Minimal genetically encoded tags for fluorescent protein labeling in living neurons

Modern light microscopy, including super-resolution techniques, has brought about a demand for small labeling tags that bring the fluorophore closer to the target. This challenge can be addressed by labeling unnatural amino acids (UAAs) with bioorthogonal click chemistry. The minimal size of the UAA and the possibility to couple the fluorophores directly to the protein of interest with single-residue precision in living cells make click labeling unique. Here, we establish click labeling in living primary neurons and use it for fixed-cell, live-cell, dual-color pulse–chase, and super-resolution microscopy of neurofilament light chain (NFL). We also show that click labeling can be combined with CRISPR/Cas9 genome engineering for tagging endogenous NFL. Due to its versatile nature and compatibility with advanced multicolor microscopy techniques, we anticipate that click labeling will contribute to novel discoveries in the neurobiology field.

Conductive Bioimprint Using Soft Lithography Technique Based on PEDOT:PSS for Biosensing

Culture platform surface topography plays an important role in the regulation of biological cell behaviour. Understanding the mechanisms behind the roles of surface topography in cell response are central to many developments in a Lab on a Chip, medical implants and biosensors. In this work, we report on a novel development of a biocompatible conductive hydrogel (CH) made of poly (3,4-ethylenedioxythiophene):polystyrene sulfonate (PEDOT:PSS) and gelatin with bioimprinted surface features. The bioimprinted CH offers high conductivity, biocompatibility and high replication fidelity suitable for cell culture applications. The bioimprinted conductive hydrogel is developed to investigate biological cells’ response to their morphological footprint and study their growth, adhesion, cell–cell interactions and proliferation as a function of conductivity. Moreover, optimization of the conductive hydrogel mixture plays an important role in achieving high imprinting resolution and conductivity. The reason behind choosing a conducive hydrogel with high resolution surface bioimprints is to improve cell monitoring while mimicking cells’ natural physical environment. Bioimprints which are a 3D replication of cellular morphology have previously been shown to promote cell attachment, proliferation, differentiation and even cell response to drugs. The conductive substrate, on the other hand, enables cell impedance to be measured and monitored, which is indicative of cell viability and spread. Two dimensional profiles of the cross section of a single cell taken via Atomic Force Microscopy (AFM) from the fixed cell on glass, and its replicas on polydimethylsiloxane (PDMS) and conductive hydrogel (CH) show unprecedented replication of cellular features with an average replication fidelity of more than 90%. Furthermore, crosslinking CH films demonstrated a significant increase in electrical conductivity from 10−6 S/cm to 1 S/cm. Conductive bioimprints can provide a suitable platform for biosensing applications and potentially for monitoring implant-tissue reactions in medical devices.

Comparison of programmed death ligand 1 immunostaining for pancreatic ductal adenocarcinoma between paired cytological and surgical samples

Objectives: Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis with surgery or chemotherapy. Programmed death ligand 1 expression (PD-L1) immunotherapy has been successful for treating lung and other cancers with PD-L1 expression. However, in many unresectable PDAC cases, cytological samples are the only available tissues for PD-L1 testing. The aim of this study is to retrospectively compare the expression of PD-L1 using cytological and surgical samples. Material and Methods: Paired formalin-fixed cell blocks and surgical samples from the same patients with confirmed diagnoses of PDAC (n = 28) were sectioned for PD-L1 immunohistochemistry. Using tumor proportion score (TPS) and combined positive score (CPS) to evaluate paired cell blocks and surgical samples, we counted and analyzed the data. Results: With TPS, the PD-L1 was expressed in 9/28 (32%) of PDAC surgical samples and in 9/28 (32%) of paired cytological samples. Overall, the PD-L1 expression had a correlation of 26/28 (93%). With CPS, the PD-L1 was expressed in 20/28 (71%) of PDAC surgical samples and in 16/28 (57%) of paired cytological samples. The PD-L1 expression had a correlation of 20/28 (71%) and a discrepancy of 8/28 (29%). The PD-L1 expression was significantly higher in moderately-differentiated PDAC than in well-differentiated with TPS. Conclusion: Cytological samples are useful for evaluating PD-L1 expression with TPS because the concordant rate was 93%. With CPS, cytological samples are limited due to the scant inflammatory cells with the concordant rate of 71%. Extensive sampling of the pancreatic tumor may improve the detection of immune cells expressing PD-L1 in cytological samples. With TPS, PD-L1 expression was significantly higher in moderate-differentiation of PDAC than in poor- and well-differentiation.

Chemical Fixation Creates Nanoscale Clusters on the Cell Surface by Aggregating Membrane Proteins

Chemical fixations have been thought to preserve the structures of the cells or tissues. However, given that the fixatives create crosslinks or aggregate proteins, there is a possibility that these fixatives create nanoscale artefacts by aggregation of membrane proteins which move around freely to some extent on the cell surface. Despite this, little research has been conducted about this problem, probably because there has been no method for observing cell surface structures at the nanoscale. In this study, we have developed a new method to observe cell surfaces stably and with high resolution using atomic force microscopy and a microporous silicon nitride membrane. We demonstrate that the size of the protrusions on the cell surface is increased after treatment with three commonly used fixatives and show that these protrusions were created by the aggregation of membrane proteins by fixatives. These results call attention when observing fixed cell surfaces at the nanoscale.

