Staphylococcus simulans biovar staphylolyticus lysostaphin efficiently cleaves Staphylococcus aureus cell walls. The protein is in late clinical trials as a topical anti-staphylococcal agent, and can be used to prevent staphylococcal growth on artificial surfaces. Moreover, the gene has been both stably engineered into and virally delivered to mice or livestock to obtain resistance against staphylococci. Here, we report the first crystal structure of mature lysostaphin and two structures of its isolated catalytic domain at 3.5, 1.78 and 1.26 Å resolution, respectively. The structure of the mature active enzyme confirms its expected organization into catalytic and cell-wall-targeting domains. It also indicates that the domains are mobile with respect to each other because of the presence of a highly flexible peptide linker. The high-resolution structures of the catalytic domain provide details of Zn2+ coordination and may serve as a starting point for the engineering of lysostaphin variants with improved biotechnological characteristics.Structured digital abstractlysostaphin by x-ray crystallography (1,2).

Semiconducting colloidal quantum dots (QDs) are nanocrystals with potential for a range of applications, [1] including fluorescent labeling in biology and medicine. [2][3][4] Different approaches have been used to adapt QDs for biological studies including methods to modify the surface of QDs, thus making them soluble in aqueous solution. This is achieved by capping the dots with mercaptoundecanoic acid (MUA), [3] by attaching proteins [5] or organic silanes [6] to the surface of the QDs, and by synthesizing hybrid nanocomposites in which the QDs are surrounded by organic shells. [7][8][9] Although some of these methods have proved to be successful, the toxicity of QDs remains a key issue for in vivo imaging. The toxicity depends on the stability of the nanocrystals in vivo where they can be subjected to a range of pH, enzyme activities and oxidizing/ reducing environments. [10] Although the level of toxicity can be partially reduced by coating the dots, [11][12][13] the stability of the capping against the changing environment remains to be established. [14] Also, to date, research on QD toxicity has mainly focused on II-VI nanocrystals. [12][13][14][15] However, since the photon emission of these dots is in the visible or nearvisible wavelength range, their use in deep-tissue imaging is limited due to scattering and small penetration of light through tissues. Light scattering is significantly reduced at wavelengths >1000 nm [16] and transmission through biological tissues tends to be higher in the wavelength range 1000-1300 nm. [17] Despite a rapidly expanding body of work on the synthesis of colloidal nanocrystals with photon emission in this wavelength region, [2,3] their biocompatibility and toxicity are still largely unexplored.Here we assess the biocompatibility of a nanocomposite based on a PbS QD entrapped in the hollow core of an apoferritin protein cage. This provides a water-soluble hybrid construct with stable and tunable fluorescence emission at wavelengths larger than 1000 nm. We report differential cytotoxicity between normal (fetal lung fibroblast cell line) and carcinoma cells (breast cancer cell lines, estrogen receptor ERþ and ERÀ) and lower levels of toxicity than those reported previously for other quantum dots in healthy cells. Native polyacrylamide gel-electrophoresis (PAGE) studies demonstrate that the PbS QDs do not alter the external surface of apoferritin or its migratory behavior. Since apoferritin provides an exterior protein coat to the QD that allows covalent attachment of other biomolecules, [18] further functionalization of the composite has the potential for several applications, including non-invasive fluorescence imaging and therapeutic tissue targeting. [19] In these studies we have used the iron storage protein ferritin (Ft) derived from horse spleen as a template for the preparation of the AFt-PbS composite. Apoferritin is obtained from ferritin molecules by reductive dissolution of its iron oxide core. [20] Apoferritin is composed of 24 polypeptide subunits, which assemble...

