DNA Framework-Guided Self-Limiting Aggregation for Highly Luminescent Metal Cluster Nanoaggregates.

The photoluminescent properties of atomically precise metal nanoclusters (MCs) have garnered significant attention in the fields of chemical sensing and biological imaging. However, the limited brightness of single-component nanoclusters hinders their practical applications, and the conventional ligand engineering approaches have proven insufficient in enhancing the emission efficiency of MCs. Here, we present a DNA framework-guided strategy to prepare highly luminescent metal cluster nanoaggregates. Our approach involves an amphiphilic DNA framework comprising a hydrophobic alkyl core and a rigid DNA framework shell, serving as a nucleation site and providing well-defined nanoconfinements for the self-limiting aggregation of MCs. Through this method, we successfully produced homogeneous MC nanoaggregates (10.1 ± 1.2 nm) with remarkable nanoscale precision. Notably, this strategy proves adaptable to various MCs, leading to a substantial enhancement in emission and quantum yield, up to 3011- and 87-fold, respectively. Furthermore, our investigation using total internal reflection fluorescence microscopy at the single-particle level uncovered a more uniform photon number distribution and higher photostability for MC nanoaggregates compared to template-free counterparts. This DNA-templating strategy introduces a conceptually innovative approach for studying the photoluminescent properties of aggregates with nanoscale precision and holds promise for constructing highly luminescent MC nanoparticles for diverse applications.

Chlorovirus PBCV-1 protein A064R has three of the transferase activities necessary to synthesize its capsid protein N-linked glycans.

Paramecium bursaria chlorella virus-1 (PBCV-1) is a large double-stranded DNA (dsDNA) virus that infects the unicellular green alga Chlorella variabilis NC64A. Unlike many other viruses, PBCV-1 encodes most, if not all, of the enzymes involved in the synthesis of the glycans attached to its major capsid protein. Importantly, these glycans differ from those reported from the three domains of life in terms of structure and asparagine location in the sequon of the protein. Previous data collected from 20 PBCV-1 spontaneous mutants (or antigenic variants) suggested that the a064r gene encodes a glycosyltransferase (GT) with three domains, each with a different function. Here, we demonstrate that: domain 1 is a ß-l-rhamnosyltransferase; domain 2 is an α-l-rhamnosyltransferase resembling only bacterial proteins of unknown function, and domain 3 is a methyltransferase that methylates the C-2 hydroxyl group of the terminal α-l-rhamnose (Rha) unit. We also establish that methylation of the C-3 hydroxyl group of the terminal α-l-Rha is achieved by another virus-encoded protein A061L, which requires an O-2 methylated substrate. This study, thus, identifies two of the glycosyltransferase activities involved in the synthesis of the N-glycan of the viral major capsid protein in PBCV-1 and establishes that a single protein A064R possesses the three activities needed to synthetize the 2-OMe-α-l-Rha-(1→2)-ß-l-Rha fragment. Remarkably, this fragment can be attached to any xylose unit.

The N-glycan structures of the antigenic variants of chlorovirus PBCV-1 major capsid protein help to identify the virus-encoded glycosyltransferases.

The chlorovirus Paramecium bursaria chlorella virus 1 (PBCV-1) is a large dsDNA virus that infects the microalga Chlorella variabilis NC64A. Unlike most other viruses, PBCV-1 encodes most, if not all, of the machinery required to glycosylate its major capsid protein (MCP). The structures of the four N-linked glycans from the PBCV-1 MCP consist of nonasaccharides, and similar glycans are not found elsewhere in the three domains of life. Here, we identified the roles of three virus-encoded glycosyltransferases (GTs) that have four distinct GT activities in glycan synthesis. Two of the three GTs were previously annotated as GTs, but the third GT was identified in this study. We determined the GT functions by comparing the WT glycan structures from PBCV-1 with those from a set of PBCV-1 spontaneous GT gene mutants resulting in antigenic variants having truncated glycan structures. According to our working model, the virus gene a064r encodes a GT with three domains: domain 1 has a ß-l-rhamnosyltransferase activity, domain 2 has an α-l-rhamnosyltransferase activity, and domain 3 is a methyltransferase that decorates two positions in the terminal α-l-rhamnose (Rha) unit. The a075l gene encodes a ß-xylosyltransferase that attaches the distal d-xylose (Xyl) unit to the l-fucose (Fuc) that is part of the conserved N-glycan core region. Last, gene a071r encodes a GT that is involved in the attachment of a semiconserved element, α-d-Rha, to the same l-Fuc in the core region. Our results uncover GT activities that assemble four of the nine residues of the PBCV-1 MCP N-glycans.

Synthesis of the Highly Branched Hexasaccharide Core of Chlorella Virus N-Linked Glycans.

