Determining Macromolecular Structures Using Cryo-Electron Microscopy.

Structural insights into macromolecular and protein complexes provide key clues about the molecular basis of the function. Cryogenic electron microscopy (cryo-EM) has emerged as a powerful structural biology method for studying protein and macromolecular structures at high resolution in both native and near-native states. Despite the ability to get detailed structural insights into the processes underlying protein function using cryo-EM, there has been hesitancy amongst plant biologists to apply the method for biomolecular interaction studies. This is largely evident from the relatively fewer structural depositions of proteins and protein complexes from plant origin in electron microscopy databank. Even though the progress has been slow, cryo-EM has significantly contributed to our understanding of the molecular biology processes underlying photosynthesis, energy transfer in plants, besides viruses infecting plants. This chapter introduces sample preparation for both negative-staining electron microscopy (NSEM) and cryo-EM for plant proteins and macromolecular complexes and data analysis using single particle analysis for beginners.

Electron Microscopy Methods for Phage-Based Study.

Electron microscopy (EM) techniques play a vital role in virology research including phage discovery and their identification. The use of different staining protocols based on the concept of negative staining is one of the most important steps in the EM processing. This chapter will summarize the widely used EM protocols in phage research, their advantages, and limitations. Phage-based therapy, especially recently developed nanoparticle-phage conjugates, are expected to find clinical significance in the antimicrobial resistance (AMR) epidemic. EM techniques are important to characterize these conjugates and we will also discuss the methods here.

Comparing the accuracy of positive and negative indocyanine green staining in guiding laparoscopic anatomical liver resection: protocol for a randomised controlled trial.

INTRODUCTION: Knowledge of the clinical liver anatomy has evolved with advanced imaging modalities and laparoscopic surgery. Therefore, precise anatomical resection knowledge has become the standard treatment for primary and secondary liver cancer. Segmentectomy, a parenchymal-preserving approach, is regarded as an option for anatomical resections in patients with impaired liver. Indocyanine green (ICG) staining is a promising method for understanding the anatomical borders of the liver segments. There are two methods of ICG staining (positive and negative), and the superiority of either approach has not been determined to date. METHODS AND ANALYSIS: This is a prospective randomised controlled superiority clinical trial performed in a single centre tertiary hospital in Japan. A comparison between the accuracy of positive and negative ICG staining in guiding laparoscopic anatomical liver resection is planned in this study. Possible candidates are patients with liver malignant tumours in whom laparoscopic monosegmentectomy or subsegmentectomy is planned. Fifty patients will be prospectively allocated into the following two groups: group A, ICG-negative staining group, and group B, ICG-positive staining group. The optimal dose of ICG for positive staining will be determined during the preparation phase. To assess the ability of the ICG fluorescence guidance in anatomical resection, the primary endpoint is the success rate of ICG staining, which consists of a SOS based on three components: superficial demarcation in the liver surface, visualisation of the parenchymal borders and consistency with the preoperative three-dimensional simulation. The secondary endpoints are the evaluation of short-term surgical outcomes and recurrence-free survival. ETHICS AND DISSEMINATION: The study was approved by Ageo Central General Hospital Clinical Research Ethical Committee (No: 1044) and it carried out following the Declaration of Helsinki (2013 revision). Informed consent will be taken from the patients before participating. The findings will be disseminated through peer-reviewed publications, scientific meetings and conferences. TRIAL REGISTRATION NUMBER: UMIN000049815.

Fluorescent Laparoscopic Central Hepatectomy for Liver Cancer Using Indocyanine Green Negative Staining.

