Missense mutations in the central domains of cardiac myosin binding protein-C and their potential contribution to hypertrophic cardiomyopathy.

Myosin binding protein-C (MyBP-C) is a multidomain protein that regulates muscle contraction. Mutations in MYBPC3, the gene encoding for the cardiac variant (henceforth called cMyBP-C), are amongst the most frequent causes of hypertrophic cardiomyopathy. Most mutations lead to a truncated version of cMyBP-C, which is most likely unstable. However, missense mutations have also been reported, which tend to cluster in the central domains of the cMyBP-C molecule. This suggests that these central domains are more than just a passive spacer between the better characterized N- and C-terminal domains. Here, we investigated the potential impact of four different missense mutations, E542Q, G596R, N755K, and R820Q, which are spread over the domains C3 to C6, on the function of MyBP-C on both the isolated protein level and in cardiomyocytes in vitro. Effect on domain stability, interaction with thin filaments, binding to myosin, and subcellular localization behavior were assessed. Our studies show that these missense mutations result in slightly different phenotypes at the molecular level, which are mutation specific. The expected functional readout of each mutation provides a valid explanation for why cMyBP-C fails to work as a brake in the regulation of muscle contraction, which eventually results in a hypertrophic cardiomyopathy phenotype. We conclude that missense mutations in cMyBP-C must be evaluated in context of their domain localization, their effect on interaction with thin filaments and myosin, and their effect on protein stability to explain how they lead to disease.

Fluorescent protein-based Zn2+ sensors reveal distinct responses of aerobic and anaerobic Escherichia coli cultures to excess Zn2.

Zinc ions are required by all known organisms. Maintaining zinc homeostasis by preventing toxic overload while ensuring sufficient acquisition for cellular functions is crucial for survival and growth of bacteria. Bacteria, however, frequently encounter and must survive in various environments. During infection in host animals, for example, bacteria are exposed to acidic conditions in the stomach and anaerobic conditions in the intestines, but the effects of oxygen on zinc homeostasis in Escherichia coli have not been well-studied. Previously, we reported a flavin-binding fluorescent protein-based zinc sensor, CreiLOVN41C, which can respond to changes in labile Zn2+ levels in bacteria under both aerobic and anaerobic conditions. Here, we combined the use of CreiLOVN41C with established oxygen-dependent fluorescent protein-based sensors, inductively coupled plasma-mass spectrometry, and growth curves to evaluate how oxygen levels affect zinc uptake in E. coli. Inductively coupled plasma-mass spectrometry results showed that cells grown aerobically with added zinc acquired more zinc, but no additional zinc was accumulated when cells were grown anaerobically. Using oxygen-independent CreiLOVN41C and the oxygen-dependent ZapCY series of sensors, intracellular labile zinc was detected in E. coli grown with varied zinc under varied conditions. Although little to no endogenous zinc was detected by any sensor in E. coli cells grown with up to 2 mM added zinc, CreiLOVN41C revealed that when Zn2+ was added and detected by cells in real-time, anaerobic cells required more Zn2+ to similarly saturate the sensor. Overall, this work reveals that zinc uptake in E. coli is impacted by oxygen levels during cell growth.

Improvement of a low-cost protocol for a simultaneous comparative evaluation of hydrolytic activity between sessile and planktonic cells: Candida albicans as a study model.

Candida albicans is often implicated in nosocomial infections with fatal consequences. Its virulence is contributed to hydrolytic enzymes and biofilm formation. Previous research focused on studying these virulence factors individually. Therefore, this study aimed to investigate the impact of biofilm formation on the hydrolytic activity using an adapted low-cost method. Eleven strains of C. albicans were used. The biofilms were formed on pre-treated silicone discs using 24-well plates and then deposited on the appropriate agar to test each enzyme, while the planktonic cells were conventionally seeded. Biofilms were analysed using Raman spectroscopy, fluorescent and scanning electron microscopy. The adapted method provided an evaluation of hydrolytic enzymes activity in C. albicans biofilm and showed that sessile cells had a higher phospholipase and proteinase activities compared with planktonic cells. These findings were supported by spectroscopic and microscopic analyses, which provided valuable insights into the virulence mechanisms of C. albicans during biofilm formation.

