Professional competence required in advanced practice nursing in critical care: An exploratory qualitative study.

AIM: To identify the required competencies of advanced practice nurses (APNs) working with patients in critical care units in Norway. DESIGN: An exploratory qualitative design. METHODS: Four focus group interviews were performed with 18 nurses who worked in critical care units. The data were examined by inductive content analysis following Graneheim and Lundman's approach. FINDINGS: Our study found that APNs in critical care require the following professional competencies to meet the needs of patients characterised by greater age, comorbidities and increased complexity: (1) intrapersonal skills as revealed in the subthemes of self-awareness; motivation and commitment; strong mental health and upholding ethical standards, (2) advanced clinical decision-making skills as identified in the subthemes of integration of theory and practice; complex practical and technical skills; dealing with increased delegated responsibility and taking the lead in managing increased practice complexity and (3) interpersonal skills, including peer guidance, practising collaboratively and the ability to position oneself.

Predictive Modeling of Induction-Hardened Depth Based on the Barkhausen Noise Signal.

A non-destructive verification method was explored in this work using the Barkhausen noise (BN) method for induction hardening depth measurements. The motive was to investigate the correlation between the hardness depth, microstructure, and the Barkhausen noise signal for an induction hardening process. Steel samples of grade C45 were induction-hardened to generate different hardness depths. Two sets of samples were produced in two different induction hardening equipment for generating the model and verification. The produced samples were evaluated by BN measurements followed by destructive verification of the material properties. The results show great potential for the several BN parameters, especially the magnetic voltage sweep slope signal, which has strong correlation with the hardening depth to depth of 4.5 mm. These results were further used to develop a multivariate predictive model to assess the hardness depth to 7 mm, which was validated on an additional dataset that was holdout from the model training.

Amelioration of systemic inflammation via the display of two different decoy protein receptors on extracellular vesicles.

Extracellular vesicles (EVs) can be functionalized to display specific protein receptors on their surface. However, surface-display technology typically labels only a small fraction of the EV population. Here, we show that the joint display of two different therapeutically relevant protein receptors on EVs can be optimized by systematically screening EV-loading protein moieties. We used cytokine-binding domains derived from tumour necrosis factor receptor 1 (TNFR1) and interleukin-6 signal transducer (IL-6ST), which can act as decoy receptors for the pro-inflammatory cytokines tumour necrosis factor alpha (TNF-α) and IL-6, respectively. We found that the genetic engineering of EV-producing cells to express oligomerized exosomal sorting domains and the N-terminal fragment of syntenin (a cytosolic adaptor of the single transmembrane domain protein syndecan) increased the display efficiency and inhibitory activity of TNFR1 and IL-6ST and facilitated their joint display on EVs. In mouse models of systemic inflammation, neuroinflammation and intestinal inflammation, EVs displaying the cytokine decoys ameliorated the disease phenotypes with higher efficacy as compared with clinically approved biopharmaceutical agents targeting the TNF-α and IL-6 pathways.

Engineered extracellular vesicle decoy receptor-mediated modulation of the IL6 trans-signalling pathway in muscle.

The cytokine interleukin 6 (IL6) is a key mediator of inflammation that contributes to skeletal muscle pathophysiology. IL6 activates target cells by two main mechanisms, the classical and trans-signalling pathways. While classical signalling is associated with the anti-inflammatory activities of the cytokine, the IL6 trans-signalling pathway mediates chronic inflammation and is therefore a target for therapeutic intervention. Extracellular vesicles (EVs) are natural, lipid-bound nanoparticles, with potential as targeted delivery vehicles for therapeutic macromolecules. Here, we engineered EVs to express IL6 signal transducer (IL6ST) decoy receptors to selectively inhibit the IL6 trans-signalling pathway. The potency of the IL6ST decoy receptor EVs was optimized by inclusion of a GCN4 dimerization domain and a peptide sequence derived from syntenin-1 which targets the decoy receptor to EVs. The resulting engineered EVs were able to efficiently inhibit activation of the IL6 trans-signalling pathway in reporter cells, while having no effect on the IL6 classical signalling. IL6ST decoy receptor EVs, were also capable of blocking the IL6 trans-signalling pathway in C2C12 myoblasts and myotubes, thereby inhibiting the phosphorylation of STAT3 and partially reversing the anti-differentiation effects observed when treating cells with IL6/IL6R complexes. Treatment of a Duchenne muscular dystrophy mouse model with IL6ST decoy receptor EVs resulted in a reduction in STAT3 phosphorylation in the quadriceps and gastrocnemius muscles of these mice, thereby demonstrating in vivo activity of the decoy receptor EVs as a potential therapy. Taken together, this study reveals the IL6 trans-signalling pathway as a promising therapeutic target in DMD, and demonstrates the therapeutic potential of IL6ST decoy receptor EVs.