Laparoscopic hepatectomy is an important treatment method for liver cancer. In the past, the resection boundary was usually determined by intraoperative ultrasound, important vascular structures, and surgeon experience. With the development of anatomical hepatectomy, visual surgery technology has gradually been applied to this type of surgery, particularly indocyanine green (ICG)-guided anatomical hepatectomy. As ICG can be specifically ingested by hepatocytes and used for fluorescence tracing, negative staining techniques have been applied according to different tumor positions. Under ICG fluorescent guidance, the surface boundary and deep resection plane can be more accurately displayed during liver resection. Thus, the tumor-bearing liver segment can be anatomically removed, which helps to avoid damage to important vessels and reduce ischemia or congestion of the remaining liver tissue. Finally, the incidence of postoperative biliary fistula and liver dysfunction is reduced; therefore, a better prognosis is obtained after the resection of liver cancer. Centrally located liver cancer is usually defined as a tumor located at segments 4, 5, or 8 that requires resection of the middle section of the liver. These are among the most difficult hepatectomies to perform because of the large surgical wounds and multiple vessel transections. Based on the specific tumor location, we formulated the required resection ranges by designing personalized fluorescent staining strategies. By completing anatomical resection based on the portal territory, this work aims to achieve the best therapeutic effect.

Imaging of Liposomes by Negative Staining Transmission Electron Microscopy and Cryogenic Transmission Electron Microscopy.

Morphological characteristics of liposomes, such as size and lamellarity directly impact their quality and biological performance of encapsulated drug. Gaining insights into these parameters may also help ensure identification and utilization of most efficient process parameters for liposomes manufacturing. Direct imaging of such self-assembling colloidal structures, although challenging, is feasible through transmission electron microscopy (TEM) which uses nanometer scale wavelength of electrons for illumination, enabling an accurate assessment of the morphological characteristics of liposomes. This chapter will provide background information on the working principle and general sample preparation procedure for the two most commonly used TEM techniques for imaging liposomes, viz. negative staining transmission electron microscopy and cryogenic transmission electron microscopy.

Parenchymal Sparing Laparoscopic Segmentectomy III and IV with Indocyanine Green Fluorescence Negative Stain Method Using Glisson Pedicle Approach.

BACKGROUND: To minimize the loss of functional liver volume in cases of severe cirrhosis and repeat hepatectomy for recurrence of hepatocellular carcinoma (HCC), anatomical hepatectomy is gradually extended from major to minor hepatectomy (Miyama et al. in Cancers (Basel):13, 2021; Ishizawa et al. in Ann Surg 256:959-964, 2012). For local located HCC, (sub)segmentectomy can yet be regarded as a choice instead of hemihepatectomy. Indocyanine green (ICG) has been used for tumor location, navigation of resected margin and liver segment, and identification of bile leakage. Negative stain that ICG dye was administered intravenously after occluding the target portal pedicle is more applicable to sectionectomy or hemihepatectomy, especially in cases where multiple target pedicles exist or portal vein puncture is difficult to carry out to achieve anatomic resection. Herein, we present a video of laparoscopic segmentectomy III and IV with ICG fluorescence negative stain using Glisson Pedicle approach. METHOD: A 49-year-old woman with hepatitis B related cirrhosis for 2 years was referred for treatment of a single nodule in segment IV invading the umbilical portion of left portal vein. The preoperative alpha-fetoprotein (AFP) was 442 ng/ml and protein induced by vitamin K absence or antagonist-II (PIVKA-II) was 122 mAu/ml. Liver function was Child-Pugh A and indocyanine green retention test at 15 min (ICG-R15) was 9.2%. The surgical procedure involved the following steps: (1) Extrahepatic Glisson pedicle dissection based on Laennec's s capsule (Sugioka et al. in J Hepatobiliary Pancreat Sci 24:17-23, 2017) was performed for isolation of the pedicles towards segments III and IV in the umbilical fossa. (2) Demarcation line was revealed and ICG (1 ml, 5 mg/l) was administered intravenously for the negative stain after dividing the target pedicles. (3) Parenchyma transection was performed along the border of the negative staining area in the cranial and caudal direction. RESULTS: Operative time was 220 min and blood loss was 150 ml with no transfusion. HCC sized 2.5 cm*1.7 cm*1.2 cm was confirmed in histopathology with a free margin and no microvascular invasion. The fibrosis of the liver parenchyma was S4 based on Ishak system. The patient was discharged on the postoperative day 6 without any complications. No recurrence in residual liver was noted on the CT scan at 9 months during follow-up. CONCLUSION: Laparoscopic segmentectomy III and IV is an effective procedure for HCC especially in cases with demands of hepatic parenchymal preservation. ICG navigation and Glisson Pedicle approach may be particularly helpful.

