Use Of Fluorescence Research Articles

Dual-mode sensing platform for Bisphenol A detection via bifunctional CsPbBr3@Cu-MOF assisted fluorescence and colorimetric analysis

BackgroundBisphenol A (BPA) has been identified as an endocrine disruptor with numerous detrimental effects on human health. There is an urgent need to develop fluorescence/colorimetric dual-mode sensing approaches with expanded detection linear range, increased accuracy, and enhanced application flexibility for BPA detection. The utilization of fluorescence and colorimetric signals in point-of-care applications and real-time sensitive sensing further highlights the significance of developing novel and efficient fluorescence/colorimetric dual-mode sensing platform with high-efficiency probes. ResultsHerein, a fluorescence and colorimetric dual-mode aptasensor was developed by using CsPbBr3@Cu-MOF as the aptamer immobilization matrix and signal generator. CsPbBr3 was functionalized with Cu-MOF using a simple two-step strategy. This strategy involved in-situ formation and modified ligand-assisted precipitation technique with 4,4′-bipyridine (4,4-Bpy) serving as the bifunctional linker. The resulting CsPbBr3@Cu-MOF exhibited improved stability in water and enhanced fluorescence. Additionally, it functioned as peroxidase mimetic to oxidize 3,3′,5,5′-tetramethyl benzidine (TMB), leading to a colorimetric change from colorless to blue. In the presence of BPA, aptamers were removed from CsPbBr3@Cu-MOF. Consequently, the fluorescence and peroxidase-activity of CsPbBr3@Cu-MOF were recovered, resulting in the enhanced fluorescence intensity and color change of TMB. Using this system, the proposed aptasensor demonstrated detection ranges of 1.0–80.0 nM with a LOD of 0.60 nM for the colorimetric method, and a linearity range of 0.1–100 nM with a LOD of 0.02 nM for the fluorescence method. SignificanceThe obtained CsPbBr3@Cu-MOF composites showed excellent fluorescence properties, good peroxidase-like activity, and aqueous stability. Furthermore, the proposed dual-mode aptasensor demonstrated simplicity, cost-effectiveness and good anti-interference abilities. This can be extended to the construction of other dual-mode sensors by changing aptamers and provides novel insights on the potential applications of perovskites in bioanalysis.

The Alphabet Soup of Microscopy: An Introduction to Advanced Imaging Techniques. Part II: What the FLIP, FLAP, FRAP, FRET, and FLIM is Going On?

Abstract The use of fluorescence in imaging has been a pivotal factor in advancing our scientific understanding. As microscopy continues to evolve, the terminology used to describe these techniques becomes increasingly complex, often resulting in a bewildering array of acronyms that resemble alphabet soup. Among the most prominent acronyms are those associated with advanced fluorescence microscopy techniques: Fluorescence Loss in Photobleaching (FLIP), Fluorescence Localization after Photobleaching (FLAP), Fluorescence Recovery after Photobleaching (FRAP), Förster Resonance Energy Transfer (FRET), and Fluorescence Lifetime Imaging Microscopy (FLIM). Each of these methods provides invaluable insights into molecular dynamics and interactions within living cells and tissues. This article, Part 2 of the Alphabet Soup of Microscopy series, aims to clarify these widely used fluorescence microscopy techniques and to illuminate their contributions to our understanding of cellular processes.

Synthetic Chemistry and Microelectrode Arrays for Point of Care Diagnostics

Molecular diagnostics typically require a molecular interaction that identifies a specific target, a device that detects, quantifies, and records that interaction, and an interface between those two elements. Each feature is critical for the success of the device. Yet while the first two are matters of intense interest an research, the surface itself is often ignored. This is problematic because it provides the platform for the whole "experiment". For a "point-of-care" diagnostic, the surface on the device must be stable both in terms of chemistry and time while still allowing the signal from the molecular interaction to be sensed and recorded.With this in mind, we have been focusing on the use of diblock copolymers as molecular surfaces for the construction of "point-of-care" diagnostics. We have developed a toolbox of chemical reactions that can be used site-selectively on such surfaces, and we have recently demonstrated that the approach taken is compatible with the use of DNA-aptamers to do multiplex sensing on a high density microelectrode array using a surface that is stable for a year. We have also illustrated how the quality of the signaling study conducted on an array is dependent on optimization of the chemical reaction.But exactly how good are the chemical reactions employed on the arrays to build a surface in a site-selective manner? One can tell with the use of fluorescence based studies that reactions happen where you want then to happen, but are there background reactions that can be problematic for a multiplex sensing experiment, do the reactions happen in high yield, and how much of an expensive, difficult to obtain biological reagent might be lost due to the confinement strategy? Are some reactions better than others in this regard? In short, if we are to optimize the surfaces constructed on a diagnostic device, then we need not only have tools in place for conducting synthetic chemistry on the device, but also the tools in place necessary to analyze those reactions. Understanding the chemistry that can and/or does occur at any electrode in a high density array requires more than looking at pretty pictures of fluorescent spots. In the talk to be given, we will utilize a combination of a "Kenner-safety-catch" linker strategy and molecular biology techniques to answer key questions about the synthetic chemistry used to construct the DNA-aptamer based multiplex sensor mentioned above.

