Endogenous Labeling for Light Microscopy during HIV-1 Immune Responses.

New approaches in single molecule spectroscopy and microscopy are able to resolve the spatial and temporal resolution of T cell receptor signaling in the context of immune responses to HIV-1 infection. These approaches need to be complemented with novel techniques that endogenously tag the protein or proteins of interest, yet avoid overexpression, to image protein dynamics under physiological conditions.

Digital mucous cyst: Altered epidermal mucin as a clue to diagnosis.

BACKGROUND: Digital mucous cyst (DMC) is histopathologically characterized by accumulation of mucin in the dermis. Some cases of DMC also show epidermal mucin, the histopathologic appearance and staining properties of which have not been described in detail. METHODS: A total of 24 cases of DMC were investigated by routine hematoxylin-eosin (H&E) and Alcian blue stains in addition to AE1/AE3 immunohistochemistry. RESULTS: Nine out of the 24 cases of DMC showed epidermal mucin. As the epidermal mucin migrates upward within the epidermis, it transforms from a flocculent granular substance into one or several solid horizontal plugs with a more homogeneous appearance and incorporates cytoplasmic fragments of keratinocytes/corneocytes. The homogeneous mucin plugs stain eosinophilic or amphophilic with an H&E formulation using hematoxylin 7212 and basophilic with Gill 3 or Harris's hematoxylin. The eosinophilic staining is enhanced when the eosin solution contains phloxine. CONCLUSIONS: The variably eosinophilic, amphophilic, or basophilic staining of epidermal mucin can be explained by its composition of basophilic mucin and eosinophilic debris from cytoplasmic fragments. The eosinophilic staining of mucin has not been reported before and can be diagnostically important because it may be mistaken for serum exudate.

Varying practices in tumor regression grading of gastrointestinal carcinomas after neoadjuvant therapy: results of an international survey.

Tumor regression grading is routinely performed on neoadjuvantly treated gastrointestinal cancer resections. Challenges in tumor regression grading include grossing standards, multiple grading systems, and difficulty interpreting therapy-induced changes. We surveyed gastrointestinal pathologists around the world for their practices in handling neoadjuvantly treated gastrointestinal cancer specimens and reporting tumor regression using a 23-question online survey. Topics addressed grossing, histologic work-up, tumor regression grading systems, and degree of difficulty identifying and estimating residual cancer within treatment effect. Two-hundred three responses were received, including 173 participants who completed the entire questionnaire. Fifty percent of the participants were from Europe, 29% from North America, 10% from Australia, and 11% from other continents. Ninety-five percent routinely report a tumor regression grade and 92% have standardized grossing and histologic work-up: 27% always completely embed the entire tumor bed, 54% embed the complete tumor site if not a grossly apparent, large mass. Fifty-nine percent use hematoxylin & eosin alone for assessment; the remaining use additional stains. In North America and Australia, the American Joint Committee on Cancer (AJCC)/College of American Pathologists (CAP)/Ryan system is routinely used for gastroesophageal (71%) and rectal carcinomas (77%). In Europe, the Mandard system is common (36%) for gastroesophageal tumors, followed by AJCC/CAP/Ryan (22%), and Becker (10%); for rectal CA, the Dworak system (30%) is followed by AJCC/CAP/Ryan (24%) and Mandard (14%). This regional differences were significant (p < 0.001 each). Fifty-one percent prefer a four-tiered system. Sixty-six percent think that regressive changes in lymph nodes should be part of a regression grade. Sixty-nine percent consider identifying residual tumor straight-forward, but estimating therapy-induced fibrosis difficult (57%). Free comments raised issues of costs for work-up and clinical relevance. In conclusion, this multinational survey provides a comprehensive overview of grossing and histologic work-up with regards to tumor regression grading in gastrointestinal cancers with partly significant regional differences particularly between North America and Europe.

Micro-staining microbes: An alternative to traditional staining of microbiological specimens using microliter volumes of reagents.

Microbial staining techniques are widely employed in clinical and academic laboratories for classifying and identifying microorganisms derived from clinical, food and environmental samples. Staining allows for the rapid visualization and determination of many morphological characteristics of microorganisms, used for their identification and classification. Over the past century, staining techniques such as the Gram stain, the Capsule stain, the Acid-fast stain and the Endospore stain, have seen few advances, and manual staining remains the gold standard. Typical instructions for these staining procedures recommend 'flooding' glass slides with milliliter volumes of dye, resulting in large volumes of hazardous waste. Here we present micro-staining, a simple alternative to flooding that utilizes microliter volumes of dye. Micro-staining minimizes the volume of waste generated, leads to significant cost savings for the laboratory, requires limited training, and produces results with equivalent quality to traditional stains.

Strategic labelling approaches for RNA single-molecule spectroscopy.

