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High speed electrospinning (HSES), compatible with pharmaceutical industry, was used to demonstrate the viability of the preparation of drug-loaded polymer nanofibers with radically ...higher productivity than the known single-needle electrospinning (SNES) setup. Poorly water-soluble itraconazole (ITRA) was formulated with PVPVA64 matrix polymer using four different solvent-based methods such as HSES, SNES, spray drying (SD) and film casting (FC). The formulations were assessed in terms of improvement in the dissolution rate of ITRA (using a “tapped basket” dissolution configuration) and analysed by SEM, DSC and XRPD. Despite the significantly increased productivity of HSES, the obtained morphology was very similar to the SNES nanofibrous material. ITRA transformed into an amorphous form, according to the DSC and XRPD results, in most cases except the FC samples. The limited dissolution of crystalline ITRA could be highly improved: fast dissolution occurred (>90% within 10min) in the cases of both (the scaled-up and the single-needle) types of electrospun fibers, while the improvement in the dissolution rate of the spray-dried microspheres was significantly lower. Production of amorphous solid dispersions (ASDs) with the HSES system proved to be flexibly scalable and easy to integrate into a continuous pharmaceutical manufacturing line, which opens new routes for the development of industrially relevant nanopharmaceuticals.
Abstract Evidence has been accumulating that elements of the vertebrate pituitary adenylate cyclase-activating polypeptide (PACAP) system are missing in non-chordate genomes, which is at odds with ...the partial sequence-, immunohistochemical-, and physiological data in the literature. Multilevel experiments were performed on the great pond snail ( Lymnaea stagnalis ) to explore the role of PACAP in invertebrates. Screening of neuronal transcriptome and genome data did not reveal homologs to the elements of vertebrate PACAP system. Despite this, immunohistochemical investigations with an anti-human PAC 1 receptor antibody yielded a positive signal in the neuronal elements in the heart. Although Western blotting of proteins extracted from the nervous system found a relevant band for PACAP-38, immunoprecipitation and mass spectrometric analyses revealed no corresponding peptide fragments. Similarly to the effects reported in vertebrates, PACAP-38 significantly increased cAMP synthesis in the heart and had a positive ionotropic effect on heart preparations. Moreover, it significantly modulated the effects of serotonin and acetylcholine. Homologs to members of Cluster B receptors, which have shared common evolutionary origin with the vertebrate PACAP receptors, PTHRs, and GCGRs, were identified and shown not to be expressed in the heart, which does not support a potential role in the mediation of PACAP-induced effects. Our findings support the notion that the PACAP system emerged after the protostome-deuterostome divergence. Using antibodies against vertebrate proteins is again highlighted to have little/no value in invertebrate studies. The physiological effects of vertebrate PACAP peptides in protostomes, no matter how similar they are to those in vertebrates, should be considered non-specific.
Abstract
The COVID-19 pandemic has demonstrated the need for massively-parallel, cost-effective tests monitoring viral spread. Here we present SARSeq,
saliva analysis by RNA sequencing
, a method to ...detect SARS-CoV-2 and other respiratory viruses on tens of thousands of samples in parallel. SARSeq relies on next generation sequencing of multiple amplicons generated in a multiplexed RT-PCR reaction. Two-dimensional, unique dual indexing, using four indices per sample, enables unambiguous and scalable assignment of reads to individual samples. We calibrate SARSeq on SARS-CoV-2 synthetic RNA, virions, and hundreds of human samples of various types. Robustness and sensitivity were virtually identical to quantitative RT-PCR. Double-blinded benchmarking to gold standard quantitative-RT-PCR performed by human diagnostics laboratories confirms this high sensitivity. SARSeq can be used to detect Influenza A and B viruses and human rhinovirus in parallel, and can be expanded for detection of other pathogens. Thus, SARSeq is ideally suited for differential diagnostic of infections during a pandemic.
The low reproducibility rate in social sciences has produced hesitation among researchers in accepting published findings at their face value. Despite the advent of initiatives to increase ...transparency in research reporting, the field is still lacking tools to verify the credibility of research reports. In the present paper, we describe methodologies that let researchers craft highly credible research and allow their peers to verify this credibility. We demonstrate the application of these methods in a multi-laboratory replication of Bem's Experiment 1 (Bem 2011
, 407-425. (doi:10.1037/a0021524)) on extrasensory perception (ESP), which was co-designed by a consensus panel including both proponents and opponents of Bem's original hypothesis. In the study we applied direct data deposition in combination with born-open data and real-time research reports to extend transparency to protocol delivery and data collection. We also used piloting, checklists, laboratory logs and video-documented trial sessions to ascertain as-intended protocol delivery, and external research auditors to monitor research integrity. We found 49.89% successful guesses, while Bem reported 53.07% success rate, with the chance level being 50%. Thus, Bem's findings were not replicated in our study. In the paper, we discuss the implementation, feasibility and perceived usefulness of the credibility-enhancing methodologies used throughout the project.
