Permeability evolution of Bentheim Sandstone at simulated georeservoir conditions.

Bentheim Sandstone is considered a suitable conventional georeservoir rock even at great depth because of its mineral composition, homogeneity, micro- and macrostructure, and is also used as a reference material in rock deformation tests. However, a full characterization of the permeability at representative depths has never been performed. Here we report new experimental data where the permeability of Bentheim Sandstone is measured both with a simultaneous variation and with a sequential variation of three different variables to simulate georeservoir conditions. The results indicate a decrease in permeability with simulated increasing depth until 2-3 km, followed by a partial permeability recovery until 4-5 km depth. During the exhumation path, initially, permeability is unaffected, but at shallow depths, a sharp increase in permeability is observed, likely due to microcracking. These variations are a consequence of a complex interaction between stress, pore pressure and temperature, highlighting the importance of experiments considering all three variables when studying the evolution of permeability at depth. These results will aid with the accurate estimation of permeability at different georeservoir conditions.

A subclass of the IS1202 family of bacterial insertion sequences targets XerCD recombination sites.

We describe here a new family of IS which are related to IS1202, originally isolated from Streptococcus pneumoniae in the mid-1990s and previously tagged as an emerging IS family in the ISfinder database. Members of this family have impacted some important properties of their hosts. We describe here another potentially important property of certain family members: specific targeting of xrs recombination sites. The family could be divided into three subgroups based on their transposase sequences and the length on the target repeats (DR) they generate on insertion: subgroup IS1202 (24-29 bp); ISTde1 (15-18 bp); and ISAba32 (5-6 bp). Members of the ISAba32 subgroup were repeatedly found abutting Xer recombinase recombination sites (xrs), separated by an intervening copy of a DR. These xrs sites, present in multiple copies in a number of Acinetobacter plasmids flanking antibiotic resistance genes, were proposed to form a new type of mobile genetic element using the chromosomally-encoded XerCD recombinase for mobility. Transposase alignments identified subgroup-specific indels which may be responsible for the differences in the transposition properties of the three subgroups (i.e. DR length and target specificity). We propose that this collection of IS be classed as a new insertion sequence family: the IS1202 family composed of three subgroups, only one of which specifically targets plasmid-borne xrs. We discuss the implications of xrs targeting for gene mobility.

Retrospective Cohort Study of Safety Outcomes Associated with Opioid Rotations to Buprenorphine.

The objective of this study was to understand the effect buprenorphine rotations have on respiratory risk and other safety outcomes. This was a retrospective observational study evaluating Veterans who underwent an opioid rotation from full-agonist opioids to buprenorphine products or to alternative opioids. The primary endpoint was change in the Risk Index for Overdose or Serious Opioid-induced Respiratory Depression (RIOSORD) score from baseline to six months post-rotation. Median baseline RIOSORD scores were 26.0 and 18.0 in the Buprenorphine Group and the Alternative Opioid Group, respectively. There was no statistically significant difference between groups in baseline RIOSORD score. At six months post-rotation, median RIOSORD scores were 23.5 and 23.0 in the Buprenorphine Group and Alternative Opioid Group, respectively. The difference in change in RIOSORD scores between groups was not statistically significant (p = 0.23). However, based on changes in RIOSORD risk class, an 11% and 0% decrease in respiratory risk was observed in the Buprenorphine and Alternative Opioid groups, respectively. This finding may be considered clinically significant given a change in risk was observed as predicted by RIOSORD score. Further research is needed to clarify the effect that opioid rotations have on respiratory depression risk and other safety outcomes.

Response from Varani et al. to "Comment on 'the IS6 family, a clinically important group of insertion sequences including IS26' by Ruth M. Hall".

