Indocyanine Green Fluorescence-Guided Laparoscopic Anatomical Segmentectomy of Liver Segment 6: Surgical Strategy and Technical Details.

BACKGROUND: Because of the complex anatomy of the right posterior hepatic pedicle, there have been few reports on standardized laparoscopic portal territory staining-guided anatomical resection of liver segment 6 (LPTAR-S6). This study aimed to elucidate the indocyanine green (ICG) fluorescence staining methods for LPTAR-S6. PATIENTS AND METHODS: LPTAR-S6 can be performed using positive and negative fluorescence staining approaches. We implemented these two approaches for patients with hepatocellular carcinoma. Descriptions of the surgical strategy and technical details are presented. RESULTS: Two patients safely underwent LPTAR-S6 using a preoperative three-dimensional reconstruction plan. The intraoperative ICG fluorescence staining effect was satisfactory, and the anatomical landmarks were fully exposed. CONCLUSIONS: A detailed preoperative three-dimensional reconstruction plan, complete intraoperative application of real-time laparoscopic ultrasound guidance, and ICG fluorescence staining can result in accurate transection of the liver parenchyma during LPTAR-S6.

Advantages of laparoscopic segmentectomy of the liver using ICG fluorescent navigation by the negative staining method: A comparison with open procedure.

Aim: Laparoscopic segmentectomy (LS) using indocyanine green (ICG) fluorescence navigation with negative staining method has potential for performing accurate and safe anatomical excision. This study aimed to evaluate the significance of LS using ICG fluorescence navigation compared with open segmentectomy (OS). Methods: Eighty-seven patients who underwent anatomical segmentectomies were evaluated for OS (n = 44) and LS (n = 43). The Glissonean pedicle approach was performed using either extra- or intrahepatic method, depending on the location of segment in LS. After clamping pedicle, negative staining method was performed. Liver transection was done along intersegmental plane visualizing by overlay mode of ICG camera. Surgical outcomes were compared between two groups. Correlation between predicted resecting liver volume (PRLV) calculated using volumetry and actual resected liver volume (ARLV) was assessed in two groups. Results: Patients who underwent LS showed better outcomes in operative time, blood loss, and length of hospital stay. There were significantly fewer Grade II and Grade III or higher postoperative complications in LS group. Both values of AST (p < 0.001) and ALT (p < 0.001) on postoperative day 1 were significantly lower in LS group than in OS group. PRLV and ARLV were more strongly correlated in LS (r = 0.896) than in OS (r = 0.773). The difference between PRLV and ARLV was significantly lower in LS group than in OS group (p = 0.022), and this trend was particularly noticeable in posterosuperior segment (p = 0.008) than in anterolateral segment (p = 0.811). Conclusion: LS using ICG navigation allows precise resection and may contribute to safer short-term outcomes than OS, particularly in posterosuperior segment.

Oligomerization states of the Mycobacterium tuberculosis RNA polymerase core and holoenzymes.

During the past few decades, a wealth of knowledge has been made available for the transcription machinery in bacteria from the structural, functional and mechanistic point of view. However, comparatively little is known about the homooligomerization of the multisubunit M. tuberculosis RNA polymerase (RNAP) enzyme and its functional relevance. While E. coli RNAP has been extensively studied, many aspects of RNAP of the deadly pathogenic M. tuberculosis are still unclear. We used biophysical and biochemical methods to study the oligomerization states of the core and holoenzymes of M. tuberculosis RNAP. By size exclusion chromatography and negative staining Transmission Electron Microscopy (TEM) studies and quantitative analysis of the TEM images, we demonstrate that the in vivo reconstituted RNAP core enzyme (α2ßß'ω) can also exist as dimers in vitro. Using similar methods, we also show that the holoenzyme (core + σA) does not dimerize in vitro and exist mostly as monomers. It is tempting to suggest that the oligomeric changes that we see in presence of σA factor might have functional relevance in the cellular process. Although reported previously in E. coli, to our knowledge we report here for the first time the study of oligomeric nature of M. tuberculosis RNAP in presence and absence of σA factor.

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.

Investigation of the Efficacy of Lanthanoid Heavy Metal Acetates as Electron Staining Reagents for Biomembrane Vesicles.

