Alternative splicing of its 5'-UTR limits CD20 mRNA translation and enables resistance to CD20-directed immunotherapies.

Aberrant skipping of coding exons in CD19 and CD22 compromises the response to immunotherapy in B-cell malignancies. Here, we showed that the MS4A1 gene encoding human CD20 also produces several messenger RNA (mRNA) isoforms with distinct 5' untranslated regions. Four variants (V1-4) were detected using RNA sequencing (RNA-seq) at distinct stages of normal B-cell differentiation and B-lymphoid malignancies, with V1 and V3 being the most abundant. During B-cell activation and Epstein-Barr virus infection, redirection of splicing from V1 to V3 coincided with increased CD20 positivity. Similarly, in diffuse large B-cell lymphoma, only V3, but not V1, correlated with CD20 protein levels, suggesting that V1 might be translation-deficient. Indeed, the longer V1 isoform contained upstream open reading frames and a stem-loop structure, which cooperatively inhibited polysome recruitment. By modulating CD20 isoforms with splice-switching morpholino oligomers, we enhanced CD20 expression and anti-CD20 antibody rituximab-mediated cytotoxicity in a panel of B-cell lines. Furthermore, reconstitution of CD20-knockout cells with V3 mRNA led to the recovery of CD20 positivity, whereas V1-reconstituted cells had undetectable levels of CD20 protein. Surprisingly, in vitro CD20-directed chimeric antigen receptor T cells were able to kill both V3- and V1-expressing cells, but the bispecific T-cell engager mosunetuzumab was only effective against V3-expressing cells. To determine whether CD20 splicing is involved in immunotherapy resistance, we performed RNA-seq on 4 postmosunetuzumab follicular lymphoma relapses and discovered that in 2 of them, the downregulation of CD20 was accompanied by a V3-to-V1 shift. Thus, splicing-mediated mechanisms of epitope loss extend to CD20-directed immunotherapies.

Individual combinations of danger signals synergistically increase FVIII product immunogenicity.

INTRODUCTION: The most severe side effect in haemophilia A treatment is the development of antifactor VIII antibodies, also called inhibitors. Why inhibitors develop in a proportion of treated patients while others are unaffected still remains unanswered. The presence of immunological danger signals, associated with events such as infection or surgery, has been proposed to play a role. Previous studies demonstrated that the presence of the bacterial molecule lipopolysaccharide (LPS) can synergistically increase the activation of human DC and subsequent T cell activation by FVIII. AIM AND METHODS: In the present study, we investigated whether a combination of two danger signals can further increase immune cell activation by FVIII. For this, human in vitro differentiated DC that were treated with combinations of danger signals were co-cultured with autologous primary T cells, and T cell proliferation was analysed. RESULTS: Interestingly, by combining LPS with a second danger signal, lower LPS concentrations were sufficient to synergistically increase DC and subsequent T cell activation by FVIII. Of note, a combination of LPS and the double-stranded RNA, polyinosinic-polycytidylic acid (poly(I:C)), was most potent in increasing FVIII immunogenicity, followed by LPS + R848 (resiquimod). However, a combination of LPS and the bacterial lipopeptide Pam3CysSK4 did not induce increased immune cell activation by FVIII. CONCLUSION: Thus, individual combinations of danger signals can increase FVIII product immunogenicity. This should be considered in the treatment routine of haemophilia A patients.

Composition and physicochemical properties of dried berry pomace.

BACKGROUND: Berry pomace is a valuable but little used by-product of juice manufacturing. When processed to a stable fruit powder, the composition differs from that of the whole fruit. To facilitate application in foods, a detailed knowledge of its composition and physicochemical properties is essential. RESULTS: Blackcurrant, redcurrant, chokeberry, rowanberry and gooseberry were selected for analysis. All pomace powders had a high fibre content (> 550 g kg-1 ) and a fat content of up to 200 g kg-1 . Despite identical milling conditions, the particle sizes of the pomace powders varied. This can be traced back to seed content and brittleness, which also becomes apparent with respect to surface characteristics. Blackcurrant pomace powder differed from other varieties in terms of its low water-binding capacity (3.2 g g-1 ) and a moderate moisture uptake, whereas chokeberry pomace powder showed the highest polyphenol content and rowanberry pomace powder was rich in flavonols. CONCLUSION: The results obtained in the present study provide a comprehensive overview of the properties of berry pomace powder and allow conclusions to be made regarding their applicability for use in complex food systems. © 2018 Society of Chemical Industry.

