Semi-automatic standardized analysis method to objectively evaluate near-infrared fluorescent dyes in image-guided surgery.

Significance: Near-infrared fluorescence imaging still lacks a standardized, objective method to evaluate fluorescent dye efficacy in oncological surgical applications. This results in difficulties in translation between preclinical to clinical studies with fluorescent dyes and in the reproduction of results between studies, which in turn hampers further clinical translation of novel fluorescent dyes. Aim: Our aim is to develop and evaluate a semi-automatic standardized method to objectively assess fluorescent signals in resected tissue. Approach: A standardized imaging procedure was designed and quantitative analysis methods were developed to evaluate non-targeted and tumor-targeted fluorescent dyes. The developed analysis methods included manual selection of region of interest (ROI) on white light images, automated fluorescence signal ROI selection, and automatic quantitative image analysis. The proposed analysis method was then compared with a conventional analysis method, where fluorescence signal ROIs were manually selected on fluorescence images. Dice similarity coefficients and intraclass correlation coefficients were calculated to determine the inter- and intraobserver variabilities of the ROI selections and the determined signal- and tumor-to-background ratios. Results: The proposed non-targeted fluorescent dyes analysis method showed statistically significantly improved variabilities after application on indocyanine green specimens. For specimens with the targeted dye SGM-101, the variability of the background ROI selection was statistically significantly improved by implementing the proposed method. Conclusion: Semi-automatic methods for standardized quantitative analysis of fluorescence images were successfully developed and showed promising results to further improve the reproducibility and standardization of clinical studies evaluating fluorescent dyes.

Intraoperative molecular imaging of colorectal lung metastases with SGM-101: a feasibility study.

PURPOSE: Metastasectomy is a common treatment option for patients with colorectal lung metastases (CLM). Challenges exist with margin assessment and identification of small nodules, especially during minimally invasive surgery. Intraoperative fluorescence imaging has the potential to overcome these challenges. The aim of this study was to assess feasibility of targeting CLM with the carcinoembryonic antigen (CEA) specific fluorescent tracer SGM-101. METHODS: This was a prospective, open-label feasibility study. The primary outcome was the number of CLM that showed a true positive fluorescence signal with SGM-101. Fluorescence positive signal was defined as a signal-to-background ratio (SBR) ≥ 1.5. A secondary endpoint was the CEA expression in the colorectal lung metastases, assessed with the immunohistochemistry, and scored by the total immunostaining score. RESULTS: Thirteen patients were included in this study. Positive fluorescence signal with in vivo, back table, and closed-field bread loaf imaging was observed in 31%, 45%, and 94% of the tumors respectively. Median SBRs for the three imaging modalities were 1.00 (IQR: 1.00-1.53), 1.45 (IQR: 1.00-1.89), and 4.81 (IQR: 2.70-7.41). All tumor lesions had a maximum total immunostaining score for CEA expression of 12/12. CONCLUSION: This study demonstrated the potential of fluorescence imaging of CLM with SGM-101. CEA expression was observed in all tumors, and closed-field imaging showed excellent CEA specific targeting of the tracer to the tumor nodules. The full potential of SGM-101 for in vivo detection of the tracer can be achieved with improved minimal invasive imaging systems and optimal patient selection. TRIAL REGISTRATION: The study was registered in ClinicalTrial.gov under identifier NCT04737213 at February 2021.

Quantification of indocyanine green near-infrared fluorescence bowel perfusion assessment in colorectal surgery.