Transmission electron microscopy (TEM) has revolutionized our understanding of protein structures by enabling atomic-resolution visualization without the need for crystallography, thanks to advancements in cryo-TEM and single particle analysis methods. However, conventional electron microscopy remains relevant for studying stained samples, as it allows the practical determination of optimal conditions through extensive experimentation. TEM also facilitates the examination of supramolecular complexes encompassing proteins, lipids, and nucleic acids. In this study, we investigated the applicability of lanthanoid reagents as electron-staining alternatives to uranyl acetate, which is globally regulated as a nuclear fuel material. We focus on a model biomembrane vesicle system, the chromatophores from the purple photosynthetic eubacterium Rhodospirillum rubrum, which integrate proteins and lipids. Through density distribution analysis of electron micrographs, we evaluated the efficacy of various lanthanoid acetates and found that triacetates of neodymium, samarium, and gadolinium exhibited similar staining effectiveness to uranyl acetate. Additionally, triacetates of praseodymium, erbium, and lutetium, followed by europium show promising results as secondary candidates. Our findings suggest that lanthanoid transition heavy metal acetates could serve as viable alternatives for electron staining in TEM, offering potential advantages over uranyl acetate.

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.

Latest Findings on Minimally Invasive Anatomical Liver Resection.

Minimally invasive liver resection (MILR) is being widely utilized owing to recent advancements in laparoscopic and robot-assisted surgery. There are two main types of liver resection: anatomical (minimally invasive anatomical liver resection (MIALR)) and nonanatomical. MIALR is defined as a minimally invasive liver resection along the respective portal territory. Optimization of the safety and precision of MIALR is the next challenge for hepatobiliary surgeons, and intraoperative indocyanine green (ICG) staining is considered to be of considerable importance in this field. In this article, we present the latest findings on MIALR and laparoscopic anatomical liver resection using ICG at our hospital.

Expression and purification of snustorr snarlik protein from Plutella xylostella.

Snustorr snarlik (Snsl) is a type of extracellular protein essential for insect cuticle formation and insect survival, but is absent in mammals, making it a potential selective target for pest control. Here, we successfully expressed and purified the Snsl protein of Plutella xylostella in Escherichia coli. Two truncated forms of Snsl protein, Snsl 16-119 and Snsl 16-159, were expressed as a maltose-binding protein (MBP) fusion protein and purified to a purity above 90% after a five-step purification protocol. Snsl 16-119, forming stable monomer in solution, was crystallized, and the crystal was diffracted to a resolution of ∼10 Å. Snsl 16-159, forming an equilibrium between monomer and octamer in solution, was shown to form rod-shaped particles on negative staining electron-microscopy images. Our results lay a foundation for the determination of the structure of Snsl, which would improve our understanding of the molecular mechanism of cuticle formation and related pesticide resistance and provide a template for structure-based insecticide design.

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.

Insights into the oligomeric structure of the HIV-1 Vpu protein.

The HIV-1-encoded protein Vpu forms an oligomeric ion channel/pore in membranes and interacts with host proteins to support the virus lifecycle. However, Vpu molecular mechanisms are currently not well understood. Here, we report on the Vpu oligomeric organization under membrane and aqueous conditions and provide insights into how the Vpu environment affects the oligomer formation. For these studies, we designed a maltose-binding protein (MBP)-Vpu chimera protein and produced it in E. coli in soluble form. We analyzed this protein using analytical size-exclusion chromatography (SEC), negative staining electron microscopy (nsEM), and electron paramagnetic resonance (EPR) spectroscopy. Surprisingly, we found that MBP-Vpu formed stable oligomers in solution, seemingly driven by Vpu transmembrane domain self-association. A coarse modeling of nsEM data as well as SEC and EPR data suggests that these oligomers most likely are pentamers, similar to what was reported regarding membrane-bound Vpu. We also noticed reduced MBP-Vpu oligomer stability upon reconstitution of the protein in ß-DDM detergent and mixtures of lyso-PC/PG or DHPC/DHPG. In these cases, we observed greater oligomer heterogeneity, with MBP-Vpu oligomeric order generally lower than in solution; however, larger oligomers were also present. Notably, we found that in lyso-PC/PG, above a certain protein concentration, MBP-Vpu assembles into extended structures, which had not been reported for Vpu. Therefore, we captured various Vpu oligomeric forms, which can shed light on Vpu quaternary organization. Our findings could be useful in understanding Vpu organization and function in cellular membranes and could provide information regarding the biophysical properties of single-pass transmembrane proteins.

Isolation and Visualization of Gliding Motility Machinery in Bacteroidota.