The interaction of glucocorticoids and progesterone distinctively affects epithelial sodium transport.

PURPOSE: Glucocorticoids and progesterone exert stimulatory effects on epithelial Na(+) transport, including increased mRNA expression of the participating ion transporters (epithelial Na(+) channels [ENaC] and Na,K-ATPases) and their electrophysiological activity. Fetuses threatened by preterm labor may receive high doses of glucocorticoids to stimulate lung maturation and are naturally exposed to high levels of female sex steroids. However, it is still unknown how the combination of both hormones influences the epithelial Na(+) transport, which is crucial for alveolar fluid clearance. METHODS: Fetal distal lung epithelial cells were incubated in media supplemented with dexamethasone and progesterone. Real-time qPCR and Ussing chamber analysis were used to determine the effects on ENaC mRNA expression and channel activity. In addition, the specific progesterone receptor antagonist (PF-02367982) and the glucocorticoid receptor antagonist mifepristone were used to identify the involved hormone receptors. RESULTS: Both dexamethasone and progesterone increased ENaC subunit expression and channel activity. However, the combination of dexamethasone and progesterone reduced the α- and γ-ENaC subunit expression compared to the effect of dexamethasone alone. Furthermore, higher dexamethasone concentrations in combination with progesterone also significantly reduced Na(+) transport in Ussing chamber measurements. Hormone receptor antagonists showed that inhibition of the progesterone receptor increased the mRNA expression of α- and γ-ENaC, whereas mifepristone decreased mRNA expression of all ENaC subunits. CONCLUSION: Glucocorticoids and progesterone individually increase ENaC mRNA expression; however, the combination of both hormones decreases the stimulatory effects of dexamethasone on Na(+) transport and ENaC mRNA expression.

Quality-Assured Analysis of PIK3CA Mutations in Hormone Receptor-Positive/Human Epidermal Growth Factor Receptor 2-Negative Breast Cancer Tissue.

In precision oncology, reliable testing of predictive molecular biomarkers is a prerequisite for optimal patient treatment. Interlaboratory comparisons are a crucial tool to verify diagnostic performance and reproducibility of one's approach. Herein is described the design and results of the first recurrent, internationally performed PIK3CA (phosphatidylinositol-4,5-bisphosphate 3 kinase catalytic subunit α) breast cancer tissue external quality assessment (EQA), organized by German Quality in Pathology GmbH and started in 2021. After the internal pretesting phase performed by the (lead) panel institutes, in both 2021 and 2022, each EQA test set comprised n = 10 tissue samples of hormone receptor-positive, human epidermal growth factor receptor 2-negative invasive breast cancer that had to be analyzed and reported by the participants. In 2021, the results were evaluated separately for German-speaking countries (part 1) and international laboratories (part 2). In 2022, the EQA was performed across the European Union. The EQA success rates were 84.6% (n = 11/13), 88.6% (n = 39/44), and 87.9% (n = 29/33) for EQA 2021 part 1, part 2, and EQA 2022, respectively. The most commonly used methods were next-generation sequencing and mutation-/allele-specific qualitative PCR-based assays. In summary, this recurrent PIK3CA EQA proved to be a suitable approach to obtain an international overview of methods used for PIK3CA mutation analysis, to evaluate them qualitatively, and identify the strengths and weaknesses of individual methods.

Pulmonary function after anterior double thoracotomy approach versus posterior surgery with costectomies in idiopathic thoracic scoliosis.

