Theoretical framework and experimental solution for the air-water interface adsorption problem in cryoEM.

As cryogenic electron microscopy (cryoEM) gains traction in the structural biology community as a method of choice for determining atomic structures of biological complexes, it has been increasingly recognized that many complexes that behave well under conventional negative-stain electron microscopy tend to have preferential orientation, aggregate or simply mysteriously "disappear" on cryoEM grids, but the reasons for such misbehavior are not well understood, limiting systematic approaches to solving the problem. Here, we have developed a theoretical formulation that explains these observations. Our formulation predicts that all particles migrate to the air-water interface (AWI) to lower the total potential surface energy - rationalizing the use of surfactant, which is a direct solution to reducing the surface tension of the aqueous solution. By conducting cryogenic electron tomography (cryoET) with the widely-tested sample, GroEL, we demonstrate that, in a standard buffer solution, nearly all particles migrate to the AWI. Gradual reduction of the surface tension by introducing surfactants decreased the percentage of particles exposed to the surface. By conducting single-particle cryoEM, we confirm that applicable surfactants do not damage the biological complex, thus suggesting that they might offer a practical, simple, and general solution to the problem for high-resolution cryoEM. Application of this solution to a real-world AWI adsorption problem with a more challenging membrane protein, namely, the ClC-1 channel, has led to its first near-atomic structure using cryoEM.

Theoretical framework and experimental solution for the air-water interface adsorption problem in cryoEM.

As cryogenic electron microscopy (cryoEM) gains traction in the structural biology community as a method of choice for determining atomic structures of biological complexes, it has been increasingly recognized that many complexes that behave well under conventional negative-stain electron microscopy tend to have preferential orientation, aggregate or simply mysteriously "disappear" on cryoEM grids. However, the reasons for such misbehavior are not well understood, which limits systematic approaches to solving the problem. Here, we have developed a theoretical formulation that explains these observations. Our formulation predicts that all particles migrate to the air-water interface (AWI) to lower the total potential surface energy-rationalizing the use of surfactant, which is a direct solution to reduce the surface tension of the aqueous solution. By performing cryogenic electron tomography (cryoET) on the widely-tested sample, GroEL, we demonstrate that, in a standard buffer solution, nearly all particles migrate to the AWI. Gradually reducing the surface tension by introducing surfactants decreased the percentage of particles exposed to the surface. By conducting single-particle cryoEM, we confirm that suitable surfactants do not damage the biological complex, thus suggesting that they might provide a practical, simple, and general solution to the problem for high-resolution cryoEM. Applying this solution to a real-world AWI adsorption problem involving a more challenging membrane protein, namely, the ClC-1 channel, has resulted in its near-atomic structure determination using cryoEM.

Asymmetric reconstruction of mammalian reovirus reveals interactions among RNA, transcriptional factor µ2 and capsid proteins.

Mammalian reovirus (MRV) is the prototypical member of genus Orthoreovirus of family Reoviridae. However, lacking high-resolution structures of its RNA polymerase cofactor µ2 and infectious particle, limits understanding of molecular interactions among proteins and RNA, and their contributions to virion assembly and RNA transcription. Here, we report the 3.3 Å-resolution asymmetric reconstruction of transcribing MRV and in situ atomic models of its capsid proteins, the asymmetrically attached RNA-dependent RNA polymerase (RdRp) λ3, and RdRp-bound nucleoside triphosphatase µ2 with a unique RNA-binding domain. We reveal molecular interactions among virion proteins and genomic and messenger RNA. Polymerase complexes in three Spinoreovirinae subfamily members are organized with different pseudo-D3d symmetries to engage their highly diversified genomes. The above interactions and those between symmetry-mismatched receptor-binding σ1 trimers and RNA-capping λ2 pentamers balance competing needs of capsid assembly, external protein removal, and allosteric triggering of endogenous RNA transcription, before, during and after infection, respectively.

Virtual Screening and Experimental Testing of B1 Metallo-ß-lactamase Inhibitors.