Many members of the phylum Bacteroidota (formerly called Bacteroidetes) adhere to and move on solid surfaces. This type of bacterial motility is called gliding and does not involve the conventional bacterial motility machinery, such as flagella and pili. To understand the mechanism of gliding motility of some Bacteroidota bacteria such as a soil bacterium Flavobacterium johnsoniae and a marine bacterium Saprospira grandis, the gliding motility machines of these two bacteria have been analyzed by electron microscopy with negative staining. Here, we describe methods to directly observe the gliding motility machinery in Bacteroidota by transmission electron microscopy.

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.

Targeting of bone morphogenetic protein complexes to heparin/heparan sulfate glycosaminoglycans in bioactive conformation.

Bone morphogenetic proteins (BMP) are powerful regulators of cellular processes such as proliferation, differentiation, and apoptosis. However, the specific molecular requirements controlling the bioavailability of BMPs in the extracellular matrix (ECM) are not yet fully understood. Our previous work showed that BMPs are targeted to the ECM as growth factor-prodomain (GF-PD) complexes (CPLXs) via specific interactions of their PDs. We showed that BMP-7 PD binding to the extracellular microfibril component fibrillin-1 renders the CPLXs from an open, bioactive V-shape into a closed, latent ring shape. Here, we show that specific PD interactions with heparin/heparan sulfate glycosaminoglycans (GAGs) allow to target and spatially concentrate BMP-7 and BMP-9 CPLXs in bioactive V-shape conformation. However, targeting to GAGs may be BMP specific, since BMP-10 GF and CPLX do not interact with heparin. Bioactivity assays on solid phase in combination with interaction studies showed that the BMP-7 PD protects the BMP-7 GF from inactivation by heparin. By using transmission electron microscopy, molecular docking, and site-directed mutagenesis, we determined the BMP-7 PD-binding site for heparin. Further, fine-mapping of the fibrillin-1-binding site within the BMP-7 PD and molecular modeling showed that both binding sites are mutually exclusive in the open V- versus closed ring-shape conformation. Together, our data suggest that targeting exquisite BMP PD-binding sites by extracellular protein and GAG scaffolds integrates BMP GF bioavailability in a contextual manner in development, postnatal life, and connective tissue disease.

Negative Staining Transmission Electron Microscopy of HIV Viral Particles Permeabilized with PFO and Capsid Stabilized with IP6.

The human immunodeficiency virus 1 (HIV-1) consists of a viral membrane surrounding the conical capsid. The capsid is a protein container assembled from approximately 1,500 copies of the viral capsid protein (CA), functioning as a reaction and transport chamber for the viral genome after cell entry. Transmission electron microscopy (TEM) is a widely used technique for characterizing the ultrastructure of isolated viral capsids after removal of the viral membrane, which otherwise hinders negative staining of structures inside the viral particle for TEM. Here, we provide a protocol to permeabilize the membrane of HIV-1 particles using a pore-forming toxin for negative staining of capsids, which are stabilized with inositol hexakisphosphate to prevent premature capsid disassembly. This approach revealed the pleomorphic nature of capsids with a partially intact membrane surrounding them. The permeabilization strategy using pore-forming toxins can be readily applied to visualize the internal architecture of other enveloped viruses using TEM. Graphical abstract.

Correct Identification of the Core-Shell Structure of Cell Membrane-Coated Polymeric Nanoparticles.

Transmission electron microscopy (TEM) observations of negatively stained cell membrane (CM)-coated polymeric nanoparticles (NPs) reveal a characteristic core-shell structure. However, negative staining agents can create artifacts that complicate the determination of the actual NP structure. Herein, it is demonstrated with various bare polymeric core NPs, such as poly(lactic-co-glycolic acid) (PLGA), poly(ethylene glycol) methyl ether-block-PLGA, and poly(caprolactone), that certain observed core-shell structures are actually artifacts caused by the staining process. To address this issue, fluorescence quenching was applied to quantify the proportion of fully coated NPs and statistical TEM analysis was used to identify and differentiate whether the observed core-shell structures of CM-coated PLGA (CM-PLGA) NPs are due to artifacts or to the CM coating. Integrated shells in TEM images of negatively stained CM-PLGA NPs are identified as artifacts. The present results challenge current understanding of the structure of CM-coated polymeric NPs and encourage researchers to use the proposed characterization approach to avoid misinterpretations.

Thermal Structure Transformation of Methylammonium Vanadate and it's Application as a Negative Staining Reagent for Observing SARS-CoV-2.

