6% among tourist travelers. This study shows that returned children, who are sick enough to go to the emergency room, present with a broad spectrum of travel-associated morbidities, mainly diarrheal illness (39%), respiratory (28.7%), and febrile/systemic illness (13.4%). Some 12 (3.6%) of children presenting with travel-associated illness have potential serious diseases requiring hospitalization. Eleven of the 12 children presenting with serious

illness were VFR or immigrant children. Our study has certain limitations; patients included in the study do not necessarily represent the whole population of Zürich. Many ill-returned children will visit pediatricians or general practitioners, and some children will present in the emergency INK-128 room due to an inadequate access to Bleomycin molecular weight the primary health care system. Furthermore, the number of travelers returning in good health is also unknown. Therefore, incidence rates or relative risks cannot be estimated. Similarly, patients with mild or self-limiting disease are more likely to see a general practitioner. On the other hand, Zürich is a large city with a mixed sociocultural population, and many of these patients may prefer to go to a more anonymous University Children’s Hospital, particularly if they do not have a regular general practitioner.2 Only 0.8% (328 of 40,486) of all emergency consultations had a travel-related reason. Nevertheless,

Teicoplanin the travel history is essential in ascertaining the possible cause

of disease and in the selection of the appropriate diagnosis. Recently, a global analysis of ill-returned children showed that diarrhea is the leading diagnosis in returned children, and our study confirms this finding.1 The fore-mentioned global analysis, however, shows significant dermatological proportional morbidity that was not observed in the Zürich collective. Our analysis is thus particularly valuable for physicians and pediatricians in the Central European setting. Another report shows no significant difference in the incidence of morbid episodes between children and adults, except for fever which is diagnosed more frequently in children.3 The study confirms that many of the diagnosed illnesses post travel are commonplace and of short duration. Travelers’ diarrhea affects over 50% of travelers and can disrupt holidays.4 The most frequent bacterial pathogens of travelers’ diarrhea are Escherichia coli, Campylobacter, Shigella, and Salmonella.3–6 As diarrhea was the most frequent illness in children in this study, this theme is important for inclusion in the pre-travel consultation. Parents should be prepared to treat mild diarrhea with oral rehydration and additionally loperamide for older children.7,8 In this study, two malaria cases were found, both from Ghana in VFR travelers. As a priority, malaria should be ruled out in children with febrile illness returning from malaria endemic areas.

The overall percentage of the study group who could be classified regarding the absence and presence of cirrhosis was 64%. Thus, if the two cut-offs for the diagnosis of cirrhosis were combined, this would allow the majority of patients to receive a definitive diagnosis; the remaining patients with inconclusive results would have to be screened for cirrhosis by other Ku-0059436 clinical trial means. In summary, a combination of data

routinely available in clinical practice, namely AST and platelet count, and serum levels of MMP-2 resulted in high diagnostic accuracy for the diagnosis of fibrosis in HIV/HCV-coinfected patients. The sequential application of APRI followed by MMP-2 levels allowed the majority of patients to be classified for the absence and presence of F≥2. This approach could represent an alternative for the evaluation of fibrosis in settings where liver biopsy or TE is not accessible. However, NU7441 cell line despite

its high diagnostic yield, use of the MAPI will leave a number of patients with intermediate results who will need additional testing to stage fibrosis. This study was partly supported by grants from Consejería de Salud, Junta de Andalucía (exp 43/05 and exp 48/07). The authors wish to thank the Ministerio de Sanidad y Consumo, Instituto de Salud Carlos III, Red de SIDA of Spain for their support (ISCIII-RETIC RD06/006). JAP is the receptor of an intensification grant from the Fundación Progreso y Salud of the Consejería de Salud de la Junta de Andalucía (Reference AI-0021). “
“Using new sensitive quantitative polymerase chain reaction (PCR) assays, cytomegalovirus (CMV) DNA is often detectable in the plasma of immunosuppressed patients. We investigated the prognostic value of a positive CMV DNA test for the development of CMV end-organ disease, other AIDS-defining events and mortality. A survival analysis was performed, using the Kaplan–Meier method and Cox proportional hazards models, for patients prospectively followed in the Swiss HIV Cohort Study, from January 1996 to December 2007, who were CMV-seropositive, had a CD4 count of ≤100 cells/μL, and

