of proteomics, signal transduction, diagnostics and drug development to address questions almost unimaginable with biochemical methods and conventional microscopies. However, the underlying physics of FRET often scares biologists. Therefore, in this review, our goal is to introduce FRET to non-physicists in a lucid manner. We will also discuss our contributions to various FRET methodologies based on microscopy and flow cytometry, while describing its application for determining the molecular heterogeneity of the plasma membrane in various cell types. Full article