Heterologous Expression and Characterization of a pH-Stable Chitinase from Micromonospora aurantiaca with a Potential Application in Chitin Degradation.

To promote the bioconversion of marine chitin waste into value-added products, we expressed a novel pH-stable Micromonospora aurantiaca-derived chitinase, MaChi1, in Escherichia coli and subsequently purified, characterized, and evaluated it for its chitin-converting capacity. Our results indicated that MaChi1 is of the glycoside hydrolase (GH) family 18 with a molecular weight of approximately 57 kDa, consisting of a GH18 catalytic domain and a cellulose-binding domain. We recorded its optimal activity at pH 5.0 and 55 °C. It exhibited excellent stability in a wide pH range of 3.0-10.0. Mg2+ (5 mM), and dithiothreitol (10 mM) significantly promoted MaChi1 activity. MaChi1 exhibited broad substrate specificity and hydrolyzed chitin, chitosan, cellulose, soluble starch, and N-acetyl chitooligosaccharides with polymerization degrees ranging from three to six. Moreover, MaChi1 exhibited an endo-type cleavage pattern, and it could efficiently convert colloidal chitin into N-acetyl-D-glucosamine (GlcNAc) and (GlcNAc)2 with yields of 227.2 and 505.9 mg/g chitin, respectively. Its high chitin-degrading capacity and exceptional pH tolerance makes it a promising tool with potential applications in chitin waste treatment and bioactive oligosaccharide production.

Particle fusion of super-resolution data reveals the unit structure of Nup96 in Nuclear Pore Complex.

Single molecule localization microscopy offers resolution nearly down to the molecular level with specific molecular labelling, and is thereby a promising tool for structural biology. In practice, however, the actual value to this field is limited primarily by incomplete fluorescent labelling of the structure. This missing information can be completed by merging information from many structurally identical particles in a particle fusion approach similar to cryo-EM single-particle analysis. In this paper, we present a data analysis of particle fusion results of fluorescently labelled Nup96 nucleoporins in the Nuclear Pore Complex to show that Nup96 occurs in a spatial arrangement of two rings of 8 units with two Nup96 copies per unit giving a total of 32 Nup96 copies per pore. We use Artificial Intelligence assisted modeling in Alphafold to extend the existing cryo-EM model of Nup96 to accurately pinpoint the positions of the fluorescent labels and show the accuracy of the match between fluorescent and cryo-EM data to be better than 3 nm in-plane and 5 nm out-of-plane.

A Noise-Tolerating Gene Association Network Uncovering an Oncogenic Regulatory Motif in Lymphoma Transcriptomics.

In cancer genomics research, gene expressions provide clues to gene regulations implicating patients' risk of survival. Gene expressions, however, fluctuate due to noises arising internally and externally, making their use to infer gene associations, hence regulation mechanisms, problematic. Here, we develop a new regression approach to model gene association networks while considering uncertain biological noises. In a series of simulation experiments accounting for varying levels of biological noises, the new method was shown to be robust and perform better than conventional regression methods, as judged by a number of statistical measures on unbiasedness, consistency and accuracy. Application to infer gene associations in germinal-center B cells led to the discovery of a three-by-two regulatory motif gene expression and a three-gene prognostic signature for diffuse large B-cell lymphoma.

Clathrin coats partially preassemble and subsequently bend during endocytosis.

Eukaryotic cells use clathrin-mediated endocytosis to take up a large range of extracellular cargo. During endocytosis, a clathrin coat forms on the plasma membrane, but it remains controversial when and how it is remodeled into a spherical vesicle. Here, we use 3D superresolution microscopy to determine the precise geometry of the clathrin coat at large numbers of endocytic sites. Through pseudo-temporal sorting, we determine the average trajectory of clathrin remodeling during endocytosis. We find that clathrin coats assemble first on flat membranes to 50% of the coat area before they become rapidly and continuously bent, and this mechanism is confirmed in three cell lines. We introduce the cooperative curvature model, which is based on positive feedback for curvature generation. It accurately describes the measured shapes and dynamics of the clathrin coat and could represent a general mechanism for clathrin coat remodeling on the plasma membrane.

Nanoscale structural organization and stoichiometry of the budding yeast kinetochore.

Proper chromosome segregation is crucial for cell division. In eukaryotes, this is achieved by the kinetochore, an evolutionarily conserved multiprotein complex that physically links the DNA to spindle microtubules and takes an active role in monitoring and correcting erroneous spindle-chromosome attachments. Our mechanistic understanding of these functions and how they ensure an error-free outcome of mitosis is still limited, partly because we lack a complete understanding of the kinetochore structure in the cell. In this study, we use single-molecule localization microscopy to visualize individual kinetochore complexes in situ in budding yeast. For major kinetochore proteins, we measured their abundance and position within the metaphase kinetochore. Based on this comprehensive dataset, we propose a quantitative model of the budding yeast kinetochore. While confirming many aspects of previous reports based on bulk imaging, our results present a unifying nanoscale model of the kinetochore in budding yeast.

Maximum-likelihood model fitting for quantitative analysis of SMLM data.

