The experience of Polish individuals who stutter based on the OASES outcomes.

BACKGROUND: Prior research has shown that stuttering is a complex and individualized condition. The Overall Assessment of the Speaker's Experience of Stuttering (OASES; Yaruss & Quesal, 2016) is a well-researched tool that measures the impact of stuttering on an individual's life. This study has used the Polish version of the OASES to examine the experience of stuttering among Polish people who stutter. METHOD: The original, English version of the OASES was translated into Polish. Reliability and validity for the Polish version were evaluated. Comparisons were made between samples from Poland and the United States for all of the sections and for the overall results of the OASES-S, OASES-T, and OASES-A. To explore the structure of the stuttering experience, a factor structure of the OASES was conducted. RESULTS: Like other versions of the OASES, the OASES-Polish (OASES-PL) demonstrated good reliability and validity. Cross-cultural comparisons have shown that Polish school-age children had significantly lower knowledge and awareness of stuttering than children in the United States (USA). Factor analysis further revealed that the structure of the experience of stuttering is similar across all age groups, but the importance of the particular aspects of stuttering varies at different stages of life. CONCLUSION: The OASES-PL is a reliable and valid instrument for measuring the impact of stuttering on Polish people who stutter and can therefore be used as a clinical tool. Polish results were relatively similar to those from the USA, though there were subtle cross-cultural differences that are worthy of further exploration. EDUCATIONAL OBJECTIVES: After reading the article, the participant will be able: (1) to describe the diverse experiences of Polish people who stutter at different ages, (2) to explain the importance of quality of life analysis in diagnosis and speech therapy with people who stutter, and (3) to explain the breadth of the stuttering phenomenon among Polish individuals who stutter.

One More Step towards a Circular Economy for Thermal Insulation Materials-Development of Composites Highly Filled with Waste Polyurethane (PU) Foam for Potential Use in the Building Industry.

The rapid development of the building sector has created increased demand for novel materials and technologies, while on the other hand resulting in the generation of a severe amount of waste materials. Among these are polyurethane (PU) foams, which are commonly applied as thermal insulation materials. Their management is a serious industrial problem, due to, for example, their complex chemical composition. Although some chemical and thermochemical methods of PU foam recycling are known, their broader use is limited due to requirements related to the complexity and safety of their installation, thus implicating high costs. Therefore, material recycling poses a promising alternative. The incorporation of waste PU foams as fillers for polymer composites could make it possible to take advantage of their structure and performance. Herein, polypropylene-based composites that were highly filled with waste PU foam and modified using foaming agents were prepared and analyzed. Depending on the foam loading and the foaming agent applied, the apparent density of material was reduced by as much as 68%. The efficient development of a porous structure, confirmed by scanning electron microscopy and high-resolution computed micro-tomography, enabled a 64% decrease in the thermal conductivity coefficient. The foaming of the structure affected the mechanical performance of composites, resulting in a deterioration of their tensile and compressive performance. Therefore, developing samples of the analyzed composites with the desired performance would require identifying the proper balance between mechanical strength and economic, as well as ecological (share of waste material in composite, apparent density of material), considerations.

Preclinical development of a molecular clamp-stabilised subunit vaccine for severe acute respiratory syndrome coronavirus 2.

OBJECTIVES: Efforts to develop and deploy effective vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue at pace. Here, we describe rational antigen design through to manufacturability and vaccine efficacy of a prefusion-stabilised spike (S) protein, Sclamp, in combination with the licensed adjuvant MF59 'MF59C.1' (Seqirus, Parkville, Australia). METHODS: A panel recombinant Sclamp proteins were produced in Chinese hamster ovary and screened in vitro to select a lead vaccine candidate. The structure of this antigen was determined by cryo-electron microscopy and assessed in mouse immunogenicity studies, hamster challenge studies and safety and toxicology studies in rat. RESULTS: In mice, the Sclamp vaccine elicits high levels of neutralising antibodies, as well as broadly reactive and polyfunctional S-specific CD4+ and cytotoxic CD8+ T cells in vivo. In the Syrian hamster challenge model (n = 70), vaccination results in reduced viral load within the lung, protection from pulmonary disease and decreased viral shedding in daily throat swabs which correlated strongly with the neutralising antibody level. CONCLUSION: The SARS-CoV-2 Sclamp vaccine candidate is compatible with large-scale commercial manufacture, stable at 2-8°C. When formulated with MF59 adjuvant, it elicits neutralising antibodies and T-cell responses and provides protection in animal challenge models.

