High interindividual variability in LDL-cholesterol reductions after inclisiran administration in a real-world multicenter setting in Germany.

BACKGROUND AND AIMS: Low-density lipoprotein cholesterol (LDL-C) is the main therapeutic target in the treatment of hypercholesterolemia. Small interfering RNA (siRNA) inclisiran is a new drug, which targets PCSK9 mRNA in the liver, reducing concentrations of circulating LDL-C. In randomized trials, inclisiran demonstrated a substantial reduction in LDL-C. The German Inclisiran Network (GIN) aims to evaluate LDL-C reductions in a real-world cohort of patients treated with inclisiran in Germany. METHODS: Patients who received inclisiran in 14 lipid clinics in Germany for elevated LDL-C levels between February 2021 and July 2022 were included in this analysis. We described baseline characteristics, individual LDL-C changes (%) and side effects in 153 patients 3 months (n = 153) and 9 months (n = 79) after inclisiran administration. RESULTS: Since all patients were referred to specialized lipid clinics, only one-third were on statin therapy due to statin intolerance. The median LDL-C reduction was 35.5% at 3 months and 26.5% at 9 months. In patients previously treated with PCSK9 antibody (PCSK9-mAb), LDL-C reductions were less effective than in PCSK9-mAb-naïve patients (23.6% vs. 41.1% at 3 months). Concomitant statin treatment was associated with more effective LDL-C lowering. There was a high interindividual variability in LDL-C changes from baseline. Altogether, inclisiran was well-tolerated, and side effects were rare (5.9%). CONCLUSION: In this real-world patient population referred to German lipid clinics for elevated LDL-C levels, inclisiran demonstrated a high interindividual variability in LDL-C reductions. Further research is warranted to elucidate reasons for the interindividual variability in drug efficacy.

Assessing the validity of a helmet fit checklist in a sample of youth football players.

The dynamics of American youth football make it critical to ensure that helmets are appropriately fit to decrease the risk of injuries. Currently, there is only one researcher-developed checklist to determine helmet fit, and psychometric testing is lacking; therefore, the aim of this work was to determine the validity of the checklist. The 13-item checklist was used to measure helmet fit in 267 youth football players prior to the start of the season. Using a Principal Components Analysis to assess validity, a 5-component model was found explaining 58% of the available variance. These results suggest that a single, summative score should not be used for this checklist; rather five scores should be calculated for each component (stability, snugness, size, integrity, and accessory). A more practical and valid tool to assess fit, such as a sub-sectioned chronological American football-specific checklist, can better assist coaches/administrators responsible for helmet fit and player safety.

Comparison of overwintering survival and fertility of Zaprionus indianus (Diptera: Drosophilidae) flies from native and invaded ranges.

Zaprionus indianus is a fly species native to the Afrotropical biogeographic region that invaded the South American continent 20 years ago. Its southernmost record is 34°S in areas with temperate climates with cold winters. To better understand its invasion biology, we investigated physiological responses to winter-like abiotic conditions that may be relevant in Z. indianus geographic expansion. We characterized Z. indianus females reproductive traits (ovarian maturation and fertility) and survival in response to cold treatments with summer-like and winter-like photoperiods. We also compared these traits between native (Yokadouma, Africa) and invasive (Yuto, South America) range wild-derived flies. We showed that Z. indianus females have the ability to arrest ovarian maturation and maintain fertility following recovery from cold stress. The critical temperature for ovarian maturation of this species was estimated at c. 13 °C, an intermediate value between those of tropical and temperate drosophilid species. Wild-derived females from Yuto responded to winter-like photoperiod by slowing down ovarian maturation at low but permissive temperatures of 14 °C and 16 °C and also delayed the start of oviposition after cold treatment. Yuto flies also survived better and recovered 20% faster from chill coma than flies from Yokadouma. These results are consistent with a scenario of local adaptations or phenotypic plasticity in the invaded range, and suggest that photoperiod could act as modulator of ovarian arrest. Conversely, the fact that native range flies showed higher fertility after cold recovery than females from invaded range is not indicative of local adaptation. All in all, our findings report a set of physiological responses that would enable Z. indianus expansion to temperate and cold areas, but also results that are compatible with a limitation to the invasion process.