The IS6 family of insertion sequences is a large but coherent group which was originally named to avoid confusion between a number of identical or nearly identical IS that were identified at about the same time and given different names (IS15D, IS26, IS46, IS140, IS160, IS176). The underlying common mechanistic feature of all IS6 family members which have been investigated is that they appear to transpose by replicative transposition and form pseudo compound transposons with the flanking IS in direct repeat and in which associated genes are simply transferred to the target replicon and lost from the donor.In the accompanying letter Hall raises a number of very serious and wide-ranging criticisms of our recent review article concerning the IS6 family of insertion sequences. She clearly feels that we have undervalued her work and that we question or ignore certain of her in vivo results. This impression is almost certainly the result of the standard of proof we generally apply to mechanistic aspects of transposition where we think it important to identify transposition intermediates including the types of synaptic, strand cleavage and strand transfer complexes involved.

Characterizing Patients after Opioid Taper in a VA Medical Center.

To identify potential areas for intervention and gain insight on current practice in patients who are tapered to zero morphine equivalent daily doses (MEDD) through the Pharmacy Pain E-Consult at a Veterans Healthcare System. This was done by describing the types of follow-up care and resources utilized by patients. This project is a retrospective chart review of Veterans with non-cancer pain on chronic-opioid therapy consulted to the pharmacy e-consult service and tapered to 0 MEDD. Descriptive statistics are collected one year pre-taper and one year post-taper. One year post-taper, approximately one-third of the patients were re-started on opioid therapy. However, average MEDD overall was significantly decreased one year post-taper compared to pre-taper. Average pain scores were not significantly different pre-taper compared to post-taper. Non-opioid medications generally increased post-taper. Follow-up with mental health and pain management clinical pharmacy specialists decreased post-taper. Tapering to zero MEDD did not lead to a significant increase in pain one-year post-taper; however, approximately 33% of patients were re-started on opioids within one year post-taper. Average MEDD scores decreased post-taper as expected but with effects on mental health being largely unknown, we believe that further study in this area will help us better support patients.

The IS6 family, a clinically important group of insertion sequences including IS26.

The IS6 family of bacterial and archaeal insertion sequences, first identified in the early 1980s, has proved to be instrumental in the rearrangement and spread of multiple antibiotic resistance. Two IS, IS26 (found in many enterobacterial clinical isolates as components of both chromosome and plasmids) and IS257 (identified in the plasmids and chromosomes of gram-positive bacteria), have received particular attention for their clinical impact. Although few biochemical data are available concerning the transposition mechanism of these elements, genetic studies have provided some interesting observations suggesting that members of the family might transpose using an unexpected mechanism. In this review, we present an overview of the family, the distribution and phylogenetic relationships of its members, their impact on their host genomes and analyse available data concerning the particular transposition pathways they may use. We also provide a mechanistic model that explains the recent observations on one of the IS6 family transposition pathways: targeted cointegrate formation between replicons.

Comparing Effectiveness of Gabapentin and Pregabalin in Treatment of Neuropathic Pain: A Retrospective Cohort of Palliative Care Outpatients.

Gabapentin and pregabalin are often considered first line treatment options for various neuropathic pain conditions. The purpose of this retrospective cohort study was to compare clinically meaningful pain reduction and other relevant outcomes among patients prescribed either gabapentin or pregabalin at the University of Arkansas for Medical Sciences (UAMS) Palliative Care Clinic (PCC). The primary endpoint was a significant improvement in pain within six months of initiating either gabapentin or pregabalin. Secondary endpoints included the average number of pills per day as documented at last visit in the study period, the incidence of gabapentinoid dose reductions due to adverse effects, and the incidence of discontinuation of a gabapentinoid due to adverse effects. 222 patients were included in the gabapentin group and 30 patients were included in the pregabalin group. There was not a statistically significant difference between the groups in the primary outcome of pain reduction (p = 0.43). Dose reductions due to adverse effects were statistically significantly (p = 0.03) higher in the gabapentin group than the pregabalin group. The average number of pills per day by a patient in the gabapentin group was 1.8 pills higher than the pregabalin group which was statistically significant (p = 0.01). The results of this analysis support the notion that there is no significant difference in meaningful pain reduction with gabapentin versus pregabalin. This study demonstrates that pregabalin may afford better tolerability and lower pill burden compared to gabapentin.