Quantitative data analysis is important for any single-molecule localization microscopy (SMLM) workflow to extract biological insights from the coordinates of the single fluorophores. However, current approaches are restricted to simple geometries or require identical structures. Here, we present LocMoFit (Localization Model Fit), an open-source framework to fit an arbitrary model to localization coordinates. It extracts meaningful parameters from individual structures and can select the most suitable model. In addition to analyzing complex, heterogeneous and dynamic structures for in situ structural biology, we demonstrate how LocMoFit can assemble multi-protein distribution maps of six nuclear pore components, calculate single-particle averages without any assumption about geometry or symmetry, and perform a time-resolved reconstruction of the highly dynamic endocytic process from static snapshots. We provide extensive simulation and visualization routines to validate the robustness of LocMoFit and tutorials to enable any user to increase the information content they can extract from their SMLM data.

Global fitting for high-accuracy multi-channel single-molecule localization.

Multi-channel detection in single-molecule localization microscopy greatly increases information content for various biological applications. Here, we present globLoc, a graphics processing unit based global fitting algorithm with flexible PSF modeling and parameter sharing, to extract maximum information from multi-channel single molecule data. As signals in multi-channel data are highly correlated, globLoc links parameters such as 3D coordinates or photon counts across channels, improving localization precision and robustness. We show, both in simulations and experiments, that global fitting can substantially improve the 3D localization precision for biplane and 4Pi single-molecule localization microscopy and color assignment for ratiometric multicolor imaging.

Quantitative Data Analysis in Single-Molecule Localization Microscopy.

Super-resolution microscopy, and specifically single-molecule localization microscopy (SMLM), is becoming a transformative technology for cell biology, as it allows the study of cellular structures with nanometer resolution. Here, we review a wide range of data analyses approaches for SMLM that extract quantitative information about the distribution, size, shape, spatial organization, and stoichiometry of macromolecular complexes to guide biological interpretation. We present a case study using the nuclear pore complex as an example that highlights the power of combining complementary approaches by identifying its symmetry, ringlike structure, and protein copy number. In face of recent technical and computational advances, this review serves as a guideline for selecting appropriate analysis tools and controls to exploit the potential of SMLM for a wide range of biological questions.

Publisher Correction: Nuclear pores as versatile reference standards for quantitative superresolution microscopy.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Nuclear pores as versatile reference standards for quantitative superresolution microscopy.

Quantitative fluorescence and superresolution microscopy are often limited by insufficient data quality or artifacts. In this context, it is essential to have biologically relevant control samples to benchmark and optimize the quality of microscopes, labels and imaging conditions. Here, we exploit the stereotypic arrangement of proteins in the nuclear pore complex as in situ reference structures to characterize the performance of a variety of microscopy modalities. We created four genome edited cell lines in which we endogenously labeled the nucleoporin Nup96 with mEGFP, SNAP-tag, HaloTag or the photoconvertible fluorescent protein mMaple. We demonstrate their use (1) as three-dimensional resolution standards for calibration and quality control, (2) to quantify absolute labeling efficiencies and (3) as precise reference standards for molecular counting. These cell lines will enable the broader community to assess the quality of their microscopes and labels, and to perform quantitative, absolute measurements.

Depth-dependent PSF calibration and aberration correction for 3D single-molecule localization.

Three-dimensional single molecule localization microscopy relies on the fitting of the individual molecules with a point spread function (PSF) model. The reconstructed images often show local squeezing or expansion in z. A common cause is depth-induced aberrations in conjunction with an imperfect PSF model calibrated from beads on a coverslip, resulting in a mismatch between measured PSF and real PSF. Here, we developed a strategy for accurate z-localization in which we use the imperfect PSF model for fitting, determine the fitting errors and correct for them in a post-processing step. We present an open-source software tool and a simple experimental calibration procedure that allow retrieving accurate z-positions in any PSF engineering approach or fitting modality, even at large imaging depths.

Mathematical guide to minimize donor size in full-thickness skin grafting.

BACKGROUND: Full-thickness skin grafting is a common repair option in dermatologic surgery. Generally, a 3:1 ellipse is designed for the donor area. This results in occasional large donor site defects. A new formula to guide donor site design would potentially create smaller donor defects. OBJECTIVE: To design a mathematical formula to facilitate smaller donor incisions. METHODS: A geometric analysis was undertaken to design a practical formula to create smaller defects. This strategy was then used in a case series of 23 patients with large (average 11 cm in diameter) defects [This sentence was corrected after online publication on June 29, 2009: (average 16 x 11 cm) changed to read (average 11 cm in diameter).]. A geometrical analysis was used to design the best way to divide donor grafts to minimize tissue loss. RESULTS: A formula called for the diameter of the primary defect to be multiplied by 3/2 and 2/3 to design a donor ellipse length and width, respectively. The resulting graft was divided into two equal sizes, using a tangential acute angle. The width and the area of the donor were significantly smaller, so the donor site was repaired using a shorter and faster repair. CONCLUSION: Skin graft harvest may be accomplished using a smaller donor graft than previously described using the formula reported here. This will probably reduce surgical morbidity, time, and expense. The authors have indicated no significant interest with commercial supporters.