Digital live imaging of intraoperative electrocochleography during cochlear implantation: the first 50 patients.

INTRODUCTION: Real-time visualization of intraoperative electrocochleography (ECochG) potentials via a digital microscope during cochlear implantation can provide direct feedback during electrode insertion. The aim of this prospective, randomized study of 50 patients was to obtain long-term data with a focus on residual hearing preservation and speech understanding. MATERIAL AND METHODS: Cochlear implantations were performed in 50 patients (26 female, 24 male) with residual hearing using a digital microscope. Patients were randomized into two groups. Intraoperative ECochG potentials were either displayed directly in the surgeon's field of view (picture-in-picture display, PiP) or not directly in the field of view (without picture-in-picture display, without PiP). Residual hearing preservation and speech comprehension were recorded within a 1-year follow-up period, compared between groups (PiP versus without PiP) and to a control group of 26 patients implanted without ECochG. RESULTS: Mean insertion time was significantly longer in the picture-in-picture group (p = 0.025). Residual hearing preservation after 6 weeks at 250 Hz was significantly better in the picture-in-picture group (p = 0.017). After one year, 76% of patients showed residual hearing in the picture-in-picture group (62% without picture-in-picture technique, p = n.s.). Use of the picture-in-picture technique resulted in better long-term pure tone residual hearing preservation at 250, 500, and 1000 Hz. Speech intelligibility improved by 46% in the picture-in-picture group (38% without picture-in-picture). DISCUSSION: This study is the first to describe long-term results in a large cohort of cochlear implant patients in whom digital visualization of intraoperative ECochG was used. Our results show that visualization of intraoperative ECochG has a positive effect on residual hearing preservation.

Evaluating HIV-1 Infectivity and Virion Maturation across Varied Producer Cells with a Novel FRET-Based Detection and Quantification Assay.

The maturation of HIV-1 virions is a crucial process in viral replication. Although T-cells are a primary source of virus production, much of our understanding of virion maturation comes from studies using the HEK293T human embryonic kidney cell line. Notably, there is a lack of comparative analyses between T-cells and HEK293T cells in terms of virion maturation efficiency in existing literature. We previously developed an advanced virion visualization system based on the FRET principle, enabling the effective distinction between immature and mature virions via fluorescence microscopy. In this study, we utilized pseudotyped, single-round infectious viruses tagged with FRET labels (HIV-1 Gag-iFRET∆Env) derived from Jurkat (a human T-lymphocyte cell line) and HEK293T cells to evaluate their virion maturation rates. HEK293T-derived virions demonstrated a maturity rate of 81.79%, consistent with other studies and our previous findings. However, virions originating from Jurkat cells demonstrated a significantly reduced maturation rate of 68.67% (p < 0.0001). Correspondingly, viruses produced from Jurkat cells exhibited significantly reduced infectivity compared to those derived from HEK293T cells, with the relative infectivity measured at 65.3%. This finding is consistent with the observed relative maturation rate of viruses produced by Jurkat cells. These findings suggest that initiation of virion maturation directly correlates with viral infectivity. Our observation highlights the dynamic nature of virus-host interactions and their implications for virion production and infectivity.

Structural Description of Chiral E-Tiling DNA Nanotubes with the Chiral Indices (n,m) and Handedness Defined by Microscopic Imaging.

In structural DNA nanotechnology, E-tiling DNA nanotubes are evidenced to be homogeneous in diameter and thus have great potential in biomedical applications such as cellular transport and communication, transmembrane ion/molecule channeling, and drug delivery. However, a precise structural description of chiral DNA nanotubes with chiral parameters was lacking, thus greatly hindering their application breadth and depth, until we recently raised and partly solved this problem. In this perspective, we summarize recent progress in defining the chiral indices and handedness of E-tiling DNA nanotubes by microscopic imaging, especially atomic force microscopy (AFM) imaging. Such a detailed understanding of the chiral structures of E-tiling DNA nanotubes will be very helpful in the future, on the one hand for engineering DNA nanostructures precisely, and, on the other, for realizing specific physicochemical properties and biological functions successfully.

