Broadening the Scope of Sortase-Mediated Ligations using Natural Sortase Homologs

Sortase-mediated ligations have become an attractive option for protein modification chemistry, enabling the synthesis of a wide range of non-natural polypeptide derivatives. In an effort to expand the scope of this methodology, we have been characterizing the in vitro reactivity of a panel of natural sortase homologs. Here we present our studies on the substrate and nucleophile tolerance of sortases from a range of bacterial species. Notable findings include that sortase A from Streptococcus pneumoniae (SrtApneu) shows a high degree of substrate promiscuity, allowing this enzyme to process a range of substrate variations that deviate from the LPXTG substrate motif typically associated with sortase-mediated methods. In addition, this enzyme has the ability to accept an expanded range of primary amine nucleophiles. To demonstrate the utility of this expanded substrate scope, we have also succeeded in using SrtApneu to site-specifically modify the N-terminal serine residue of Dermcidin (DCD-1L). Overall, these results demonstrate that naturally occurring sortases represent a viable approach for the continued development of sortase-mediated protein modification

Evaluating Nucleophile and Substrate Specificities of Sortase A Homologs for Orthogonal Reactivity

Enzymes have become an attractive option for protein modification chemistry due to the remarkable site-specificity they afford. Of particular interest is sortase A from taphylococcus aureus (SrtAaur), which has garnered attention for its ability to install a variety of non-natural modifications to a conserved oligopeptide substrate. In addition to SrtAaur it has become apparent that sortase A homologs exist in other bacterial strains, each of which is potentially a novel catalyst for protein engineering. Previous work has demonstrated that eight representative sortase A homologs exhibit unique specificities for synthetic peptide substrates, capable of identifying characteristic combinations of amino acids in the “sorting motif.” Presented here is a nucleophile profile of the most promiscuous sortase A homolog investigated, that from Streptococcus pneumoniae (SrtApneu). Exhibiting unique specificities, this SrtA variant may enable unique protein modification chemistry

Life Cycle Assessment of Pavements: A Critical Review of Existing Literature and Research

This report provides a critical review of existing literature and modeling tools related to life-cycle assessment (LCA) applied to pavements. The review finds that pavement LCA is an expanding but still limited research topic in the literature, and that the existing body of work exhibits methodological deficiencies and incompatibilities that serve as barriers to the widespread utilization of LCA by pavement engineers and policy makers. This review identifies five key issues in the current body of work: inconsistent functional units, improper system boundaries, imbalanced data for asphalt and cement, use of limited inventory and impact assessment categories, and poor overall utility. This review also identifies common data and modeling gaps in pavement LCAs that should be addressed in future work. These gaps include: the use phase (rolling resistance, albedo, carbonation, lighting, leachate, and tire wear and emissions), asphalt fumes, feedstock energy of bitumen, traffic delay, the maintenance phase, and the end-of-life phase. This review concludes with a comprehensive list of recommendations for future research, which shed light on where improvements in knowledge can be made that will benefit the accuracy and comprehensiveness of pavement LCAs moving forward

Progress Towards the Substrate-bound Structure of Streptococcus pneumoniae Sortase A

Sortases are cysteine transpeptidases found primarily on the cell surface of Gram-positive bacteria. Sortase-mediated ligations have become an attractive option for protein modification chemistry, enabling the synthesis of a wide range of non-natural polypeptide derivatives. Attempts at understanding how these enzymes recognize and bind substrates are integral to furthering their usefulness in protein engineering and, potentially, treatment of bacterial diseases. However, the variable substrate specificity and activity between homologs of these enzymes is not yet fully understood. Of specific interest to us is sortase A from Streptococcus pneumoniae (SrtApneu), as it demonstrates a broad substrate tolerance not observed in other sortase A homologs. Correspondingly, we have made advances towards characterizing a substrate bound structure of SrtApneu in an effort to further understand its unique substrate promiscuity, deviating from the canonical LPXTG sorting signal. Our strategy initially involved generating a non-cleavable peptide analog capable of docking into the active site, however, synthesis of a ketomethylene-linked dipeptide isostere and its insertion into a peptide via solid phase peptide synthesis proved to be more challenging than we anticipated. We revised our approach by designing a substrate harboring an LPACG sorting motif. Peptide preparations with a thiopyridine leaving group favorably facilitated disulfide bridging between the active site and sorting motif cysteines, allowing for elucidation of a SrtApneu structure displaying key interactions that allow the enzyme to recognize a wide-variety of substrates. To this end, we have utilized x-ray crystallography and solution NMR in an attempt to characterize SrtApneu with a bound substrate analog. Although we were unsuccessful, this work has established a foundation for future efforts toward determining the substrate-bound structure of SrtApneu

Online media scans: Applying systematic review techniques to assess statewide human papillomavirus vaccination activities

Background. Although the human papillomavirus (HPV) vaccine has been approved for use in adolescents in the US for over a decade, vaccination uptake remains low. Of concern, HPV vaccine coverage is below the national average in Minnesota, USA. To understand the reach of current HPV programming and research, we use an online media scan; this method may be applied to other jurisdictions to gain insight about various public health issues.Design and Methods. This online media scan describes the nature and scope of ongoing activities to increase HPV vaccination in Minnesota. The media scan included: a) structured internet searches of HPV vaccine health education/promotion activities ongoing in Minnesota since 2013, and b) searches in research databases of the published literature on HPV vaccination in Minnesota from 2013 to 2018. Results. Searches resulted in 880 online and 142 research article matches, with 40 and 36 meeting selection criteria. Results were categorized by activities focusing on race/ethnicity, sex, health providers, parents, lesbian, gay, bisexual, transgender and queer or questioning (LGBTQ) populations, geographic location, catchup vaccination, and insurance status. Most activities were statewide (52% health education/promotion and 35% research), followed by activities located in entirely urban areas (15% health education/promotion and 41% research) with only 6% of health education/promotion activities and 2% of research activities carried out in entirely rural areas.Conclusions. A range of local and statewide HPV vaccine health education/promotion and research activities were identified in Minnesota. Several efforts partnered with American Indian and Somali/Somali-American communities, but fewer activities focused on HPV vaccination among LGBTQ youth and HPV vaccination in rural areas