had a plasma sample available for the measurement 17-DMAG (Alvespimycin) HCl of baseline CMV DNA with an ultrasensitive PCR. The outcome analysed was an AIDS-defining event, including CMV end-organ disease, or death. Variables analysed at the time of CMV measurement were demographic variables, CD4 cell counts, HIV-1 RNA loads, and use and type of highly active antiretroviral therapy (HAART). Of 1128 patients, 208 (18%) presented an AIDS-defining event and 246 (22%) died. A total of 368 patients (34% of samples) had detectable CMV DNA at baseline, with DNA concentrations of up to 38 800 copies/mL. In the multivariate analysis, CMV DNA predicted evolution not only towards CMV end-organ disease [hazard ratio (HR) 12.6; 95% confidence interval (CI) 4.27–37.41], but also towards other AIDS-defining events (HR 2.6; 95% CI 1.60–4.33) and death (HR 1.

IMC captures heat flow in the microwatt (μW) range and enables detection of the metabolic heat evolved from ca. 10 000 mammalian cells or ca. 100 000 bacteria (Braissant et al., 2010). Thus, IMC has the potential to provide real-time quantitative data on metabolic activity, aggregation, and biomass formation in biofilms in situ. The sensitivity of IMC has been exploited in evaluating Ku0059436 metabolism and growth of living cells in culture in medical and environmental microbiology (Howell et al., 2012). While IMC

has been applied to study the co-aggregation of different strains of biofilm-forming bacteria (Postollec et al., 2003), studies that focus on the use of this technique for investigating in vitro multispecies biofilms are scarce. The purpose of this study was to characterize a peri-implantitis-related biofilm by well-established commonly used microscopic methods and to complement this information using IMC to determine various measures PS-341 research buy of the metabolic activity. A three-species biofilm was allowed to form on surfaces of protein-coated titanium disks in a newly developed anaerobic flow chamber system. The selected bacterial species were an early colonizer, Streptococcus sanguinis; a pathogenic bridging organism, Fusobacterium nucleatum; and a common periodontal and peri-implant pathogen, Porphyromonas gingivalis (Quirynen et al.,

Rebamipide 2006; Fürst et al., 2007; Heuer et al., 2007). Streptococcus sanguinis (DSM 20068), F. nucleatum (ATCC 10953), and P. gingivalis (DSM 20709) were used for the biofilm formation. A 10 μL inoculum of S. sanguinis in skim milk solution (stored at −20 °C) was suspended in 5 mL Schaedler broth (BBL™; Becton Dickinson, Basel, Switzerland) and incubated aerobically at 37 °C for 8 h. The bacterial suspension was used

as an inoculum for a new subculture (1 : 50), which was incubated aerobically at 37 °C for 16 h. The culture was ultrasonicated for 30 s (22.5 W; Vibracell, Sonics & Materials, Newtown, CT), centrifuged at 5700 g for 5 min at room temperature, washed with physiological saline, and harvested by centrifugation. The S. sanguinis cells were resuspended in simulated body fluid (Cho et al., 1995) to a density of 1.1 × 108 ± 6.2 × 107 CFU mL−1. Fusobacterium nucleatum and P. gingivalis were maintained in Microbank® blue vials (Chemie Brunschwig AG, Basel, Switzerland) at −70 °C. One pearl of each frozen culture was inoculated into 10 mL thioglucolate aliquots (Biomerieux SA, Geneva, Switzerland), enriched with 5 μg mL−1 hemin (Fluka, Buchs, Switzerland) and 0.5 μg mL−1 menadione (VWR International, Dietikon, Switzerland), and incubated anaerobically at 37 °C for 96 h. The cultures were harvested; F. nucleatum and P. gingivalis were suspended to a density of 3.2 × 107 ± 1.9 × 106 CFU mL−1 and 2.1 × 109 ± 9.3 × 108 CFUmL−1, respectively.