Upregulation of HIF1-α via an NF-κB/COX2 pathway confers proliferative dominance of HER2-negative ductal carcinoma in situ cells in response to inflammatory stimuli.

There are data to suggest that some ductal carcinoma in situ (DCIS) may evolve through an evolutionary bottleneck, where minor clones susceptible to the imposed selective pressure drive disease progression. Here, we tested the hypothesis that an impact of the inflammatory environment on DCIS evolution is HER2-dependent, conferring proliferative dominance of HER2-negative cells. In tissue samples, density of tumour-infiltrating immune cells (TIICs) was associated only with high tumour nuclear grade, but in 9% of predominantly HER2-negative cases, the presence of tumoral foci ('hot-spots') of basal-like cells with HIF1-α activity adjacent to the areas of dense stromal infiltration was noted. Results of in vitro analyses further demonstrated that IL-1ß and TNF-α as well as macrophage-conditioned medium triggered phosphorylation of NF-κB and subsequent upregulation of COX2 and HIF1-α, exclusively in HER2-negative cells. Treatment with both IL-1ß and TNF-α resulted in growth stimulation and inhibition of HER2-negative and HER2-positive cells, respectively. Moreover, ectopic overexpression of HIF1-α rescued HER2-positive cells from the negative effect of IL-1ß and TNF-α on cell growth. Our data provide novel insight into the molecular basis of HER2-dependent proliferation of DCIS cells and indicate the NF-κB/COX2 → HIF1-α signalling axis as a dominant mechanism of DCIS evolution induced by inflammatory microenvironment. Presented findings also highlight the clinical significance of heterogeneity of DCIS tumours and suggest that HIF1-α might be considered as a predictive marker of disease progression.

Ossifying fasciitis at an extraordinary site - a case report and analysis of diagnostic pitfalls.

Ossifying fasciitis is a very rare disease of reactive character; however, it can mimic malignant lesions, especially osteosarcoma. We report a case of a 30-year-old woman, who experienced a rapidly growing painful lesion of the left knee joint, preceded by a trauma. The tumor was resected, and the histopathological image suggested a malignant lesion with features of an osteosarcoma. A detailed correlation with a clinicopathological and radiological analysis led to the final diagnosis of ossifying fasciitis at an extraordinary site of patellar retinaculum. Our case shows that the close similarity between ossifying fasciitis and osteosarcoma may be challenging.

Proteomics reveals the in vitro protein digestibility of seven transmembrane enzymes from the docosahexaenoic acid biosynthesis pathway.

The measurement of protein digestibility is one of the key steps in determining the safety of a genetically modified crop that has been traditionally accomplished using antibodies. Membrane proteins are often extremely difficult to express with replicated authentic tertiary structure in heterologous systems. As a result raising antibodies for use in safety assessment may not be feasible. In this study, LC-MS based proteomics was used to characterise seven transmembrane enzymes from the docosahexaenoic acid biosynthetic pathway that had been introduced into canola. The application of a two-stage digestion strategy involving simulated gastric fluid followed by trypsin enabled the measurement of protein digestibility in vitro. Tryptic peptide markers that spanned the length of each desaturase protein were monitored and showed that these proteins were readily degraded (>95% within 5 min) and highlighted regions of the elongase enzymes that showed limited resistance to simulated gastric digestion. Traditional gel-based and Western blotting analysis of ω3-desaturase and Δ6-elongase revealed rapid hydrolysis of the intact proteins within seconds and no fragments (>3 kDa) remained after 60 min, complementing the novel approach described herein. The LC-MS approach was sensitive, selective and did not require the use of purified proteins.