Toxin-Antitoxin Gene Pairs Found in Tn3 Family Transposons Appear To Be an Integral Part of the Transposition Module.

Much of the diversity of prokaryotic genomes is contributed by the tightly controlled recombination activity of transposons (Tns). The Tn3 family is arguably one of the most widespread transposon families. Members carry a large range of passenger genes incorporated into their structures. Family members undergo replicative transposition using a DDE transposase to generate a cointegrate structure which is then resolved by site-specific recombination between specific DNA sequences (res) on each of the two Tn copies in the cointegrate. These sites also carry promoters controlling expression of the recombinase and transposase. We report here that a number of Tn3 members encode a type II toxin-antitoxin (TA) system, typically composed of a stable toxin and a labile antitoxin that binds the toxin and inhibits its lethal activity. This system serves to improve plasmid maintenance in a bacterial population and, until recently, was believed to be associated with bacterial persistence. At least six different TA gene pairs are associated with various Tn3 members. Our data suggest that several independent acquisition events have occurred. In contrast to most Tn3 family passenger genes, which are generally located away from the transposition module, the TA gene pairs abut the res site upstream of the resolvase genes. Although their role when part of Tn3 family transposons is unclear, this finding suggests a potential role for the embedded TA in stabilizing the associated transposon with the possibility that TA expression is coupled to expression of transposase and resolvase during the transposition process itself.IMPORTANCE Transposable elements (TEs) are important in genetic diversification due to their recombination properties and their ability to promote horizontal gene transfer. Over the last decades, much effort has been made to understand TE transposition mechanisms and their impact on prokaryotic genomes. For example, the Tn3 family is ubiquitous in bacteria, molding their host genomes by the paste-and-copy mechanism. In addition to the transposition module, Tn3 members often carry additional passenger genes (e.g., conferring antibiotic or heavy metal resistance and virulence), and three were previously known to carry a toxin-antitoxin (TA) system often associated with plasmid maintenance; however, the role of TA systems within the Tn3 family is unknown. The genetic context of TA systems in Tn3 members suggests that they may play a regulatory role in ensuring stable invasion of these Tns during transposition.

Detection and Characterization of Transposons in Bacteria.

Bacterial transposons, through their ability to transfer DNA sequences from one position in the genome to another, play a central role in the shape and the evolution of genomes. Extensive studies have been performed during the last five decades to understand the molecular mechanisms involved in the transposition of a variety of elements. Among the methods used, the papillation and the mating out coupled to arbitrary primed PCR assays described in this chapter are widely used as very powerful approaches to detect and characterize transposition events in vivo.

Testing implementation facilitation of a primary care-based collaborative care clinical program using a hybrid type III interrupted time series design: a study protocol.

BACKGROUND: Dissemination of evidence-based practices that can reduce morbidity and mortality is important to combat the growing opioid overdose crisis in the USA. Research and expert consensus support reducing high-dose opioid therapy, avoiding risky opioid-benzodiazepine combination therapy, and promoting multi-modal, collaborative models of pain care. Collaborative care interventions that support primary care providers have been effective in medication tapering. We developed a patient-centered Primary Care-Integrated Pain Support (PIPS) collaborative care clinical program based on effective components of previous collaborative care interventions. Implementation facilitation, a multi-faceted and dynamic strategy involving the provision of interactive problem-solving and support during implementation of a new program, is used to support key organizational staff throughout PIPS implementation. The primary aim of this study is to evaluate the effectiveness of the implementation facilitation strategy for implementing and sustaining PIPS in the Veterans Health Administration (VHA). The secondary aim is to examine the effect of the program on key patient-level clinical outcomes-transitioning to safer regimens and enhancing access to complementary and integrative health treatments. The tertiary aim is to determine the categorical costs and ultimate budget impact of PIPS implementation. METHODS: This multi-site study employs an interrupted time series, hybrid type III design to evaluate the effectiveness of implementation facilitation for a collaborative care clinical program-PIPS-in primary care clinics in three geographically diverse VHA health care systems (sites). Participants include pharmacists and allied staff involved in the delivery of clinical pain management services as well as patients. Eligible patients are prescribed either an outpatient opioid prescription greater than or equal to 90 mg morphine equivalent daily dose or a combination opioid-benzodiazepine regimen. They must also have an upcoming appointment in primary care. The Consolidated Framework for Implementation Research will guide the mixed methods work across the formative evaluation phases and informs the selection of activities included in implementation facilitation. The RE-AIM framework will be used to assess Reach, Effectiveness, Adoption, Implementation, and Maintenance of PIPS. DISCUSSION: This implementation study will provide important insight into the effectiveness of implementation facilitation to enhance uptake of a collaborative care program in primary care, which targets unsafe opioid prescribing practices.

