First Detailed Genetic Characterization of the Structural Organization of Type III Arginine Catabolic Mobile Elements Harbored by Staphylococcus epidermidis by Using Whole-Genome Sequencing.

The type III arginine catabolic mobile element (ACME) was detected in three Staphylococcus epidermidis oral isolates recovered from separate patients (one healthy, one healthy with dental implants, and one with periodontal disease) based on ACME-arc-operon- and ACME-opp3-operon-directed PCR. These isolates were subjected to whole-genome sequencing to characterize the precise structural organization of ACME III for the first time, which also revealed that all three isolates were the same sequence type, ST329.

Poly(Ethylene glycol)-based backbones with high peptide loading capacities.

Polymer-peptide conjugates are a promising class of compounds, where polymers can be used to overcome some of the limitations associated with peptides intended for therapeutic and/or diagnostic applications. Linear polymers such as poly(ethylene glycol) can be conjugated through terminal moieties and have therefore limited loading capacities. In this research, functionalised linear poly(ethylene glycol)s are utilised for peptide conjugation, to increase their potential loading capacities. These poly(ethylene glycol) derivatives are conjugated to peptide sequences containing representative side-chain functionalised amino acids, using different conjugation chemistries, including copper-catalysed azide-alkyne cycloaddition, amide coupling and thiol-ene reactions. Conjugation of a sequence containing the RGD motif to poly(allyl glycidyl ether) by the thiol-ene reaction, provided a conjugate which could be used in platelet adhesion studies.

Transcending the Gender Binary under International Law: Advancing Health-Related Human Rights for Trans* Populations.

Despite a recent wave in global recognition of the rights of transgender and gender-diverse populations, referred to in this text by the umbrella label of trans*, international law continues to presume a cisgender binary definition of gender - dismissing the lived realities of trans* individuals throughout the world. This gap in international legal recognition and protection has fundamental implications for health, where trans* persons have been and continue to be subjected to widespread discrimination in health care, longstanding neglect of health needs, and significant violations of bodily autonomy.

Evidence for local and international spread of Mycobacterium avium subspecies paratuberculosis through whole genome sequencing of isolates from the island of Ireland.

We describe application of whole genome sequencing (WGS) to a collection of 197 Mycobacterium avium subsp paratuberculosis (MAP) isolates gathered from 122 cattle herds across 27 counties of the island of Ireland. We compare WGS to MAP diversity quantified using mycobacterial interspersed random unit - variable number tandem repeats (MIRU-VNTR). While MIRU-VNTR showed only two major types, WGS could split the 197 isolates into eight major groups. We also found six isolates corresponding to INMV 13, a novel MIRU-VNTR type for Ireland. Evidence for dispersal of MAP across Ireland via cattle movement could be discerned from the data, with mixed infections present in several herds. Furthermore, comparisons of MAP WGS data from Ireland to data from Great Britain and continental Europe revealed many instances of close genetic similarity and hence evidence for international transmission of infection. BEAST MASCOT structured coalescent analyses, with relaxed and strict molecular clocks, estimated the substitution rate to be 0.10-0.13 SNPs/site/year and disclosed greater transitions per lineage per year from Europe to Ireland, indicating transmission into Ireland. Our work therefore reveals new insight into the seeding of MAP infection across Ireland, highlighting how WGS can inform policy formulation to ultimately control MAP transmission at local, national and international scales.

Assessing suitability for sacubitril-valsartan therapy in an Irish cohort: challenges and opportunities.

INTRODUCTION: Sacubitril-valsartan has been shown by the PARADIGM-HF trial to decrease hospital admissions and improve mortality in patients with heart failure with reduced ejection fraction. The PARADIGM trial had stringent exclusion criteria. It is not known how applicable these trial criteria are to real-life practice. In this study, we sought to determine the percentage of patients eligible for sacubitril-valsartan therapy in a level 3 hospital without a dedicated heart failure service. METHODS: All patients discharged from our service with a diagnosis of congestive cardiac using our hospital in-patient enquiry (HIPE) system underwent hierarchal analysis. In order to be deemed eligible for sacubitril-valsartan therapy, patients had to meet PARADIGM-HF inclusion criteria. RESULTS: Our 143 patients represented a more clinically unwell, elderly cohort than the PARADIGM trial study population. Only 24 patients (16.66%) had an ejection fraction of 40% or less. Our results indicate that only 4/143 patients in a real-world setting (2.79%) were eligible for sacubitril-valsartan therapy at the point of discharge as per the PARADIGM-HF study criteria. This is primarily due to the higher than expected percentage of patients in our cohort with an ejection fraction of over 40% (n = 120) and the low percentage of patients on therapeutic doses of ACEI/ARB (n = 15). CONCLUSIONS: Our study showed that a smaller than expected proportion of our patients in real-world practice are suitable for sacubitril-valsartan therapy at discharge. Most patients were in the HFPEF cohort which does not currently have evidence for treatment with sacubitril-valsartan. Low rates of prescribing of basic heart failure medicatons and the absence of dedicated heart failure services in a non-tertiary centre may explain the poor compliance observed. Improving guideline adherence and increasing awareness of evidence-based medication use at primary and secondary care levels would be of benefit to Irish heart failure patients.