Application of hydroxypropyl methylcellulose as a protective agent against magnesium stearate induced crystallization of amorphous itraconazole.

Itraconazole is a fungicide drug which has low bioavailability due to its poor water solubility. Amorphous solid dispersion (ASD) is a tool that has the potential to greatly increase the dissolution rate and extent of compounds. In this work, the dissolution of tablets containing the ASD of itraconazole with either hydroxypropyl methylcellulose (HPMC) or vinylpyrrolidone-vinyl acetate copolymer (PVPVA) was compared in order to find a formulation which can prevent the drug from the precipitation caused by magnesium stearate. Formulations containing the PVPVA-based ASD with HPMC included in various forms could reach 90% dissolution in 2 h, while HPMC-based ASDs could release 100% of the drug. However, HPMC-based ASD had remarkably poor grindability and low bulk density, which limited its processability and applicability. The latter issue could be resolved by roller compacting the ASD, which significantly increases the bulk density and the flowability of the powder blends used for tableting. This roller compaction step might be a base for the industrial application of HPMC-based, electrospun ASDs.

Detailed stability investigation of amorphous solid dispersions prepared by single-needle and high speed electrospinning.

In this research the long-term stability (one year) of amorphous solid dispersions (ASDs) prepared by high speed electrospinning was investigated at 25 °C/60% relative humidity (RH) (closed conditions) and 40 °C/75% RH (open conditions). Single needle electrospinning and film casting were applied as reference technologies. Itraconazole (ITR) was used as the model API in 40% concentration and the ASDs consisted of either one of the following polymers as a comparison: polyvinylpyrrolidone-vinyl acetate 6:4 copolymer (no hydrogen bonds between API and polymer) and hydroxypropyl methylcellulose (possible hydrogen bonds between oxo or tertiary nitrogen function of API and hydroxyl moiety of polymer). DSC, XRPD and dissolution characteristics of samples at 0, 3 and 12 months were investigated. In addition, Raman maps of certain electrospun ASDs were assessed to investigate crystallinity. A new chemometric method, based on Multivariate Curve Resolution-Alternating Least Squares algorithm, was developed to calculate the spectrum of amorphous ITR in the matrices and to determine the crystalline/amorphous ratio of aged samples. As it was expected ITR in single needle electrospun SDs was totally amorphous at the beginning, in addition hydroxypropyl methylcellulose could keep ITR in this form at 40 °C/75% RH up to one year due to the hydrogen bonds and high glass transition temperature of the SD. In polyvinylpyrrolidone-vinyl acetate matrix ITR remained amorphous at 25 °C/60% RH throughout one year. Materials prepared by scaled-up, high throughput version of electrospinning, which is compatible with pharmaceutical industry, also gained the same quality. Therefore these ASDs are industrially applicable and with an appropriate downstream process it would be possible to bring them to the market.

Usefulness of a novel Caco-2 cell perfusion system II. Characterization of monolayer properties and peptidase activity.

In this study, the enzymatic activity and the influence of support filters and extracellular matrix proteins on the differentiation of Caco-2 cells grown in a perfusion system (Minucells and MinutissueTM) were examined and compared to traditional culturing approaches. Differences were observed regarding the differentiation of Caco-2 cells using the traditional approach and perfusion system such that the cell monolayers grown in a perfusion system showed a significant increase in dipeptidase activities (18.20 +/- 0.43nmol x min(-1) x cm(-2)) compared to the cells cultivated using the 21-day protocol (9.45 +/- 0.50 nmol x min(-1) x cm(-2)). The peptidase activity of Caco-2 cells was strikingly inhibited when Matrigel extracellular protein was used for coating polycarbonate support filters. While the enzymatic activities of the cell monolayers differentiated in the perfusion system were up-regulated, the transepithelial electrical resistance values of the cell monolayers (171 +/- 52 and 251 +/- 62 omega x cm2 for polycarbonate and polyester, respectively) decreased compared to the traditional Snapwell inserts (644 +/- 119 omega x cm2). The results suggested that the perfusion systems were useful permeability models which reduce workload, resources and manpower needed to obtain useful Caco-2 monolayers. In addition, the approach offers an efficient tool for long-term culturing of highly differentiated Caco-2 cell monolayers.