BACKGROUND: Indocyanine green near-infrared fluorescence bowel perfusion assessment has shown its potential benefit in preventing anastomotic leakage. However, the surgeon's subjective visual interpretation of the fluorescence signal limits the validity and reproducibility of the technique. Therefore, this study aimed to identify objective quantified bowel perfusion patterns in patients undergoing colorectal surgery using a standardized imaging protocol. METHOD: A standardized fluorescence video was recorded. Postoperatively, the fluorescence videos were quantified by drawing contiguous region of interests (ROIs) on the bowel. For each ROI, a time-intensity curve was plotted from which perfusion parameters (n = 10) were derived and analyzed. Furthermore, the inter-observer agreement of the surgeon's subjective interpretation of the fluorescence signal was assessed. RESULTS: Twenty patients who underwent colorectal surgery were included in the study. Based on the quantified time-intensity curves, three different perfusion patterns were identified. Similar for both the ileum and colon, perfusion pattern 1 had a steep inflow that reached its peak fluorescence intensity rapidly, followed by a steep outflow. Perfusion pattern 2 had a relatively flat outflow slope immediately followed by its plateau phase. Perfusion pattern 3 only reached its peak fluorescence intensity after 3 min with a slow inflow gradient preceding it. The inter-observer agreement was poor-moderate (Intraclass Correlation Coefficient (ICC): 0.378, 95% CI 0.210-0.579). CONCLUSION: This study showed that quantification of bowel perfusion is a feasible method to differentiate between different perfusion patterns. In addition, the poor-moderate inter-observer agreement of the subjective interpretation of the fluorescence signal between surgeons emphasizes the need for objective quantification.

Climate change threats to the global functional diversity of freshwater fish.

Climate change impacts on freshwater ecosystems and freshwater biodiversity show strong spatial variability, highlighting the importance of a global perspective. While previous studies on biodiversity mostly focused on species richness, functional diversity, which is a better predictor of ecosystem functioning, has received much less attention. This study aims to comprehensively assess climate change threats to the functional diversity of freshwater fish across the world, considering three complementary metrics-functional richness, evenness and divergence. We built on existing spatially explicit projections of geographical ranges for 11,425 riverine fish species as affected by changes in streamflow and water temperature extremes at four warming levels (1.5°C, 2.0°C, 3.2°C and 4.5°C). To estimate functional diversity, we considered the following four continuous, morphological and physiological traits: relative head length, relative body depth, trophic level and relative growth rate. Together, these traits cover five ecological functions. We treated missing trait values in two different ways: we either removed species with missing trait values or imputed them. Depending on the warming level, 6%-25% of the locations globally face a complete loss of functional diversity when assuming no dispersal (6%-17% when assuming maximal dispersal), with hotspots in the Amazon and Paraná River basins. The three facets of functional diversity do not always follow the same pattern. Sometimes, functional richness is not yet affected despite species loss, while functional evenness and divergence are already reducing. Other times, functional richness reduces, while functional evenness and/or divergence increase instead. The contrasting patterns of the three facets of functional diversity show their complementarity among each other and their added value compared to species richness. With increasing climate change, impacts on freshwater communities accelerate, making early mitigation critically important.

A quantitative assessment of perfusion of the gastric conduit after oesophagectomy using near-infrared fluorescence with indocyanine green.

INTRODUCTION: Anastomotic leakage is a severe complication after oesophageal resection with gastric conduit reconstruction. Poor perfusion of the gastric conduit plays an important role in the development of anastomotic leakage. Quantitative near-infrared (NIR) fluorescence angiography with indocyanine green (ICG-FA) is an objective technique that can be used for perfusion assessment. This study aims to assess perfusion patterns of the gastric conduit with quantitative ICG-FA. METHODS: In this exploratory study, 20 patients undergoing oesophagectomy with gastric conduit reconstruction were included. A standardized NIR ICG-FA video of the gastric conduit was recorded. Postoperatively, the videos were quantified. Primary outcomes were the time-intensity curves and nine perfusion parameters from contiguous regions of interest on the gastric conduit. A secondary outcome was the inter-observer agreement of subjective interpretation of the ICG-FA videos between six surgeons. The inter-observer agreement was tested with an intraclass correlation coefficient (ICC). RESULTS: In a total of 427 curves, three distinct perfusion patterns were recognized: pattern 1 (steep inflow, steep outflow); pattern 2 (steep inflow, minor outflow); and pattern 3 (slow inflow, no outflow). All perfusion parameters were significantly different between the perfusion patterns. The inter-observer agreement was poor - moderate (ICC:0.345,95%CI:0.164-0.584). DISCUSSION: This was the first study to describe perfusion patterns of the complete gastric conduit after oesophagectomy. Three distinct perfusion patterns were observed. The poor inter-observer agreement of the subjective assessment underlines the need for quantification of ICG-FA of the gastric conduit. Further studies should evaluate the predictive value of perfusion patterns and parameters on anastomotic leakage.