Previous studies have described the structure and function of the insular cortex in terms of spatially continuous gradients. Here we assess how spatial features of insular resting state functional ...organization correspond to individual pain sensitivity. From a previous multicenter study, we included 107 healthy participants, who underwent resting state functional MRI scans, T1-weighted scans and quantitative sensory testing on the left forearm. Thermal and mechanical pain thresholds were determined. Connectopic mapping, a technique using non-linear representations of functional organization was employed to describe functional connectivity gradients in both insulae. Partial coefficients of determination were calculated between trend surface model parameters summarizing spatial features of gradients, modal and modality-independent pain sensitivity. The dominant connectopy captured the previously reported posteroanterior shift in connectivity profiles. Spatial features of dominant connectopies in the right insula explained significant amounts of variance in thermal (R
= 0.076; p < 0.001 and R
= 0.031; p < 0.029) and composite pain sensitivity (R
= 0.072; p < 0.002). The left insular gradient was not significantly associated with pain thresholds. Our results highlight the functional relevance of gradient-like insular organization in pain processing. Considering individual variations in insular connectopy might contribute to understanding neural mechanisms behind pain and improve objective brain-based characterization of individual pain sensitivity.
Purpose Temporomandibular disorders (TMDs) are frequent stomatological disorders. However, their treatment is controversial. Therefore, we compared the efficacy of combination therapy (splint therapy ...along with physiotherapy, manual therapy, and counseling) with physiotherapy, manual therapy, and counseling alone. The extent of mouth opening and pain perception were the outcomes.Study selection Systematic searches for English publications were performed using four major literature databases (Cochrane Library, EMBASE, PubMed, and Web of Science). We included randomized controlled trials. We calculated mean differences with 95% confidence interval (CI) for pain perception and maximum mouth opening (MMO) for the two groups. The Hartung-Knapp adjustment was used for cases comprising at least five studies.Results Six articles were included in the pain perception category, and four were reviewed for MMO at baseline. Four articles assessed pain perception, and two assessed MMO at 1 month. Five articles were analyzed upon comparing pain perception at baseline and 1-month follow-up. The mean difference was -2.54 95% CI: -3.38; to -1.70 in the intervention group and -2.33 95% CI: -4.06; to -0.61 in the control group. Two articles were analyzed upon comparing MMO at baseline and 1-month follow-up. The mean difference in the intervention group was 3.69 95% CI: -0.34; 7.72, whereas that in the control group was 3.62 95% CI: -3.43; 10.67.Conclusions Both therapies can be used in the management of myogenic TMD. Due to the marginal differences between the baseline and 1-month values, our results could not confirm the efficacy of combination therapy.
Background
Haemostasis is a crucial process by which the body stops bleeding. It is achieved by the formation of a platelet plug, which is strengthened by formation of a fibrin mesh mediated by the ...coagulation cascade. In proinflammatory and prothrombotic conditions, multiple interactions of the complement system and the coagulation cascade are known to aggravate thromboinflammatory processes and increase the risk of arterial and venous thrombosis. Whether those interactions also play a relevant role during the physiological process of haemostasis is not yet completely understood. The aim of this study was to investigate the potential role of complement components and activation during the haemostatic response to mechanical vessel injury.
Methods
We used a microvascular bleeding model that simulates a blood vessel, featuring human endothelial cells, perfusion with fresh human whole blood, and an inducible mechanical injury to the vessel. We studied the effects of complement inhibitors against components of the lectin (MASP-1, MASP-2), classical (C1s), alternative (FD) and common pathways (C3, C5), as well as a novel triple fusion inhibitor of all three complement pathways (TriFu). Effects on clot formation were analysed by recording of fibrin deposition and the platelet activation marker CD62P at the injury site in real time using a confocal microscope.