Quantitation of seven transmembrane proteins from the DHA biosynthesis pathway in genetically engineered canola by targeted mass spectrometry.

Examining tissue-specific expression and the measurement of protein abundance are important steps when assessing the performance of genetically engineered crops. Liquid chromatography-mass spectrometry offers many advantages over traditional methods for protein quantitation, especially when dealing with transmembrane proteins that are often difficult to express or generate antibodies against. In this study, discovery proteomics was used to detect the seven transgenic membrane-bound enzymes from the docosahexaenoic acid (DHA) biosynthetic pathway that had been engineered into canola. Subsequently, a targeted LC-MS/MS method for absolute quantitation was developed and applied to the simultaneous measurement of the seven DHA biosynthetic pathway enzymes in genetically modified canola grown across three sites. The results of this study demonstrated that the enzymatic proteins that drive the production of DHA using seed-specific promoters were detected only in mature and developing seed of DHA canola. None of the DHA biosynthesis pathway proteins were detected in wild-type canola planted in the same site or in the non-seed tissues of the transgenic canola, irrespective of the sampling time or the tissues tested. This study describes a streamlined approach to simultaneously measure multiple membrane-bound proteins in planta.

Stabilization of a prokaryotic LAT transporter by random mutagenesis.

The knowledge of three-dimensional structures at atomic resolution of membrane transport proteins has improved considerably our understanding of their physiological roles and pathological implications. However, most structural biology techniques require an optimal candidate within a protein family for structural determination with (a) reasonable production in heterologous hosts and (b) good stability in detergent micelles. SteT, the Bacillus subtilis L-serine/L-threonine exchanger is the best-known prokaryotic paradigm of the mammalian L-amino acid transporter (LAT) family. Unfortunately, SteT's lousy stability after extracting from the membrane prevents its structural characterization. Here, we have used an approach based on random mutagenesis to engineer stability in SteT. Using a split GFP complementation assay as reporter of protein expression and membrane insertion, we created a library of 70 SteT mutants each containing random replacements of one or two residues situated in the transmembrane domains. Analysis of expression and monodispersity in detergent of this library permitted the identification of evolved versions of SteT with a significant increase in both expression yield and stability in detergent with respect to wild type. In addition, these experiments revealed a correlation between the yield of expression and the stability in detergent micelles. Finally, and based on protein delipidation and relipidation assays together with transport experiments, possible mechanisms of SteT stabilization are discussed. Besides optimizing a member of the LAT family for structural determination, our work proposes a new approach that can be used to optimize any membrane protein of interest.

Assessment of membrane protein expression and stability using a split green fluorescent protein reporter.

Membrane proteins are challenging targets for structural biologists. Finding optimal candidates for such studies requires extensive and laborious screening of protein expression and/or stability in detergent. The use of green fluorescent protein (GFP) as a reporter has enormously facilitated these studies; however, its 238 residues can potentially alter the intrinsic properties of the target (e.g., expression or stability). With the aim of minimizing undesired effects of full-length GFP, here we describe the utility of a split GFP reporter during precrystallization studies of membrane proteins. GFP fluorescence appeared by complementation of the first 15 residues of GFP (GFP(11)) (fused to the C terminus of a membrane protein target) with the remaining nonfluorescent GFP (GFP(1-10)). The signal obtained after sequential expression of SteT (l-serine/l-threonine exchanger of Bacillus subtilis) fused to GFP(11) followed by GFP(1-10) specifically measured the protein fraction inserted into the Escherichia coli cytoplasmic membrane, thereby discarding protein aggregates confined as inclusion bodies. Furthermore, in vitro complementation of purified SteT-GFP(11) with purified GFP(1-10) was exploited to rapidly assess the stability of wild-type and G294V mutant versions of SteT-GFP(11) following detergent solubilization and purification. This method can be applied in a medium- to high-throughput manner with multiple samples.

Algorithm for the management of endoscopic perforations: a quality improvement project.