Assessment of Surgical Strategies for Pilonidal Sinus Disease in the Netherlands.

Purpose Pilonidal sinus disease (PSD) is a subcutaneous infection of the sacrococcygeal region due to entrapment of hair and/or debris. International guidelines recommend minimally invasive techniques and flap techniques. A Dutch guideline for the treatment of PSD is not available and this may lead to practice variation. The aim of this study was to perform a national survey on the surgical treatment of PSD in the Netherlands. Method An online survey was sent by e-mail to all surgeons and surgical residents of the Dutch Association for Surgeons. Respondents were asked to reflect on their preferences in the treatment of PSD, their perceived satisfaction with this treatment, and the need for national guidelines. Results A total of 819 (48.6%) of 1684 invitees responded to the survey, of whom 615 (37%) met the inclusion criteria. Traditional excision techniques were most frequently performed for all types of PSD (50.7%) followed by flap techniques (22.6%) and minimally invasive techniques (22%). Only 22.6% of the participants were satisfied with the current treatment and 82% supported the development of a national guideline. Conclusion Traditional excision techniques are most frequently performed for PSD in the Netherlands but the majority of surgeons and surgical residents are not satisfied with the current treatment. There is a demand for a national guideline.

Intraoperative Near-Infrared Fluorescence Imaging with Indocyanine Green for Identification of Gastrointestinal Stromal Tumors (GISTs), a Feasibility Study.

BACKGROUND: Optimal intraoperative tumor identification of gastrointestinal stromal tumors (GISTs) is important for the quality of surgical resections. This study aims to assess the potential of near-infrared fluorescence (NIRF) imaging with indocyanine green (ICG) to improve intraoperative tumor identification. METHODS: Ten GIST patients, planned to undergo resection, were included. During surgery, 10 mg of ICG was intravenously administered, and NIRF imaging was performed at 5, 10, and 15 min after the injection. The tumor fluorescence intensity was visually assessed, and tumor-to-background ratios (TBRs) were calculated for exophytic lesions. RESULTS: Eleven GIST lesions were imaged. The fluorescence intensity of the tumor was visually synchronous and similar to the background in five lesions. In one lesion, the tumor fluorescence was more intense than in the surrounding tissue. Almost no fluorescence was observed in both the tumor and healthy peritoneal tissue in two patients with GIST lesions adjacent to the liver. In three GISTs without exophytic growth, no fluorescence of the tumor was observed. The median TBRs at 5, 10, and 15 min were 1.0 (0.4-1.2), 1.0 (0.5-1.9), and 0.9 (0.7-1.2), respectively. CONCLUSION: GISTs typically show similar fluorescence intensity to the surrounding tissue in NIRF imaging after intraoperative ICG administration. Therefore, intraoperatively administered ICG is currently not applicable for adequate tumor identification, and further research should focus on the development of tumor-specific fluorescent tracers for GISTs.

Fluorescence-guided surgery in colorectal cancer; A review on clinical results and future perspectives.

BACKGROUND: Colorectal cancer is the fourth most diagnosed malignancy worldwide and surgery is one of the cornerstones of the treatment strategy. Near-infrared (NIR) fluorescence imaging is a new and upcoming technique, which uses an NIR fluorescent agent combined with a specialised camera that can detect light in the NIR range. It aims for more precise surgery with improved oncological outcomes and a reduction in complications by improving discrimination between different structures. METHODS: A systematic search was conducted in the Embase, Medline and Cochrane databases with search terms corresponding to 'fluorescence-guided surgery', 'colorectal surgery', and 'colorectal cancer' to identify all relevant trials. RESULTS: The following clinical applications of fluorescence guided surgery for colorectal cancer were identified and discussed: (1) tumour imaging, (2) sentinel lymph node imaging, (3) imaging of distant metastases, (4) imaging of vital structures, (5) imaging of perfusion. Both experimental and FDA/EMA approved fluorescent agents are debated. Furthermore, promising future modalities are discussed. CONCLUSION: Fluorescence-guided surgery for colorectal cancer is a rapidly evolving field. The first studies show additional value of this technique regarding change in surgical management. Future trials should focus on patient related outcomes such as complication rates, disease free survival, and overall survival.