INTRODUCTION: The surgical approach in the treatment of idiopathic thoracic scoliosis depends on the type of curve involved. In anterior correction, the rib hump is corrected by derotating the thoracic spine. In posterior scoliosis surgery, additional rib hump resection is sometimes necessary to achieve an optimal cosmetic result. The aim of this study was to compare pulmonary function in these two patient groups. MATERIALS AND METHODS: Forty patients in the anterior group (A) were treated with standard double thoracotomy, with an anterior derotation spondylodesis and a primary stable dual-rod system. The posterior group (P) included 29 patients who were treated with a pedicle screw-based posterior instrumentation spondylodesis, with additional rib hump resection. Pulmonary function was evaluated preoperatively, on the 12th postoperative day, and at 3, 6, 12 and 24 months during the follow-up. RESULTS: The patients' mean age was 15 years in group A and 19 in group P with a standard deviation 8.7 years and a significant difference. With regard to body height or weight there were no significant differences between the two groups. In group A, the deterioration in pulmonary function immediately after the operation (from [Formula: see text] 75.3 %/71.3 % preoperatively to 38.5 %/36.1 % postoperatively) was clearer than in group P ([Formula: see text] 71.6 %/65.7 % preoperatively to 47.7 %/48.4 % postoperatively). During a follow-up period of 3 months, the values improved in both groups in comparison with the values immediately after the operation. Up to the 2 year follow-up, pulmonary function in the posterior and anterior groups corresponded to the preoperative values, with no significant differences. There was a trend toward moderately increased values in the posterior group and moderately decreased values in the anterior group at the 2-year follow-up examination, in comparison with the preoperative baseline, but without a statistically significant difference. Two major complications occurred in the anterior group, with reintubation and several bronchoscopy examinations due to atelectasis. CONCLUSION: The severe deterioration in group A is caused by the substantial trauma with double thoracotomy in contrast to rib hump resection. For patients with severe restrictive pulmonary distress, posterior instrumentation in combination with rib hump resection would be preferable to an anterior procedure involving double thoracotomy. Respiratory physiotherapy exercise should be administered in order to minimise postoperative pulmonary distress. In conclusion opening of the chest wall leads to deterioration of pulmonary function with improvement to the preoperative values after 6 months in the posterior and after 24 months in the anterior group.

Correlation of acromial morphology with impingement syndrome and rotator cuff tears.

BACKGROUND AND PURPOSE: Indications for acromioplasty are based on clinical symptoms and are generally supported by typical changes in acromial morphology on standard radiographs. We evaluated 5 commonly used radiographic parameters of acromial morphology and assessed the association between different radiographic characteristics on the one hand and subacromial impingement or rotator cuff tears on the other. PATIENTS AND METHODS: We measured acromial type (Bigliani), acromial slope (AS), acromial tilt (AT), lateral acromial angle (LAA), and acromion index (AI) on standard radiographs from 50 patients with full-thickness supraspinatus tendon tears, 50 patients with subacromial impingement, and 50 controls without subacromial pathology. RESULTS: The acromial type according to Bigliani was not associated with any particular cuff lesion. A statistically significant difference between controls and impingement patients was found for AS. AT of controls was significantly smaller than that of impingement patients and cuff-tear patients. LAA of cuff-tear patients differed significantly from that of controls and impingement patients, but LAA of controls was not significantly different from that of impingement patients. Differences between impingement patients and cuff-tear patients were also significant. AI of controls was significantly lower than of impingement patients and of cuff-tear patients. A good correlation was found between acromial type and AS. INTERPRETATION: A low lateral acromial angle and a large lateral extension of the acromion were associated with a higher prevalence of impingement and rotator cuff tears. An extremely hooked anterior acromion with a slope of more than 43° and an LAA of less than 70° only occurred in patients with rotator cuff tears.

Multifaceted Tissue-Protective Functions of Polyvalent Immunoglobulin Preparations in Severe Infections-Interactions with Neutrophils, Complement, and Coagulation Pathways.