Negative Staining Electron Microscopy for Morpho-Functional Characterization of Purified Golgi Membranes from Mammalian Cells.

The Golgi apparatus is a highly dynamic organelle that controls lipid and protein sorting in the endocytic and exocytic cellular pathways. Perturbation of the lipid homeostasis or of the molecular machineries that regulate membrane remodeling/trafficking events on the Golgi membranes can dramatically change the morphology and functions of the Golgi apparatus. So far, several approaches have been described to characterize and define the Golgi morphology in intact cells and in vitro. Here, we describe the application of negative staining (NS) electron microscopy (EM) on purified Golgi membranes from HeLa cells. This approach allows to quantify and functionally characterize membrane remodeling events upon specific treatments that alter the Golgi morphology.

Preyssler-type phosphotungstate is a new family of negative-staining reagents for the TEM observation of viruses.

Transmission electron microscopy (TEM) is an essential method in virology because it allows for direct visualization of virus morphology at a nanometer scale. Negative staining to coat virions with heavy metal ions must be performed before TEM observations to achieve sufficient contrast. Herein, we report that potassium salts of Preyssler-type phosphotungstates (K(15-n)[P5W30O110Mn+], M = Na+, Ca2+, Ce3+, Eu3+, Bi3+, or Y3+) are high-performance negative staining reagents. Additionally, we compare the staining abilities of these salts to those of uranyl acetate and Keggin-type phosphotungstate. The potassium salt of Preyssler-type phosphotungstates has the advantage of not requiring prior neutralization because it is a neutral compound. Moreover, the potassium counter-cation can be protonated by a reaction with H+-resin, allowing easy exchange of protons with other cations by acid-base reaction. Therefore, the counter-cations can be changed. Encapsulated cations can also be exchanged, and clear TEM images were obtained using Preyssler-type compounds with different encapsulated cations. Preyssler-type phosphotungstates may be superior negative staining reagents for observing virus. Polyoxotungstates (tungsten-oxide molecules with diverse molecular structures and properties) are thus promising tools to develop negative staining reagents for TEM observations.

Laparoscopic Segmentectomy IV Using Hepatic Round Ligament Approach Combined with Fluorescent Negative Staining Method.

BACKGROUND: Laparoscopic segmentectomy, which maximizes the preservation of the functional hepatic reserve and the possibility for future repeat hepatectomy while ensuring adequate surgical margin, is a feasible alternative to hemihepatectomy for hepatocellular carcinoma (HCC) (Vigano et al. in Ann Surg 270(5):842-851, 2019, Ishizawa et al. in Ann Surg 256(6):959-964, 2012). Herein, we present a video of laparoscopic segmentectomy IV for HCC using hepatic round ligament approach combined with fluorescent negative staining method. PATIENT AND METHODS: A 44-year-old male with history of chronic hepatitis B virus (HBV) infection for 22 months was referred for treatment of a single HCC in segment IV. The procedure was performed according to the following steps: (1) lowering the hilar plate based on Laennec's capsule (Sugioka et al. in J Hepatobiliary Pancreat Sci 24(1):17-23, 2017) after cholecystectomy; (2) cutting the Glisson's pedicles to segment IV along the fissure for the round ligament; (3) the first parenchyma transection was along the falciform ligament, while cutting some deep pedicles to segment IV; (4) clamping the left Glisson's pedicle and using fluorescent negative staining method (Abo et al. in Eur J Surg Oncol 41(2):257-264, 2015, Funamizu et al. in J Hepatobiliary Pancreat Sci, 2021, Xu et al. in Surg Endosc 34(10):4683-4691, 2020); (5) the second parenchyma transection was performed along the boundary of negative fluorescence region to expose the middle hepatic vein (MHV) using a combination of cranial and caudal approaches. RESULTS: The operative time was 190 min, and blood loss during operation was 80 mL. The histopathologic examination showed a solitary HCC, 2.5 cm in diameter, with negative surgical margin and no microvascular invasion. The patient had an uneventful postoperative recovery and was discharged on postoperative day 5. CONCLUSION: The round ligament approach combined with fluorescent negative staining method for laparoscopic anatomic segmentectomy IV is a feasible and effective technique.