Are degenerative spondylolisthesis and further slippage postoperatively really issues in spinal stenosis surgery?

Background and aims Opinions diverge concerning the prognostic importance of preoperative degenerative spondylolisthesis in patients with lumbar spinal stenosis, as well as the significance of further slippage post-operatively following decompression alone. However, a slip is only one among several factors related to the topic, e.g. duration and intensity of back and leg pain, pre-operative walking ability, number of levels operated and not least the experience of the surgeon. Our aim was to take all of the above-mentioned factors into consideration when analysing the patients' clinical outcome, reported as Change in back pain, Change in leg pain, Overall satisfaction and Change in walking ability, with special emphasis on the possible importance of pre- and/or post-operative degenerative spondylolisthesis. Methods We studied 200 consecutive patients, mean follow-up time 81 months (range 62-108). Before treatment and on the follow-up occasion all patients answered the SF-36 questionnaire and assessed their back and leg pain on a visual analogue scale (VAS). At follow-up the patients were asked about possible changes in back and leg pain (completely free, much better, somewhat better, unchanged, somewhat worse, much worse) and whether they were; satisfied with the outcome, in doubt or not satisfied. Before treatment and at follow-up the presence or not of degenerative spondylolisthesis was determined in the lateral view on a plain X-ray or MRI. By use of a microsurgical technique decompression was achieved in all patients by bilateral laminotomy not sparing the midline ligaments, irrespective of a degenerative spondylolisthesis or not. Eight surgeons with different surgical experience performed the operations. Four separate multivariate analyses were conducted, one for each clinical outcome. The Lasso method was used for variable selection and multiple imputation was applied to handle missing values. Results At follow-up 78.5% of the patients were completely satisfied with the outcome. Minimal clinical important difference (MCID) was achieved for 69% of the patients. Before surgery 28 patients were able to walk more than 1 km compared to 111 at follow-up. The reoperation rate at 6.8 years was 12% further decompressions and 2.5% fusions at the index level. Post-operative slippage was equally common in patients with and without a preoperative slip (around 30%). There were no notable differences in outcome in patients with and without a preoperative slip and no effect of further slippage at the index or another level post-operatively. Nor could the statistical analysis show any of the other covariates (age, gender, duration and intensity of back and leg pain, pre-operative walking ability or number of levels operated) to be of statistically significant importance for predicting the outcome. In the univariate statistical analysis differences were found between the patients of individual surgeons regarding satisfaction, pain improvement, and reoperation rates in favour of surgical experience, which were, however, not statistically significant in the multivariate analysis. Conclusions None of the covariates, including pre-operative spondylolisthesis and further slippage post-operatively, were statistically significant for predicting the clinical outcome. Implication Our results provide no evidence for adding fusion to the decompression.

Statistical Evaluation of Barkhausen Noise Testing (BNT) for Ground Samples.

Barkhausen noise testing (BNT) is a nondestructive method for investigating many properties of ferromagnetic materials. The most common application is the monitoring of grinding burns caused by introducing locally high temperatures while grinding. Other features, such as microstructure, residual stress changes, hardening depth, and so forth, can be monitored as well. Nevertheless, because BNT is a method based on a complex magnetoelectric phenomenon, it is not yet standardized. Therefore, there is a need to study the traceability and stability of the measurement method. This study aimed to carry out a statistical analysis of ferromagnetic samples after grinding processes by the use of BNT. The first part of the experiment was to grind samples in different facilities (Sweden and Finland) with similar grinding parameters, different grinding wheels, and different hardness values. The second part was to evaluate measured BNT parameters to determine significant factors affecting BNT signal value. The measurement data from the samples were divided into two different batches according to where they were manufactured. Both grinding batches contained measurement data from three different participants. The main feature for calculation was the root-mean-square (RMS) value. The first processing step was to normalize the RMS values for all the measurements. A standard analysis of variance (ANOVA) was applied for the normalized dataset. The ANOVA showed that the grinding parameters had a significant impact on the BNT signal value, while the other investigated factors (e.g., participant) were negligible. The reasons for this are discussed at the end of the paper.