A Fluorescence-based Method to Reaccess Root Canals in Endodontically Treated Teeth: A Micro–Computed Tomography Tridimensional Assessment

IntroductionThe aim of this study was to evaluate the volume of dentin removal and the volume of remnants of restorative material after the removal of an esthetic restorative coronal set and cervical barrier in endodontically treated mandibular molars with the aid of different magnification methods using 3-dimensional (3D) micro–computed tomographic (micro-CT) morphometric analysis. MethodsA sample of 30 mandibular first molars (N = 30) was used. All teeth were endodontically treated, and the specimens were initially scanned using micro-CT imaging and reconstructed. The molars were filled by a single-cone technique, and immediately the material at the initial 2-mm cervical level was removed. Cervical barriers were confected using ionomer glass cement with fluorescein 0.1%, filling the 2 mm at the cervical level of the canals and an additional 2 mm as the base. The coronal restoration set was performed using esthetic resin composites. A simulated tooth aging process was performed with 20,000 thermocycling cycles. The sample was distributed into the following 3 groups (n = 10) for the removal of the restoration set and cervical barrier with diamond burs based on the magnification aid: no magnification aid (naked eye), operative microscope aid, and REVEAL device (Design for Vision Inc, Bohemia, NY) aid. After removal, the final 3D micro-CT scanning and reconstruction were conducted with the same parameters as the initial scanning, and superposition of the final and initial scanning was performed. Morphometric analysis was conducted using CTAn software (Bruker microCT, Kontich, Belgium) to assess the volume of remnant restorative material (mm³), the volume of dentin removal (mm³), and the direction and site of dentin removal. Data were analyzed using 1-Way analysis of variance (P < .05). ResultsThe REVEAL group showed better results regarding the volume of remnant material (3.17 ± 1.65) and the percentage of dentin removal (2.56 ± 1.34). The microscope group showed no statistical difference compared with the REVEAL group regarding dentin removal (3.30 ± 1.48) and was statistically similar to the naked eye group in the volume of remnant material (9.63 ± 4.33). The naked eye group showed the worst results for the volume of remnant material (7.60 ± 2.68) and the percentage of dentin removal (6.60 ± 3.70). ConclusionsThe use of fluorescence associated with magnification was the method that presented the best results, with lower percentages of dentin removal and smaller volumes of remaining restorative material. This is an innovative technology in endodontics that shows potential to overcome the challenge of reaccessing root canals in the context of endodontic retreatment.

Usefulness of fluorescence imaging with indocyanine green for evaluation of bowel perfusion in the urgency setting: a systematic review and meta-analysis.

Fluorescence imaging with indocyanine green (ICG) has been extensively utilized to assess bowel perfusion in oncologic surgery. In the emergency setting, there are many situations in which bowel perfusion assessment is required. Large prospective studies or RCTs evaluating feasibility, safety and utility of ICG in the emergency setting are lacking. The primary aim is to assess the usefulness of ICG for evaluation of bowel perfusion in the emergency setting. The manuscript was drafted following the recommendations of Preferred Reporting Items for Systematic Reviews and Meta-Analyses statement (PRISMA). A systematic literature search was carried out through Pubmed, Scopus, and the ISI Web of Science. Assessment of included study using the methodological index for nonrandomized studies (MINORS) was calculated. The meta-analysis was carried out in line with recommendations from the Cochrane Collaboration and Meta-analysis of Observational Studies in Epidemiology guidelines, and the Mantel-Haenszel random effects model was used to calculate effect sizes. 10093 papers were identified. Eighty-four were reviewed in full-text, and 78 were excluded: 64 were case reports; 10 were reviews without original data; 2 were letters to the editor; and 2 contained unextractable data. Finally, six studies 22-27 were available for quality assessment and quantitative synthesis. The probability of reoperation using ICG fluorescence angiography resulted similar to the traditional assessment of bowel perfusion with a RD was -0.04 (95% CI: -0.147 to 0.060). The results were statistically significant P =0.029, although the heterogeneity was not negligible with a 59.9% of the I2 index. No small study effect or publication bias were found. This first metanalysis on the use of IGC fluorescence for ischemic bowel disease showed that this methodology is a safe and feasible tool in the assessment of bowel perfusion in the emergency setting. This topic should be further investigated in high-quality studies.