Consistently, low-frequency faces specifically

activate the subcortical visual pathway, including the superior colliculus, pulvinar and amygdala (Vuilleumier et al., 2003). Furthermore, residual visual ability Ribociclib was tuned to low spatial frequency in a patient with blindsight due to lesions in the visual cortical areas (Sahraie et al., 2002). This fast activation of the pulvinar might be due to direct inputs from the superior colliculus, contributing to the ability of newborns to orient toward faces. The present study provides neurophysiological evidence of pulvinar involvement in fast and coarse facial information processing. The second hypothesis proposes that interactive activity based on reciprocal connections between the subcortical and cortical areas is important for stimulus recognition and attention (Bullier, 2001;

Pessoa & Adolphs, 2010). These cortico-pulvino-cortical circuits might be involved in coordinating and amplifying signals, and improving signal-to-noise ratios (Shipp, 2003; Pessoa & Adolphs, 2010), as well as modulating interactions between oscillatory processes in different cortical areas, which contributes to visual attention (Serences & Yantis, 2006; Saalmann & Kastner, 2009). Our results here indicate that pulvinar neurons detect face-like patterns in epoch 1, while they categorize the visual stimuli into one of the five stimulus categories in epoch 2. Furthermore, the amount of stimulus information conveyed by the pulvinar neurons and the number of stimulus-differential neurons was higher in epoch 2 than in RGFP966 ic50 epoch 1. These results indicate that all pulvinar neurons become more sensitive to other categories of stimuli after epoch 1 (i.e. epoch 2 or later), during which cortical neurons also become active (for response latencies of cortical neurons, see a review by Lamme & Roelfsema, 2000).

These findings suggest that pulvinar responsiveness to a variety of stimuli in epoch 2 might be due to reciprocal connections with cortical areas with similar response latencies. Consistent with this, a neuropsychological study of human patients with pulvinar lesions suggests that the pulvinar is involved in enhancing stimulus saliency (Snow et al., 2009), which might contribute to neural computation in an early stage of stimulus categorization (Meeren et al., 2008). Our results provide direct neurophysiological evidence that pulvinar neurons respond to face-like patterns with short latencies, which seems to be consistent with the view that the pulvinar nuclei comprise a subcortical pathway that rapidly processes coarse facial information. Following the initial recognition of the facial stimulus, the population activity of the pulvinar neurons participates in classifying the facial pattern, with a concomitant increase in the amount of information processed.

The finding of a larger amplitude of the N1 component over the right as compared with the left hemisphere sites and of a more widespread group difference in the N1 peak amplitude over the right hemisphere in our RO4929097 study is noteworthy. Although lateralization effects in ERP results should be interpreted with caution, our results do agree with reports of greater right hemisphere involvement in the processing of spectral information and of timbre in particular (e.g. Belin et al., 2000; Zatorre & Belin, 2001; von Kriegstein

et al., 2003). While the N1 enhancement in musicians was present to all sound types, the relationship between its peak amplitude and measures of musical proficiency was limited to the NAT condition. More specifically, individuals who rated their own musical ability more highly had a larger N1 peak amplitude to both music

and voice deviants. Additionally, individuals with higher MAP scores had higher N1 peak amplitude to music deviants. A similar but weaker relationship was also present between MAP click here scores and N1 to voice deviants. A relationship between N1 and either the age at onset of training or the duration of training was not significant. In part this may be due to the fact that we tested amateur musicians, who on average started their training later than what would be typical for professional musicians. Overall, however, reports of correlation between either the age at the onset of musical training or the duration Dimethyl sulfoxide of such training and the enhancement of early ERP responses are not consistent (e.g. Pantev et al., 1998; Shahin et al., 2003; Musacchia et al., 2007). Our evaluation of timbre encoding in musicians and non-musicians has its limitations. Our main task probed the ability of the two groups of participants to resist distraction

and did not measure overt timbre perception. Therefore, whether enhanced N1 peak amplitude to complex sounds in musicians actually translates into better timbre identification and/or discrimination requires future studies. Related to the above point is the fact that the design of our study required that we use only a small set of sounds to represent vocal and musical timbres. In contrast, studies of the FTPV component used a large range of vocal and non-vocal sounds. Future studies that use a larger set of timbre examples and focus on the FTPV component may help determine whether musicians’ neural encoding of voices as a perceptual category (compared with voices’ acoustic properties as in the current study) is superior to that in non-musicians. In summary, musicians showed an enhanced N1 ERP component not only to musical and vocal sounds but also to never before heard spectrally-rotated sounds.