A sample-preparation-free, point-of-care testing system for in situ detection of bovine mastitis.

We present a highly integrated point-of-care testing (POCT) device capable of immediately and accurately screening bovine mastitis infection based on somatic cell counting (SCC). The system primarily consists of a homemade cell-counting chamber and a miniature fluorescent microscope. The cell-counting chamber is pre-embedded with acridine orange (AO) in advance, which is simple and practical. And then SCC is directly identified by microscopic imaging analysis to evaluate the bovine mastitis infection. Only 4 µL of raw bovine milk is required for a simple sample testing and accurate SCC. The entire assay process from sampling to result in presentation is completed quickly within 6 min, enabling instant "sample-in and answer-out." Under laboratory conditions, we mixed bovine leukocyte suspension with whole milk and achieved a detection limit as low as 2.12 × 104 cells/mL on the system, which is capable of screening various types of clinical standards of bovine milk. The fitting degrees of the proposed POCT system with manual fluorescence microscopy were generally consistent (R2 > 0.99). As a proof of concept, four fresh milk samples were used in the test. The average accuracy of somatic cell counts was 98.0%, which was able to successfully differentiate diseased cows from healthy ones. The POCT system is user-friendly and low-cost, making it a potential tool for on-site diagnosis of bovine mastitis in resource-limited areas.

Study on Rapid Detection of Pesticide Residues in Shanghaiqing Based on Analyzing Near-Infrared Microscopic Images.

Aiming at guiding agricultural producers to harvest crops at an appropriate time and ensuring the pesticide residue does not exceed the maximum limit, the present work proposed a method of detecting pesticide residue rapidly by analyzing near-infrared microscopic images of the leaves of Shanghaiqing (Brassica rapa), a type of Chinese cabbage with computer vision technology. After image pre-processing and feature extraction, the pattern recognition methods of K nearest neighbors (KNN), naïve Bayes, support vector machine (SVM), and back propagation artificial neural network (BP-ANN) were applied to assess whether Shanghaiqing is sprayed with pesticides. The SVM method with linear or RBF kernel provides the highest recognition accuracy of 96.96% for the samples sprayed with trichlorfon at a concentration of 1 g/L. The SVM method with RBF kernel has the highest recognition accuracy of 79.16~84.37% for the samples sprayed with cypermethrin at a concentration of 0.1 g/L. The investigation on the SVM classification models built on the samples sprayed with cypermethrin at different concentrations shows that the accuracy of the models increases with the pesticide concentrations. In addition, the relationship between the concentration of the cypermethrin sprayed and the image features was established by multiple regression to estimate the initial pesticide concentration on the Shanghaiqing leaves. A pesticide degradation equation was established on the basis of the first-order kinetic equation. The time for pesticides concentration to decrease to an acceptable level can be calculated on the basis of the degradation equation and the initial pesticide concentration. The present work provides a feasible way to rapidly detect pesticide residue on Shanghaiqing by means of NIR microscopic image technique. The methodology laid out in this research can be used as a reference for the pesticide detection of other types of vegetables.

Complement receptor 3 mediates both sinking phagocytosis and phagocytic cup formation via distinct mechanisms.