Perforations are an uncommon but serious complication of endoscopy. Although they are well recognized, no universally accepted strategy for their management exists. The need for management algorithms in situations that call for multiple interventions in a short time, with coordinated effort encompassing multiple providers from different specialties, has long been recognized, but no such clinical care pathway has been developed for the management of endoscopic perforations. Since perforations are uncommon, a predetermined plan of action can streamline patient management. Furthermore, such a plan demonstrates preparedness on the part of the gastroenterologist. We developed an endoscopic perforation management strategy based on the best available scientific evidence and our specific resources. We report our experience in the hope that it may form a useful framework for gastroenterologists attempting to do the same at their own institution.

Molecular basis of substrate-induced permeation by an amino acid antiporter.

Transporters of the amino acid, polyamine and organocation (APC) superfamily play essential roles in cell redox balance, cancer, and aminoacidurias. The bacterial L-arginine/agmatine antiporter, AdiC, is the main APC structural paradigm and shares the "5 + 5 inverted repeat" fold found in other families like the Na(+)-coupled neurotransmitter transporters. The available AdiC crystal structures capture two states of its transport cycle: the open-to-out apo and the outward-facing Arg(+)-bound occluded. However, the role of Arg(+) during the transition between these two states remains unknown. Here, we report the crystal structure at 3.0 Å resolution of an Arg(+)-bound AdiC mutant (N101A) in the open-to-out conformation, completing the picture of the major conformational states during the transport cycle of the 5 + 5 inverted repeat fold-transporters. The N101A structure is an intermediate state between the previous known AdiC conformations. The Arg(+)-guanidinium group in the current structure presents high mobility and delocalization, hampering substrate occlusion and resulting in a low translocation rate. Further analysis supports that proper coordination of this group with residues Asn101 and Trp293 is required to transit to the occluded state, providing the first clues on the molecular mechanism of substrate-induced fit in a 5 + 5 inverted repeat fold-transporter. The pseudosymmetry found between repeats in AdiC, and in all fold-related transporters, restraints the conformational changes, in particular the transmembrane helices rearrangements, which occur during the transport cycle. In AdiC these movements take place away from the dimer interface, explaining the independent functioning of each subunit.

Complications during natural orifice translumenal endoscopic surgery: endoscopic management of splenic laceration and hemorrhage.

BACKGROUND: Accidental splenic laceration and hemorrhage during natural orifice translumenal endoscopic surgery (NOTES) can lead to life-threatening consequences. The NOTES approach may need to be aborted in these circumstances for a standard laparoscopy or laparotomy. AIM: To determine the feasibility of endoscopically managing intraoperative splenic laceration and hemorrhage during NOTES using standard endoscopic tools. METHODS: Nine pigs underwent transcolonic endoscopic surgery, and 18 intentional splenic lacerations were made. Animals were treated as follows: (1) control group with no therapy (n = 3), (2) endoscopic tamponade/packing (n = 3), and (3) endoscopic hemostasis with bipolar cautery (n = 12). A blinded second endoscopist performed NOTES exploration and attempted to identify the site and treat the laceration in 3 cases. The colonic incision was closed using endoclips in the survival studies. Necropsy was performed immediately after surgery in acute cases and at the end of 1 week in the survival cases. RESULTS: Bleeding persisted beyond 10 minutes in all control cases without therapy. In the tamponade group, bleeding persisted beyond 17 minutes in 2 and a large clot formed at 12 minutes in 1 case that precluded further assessment. Bleeding was controlled endoscopically using standard bipolar cautery in all animals (mean time: 12 minutes). All lacerations were identified and managed by the blinded endoscopist. Survival animals had an uncomplicated postoperative course. No bleeding was seen at necropsy. CONCLUSION: We demonstrate the management of intraoperative splenic hemorrhage during NOTES using standard endoscopic tools. The site of splenic bleeding could be correctly identified and treated in a blinded fashion.

Role of transmembrane domain 8 in substrate selectivity and translocation of SteT, a member of the L-amino acid transporter (LAT) family.