In Vivo Labeling and Detection of Circulating Tumor Cells in Mice Using OTL38

PurposeWe recently developed an optical instrument to non-invasively detect fluorescently labeled circulating tumor cells (CTCs) in mice called ‘Diffuse in vivo Flow Cytometry’ (DiFC). OTL38 is a folate receptor (FR) targeted near-infrared (NIR) contrast agent that is FDA approved for use in fluorescence guided surgery of ovarian and lung cancer. In this work, we investigated the use OTL38 for in vivo labeling and detection of FR + CTCs with DiFC.ProceduresWe tested OTL38 labeling of FR + cancer cell lines (IGROV-1 and L1210A) as well as FR- MM.1S cells in suspensions of Human Peripheral Blood Mononuclear cells (PBMCs) in vitro. We also tested OTL38 labeling and NIR-DIFC detection of FR + L1210A cells in blood circulation in nude mice in vivo.Results62% of IGROV-1 and 83% of L1210A were labeled above non-specific background levels in suspensions of PBMCs in vitro compared to only 2% of FR- MM.1S cells. L1210A cells could be labeled with OTL38 directly in circulation in vivo and externally detected using NIR-DiFC in mice with low false positive detection rates.ConclusionsThis work shows the feasibility of labeling CTCs in vivo with OTL38 and detection with DiFC. Although further refinement of the DiFC instrument and signal processing algorithms and testing with other animal models is needed, this work may eventually pave the way for human use of DiFC.

Characteristics and literature review of ETV6::ABL1 fusion gene-positive acute myeloid leukemia.

To describe the features of ETV6::ABL1 AML as well as the clinical treatment and outcomes. Clinical data were collected from three patients diagnosed with ETV6::ABL1 AML at Hebei Yanda Lu Daopei Hospital and Beijing Lu Daopei Hospital. Their clinical and laboratory features were analyzed, and the treatment process and outcomes were described. Ten reported cases of ETV6::ABL1 AML from the literature were also included for analysis. The median age of the patients was 34years, and 2 patients were male. No patient had a history of blood disorders before diagnosis. After relapse, they were referred to our hospital, where the ETV6::ABL1 gene was detected. Unfortunately, Patient 1 died rapidly after leukemia relapse due to severe infection. Patients 2 and 3 received salvage therapy with a dasatinib-containing regimen, followed by allo-HSCT, and are currently alive and disease-free. ETV6::ABL1 is a rare but recurrent genetic aberration in AML, and the combined use of fluorescence in situ hybridization and PCR can better identify this fusion gene. Patients carrying ETV6::ABL1 have a high relapse rate and a poor prognosis. TKIs are a reasonable treatment option for this group, and allo-HSCT may be curative.

Optical Methods for the Study of Brain Metabolism In Situ.

From its inception, the International Society of Oxygen Transport to Tissue (ISOTT) included several core members who were intrigued by the possibility of using light to probe the metabolic state of the cerebral cortex. This 50th anniversary review will focus on optical methods (UV, Visible, NIR) used to study brain oxygen metabolism in the intact brain. Chance, Lübbers, and Jöbsis did fundamental work in the 1970s and 1980s. These investigators spearheaded the use of fluorescence of pyridine nucleotides (e.g., NADH) and flavoprotein, and visible and near-infrared spectroscopy of haemoglobin amount and saturation, as well as the mitochondrial cytochrome chain. Two of our society awards are named for these pioneers and founders. By the new millennium, their many scientific descendants (although still too few for the task at hand) have been productive and moved the field forward. Technical advances in materials, detectors, devices, and computer power have made great advances possible. Physiological theory and understanding of cell and molecular mechanisms still lag, providing fertile territory for future goals. There are at least two major directions currently that will certainly continue to illuminate brain metabolism (obvious-and well worn-pun intended). These are the burgeoning NIR instrumentation variations, and second, the tremendous potential of two-photon phosphorescence lifetime microscopy. These advances have been introduced through the years and continue to be advanced at our annual ISOTT symposia.