Most single-molecule techniques observing RNA in vitro or in vivo require fluorescent labels that have to be connected to the RNA of interest. In recent years, a plethora of methods has been developed to achieve site-specific labelling, in many cases under near-native conditions. Here, we review chemical as well as enzymatic labelling methods that are compatible with single-molecule fluorescence spectroscopy or microscopy and show how these can be combined to offer a large variety of options to site-specifically place one or more labels in an RNA of interest. By either chemically forming a covalent bond or non-covalent hybridization, these techniques are prerequisites to perform state-of-the-art single-molecule experiments.

Waltzing around Sacred Cows on the Way to the Future.

Our mostly manual, agar-based clinical microbiology laboratory is slowly but steadily being redefined by automation and innovation. Ironically, the oldest test, the Gram stain test, is still manually read and interpreted by trained personnel. In a proof-of-concept study, Smith et al. (J. Clin. Microbiol. 56:e01521-17, 2018, https://doi.org/10.1128/JCM.01521-17) used computer imaging with a deep convolutional neural network to examine and interpret Gram-stained slides from positive blood culture bottles. In light of the shortage of medical technologists/microbiologists and the need for results from positive blood culture bottles 24/7, this paper paves the way for the next innovations for the clinical microbiology laboratory of the future.

Recent Advances and Future Prospects of Aggregation-induced Emission Carbohydrate Polymers.

Aggregation-induced emission (AIE) is an abnormal phenomenon that has sparked great attention for diverse applications in different fields. In particular, the fabrication and biological imaging applications of AIE-active fluorescent organic nanoparticles (FONs) have become a focus in the emerging and promising fields. A large number of AIE-active polymeric nanoprobes have recently been fabricated through different strategies. The advances and progress in this direction have also recently been summarized by some groups. However, the fabrication and biomedical applications of AIE-active FONs based on carbohydrate polymers and AIE-active dyes are quite rare and limited. In this feature article, the recently reported AIE-active FONs with different structures and applications based on AIE-active dyes and carbohydrate polymers are highlighted, and the major current limitations and development tendencies are also discussed.

MIAQuant, a novel system for automatic segmentation, measurement, and localization comparison of different biomarkers from serialized histological slices.

In the clinical practice, automatic image analysis methods quickly quantizing histological results by objective and replicable methods are getting more and more necessary and widespread. Despite several commercial software products are available for this task, they are very little flexible, and provided as black boxes without modifiable source code. To overcome the aforementioned problems, we employed the commonly used MATLAB platform to develop an automatic method, MIAQuant, for the analysis of histochemical and immunohistochemical images, stained with various methods and acquired by different tools. It automatically extracts and quantifies markers characterized by various colors and shapes; furthermore, it aligns contiguous tissue slices stained by different markers and overlaps them with differing colors for visual comparison of their localization. Application of MIAQuant for clinical research fields, such as oncology and cardiovascular disease studies, has proven its efficacy, robustness and flexibility with respect to various problems; we highlight that, the flexibility of MIAQuant makes it an important tool to be exploited for basic researches where needs are constantly changing. MIAQuant software and its user manual are freely available for clinical studies, pathological research, and diagnosis.

Molecular Imaging in Preclinical Models of IBD with Nuclear Imaging Techniques: State-of-the-Art and Perspectives.

Inflammatory bowel disease (IBD), which includes ulcerative colitis and Crohn's disease, is characterized by chronic unregulated inflammation of the intestinal mucosa of the gastrointestinal tract. To date, this pathology has no cure. Colonoscopy and biopsies are the current gold standard diagnostic tools. However, being a chronic disease, IBD requires continuous follow-up to check for disease progress, treatment response, and remission. Unfortunately, these 2 diagnostic procedures are invasive and generally unable to show the cellular and molecular changes that take place in vivo. In this context, it is clear that there is a strong need for optimized noninvasive imaging techniques able to overcome the aforementioned limitations. This review aims to bring to light the scientific advancements that have been achieved so far in nuclear medicine in relation to tracking of immune cells involved in the preclinical models of IBD. In particular, this review will explore the advantages and limitations of the radiopharmaceuticals that aim to track whole cells like neutrophils, those that involve the radiolabeling of immune cell substrates or available human IBD medical therapies, and those that aim to track cell signaling molecules (e.g., cytokines and cell adhesion molecules). After a detailed critical summary of the state-of-the art, the challenges and perspectives of molecular imaging applied to IBD studies will be analyzed. Special attention will be paid to the translational potential of the described techniques and on the potential impact of these innovative approaches on the drug discovery pipelines and their contribution to the evolution of personalized medicine.

News from the Biological Stain Commission, No. 17.

In the 17(th) issue of News from the Biological Stain Commission (BSC) under the heading of Regulatory affairs, the Biological Stain Commission's International Affairs Committee presents information from the 20(th) meeting of ISO/TC 212 Clinical laboratory testing and in vitro diagnostic test systems held on October 15 - 17, 2014 in Toronto, Canada, and from the 29(th) meeting of CEN/TC 140 In vitro diagnostic medical devices held on February 3, 2015 in Berlin, Germany.