Severe infections induce immune defense mechanisms and initial tissue damage, which produce an inflammatory neutrophil response. Upon dysregulation of these responses, inflammation, further tissue damage, and systemic spread of the pathogen may occur. Subsequent vascular inflammation and activation of coagulation processes may cause microvascular obstruction at sites distal to the primary site of infection. Low immunoglobulin (Ig) M and IgG levels have been detected in patients with severe infections like sCAP and sepsis, associated with increased severity and mortality. Based on Ig's modes of action, supplementation with polyvalent intravenous Ig preparations (standard IVIg or IgM/IgA-enriched Ig preparations) has long been discussed as a treatment option for severe infections. A prerequisite seems to be the timely administration of Ig preparations before excessive tissue damage has occurred and coagulopathy has developed. This review focuses on nonclinical and clinical studies that evaluated tissue-protective activities resulting from interactions of Igs with neutrophils, complement, and the coagulation system. The data indicate that coagulopathy, organ failure, and even death of patients can possibly be prevented by the timely combined interactions of (natural) IgM, IgA, and IgG with neutrophils and complement.

Alternative splicing of its 5'-UTR limits CD20 mRNA translation and enables resistance to CD20-directed immunotherapies.

Aberrant skipping of coding exons in CD19 and CD22 compromises responses to immunotherapy for B-cell malignancies. Here, we show that the MS4A1 gene encoding human CD20 also produces several mRNA isoforms with distinct 5' untranslated regions (5'-UTR). Four variants (V1-4) were detectable by RNA-seq in distinct stages of normal B-cell differentiation and B-lymphoid malignancies, with V1 and V3 being the most abundant by far. During B-cell activation and Epstein-Barr virus infection, redirection of splicing from V1 to V3 coincided with increased CD20 positivity. Similarly, in diffuse large B-cell lymphoma only V3, but not V1, correlated with CD20 protein levels, suggesting that V1 might be translation-deficient. Indeed, the longer V1 isoform was found to contain upstream open reading frames (uORFs) and a stem-loop structure, which cooperatively inhibited polysome recruitment. By modulating CD20 isoforms with splice-switching Morpholino oligomers, we enhanced CD20 expression and anti-CD20 antibody rituximab-mediated cytotoxicity in a panel of B-cell lines. Furthermore, reconstitution of CD20-knockout cells with V3 mRNA led to the recovery of CD20 positivity, while V1-reconstituted cells had undetectable levels of CD20 protein. Surprisingly, in vitro CD20-directed CAR T cells were able to kill both V3- and V1-expressing cells, but the bispecific T cell engager mosunetuzumab was only effective against V3-expressing cells. To determine whether CD20 splicing is involved in immunotherapy resistance, we performed RNA-seq on four post-mosunetuzumab follicular lymphoma relapses and discovered that in two of them downregulation of CD20 was accompanied by the V3-to-V1 shift. Thus, splicing-mediated mechanisms of epitope loss extend to CD20-directed immunotherapies. Key Points: In normal & malignant human B cells, CD20 mRNA is alternatively spliced into four 5'-UTR isoforms, some of which are translation-deficient.The balance between translation-deficient and -competent isoforms modulates CD20 protein levels & responses to CD20-directed immunotherapies. Explanation of Novelty: We discovered that in normal and malignant B-cells, CD20 mRNA is alternatively spliced to generate four distinct 5'-UTRs, including the longer translation-deficient V1 variant. Cells predominantly expressing V1 were still sensitive to CD20-targeting chimeric antigen receptor T-cells. However, they were resistant to the bispecific anti-CD3/CD20 antibody mosunetuzumab, and the shift to V1 were observed in CD20-negative post-mosunetuzumab relapses of follicular lymphoma.

Publication rate of abstracts presented at the Annual Congress of the Spine Society of Europe (years 2000-2003).