Molecular cytogenetics of four species of Calendula (Asteraceae)

For the first time, a comparative molecular cytogenetic study of four species of Calendula L. (Asteraceae): C. officinalis, C. stellata, C. tripterocarpa, and C. arvensis was carried out. In each species, chromosome numbers were determined: C. stellata (2n = 2х = 14), C. officinalis (2n = 4x = 32), C. tripterocarpa (2n = 2x = 30), and C. arvensis (2n = 4x = 44), and also specific chromosome localization of 45S and 5S rDNA clusters was revealed with the use of fluorescence in situ hybridization. An additional polymorphic minor 45S rDNA hybridization signal was found in the C. officinalis karyotype, which was located median in the short arm of one chromosome. The patterns of chromosome distribution of the major sites of 45S and 5S rDNA in karyotypes of the studied species confirmed the hybrid origin of C. arvensis (2n = 4x = 44) which could be a result of the introgressive hybridization of two other species: C. stellata (2n = 2x = 14) and C. tripterocarpa (2n = 2x = 30) during speciation.

A Comparative Study of Clinical Characteristics, Cytogenetic Abnormalities and Outcomes Among Multiple Myeloma, Primary Light-Chain Amyloidosis and Multiple Myeloma with Concurrent AL Amyloidosis

Introduction: Multiple myeloma (MM) and light-chain (AL) amyloidosis are both plasma cell dyscrasias and may coexist in the same patients, contributing to distinct clinical outcomes. Although they share a similar spectrum of cytogenetic abnormalities (CA), the distribution and prognostic value of these aberrations are varied. The use of fluorescence in situ hybridization (FISH) in predicting outcomes for MM is well established, while the prognostic value of CA in AL amyloidosis, notably in MM with concurrent AL amyloidosis, is still evolving. This study aimed to evaluate the outcomes of patients with MM, AL amyloidosis and coexistence of the two disorders, and address the landscape and prognostic implication of CA in patients with these 3 types of plasma cell dyscrasias. Methods: A total of 902 consecutive patients with newly diagnosed MM between January 2007 and October 2021 or AL amyloidosis between January 2007 and June 2023 were retrospectively included. Patients were classified as MM alone, MM with coexistent AL amyloidosis (MM-AL) or primary AL amyloidosis alone (pAL-alone). The presence of symptomatic MM was based on the International Myeloma Working Group (IMWG) criteria and the diagnosis of AL amyloidosis was confirmed by Congo-red-positive biopsy and immunoelectron microscopy study of the amyloid. Demographic and clinical characteristics, FISH results, along with real-world effectiveness outcomes including best hematologic response, overall survival (OS), and progression free survival (PFS) were assessed. Results: Of the 902 patients enrolled, 658 patients were in the MM-alone group, 99 in the MM-AL group and 145 in the pAL-alone group. The three groups share a similar incidence rate of CA, while the prevalence of t(11;14) was significantly higher in pAL-alone group than MM-AL and MM-alone group (41.0% vs. 24.5% vs. 16.6%, p&amp;lt;0.001), and the prevalence of del13q14, gain1q21 and IMWG high-risk cytogenetic abnormalities [HRCA, including t(4;14), t(14;16) and del17p] decrease in turn in MM-alone, MM-AL and pAL-alone group(del13q14, 46.4% vs. 38.1% vs. 29.7%, p=0.005; gain1q21, 52.6% vs. 46.4% vs. 27.8%, p&amp;lt;0.001; HRCA, 27.5 % vs. 17.2 vs. 7.6%, p&amp;lt;0.001) (Table). In patients with AL amyloidosis with or without coexisting MM, presence of del13q14 was associated with higher level of baseline bone marrow plasmacytosis and difference in involved and uninvolved free light chain (dFLC). Compared with the MM-alone group, a significantly lower proportion of the MM-AL group achieved hematologic response (90.4% vs. 67.7%, p&amp;lt;0.001) and very good partial remission (VGPR, 44.6% vs. 68.9%, p&amp;lt;0.001). The OS of patients with MM-AL was significantly inferior to patients with MM-alone (median, 25.2 months vs. 96.8 months, p&amp;lt;0.001) or pAL-alone (median, 25.2 months vs. not reached, p&amp;lt;0.001 )(Figure B), while patients with MM-alone has shorter PFS than AL amyloidosis irrespective of the presence or absence of MM (MM-alone vs. pAL-alone: median, 56.8 months vs. not reached, p=0.007; MM-alone vs. MM-AL: median, 56.8 months vs. not reached, p=0.515) (Figure A). Other than MM-alone group, no significant difference in PFS and OS was found between pAL patients with and without HRCA, as well as the MM-AL group, indicating the difference in prognostic implications of common HRCAs among the three groups. When stratified by the type of plasma cell disorders and status of t(11;14) (a CA that strongly related to poor outcome of pAL), patients with MM-AL and t(11;14) presented the worst OS (median, 8.2 months, p&amp;lt;0.001) (Figure C). Conclusion: This comparative study presented an apparent discrepancy in distribution and prognostic role of CA in different plasma cell dyscrasias. Comparing with patients with MM-alone and pAL-alone, MM-AL patients, notably with the presence of t(11;14), had the poorest survival.

