Assigning a function to a conserved archaeal metallo-β-lactamase from Haloferax volcanii

The metallo-β-lactamase family of enzymes comprises a large group of proteins with diverse functions in the metabolism of the cell. Among others, this superfamily contains proteins which are involved in DNA and RNA metabolism, acting as nucleases in e.g. repair and maturation. Many proteins have been annotated in prokaryotic genomes as being potential metallo-β-lactamases, but very often the function has not been proven. The protein HVO_2763 from Haloferax volcanii is such a potential metallo-β-lactamase. HVO_2763 has sequence similarity to the metallo-β-lactamase tRNase Z, a tRNA 3′ processing endonuclease. Here, we report the characterisation of this metallo-β-lactamase HVO_2763 in the halophilic archaeon Haloferax volcanii. Using different in vitro assays with the recombinant HVO_2763, we could show that the protein does not have tRNA 3′ processing or exonuclease activity. According to transcriptome analyses of the HVO_2763 deletion strain, expression of proteins involved in membrane transport is downregulated in the mutant. Therefore, HVO_2763 might be involved directly or indirectly in membrane transport

Occurrence of Antibiotic-Resistant Bacteria in Therapy Pools and Surrounding Surfaces

The number of patients colonized with antibiotic-resistant bacteria is increasing in health care facilities. Because transmission of antibiotic-resistant bacteria is feared, there exist reports that the affected patients are frequently excluded from hydrotherapy, which is a non-invasive and beneficial treatment used for patients with different diseases. Data from the literature suggest that deficient water disinfection measures exist, which are not always sufficient to kill all released bacteria. If the pool water is not disinfected properly, it may also infect the bathers. Immunocompromised patients are particularly susceptible to be infected with (antibiotic-resistant) bacteria. In order to determine the distribution of antibiotic-resistant bacteria in the pool water treatment system and the pool environment and to estimate the associated transmission risk we analyzed samples from eleven health care facilities. Antibiotic-resistant bacteria were found in the water and surface samples collected. One hundred and two antibiotic-resistant isolates from water samples and 307 isolates from surrounding surfaces were obtained, respectively. The majority of the isolates belonged to non-fermenting Gram-negative rods, like Pseudomonas spp. Some isolates were resistant to a wide range of the tested antibiotics. The results indicate a relation between the number of isolates in water samples and the number of patients using the pools in combination with deficiencies in water treatment. In the pool environment the highest number of isolates was obtained from barefoot areas and floor cleaning equipment

Treatment of end-stage peripheral artery disease by neuromodulation

Background: Neuromodulation is a therapeutic option to improve limb salvage in end-stage peripheral arterial disease (PAD), but there is no consensus on its indication for spinal cord stimulation (SCS) in PAD patients. Objective: The aim of this study was to present the outcome of end-stage PAD patients treated with SCS. Methods: This study is a retrospective analysis based on a local prospective registry. Neuromodulation was performed if there was: 1) no revascularisation option, 2) no septicemia, 3) and Rutherford stage 4-6. The primary endpoint of the study was limb salvage. Secondary endpoints were reduction in pain or simply pain reduction pain (assessed using a visual anlog scale/VAS) and improvement in walking distance. Results: Limb salvage was reached in 30/34 patients (88%). Patients reported a significant reduction in pain on the 10-point VAS scale from baseline (median = 7.5, IQR = 7-8) to follow-up at 2 years (median = 0, IQR 0-2.75), p < 0.001. Walking distance also improved from preoperative (median = 50 m, IQR = 20-50 m) to follow-up at 2 years (median = 150 m, IQR 50-272 m), p < 0.001. Results: SCS implantation in patients with end-stage PAD can enable limb salvage in a high percentage of cases and increase mobility due to pain reduction. The role of microcirculation in these improvements needs to be investigated in further studies. Keywords: Neuromodulation; chronic critical limb ischemia; end-stage peripheral artery disease; spinal cord stimulation

Design of Light-Controlled Protein Conformations and Functions

In recent years, interest in controlling protein function with light has increased. Light offers a number of unique advantages over other methods, including spatial and temporal control and high selectivity. Here, we describe a general protocol for engineering a protein to be controllable with light via reaction with an exogenously introduced photoisomerizable small molecule and illustrate our protocol with two examples from the literature: the engineering of the calcium affinity of the cell–cell adhesion protein cadherin, which is an example of a protein that switches from a native to a disrupted state, and the engineering of the opening and closing of the chaperonin Mm-cpn, an example of a switch between two functional states. This protocol guides the user from considering which proteins may be most amenable to this type of engineering, to considerations of how and where to make the desired changes, to the assays required to test for functionality