Use of Methadone to Reverse Opioid Escalation in a Patient With Surgical Pain.

Acute pain is a prevalent issue for patients recovering from surgical procedures. Methadone has been recognized as a unique option for treatment of surgical pain due to its multiple mechanisms of analgesia and its potential to decrease tolerance to other opioids. Studies of methadone use in postoperative settings are sparse in part due to safety concerns, such as complex pharmacokinetics, risk of respiratory depression, and association with arrhythmias. In this case study of a 70-year-old male with postsurgical abdominal pain, methadone utilization over a period of 9 days resulted in patient-reported analgesia and aided in de-escalating overall opioid use. More studies are needed to develop guidance on how methadone can be used to relieve pain following surgical procedures.

Targeting IS608 transposon integration to highly specific sequences by structure-based transposon engineering.

Transposable elements are efficient DNA carriers and thus important tools for transgenesis and insertional mutagenesis. However, their poor target sequence specificity constitutes an important limitation for site-directed applications. The insertion sequence IS608 from Helicobacter pylori recognizes a specific tetranucleotide sequence by base pairing, and its target choice can be re-programmed by changes in the transposon DNA. Here, we present the crystal structure of the IS608 target capture complex in an active conformation, providing a complete picture of the molecular interactions between transposon and target DNA prior to integration. Based on this, we engineered IS608 variants to direct their integration specifically to various 12/17-nt long target sites by extending the base pair interaction network between the transposon and the target DNA. We demonstrate in vitro that the engineered transposons efficiently select their intended target sites. Our data further elucidate how the distinct secondary structure of the single-stranded transposon intermediate prevents extended target specificity in the wild-type transposon, allowing it to move between diverse genomic sites. Our strategy enables efficient targeting of unique DNA sequences with high specificity in an easily programmable manner, opening possibilities for the use of the IS608 system for site-specific gene insertions.

The Birth and Demise of the ISApl1-mcr-1-ISApl1 Composite Transposon: the Vehicle for Transferable Colistin Resistance.

The origin and mobilization of the ~2,609-bp DNA segment containing the mobile colistin resistance gene mcr-1 continue to be sources of uncertainty, but recent evidence suggests that the gene originated in Moraxella species. Moreover mcr-1 can be mobilized as an ISApl1-flanked composite transposon (Tn6330), but many sequences have been identified without ISApl1 or with just a single copy (single ended). To further clarify the origins and mobilization of mcr-1, we employed the Geneious R8 software suite to comprehensively analyze the genetic environment of every complete mcr-1 structure deposited in GenBank as of this writing (September 2017) both with and without associated ISApl1 (n = 273). This revealed that the 2,609-bp mcr-1 structure was likely mobilized from a close relative of a novel species of Moraxella containing a chromosomal region sharing >96% nucleotide identity with the canonical sequence. This chromosomal region is bounded by AT and CG dinucleotides, which have been described on the inside ends (IE) of all intact Tn6330 described to date and represent the ancestral 2-bp target site duplications (TSDs) generated by ISApl1 transposition. We further demonstrate that all mcr-1 structures with just one ISApl1 copy or with no ISApl1 copies were formed by deletion of ISApl1 from the ancestral Tn6330, likely by a process related to the "copy-out-paste-in" transposition mechanism. Finally, we show that only the rare examples of single-ended structures that have retained a portion of the excised downstream ISApl1 including the entire inverted right repeat might be capable of mobilization.IMPORTANCE A comprehensive analysis of all intact mcr-1 sequences in GenBank was used to identify a region on the chromosome of a novel Moraxella species with remarkable homology to the canonical mcr-1 structure and that likely represents the origin of this important gene. These data also demonstrate that all mcr-1 structures lacking one or both flanking ISApl1 were formed from ancestral composite transposons that subsequently lost the insertion sequences by a process of abortive transposition. This observation conclusively shows that mobilization of mcr-1 occurs as part of a composite transposon and that structures lacking the downstream ISApl1 are not capable of mobilization.