Transmission Electron Microscopic Methods for Plant Virology.

Transmission electron microscopy (TEM) is an important tool for observing the ultrastructure of plant virions and their host cells. The two main applicable TEM technologies used in plant virology are negative staining and ultrathin section. Negative staining is mainly used to observe the high-resolution structure of virus particles under a transmission electron microscope. Sample preparation for negative staining is convenient and fast, making it suitable for studying the virions in crude sap or purified solution. A modification of negative staining, by combining immunological reaction, named as technique of immuno-negative staining, is used to enrich or identify viruses. Ultrathin section is used for ultrastructural cytopathological studies in the virus-infected host cells, including the morphology of virus particles, the structure of viral induced inclusion bodies, the subcellular distribution of virions and the structural alteration of the host cell induced by viral infection. Such information is valuable to analyze the behavior of virus in replication, assembly, and intercellular transportation, and thus to understand the viral infection cycle. The present chapter describes the operation details of negative staining and ultrathin section TEM.

Laparoscopic anatomical liver resection for malignancies using positive or negative staining technique with intraoperative indocyanine green-fluorescence imaging.

BACKGROUND: Indications for a minimally invasive resections are increasing worldwide, but respecting anatomical planes during intraparenchymal transection is demanding. Intraoperative ICG fluorescence staining of liver parenchyma has been introduced as a tool for real-time intraoperative guidance. The aim of this study is to make a systematic review of the current relevant literature on indications, techniques, and results of laparoscopic anatomical liver resection (LALR) using intraoperative indocyanine green (ICG) fluorescence for positive and negative staining of liver segments in patients affected by liver malignancies. METHODS: Electronic bibliographical databases (MEDLINE and PubMed) were searched according to the PRISMA criteria. English language articles meeting the selection criteria and published until June 2020 were retrieved and reviewed. RESULTS: a total of 86 articles were initially found and 11 articles were finally included in the analysis with a total of 83 patients treated. Sixty-two patients (74.6%) underwent mono-segmentectomies. Thirty-five patients (42.1%) underwent the positive staining technique, and forty-eight patients (57.8%) the negative staining technique. CONCLUSIONS: The positive or negative indocyanine green staining technique with real-time fluorescence guidance is an emerging and promising approach. However, the technique has to be standardized and advantages in terms of oncologic results still need validation in further studies.

Refining the Removal of Perfluorocarbon Liquid Remnants Through Negative Staining With Vital Dyes.

The complete removal of perfluorocarbon liquid (PFCL) after complex vitreoretinal surgery can be a challenging task. However, it remains a crucial step to avoid several ocular complications associated with retained PFCL. In this report, the authors describe a simple and effective technique to highlight the presence of retained PFCL bubbles in the epiretinal space through negative staining with vital dyes. [Ophthalmic Surg Lasers Imaging Retina. 2021;52:153-154.].

Double cone-unit laparoscopic hepatic resection using indocyanine green negative counterstaining (with video).

A hepatic cone-unit represents an anatomical unit dominated by a smaller Glissonean pedicle. Anatomical resection of a tumor located in an intersegmental plane is challenging, but could be achieved effectively by performing multiple cone-unit resection. We performed double cone-unit laparoscopic resection of hepatocellular carcinoma located on the intersegmental plane between segments 6a, b. The liver parenchyma covering the posterior Glissonean pedicle was divided along Rouviere's sulcus, the Glissonean branches of segments 6a, b were isolated and ligated, and indocyanine green (ICG) negative counterstaining was performed. The hepatic parenchyma was dissected along the demarcation line to identify the right hepatic vein and the double cone-unit resection was then completed with a negative surgical margin. Thus, double cone-unit laparoscopic hepatectomy with ICG negative counterstaining may be a feasible option for tumors located in an intersegmental plane.

Purification of Human Complement Component C4 and Sample Preparation for Structural Biology Applications.