Anti-MOG autoantibody–associated schizophreniform psychosis

Objective: Autoimmune mechanisms are related to disease development in a subgroup of patients with psychosis. The contribution of immunoglobulin G (IgG) antibodies against myelin oligodendrocyte glycoprotein (MOG) is mainly unclear in this context. Methods: Therefore, two patients with psychosis and anti-MOG antibodies—detected in fixed cell-based and live cell-based assays—are presented. Results: Patient 1 suffered from late-onset psychosis with singular white matter lesions in MRI and intermittent EEG slowing. Patient 2 suffered from a chronic paranoid-hallucinatory disorder with intermittent confusional states, non-specific white matter alterations on MRI, a disorganized alpha rhythm on EEG and elevated cerebrospinal-fluid protein. Both patients had anti-MOG antibody titers of 1:320 in serum (reference<1:20). Conclusion: The arguments for and against a causal role for anti-MOG antibodies are discussed. The antibodies could be relevant, but due to moderate titers, they may have caused a rather “subtle clinical picture” consisting of psychosis instead of “classical” MOG encephalomyelitis.

Overlap Intensity: An ImageJ Macro for Analyzing the HIV-1 In Situ Uncoating Assay

Capsid uncoating is at the crossroads of early steps in HIV-1 replication. In recent years, the development of novel assays has expanded how HIV-1 uncoating can be studied. In the in situ uncoating assay, dual fluorescently labelled virus allows for the identification of fused viral cores. Antibody staining then detects the amount of capsid associated with each viral core at different times post-infection. Following fixed cell imaging, manual counting can be used to assess the fusion state and capsid signal for each viral core, but this method can introduce bias with increased time of analysis. To address these limitations, we developed the Overlap Intensity macro in ImageJ. This macro automates the detection of viral cores and quantification of overlapping fusion and capsid signals. We demonstrated the high accuracy of the macro by comparing core detection to manual methods. Analysis of an in situ uncoating assay further verified the macro by detecting progressive uncoating as expected. Therefore, this macro improves the accessibility of the in situ uncoating assay by replacing time-consuming manual methods or the need for expensive data analysis software. Beyond the described assay, the Overlap Intensity macro includes adjustable settings for use in other methods requiring quantification of overlapping fluorescent signals.

Deep learning-enhanced morphological profiling predicts cell fate dynamics in real-time in hPSCs

Predicting how stem cells become patterned and differentiated into target tissues is key for optimising human tissue design. Here, we established DEEP-MAP - for deep learning-enhanced morphological profiling - an approach that integrates single-cell, multi-day, multi-colour microscopy phenomics with deep learning and allows to robustly map and predict cell fate dynamics in real-time without a need for cell state-specific reporters. Using human pluripotent stem cells (hPSCs) engineered to co-express the histone H2B and two-colour FUCCI cell cycle reporters, we used DEEP-MAP to capture hundreds of morphological- and proliferation-associated features for hundreds of thousands of cells and used this information to map and predict spatiotemporally single-cell fate dynamics across germ layer cell fates. We show that DEEP-MAP predicts fate changes as early or earlier than transcription factor-based fate reporters, reveals the timing and existence of intermediate cell fates invisible to fixed-cell technologies, and identifies proliferative properties predictive of cell fate transitions. DEEP-MAP provides a versatile, universal strategy to map tissue evolution and organisation across many developmental and tissue engineering contexts.

Quantitative single molecule RNA-FISH and RNase-free cell wall digestion in Neurospora crassa.

Single molecule RNA-FISH (smFISH) is a valuable tool for analysis of mRNA spatial patterning in fixed cells that is underutilized in filamentous fungi. A primary complication for fixed-cell imaging in filamentous fungi is the need for enzymatic cell wall permeabilization, which is compounded by considerable variability in cell wall composition between species. smFISH adds another layer of complexity due to a requirement for RNase free conditions. Here, we describe the cloning, expression, and purification of a chitinase suitable for supplementation of a commercially available RNase-free enzyme preparation for efficient permeabilization of the Neurospora cell wall. We further provide a method for smFISH in Neurospora which includes a tool for generating numerical data from images that can be used in downstream customized analysis protocols.

The Consequences of Differential Origin Licensing Dynamics

MCM complexes are loaded onto chromosomes to license DNA replication origins in G1 phase of the cell cycle, but it is not yet known how mammalian MCM complexes are adequately distributed to both euchromatin and heterochromatin. To address this question, we combined time-lapse live-cell imaging with fixed cell immunofluorescence imaging of single human cells to quantify the relative rates of MCM loading in heterochromatin and euchromatin at different times within G1. We report here that MCM loading in euchromatin is faster than in heterochromatin in very early G1, but surprisingly, heterochromatin loading accelerates faster than euchromatin in middle and late G1. These different loading dynamics require ORCA-dependent differences in ORC distribution during G1. A consequence of heterochromatin origin licensing dynamics is that cells experiencing a truncated G1 phase from premature Cyclin E expression enter S phase with underlicensed heterochromatin, and DNA damage accumulates preferentially in heterochromatin in the subsequent S/G2 phase. Thus G1 length is critical for sufficient MCM loading, particularly in heterochromatin, to ensure complete genome duplication and to maintain genome stability.