Known knowns, known unknowns and unknown unknowns in prokaryotic transposition.

Although the phenomenon of transposition has been known for over 60 years, its overarching importance in modifying and streamlining genomes took some time to recognize. In spite of a robust understanding of transposition of some TE, there remain a number of important TE groups with potential high genome impact and unknown transposition mechanisms and yet others, only recently identified by bioinformatics, yet to be formally confirmed as mobile. Here, we point to some areas of limited understanding concerning well established important TE groups with DDE Tpases, to address central gaps in our knowledge of characterised Tn with other types of Tpases and finally, to highlight new potentially mobile DNA species. It is not exhaustive. Examples have been chosen to provide encouragement in the continued exploration of the considerable prokaryotic mobilome especially in light of the current threat to public health posed by the spread of multiple AbR.

Wave-particle energy exchange directly observed in a kinetic Alfvén-branch wave.

Alfvén waves are fundamental plasma wave modes that permeate the universe. At small kinetic scales, they provide a critical mechanism for the transfer of energy between electromagnetic fields and charged particles. These waves are important not only in planetary magnetospheres, heliospheres and astrophysical systems but also in laboratory plasma experiments and fusion reactors. Through measurement of charged particles and electromagnetic fields with NASA's Magnetospheric Multiscale (MMS) mission, we utilize Earth's magnetosphere as a plasma physics laboratory. Here we confirm the conservative energy exchange between the electromagnetic field fluctuations and the charged particles that comprise an undamped kinetic Alfvén wave. Electrons confined between adjacent wave peaks may have contributed to saturation of damping effects via nonlinear particle trapping. The investigation of these detailed wave dynamics has been unexplored territory in experimental plasma physics and is only recently enabled by high-resolution MMS observations.

The impact of insertion sequences on bacterial genome plasticity and adaptability.

Transposable elements (TE), small mobile genetic elements unable to exist independently of the host genome, were initially believed to be exclusively deleterious genomic parasites. However, it is now clear that they play an important role as bacterial mutagenic agents, enabling the host to adapt to new environmental challenges and to colonize new niches. This review focuses on the impact of insertion sequences (IS), arguably the smallest TE, on bacterial genome plasticity and concomitant adaptability of phenotypic traits, including resistance to antibacterial agents, virulence, pathogenicity and catabolism. The direct consequence of IS transposition is the insertion of one DNA sequence into another. This event can result in gene inactivation as well as in modulation of neighbouring gene expression. The latter is usually mediated by de-repression or by the introduction of a complete or partial promoter located within the element. Furthermore, transcription and transposition of IS are affected by host factors and in some cases by environmental signals offering the host an adaptive strategy and promoting genetic variability to withstand the environmental challenges.

Unlocking Tn3-family transposase activity in vitro unveils an asymetric pathway for transposome assembly.