Current Intraoperative Imaging Techniques to Improve Surgical Resection of Laryngeal Cancer: A Systematic Review.

Laryngeal cancer is a prevalent head and neck malignancy, with poor prognosis and low survival rates for patients with advanced disease. Treatment consists of unimodal therapy through surgery or radiotherapy in early staged tumors, while advanced stage tumors are generally treated with multimodal chemoradiotherapy or (total) laryngectomy followed by radiotherapy. Still, the recurrence rate for advanced laryngeal cancer is between 25 and 50%. In order to improve surgical resection of laryngeal cancer and reduce local recurrence rates, various intraoperative optical imaging techniques have been investigated. In this systematic review, we identify these technologies, evaluating the current state and future directions of optical imaging for this indication. Narrow-band imaging (NBI) and autofluorescence (AF) are established tools for early detection of laryngeal cancer. Nonetheless, their intraoperative utility is limited by an intrinsic inability to image beyond the (sub-)mucosa. Likewise, contact endoscopy (CE) and optical coherence tomography (OCT) are technically cumbersome and only useful for mucosal margin assessment. Research on fluorescence imaging (FLI) for this application is sparse, dealing solely with nonspecific fluorescent agents. Evidently, the imaging modalities that have been investigated thus far are generally unsuitable for deep margin assessment. We discuss two optical imaging techniques that can overcome these limitations and suggest how they can be used to achieve adequate margins in laryngeal cancer at all stages.

Generation of dyskeratosis congenita-like hematopoietic stem cells through the stable inhibition of DKC1.

Dyskeratosis congenita (DC) is a rare telomere biology disorder, which results in different clinical manifestations, including severe bone marrow failure. To date, the only curative treatment for the bone marrow failure in DC patients is allogeneic hematopoietic stem cell transplantation. However, due to the toxicity associated to this treatment, improved therapies are recommended for DC patients. Here, we aimed at generating DC-like human hematopoietic stem cells in which the efficacy of innovative therapies could be investigated. Because X-linked DC is the most frequent form of the disease and is associated with an impaired expression of DKC1, we have generated DC-like hematopoietic stem cells based on the stable knock-down of DKC1 in human CD34+ cells with lentiviral vectors encoding for DKC1 short hairpin RNAs. At a molecular level, DKC1-interfered CD34+ cells showed a decreased expression of TERC, as well as a diminished telomerase activity and increased DNA damage, cell senescence, and apoptosis. Moreover, DKC1-interfered human CD34+ cells showed defective clonogenic ability and were incapable of repopulating the hematopoiesis of immunodeficient NSG mice. The development of DC-like hematopoietic stem cells will facilitate the understanding of the molecular and cellular basis of this inherited bone marrow failure syndrome and will serve as a platform to evaluate the efficacy of new hematopoietic therapies for DC.

A primer to gene therapy: Progress, prospects, and problems.

Genetic therapies based on gene addition have witnessed a variety of clinical successes and the first therapeutic products have been approved for clinical use. Moreover, innovative gene editing techniques are starting to offer new opportunities in which the mutations that underlie genetic diseases can be directly corrected in afflicted somatic cells. The toolboxes underpinning these DNA modifying technologies are expanding with great pace. Concerning the ongoing efforts for their implementation, viral vector-based gene delivery systems have acquired center-stage, providing new hopes for patients with inherited and acquired disorders. Specifically, the application of genetic therapies using viral vectors for the treatment of inborn metabolic disorders is growing and clinical applications are starting to appear. While the field has matured from the technology perspective and has yielded efficacious products, it is the perception of many stakeholders that from the regulatory side further developments are urgently needed. In this review, we summarize the features of state-of-the-art viral vector systems and the corresponding gene-centered therapies they seek to deliver. Moreover, a brief summary is also given on emerging gene editing approaches built on CRISPR-Cas9 nucleases and, more recently, nickases, including base editors and prime editors. Finally, we will point at some regulatory aspects that may deserve further attention for translating these technological developments into actual advanced therapy medicinal products (ATMPs).