Activated complement component C4 (C4b) is the nonenzymatic component of the classical pathway (CP) convertases of the complement system. Preparation of C4 and C4b samples suitable for structural biology studies is challenging due to low yields and complexity of recombinant C4 production protocols reported so far and heterogeneity of C4 in native sources. Here we present a purification protocol for human C4 and describe sample preparation methods for structural investigation of C4 and its complexes by crystallography, small angle X-ray scattering, and electron microscopy.

Evaluation of accuracy of laparoscopic liver mono-segmentectomy using the Glissonian approach with indocyanine green fluorescence negative staining by comparing estimated and actual resection volumes: A single-center retrospective cohort study.

BACKGROUND/PURPOSE: Laparoscopic liver mono-segmentectomy (LLMS) may improve patient outcomes, but it is difficult and its accuracy and safety are unknown. We evaluated the accuracy of LLMS using Glissonian approach with indocyanine green fluorescence (ICG) negative staining. METHODS: Seventy-four patients eligible for LLMS except for segment 1 were enrolled. Preoperative three-dimensional CT-based surgical simulation was used to determine estimated liver resection volume (ELRV), which was compared with modified actual liver resection volume (ALRV) obtained from actual liver resection mass. The LLMS accuracy was also evaluated based on operator's experience (attending surgeon [AS] or trainee surgeon [TS]). RESULTS: Estimated liver resection volumes significantly correlated with ALRVs (r = .82) in all cases. Moreover, TS-conducted LLMS also showed acceptable difference between ELRV and ALRV compared with AS-conducted LLMS. There were no intergroup differences in estimated blood loss, operation time, time of Pringle maneuver, postoperative complications, and length of postoperative hospitalization (P < .05). Moreover, R0 resection was comparable between the AS and TS groups. CONCLUSIONS: Laparoscopic liver mono-segmentectomy with Glissonian approach using ICG negative imaging ensured safe and accurate procedure owing to facilitated visualization of the resection line. Our approach was effective in avoiding postoperative liver dysfunction and securing radical resection. In addition, it might be helpful in TS education of LLMS.

Laparoscopic anatomical portal territory hepatectomy using Glissonean pedicle approach (Takasaki approach) with indocyanine green fluorescence negative staining: how I do it.

BACKGROUND: Laparoscopic anatomical resection (LAR) is a highly challenging procedure. This study aimed to describe our experience of the LAR with an indocyanine green fluorescence negative staining (ICGNS) by the Glissonean pedicle transection (Takasaki) approach. METHODS: From April 2017 to December 2019, 43 consecutive patients underwent LAR with ICGNS strategy in our medical team. The details of the ICGNS strategy were described. The demographic and clinicopathological data of the included patients were retrospectively analyzed. RESULTS: The extent of resections included right hemihepatectomy (n = 12), left hemihepatectom (n = 4), left lateral sectionectomy (n = 3), Right anterior sectionectomy (n = 3), Right posterior sectionectomy (n = 6), central hepatectomy (n = 2), single anterolateral segmentectomy (n = 5), single posterosuperior segmentectomy (n = 6), and bisegmentectomy (n = 2). The mean operation time was 212 ± 53 min, and the median estimated blood loss was 200 (100-300) ml. The overall complication rate was 30.2% (grade I, 14%; grade II, 14%; grade III, 2.3%). The median duration of postoperative hospital stay was 6 (4-7) days. CONCLUSION: ICGNS is a safe and feasible LAR strategy that greatly facilitates selecting the liver transection plane, although its benefits need to be verified by large-sample comparative studies.

Progress Towards CryoEM: Negative-Stain Procedures for Biological Samples.

In recent years, electron cryo-microscopy (CryoEM) has become a powerful method for the high-resolution studies of biological macromolecules. While CryoEM experiments can begin without additional microscopy steps, negative-stain EM can tremendously minimize CryoEM screening. Negative-stain is a quick method that can be used to screen for robust biochemical conditions, the integrity, binding, and composition of samples and to get an estimation of sample grid concentration. For some applications, the map resolutions potentially afforded by stain may be as biologically informative as in CryoEM. Here, I describe the benefits and pitfalls of negative-stain EM, with particular emphasis on Uranyl stains with the main goal of screening in advance of CryoEM. In addition, I provide a materials list, detailed protocol and possible adjustments for the use of stains for biological samples requiring imaging and/or diffraction-based methods of EM.