ETV6-ABL1 Fusion Gene Was Associated with a Poor Prognosis in Patients with Acute Myeloid Leukemia

Background:Acute myeloid leukemia (AML) is commonly characterized by chromosomal abnormalities that result in the formation of chimeric genes involved in leukemogenesis. ETV6-ABL1-a fusion gene results from a complex rearrangement involving translocation, inversion, or insertion of ETV6 into chromosomal band 9q34 or ABL1 into 12p13. This rare but recurrent genetic aberration is found in AML patients and is associated with a poor prognosis. Methods:A retrospective review was conducted of three relapsed AML patients with ETV6-ABL1 fusion genes screened by multiplex RT-PCR who were treated at Beijing Lu Daopei Hospital and Hebei Yanda Lu Daopei Hospital between November 2019 and January 2022. Results:At diagnosis, the median age was 32 years (range 32-42 years), and two patients were male(Pt.2,3). AML subtypes included M2 (Pt.2) and M4 (Pt.1,3), and no patient had a prior history of myeloproliferative neoplasms. Clinical and laboratory characteristics at diagnosis included: hepatosplenomegaly (Pt.3), lymphadenopathy (Pt.1), leukocytosis (median 209.71×10 9 /L, range, 60.28-687.2×10 9/L),but no significant eosinophilia(≥1.5×10 9/L). Cytogenetic analyses showed a normal karyotype in all three patients. FLT3-ITD gene mutation was found in two patients(Pt.1,2). All there patients received the same IA regimen(idarubicincytarabine) as induction chemotherapy and achieved complete remission, followed by multi-agent chemotherapy as consolidation treatment.Pt.1 received additional targeted therapy with sorafenib. However, after a median duration of remission (DOR) of 4 months (range 3-8 months), all patients experienced relapse and sought for further therapy at our hospital. Upon re-evaluation by laboratory tests, ETV6-ABL1 fusion gene was detected by multiplex RT-PCR, and additional NUP98-NSD1 fusion was also found in Pt.2. FLT3-ITD gene mutation was confirmed in Pt. 1 and Pt. 2, but the characteristic t(9;12) (q34;p13) chromosomal abnormalities were not present in any of the patients. Pt. 2 presented with an abnormal karyotype of 46, XY, t(3;17)(q12;p11.2),add (10)(q22)[5] / 46,XY[15], while the rest 2 patients had a normal karyotype. Pt.1 died rapidly from serious infection after relapse. Pt. 2 and Pt. 3 received dasatinib-containing combination regimens as salvage therapy and achieved partial remission. Pt. 2 underwent haploidentical allogeneic hematopoietic stem cell transplantation (allo-HSCT) with an enhanced myeloablative conditioning regimen in March 2021, and has maintained molecular remission until now. Pt. 3 is currently undergoing HSCT preparation. Conclusion: ETV6-ABL1 fusion gene is a rare but recurrent genetic aberration in AML, and its rearrangement is not uniform across eachpatient, typically involving cryptic insertions. Routine chromosome G-banding analysis may not identify this fusion gene, and the combined use of fluorescence in situ hybridization and PCR is recommended for better detection for ETV6-ABL1. Patients with this fusion gene have a high relapse rate and a poor prognosis. Tyrosine kinase inhibitors (eg. Dasatinib)are a reasonable treatment option, and allo-HSCT may be considered for potential cure.

Toward the Detection Limit of Electrochemistry: Studying Anodic Processes with a Fluorogenic Reporting Reaction.