Photoaquation mechanism of hexacyanoferrate(II) ions: ultrafast 2D UV and transient visible and IR spectroscopies

Ferrous iron(II) hexacyanide in aqueous solutions is known to undergo photoionization and photoaquation reactions depending on the excitation wavelength. To investigate this wavelength dependence, we implemented ultrafast two-dimensional UV transient absorption spectroscopy, covering a range from 280 to 370 nm in both excitation and probing, along with UV pump/visible probe or time-resolved infrared (TRIR) transient absorption spectroscopy and density functional theory (DFT) calculations. As far as photoaquation is concerned, we find that excitation of the molecule leads to ultrafast intramolecular relaxation to the lowest triplet state of the [Fe(CN)6]4– complex, followed by its dissociation into CN– and [Fe(CN)5]3– fragments and partial geminate recombination, all within <0.5 ps. The subsequent time evolution is associated with the [Fe(CN)5]3– fragment going from a triplet square pyramidal geometry, to the lowest triplet trigonal bipyramidal state in 3–4 ps. This is the precursor to aquation, which occurs in ∼20 ps in H2O and D2O solutions, forming the [Fe(CN)5(H2O/D2O)]3– species, although some aquation also occurs during the 3–4 ps time scale. The aquated complex is observed to be stable up to the microsecond time scale. For excitation below 310 nm, the dominant channel is photooxidation with a minor aquation channel. The photoaquation reaction shows no excitation wavelength dependence up to 310 nm, that is, it reflects a Kasha Rule behavior. In contrast, the photooxidation yield increases with decreasing excitation wavelength. The various intermediates that appear in the TRIR experiments are identified with the help of DFT calculations. These results provide a clear example of the energy dependence of various reactive pathways and of the role of spin-states in the reactivity of metal complexes

Prevalence of nuclear and mitochondrial DNA mutations related to adult mitochondrial disease.

OBJECTIVE: The prevalence of mitochondrial disease has proven difficult to establish, predominantly as a result of clinical and genetic heterogeneity. The phenotypic spectrum of mitochondrial disease has expanded significantly since the original reports that associated classic clinical syndromes with mitochondrial DNA (mtDNA) rearrangements and point mutations. The revolution in genetic technologies has allowed interrogation of the nuclear genome in a manner that has dramatically improved the diagnosis of mitochondrial disorders. We comprehensively assessed the prevalence of all forms of adult mitochondrial disease to include pathogenic mutations in both nuclear and mtDNA. METHODS: Adults with suspected mitochondrial disease in the North East of England were referred to a single neurology center from 1990 to 2014. For the midyear period of 2011, we evaluated the minimum prevalence of symptomatic nuclear DNA mutations and symptomatic and asymptomatic mtDNA mutations causing mitochondrial diseases. RESULTS: The minimum prevalence rate for mtDNA mutations was 1 in 5,000 (20 per 100,000), comparable with our previously published prevalence rates. In this population, nuclear mutations were responsible for clinically overt adult mitochondrial disease in 2.9 per 100,000 adults. INTERPRETATION: Combined, our data confirm that the total prevalence of adult mitochondrial disease, including pathogenic mutations of both the mitochondrial and nuclear genomes (≈1 in 4,300), is among the commonest adult forms of inherited neurological disorders. These figures hold important implications for the evaluation of interventions, provision of evidence-based health policies, and planning of future services

Mortality and cardiovascular diseases risk in patients with Barrett's oesophagus:a population-based nationwide cohort study

Background: Patients with Barrett's oesophagus may be at increased risk of mortality overall, and cardiovascular disease has been suggested as the main underlying cause of death. Aim: To examine cause-specific mortality and risk of cardiovascular events among patients with Barrett's oesophagus. Methods: Utilising existing Danish data sources (1997–2011), we identified all patients with histologically verified Barrett's oesophagus (n = 13 435) and 123 526 members of the general population matched by age, sex and individual comorbidities. We calculated cause-specific mortality rates and incidence rates of cardiovascular diseases. We then compared rates between patients with Barrett's oesophagus and the general population comparison cohort, using stratified Cox proportional hazard regression. Results: Patients with Barrett's oesophagus had a 71% increased risk of overall mortality. The cause-specific mortality rate per 1000 person-years for patients with Barrett's oesophagus was 8.5 for cardiovascular diseases, 14.7 for non-oesophageal cancers, and 5.4 for oesophageal cancer. Compared to the general population cohort, corresponding hazard ratios were 1.26 (95% confidence interval (CI): 1.15–1.38), 1.77 (95% CI: 1.65–1.90), and 19.4 (95% CI: 16.1–23.4), respectively. The incidence rates of cardiovascular diseases per 1000 person-years for Barrett's oesophagus patients and for persons from the general population cohort, respectively, varied from 0.4 and 0.2 for subarachnoid bleeding (hazard ratio 1.10, 95% CI: 0.87–1.39) to 8.1 and 5.9 for congestive heart failure (hazard ratio 1.33, 95% CI: 1.21–1.46). Conclusion: Prophylactic measures targeted at cardiovascular diseases and non-oesophageal cancers potentially could be more important than measures against oesophageal cancer, for improving prognosis among patients with Barrett's oesophagus