A clinical isolate of Streptococcus pneumoniae was transformed with a plasmid containing the lux operon of Photorhabdus luminescens that had been modified to function in gram-positive bacteria. Cells containing this plasmid produced light stably and constitutively, without compromising the growth rate. Light output was correlated with measurements of optical density and viable counts during exponential growth and provided a sensitive, real-time measure of the pharmacodynamics of the fluoroquinolone gemifloxacin.The luxCDABE operon of Photorhabdus luminescens has been expressed successfully in a variety of gram-negative bacteria, thereby generating a bioluminescence phenotype that does not require the addition of exogenous substrate (11,16). Light output from these bioluminescent bacteria is a highly sensitive reporter of metabolic activity (10) and can therefore be used to monitor the real-time effects of antimicrobials on bacterial metabolism (14,15). Moreover, in experimental systems in which a strong correlation between bioluminescence and viable counts can be demonstrated, measurement of bioluminescence offers a rapid, alternative method for monitoring bacterial viability (10,14).Recent publications (3, 13) describe modifications to the lux operon that enable it to be expressed efficiently in the grampositive bacterium Staphylococcus aureus. In this paper, we describe the construction of a derivative of the Streptococcus spp.-Escherichia coli shuttle vector pVA838 in which the modified lux operon described by Qazi et al. (13) was placed under the control of a constitutive promoter from S. pneumoniae. Introduction of this plasmid into a clinical isolate of S. pneumoniae conferred a highly stable bioluminescence phenotype. We have examined the relationship between bioluminescence and other parameters used to monitor growth and viability and evaluated the use of bioluminescence as a means of monitoring the pharmacodynamic effects of gemifloxacin on S. pneumoniae. Gemifloxacin is a new fluoroquinolone which is potent against a wide spectrum of gram-positive and gram-negative pathogens (12).Bacterial strains and plasmids. S. pneumoniae strain SMH 11622 (6) was a clinical isolate from Southmead Hospital (Bristol, United Kingdom). E. coli DH5␣ was used as a host for propagating plasmids. Plasmid pSB2025 carries the lux operon from P. luminescens, which was modified for expression in gram-positive bacteria and cloned into pSL1190 (13). All other constructs were derivatives of pBluescript or the Streptococcus-E. coli shuttle vector pVA838 (9).Construction of a plasmid expressing the lux genes in S. pneumoniae. In a previous attempt to generate a bioluminescent transformant of S. pneumoniae, the cloned luxAB genes from Vibrio harveyi (5) were introduced into pVA838 as a BamHI-SalI fragment via pBluescript (Fig. 1). In order to gain expression from this construct, the constitutive promoter of the S. pneumoniae aminopterin resistance operon (ami) (1) was fused to the 5Ј end of luxA. This was achieved by subcloning the a...

Background Extensive literature support telehealth as a supplement or adjunct to in-person care for the management of chronic conditions such as congestive heart failure (CHF) and type 2 diabetes mellitus (T2DM). Evidence is needed to support the use of telehealth as an equivalent and equitable replacement for in-person care and to assess potential adverse effects. Objective We conducted a systematic review to address the following question: among adults, what is the effect of synchronous telehealth (real-time response among individuals via phone or phone and video) compared with in-person care (or compared with phone, if synchronous video care) for chronic management of CHF, chronic obstructive pulmonary disease, and T2DM on key disease-specific clinical outcomes and health care use? Methods We followed systematic review methodologies and searched two databases (MEDLINE and Embase). We included randomized or quasi-experimental studies that evaluated the effect of synchronously delivered telehealth for relevant chronic conditions that occurred over ≥2 encounters and in which some or all in-person care was supplanted by care delivered via phone or video. We assessed the bias using the Cochrane Effective Practice and Organization of Care risk of bias (ROB) tool and the certainty of evidence using the Grading of Recommendations Assessment, Development, and Evaluation. We described the findings narratively and did not conduct meta-analysis owing to the small number of studies and the conceptual heterogeneity of the identified interventions. Results We identified 8662 studies, and 129 (1.49%) were reviewed at the full-text stage. In total, 3.9% (5/129) of the articles were retained for data extraction, all of which (5/5, 100%) were randomized controlled trials. The CHF study (1/5, 20%) was found to have high ROB and randomized patients (n=210) to receive quarterly automated asynchronous web-based review and follow-up of telemetry data versus synchronous personal follow-up (in-person vs phone-based) for 1 year. A 3-way comparison across study arms found no significant differences in clinical outcomes. Overall, 80% (4/5) of the studies (n=466) evaluated synchronous care for patients with T2DM (ROB was judged to be low for 2, 50% of studies and high for 2, 50% of studies). In total, 20% (1/5) of the studies were adequately powered to assess the difference in glycosylated hemoglobin level between groups; however, no significant difference was found. Intervention design varied greatly from remote monitoring of blood glucose combined with video versus in-person visits to an endocrinology clinic to a brief, 3-week remote intervention to stabilize uncontrolled diabetes. No articles were identified for chronic obstructive pulmonary disease. Conclusions This review found few studies with a variety of designs and interventions that used telehealth as a replacement for in-person care. Future research should consider including observational studies and studies on additional highly prevalent chronic diseases.

The pneumococcus is the principle cause of bacterial pneumonia and also a major cause of bacterial meningitis. The mechanisms and sites of pneumococcal adherence and invasion of the respiratory tract in vivo are not clear however. We have made pneumococci expressing green fluorescent protein (GFP) and used it to trace pneumococcal adherence and invasion in vivo. By using GFP pneumococci we have shown bacterial adherence and invasion of broncho-epithelial cells in vivo by 4 h post-infection, with increases in pneumococcal invasiveness by 24 h. Using confocal image analysis we have shown varying levels of pneumococcal penetration and internalisation into host cells, as well as translocation through epithelial layers. To our knowledge this is the first report of pneumococcal invasion and cellular translocation in vivo.