Nonlinear optical response of heme solutions.

Heme is the prosthetic group for cytochrome that exists in nearly all living organisms and serves as a vital component of human red blood cells (RBCs). Tunable optical nonlinearity in suspensions of RBCs has been demonstrated previously, however, the nonlinear optical response of a pure heme (without membrane structure) solution has not been studied to our knowledge. In this work, we show optical nonlinearity in two common kinds of heme (i.e., hemin and hematin) solutions by a series of experiments and numerical simulations. We find that the mechanism of nonlinearity in heme solutions is distinct from that observed in the RBC suspensions where the nonlinearity can be easily tuned through optical power, concentration, and the solution properties. In particular, we observe an unusual phenomenon wherein the heme solution exhibits negative optical nonlinearity and render self-collimation of a focused beam at specific optical powers, enabling shape-preserving propagation of light to long distances. Our results may have potential applications in optical imaging and medical diagnosis through blood.

The Golgi microtubules regulate single cell durotaxis.

Current understandings on cell motility and directionality rely heavily on accumulated investigations of the adhesion-actin cytoskeleton-actomyosin contractility cycles, while microtubules have been understudied in this context. Durotaxis, the ability of cells to migrate up gradients of substrate stiffness, plays a critical part in development and disease. Here, we identify the pivotal role of Golgi microtubules in durotactic migration of single cells. Using high-throughput analysis of microtubule plus ends/focal adhesion interactions, we uncover that these non-centrosomal microtubules actively impart leading edge focal adhesion (FA) dynamics. Furthermore, we designed a new system where islands of higher stiffness were patterned within RGD peptide coated polyacrylamide gels. We revealed that the positioning of the Golgi apparatus is responsive to external mechanical cues and that the Golgi-nucleus axis aligns with the stiffness gradient in durotaxis. Together, our work unveils the cytoskeletal underpinning for single cell durotaxis. We propose a model in which the Golgi-nucleus axis serves both as a compass and as a steering wheel for durotactic migration, dictating cell directionality through the interaction between non-centrosomal microtubules and the FA dynamics.

Golgi-associated microtubules are fast cargo tracks and required for persistent cell migration.

Microtubules derived from the Golgi (Golgi MTs) have been implicated to play critical roles in persistent cell migration, but the underlying mechanisms remain elusive, partially due to the lack of direct observation of Golgi MT-dependent vesicular trafficking. Here, using super-resolution stochastic optical reconstruction microscopy (STORM), we discovered that post-Golgi cargos are more enriched on Golgi MTs and also surprisingly move much faster than on non-Golgi MTs. We found that, compared to non-Golgi MTs, Golgi MTs are morphologically more polarized toward the cell leading edge with significantly fewer inter-MT intersections. In addition, Golgi MTs are more stable and contain fewer lattice repair sites than non-Golgi MTs. Our STORM/live-cell imaging demonstrates that cargos frequently pause at the sites of both MT intersections and MT defects. Furthermore, by optogenetic maneuvering of cell direction, we demonstrate that Golgi MTs are essential for persistent cell migration but not for cells to change direction. Together, our study unveils the role of Golgi MTs in serving as a group of "fast tracks" for anterograde trafficking of post-Golgi cargos.

A novel therapeutic drug for colon cancer: EpCAM scFv-truncated protamine (tp)-siRNA.

Colon cancer is a type of malignant tumor that causes considerable mortality worldwide. Epithelial cellular adhesion molecule (EpCAM), a tumor-associated antigen of colon tumors, is a target for colon cancer therapy. EpCAM-specific monoclonal antibodies (mAbs) have been applied in human colon cancer since the 1990s; however, the therapeutic effects are limited. EpCAM activates nuclear signaling pathways by releasing its intracellular domain (EpICD). The released EpICD stimulates the Wnt/ß-catenin signaling pathway, which is also strongly associated with tumorigenesis. EpCAM is also a target gene of the Wnt/ß-catenin signaling pathway. EpCAM and the Wnt/ß-catenin signaling pathway form a functional interaction cycle in colon cancer. Thus, we propose a new therapeutic drug for colon cancer: an EpCAM single-chain fragment variable antibody (scFv)-truncated protamine-siRNA. EpCAM scFv can recognize and bind colon cancer cells through its EpCAM antigen activity. Furthermore, the specific siRNA transferred into colon cancer cells specifically inhibits Wnt/ß-catenin signal transmission. Therefore, this new drug may efficiently interrupt the functional cycle between EpCAM and Wnt/ß-catenin signaling and be an effective therapeutic strategy for colon cancer.

Rapid, artifact-reduced, image reconstruction for super-resolution structured illumination microscopy.