Bioinspired Non-Heme Iron Complexes: The Evolution of Facial N, N, O Ligand Design.

Iron-containing metalloenzymes that contain the 2-His-1-Carboxylate facial triad at their active site are well known for their ability to activate molecular oxygen and catalyse a broad range of oxidative transformations. Many of these reactions are synthetically challenging, and developing small molecular iron-based catalysts that can achieve similar reactivity and selectivity remains a long-standing goal in homogeneous catalysis. This review focuses on the development of bioinspired facial N,N,O ligands that model the 2-His-1-Carboxylate facial triad to a greater degree of structural accuracy than many of the polydentate N-donor ligands commonly used in this field. By developing robust, well-defined N,N,O facial ligands, an increased understanding could be gained of the factors governing enzymatic reactivity and selectivity.

Integrating gene delivery and gene-editing technologies by adenoviral vector transfer of optimized CRISPR-Cas9 components.

Enhancing the intracellular delivery and performance of RNA-guided CRISPR-Cas9 nucleases (RGNs) remains in demand. Here, we show that nuclear translocation of commonly used Streptococcus pyogenes Cas9 (SpCas9) proteins is suboptimal. Hence, we generated eCas9.4NLS by endowing the high-specificity eSpCas9(1.1) nuclease (eCas9.2NLS) with additional nuclear localization signals (NLSs). We demonstrate that eCas9.4NLS coupled to prototypic or optimized guide RNAs achieves efficient targeted DNA cleavage and probe the performance of SpCas9 proteins with different NLS compositions at target sequences embedded in heterochromatin versus euchromatin. Moreover, after adenoviral vector (AdV)-mediated transfer of SpCas9 expression units, unbiased quantitative immunofluorescence microscopy revealed 2.3-fold higher eCas9.4NLS nuclear enrichment levels than those observed for high-specificity eCas9.2NLS. This improved nuclear translocation yielded in turn robust gene editing after nonhomologous end joining repair of targeted double-stranded DNA breaks. In particular, AdV delivery of eCas9.4NLS into muscle progenitor cells resulted in significantly higher editing frequencies at defective DMD alleles causing Duchenne muscular dystrophy (DMD) than those achieved by AdVs encoding the parental, eCas9.2NLS, protein. In conclusion, this work provides a strong rationale for integrating viral vector and optimized gene-editing technologies to bring about enhanced RGN delivery and performance.

Genomic Engineering in Human Hematopoietic Stem Cells: Hype or Hope?

Many gene editing techniques are developed and tested, yet, most of these are optimized for transformed cell lines, which differ from their primary cell counterparts in terms of transfectability, cell death propensity, differentiation capability, and chromatin accessibility to gene editing tools. Researchers are working to overcome the challenges associated with gene editing of primary cells, namely, at the level of improving the gene editing tool components, e.g., the use of modified single guide RNAs, more efficient delivery of Cas9 and RNA in the ribonucleoprotein of these cells. Despite these efforts, the low efficiency of proper gene editing in true primary cells is an obstacle that needs to be overcome in order to generate sufficiently high numbers of corrected cells for therapeutic use. In addition, many of the therapeutic candidate genes for gene editing are expressed in more mature blood cell lineages but not in the hematopoietic stem cells (HSCs), where they are tightly packed in heterochromatin, making them less accessible to gene editing enzymes. Bringing HSCs in proliferation is sometimes seen as a solution to overcome lack of chromatin access, but the induction of proliferation in HSCs often is associated with loss of stemness. The documented occurrences of off-target effects and, importantly, on-target side effects also raise important safety issues. In conclusion, many obstacles still remain to be overcome before gene editing in HSCs for gene correction purposes can be applied clinically. In this review, in a perspective way, we will discuss the challenges of researching and developing a novel genetic engineering therapy for monogenic blood and immune system disorders.

A Small Key for a Heavy Door: Genetic Therapies for the Treatment of Hemoglobinopathies.