Body size in Drosophila: genetic architecture, allometries and sexual dimorphism.

Even though substantial progress has been made to elucidate the physiological and environmental factors underpinning differences in body size, little is known about its genetic architecture. Furthermore, all animal species bear a specific relationship between the size of each organ and overall body size, so different body size traits should be investigated as well as their sexual dimorphism that may have an important impact on the evolution of body size. We have surveyed 191 co-isogenic lines of Drosophila melanogaster, each one of them homozygous for a single P-element insertion, and assessed the effects of mutations on different body size traits compared to the P-element-free co-isogenic control. Nearly 60% of the lines showed significant differences with respect to the control for these traits in one or both sexes and almost 35% showed trait- and sex-specific effects. Candidate gene mutations frequently increased body size in males and decreased it in females. Among the 92 genes identified, most are involved in development and/or metabolic processes and their molecular functions principally include protein-binding and nucleic acid-binding activities. Although several genes showed pleiotropic effects in relation to body size, few of them were involved in the expression of all traits in one or both sexes. These genes seem to be important for different aspects related to the general functioning of the organism. In general, our results indicate that the genetic architecture of body size traits involves a large fraction of the genome and is largely sex and trait specific.

Parallel artificial membrane permeability assay (PAMPA) combined with a 10-day multiscreen Caco-2 cell culture as a tool for assessing new drug candidates.

The parallel artificial membrane permeability assay (PAMPA) is extensively used for the evaluation of early drug candidates. It is high throughput, low cost and is amenable to automation. This method has been shown useful in assessing transmembrane, non-energy dependent, diffusion of drugs such that reasonable predictability with in vivo (passive) absorption is possible. Cell cultures mimicking the gastrointestinal tract such as the CACO-2 cultures have the advantage of taking into account other transport mechanism including paracellular and carrier-mediated uptake but are lower throughput and labor-intensive. In this study, the applicability of two high throughput permeability assays namely PAMPA (PSR4p, pION Inc.) and 96-well Caco-2 cell assay (MultiScreen, Millipore) were used to rank drug permeability as well as to predict passive and active drug absorption/secretion for a series of marketed drugs as well as a collection of structurally diverse drug candidates. CACO-2 cells were cultured using MultiScreen hardware over a period of 10 days with the integrity of the cells assessed using transepithelial electrical resistance (TEER) and by the ability of the monolayer to the transport a paracellular marker, sodium fluorescence. Effective permeability (Peff) data were calculated using spectrophotometric data and were binned based on a pre-defined cut-off values as either highly and poorly permeable. A comparison of a well characterized drug training set indicate at least 85% concordance between the data generated from PAMPA and Caco-2 MultiScreen. The values obtained using the MultiScreen approach were also similar to data obtained from the literature using the conventional 21-day Caco-2 cell assay. Differences between PAMPA and CACO-2 ranking were useful indicators of either drug efflux (PAMPA (Peff) > CACO-2 (Peff)) or absorptive transport (CACO-2 (Peff) > PAMPA (Peff)). These results indicate that PAMPA combined with the MultiScreen Caco-2 cell culture may be a useful high throughput screening for predicting passive diffusion and active transport of new drugs.

Genotype by environment interactions in viability and developmental time in populations of cactophilic Drosophila.

The genetic and ecological basis of viability and developmental time differences between Drosophila buzzatii and D. koepferae were analysed using the isofemale line technique. Several isofemale lines were sampled from pairs of allopatric/sympatric populations of each species. Flies were reared in media prepared with decaying tissues of two of the main natural cactus hosts of each species. This experimental design enabled us to evaluate the relative contribution of phenotypic plasticity, genetic variation and genotype by environment interaction (G x E) to total phenotypic variation for two fitness traits, viability and developmental time. Our results revealed significant G x E in both traits, suggesting that the maintenance of genetic variation can be explained, at least in part, by diversifying selection in different patches of a heterogeneous environment in both species. However, the relative importance of the factors involved in the G x E varied between traits and populations within species. For viability, the G x E can be mainly attributed to changes in the rank order of lines across cacti. However, the pattern was different for developmental time. In D. buzzatii the G x E can be mainly accounted for by changes in among line variance across cacti, whereas changes in the rank order of lines across cacti was the main component in D. koepferae. These dissimilar patterns of variation between traits and species suggest that the evolutionary forces shaping genetic variation for developmental time and viability vary between populations within species and between species.