Results
With the inhibitors targeting MASP-2 or C1s, no significant reduction of fibrin formation was observed, while platelet activation was significantly reduced in the presence of the FD inhibitor. Both common pathway inhibitors targeting C3 or C5, respectively, were associated with a substantial reduction of fibrin formation, and platelet activation was also reduced in the presence of the C3 inhibitor. Triple inhibition of all three activation pathways at the C3-convertase level by TriFu reduced both fibrin formation and platelet activation. When several complement inhibitors were directly compared in two individual donors, TriFu and the inhibitors of MASP-1 and C3 had the strongest effects on clot formation.
Conclusion
The observed impact of complement inhibition on reducing fibrin clot formation and platelet activation suggests a role of the complement system in haemostasis, with modulators of complement initiation, amplification or effector functions showing distinct profiles. While the interactions between complement and coagulation might have evolved to support haemostasis and protect against bleeding in case of vessel injury, they can turn harmful in pathological conditions when aggravating thromboinflammation and promoting thrombosis.
Noninvasive diagnostic tools for right ventricular (RV) dysfunction measurements are increasingly being used, although their association with the pathologic mechanisms of dysfunction is poorly ...understood. Although investigations have focused mainly on RV systolic function, RV diastolic function remains mostly neglected. The aim of this study was to test which echocardiographic parameters best reflect RV diastolic function in mice.
Pulmonary artery banding (PAB) was used to induce RV pressure overload in mice. Transthoracic echocardiography and invasive hemodynamic measurements were performed after 3 weeks in PAB and sham-operated mice. Subsequently, the hearts were investigated by histology and analyzed for gene expression.
PAB-induced pressure overload (RV systolic pressure PAB 52.6 ± 11.8 mm Hg vs sham 27.0 ± 2.7 mm Hg) resulted in RV hypertrophy and remodeling, as reflected by increased Fulton index (PAB 0.37 ± 0.05 vs sham 0.25 ± 0.02, P = .001). Masson's trichrome staining revealed increased interstitial fibrosis (PAB 12.25 ± 3.12% vs sham 3.97 ± 1.58%, P = .002). This was associated with significant systolic RV dysfunction as demonstrated by reduced contractility index and diastolic dysfunction as demonstrated by end-diastolic pressure (PAB 2.66 ± 0.83 mm Hg vs sham 1.49 ± 0.50 mm Hg, P < .001) and τ (PAB 40.0 ± 16.1 msec vs sham 13.0 ± 3.5 msec, P < .001). Messenger ribonucleic acid expression of β-myosin heavy chain, atrial and brain natriuretic peptides, collagen family members was elevated, and the sarco/endoplasmic reticulum Ca(2+)-ATPase was decreased. Echocardiography revealed significant increases in RV free wall thickness and isovolumic relaxation time and a decrease in left ventricular eccentricity index, E', and tricuspid annular plane systolic excursion. Isovolumic relaxation time and E' were significantly correlated with end-diastolic pressure (rs = 0.511 and -0.451) and τ (rs = 0.739 and -0.445, respectively). Moreover, E' was negatively correlated with the degree of RV fibrosis (rs = -0.717).
Within 3 weeks, PAB causes pressure overload-induced RV hypertrophy and remodeling with compensated systolic and diastolic dysfunction in mice. RV free wall thickness, tricuspid annular plane systolic excursion, E', E/E' ratio, and isovolumic relaxation time appear to be the most reliable echocardiographic parameters for the assessment of RV dysfunction.
Chronic pancreatitis is an inflammatory disorder of the pancreas. We analyzed CPA1, encoding carboxypeptidase A1, in subjects with nonalcoholic chronic pancreatitis (cases) and controls in a German ...discovery set and three replication sets. Functionally impaired variants were present in 29/944 (3.1%) German cases and 5/3,938 (0.1%) controls (odds ratio (OR) = 24.9, P = 1.5 × 10(-16)). The association was strongest in subjects aged ≤ 10 years (9.7%; OR = 84.0, P = 4.1 × 10(-24)). In the replication sets, defective CPA1 variants were present in 8/600 (1.3%) cases and 9/2,432 (0.4%) controls from Europe (P = 0.01), 5/230 (2.2%) cases and 0/264 controls from India (P = 0.02) and 5/247 (2.0%) cases and 0/341 controls from Japan (P = 0.013). The mechanism by which CPA1 variants confer increased pancreatitis risk may involve misfolding-induced endoplasmic reticulum stress rather than elevated trypsin activity, as is seen with other genetic risk factors for this disease.
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