Super-resolution structured illumination microscopy (SR-SIM) is finding increasing application in biomedical research due to its superior ability to visualize subcellular dynamics in living cells. However, during image reconstruction artifacts can be introduced and when coupled with time-consuming postprocessing procedures, limits this technique from becoming a routine imaging tool for biologists. To address these issues, an accelerated, artifact-reduced reconstruction algorithm termed joint space frequency reconstruction-based artifact reduction algorithm (JSFR-AR-SIM) was developed by integrating a high-speed reconstruction framework with a high-fidelity optimization approach designed to suppress the sidelobe artifact. Consequently, JSFR-AR-SIM produces high-quality, super-resolution images with minimal artifacts, and the reconstruction speed is increased. We anticipate this algorithm to facilitate SR-SIM becoming a routine tool in biomedical laboratories.

Single-cell transcriptomic dissection of the cellular and molecular events underlying the triclosan-induced liver fibrosis in mice.

BACKGROUND: Triclosan [5-chloro-2-(2,4-dichlorophenoxy) phenol, TCS], a common antimicrobial additive in many personal care and health care products, is frequently detected in human blood and urine. Therefore, it has been considered an emerging and potentially toxic pollutant in recent years. Long-term exposure to TCS has been suggested to exert endocrine disruption effects, and promote liver fibrogenesis and tumorigenesis. This study was aimed at clarifying the underlying cellular and molecular mechanisms of hepatotoxicity effect of TCS at the initiation stage. METHODS: C57BL/6 mice were exposed to different dosages of TCS for 2 weeks and the organ toxicity was evaluated by various measurements including complete blood count, histological analysis and TCS quantification. Single cell RNA sequencing (scRNA-seq) was then carried out on TCS- or mock-treated mouse livers to delineate the TCS-induced hepatotoxicity. The acquired single-cell transcriptomic data were analyzed from different aspects including differential gene expression, transcription factor (TF) regulatory network, pseudotime trajectory, and cellular communication, to systematically dissect the molecular and cellular events after TCS exposure. To verify the TCS-induced liver fibrosis, the expression levels of key fibrogenic proteins were examined by Western blotting, immunofluorescence, Masson's trichrome and Sirius red staining. In addition, normal hepatocyte cell MIHA and hepatic stellate cell LX-2 were used as in vitro cell models to experimentally validate the effects of TCS by immunological, proteomic and metabolomic technologies. RESULTS: We established a relatively short term TCS exposure murine model and found the TCS mainly accumulated in the liver. The scRNA-seq performed on the livers of the TCS-treated and control group profiled the gene expressions of > 76,000 cells belonging to 13 major cell types. Among these types, hepatocytes and hepatic stellate cells (HSCs) were significantly increased in TCS-treated group. We found that TCS promoted fibrosis-associated proliferation of hepatocytes, in which Gata2 and Mef2c are the key driving TFs. Our data also suggested that TCS induced the proliferation and activation of HSCs, which was experimentally verified in both liver tissue and cell model. In addition, other changes including the dysfunction and capillarization of endothelial cells, an increase of fibrotic characteristics in B plasma cells, and M2 phenotype-skewing of macrophage cells, were also deduced from the scRNA-seq analysis, and these changes are likely to contribute to the progression of liver fibrosis. Lastly, the key differential ligand-receptor pairs involved in cellular communications were identified and we confirmed the role of GAS6_AXL interaction-mediated cellular communication in promoting liver fibrosis. CONCLUSIONS: TCS modulates the cellular activities and fates of several specific cell types (including hepatocytes, HSCs, endothelial cells, B cells, Kupffer cells and liver capsular macrophages) in the liver, and regulates the ligand-receptor interactions between these cells, thereby promoting the proliferation and activation of HSCs, leading to liver fibrosis. Overall, we provide the first comprehensive single-cell atlas of mouse livers in response to TCS and delineate the key cellular and molecular processes involved in TCS-induced hepatotoxicity and fibrosis.

Multi-color structured illumination microscopy for live cell imaging based on the enhanced image recombination transform algorithm.

Structured illumination microscopy (SIM) has attracted considerable interest in super-resolution, live-cell imaging because of its low light dose and high imaging speed. Obtaining a high-quality reconstruction image in SIM depends on the precise determination of the parameters of the fringe illumination pattern. The image recombination transform (IRT) algorithm is superior to other algorithms in obtaining the precise initial phase without any approximation, which is promising to provide a considerable solution to address the difficulty of initial phase estimation at low-modulation-depth conditions. However, the IRT algorithm only considers a phase shift of π∕2, which limits its applications in general scenarios. In this letter, we present a general form of IRT algorithm suitable for arbitrary phase shifts, providing a powerful tool for parameter estimation in low signal-to-noise cases. To demonstrate the effectiveness of the enhanced IRT algorithm, we constructed a multicolor, structured illumination microscope and studied at super-resolution, the cargo traffic in HRPE cells, and monitored the movement of mitochondrial structures and microtubules in COS-7 cells. The custom SIM system using the enhanced IRT algorithm allows multicolor capability and a low excitation intensity fluorescence imaging less than 1 W/cm2. High-quality super-resolution images are obtained, which demonstrates the utility of this approach in imaging in the life sciences.