The microcin undergoes post-translational modification with a trimer of N-(2,3-dihydroxybenzoyl) linked to the C-terminal serine residue by a β-d-glucose. This modification which has been shown to bind iron mimics a catechol-type siderophore and significantly increases the toxicity of the peptide (Thomas et al., 2004; Destoumieux-Garzón et al., 2006). A number of bacterial pathogens with specific mammalian hosts possess systems for

directly obtaining iron from host proteins such as transferrin and lactoferrin, which sequester free iron in the body’s extracellular fluids. The most thoroughly characterized of these systems is the transferrin transport system of Neisseria gonorrhoeae and Neisseria meningitidis (Noinaj et al., 2012). Transferrin-binding protein A (TbpA),

a 100 kDa integral outer membrane protein and transferrin-binding protein ABT-199 price B (TbpB) an 80 kDa membrane anchored coreceptor form the basis of this system. TbpA, a TonB-dependent receptor strongly binds transferrin and acts as the conduit for transport of the liberated ferric iron across the outer membrane; however, it lacks the ability to distinguish between the apo and holo forms of the protein (Moraes et al., 2009). The coreceptor TbpB has a strong affinity for the iron loaded transferrin Dasatinib purchase only and acts synergistically with TbpA, considerably increasing the efficiency of iron import (Anderson et al., 1994). Following binding and extraction of iron, apo-transferrin is released from the complex (Lee & Schryvers, 1988). The importance of

this system for fitness is demonstrated by the fact that its inactivation renders N. gonorrhoeae avirulent (Cornelissen et al., 1998). The majority of iron in a mammalian host is stored intracellularly as haemoglobin (Rohde et al., 2002). As such, haemoglobin P-type ATPase and the haem it contains represent an important iron source for invading pathogens (Wandersman & Stojiljkovic, 2000). As a result, pathogenic bacteria commonly secrete haemolysins and cytolysins that lyse host cells and release haemoglobin and other haemoproteins (Krewulak & Vogel, 2008). Uptake of the liberated haem is then achieved by a number of specialized systems, which in Gram-negative bacteria generally consist of a TonB-dependent outer membrane receptor, a periplasmic-binding protein and an ABC transporter (Tong & Guo, 2009). An example of a system, where a cell surface receptor directly acquires free or protein-bound haem, is the two-component HpuA/B system of N. meningitidis, which is evolutionarily and mechanistically related to the transferrin-binding system discussed earlier (Rohde et al., 2002). A second system indentified in a number of Gram-negative bacteria and characterized in the opportunistic pathogens P.

S.), The Danish Research Council for Nature and Universe (funding for A.J.) and the Villum Kann Rasmussen Foundation (funding for A.R.J.). We acknowledge Lasse Gudmundsson and Spire Kiersgaard (both from GEUS) for their assistance with the field work and Patricia Simpson for her excellent help during the writing of the manuscript. “
“Dekkera bruxellensis is the major contaminant yeast in the wine industry worldwide. Here, we present the draft genome sequence of D. bruxellensis LAMAP2480 isolated from a Chilean wine. Genomic evidence reveals shared and exclusive genes potentially involved

in colonization and survival during alcoholic fermentation. “
“The prevalence of drug-resistance in Mycobacterium tuberculosis is already having a negative impact on the control of tuberculosis. We report the draft genome sequences of two super-extensively drug-resistant M. tuberculosis isolates from China, selleck chemicals llc FJ05194 (lineage 2) and GuangZ0019 (lineage 4), and compare them with the H37Rv reference

strain to identify possible sources of genetic variation associated with their extensive drug resistance. Our results suggest that their extensive drug resistance probably Wnt inhibitor results from the stepwise accumulation of resistances to individual drugs. “
“We report draft genome sequence of Ochrobactrum intermedium strain 229E concurrent with Helicobacter pylori in urease positive gastric biopsy of non-ulcer dyspeptic individual from Southern part of India. Since the role of Ochrobactrum in human gastric environment is poorly understood, comprehensive pathological, microbiological, and genome level understanding are necessary to evaluate its association with H. pylori in the gastric niche. Comparative analysis of O. intermedium 299E strain revealed functional similarities with virulence related gene clusters present in H. pylori genomes, which probably might aid in its ability to persist in the

human gastric mucosa. However, H.pylori specific vacuolating cytotoxin (vacA) involved in vacuolization, cytotoxicity, and T-cell inhibition was absent in the O. intermedium 229E genome. Taken together, O. intermedium 229E shared STK38 numerous features like secretion system, urease, and flagella with H.pylori genome sequence that might aid concurrence in the gastric niche. “
“Methicillin-resistant Staphylococcus aureus (MRSA) is an increasing cause of serious infection, both in the community and hospital settings. Despite sophisticated strategies and efforts, the antibiotic options for treating MRSA infection have been narrowed due to the limited number of newly developed antimicrobials. Herein, we analyze the completely sequenced genome of a novel virulent phage YMC/09/04/R1988 MRSA BP as a potential alternative anti-MRSA agent, which lysed clinical isolates from a patient admitted to the hospital due to hip disarticulation. The phage contains a linear double-stranded DNA genome of 44 459 bp in length, with 33.