[Visualization and Functional Regulation of Live Cell Proteins Based on Labeling Probe Design].

  There are several approaches to understanding the physiological roles of biomolecules: (1) by observing the localization or activities of biomolecules (based on microscopic imaging experiments with fluorescent proteins or fluorescent probes) and (2) by investigating the cellular response via activation or suppression of functions of the target molecule (by using inhibitors, antagonists, siRNAs, etc.). In this context, protein-labeling technology serves as a powerful tool that can be used in various experiments, such as for fluorescence imaging of target proteins. Recently, we developed a protein-labeling technology that uses a mutant ß-lactamase (a bacterial hydrolase) as the tag protein. In this protein-labeling technology, also referred to as the BL-tag technology, various ß-lactam compounds were used as specific ligands that were covalently labeled to the tag. One major advantage of this labeling technology is that various functions can be carried out by suitably designing both the functional moieties such as the fluorophore and the ß-lactam ligand structure. In this review, we briefly introduce the BL-tag technology and describe our future outlook for this technology, such as in fluorescence imaging of biomolecules and functional regulation of cellular proteins in living cells.

Illumination of growth, division and secretion by metabolic labeling of the bacterial cell surface.

The cell surface is the essential interface between a bacterium and its surroundings. Composed primarily of molecules that are not directly genetically encoded, this highly dynamic structure accommodates the basic cellular processes of growth and division as well as the transport of molecules between the cytoplasm and the extracellular milieu. In this review, we describe aspects of bacterial growth, division and secretion that have recently been uncovered by metabolic labeling of the cell envelope. Metabolite derivatives can be used to label a variety of macromolecules, from proteins to non-genetically-encoded glycans and lipids. The embedded metabolite enables precise tracking in time and space, and the versatility of newer chemoselective detection methods offers the ability to execute multiple experiments concurrently. In addition to reviewing the discoveries enabled by metabolic labeling of the bacterial cell envelope, we also discuss the potential of these techniques for translational applications. Finally, we offer some guidelines for implementing this emerging technology.

Intein applications: from protein purification and labeling to metabolic control methods.

The discovery of inteins in the early 1990s opened the door to a wide variety of new technologies. Early engineered inteins from various sources allowed the development of self-cleaving affinity tags and new methods for joining protein segments through expressed protein ligation. Some applications were developed around native and engineered split inteins, which allow protein segments expressed separately to be spliced together in vitro. More recently, these early applications have been expanded and optimized through the discovery of highly efficient trans-splicing and trans-cleaving inteins. These new inteins have enabled a wide variety of applications in metabolic engineering, protein labeling, biomaterials construction, protein cyclization, and protein purification.

Histochemistry: historical development and current use in pathology.

We describe the history of the histochemical stains that contributed most to the development of modern pathology during the last two centuries. Histochemical stains are presented in a list, which provides the essential information about year, country and main use of each to enable the reader to follow the chronological and geographical history of histochemistry. In addition to the historical evaluation of histochemistry development, we investigate how many classical histochemical stains survive in a modern laboratory of pathology and how often they are used for diagnostic practice compared to immunohistochemical (IHC) techniques. A ratio of about one histochemical reaction to 13 IHC reactions was tabulated. Finally, our data make it possible to define different cultural approaches to the terminology of histochemical and IHC stains: the former were based on eponyms, which link the stain with the name of its inventor, while the latter use a more impersonal biological terminology.

Neuronal morphology goes digital: a research hub for cellular and system neuroscience.

The importance of neuronal morphology in brain function has been recognized for over a century. The broad applicability of "digital reconstructions" of neuron morphology across neuroscience subdisciplines has stimulated the rapid development of numerous synergistic tools for data acquisition, anatomical analysis, three-dimensional rendering, electrophysiological simulation, growth models, and data sharing. Here we discuss the processes of histological labeling, microscopic imaging, and semiautomated tracing. Moreover, we provide an annotated compilation of currently available resources in this rich research "ecosystem" as a central reference for experimental and computational neuroscience.

Digital image analysis: a review of reproducibility, stability and basic requirements for optimal results.

Digital image analysis (DIA) is increasingly implemented in histopathological research to facilitate truly quantitative measurements, decrease inter-observer variation and reduce hands-on time. Originally, efforts were made to enable DIA to reproduce manually obtained results on histological slides optimized for light microscopy and the human eye. With improved technical methods and the acknowledgement that computerized readings are different from analysis by human eye, recognition has been achieved that to really empower DIA, histological slides must be optimized for the digital 'eye', with reproducible results correlating with clinical findings. In this review, we focus on the basic expectations and requirements for DIA to gain wider use in histopathological research and diagnostics. With a reference to studies that specifically compare DIA with conventional methods, this review discusses reproducibility, application of stereology-based quantitative measurements, time consumption, optimization of histological slides, regions of interest selection and recent developments in staining and imaging techniques.