A peptide-based vector for efficient gene transfer in vitro and in vivo.

Finding suitable nonviral delivery vehicles for nucleic acid-based therapeutics is a landmark goal in gene therapy. Cell-penetrating peptides (CPPs) are one class of delivery vectors that has been exploited for this purpose. However, since CPPs use endocytosis to enter cells, a large fraction of peptides remain trapped in endosomes. We have previously reported that stearylation of amphipathic CPPs, such as transportan 10 (TP10), dramatically increases transfection of oligonucleotides in vitro partially by promoting endosomal escape. Therefore, we aimed to evaluate whether stearyl-TP10 could be used for the delivery of plasmids as well. Our results demonstrate that stearyl-TP10 forms stable nanoparticles with plasmids that efficiently enter different cell-types in a ubiquitous manner, including primary cells, resulting in significantly higher gene expression levels than when using stearyl-Arg9 or unmodified CPPs. In fact, the transfection efficacy of stearyl-TP10 almost reached the levels of Lipofectamine 2000 (LF2000), however, without any of the observed lipofection-associated toxicities. Most importantly, stearyl-TP10/plasmid nanoparticles are nonimmunogenic, mediate efficient gene delivery in vivo, when administrated intramuscularly (i.m.) or intradermally (i.d.) without any associated toxicity in mice.

Circular RNA interference effector molecules (WO10084371).

Just over a decade after the discovery of RNA interference (RNAi), the RNAi field has begun travelling the bumpy road towards the clinic. Notwithstanding this extraordinarily rapid progress, and despite holding great promise for substantial future clinical impact, RNAi-inducing agents unfortunately exhibit certain physico-chemical and pharmacokinetic drawbacks. The patent application WO10084371 utilizes the exquisite biological mechanism of auto-catalytic intron splicing to generate circularized interfering RNAs (ciRNAs) in order to remedy the issue of rapid nuclease-mediated degradation of RNAi-inducers. The present patent evaluation assesses the utility of the ciRNAs in light of commonly used nucleotide modification strategies for modulating the properties of siRNAs, as well as scrutinizes the experimental data substantiating the patent application in question. The ciRNAs disclosed in WO10084371 exhibit potency on par with exogenously introduced short hairpin RNAs, albeit displaying increased exonuclease resistance. However, experimental validation as to the exact silencing mechanism is sparse, although the potential Dicer-substrate function of the ciRNAs is indeed a promising aspect. Despite the novel, elegant approach of WO10084371, the current gold standard nucleotide modifications appear to deliver sufficient stability for therapeutic applications, meaning that solving the issue of siRNA delivery still remains the major hurdle for clinical success.

Toxicity methods for CPPs.

CPPs have for numerous years been utilized as delivery vectors of various pharmaceutically interesting cargoes, both in vitro and in vivo. As CPPs are gradually approaching the bedsides, investigating toxicity associated with these highly interesting peptides becomes increasingly important and thorough initial assessment of cytotoxicity in vitro is a first step towards advancing these delivery vehicles in to the clinics. The present chapter describes protocols for four cytotoxicity assays in order to provide a toolbox for toxicity assessment of CPPs. The foci lie on membrane integrity (deoxyglucose leakage and propidium iodide assays) and cell viability (the MTT assay), but the chapter also provides a protocol for assessing an important parameter for future clinical applications, namely the hemolytic properties of CPPs.

Application of PepFect peptides for the delivery of splice-correcting oligonucleotides.