A long-standing hypothesis is that complement receptors (CRs), especially CR3, mediate sinking phagocytosis, but evidence is lacking. Alternatively, CRs have been reported to induce membrane ruffles or phagocytic cups, akin to those induced by Fcγ receptors (FcγRs), but the details of these events are unclear. Here we used real-time 3D imaging and knockout mouse models to clarify how particles (human red blood cells) are internalized by resident peritoneal F4/80+ cells (macrophages) via CRs and/or FcγRs. We first show that FcγRs mediate highly efficient, rapid (2-3 min) phagocytic cup formation, which is completely abolished by deletion or mutation of the FcR γ-chain or conditional deletion of the signal transducer Syk. FcγR-mediated phagocytic cups robustly arise from any point of cell-particle contact, including filopodia. In the absence of CR3, FcγR-mediated phagocytic cups exhibit delayed closure and become aberrantly elongated. Independent of FcgRs, CR3 mediates sporadic ingestion of complement-opsonized particles by rapid phagocytic cup-like structures, typically emanating from membrane ruffles and largely prevented by deletion of the immunoreceptor tyrosine-based activation motif (ITAM) adaptors FcR γ-chain and DAP12 or Syk. Deletion of ITAM adaptors or Syk clearly revealed that there is a slow (10-25 min) sinking mode of phagocytosis via a restricted orifice. In summary, we show that (1) CR3 indeed mediates a slow sinking mode of phagocytosis, which is accentuated by deletion of ITAM adaptors or Syk, (2) CR3 induces phagocytic cup-like structures, driven by ITAM adaptors and Syk, and (3) CR3 is involved in forming and closing FcγR-mediated phagocytic cups.

A fluorescent reporter system enables spatiotemporal analysis of host cell modification during herpes simplex virus-1 replication.

Herpesviruses are large and complex viruses that have a long history of coevolution with their host species. One important factor in the virus-host interaction is the alteration of intracellular morphology during viral replication with critical implications for viral assembly. However, the details of this remodeling event are not well understood, in part because insufficient tools are available to deconstruct this highly heterogeneous process. To provide an accurate and reliable method of investigating the spatiotemporal dynamics of virus-induced changes to cellular architecture, we constructed a dual-fluorescent reporter virus that enabled us to classify four distinct stages in the infection cycle of herpes simplex virus-1 at the single cell level. This timestamping method can accurately track the infection cycle across a wide range of multiplicities of infection. We used high-resolution fluorescence microscopy analysis of cellular structures in live and fixed cells in concert with our reporter virus to generate a detailed and chronological overview of the spatial and temporal reorganization during viral replication. The highly orchestrated and striking relocation of many organelles around the compartments of secondary envelopment during transition from early to late gene expression suggests that the reshaping of these compartments is essential for virus assembly. We furthermore find that accumulation of HSV-1 capsids in the cytoplasm is accompanied by fragmentation of the Golgi apparatus with potential impact on the late steps of viral assembly. We anticipate that in the future similar tools can be systematically applied for the systems-level analysis of intracellular morphology during replication of other viruses.

Chemokine receptor CXCR4 oligomerization is disrupted selectively by the antagonist ligand IT1t.

CXCR4, a member of the family of chemokine-activated G protein-coupled receptors, is widely expressed in immune response cells. It is involved in both cancer development and progression as well as viral infection, notably by HIV-1. A variety of methods, including structural information, have suggested that the receptor may exist as a dimer or an oligomer. However, the mechanistic details surrounding receptor oligomerization and its potential dynamic regulation remain unclear. Using both biochemical and biophysical means, we confirm that CXCR4 can exist as a mixture of monomers, dimers, and higher-order oligomers in cell membranes and show that oligomeric structure becomes more complex as receptor expression levels increase. Mutations of CXCR4 residues located at a putative dimerization interface result in monomerization of the receptor. Additionally, binding of the CXCR4 antagonist IT1t-a small drug-like isothiourea derivative-rapidly destabilizes the oligomeric structure, whereas AMD3100, another well-characterized CXCR4 antagonist, does not. Although a mutation that regulates constitutive activity of CXCR4 also results in monomerization of the receptor, binding of IT1t to this variant promotes receptor dimerization. These results provide novel insights into the basal organization of CXCR4 and how antagonist ligands of different chemotypes differentially regulate its oligomerization state.

Advances in Molecular and Microscale Characterization of Soil Organic Matter: Current Limitations and Future Prospects.