INTRODUCTION: The quality of presentations at medical conferences is of major importance. The publication rate (PR) following congress presentation is an indicator of the extent and quality of a scientific society's activity. The purpose of this study was to investigate publication rates in the Spine Society of Europe (SSE), compare them with the results for American spine societies, and determine factors affecting publication. MATERIALS AND METHODS: All 839 abstracts of podium and poster presentations at SSE congresses held in 2000-2003 were investigated. PRs in peer-reviewed journals within a period of 5 years were assessed. Subgroup analyses were performed for different study types. The consistency of abstracts with publications was also analyzed. RESULTS: The overall PR was 37.8%, with a mean of 17.7 ± 15.7 months between congress and publication and a mean impact factor of 1.8 ± 1.0 at the time of publication. Comparatively high PRs were found for podium presentations versus posters, studies with higher versus lower levels of evidence, experimental versus clinical studies, prospective versus retrospective studies, randomized versus nonrandomized studies, studies reporting significant main results versus those without, and multicenter studies versus single-center studies. Biomechanical studies also achieved high PRs. CONCLUSION: The PR was similar to that of NASS (40%) and only slightly inferior to that of SRS (47%) and ISSLS (45%). This shows the high quality of presentations at SSE congresses. The fate of unpublished abstracts is worth further consideration. It is questionable whether it is acceptable to cite abstracts that have not passed a journal's peer-review process and to implement their results in clinical practice.

Modulation of CD22 Protein Expression in Childhood Leukemia by Pervasive Splicing Aberrations: Implications for CD22-Directed Immunotherapies.

Downregulation of surface epitopes causes postimmunotherapy relapses in B-lymphoblastic leukemia (B-ALL). Here we demonstrate that mRNA encoding CD22 undergoes aberrant splicing in B-ALL. We describe the plasma membrane-bound CD22 Δex5-6 splice isoform, which is resistant to chimeric antigen receptor (CAR) T cells targeting the third immunoglobulin-like domain of CD22. We also describe splice variants skipping the AUG-containing exon 2 and failing to produce any identifiable protein, thereby defining an event that is rate limiting for epitope presentation. Indeed, forcing exon 2 skipping with morpholino oligonucleotides reduced CD22 protein expression and conferred resistance to the CD22-directed antibody-drug conjugate inotuzumab ozogamicin in vitro. Furthermore, among inotuzumab-treated pediatric patients with B-ALL, we identified one nonresponder in whose leukemic blasts Δex2 isoforms comprised the majority of CD22 transcripts. In a second patient, a sharp reduction in CD22 protein levels during relapse was driven entirely by increased CD22 exon 2 skipping. Thus, dysregulated CD22 splicing is a major mechanism of epitope downregulation and ensuing resistance to immunotherapy. SIGNIFICANCE: The mechanism(s) underlying downregulation of surface CD22 following CD22-directed immunotherapy remains underexplored. Our biochemical and correlative studies demonstrate that in B-ALL, CD22 expression levels are controlled by inclusion/skipping of CD22 exon 2. Thus, aberrant splicing of CD22 is an important driver/biomarker of de novo and acquired resistance to CD22-directed immunotherapies. See related commentary by Bourcier and Abdel-Wahab, p. 87. This article is highlighted in the In This Issue feature, p. 85.

Sagittal balance of thoracic lordoscoliosis: anterior dual rod instrumentation versus posterior pedicle screw fixation.

Posterior pedicle screw fixation is now the standard treatment for surgical correction of idiopathic scoliosis and has largely replaced anterior techniques, but there have been reports describing a lordogenic effect of segmental pedicle screw instrumentation in the thoracic spine. This clinical study compared anterior dual rod instrumentation with posterior pedicle screw fixation for idiopathic thoracic lordoscoliosis, including 42 patients (7 male, 35 female; average age 16 years, range 12-34) who underwent posterior pedicle screw fixation (n = 20) or anterior dual rod instrumentation (n = 22) at two centers. The average follow-up period was 33 months (24-108 months). Inclusion criteria were a diagnosis of adolescent idiopathic scoliosis with a structural thoracic curve (Lenke 1-3) and thoracic hypokyphosis (T4-T12 < 20°). The main thoracic curve magnitude and sagittal profile on standing radiographs were evaluated. Thoracic kyphosis was significantly restored from preoperatively 10.2° to 23.4° postoperatively in the anterior group and from 7.6° to 12.9° in the posterior group (P < 0.005). Kyphosis improved significantly better in the anterior group than in the posterior group (P < 0.005). The preoperative and postoperative main thoracic curve values were 63° (48-80°) and 25.2° in the anterior group and 60.6° (50-88°) and 23.6° in the posterior group, with no significant differences between the groups. No neurological or other severe complications were observed. Anterior dual rod instrumentation in patients with thoracic lordoscoliosis allows significantly better restoration of thoracic kyphosis than posterior pedicle screw instrumentation.

