Construction of a single polypeptide that matures and exports the lasso peptide microcin J25.

Roped in: The lasso peptide microcin J25 (MccJ25) is matured by two enzymes and is exported by a putative ABC transporter. We probed the function of the maturation enzymes using mutagenesis. We demonstrate that fusions of the enzymes with intervening linkers can produce MccJ25. Even a 151 kDa tripartite fusion between the ABC transporter and the two enzymes is capable of producing and exporting MccJ25.

Risk factors of hospitalization and readmission of patients with COPD in Hong Kong population: analysis of hospital admission records.

BACKGROUND: Chronic Obstructive Pulmonary Disease (COPD) accounts for around 4% of all public hospital annual admissions in Hong Kong. By year 2020, COPD will be ranked fifth among the conditions with the highest burden to the society. This study identifies admission and unplanned readmission of COPD patients, factors affecting unplanned readmission, and estimates its cost burden on the public healthcare system in Hong Kong. METHODS: This is a retrospective study analyzing COPD admissions to all public hospitals in Hong Kong. All admission episodes to acute medical wards with the principal diagnosis of COPD (ICD-9:490-492, 494-496) from January 2006 to December 2007 were captured. Unplanned readmission was defined as an admission which followed a previous admission within 30 days. RESULTS: In 2006 and 2007, 65497 (8.0%) of episodes from medical wards were identified as COPD admissions, and among these, 15882 (24.2%) were unplanned readmissions. The mean age of COPD patients was 76.81 ± 9.59 years and 77% were male. Unplanned readmission was significantly associated with male gender, receiving public assistance and living in nursing homes while no association was found with the Charlson comorbidity index. Patients who were readmitted unplanned had a significant longer acute length of stay (ß = 0.3894, P < 0.001) after adjustment for other covariates. CONCLUSIONS: Unplanned readmission of COPD patients has a huge impact on the public healthcare system. A systematic approach in programme provision and a good discharge planning process targeting on COPD patients who are at high risk of unplanned readmission are essential.

Much of the microcin J25 leader peptide is dispensable.

The antimicrobial peptide microcin J25 (MccJ25) is matured by two enzymes, McjB and McjC, from a 58 amino acid (aa) preprotein, McjA, into its final 21 aa lasso topology. Herein we have investigated the role of the leader peptide of McjA and found that only the eight C-terminal amino acids of this leader peptide are required for maturation of MccJ25. There is a high content of lysine residues in the McjA leader peptide, but herein we also demonstrate that these charged amino acids do not play a major role in the maturation of MccJ25. Alanine scanning mutagenesis studies revealed that the Thr-35 residue in the leader peptide is critical for correct processing of McjA into mature MccJ25. In the absence of detailed structural and biochemical data about McjB and McjC, these studies allow us to propose a putative role for the leader peptide as a simple motif for docking of the McjA preprotein in the maturation enzymes.

Engineered gene clusters for the production of the antimicrobial peptide microcin J25.

Microcin J25 (MccJ25) is an antimicrobial peptide produced by isolates of Escherichia coli with activity against closely related species. Production and export of mature MccJ25 in E. coli requires four genes that are organized on a plasmid-borne cluster in natural producer strains. In these strains, MccJ25 production does not commence until the cells reach stationary phase, and, according to previous literature, the highest titers of MccJ25 are obtained from cells grown in nutrient-poor medium. We sought to design an engineered MccJ25 gene cluster that alleviated the growth phase and media limitations of the natural cluster. In contrast to previous reports, we observe here that production of MccJ25 from its natural cluster is efficient in rich media, such as Luria-Bertani (LB). The engineered gene cluster functions in several E. coli strains and produces titers of MccJ25 that are moderately increased (1.5- to 2-fold) relative to the natural cluster. RT-PCR experiments and translational GFP fusion experiments confirm that the engineered cluster produces MccJ25 throughout exponential phase. Furthermore, we provide evidence that control of the natural MccJ25 gene cluster is at the transcriptional level. The observations herein provide design parameters for large-scale production of MccJ25 for biotechnological applications.

Epitope Mapping Using Yeast Display and Next Generation Sequencing.