Robotic ICG guided anatomical liver resection in a multi-centre cohort: an evolution from "positive staining" into "negative staining" method.

BACKGROUND: Laparoscopic major anatomical liver resection is challenging. The robotic liver resection (RLR) approach, with Firefly indocyanine green (ICG) imaging, was proposed to overcome the limitations of laparoscopy. The aim of this multi-centre international study was to evaluate the use of Firefly ICG imaging in anatomical RLR. METHODS: A retrospective study of consecutive patients undergoing RLR anatomical resection with intra-operative ICG administration from January 2015 to July 2018 were enrolled. Patients who underwent simultaneous or en-bloc resections of other organs were excluded. RESULTS: A total of 52 patients were recruited of which 32 patients were healthy donors, 17 with malignancy and 3 for benign conditions. 12 patients had cirrhosis. 28 patients underwent a right hepatectomy (53.8%) with left hepatectomy performed with 18 patients. 40 patients underwent negative staining and 12 patients via direct portal vein injection for positive staining. ICG demarcation line was visualized in 43 patients and was clearer than the ischaemic demarcation line in 29 patients. All resections for malignancy had clear margins. There were no 30-day/inpatient mortalities. CONCLUSION: Robotic ICG guided hepatectomy technique for anatomical liver resection is safe, feasible and has the benefit for improved visualization in healthy donors and cirrhotic patients.

An atypical BRCT-BRCT interaction with the XRCC1 scaffold protein compacts human DNA Ligase IIIα within a flexible DNA repair complex.

The XRCC1-DNA ligase IIIα complex (XL) is critical for DNA single-strand break repair, a key target for PARP inhibitors in cancer cells deficient in homologous recombination. Here, we combined biophysical approaches to gain insights into the shape and conformational flexibility of the XL as well as XRCC1 and DNA ligase IIIα (LigIIIα) alone. Structurally-guided mutational analyses based on the crystal structure of the human BRCT-BRCT heterodimer identified the network of salt bridges that together with the N-terminal extension of the XRCC1 C-terminal BRCT domain constitute the XL molecular interface. Coupling size exclusion chromatography with small angle X-ray scattering and multiangle light scattering (SEC-SAXS-MALS), we determined that the XL is more compact than either XRCC1 or LigIIIα, both of which form transient homodimers and are highly disordered. The reduced disorder and flexibility allowed us to build models of XL particles visualized by negative stain electron microscopy that predict close spatial organization between the LigIIIα catalytic core and both BRCT domains of XRCC1. Together our results identify an atypical BRCT-BRCT interaction as the stable nucleating core of the XL that links the flexible nick sensing and catalytic domains of LigIIIα to other protein partners of the flexible XRCC1 scaffold.

Analysis of Recombinant Adeno-Associated Virus (rAAV) Sample Morphology Using Negative Staining and High-Resolution Electron Microscopy.

Negative staining is a simple and rapid method for studying the morphology and ultrastructure of small particulate specimens (e.g., viruses, bacteria, cell fragments, and isolated macromolecules such as proteins and nucleic acids). The technique described in this protocol involves allowing particles or fragments of cells to settle onto a support film, then applying a drop of metal salt solution to the adherent particulate specimen. The stain penetrates the interstices of the particles to bring out detail. In this situation, the preparation dries rapidly. The dissolved substance precipitates out of solution in an amorphous condition at the 0.1-nm level, and it is deposited over the support film and exposed surface of the specimen. The theoretical requirements of a good negative staining are a substance (1) of high density to provide high contrast, (2) at high solubility so that the stain does not come out of solution prematurely but does so only at the final stage of drying, (3) of high melting point and boiling point so that the material does not evaporate at high temperatures induced by the electron beam, and (4) in which the precipitate should be essentially amorphous down to the limit of resolution.