The global rise of metallo-ß-lactamases (MBLs) is problematic due to their ability to inactivate most ß-lactam antibiotics. MBL inhibitors that could be coadministered with and restore the efficacy of ß-lactams are highly sought after. In this study, we employ virtual screening of candidate MBL inhibitors without thiols or carboxylates to avoid off-target effects using the Avalanche software package, followed by experimental validation of the selected compounds. As target enzymes, we chose the clinically relevant B1 MBLs NDM-1, IMP-1, and VIM-2. Among 32 compounds selected from an approximately 1.5 million compound library, 6 exhibited IC50 values less than 40 µM against NDM-1 and/or IMP-1. The most potent inhibitors of NDM-1, IMP-1, and VIM-2 had IC50 values of 19 ± 2, 14 ± 1, and 50 ± 20 µM, respectively. While chemically diverse, the most potent inhibitors all contain combinations of hydroxyl, ketone, ester, amide, or sulfonyl groups. Docking studies suggest that these electron-dense moieties are involved in Zn(II) coordination and interaction with protein residues. These novel scaffolds could serve as the basis for further development of MBL inhibitors. A procedure for renaming NDM-1 residues to conform to the class B ß-lactamase (BBL) numbering scheme is also included.

Azolylthioacetamides as a potent scaffold for the development of metallo-ß-lactamase inhibitors.

In an effort to develop new inhibitors of metallo-ß-lactamases (MßLs), twenty-eight azolylthioacetamides were synthesized and assayed against MßLs. The obtained benzimidazolyl and benzioxazolyl substituted 1-19 specifically inhibited the enzyme ImiS, and 10 was found to be the most potent inhibitor of ImiS with an IC50 value of 15 nM. The nitrobenzimidazolyl substituted 20-28 specifically inhibited NDM-1, with 27 being the most potent inhibitor with an IC50 value of 170 nM. Further studies with 10, 11, and 27 revealed a mixed inhibition mode with competitive and uncompetitive inhibition constants in a similar range as the IC50 values. These inhibitors resulted in a 2-4-fold decrease in imipenem MIC values using E. coli cells producing ImiS or NDM-1. While the source of uncompetitive (possibly allosteric) inhibition remains unclear, docking studies indicate that 10 and 11 may interact orthosterically with Zn2 in the active site of CphA, while 27 could bridge the two Zn(II) ions in the active site of NDM-1 via its nitro group.

Primary total hip arthroplasty using 3rd generation ceramic-on-ceramic articulation.

PURPOSE: Ceramic-on-ceramic (CoC) is currently a popular bearing combination in young patients in primary total hip arthroplasty (THA). The purpose of this study was to evaluate clinical and radiographic results and complications of cementless THA with 3rd generation CoC articulation. MATERIALS AND METHODS: From April 2001 to January 2008, 310 primary THAs were performed in 300 patients using 3rd generation CoC articulation. The mean follow-up period was 8.9 years and the mean age at index surgery was 54.6 years. Patient clinical outcome was evaluated with the Harris Hip Score. Radiographic evaluations was performed to analyse osteolysis, implant fixation and loosening. RESULTS: Mean Harris Hip Score at last follow-up was 95.4 (76-100). Radiographic analysis demonstrated no evidence of stem or cup loosening and there were no cases of osteolysis. Ceramic wear was not detectable on the plain radiograph. Complications requiring revision occurred in 12 cases; 2 ceramic head fractures, 4 dislocations, 2 deep infections and 4 cases of periprosthetic fracture. The cohort had an overall revision rate of 3.9%. CONCLUSIONS: Clinical outcomes using cementless THA with 3rd generation CoC articulation were satisfactory. Although the mechanical properties of ceramic materials have improved, there are still problems such as ceramic fracture and squeaking. More clinical study and investigation for alternative bearing are necessary to reduce complications. 4th generation CoC or ceramic on cross linked polyethylene may address some of these issues.

Triazolylthioacetamide: A Valid Scaffold for the Development of New Delhi Metallo-ß-Lactmase-1 (NDM-1) Inhibitors.