Chlorella viruses produce N-linked glycoproteins with carbohydrate moieties that differ in structure from all other N-linked glycans. In addition, unlike most viruses, these organisms do not hijack the biosynthetic machinery of the host to make glycocoproteins; instead, they produce their own carbohydrate-processing enzymes. A better understanding of the function and assembly of these fascinating and structurally-unprecedented glycans requires access to probe molecules. This work describes the first synthesis of a chlorella virus N-linked glycan, a highly branched hexasaccharide that contains the pentasaccharide present in all of the >15 structures reported to date. The target molecule includes a glucosyl-asparagine linkage and a "hyperbranched" fucose residue in which all of the hydroxyl groups are glycosylated. Both convergent and linear approaches were investigated with the latter being successful in providing the target in 16 steps and 13 % overall yield.

Synthesis of a Highly Branched Nonasaccharide Chlorella Virus N-Glycan Using a "Counterclockwise" Assembly Approach.

Chloroviruses produce a capsid protein containing N-linked glycans differing in structure from those found in all other organisms. These species feature a core "hyper-branched" fucose residue in which every hydroxyl group is glycosylated. We describe the synthesis of a nonasaccharide from Paramecium bursaria chlorella virus 1, one of most complex chlorovirus N-glycans reported, using a "counterclockwise" strategy involving the sequential addition of trisaccharide, disaccharide, and monosaccharide motifs to a trisaccharide containing the core fucose residue.

The miR-200 family as prognostic markers in clear cell renal cell carcinoma.

OBJECTIVES: microRNAs (miRNAs) are small noncoding RNAs that regulate gene expression by mRNA cleavage or translational repression. The miR-200 family is involved in the regulation of various tumor biologic processes including apoptosis, proliferation, invasion, and metastasis. They function mainly as tumor suppressors. In this study, we aim to validate the prognostic significance of miR-200 family using large cohort of primary clear cell renal cell carcinoma (ccRCC) and matched normal tissue and to explore the role of miR-200 family in RCC pathogenesis and progression. MATERIALS AND METHODS: We analyzed the expression of 3 members of the miR-200 family; miR-141, miR-200b, and miR-200c, between primary ccRCC, matched normal renal tissues, and nonmatched metastatic RCC. We compared clinicopathologic parameter including disease-free survival to miR-200 family expression. Additionally, we validated our results using The Cancer Genome Atlas dataset. We explored functional role of these miRNAs by bioinformatics analyses. RESULTS AND CONCLUSIONS: Expression of miR-200 family significantly decreased in cancer compared to non-neoplastic tissues. miR-141 and miR-200b were significantly down-regulated in metastatic than primary tumors. There was statistically significant negative association between all 3 miRNAs and tumor size and stage. As binary variables, univariate analyses revealed that miR-141, miR-200b, and miR-200c-positive ccRCC patients have a statistically significant lower chance of disease-recurrence or relapse and multivariate analyses showed miR-200b and miR-200c-positive patients have longer disease-free survival. We could predict disease-free survival better when 2 or more miRNAs were used as a combination. Overall survival analysis using The Cancer Genome Atlas data revealed that miR-200b-positive patients have significantly better survival. These results suggest that miR-141, miR-200b, and miR-200c are independent prognostic markers for ccRCC. Targets of these miRNAs are associated with pathways related to cancer invasion and metastasis, including TRAIL pathway, VEGF and VEGFR signaling network, and epithelial-mesenchymal transition.

In Situ Confocal Raman Microscopy of Hydrated Early Stages of Bacterial Biofilm Formation on Various Surfaces in a Flow Cell.

Bacterial biofilms are precursors to biofouling by other microorganisms. Understanding their initiation may allow us to design better ways to inhibit them, and thus to inhibit subsequent biofouling. In this study, the ability of confocal Raman microscopy to follow the initiation of biofouling by a marine bacterium, Pseudoalteromonas sp. NCIMB 2021 (NCIMB 2021), in a flow cell, using optical and confocal Raman microscopy, was investigated. The base of the flow cell comprised a cover glass. The cell was inoculated and the bacteria attached to, and grew on, the cover glass. Bright field images and Raman spectra were collected directly from the hydrated biofilms over several days. Although macroscopically the laser had no effect on the biofilm, within the first 24 h cells migrated away from the position of the laser beam. In the absence of flow, a buildup of extracellular substances occurred at the base of the biofilm. When different coatings were applied to cover glasses before they were assembled into the flow cells, the growth rate, structure, and composition of the resulting biofilm was affected. In particular, the ratio of Resonance Raman peaks from cytochrome c (CC) in the extracellular polymeric substances, to the Raman phenylalanine (Phe) peak from protein in the bacteria, depended on both the nature of the surface and the age of the biofilm. The ratios were highest for 24 h colonies on a hydrophobic surface. Absorption of a surfactant with an ethyleneoxy chain into the hydrophobic coating created a surface similar to that given with a simple PEG coating, where bacteria grew in colonies away from the surface rather than along the surface, and CC:Phe ratios were initially low but increased at least fivefold in the first 48 h.