System l-amino acid transporters (LAT) belong to the amino acid, polyamine, and organic cation superfamily of transporters and include the light subunits of heteromeric amino acid transporters and prokaryotic homologues. Cysteine reactivity of SteT (serine/threonine antiporter) has been used here to study the substrate-binding site of LAT transporters. Residue Cys-291, in transmembrane domain 8 (TM8), is inactivated by thiol reagents in a substrate protectable manner. Surprisingly, DTT activated the transporter by reducing residue Cys-291. Cysteine-scanning mutagenesis of TM8 showed DTT activation in the single-cysteine mutants S287C, G294C, and S298C, lining the same alpha-helical face. S-Thiolation in Escherichia coli cells resulted in complete inactivation of the single-cysteine mutant G294C. l-Serine blocked DTT activation with an EC(50) similar to the apparent K(M) of this mutant. Thus, S-thiolation abolished substrate translocation but not substrate binding. Mutation of Lys-295, to Cys (K295C) broadened the profile of inhibitors and the spectrum of substrates with the exception of imino acids. A structural model of SteT based on the structural homologue AdiC (arginine/agmatine antiporter) positions residues Cys-291 and Lys-295 in the putative substrate binding pocket. All this suggests that Lys-295 is a main determinant in the recognition of the side chain of SteT substrates. In contrast, Gly-294 is not facing the surface, suggesting conformational changes involving TM8 during the transport cycle. Our results suggest that TM8 sculpts the substrate-binding site and undergoes conformational changes during the transport cycle of SteT.

Prospective randomized blinded comparison of a short-wire endoscopic retrograde cholangiopancreatography system with traditional long-wire devices.

BACKGROUND: Short-wire endoscopic retrograde cholangiopancreatography (ERCP) systems are perceived to carry advantage over traditional long-wire devices. To date, this potential advantage has not been well documented, and gastroenterologists are confronted in everyday practice with the dilemma of choosing a particular system without the benefit of having objective comparative data. AIMS: The aim of this study was to compare the performance characteristics of the Fusion ERCP short-wire system with traditional long-wire devices. METHODS: This is a prospective, blinded, randomized, controlled trial. Patients with a clinical indication for ERCP were randomized to undergo the procedure with the Fusion short-wire system or long-wire devices. All procedures were done by one experienced endoscopist who was blinded to the outcomes of the study. The person recording the outcomes was an independent observer not involved in the procedure and was blinded to the study hypothesis. The main outcome was device exchange time. Secondary outcomes included stent insertion time, total procedure time, fluoroscopy time, cannulation time, successful cannulation of the desired duct, and complications. RESULTS: A total of 71 patients were enrolled. The short-wire system provided for significantly faster mean device exchange time (125 versus 177 s; P = 0.05) and stent insertion time (135 versus 254 s; P < 0.001) as compared with the long-wire system. A trend towards shorter total procedure time, fluoroscopy time, and cannulation time was noted with the short-wire system but did not reach statistical significance. Successful cannulation of the desired duct was achieved in all patients. Post-ERCP pancreatitis occurred in one patient in the short-wire and in two patients in the long-wire group. CONCLUSIONS: This short-wire system provides for significantly shorter device exchange and stent insertion times compared with traditional long-wire devices.

Prospective trial comparing solid-state catheter and water-perfusion triple-lumen catheter for sphincter of Oddi manometry done at the time of ERCP.