The metallo-ß-lactamases (MßLs) cleave the ß-lactam ring of ß-lactam antibiotics, conferring resistance against these drugs to bacteria. Twenty-four triazolylthioacetamides were prepared and evaluated as inhibitors of representatives of the three subclasses of MßLs. All these compounds exhibited specific inhibitory activity against NDM-1 with an IC50 value range of 0.15-1.90 µM, but no activity against CcrA, ImiS, and L1 at inhibitor concentrations of up to 10 µM. Compounds 4d and 6c are partially mixed inhibitors with K i values of 0.49 and 0.63 µM using cefazolin as the substrate. Structure-activity relationship studies reveal that replacement of hydrogen on the aromatic ring by chlorine, heteroatoms, or alkyl groups can affect bioactivity, while leaving the aromatic ring of the triazolylthiols unmodified maintains the inhibitory potency. Docking studies reveal that the typical potent inhibitors of NDM-1, 4d and 6c, form stable interactions in the active site of NDM-1, with the triazole bridging Zn1 and Zn2, and the amide interacting with Lys 211 (Lys224).

Amino Acid Thioester Derivatives: A Highly Promising Scaffold for the Development of Metallo-ß-lactamase L1 Inhibitors.

In light of the biomedical significance of metallo-ß-lactamases (MßLs), ten new mercaptoacetic acid thioester amino acid derivatives were synthesized and characterized. Biological activity assays indicated that all these synthesized compounds are very potent inhibitors of L1, exhibiting an IC50 value range of 0.018-2.9 µM and a K i value range of 0.11-0.95 µM using cefazolin as substrate. Partial thioesters also showed effective inhibitory activities against NDM-1 and ImiS with an IC50 value range of 12-96 and 3.6-65 µM, respectively. Also, all these thioesters increased susceptibility of E. coli cells expressing L1 to cefazolin, indicated by a 2-4-fold reduction in MIC of the antibiotic. Docking studies revealed potential binding modes of the two most potent L1 inhibitors to the active site in which the carboxylate group interacts with both Zn(II) ions and Ser221. This work introduces a highly promising scaffold for the development of metallo-ß-lactamase L1 inhibitors.

Meropenem and chromacef intermediates observed in IMP-25 metallo-ß-lactamase-catalyzed hydrolysis.

Metallo-ß-lactamases inactivate most ß-lactam antibacterials, and much attention has been paid to their catalytic mechanism. One issue of controversy has been whether ß-lactam hydrolysis generally proceeds through an anionic intermediate bound to the active-site Zn(II) ions or not. The formation of an intermediate has not been shown conclusively in imipenemase (IMP) enzymes to date. Here, we provide evidence that intermediates are formed during the hydrolysis of meropenem and chromacef catalyzed by the variant IMP-25 and, to a lesser degree, IMP-1.

Azolylthioacetamide: A Highly Promising Scaffold for the Development of Metallo-ß-lactamase Inhibitors.

A new scaffold, azolylthioacetamide, was constructed and assayed against metallo-ß-lactamases (MßLs). The obtained molecules specifically inhibited MßL ImiS, and 1c was found to be the most potent inhibitor, with a K i = 1.2 µM using imipenem as substrate. Structure-activity relationships reveal that the aromatic carboxyl improves inhibitory activity of the inhibitors, but the aliphatic carboxyl does not. Compounds 1c-d and 1h-i showed the best antibacterial activities against E. coli BL21(DE3) cells producing CcrA or ImiS, resulting in 32- and 8-fold reduction in MIC values, respectively; 1c and 1f-j resulted in a reduction in MIC against P. aeruginosa. Docking studies revealed that 1a, 1c, and 1d fit tightly into the substrate binding site of CphA as a proxy for ImiS with the aromatic carboxylate forming interactions with Lys224, the Zn(II) ion, the backbone of Asn233, and hydrophobic portions of the inhibitors aligning with hydrophobic patches of the protein surface.