Fixation of HIV-1 CCR5 use by IL-2 therapy may suggest a potential association between these approaches for the long-term management of individuals infected with R5 HIV-1. We would like to thank Fernanda Dorigatti (Laboraf SpA, Milano) for her support in the quantification of HIV viremia, and the HIV-positive individuals who donated their blood allowing the performance of this study. This study was supported

in part by grants (to AL and GP) of the VI° National Program of Research on AIDS of the Istituto Superiore di Sanità, Rome, Italy and by the Fondation Dormeur. “
“Despite the reported decrease in the incidence and mortality rates of central nervous system (CNS) infections after the introduction of highly active antiretroviral therapy (HAART), few studies have focused on the global incidence and the relationship of these diseases with immune reconstitution this website BIBF 1120 concentration inflammatory syndrome (IRIS) in the developed world. A descriptive cohort study of all consecutive adult HIV-infected patients with CNS opportunistic infections diagnosed between 2000 and 2010 in a tertiary hospital in Spain was carried out. Demographic, clinical, laboratory, and microbiological data were recorded. Patients were followed up until death or loss to follow-up or until 30 July 2011, when the study finished.

The significance of differences in the incidence rate between early and late HAART periods was determined using the Mantel–Haenszel test. Survival distribution was estimated using the Kaplan–Meier method. A total

of 110 cases of CNS infections were diagnosed. The incidence of CNS opportunistic infections decreased from 9 cases per 1000 HIV-infected patients per year in the early HAART period to 3.8 in the late HAART period (P = 0.04). Overall, the estimated mean survival time was 58.8 months (95% confidence interval 47.1–70.6 months). Of the 110 patients, 18 (16.4%) met the criteria of IRIS, 10 (55.6%) were paradoxical and eight (44.4%) were Quisqualic acid unmasking. IRIS was not associated with a higher mortality rate. The annual incidence of CNS infections decreased progressively during the period of study. The mortality rate associated with these diseases remains high despite HAART. The development of IRIS associated with neurological infections had no influence on prognosis. The widespread use of highly active antiretroviral therapy (HAART) has led to a dramatic decline in the incidence of new AIDS cases and most opportunistic illnesses [1-3]. In the developed world, cases of opportunistic neurological infections such as cryptococcal meningitis, tuberculous meningitis, cerebral toxoplasmosis and progressive multifocal leukoencephalopathy (PML) are nowadays becoming infrequent [4-6]. For this reason, in the last decade, most studies on opportunistic infections have been performed in limited-resource settings where their incidence is still high as a consequence of the lack of availability of HAART.

However, the dd-CPases as a group share substantial homology in their primary structures and are believed to act on peptidoglycan substrates via similar mechanisms in vitro (Baquero et al., 1996). What is not known is whether the MMD residues affect the dd-CPase activities of these PBPs, and whether these changes explain the difference in the physiological functions

of PBPs 5 and 6. To determine more exactly how the 20-amino acid MMDs of PBPs 5 and 6 contribute to the differences in the in vitrodd-CPase activities of these enzymes, we compared the enzymatic characteristics find more of four soluble PBPs (designated as ‘sPBPs’): sPBP 5, sPBP 6 and the mosaic proteins sPBP 656 and sPBP 565. The variations in enzymatic activities among these proteins help explain the basis of the different biochemical and physiological properties of the dd-CPase PBPs. Escherichia coli BL21 star (Stratagene, West Cedar Creek, TX) was used to express recombinant proteins for purification in bulk. Plasmid pT7-cPBP5 was provided as a gift by Robert A. Nicholas. Plasmids pAG6-4, pAG565-3 and pAG656-1 (9) were used to amplify the genes of sPBP 6, sPBP 565 and sPBP 656, respectively. Unless otherwise specified, restriction enzymes and DNA-modifying enzymes