The use of polymer-based electrospun nanofibers containing amorphous drug dispersions for the delivery of poorly water-soluble pharmaceuticals.

Electrostatic spinning was applied to the preparation of drug-laden nanofiber for potential use in oral and topical drug delivery. While this technique is in its infancy with regard to pharmaceutical applications, a number of recent publications suggest that it may be of high value in the formulation of poorly water-soluble drugs by combining nanotechnology and solid solution/dispersion methodologies. The purpose of this article is to describe some of these recently published applications. For immediate release oral application, a water-soluble cellulose polymer was selected (i.e., hydroxypropylmethylcellulose, HPMC) while for topical application, a nonbiodegradable, water-insoluble polymer was investigated (i.e., a segmented polyurethane, SPU). Solutions of the polymer and the drugs in appropriate solvents could be spun across various potentials (16-24 kV) generating nanofibers with diameters ranging from 300 to 2000 nm. Dissolution studies found that the non-woven fabrics derived from HPMC and containing itraconazole dissolved over a time course of minutes to hours depending on the formulation used as well as the drug/polymer ratios. Drug release from the SPU samples was dependent on the incorporated drug as well as nanostructure obtained.

Creating a sense of family in urban schools using the "sport for peace" curriculum.

Urban students often have difficulty engaging in the learning process and affiliating with others. A three-phase research design was used to examine the effectiveness of a high school physical education curriculum reform initiative entitled "Sport for Peace" to enhance student engagement and willingness to interact positively with others. Ten physical educators in six urban schools taught a traditional soccer unit (Phase I) followed by instruction and mentoring in the Sport for Peace curriculum (Phase II). In the third phase of the research, teachers developed and taught a Sport for Peace unit to their students. Data were collected using observation and interview methods and analyzed with constant comparison. Results suggested that the Sport for Peace curricular structures fostered shared responsibility for learning, trust, respect, and a sense of family. Both high- and low-skilled girls and boys felt successful and responded positively, creating a class community more conducive to engagement and participation.

Domain organization, genomic structure, evolution, and regulation of expression of the aggrecan gene family.

Proteoglycans are complex macromolecules, consisting of a polypeptide backbone to which are covalently attached one or more glycosaminoglycan chains. Molecular cloning has allowed identification of the genes encoding the core proteins of various proteoglycans, leading to a better understanding of the diversity of proteoglycan structure and function, as well as to the evolution of a classification of proteoglycans on the basis of emerging gene families that encode the different core proteins. One such family includes several proteoglycans that have been grouped with aggrecan, the large aggregating chondroitin sulfate proteoglycan of cartilage, based on a high number of sequence similarities within the N- and C-terminal domains. Thus far these proteoglycans include versican, neurocan, and brevican. It is now apparent that these proteins, as a group, are truly a gene family with shared structural motifs on the protein and nucleotide (mRNA) levels, and with nearly identical genomic organizations. Clearly a common ancestral origin is indicated for the members of the aggrecan family of proteoglycans. However, differing patterns of amplification and divergence have also occurred within certain exons across species and family members, leading to the class-characteristic protein motifs in the central carbohydrate-rich region exclusively. Thus the overall domain organization strongly suggests that sequence conservation in the terminal globular domains underlies common functions, whereas differences in the central portions of the genes account for functional specialization among the members of this gene family.

Structural and functional analysis of the chick chondroitin sulfate proteoglycan (aggrecan) promoter and enhancer region.