Prospective evaluation of physical activity in patients with idiopathic scoliosis or kyphosis receiving brace treatment.

Bracing is an established method of conservative treatment for adolescent idiopathic scoliosis and kyphosis. Compliance among adolescents is frequently inadequate due to the discomfort of wearing a brace, cosmetic issues, and fear on the part of patients and parents that bracing may reduce everyday physical activities. The aim of this prospective, controlled study was to objectify the impact of spinal bracing on daily step activity in patients receiving conservative treatment for adolescent idiopathic scoliosis (AIS) or adolescent kyphosis (AK). Forty-eight consecutive patients (mean age 13.4 ± 2.3 years), consisting of 38 AIS patients (33 girls, 5 boys) and 10 AK patients (6 girls, 4 boys) were included. Once the decision to carry out bracing had been taken and while the patients were waiting for the individual brace to be built, step activity was assessed without braces by means of step activity monitoring (SAM) for seven consecutive days. After 8 weeks of brace wearing, step activity was assessed during regular brace treatment, again for seven consecutive days. In addition, brace-wearing times were simultaneously recorded using temperature probes implanted in the braces to measure compliance. Before and during brace treatment, patients completed the Scoliosis Research Society (SRS-22) questionnaire. The SAM was worn for an average of 12.7 ± 1.5 h/day during the first measurement and 12.3 ± 1.9 h on average during the second measurement. The mean gait cycles (GCs) per day and per hour before treatment were 5,036 ± 1,465 and 395 ± 105, respectively. No significant reduction in step activity was found at the follow-up measurement during bracing, at 4,880 ± 1,529 GCs/day and 403 ± 144 GCs/h. Taking the 23-h recommended time for brace wearing as a basis (100%), patients wore the brace for 72.7 ± 27.6% of the prescribed time, indicating an acceptable level of compliance. Girls showed a higher compliance level (75.6 ± 25.6%) in comparison with boys (56.7 ± 31.9%), although the difference was not significant (P = 0.093). The SRS-22 total score showed no differences between the two measurements (2.57 ± 0.23 vs. 2.56 ± 0.28). Implementing a simultaneous and objective method of assessing step activity and brace-wearing times in everyday life proved to be feasible, and it expands the information available regarding the impact of bracing on patients' quality of life. The results clearly show that brace treatment does not negatively interfere with daily step activity in AIS and AK patients. This is an important finding that should help reduce patients' and parents' worries concerning bracing.

The Dual Role of a Polyvalent IgM/IgA-Enriched Immunoglobulin Preparation in Activating and Inhibiting the Complement System.

Activation of the complement system is important for efficient clearance of a wide variety of pathogens via opsonophagocytosis, or by direct lysis via complement-dependent cytotoxicity (CDC). However, in severe infections dysregulation of the complement system contributes to hyperinflammation. The influence of the novel IgM/IgA-enriched immunoglobulin preparation trimodulin on the complement pathway was investigated in in vitro opsonophagocytosis, binding and CDC assays. Immunoglobulin levels before and after trimodulin treatment were placed in relation to complement assessments in humans. In vitro, trimodulin activates complement and induces opsonophagocytosis, but also interacts with opsonins C3b, C4b and anaphylatoxin C5a in a concentration-dependent manner. This was not observed for standard intravenous IgG preparation (IVIg). Accordingly, trimodulin, but not IVIg, inhibited the downstream CDC pathway and target cell lysis. If applied at a similar concentration range in healthy subjects, trimodulin treatment resulted in C3 and C4 consumption in a concentration-dependent manner, which was extended in patients with severe community-acquired pneumonia. Complement consumption is found to be dependent on underlying immunoglobulin levels, particularly IgM, pinpointing their regulative function in humans. IgM/IgA provide a balancing effect on the complement system. Trimodulin may enhance phagocytosis and opsonophagocytosis in patients with severe infections and prevent excessive pathogen lysis and release of harmful anaphylatoxins.