Recently, shot noise has been shown to be an inherent part of all charge-transfer processes, leading to a practical limit of quantification of 2100 electrons (≈0.34 fC) [ Curr. Opin. Electrochem. 2020, 22, 170-177]. Attainable limits of quantification are made much larger by greater background currents and insufficient instrumentation, which restricts progress in sensing and single-entity applications. This limitation can be overcome by converting electrochemical charges into photons, which can be detected with much greater sensitivity, even down to a single-photon level. In this work, we demonstrate the use of fluorescence, induced through a closed bipolar setup, to monitor charge-transfer processes below the detection limit of electrochemical workstations. During this process, the oxidation of ferrocenemethanol (FcMeOH) in one cell is used to concurrently drive the oxidation of Amplex Red (AR), a fluorogenic redox molecule, in another cell. The spectroelectrochemistry of AR is investigated and new insights on the commonplace practice of using deprotonated glucose to limit AR photooxidation are presented. The closed bipolar setup is used to produce fluorescence signals corresponding to the steady-state voltammetry of FcMeOH on a microelectrode. Chronopotentiometry is then used to show a linear relationship between the charge passed through FcMeOH oxidation and the integrated AR fluorescence signal. The sensitivity of the measurements obtained at different timescales varies between 2200 and 500 electrons per detected photon. The electrochemical detection limit is approached using a diluted FcMeOH solution in which no faradaic current signal is observed. Nevertheless, a fluorescence signal corresponding to FcMeOH oxidation is still seen, and the detection of charges down to 300 fC is demonstrated.

Utility of Fluorescence with Indiocyanine Green (ICG) In Laparoscopic Radical Cholecystectomy En-Bloc for the Treatment of Gallbladder Cancer

Utility of Fluorescence with Indiocyanine Green (ICG) In Laparoscopic Radical Cholecystectomy En-Bloc for the Treatment of Gallbladder Cancer

The Use of Fluorescence In situ Hybridisation in the Diagnosis of Hidden Mosaicism in Egyptian Patients with Turner Syndrome.

Turner syndrome (TS) is the most common chromosomal abnormality in females. The diagnosis of TS is based on karyotyping of 30 blood lymphocytes. This technique does not rule out tissue mosaicism or low-grade mosaicism in the blood. Because of the associated risk of gonadoblastoma, mosaicism is especially important in case this involves a Y chromosome. This study was set to determine the value of additional genetic studies such as fluorescent in situ hybridisation and the inclusion of buccal cells in search for mosaicism in TS patients. This cross-sectional, descriptive study was performed in Human Genetics Department, Medical Research Institute, Alexandria University. Fluorescence in situ hybridisation technique was applied to lymphocyte cultures as well as buccal smears using centromeric probes for X and Y chromosomes. Genotype phenotype correlation was also evaluated. Descriptive study where categorical variables were described using number and percentage and continuous variables were described using mean and standard deviation. Fluorescence in situ hybridisation technique study detected hidden mosaicism in 60% of studied patients; 20% of patients had a cell line containing Y material, while 40% had variable degrees of X, XX mosaicism, and in the remaining 40% no second cell line was detected. Fluorescence in situ hybridisation study helped identify the origin of the marker to be Y in all patients. The introduction of an additional cell line helped in identifying mosaicism in patients with monosomy X. Virilisation signs were only observed among TS patients with Y cell line mosaicism. The clinical manifestations were more severe in patients with monosomy X than other mosaic cases. Molecular cytogenetic investigation for all suspected cases of TS should be considered for appropriate treatment plan and genetic counselling.

Coupled C, H, N, S and Fe biogeochemical cycles operating in the continental deep subsurface of the Iberian Pyrite Belt.

Microbial activity is a major contributor to the biogeochemical cycles that make up the life support system of planet Earth. A 613 m deep geomicrobiological perforation and a systematic multi-analytical characterization revealed an unexpected diversity associated with the rock matrix microbiome that operates in the subsurface of the Iberian Pyrite Belt (IPB). Members of 1 class and 16 genera were deemed the most representative microorganisms of the IPB deep subsurface and selected for a deeper analysis. The use of fluorescence in situ hybridization allowed not only the identification of microorganisms but also the detection of novel activities in the subsurface such as anaerobic ammonium oxidation (ANAMMOX) and anaerobic methane oxidation, the co-occurrence of microorganisms able to maintain complementary metabolic activities and the existence of biofilms. The use of enrichment cultures sensed the presence of five different complementary metabolic activities along the length of the borehole and isolated 29 bacterial species. Genomic analysis of nine isolates identified the genes involved in the complete operation of the light-independent coupled C, H, N, S and Fe biogeochemical cycles. This study revealed the importance of nitrate reduction microorganisms in the oxidation of iron in the anoxic conditions existing in the subsurface of the IPB.