First description of arginine catabolic mobile element (ACME) type VI harboring the kdp operon only in Staphylococcus epidermidis using short and long read whole genome sequencing: Further evidence of ACME diversity.

The arginine catabolic mobile element (ACME) was first described in methicillin-resistant Staphylococcus aureus and is considered to enhance transmission, persistence and survival. Subsequently ACMEs were shown to be more prevalent in the coagulase-negative Staphylococcus epidermidis. Previously, ACME types were distinguished by characteristic combinations of the arc and opp3 operons [I (arc+, opp3+), II (arc+, opp3-) and III (arc-, opp3+)] encoding an arginine deaminase pathway and oligopeptide permease transporter, respectively. Recently two novel ACME types harboring the potassium transporter-encoding operon kdp were described in oral S. epidermidis isolates [IV (arc+, opp3-, kdp+), and V (arc+, opp3+, kdp+)]. This study investigated two independent oral S. epidermidis isolates that yielded amplimers with kdp-directed primers only when subjected to ACME typing PCRs. Hybrid assemblies based on Illumina MiSeq short-read and Oxford Nanopore MinION long-read whole genome sequences revealed that both isolates harbored a sixth, novel ACME type (VI) integrated into orfX. Both ACME VIs lacked the arc and opp3 operons, harbored the kdp operon adjacent to other commonly ACME-associated genes including speG, hsd, sdr, and rep, but the structural organization of the adjacent regions were distinct. These ACMEs were flanked by different direct repeat sequences and the ACME VI-positive isolates belonged to unrelated genetic clusters. Overall these findings are indicative of independent evolution. The identification of ACME type VI further illustrates the diversity of ACME elements in S. epidermidis. The presence of ACMEs harboring kdp may confer a selective advantage on oral S. epidermidis in a potassium-rich environment such as found in dental plaque.

Poly(ethylene glycol)-Based Peptidomimetic "PEGtide" of Oligo-Arginine Allows for Efficient siRNA Transfection and Gene Inhibition.

While a wide range of experimental and commercial transfection reagents are currently available, persistent problems remain regarding their suitability for continued development. These include the transfection efficiency for difficult-to-transfect cell types and the risks of decreased cell viability that may arise from any transfection that does occur. Therefore, research is now turning toward alternative molecules that improve the toxicity profile of the gene delivery vector (GDV), while maintaining the transfection efficiency. Among them, cell-penetrating peptides, such as octa-arginine, have shown significant potential as GDVs. Their pharmacokinetic and pharmacodynamic properties can be enhanced through peptidomimetic conversion, whereby a peptide is modified into a synthetic analogue that mimics its structure and/or function, but whose backbone is not solely based on α-amino acids. Using this technology, novel peptidomimetics were developed by co- and postpolymerization functionalization of substituted ethylene oxides, producing poly(ethylene glycol) (PEG)-based peptidomimetics termed "PEGtides". Specifically, a PEGtide of the poly(α-amino acid) oligo-arginine [poly(glycidylguanidine)] was assessed for its ability to complex and deliver a small interfering ribonucleic acid (siRNA) using a range of cell assays and high-content analysis. PEGtide-siRNA demonstrated significantly increased internalization and gene inhibition over 24 h in Calu-3 pulmonary epithelial cells compared to commercial controls and octa-arginine-treated samples, with no evidence of toxicity. Furthermore, PEGtide-siRNA nanocomplexes can provide significant levels of gene inhibition in "difficult-to-transfect" mouse embryonic hypothalamic (mHypo N41) cells. Overall, the usefulness of this novel PEGtide for gene delivery was clearly demonstrated, establishing it as a promising candidate for continued translational research.