Monoclonal antibodies are the largest class of therapeutic proteins due in part to their ability to bind an antigen with a high degree of affinity and specificity. A precise determination of their epitope is important for gaining insights into their therapeutic mechanism of action and to help differentiate antibodies that bind the same antigen. Here, we describe a method to precisely and efficiently map the epitopes of multiple antibodies in parallel over the course of just several weeks. This approach is based on a combination of rational library design, yeast surface display, and next generation DNA sequencing and provides quantitative insights into the epitope residues most critical for the antibody-antigen interaction. As an example, we will use this method to map the epitopes of several antibodies that neutralize alpha toxin from Staphylococcus aureus.

Productive common light chain libraries yield diverse panels of high affinity bispecific antibodies.

The commercial success of bispecific antibodies generally has been hindered by the complexities associated with generating appropriate molecules for both research scale and large scale manufacturing purposes. Bispecific IgG (BsIgG) based on two antibodies that use an identical common light chain can be combined with a minimal set of Fc mutations to drive heavy chain heterodimerization in order to address these challenges. However, the facile generation of common light chain antibodies with properties similar to traditional monoclonal antibodies has not been demonstrated and they have only been used sparingly. Here, we describe the design of a synthetic human antibody library based on common light chains to generate antibodies with biochemical and biophysical properties that are indistinguishable to traditional therapeutic monoclonal antibodies. We used this library to generate diverse panels of well-behaved, high affinity antibodies toward a variety of epitopes across multiple antigens, including mouse 4-1BB, a therapeutically important T cell costimulatory receptor. Over 200 BsIgG toward 4-1BB were generated using an automated purification method we developed that enables milligram-scale production of BsIgG. This approach allowed us to identify antibodies with a wide range of agonistic activity that are being used to further investigate the therapeutic potential of antibodies targeting one or more epitopes of 4-1BB.

Aberrant promoter hypermethylation and silencing of the critical 3p21 tumour suppressor gene, RASSF1A, in Chinese oesophageal squamous cell carcinoma.

3p21 is an important locus harbouring critical tumour suppressor genes (TSG), which are implicated in the pathogenesis of multiple tumours, including oesophageal carcinoma. RASSF1A is a 3p21.3 candidate TSG frequently inactivated by promoter methylation in multiple tumours. We investigated RASSF1A promoter methylation and gene expression in Chinese oesophageal squamous cell carcinoma (ESCC) to compare it to data from Japanese patients. Methylation-specific PCR (MSP) showed that RASSF1A was partially methylated in 3/7 (43%) cell lines; 22/64 (34%) primary tumours and 3/64 (5%) corresponding non-tumour samples; and was not methylated in 2 immortalized normal oesophageal epithelial cell lines and 6 normal oesophageal epithelium samples. Bisulfite genome sequencing confirmed the MSP results. Promoter hypermethylation correlated well with RASSF1A mRNA down-regulation. Treatment of cell lines with 5-aza-2'-deoxycytidine activated RASSF1A mRNA expression along with promoter demethylation. RASSF1A hypermethylation in the Chinese cohort was much lower than in a published report of Japanese ESCC patients (52%) and cell lines (74%). Our own analysis of Japanese ESCC cell lines for direct comparison also detected a high frequency of RASSF1A hypermethylation (8/10; 80%) and high levels of hypermethylation at each CpG site. No significant association between RASSF1A hypermethylation and histological differentiation (p=0.953), tumour staging (p=0.117), or survival (p=0.7571) was found in Chinese ESCC, unlike the results of Japanese patients. The incidence of oesophageal cancer shows marked variation by geographic area and ethnic group; it is almost three times higher in China than in Japan, indicating possible different pathogenetic mechanisms. Our results show that RASSF1A hypermethylation in ESCC has epidemiological/ethnic differences, and suggest that Chinese ESCC may result from different pathogenetic mechanisms.

Lasso Peptide Biosynthetic Protein LarB1 Binds Both Leader and Core Peptide Regions of the Precursor Protein LarA.