The solid-state thermal structure transformation of methylammonium vanadate, (CH3NH3)VO3, from -150 °C to 350 °C is reported. Variable-temperature X-ray single-crystal structure analysis at 23, 0, -50, -100, and -150 °C reveal (CH3NH3)VO3 comprises of methylammonium cations and "snake-like" ([VO3]-)n anion chains propagating along the c-direction in the Pna21 space group. In between -150 and -100 °C, we observe a reversible structural transformation due to the re-orientation of the methylammonium cations in the crystal packing, which is also confirmed by the reversible profiles observed in differential scanning calorimetry. The methylammonium vanadate is stable until at ca. 100 °C and further heating releases methylamine and water and V2O5 is formed at ca. 275 °C . Furthermore, we show that the methylammonium vanadate can be used as a negative staining reagent for visualizing SARS-CoV-2, allowing us to discern the spike proteins from the body of the virus using transmission electron microscopy.

Preparation of RNA Polymerase Complexes for Their Analysis by Single-Particle Cryo-Electron Microscopy.

Recent technological progress revealed new prospects of high-resolution structure determination of macromolecular complexes using cryo-electron microscopy (cryo-EM) . In the field of RNA polymerase (Pol) I research, a number of cryo-EM studies contributed to understanding the highly specialized mechanisms underlying the transcription of ribosomal RNA genes . Despite a broad applicability of the cryo-EM method itself, preparation of samples for high-resolution data collection can be challenging. Here, we describe strategies for the purification and stabilization of Pol I complexes, exemplarily considering advantages and disadvantages of the methodology. We further provide an easy-to-implement protocol for the coating of EM-grids with self-made carbon support films. In sum, we present an efficient workflow for cryo-grid preparation and optimization, including early stage cryo-EM screening that can be adapted to a wide range of soluble samples for high-resolution structure determination .

Solubilization and Purification of α5ß1 Integrin from Rat Liver for Reconstitution into Nanodiscs.

Transmembrane proteins (or integral membrane proteins) are synthesized in the endoplasmic reticulum where most of them are core glycosylated prior to folding and in some cases assembly into quaternary structures. Correctly glycosylated, folded, and assembled transmembrane proteins are then shuttled to the Golgi apparatus for additional posttranslational modifications such as complex-type glycosylations, sulfation or proteolytic clipping. At the plasma membrane, the glycosylated extracellular domains are key to communicate with the cellular environment for a variety of functions, such as binding to the extracellular matrix for cell adhesion and migration, to neighboring cells for cell-cell interaction, or to extracellular components for nutrient uptake and cell signaling. Intracellular domains are essential to mediate signaling cascades, or to connect to cytosolic adaptors for internalization and intracellular compartmentalization. Despite its importance for the understanding of molecular mechanisms of transmembrane protein function, the determination of their structures has remained a challenging task. In recent years, their reconstitution in lipid nanodiscs in combination with high resolution cryo-electron microscopy has provided novel avenues to render this process more accessible. Here, we describe detailed protocols for the solubilization of heavily glycosylated α5ß1 integrin from rat livers, its purification and reconstitution into nanodiscs. At the plasma membrane of many cells, including tumor metastases, this essential heterodimeric transmembrane protein mediates the communication between extracellular matrix and cytosolic cytoskeleton in processes of cell adhesion and migration. We expect that the protocols that are described here will provide new opportunities for the determination of the full structure of α5ß1 integrin, as well as for the understanding of how interacting partners can regulate function and activity of this transmembrane protein.

Systematic Investigation of Lanthanoid Transition Heavy Metal Acetates as Electron Staining Reagents for Protein Molecules in Biological Transmission Electron Microscopy.

Cryo-electron microscopy, widely used for high-resolution protein structure determination, does not require staining. Yet negative staining with heavy metal salts such as uranyl acetate has been in persistent demand since the 1950s due to its image contrasting capabilities at room temperature with a common electron microscope. However, uranium compounds are nuclear fuel materials and are tightly controlled worldwide. Acetates of each lanthanoid series elements except promethium are prepared at the same concentration (2%(w/v)) and used as a model on horse spleen ferritin for electron microscopic analysis to systematically evaluate their effectiveness as electron staining reagents for the protein. Analysis shows that the triacetates of samarium and europium, followed by gadolinium and erbium, and then lanthanum and neodymium could function as electron staining reagents. Thulium-triacetate precipitates thin plate-like crystals and may be used for selecting better imaging fields. Of the 14 lanthanoid-triacetates examined, about half are viable alternatives to uranyl acetate as an electron staining reagent for ferritin, and there appears an optimal range in ionic sizes for promising lanthanoids. This is the first systematic investigation of lanthanoid transition heavy metal triacetates from the viewpoint of lanthanoid contraction, using density distribution histograms of electron micrographs as an indicator for comparison with uranyl acetate.