ER-mitochondria contacts promote mtDNA nucleoids active transportation via mitochondrial dynamic tubulation.

A human cell contains hundreds to thousands of mitochondrial DNA (mtDNA) packaged into nucleoids. Currently, the segregation and allocation of nucleoids are thought to be passively determined by mitochondrial fusion and division. Here we provide evidence, using live-cell super-resolution imaging, that nucleoids can be actively transported via KIF5B-driven mitochondrial dynamic tubulation (MDT) activities that predominantly occur at the ER-mitochondria contact sites (EMCS). We further demonstrate that a mitochondrial inner membrane protein complex MICOS links nucleoids to Miro1, a KIF5B receptor on mitochondria, at the EMCS. We show that such active transportation is a mechanism essential for the proper distribution of nucleoids in the peripheral zone of the cell. Together, our work identifies an active transportation mechanism of nucleoids, with EMCS serving as a key platform for the interplay of nucleoids, MICOS, Miro1, and KIF5B to coordinate nucleoids segregation and transportation.

[Colloidal gold immunochromatographic strip for rapid detection of Haemophilus influenzae].

To establish a novel colloidal gold immunochromatography assay (GICA) for rapid, sensitive and accurate detection of Haemophilus influenzae infection by using the outer membrane protein P6 as detection target. First, the linear antigen epitope located in the extracellular domain of the P6 protein (GenBank accession number: AGH02799) was predicted by bioinformatics analysis. The region (62-75 aa of the protein) with strong antigen specificity was chosen and synthesized. Two rabbits were then immunized by the polypeptides (14 aa) for production of polyclonal antibodies. Then, the recombinant P6 proteins were also obtained to produce polyclonal antibodies. Finally, based on the two antibodies, a novel colloidal GICA for detection of Haemophilus influenzae infection was established and the specificity, sensitivity, repeatability and stability of this method were evaluated. At the same time, the method was tested in clinical simulation, and the plate culture method was used to verify its accuracy. The test strip for Haemophilus influenzae infection was successfully prepared. The detection limit of the test strip was as low as 1×105 CFU/mL and the whole process can be completed within 15 minutes. The strip specifically recognized Haemophilus influenzae and did not react with nine of other common respiratory pathogens such as Streptococcus pneumoniae, Moraxella catarrhalis, Mycoplasma pneumonia, and Legionella pneumophila. And the strips could be stored at 25 °C for at least 6 months without losing sensitivity or specificity. The coincidence rate between the results of 200 clinical samples and the plate culture method was 90.5%. Haemophilus influenzae protein P6, which possessed a high degree of surface antigen accessibility and antigencity, could be used as a marker for Haemophilus influenzae detection. The immunochromatographic colloidal gold test strip which bears the features of rapidity, convenience and sensitivity provides a unique tool for the on-site surveillance and diagnosis of Haemophilus influenzae infection in clinical test.

Study of RNA Polymerase II Clustering inside Live-Cell Nuclei Using Bayesian Nanoscopy.

Nanoscale spatiotemporal clustering of RNA polymerase II (Pol II) plays an important role in transcription regulation. However, dynamics of individual Pol II clusters in live-cell nuclei has not been measured directly, prohibiting in-depth understanding of their working mechanisms. In this work, we studied the dynamics of Pol II clustering using Bayesian nanoscopy in live mammalian cell nuclei. With 50 nm spatial resolution and 4 s temporal resolution, Bayesian nanoscopy allows direct observation of the assembly and disassembly dynamics of individual Pol II clusters. The results not only provide quantifications of Pol II clusters but also shed light on the understanding of cluster formation and regulation. Our study suggests that transcription factories form on-demand and recruit Pol II molecules in their pre-elongation phase. The assembly and disassembly of individual Pol II clusters take place asynchronously. Overall, the methods developed herein are also applicable to studying a wide realm of real-time nanometer-scale nuclear processes in live cells.

T cells that trigger acute experimental autoimmune encephalomyelitis also mediate subsequent disease relapses and predominantly produce IL-17.

Earlier studies showed that donor T cells that initiated a murine adoptive EAE persisted in the CNS of the recipients throughout the subsequent relapsing cycles. To clarify the functions of the persistent donor T cells in EAE relapsing disease, anti-Thy-1 antibodies were used to deplete these cells. Results showed that such treatment abrogated subsequent relapsing cycles in these animals. In addition, it was evident that a shift in cytokine profile occurred during acute and relapsing disease phases. These results unambiguously support the appropriateness of targeting T cells with specificity for the priming antigen in design of therapeutic approaches for MS.