BACKGROUND: Sphincter of Oddi manometry is the reference standard for the diagnosis of sphincter of Oddi dysfunction. Numerous studies have established ranges of normal values as well as typical readings in pathologic conditions. All these studies have been done using a water-perfused, triple-lumen catheter (TLC). A recently approved, new generation, solid-state catheter (SSC) has potential advantages, but concerns have been raised as to whether the pressures obtained by TLCs are reproducible by SSCs. To date, no data exist on the accuracy of sphincter of Oddi pressure measurements with the new-generation SSCs. OBJECTIVE: To evaluate the accuracy of the SSC by using the TLC as the reference standard. DESIGN: Prospective crossover trial. SETTING: A tertiary academic center. PATIENTS: Thirty patients with clinical indications for sphincter of Oddi manometry. INTERVENTIONS: Sphincter of Oddi manometry with TLC and SSC in the same patient. MAIN OUTCOME MEASUREMENTS: Accuracy of sphincter of Oddi pressure measurements. RESULTS: A total of 376 pressure measurements in 47 sphincter segments (24 biliary, 23 pancreatic) were obtained. Manometry results were abnormal in 10 of 24 biliary sphincters and 12 of 23 pancreatic sphincters. There was complete agreement on the final results of the sphincter of Oddi manometry (normal/abnormal) between the TLC and SSC (accuracy 100%). A split-plot analysis of the 378 individual measurements was performed. The P value of .9966 was insignificant, consistent with no catheter effect on the measurements. LIMITATIONS: Lack of blinding. CONCLUSIONS: Measurement of sphincter of Oddi pressures with the SSC is accurate, and results were essentially identical to those of the water-perfused catheter system.

Endoscopic therapy for chronic pancreatitis: technical success, clinical outcomes, and complications.

Chronic pancreatitis (CP) can cause failure of both the exocrine and endocrine portions of the gland. Pain is the most recalcitrant clinical complaint in CP. Generally, conservative measures are first attempted to manage pain. These include cessation of alcohol use and smoking, enzyme replacement therapy, and finally, opioid analgesia. Endoscopy can be employed to treat the pain and complications due to CP. The results of the only two prospective randomized controlled trials suggest that surgery has a more durable effect than endoscopic therapy in controlling pain. Both trials suffer from severe limitations, however, and endoscopy remains the preferred approach for many patients because of its minimally invasive nature. Endoscopic ultrasound celiac plexus block has limited value in helping to control pain. More randomized trials are needed, along with further technologic innovation to improve the current treatment modalities. When considering interventional therapy for a patient with CP, a tailored and multidisciplinary therapeutic approach should be taken.

Specific mutations within the AT-rich region of a plasmid replication origin affect either origin opening or helicase loading.

Prokaryotic and eukaryotic replicons possess a distinctive region containing a higher than average number of adenine and thymine residues (the AT-rich region) where, during the process of replication initiation, the initial destabilization (opening) of the double helix takes place. In many prokaryotic origins, this region consists of repeated 13-mer motifs whose function has not yet been specified. Here we identify specific mutations within the 13-mer sequences of the broad-host-range plasmid RK2 that can result in defective origin opening or that do not affect opening but induce defects in helicase loading. We also show that after the initial recruitment of helicase at the DnaA-box sequences of the plasmid origin, the helicase is translocated to the AT-rich region in a reaction requiring specific sequence of the 13-mers and appropriate facing of the origin motifs. Our results demonstrate that specific sequences within the AT-rich region of a replication origin are required for either origin opening or helicase loading.

Positioning and the specific sequence of each 13-mer motif are critical for activity of the plasmid RK2 replication origin.

The minimal replication origin of the broad-host-range plasmid RK2, oriV, contains five iterons which are binding sites for the plasmid-encoded replication initiation protein TrfA, four DnaA boxes, which bind the host DnaA protein, and an AT-rich region containing four 13-mer sequences. In this study, 26 mutants with altered sequence and/or spacing of 13-mer motifs have been constructed and analysed for replication activity in vivo and in vitro. The data show that the replacement of oriV 13-mers by similar but not identical 13-mer sequences from Escherichia coli oriC inactivates the origin. In addition, interchanging the positions of the oriV 13-mers results in greatly reduced activity. Mutants with T/A substitutions are also inactive. Furthermore, introduction of single-nucleotide substitutions demonstrates very restricted sequence requirements depending on the 13-mer position. Only two of the mutants are host specific, functional in Pseudomonas aeruginosa but not in E. coli. Our experiments demonstrate considerable complexity in the plasmid AT-rich region architecture required for functionality. It is evident that low internal stability of this region is not the only feature contributing to origin activity. Our studies suggest a requirement for sequence-specific protein interactions within the 13-mers during assembly of replication complexes at the plasmid origin.