Lasso peptides are a member of the superclass of ribosomally synthesized and posttranslationally modified peptides (RiPPs). Like all RiPPs, lasso peptides are derived from a gene-encoded precursor protein. The biosynthesis of lasso peptides requires two enzymatic activities: proteolytic cleavage between the leader peptide and the core peptide in the precursor protein, accomplished by the B enzymes, and ATP-dependent isopeptide bond formation, accomplished by the C enzymes. In a subset of lasso peptide biosynthetic gene clusters from Gram-positive organisms, the B enzyme is split between two proteins. One such gene cluster is found in the organism Rhodococcus jostii, which produces the antimicrobial lasso peptide lariatin. The B enzyme in R. jostii is split between two open reading frames, larB1 and larB2, both of which are required for lariatin biosynthesis. While the cysteine catalytic triad is found within the LarB2 protein, LarB1 is a PqqD homologue expected to bind to the lariatin precursor LarA based on its structural homology to other RiPP leader peptide binding domains. We show that LarB1 binds to the leader peptide of the lariatin precursor protein LarA with a sub-micromolar affinity. We used photocrosslinking with the noncanonical amino acid p-azidophenylalanine and mass spectrometry to map the interaction of LarA and LarB1. This analysis shows that the LarA leader peptide interacts with a conserved motif within LarB1 and, unexpectedly, the core peptide of LarA also binds to LarB1 in several positions. A Rosetta model built from distance restraints from the photocrosslinking experiments shows that the scissile bond between the leader peptide and core peptide in LarA is in a solvent-exposed loop.

Precise and efficient antibody epitope determination through library design, yeast display and next-generation sequencing.

The ability of antibodies to bind an antigen with a high degree of affinity and specificity has led them to become the largest and fastest growing class of therapeutic proteins. Clearly identifying the epitope at which they bind their cognate antigen provides insight into their mechanism of action and helps differentiate antibodies that bind the same antigen. Here, we describe a method to precisely and efficiently map the epitopes of a panel of antibodies in parallel over the course of several weeks. This method relies on the combination of rational library design, quantitative yeast surface display and next-generation DNA sequencing and was demonstrated by mapping the epitopes of several antibodies that neutralize alpha toxin from Staphylococcus aureus. The accuracy of this method was confirmed by comparing the results to the co-crystal structure of one antibody and alpha toxin and was further refined by the inclusion of a lower-affinity variant of the antibody. In addition, this method produced quantitative insight into the epitope residues most critical for the antibody-antigen interaction and enabled the relative affinities of each antibody toward alpha toxin variants to be estimated. This affinity estimate serves as a predictor of neutralizing antibody potency and was used to anticipate the ability of each antibody to effectively bind and neutralize naturally occurring alpha toxin variants secreted by strains of S. aureus, including clinically relevant strains. Ultimately this type information can be used to help select the best clinical candidate among a set of antibodies against a given antigen.

Trends in prevalence and mortality of dementia in elderly Hong Kong population: projections, disease burden, and implications for long-term care.

Background. We describe the trends in prevalence and mortality of dementia among older people in Hong Kong over time. Projections of the number of older people with dementia through 2039 and estimation of the disease burden are also included. Methods. Prevalence data were extracted from previous studies in Hong Kong. Mortality data were obtained from the Department of Health of Hong Kong. Projections of the number of people with dementia were calculated by applying the prevalence rates of dementia obtained from previous studies to Hong Kong population projections. The burden of dementia was measured by Disability-Adjusted Life Years (DALYs). Results. The number of people aged 60 and above with dementia is projected to increase by 222%, from 103,433 in 2009 to 332,688 in 2039, with a large proportion of those living in institutions. The number of deaths due to dementia among people aged 60 and above has more than doubled between 2001 and 2009. Mortality rates for dementia have also risen. In 2006, about 286,313 DALYS were lost due to dementia. Conclusions. The information presented may be used to formulate a long-term care strategy for dementia of the ageing population in Hong Kong.

Computational design of the lasso peptide antibiotic microcin J25.

Microcin J25 (MccJ25) is a 21 amino acid (aa) ribosomally synthesized antimicrobial peptide with an unusual structure in which the eight N-terminal residues form a covalently cyclized macrolactam ring through which the remaining 13 aa tail is fed. An open question is the extent of sequence space that can occupy such an extraordinary, highly constrained peptide fold. To begin answering this question, here we have undertaken a computational redesign of the MccJ25 peptide using a two-stage sequence selection procedure based on both energy minimization and fold specificity. Eight of the most highly ranked sequences from the design algorithm, each of which contained two or three amino acid substitutions, were expressed in Escherichia coli and tested for production and antimicrobial activity. Six of the eight variants were successfully produced by E.coli at production levels comparable with that of the wild-type peptide. Of these six variants, three retain detectable antimicrobial activity, although this activity is reduced relative to wild-type MccJ25. The results here build upon previous findings that even rigid, constrained structures like the lasso architecture are amenable to redesign. Furthermore, this work provides evidence that a large amount of amino acid variation is tolerated by the lasso peptide fold.