Aggrecan is a large chondroitin sulfate proteoglycan, the expression of which is both tissue-specific and developmentally regulated. Here we report the cloning and sequencing of the 1.8-kilobase genomic 5' flanking sequence of the chick aggrecan gene and provide a functional and structural characterization of its promoter and enhancer region. Sequence analysis reveals potential Sp1, AP2, and NF-I related sites, as well as several putative transcription factor binding sites, including the cartilage-associated silencers CIIS1 and CIIS2. A number of these transcription factor binding motifs are embedded in a sequence flanked by prominent inverted repeats. Although lacking a classic TATA box, there are two instances in the 1.8-kb genomic fragment of TATA-like TCTAA sequences, as have been defined previously in other promoter regions. Primer extension and S1 protection analyses reveal three major transcription start sites, also located between the inverted repeats. Transient transfections of chick sternal chondrocytes and fibroblasts with reporter plasmids bearing progressively reduced portions of the aggrecan promoter region allowed mapping of chondrocyte-specific transcription enhancer and silencer elements that are consistent with the sequence analysis. These findings suggest the importance of this regulatory region in the tissue-specific expression of the chick aggrecan gene.

The isolation and characterization of cDNA encoding the mouse bifunctional ATP sulfurylase-adenosine 5'-phosphosulfate kinase.

Biosynthesis of the activated sulfate donor, adenosine 3'-phosphate 5'-phosphosulfate, involves the sequential action of two enzyme activities: ATP sulfurylase, which catalyzes the formation of adenosine 5'-phosphosulfate (APS) from ATP and free sulfate, and APS kinase, which subsequently phosphorylates APS to produce adenosine 3'-phosphate 5'-phosphosulfate. Oligonucleotide primers were derived from a human infant brain-expressed sequence tag putatively encoding a portion of APS kinase. Using these primers, reverse transcriptase-polymerase chain reaction was performed on mRNA from neonatal normal mice resulting in amplification of a 127-bp DNA fragment. This fragment was subsequently used to screen a mouse brain lambda gt11 cDNA library, yielding a 2.2-kb clone. Primers were designed from the 5'-end of the 2.2-kb clone, and 5'-rapid amplification of cDNA ends was used to obtain the translation start site. Sequence from the overlapping clones was assembled into a 2475-bp composite sequence, which contains a single open reading frame that translates into a 624-deduced amino acid sequence. Northern blots of total RNA from neonatal mice yielded a single message species at approximately 3.3 kb. Southern blot of genomic DNA digested with several restriction enzymes suggested the gene is present as a single copy. Comparison against sequence data bases suggested the composite sequence was a fused sulfurylase-kinase product, since the deduced amino acid sequence showed extensive homology to known separate sequences of both ATP sulfurylase and APS kinase from several sources. The first 199 amino acids corresponded to APS kinase sequence, followed by 37 distinct amino acids, which did not match any known sequence, followed by 388 amino acids that are highly homologous to known ATP sulfurylase sequences. Finally, recombinant enzyme expressed in COS-1 cells exhibited both ATP sulfurylase and APS kinase activity.

Increased lung clearance of 99mTcDTPA in allograft lung rejection. The Paris-Sud Lung Transplant Group.

To investigate whether lung 99mTc-DTPA clearance is altered during allograft lung rejection, a group of four double lung and 24 heart-lung transplant patients was studied using serial measurement of the clearance rate of aerosolized 99mTc-DTPA (DTPA-Cl), in association with pulmonary function tests, bronchoalveolar lavage, and transbronchial lung biopsies. Using histologic diagnosis as a standard, we compared 56 episodes with normal lung histology to 32 episodes with allograft lung rejection. A control group of 20 healthy nonsmokers was used to define normal DTPA-Cl. In patients with normal lung histology, DTPA-Cl was higher than in control subjects (2.62 +/- 0.25 versus 1.20 +/- 0.12 %/min; p less than 0.001). In the episodes of allograft lung rejection, DTPA-Cl increased to 3.65 +/- 0.41 %/min (p less than 0.02) as compared with episodes of normal lung histology. The change in DTPA-Cl during allograft lung rejection was correlated (r = 0.3, p less than 0.01) with the increased percentage of lymphocytes in bronchoalveolar lavage (27.8 +/- 3.5% in rejection versus 19.9 +/- 2.2% in normal histology; p less than 0.02). Sensitivity and specificity of DTPA-Cl measurement in detecting lung rejection were 69 and 82%, respectively, versus 45 and 85% for FEV1 measurement. These results suggest that DTPA-Cl monitoring could be used in conjunction with pulmonary function testing as a noninvasive approach for the detection of lung rejection.