First description of novel arginine catabolic mobile elements (ACMEs) types IV and V harboring a kdp operon in Staphylococcus epidermidis characterized by whole genome sequencing.

The arginine catabolic mobile element (ACME) was first described in the methicillin-resistant Staphylococcus aureus strain USA300 and is thought to facilitate survival on skin. To date three distinct ACME types have been characterized comprehensively in S. aureus and/or Staphylococcus epidermidis. Type I harbors the arc and opp3 operons encoding an arginine deaminase pathway and an oligopeptide permease ABC transporter, respectively, type II harbors the arc operon only, and type III harbors the opp3 operon only. To investigate the diversity and detailed genetic organization of ACME, whole genome sequencing (WGS) was performed on 32 ACME-harboring oro-nasal S. epidermidis isolates using MiSeq- and PacBio-based WGS platforms. In nine isolates the ACMEs lacked the opp3 operon, but harbored a complete kdp operon (kdpE/D/A/B/C) located a maximum of 2.8 kb upstream of the arc operon. The kdp operon exhibited 63% DNA sequence identity to the native S. aureus kdp operon. These findings identified a novel, previously undescribed ACME type (designated ACME IV), which could be subtyped (IVa and IVb) based on distinct 5' flanking direct repeat sequences (DRs). Multilocus sequence typing (MLST) sequences extracted from the WGS data identified the sequence types (STs) of the isolates investigated. Four of the nine ACME IV isolates belonged to ST153, and one to ST17, a single locus variant of ST153. A tenth isolate, identified as ST5, harbored another novel ACME type (designated ACME V) containing the kdp, arc and opp3 operons and flanked by DR_F, and DR_B but lacked any internal DRs. ACME V was colocated with a staphylococcal chromosome cassette mec (SCCmec) IV element and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) in a 116.9 kb composite island. The extensive genetic diversity of ACME in S. epidermidis has been further elucidated by WGS, revealing two novel ACME types IV and V for the first time.

Significant Enrichment and Diversity of the Staphylococcal Arginine Catabolic Mobile Element ACME in Staphylococcus epidermidis Isolates From Subgingival Peri-implantitis Sites and Periodontal Pockets.

Staphylococcus aureus and Staphylococcus epidermidis are frequent commensals of the nares and skin and are considered transient oral residents. Reports on their prevalence in the oral cavity, periodontal pockets and subgingivally around infected oral implants are conflicting, largely due to methodological limitations. The prevalence of these species in the oral cavities, periodontal pockets and subgingival sites of orally healthy individuals with/without implants and in patients with periodontal disease or infected implants (peri-implantitis) was investigated using selective chromogenic agar and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Staphylococcus epidermidis was predominant in all participant groups investigated. Its prevalence was significantly higher (P = 0.0189) in periodontal pockets (30%) than subgingival sites of healthy individuals (7.8%), and in subgingival peri-implantitis sites (51.7%) versus subgingival sites around non-infected implants (16.1%) (P = 0.0057). In contrast, S. aureus was recovered from subgingival sites of 0-12.9% of the participant groups, but not from periodontal pockets. The arginine catabolic mobile element (ACME), thought to enhance colonization and survival of S. aureus, was detected in 100/179 S. epidermidis and 0/83 S. aureus isolates screened using multiplex PCR and DNA microarray profiling. Five distinct ACME types, including the recently described types IV and V (I; 14, II; 60, III; 10, IV; 15, V; 1) were identified. ACME-positive S. epidermidis were significantly (P = 0.0369) more prevalent in subgingival peri-implantitis sites (37.9%) than subgingival sites around non-infected implants (12.9%) and also in periodontal pockets (25%) compared to subgingival sites of healthy individuals (4.7%) (P = 0.0167). To investigate the genetic diversity of ACME, 35 isolates, representative of patient groups, sample sites and ACME types underwent whole genome sequencing from which multilocus sequence types (STs) were identified. Sequencing data permitted ACME types II and IV to be subdivided into subtypes IIa-c and IVa-b, respectively, based on distinct flanking direct repeat sequences. Distinct ACME types were commonly associated with specific STs, rather than health/disease states or recovery sites, suggesting that ACME types/subtypes originated amongst specific S. epidermidis lineages. Ninety of the ACME-positive isolates encoded the ACME-arc operon, which likely contributes to oral S. epidermidis survival in the nutrient poor, semi-anaerobic, acidic and inflammatory conditions present in periodontal disease and peri-implantitis.