Soil organic matter (SOM) comprises a continuum of organic materials from granular organic debris to small organic molecules and contains more organic carbon than global vegetation and the atmosphere combined. It has remarkable effects on soil ecological functions and the global carbon cycle as well as the fate of pollutants in the terrestrial ecosystem. Therefore, characterization of SOM is an important topic in soil science, ecology, and environmental science. Chemical complexity and spatial heterogeneity are by far the two biggest challenges to our understanding of SOM. Recent developments in analytical techniques and methods provide the opportunity to reveal SOM composition at the molecular level and to observe its distribution in soils at micro- and nanoscales, which have greatly improved our understanding of SOM. This paper reviews the outstanding advances in SOM characterization regarding these two issues from target and nontarget analyses comprising molecular marker analysis, ultrahigh-resolution mass spectrometry analysis, and in situ microscopic imaging techniques such as synchrotron-based spectromicroscopy, nanoscale secondary ion mass spectrometry, and emerging electron and optical microscopic imaging techniques. However, current techniques and methods remain far from unlocking the unknown properties of SOM. We systematically point out the limitations of the current technologies and outline the future prospects for comprehensive characterization of SOM at the molecular level and micro- and nanoscales, paying particular attention to issues of environmental concern.

Biofouling of reverse osmosis membranes: assessment by surface-enhanced Raman spectroscopy and microscopic imaging.

The decline in the performance of spiral-wound reverse osmosis (SWRO) membranes is frequently due to biofouling. This study focus on qualitative and quantitative diagnosis of SWRO membrane biofouling. Bacterial counts on the different surfaces of the fouled membranes were carried out. Surface enhanced Raman spectroscopy (SERS) was performed to highlight clogging materials as well as their natures and identity. The topography of the fouled membranes and the structures of biofilms were visualized by fluorescence microscopy (FM) and scanning electron microscopy (SEM). The results indicated the presence of bacteria in the different SWRO membrane areas. Those strongly adhered were significantly higher than those weakly. It varied between 26 × 105 and 262 × 105 CFU m-2. However, SERS mapping showed different fouling levels and the thickness of the fouling layer was 5 µm. Microscopic imaging revealed biotic and abiotic deposits. These data can together allow better management of the seawater desalination process.

Intelligent Medical IoT-Enabled Automated Microscopic Image Diagnosis of Acute Blood Cancers.

Blood cancer, or leukemia, has a negative impact on the blood and/or bone marrow of children and adults. Acute lymphocytic leukemia (ALL) and acute myeloid leukemia (AML) are two sub-types of acute leukemia. The Internet of Medical Things (IoMT) and artificial intelligence have allowed for the development of advanced technologies to assist in recently introduced medical procedures. Hence, in this paper, we propose a new intelligent IoMT framework for the automated classification of acute leukemias using microscopic blood images. The workflow of our proposed framework includes three main stages, as follows. First, blood samples are collected by wireless digital microscopy and sent to a cloud server. Second, the cloud server carries out automatic identification of the blood conditions-either leukemias or healthy-utilizing our developed generative adversarial network (GAN) classifier. Finally, the classification results are sent to a hematologist for medical approval. The developed GAN classifier was successfully evaluated on two public data sets: ALL-IDB and ASH image bank. It achieved the best accuracy scores of 98.67% for binary classification (ALL or healthy) and 95.5% for multi-class classification (ALL, AML, and normal blood cells), when compared with existing state-of-the-art methods. The results of this study demonstrate the feasibility of our proposed IoMT framework for automated diagnosis of acute leukemia tests. Clinical realization of this blood diagnosis system is our future work.

In vivo two-photon microscopy reveals the contribution of Sox9+ cell to kidney regeneration in a mouse model with extracellular vesicle treatment.

Mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) have been shown to stimulate regeneration in the treatment of kidney injury. Renal regeneration is also thought to be stimulated by the activation of Sox9+ cells. However, whether and how the activation mechanisms underlying EV treatment and Sox9+ cell-dependent regeneration intersect is unclear. We reasoned that a high-resolution imaging platform in living animals could help to untangle this system. To test this idea, we first applied EVs derived from human placenta-derived MSCs (hP-MSCs) to a Sox9-CreERT2; R26mTmG transgenic mouse model of acute kidney injury (AKI). Then, we developed an abdominal imaging window in the mouse and tracked the Sox9+ cells in the inducible Sox9-Cre transgenic mice via in vivo lineage tracing with two-photon intravital microscopy. Our results demonstrated that EVs can travel to the injured kidneys post intravenous injection as visualized by Gaussia luciferase imaging and markedly increase the activation of Sox9+ cells. Moreover, the two-photon living imaging of lineage-labeled Sox9+ cells showed that the EVs promoted the expansion of Sox9+ cells in kidneys post AKI. Histological staining results confirmed that the descendants of Sox9+ cells contributed to nephric tubule regeneration which significantly ameliorated the renal function after AKI. In summary, intravital lineage tracing with two-photon microscopy through an embedded abdominal imaging window provides a practical strategy to investigate the beneficial functions and to clarify the mechanisms of regenerative therapies in AKI.