Ion Mobility Spectrometry as a Potential Tool for Flavor Control in Chocolate Manufacture.

Chocolate has a complex flavor profile composed of more than 600 volatile compounds that mainly arise from the thermo-mechanical treatment during roasting and conching. The aim of this study was to evaluate the applicability of ion mobility spectrometry (IMS), as a real-time method for process monitoring in chocolate manufacture. It is evident from the ion mobility (IM) fingerprint spectra that individual processing steps affect the signal intensities at particular drift time regions. The analysis of individual IM spectra by principal component analysis (PCA) revealed that it is possible to distinguish with respect to conching temperature and time. PCA also allowed identifying those parts of the IM spectra that were predominantly affected by the respective treatment. It was, on the basis of the IM flavor fingerprints and subsequent PCA, possible to distinguish between the different states of processing of bulk cocoa. The results of the study imply that, using appropriate post-data treatment, IMS could be used for process control in cocoa processing.

Biochemical engineering of cell surface sialic acids stimulates axonal growth.

Sialylation is essential for development and regeneration in mammals. Using N-propanoylmannosamine, a novel precursor of sialic acid, we were able to incorporate unnatural sialic acids with a prolonged N-acyl side chain (e.g., N-propanoylneuraminic acid) into cell surface glycoconjugates. Here we report that this biochemical engineering of sialic acid leads to a stimulation of neuronal cells. Both PC12 cells and cerebellar neurons showed a significant increase in neurite outgrowth after treatment with this novel sialic acid precursor. Furthermore, also the reestablishment of the perforant pathway was stimulated in brain slices. In addition, we surprisingly identified several cytosolic proteins with regulatory functions, which are differentially expressed after treatment with N-propanoylmannosamine. Because sialic acid is the only monosaccharide that is activated in the nucleus, we hypothesize that transcription could be modulated by the unnatural CMP-N-propanoylneuraminic acid and that sialic acid activation might be a general tool to regulate cellular functions, such as neurite outgrowth.

Isolation of two Δ5 polymethylene interrupted fatty acids from Podocarpus falcatus by countercurrent chromatography.

The lipids of gymnosperms frequently feature unusual polyunsaturated fatty acids (PUFAs) such as sciadonic acid (20:3Δ5,11,14) and juniperonic acid (20:4Δ5,11,14,17) showing a first double bond on C-5 which is separated from the next double bond by five methylene units. Compared to "classic" fatty acids, these fatty acids are not easily commercially available and their prices are quite high. For this reason, we wished to isolate those fatty acids from the seed oil of Podocarpus falcatus by countercurrent chromatography (CCC) after conversion of the fatty acids to methyl esters (FAMEs). The contribution of sciadonic acid (20:3Δ5,11,14) and juniperonic acid (20:4Δ5,11,14,17) in the unfractionated sample was 10% and 6% respectively, while oleic acid (18:1Δ9) and linoleic acid (18:2Δ9,12) were the major fatty acids. After a first CCC run with FAMEs from Podocarpus falcatus, fractions enriched in the target compounds were chosen for subsequent isolation by means of two subsequent CCC runs. Initially, 13mg of juniperonic acid was recovered with a purity of 92% according to analysis by gas chromatography with mass spectrometry (GC/MS). Further purification of this fraction yielded 2.7mg with a purity of 99% according to GC/MS. The isolation of sciadonic acid was hampered by high amounts of linoleic acid with the same equivalent chain length in suitable fractions of the first CCC separation. After an enrichment step by CCC, the critical pair sciadonic acid and linoleic acid was finally separated as free fatty acids. After this step, 4.4mg of sciadonic acid was recovered with 99% purity. The methodology could also be applied to isolate larger amounts of those fatty acids or for the isolation of other minor fatty acids.

Congenital myasthenic syndrome due to a novel missense mutation in the gene encoding choline acetyltransferase.