Throughout the past decades, the search for a treatment for severe hemoglobinopathies has gained increased interest within the scientific community. The discovery that ɤ-globin expression from intact HBG alleles complements defective HBB alleles underlying ß-thalassemia and sickle cell disease, has provided a promising opening for research directed at relieving ɤ-globin repression mechanisms and, thereby, improve clinical outcomes for patients. Various gene editing strategies aim to reverse the fetal-to-adult hemoglobin switch to up-regulate ɤ-globin expression through disabling either HBG repressor genes or repressor binding sites in the HBG promoter regions. In addition to these HBB mutation-independent strategies involving fetal hemoglobin (HbF) synthesis de-repression, the expanding genome editing toolkit is providing increased accuracy to HBB mutation-specific strategies encompassing adult hemoglobin (HbA) restoration for a personalized treatment of hemoglobinopathies. Moreover, besides genome editing, more conventional gene addition strategies continue under investigation to restore HbA expression. Together, this research makes hemoglobinopathies a fertile ground for testing various innovative genetic therapies with high translational potential. Indeed, the progressive understanding of the molecular clockwork underlying the hemoglobin switch together with the ongoing optimization of genome editing tools heightens the prospect for the development of effective and safe treatments for hemoglobinopathies. In this context, clinical genetics plays an equally crucial role by shedding light on the complexity of the disease and the role of ameliorating genetic modifiers. Here, we cover the most recent insights on the molecular mechanisms underlying hemoglobin biology and hemoglobinopathies while providing an overview of state-of-the-art gene editing platforms. Additionally, current genetic therapies under development, are equally discussed.

Versatile Coordination and C-C Coupling of Diphosphine-Tethered Imine Ligands with Ni(II) and Ni(0).

Ligands that can adapt their coordination mode to the electronic properties of a metal center are of interest to support catalysis or small molecule activation processes. In this context, the ability of imine moieties to bind in either an η1(N)-fashion via σ-donation of the lone pair or, less commonly, in an η2(C,N)-fashion via π-coordination is potentially attractive for the design of new metal-ligand cooperative systems. Herein, the coordination chemistry of chelating ligands with a diphosphine imine framework (PCNP) to nickel is investigated. The imine moiety binds in an η1(N)-fashion in a Ni(II)Cl2 complex. The uncommon η2(C,N)-interaction is obtained in Ni(0) complexes in the presence of a PPh3 coligand. Increasing the bulk on the phosphine side-arms in the Ni(0) complexes, by substituting phenyl for o-tolyl groups, leads to a distinct binding mode in which only one of the phosphorus atoms is coordinated. In the absence of a coligand, a mixture of two different dimeric Ni(0) complexes is formed. In one of them, the imine adopts an uncommon η1(N)η2(C,N) bridging mode of the ligand to nickel, while the second one may involve reactivity on the ligand by the formation of a new C-C bond by oxidative coupling. The latter is supported by the isolation and structural characterization of a crystalline bis-CO derivative featuring a C-C bond formed by oxidative coupling of two imine moieties.

Does the presence of ant nests matter for oviposition to a specialized myrmecophilous Maculinea butterfly?

More than 50% of the lycaenid butterflies have an ant-associated lifestyle (myrmecophily) which may vary from coexistence to specific mutualistic or even parasitic interactions. Ant-related host-plant selection and oviposition has been observed in some myrmecophilous lycaenids. Therefore, it is remarkable that there is no evidence for this behaviour in the highly specialized, obligate myrmecophilous butterflies of the genus Maculinea. In contrast with previous findings, our results provide evidence for ant-related oviposition patterns in Maculinea alcon in relation to the distribution of specific host-ant nests (i.e. Myrmica ruginodis) based on repeated egg counts during the flight period in two populations. We also show that ant-related oviposition can be counterbalanced by intraspecific competition and oviposition deterrency when host plants already carry several eggs. Therefore, the absence of a correlation between egg load and the presence of host-ant nests at the end of the flight period should be interpreted carefully Whether ovipositional cues are obtained either directly (from ants or their nests) or indirectly (from vegetation structure), and whether alternative explanations based on the phenology and growth form of host plants are possible, is discussed.