One oligonucleotide-based approach that appear very promising for the treatment of different genetic disorders are based on so-called splice-correcting oligonucleotides (SCOs) that are exploited to manipulate splicing patterns. In order to increase the bioavailability, cell-penetrating peptides (CPPs) have readily been covalently conjugated to SCOs to facilitate cellular internalization. While being a successful strategy for the delivery of uncharged oligonucleotides (ONs), it is extremely difficult to generate covalent conjugates between commonly used negatively charged ON analogs and cationic CPPs. Furthermore, high concentrations of ONs in the micromolar range are often needed to obtain biological responses, most likely as a result of endosomal entrapment of material. Therefore, exploring other vectorization methods using CPPs with endosomolytic properties are highly desired.A method of using stearyl modified CPP (i.e., TP10) analogs, named PepFect3 and PepFect4, are being described for the transfection of antisense SCOs using a simple one-step co-incubation procedure. These peptides form complexes with SCOs and efficiently promote cellular uptake by facilitating endosomal escape. This chapter describes the methods of how to form and characterize these nanoparticles and the cellular assay used to address the delivery.

Clostridial translocation peptides for promoting endosomal escape of RNAi agents (WO09083738).

Pharmacological agents harnessing the potency of RNAi (RNA interference) are currently receiving the lion's share of the attention within the drug delivery field, but their clinical utility is unfortunately hampered by their inherent physico-chemical characteristics and pharmacokinetic properties. The patent application WO09083738 pertains to RNA delivery vehicles comprising clostridial neurotoxin-derived polypeptides, for enhancing the intracellular availability of RNAi-inducing pharmacological agents, primarily siRNAs. The present patent evaluation assesses the utility of the claimed delivery vehicles, as well as the merits of the disclosure as a whole, in light of alternative siRNA delivery strategies. WO09083738 discloses the purported usefulness of clostridial translocation domains and non-cytotoxic proteases, as well as targeting moieties and numerous other constituents, for improving delivery of siRNA agents. Despite the potentially interesting delivery concept, and the numerous embodiments encompassed within the highly extensive application, the limited extent of the experimental data is surprising, in particular in view of the broad claims. Thus, considering the increasingly rigorous demands for experimental support placed on life science patent applications, additional in vitro and in vivo validation would undoubtedly have strengthened the case for WO09083738.

A stearylated CPP for delivery of splice correcting oligonucleotides using a non-covalent co-incubation strategy.

Aberrations in splicing patterns play a significant role in several diseases, and splice correction, together with other forms of gene regulation, is consequently an emerging therapeutic target. In order to achieve successful oligonucleotide transfection, efficient delivery vectors are generally necessary. In this study we present one such vector, the chemically modified cell-penetrating peptide (CPP) TP10, for efficient delivery of a splice-correcting 2'-OMe RNA oligonucleotide. Utilizing a functional splice correction assay, we assessed the transfection efficiency of non-covalent complexes of oligonucleotides and stearylated or cysteamidated CPPs. Stearylation of the CPPs Arg9 and penetratin, as well as cysteamidation of MPG and TP10, did not improve transfection, whereas the presence of an N-terminal stearyl group on TP10 improved delivery efficiency remarkably compared to the unmodified peptide. The splice correction levels observed with stearyl-TP10 are in fact in parity with the effects seen with the commercially available transfection agent Lipofectamine 2000. However, the inherent toxicity associated with cationic lipid-based transfections can be completely eliminated when using the stearylated TP10, making this vector highly promising for non-covalent delivery of negatively charged oligonucleotides.

Distinct uptake routes of cell-penetrating peptide conjugates.

Cell-penetrating peptides (CPPs) are a growing family of peptides that have opened a new avenue in drug delivery, allowing various hydrophilic macromolecules to enter cells. In accordance with most other cationic delivery vectors, CPPs seem to rely mostly on endocytosis for internalization. However, due to conflicting results the exact endocytic pathways for CPP uptake have not yet been resolved. Here, we evaluated the ability of seven CPPs, with different chemical properties, to convey peptide nucleic acids (PNAs) inside cells. Assays based on both splice correction, generating biologically active read-out, and on traditional fluorescence measurements were utilized. The same assays were employed to assess different endocytic pathways and the dependence on extracellular heparan sulfates for internalization. Both highly cationic CPPs (M918, penetratin, and Tat) and amphipathic peptides (transportan, TP10, MAP, and pVEC) were investigated in this study. Conjugate uptake relied on endocytosis for all seven peptides but splice-correcting activity varied greatly for the investigated CPPs. The exact endocytic internalization routes were evaluated through the use of well-known endocytosis inhibitors and tracers. In summary, the different chemical properties of CPPs have little correlation with their ability to efficiently deliver splice-correcting PNA. However, conjugates of polycationic and amphipathic peptides appear to utilize different internalization routes.