were from New England Biolabs (Ipswich, MA) and other chemicals and reagents were from Sigma-Aldrich (St. Louis, MO). To generate genes expressing sPBPs, the genes encoding the respective PBPs were amplified using oligonucleotide primers (from MWG Biotech Inc., High Point, NC) in such a way that JNK activity inhibition the resulting genes would express proteins devoid of their signal peptides and carboxyl-terminal amphipathic anchors. Primer pairs used for amplifications were: (1) P1 and P2 for sPBP 6, using pAG6-4 as the template; (2) P1 and P5 for sPBP 656, using pAG656-1 as the template; and (3) P3 and P4 for sPBP 565,

using pAG565-3 as the template (Table 1). The conditions for amplification with Deep Vent DNA polymerase were as follows: 94 °C for 5 min (initial denaturation), 94 °C for 1 min, 60 °C for 1 min and 72 °C for 1 min (for 30 cycles), followed by a final extension of 72 °C for 7 min. Each amplified product MRIP was cloned separately into the NdeI and HindIII sites of pT7-7 K, generating pTA6-2S (expressing sPBP 6), pTA656-2S (expressing sPBP 656) and pTA565-3S (expressing sPBP 565). Plasmids were selected by including kanamycin (50 μg mL−1) in the medium and were sequenced to confirm that no mutations had arisen (sequencing was performed by MWG Biotech Inc.). Any sequence disparities in the constructs were removed by site-directed mutagenesis and reconfirmed by sequencing. Plasmids encoding the sPBPs were transformed into E. coli BL21 star and expressed under optimal conditions as determined beforehand (not shown).

However, the dd-CPases as a group share substantial homology in their primary structures and are believed to act on peptidoglycan substrates via similar mechanisms in vitro (Baquero et al., 1996). What is not known is whether the MMD residues affect the dd-CPase activities of these PBPs, and whether these changes explain the difference in the physiological functions

of PBPs 5 and 6. To determine more exactly how the 20-amino acid MMDs of PBPs 5 and 6 contribute to the differences in the in vitrodd-CPase activities of these enzymes, we compared the enzymatic characteristics 17-AAG research buy of four soluble PBPs (designated as ‘sPBPs’): sPBP 5, sPBP 6 and the mosaic proteins sPBP 656 and sPBP 565. The variations in enzymatic activities among these proteins help explain the basis of the different biochemical and physiological properties of the dd-CPase PBPs. Escherichia coli BL21 star (Stratagene, West Cedar Creek, TX) was used to express recombinant proteins for purification in bulk. Plasmid pT7-cPBP5 was provided as a gift by Robert A. Nicholas. Plasmids pAG6-4, pAG565-3 and pAG656-1 (9) were used to amplify the genes of sPBP 6, sPBP 565 and sPBP 656, respectively. Unless otherwise specified, restriction enzymes and DNA-modifying enzymes

were from New England Biolabs (Ipswich, MA) and other chemicals and reagents were from Sigma-Aldrich (St. Louis, MO). To generate genes expressing sPBPs, the genes encoding the respective PBPs were amplified using oligonucleotide primers (from MWG Biotech Inc., High Point, NC) in such a way that check details the resulting genes would express proteins devoid of their signal peptides and carboxyl-terminal amphipathic anchors. Primer pairs used for amplifications were: (1) P1 and P2 for sPBP 6, using pAG6-4 as the template; (2) P1 and P5 for sPBP 656, using pAG656-1 as the template; and (3) P3 and P4 for sPBP 565,

using pAG565-3 as the template (Table 1). The conditions for amplification with Deep Vent DNA polymerase were as follows: 94 °C for 5 min (initial denaturation), 94 °C for 1 min, 60 °C for 1 min and 72 °C for 1 min (for 30 cycles), followed by a final extension of 72 °C for 7 min. Each amplified product Montelukast Sodium was cloned separately into the NdeI and HindIII sites of pT7-7 K, generating pTA6-2S (expressing sPBP 6), pTA656-2S (expressing sPBP 656) and pTA565-3S (expressing sPBP 565). Plasmids were selected by including kanamycin (50 μg mL−1) in the medium and were sequenced to confirm that no mutations had arisen (sequencing was performed by MWG Biotech Inc.). Any sequence disparities in the constructs were removed by site-directed mutagenesis and reconfirmed by sequencing. Plasmids encoding the sPBPs were transformed into E. coli BL21 star and expressed under optimal conditions as determined beforehand (not shown).