The Tn3 family is a widespread group of replicative transposons that are notorious for their contribution to the dissemination of antibiotic resistance and the emergence of multiresistant pathogens worldwide. The TnpA transposase of these elements catalyzes DNA breakage and rejoining reactions required for transposition. It also is responsible for target immunity, a phenomenon that prevents multiple insertions of the transposon into the same genomic region. However, the molecular mechanisms whereby TnpA acts in both processes remain unknown. Here, we have developed sensitive biochemical assays for the TnpA transposase of the Tn3-family transposon Tn4430 and used these assays to characterize previously isolated TnpA mutants that are selectively affected in immunity. Compared with wild-type TnpA, these mutants exhibit deregulated activities. They spontaneously assemble a unique asymmetric synaptic complex in which one TnpA molecule simultaneously binds two transposon ends. In this complex, TnpA is in an activated state competent for DNA cleavage and strand transfer. Wild-type TnpA can form this complex only on precleaved ends mimicking the initial step of transposition. The data suggest that transposition is controlled at an early stage of transpososome assembly, before DNA cleavage, and that mutations affecting immunity have unlocked TnpA by stabilizing the protein in a monomeric activated synaptic configuration. We propose an asymmetric pathway for coupling active transpososome assembly with proper target recruitment and discuss this model with respect to possible immunity mechanisms.

Mechanisms of Evolution in High-Consequence Drug Resistance Plasmids.

The dissemination of resistance among bacteria has been facilitated by the fact that resistance genes are usually located on a diverse and evolving set of transmissible plasmids. However, the mechanisms generating diversity and enabling adaptation within highly successful resistance plasmids have remained obscure, despite their profound clinical significance. To understand these mechanisms, we have performed a detailed analysis of the mobilome (the entire mobile genetic element content) of a set of previously sequenced carbapenemase-producing Enterobacteriaceae (CPE) from the National Institutes of Health Clinical Center. This analysis revealed that plasmid reorganizations occurring in the natural context of colonization of human hosts were overwhelmingly driven by genetic rearrangements carried out by replicative transposons working in concert with the process of homologous recombination. A more complete understanding of the molecular mechanisms and evolutionary forces driving rearrangements in resistance plasmids may lead to fundamentally new strategies to address the problem of antibiotic resistance. IMPORTANCE: The spread of antibiotic resistance among Gram-negative bacteria is a serious public health threat, as it can critically limit the types of drugs that can be used to treat infected patients. In particular, carbapenem-resistant members of the Enterobacteriaceae family are responsible for a significant and growing burden of morbidity and mortality. Here, we report on the mechanisms underlying the evolution of several plasmids carried by previously sequenced clinical Enterobacteriaceae isolates from the National Institutes of Health Clinical Center (NIH CC). Our ability to track genetic rearrangements that occurred within resistance plasmids was dependent on accurate annotation of the mobile genetic elements within the plasmids, which was greatly aided by access to long-read DNA sequencing data and knowledge of their mechanisms. Mobile genetic elements such as transposons and integrons have been strongly associated with the rapid spread of genes responsible for antibiotic resistance. Understanding the consequences of their actions allowed us to establish unambiguous evolutionary relationships between plasmids in the analysis set.

A Model for Transposition of the Colistin Resistance Gene mcr-1 by ISApl1.

Analysis of mcr-1-containing sequences identified a common ∼2,607-bp DNA segment that in many cases is flanked on one or both ends by ISApl1 We present evidence that mcr-1 is mobilized by an ISApl1 composite transposon which has, in some cases, subsequently lost one or both copies of ISApl1 We also show that mcr-1 can be mobilized in some circumstances by a single upstream copy of ISApl1 in conjunction with the remnants of a downstream ISApl1.

New Small Plasmid Harboring blaKPC-2 in Pseudomonas aeruginosa.

The aim of this study was to characterize the genetic context of blaKPC-2 in Pseudomonas aeruginosa sequence type 244 from Brazil. The blaKPC-2 gene was detected in a new small plasmid, pBH6. Complete sequencing revealed that pBH6 was 3,652 bp long and included the Tn3 resolvase and Tn3 inverted repeat (IR), a partial copy of ISKpn6, and a putative ori region but no rep genes. pBH6 replicated stably into Escherichia coli strain DH10B and P. aeruginosa strain PAO.