The genetic Ca2+ sensor GCaMP3 reveals multiple Ca2+ stores differentially coupled to Ca2+ entry in the human malaria parasite Plasmodium falciparum.

Cytosolic Ca2+ regulates multiple steps in the host-cell invasion, growth, proliferation, and egress of blood-stage Plasmodium falciparum, yet our understanding of Ca2+ signaling in this endemic malaria parasite is incomplete. By using a newly generated transgenic line of P. falciparum (PfGCaMP3) that expresses constitutively the genetically encoded Ca2+ indicator GCaMP3, we have investigated the dynamics of Ca2+ release and influx elicited by inhibitors of the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase pumps, cyclopiazonic acid (CPA), and thapsigargin (Thg). Here we show that in isolated trophozoite phase parasites: (i) both CPA and Thg release Ca2+ from intracellular stores in P. falciparum parasites; (ii) Thg is able to induce Ca2+ release from an intracellular compartment insensitive to CPA; (iii) only Thg is able to activate Ca2+ influx from extracellular media, through a mechanism resembling store-operated Ca2+ entry, typical of mammalian cells; and (iv) the Thg-sensitive Ca2+ pool is unaffected by collapsing the mitochondria membrane potential with the uncoupler carbonyl cyanide m-chlorophenyl hydrazone or the release of acidic Ca2+ stores with nigericin. These data suggest the presence of two Ca2+ pools in P. falciparum with differential sensitivity to the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase pump inhibitors, and only the release of the Thg-sensitive Ca2+ store induces Ca2+ influx. Activation of the store-operated Ca2+ entry-like Ca2+ influx may be relevant for controlling processes such as parasite invasion, egress, and development mediated by kinases, phosphatases, and proteases that rely on Ca2+ levels for their activation.

The BBSome assembly is spatially controlled by BBS1 and BBS4 in human cells.

Bardet-Biedl syndrome (BBS) is a pleiotropic ciliopathy caused by dysfunction of primary cilia. More than half of BBS patients carry mutations in one of eight genes encoding for subunits of a protein complex, the BBSome, which mediates trafficking of ciliary cargoes. In this study, we elucidated the mechanisms of the BBSome assembly in living cells and how this process is spatially regulated. We generated a large library of human cell lines deficient in a particular BBSome subunit and expressing another subunit tagged with a fluorescent protein. We analyzed these cell lines utilizing biochemical assays, conventional and expansion microscopy, and quantitative fluorescence microscopy techniques: fluorescence recovery after photobleaching and fluorescence correlation spectroscopy. Our data revealed that the BBSome formation is a sequential process. We show that the pre-BBSome is nucleated by BBS4 and assembled at pericentriolar satellites, followed by the translocation of the BBSome into the ciliary base mediated by BBS1. Our results provide a framework for elucidating how BBS-causative mutations interfere with the biogenesis of the BBSome.