Coral holobiont research needs spatial analyses at the microbial scale.

Coral holobiont research needs spatial analyses at the microbial scale.

Mind the gap—the use of sodium fluoresceine for resection of brain metastases to improve the resection rate

Introduction and purposeBrain metastases appear to be well resectable due to dissectable tumor margins, but postoperative MRI quite often depicts residual tumor with potential influence on tumor control and overall survival. Therefore, we introduced sodium fluoresceine into the routine workflow for brain metastasis resection. The aim of this study was to evaluate whether the use of fluorescence-guided surgery has an impact on postoperative tumor volume and local recurrence.Material and methodsWe retrospectively included patients who underwent surgical resection for intracranial metastases of systemic cancer between 11/2017 and 05/2021 at our institution. Tumor volumes were assessed pre- and postoperatively on T1-CE MRI. Clinical and epidemiological data as well as follow-up were gathered from our prospective database.ResultsSeventy-nine patients (33 male, 46 female) were included in this study. Median preoperative tumor volume amounted to 11.7cm3 and fluoresceine was used in 53 patients (67%). Surgeons reported an estimated gross total resection (GTR) in 95% of the cases, while early postoperative MRI could confirm GTR in 72%. Patients resected using fluoresceine demonstrated significantly lower postoperative residual tumor volumes with a difference of 0.7cm3 (p = 0.044) and lower risk of local tumor recurrence (p = 0.033). The use of fluorescence did not influence the overall survival (OS). Postoperative radiotherapy resulted in a significantly longer OS (p = 0.001).DiscussionWhile GTR rates may be overrated, the use of intraoperative fluorescence may help neurosurgeons to achieve a more radical resection. Fluoresceine seems to facilitate surgical resection and increase the extent of resection thus reducing the risk for local recurrence.

SAIBR: a simple, platform-independent method for spectral autofluorescence correction.

ABSTRACTBiological systems are increasingly viewed through a quantitative lens that demands accurate measures of gene expression and local protein concentrations. CRISPR/Cas9 gene tagging has enabled increased use of fluorescence to monitor proteins at or near endogenous levels under native regulatory control. However, owing to typically lower expression levels, experiments using endogenously tagged genes run into limits imposed by autofluorescence (AF). AF is often a particular challenge in wavelengths occupied by commonly used fluorescent proteins (GFP, mNeonGreen). Stimulated by our work in C. elegans, we describe and validate Spectral Autofluorescence Image Correction By Regression (SAIBR), a simple platform-independent protocol and FIJI plug-in to correct for autofluorescence using standard filter sets and illumination conditions. Validated for use in C. elegans embryos, starfish oocytes and fission yeast, SAIBR is ideal for samples with a single dominant AF source; it achieves accurate quantitation of fluorophore signal, and enables reliable detection and quantification of even weakly expressed proteins. Thus, SAIBR provides a highly accessible low-barrier way to incorporate AF correction as standard for researchers working on a broad variety of cell and developmental systems.