Congenital myasthenic syndromes are caused by different genetic defects affecting proteins expressed at the neuromuscular junction. Recently, the first molecular genetic defect resulting in a presynaptic congenital myasthenic syndrome has been reported: Recessive loss-of-function mutations in CHAT, the gene encoding choline acetyltransferase, were described in five congenital myasthenic syndrome families. In this study, we investigated three patients from two independent Turkish kinships. Clinically, all patients presented with moderate myasthenic symptoms including ptosis and muscle weakness with increased fatigability. Multiple episodes of sudden apnea were reported for all patients. One child suffering from a second, unrelated disorder, i.e. hepatocellular carcinoma, showed a severe myasthenic phenotype, requiring permanent ventilation. Genetically, we identified a novel missense mutation (I336T) in the CHAT gene homozygously in all three patients. Haplotype analysis revealed that the mutant allele cosegregates with the clinical phenotype in both families (maximum combined two-point LOD-score of 2.46 for D10S1793). In summary, we confirm that CHAT mutations are responsible for a clinically distinct form of congenital myasthenic syndrome, characterized by episodic apnea. Infections and stress may lead to a life-threatening failure of neuromuscular transmission in congenital myasthenic syndrome with episodic apnea. The observation of the same mutation (I336T) in two independent Turkish kinships may suggest a common origin, i.e. founder.

Variable reduction of caveolin-3 in patients with LGMD2B/MM.

Mutations in the human dysferlin gene ( DYSF) cause autosomal recessive muscular dystrophies characterized by degeneration and weakness of proximal and/or distal muscles: limb girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy (MM). Recently, an interaction between caveolin-3 and dysferlin in normal and dystrophic muscle (primary caveolin-3 deficiency; LGMD1C) was shown. In this study, clinical,morphological and genetic analysis was carried out in four independent LGMD2B/MM patients. All patients presented with an adult-onset, slowly progressive muscular dystrophy with variable involvement of proximal and distal muscles. We found complete lack of dysferlin in the four LGMD2B/MM patients. Secondary reduction of caveolin-3 was detected in three out of the four patients. Regular caveolae were detected along the basal lamina in two patients by electron microscopy. We provide further evidence that dysferlin and caveolin-3 interact in human skeletal muscle. It remains to be elucidated whether the loss of this interaction contributes to pathogenic events in muscular dystrophy.

The prevalence of rotator cuff tears: is the contralateral shoulder at risk?

BACKGROUND: Rotator cuff tears are a common cause of pain and disability of the shoulder. Information on the prevalence and identification of potential risk factors could help in early detection of rotator cuff tears and improve treatment outcome. HYPOTHESIS: Patients treated for a symptomatic rotator cuff tear on one side have a higher prevalence of rotator cuff tears and decreased shoulder function on the contralateral side compared with an age- and sex-matched group of healthy individuals. STUDY DESIGN: Case control study; Level of evidence, 3. METHODS: One group consisted of 55 patients who had been arthroscopically treated on one shoulder for rotator cuff tear (tear group). In this group, the nonoperated contralateral shoulder was examined. For comparison, the matching shoulder in a control group consisting of 55 subjectively healthy individuals matched by age (±1 year) and sex to the tear group was included. Diagnosis of a rotator cuff tear was made by ultrasound. Outcomes were measured using the Constant score. RESULTS: The prevalence of supraspinatus tears was significantly higher (P < .0001) in the tear group (67.3%) compared with the control group (11.0%). The Constant score for the activities of daily living subscale, however, was significantly lower (18.4) in the tear group compared with the control group (19.9; P = .012). No other subcategory score nor the overall score showed a significant difference. There was a significantly higher tear prevalence in the tear group of patients aged between 50 and 59 years (P < .001) and 60 and 69 years (P = .004). No tear was diagnosed in the control group in individuals younger than 60 years. CONCLUSION: Patients treated for partial and full-thickness rotator cuff tears have a significantly higher risk of having a tear on the contralateral side and have noticeable deficits in their shoulder function regarding activities of daily living even if the tear is otherwise asymptomatic.