Fluorescence assay for simultaneous quantification of CFTR ion-channel function and plasma membrane proximity.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a plasma membrane anion channel that plays a key role in controlling transepithelial fluid movement. Excessive activation results in intestinal fluid loss during secretory diarrheas, whereas CFTR mutations underlie cystic fibrosis (CF). Anion permeability depends both on how well CFTR channels work (permeation/gating) and on how many are present at the membrane. Recently, treatments with two drug classes targeting CFTR-one boosting ion-channel function (potentiators) and the other increasing plasma membrane density (correctors)-have provided significant health benefits to CF patients. Here, we present an image-based fluorescence assay that can rapidly and simultaneously estimate both CFTR ion-channel function and the protein's proximity to the membrane. We monitor F508del-CFTR, the most common CF-causing variant, and confirm rescue by low temperature, CFTR-targeting drugs and second-site revertant mutation R1070W. In addition, we characterize a panel of 62 CF-causing mutations. Our measurements correlate well with published data (electrophysiology and biochemistry), further confirming validity of the assay. Finally, we profile effects of acute treatment with approved potentiator drug VX-770 on the rare-mutation panel. Mapping the potentiation profile on CFTR structures raises mechanistic hypotheses on drug action, suggesting that VX-770 might allow an open-channel conformation with an alternative arrangement of domain interfaces. The assay is a valuable tool for investigation of CFTR molecular mechanisms, allowing accurate inferences on gating/permeation. In addition, by providing a two-dimensional characterization of the CFTR protein, it could better inform development of single-drug and precision therapies addressing the root cause of CF disease.

Domain architecture divergence leads to functional divergence in binding and catalytic domains of bacterial and fungal cellobiohydrolases.

Cellobiohydrolases directly convert crystalline cellulose into cellobiose and are of biotechnological interest to achieve efficient biomass utilization. As a result, much research in the field has focused on identifying cellobiohydrolases that are very fast. Cellobiohydrolase A from the bacterium Cellulomonas fimi (CfCel6B) and cellobiohydrolase II from the fungus Trichoderma reesei (TrCel6A) have similar catalytic domains (CDs) and show similar hydrolytic activity. However, TrCel6A and CfCel6B have different cellulose-binding domains (CBDs) and linkers: TrCel6A has a glycosylated peptide linker, whereas CfCel6B's linker consists of three fibronectin type 3 domains. We previously found that TrCel6A's linker plays an important role in increasing the binding rate constant to crystalline cellulose. However, it was not clear whether CfCel6B's linker has similar function. Here we analyze kinetic parameters of CfCel6B using single-molecule fluorescence imaging to compare CfCel6B and TrCel6A. We find that CBD is important for initial binding of CfCel6B, but the contribution of the linker to the binding rate constant or to the dissociation rate constant is minor. The crystal structure of the CfCel6B CD showed longer loops at the entrance and exit of the substrate-binding tunnel compared with TrCel6A CD, which results in higher processivity. Furthermore, CfCel6B CD showed not only fast surface diffusion but also slow processive movement, which is not observed in TrCel6A CD. Combined with the results of a phylogenetic tree analysis, we propose that bacterial cellobiohydrolases are designed to degrade crystalline cellulose using high-affinity CBD and high-processivity CD.

AggreCount: an unbiased image analysis tool for identifying and quantifying cellular aggregates in a spatially defined manner.

Protein quality control is maintained by a number of integrated cellular pathways that monitor the folding and functionality of the cellular proteome. Defects in these pathways lead to the accumulation of misfolded or faulty proteins that may become insoluble and aggregate over time. Protein aggregates significantly contribute to the development of a number of human diseases such as amyotrophic lateral sclerosis, Huntington's disease, and Alzheimer's disease. In vitro, imaging-based, cellular studies have defined key biomolecular components that recognize and clear aggregates; however, no unifying method is available to quantify cellular aggregates, limiting our ability to reproducibly and accurately quantify these structures. Here we describe an ImageJ macro called AggreCount to identify and measure protein aggregates in cells. AggreCount is designed to be intuitive, easy to use, and customizable for different types of aggregates observed in cells. Minimal experience in coding is required to utilize the script. Based on a user-defined image, AggreCount will report a number of metrics: (i) total number of cellular aggregates, (ii) percentage of cells with aggregates, (iii) aggregates per cell, (iv) area of aggregates, and (v) localization of aggregates (cytosol, perinuclear, or nuclear). A data table of aggregate information on a per cell basis, as well as a summary table, is provided for further data analysis. We demonstrate the versatility of AggreCount by analyzing a number of different cellular aggregates including aggresomes, stress granules, and inclusion bodies caused by huntingtin polyglutamine expansion.