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Two accessory loop regions that are present in numerous variants of New Delhi metallo-β-lactamases (NDM) are important for the enzymatic activity. The first one is a flexible loop L3 that is located near the active site and is thought to play an important role in the catalytic process. The second region, Ω loop is located close to a structural element that coordinates two essential zinc ions. Both loops are not involved in any specific interactions with a substrate. Herein, we investigated how the length and hydrophobicity of loop L3 influence the enzymatic activity of NDMs, by analyzing mutants of NDM-1 with various deletions/point mutations within the L3 loop. We also investigated NDM variants with sequence variations/artificial deletions within the Ω loop. For all these variants we determined kinetic parameters for the hydrolysis of ampicillin, imipenem, and a chromogenic cephalosporin (CENTA). None of the mutations in the L3 loop completely abolished the enzymatic activity of NDM-1. Our results suggest that various elements of the loop play different roles in the hydrolysis of different substrates and the flexibility of the loop seems necessary to fulfill the requirements imposed by various substrates. Deletions within the Ω loop usually enhanced the enzymatic activity, particularly for the hydrolysis of ampicillin and imipenem. However, the exact role of the Ω loop in the catalytic reaction remains unclear. In our kinetic tests, the NDM enzymes were inhibited in the β-lactamase reaction by the CENTA substrate. We also present the X-ray crystal structures of the NDM-1, NDM-9 and NDM-12 proteins., Competing Interests: Declaration of competing interest None declared., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Two new dimeric Zn(II) ([{ZnL 1 (DMSO 2 )} 2 ]·DMSO (1), [{ZnL 2 Cl} 2 ] (2)) and a novel tetrameric Zn(II) complex ([(Zn 2 L 3 ) 2 (μ-OAc) 2 (μ 3 -O) 2 ] (3)), where H 2 L 1  = 4-(p-methoxyphenyl) thiosemicarbazone of o-hydroxynapthaldehyde, HL 2  = 4-(p-methoxyphenyl)thiosemicarbazone of benzoyl pyridine and H 2 L 3  = 4-(p-chlorophenyl)thiosemicarbazone of o-vanillin are reported. Ligands and their complexes were characterized by spectroscopic and single crystal X-ray diffraction techniques. In addition, the complexes exhibited good binding affinity towards HSA (10 12  M -1 ), which is supported by their ability to quench the tryptophan fluorescence emission spectra of HSA. The complexes were also screened for their DNA binding propensity through UV-vis absorption titration, circular dichroism and fluorescence spectral studies. Results show that they effectively interact with CT-DNA through an intercalative mode of binding, with binding constants ranging from 10 3 to 10 4  M -1 . Among the three complexes 1 has the highest binding affinity towards CT-DNA. Further, the phosphatase activity was evaluated using bis(2,4-dinitrophenyl)phosphate (BDNPP) as substrate, however, the complexes did not yield any measurable catalytic activity. Nevertheless the complexes showed significant cytotoxic potential against HeLa and HT-29 cancer cell lines that was assessed through MTT assay and DAPI staining. Remarkably, complex 1 showed better activity than cisplatin against HT-29 cell line., Competing Interests: Declaration of competing interest All the authors' declare no conflict of interest., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Thermotoga maritima is a hyperthermophilic bacterium but its genome encodes a number of archaeal proteins including S-adenosyl-L-homocysteine hydrolase (SAHase), which regulates cellular methylation reactions. The question of proper folding and activity of proteins of extremophilic origin is an intriguing problem. When expressed in E.coli and purified (as a homotetramer) at room temperature, the hyperthermophilic SAHase from T.maritima was inactive. ITC study indicated that the protein undergoes heat-induced conformational changes, and enzymatic activity assays demonstrated that these changes are required to attain enzymatic activity. To explain the mechanism of thermal activation, two crystal structures of the inactive form of T. maritima SAHase (iTmSAHase) were determined for an incomplete binary complex with the reduced cofactor (NADH), and in a mixture of binary complexes with NADH and with adenosine. In contrast to active SAHases, in iTmSAHase only two of the four subunits contain a bound cofactor, predominantly in its non-reactive, reduced state. Moreover, the closed-like conformation of the cofactor-containing subunits precludes substrate delivery to the active site. The two other subunits cannot be involved in the enzymatic reaction either; although they have an open-like conformation, they do not contain the cofactor, whose binding site may be occupied by an adenosine molecule. The results suggest that this enzyme, when expressed in mesophilic cells, is arrested in the activity-incompatible conformation revealed by its crystal structures., (Copyright © 2017 Elsevier B.V. All rights reserved.)

To assist ongoing investigations of the limits of the tradeoff between spatial resolution and noise in PET imaging, several PET instruments based on silicon-pad detectors have been developed. The latest is a segment of a dual-ring device to demonstrate that excellent reconstructed image resolution can be achieved with a scanner that uses high-resolution detectors placed close to the object of interest or surrounding a small field-of-view in combination with detectors having modest resolution at larger radius. The outer ring of our demonstrator comprises conventional BGO block detectors scavenged from a clinical PET scanner and located at a 500mm radius around a 50mm diameter field-of-view. The inner detector-in contrast to the high-Z scintillator typically used in PET-is based on silicon-pad detectors located at 70mm nominal radius. Each silicon detector has 512 1.4mm x 1.4mm x 1mm detector elements in a 16 x 32 array and is read out using VATA GP7 ASICs (Gamma Medica-Ideas, Northridge, CA). Even though virtually all interactions of 511 keV annihilation photons in silicon are Compton-scatter, both high spatial resolution and reasonable sensitivity appears possible. The system has demonstrated resolution of ~0.7mm FWHM with Na-22 for coincidences having the highest intrinsic resolution (silicon-silicon) and 5-6mm FWHM for the lowest resolution BGO-BGO coincidences. Spatial resolution for images reconstructed from the mixed silicon-BGO coincidences is ~1.5mm FWHM demonstrating the "magnifying-glass" concept.