P-107 A Novel Sperm Selection Technique for Embryos of the Desired Sex

Abstract Study question Can a novel sperm selection technique (GST) yield higher rates of embryos and offspring of the desired sex in couples undergoing ICSI with PGT-A? Summary answer GST consistently enriched spermatozoa, resulting in a higher proportion of embryos and offspring of the desired sex without impairing clinical outcomes or offspring health. What is known already Although various methods to select sex-specific spermatozoa have been proposed over the years, many of these techniques have been shown to have varying degrees of success in addition to being time-consuming and costly. Moreover, the use of fluorescence and electrical charges in some of these methods has raised concerns about their potential contribution to congenital malformations. Here, we tested a novel sperm selection method aimed at achieving a higher proportion of embryos of the desired sex, without compromising clinical outcome or offspring health. Study design, size, duration Over a 6-year period, ejaculates from male partners of couples (n = 109) undergoing ICSI with PGT-A were processed using GST to enrich spermatozoa for the couples’ preferred sex. Standard sperm processing was carried out for couples undergoing ICSI exclusively to assess conceptus aneuploidy, comprising the control group (n = 1,261). The proportion of male and female spermatozoa in the initial and selected specimens, PGT-A results, and ICSI outcomes were compared between the two groups. Participants/materials, setting, methods A total of 1,370 couples were treated in 2,483 ICSI cycles. Standard sperm processing was performed for 1,261 couples who did not have an offspring sex preference. For 109 consenting couples, GST was used to enrich spermatozoa for their desired sex (IRB 1306014043). To confirm sex enrichment, ≥1,000 sperm cells were screened by fluorescent in-situ hybridization (FISH) for 9 chromosomes. The couples’ PGT-A results and ICSI outcomes were compared between the control and GST cohorts. Main results and the role of chance For the control cohort (n = 1,261), ejaculates were processed in the standard fashion. Spermatozoa sex ratio was unaffected. These couples (maternal age, 37.1±4yrs; paternal age, 39.1±6yrs) underwent 2,356 ICSI cycles (1.2±1), yielding an 80.9% fertilization rate (14,830/18,321). PGT-A results confirmed that 46.6% (n = 760) of their embryos were female and 53.4% (n = 872) were male. They achieved a 76.3% (725/950) implantation rate and a 64.9% (617/950) clinical pregnancy rate resulting in 569 healthy deliveries (48% female, 52% male). From the study cohort (n = 109), 60 couples desired a female and 49 desired a male child. Those who desired female offspring (maternal age, 37.9±4yrs; paternal age, 40.8±6yrs) obtained an 81.6% sperm sex enrichment, per FISH. They underwent 74 ICSI cycles and achieved a 77.6% (592/763) fertilization rate resulting in 78.1% (235/301) female embryos that generated a 79.3% (23/29) implantation rate, yielding 16 singleton deliveries of healthy female offspring that are developing normally. The 49 couples (maternal age, 37.6±3yrs; paternal age, 40.8±5yrs) preferring male offspring obtained an 80.8% sperm sex enrichment. They underwent 53 ICSI cycles and achieved a 74.7% (481/644) fertilization rate with an equivalent proportion of male embryos (231/292, 79.1%). Their implantation rate was 90.9% (20/22), yielding 14 healthy male singletons, all developing normally. Limitations, reasons for caution Although our sperm sex selection method does not guarantee offspring of a specific sex, it allowed couples participating in the study to obtain a greater proportion of conceptuses of their desired genotype. This method does not aim to replace PGT-A, but rather reduce embryo wastage. Wider implications of the findings Semen specimens processed by GST yielded satisfactory fertilization and embryo development, comparable to those from the control cohort. Moreover, offspring health was not negatively affected. These encouraging findings indicate that our method is safe and can consistently enrich for the desired embryo sex in a reliable and ethically palatable manner. Trial registration number n/a

The Role of Indocyanine Green Fluorescence in Rectal Cancer Robotic Surgery: A Narrative Review.

Simple SummarySurgery remains the only curative treatment for rectal cancer, despite the progress in oncological therapies. The widespread use of robotic surgery drastically changed the approach to rectal cancer, reducing the burden of the procedure on the patient’s quality of life and allowing a faster recovery. Indocyanine green fluorescence has shown promising results in reducing severe surgical complications, such as anastomotic leak, that can delay the beginning of further chemo-radio treatments. The purpose of this descriptive review is to analyze the impact of indocyanine green fluorescence when applied in robotic surgery on short-term surgical outcomes for rectal cancer, providing a picture of the current literature on the issue, highlighting the heterogeneity of protocols and focusing on possible future development.Background: In rectal cancer surgery, anastomotic leakage (AL) remains the most feared complication, with a frequency of up to 30% in non-high-volume centers. The preservation of proper vascularization is a key factor for successful anastomosis. The use of fluorescence with indocyanine green (ICG) as an intraoperative method to verify optimal perfusion is becoming an interesting tool in rectal surgery. Today, robotic surgery, together with the use of the intraoperative evaluation of the perfusion with ICG, could be a real strategy to deal with AL, allowing for a more delicate and less traumatic surgical technique. This strategy may allow for an extremely accurate surgery, and for optimal control of the proper vascularization of the rectum. Methods: The purpose of this descriptive review is to analyze the impact of fluorescence and robotic surgery on short-term surgical outcomes for rectal cancer. Results: We performed a systematic literature search using the PubMed, Embase and Cochrane library databases. The primary endpoints were to evaluate the application of ICG fluorescence in robotic rectal surgery and the rate of anastomotic leakage when using these technological implementations. The secondary endpoints were to evaluate the dosage of ICG and the timing of application by different surgeons. Conclusions: ICG fluorescence is an inexpensive and quick method to assess bowel perfusion, providing immediate feedback to the surgeon, even if its role has not been proven. A quantitative system must be systematically introduced to minimize the subjectiveness of the visualized image.