Chick cartilage chondroitin sulfate proteoglycan core protein. II. Nucleotide sequence of cDNA clone and localization of the S103L epitope.

A lambda gt11 expression library containing cDNA from total chick embryo was screened with S103L, a rat monoclonal antibody which reacts specifically with the core protein of the chick cartilage chondroitin sulfate proteoglycan. One clone was identified which produced a 220-kDa beta-galactosidase/S103L-binding fusion protein. Sequencing the entire 1.5-kilobase cDNA insert showed that it contained a single open reading frame, which encoded a portion of the proteoglycan core protein from the chondroitin sulfate domain. This was confirmed by comparison with amino acid sequence data from peptide CS-B, which was derived from the chondroitin sulfate domain (Krueger, R.C., Jr., Fields, T. A., Hildreth, J., IV, and Schwartz, N.B. (1990) J. Biol. Chem. 265, 12075-12087). Furthermore, the 3' end of the insert overlapped with 23 bases at the 5' end of the published sequence for the C-terminal globular domain (Sai, S., Tanaka, T., Kosher, R. A., and Tanzer, M. L. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 5081-5085), which oriented this clone, as well as the CS peptide, along the protein core. The cDNA insert hybridized with a 9-kilobase mRNA from sternal chondrocytes as well as a similar sized message in brain but did not hybridize to any message from rat chondrosarcoma or from undifferentiated limb bud mesenchyme. In further studies, the fusion protein as well as a cyanogen bromide fragment (70 kDa) derived from it were isolated and shown to react with S103L, indicating that cleavage at methionine residues does not disrupt the antibody recognition site. Purification and N-terminal sequencing of the antigenic CNBr fragment derived from the fusion protein revealed that its N terminus is preceded by a methionine in the fusion protein and overlaps with the N terminus of peptide CS-B. As peptide CS-B is not recognized by S103L and the C terminus of peptide CS-B lies beyond the proteoglycan portion of the antigenic CNBr fragment, the S103L epitope is either contained within the 11 amino acids preceding the N terminus of peptide CS-B or it spans the clostripain cleavage site at the origin of the N terminus of peptide CS-B.

Intravenous digital subtraction angiography and lung imaging: compared value in the diagnosis of pulmonary embolism.

In a prospective study, 42 consecutive patients with clinically suspected pulmonary embolism underwent ventilation-perfusion (V-Q) lung imaging and digital subtraction angiography (DSA) concurrently with selective conventional pulmonary angiography (CPA). Thirty-eight studies achieved within 24 hours were reviewed independently by two pairs of observers. The findings were compared using CPA as the gold standard. V-Q lung imaging had a high percentage of indeterminate results, but none were false negative nor false positive. DSA had a lower percentage of indeterminate results but missed four of the 25 positive cases and erroneously affirmed the presence of pulmonary embolism in three cases. Therefore, the authors think that V-Q lung imaging should remain the screening examination of choice for evaluating patients with suspected pulmonary embolism.

Recurrence of pulmonary embolism during anticoagulant treatment: a prospective study.

The risk of early recurrence of pulmonary embolism in patients with venous thromboembolic disease treated by anticoagulants is not well established. To determine the risk linked to contemporary proximal deep venous thrombosis, a prospective study was organised to give clinical and scintigraphic surveillance to 50 patients with angiographically proved pulmonary embolism plus phlebographically proved proximal deep vein thrombosis during the first 15 days of anticoagulant treatment. Perfusion lung scans were performed initially and on days 3, 7, and 15. Only two patients had a recurrence of pulmonary embolism during this period; both episodes were revealed by new symptoms, and one recurrence was fatal. The systematic performance of angiography in four patients found to have new scintigraphic defects led to the diagnosis of "spurious scintigraphic recurrence" in three of them. It is concluded that (a) adjusted anticoagulant treatment showed an effectiveness of 96% for preventing early recurrence of pulmonary embolism in this group of supposed high risk patients, and (b) in patients with recent pulmonary embolism new defects on systematic perfusion lung scans are not specific indicators of recurrent pulmonary embolism.