Scalable protein production by Komagataella phaffii enabled by ARS plasmids and carbon source-based selection.

BACKGROUND: Most recombinant Komagataella phaffii (Pichia pastoris) strains for protein production are generated by genomic integration of expression cassettes. The clonal variability in gene copy numbers, integration loci and consequently product titers limit the aptitude for high throughput applications in drug discovery, enzyme engineering or most comparative analyses of genetic elements such as promoters or secretion signals. Circular episomal plasmids with an autonomously replicating sequence (ARS), an alternative which would alleviate some of these limitations, are inherently unstable in K. phaffii. Permanent selection pressure, mostly enabled by antibiotic resistance or auxotrophy markers, is crucial for plasmid maintenance and hardly scalable for production. The establishment and use of extrachromosomal ARS plasmids with key genes of the glycerol metabolism (glycerol kinase 1, GUT1, and triosephosphate isomerase 1, TPI1) as selection markers was investigated to obtain a system with high transformation rates that can be directly used for scalable production processes in lab scale bioreactors. RESULTS: In micro-scale deep-well plate experiments, ARS plasmids employing the Ashbya gossypii TEF1 (transcription elongation factor 1) promoter to regulate transcription of the marker gene were found to deliver high transformation efficiencies and the best performances with the reporter protein (CalB, lipase B of Candida antarctica) for both, the GUT1- and TPI1-based, marker systems. The GUT1 marker-bearing strain surpassed the reference strain with integrated expression cassette by 46% upon re-evaluation in shake flask cultures regarding CalB production, while the TPI1 system was slightly less productive compared to the control. In 5 L bioreactor methanol-free fed-batch cultivations, the episomal production system employing the GUT1 marker led to 100% increased CalB activity in the culture supernatant compared to integration construct. CONCLUSIONS: For the first time, a scalable and methanol-independent expression system for recombinant protein production for K. phaffii using episomal expression vectors was demonstrated. Expression of the GUT1 selection marker gene of the new ARS plasmids was refined by employing the TEF1 promoter of A. gossypii. Additionally, the antibiotic-free marker toolbox for K. phaffii was expanded by the TPI1 marker system, which proved to be similarly suited for the use in episomal plasmids as well as integrative expression constructs for the purpose of recombinant protein production.

Comparison of CRISPR-MAD7 and CRISPR-Cas9 for Gene Disruptions in Komagataella phaffii.

CRISPR (clustered regularly interspaced short palindromic repeats)-based technologies are powerful, programmable tools for site-directed genome modifications. After successful adaptation and efficient use of CRISPR-Cas9 for genome engineering in methylotrophic yeast Komagataella phaffii, a broader variety of employable endonucleases was desired to increase the experimental flexibility and to provide alternatives in case there are specific legal restrictions in industrial research due to the intellectual property rights (IPRs) of third parties. MAD7, an engineered Class 2 Type V Cas nuclease, was promoted as a royalty-free alternative for academic and industrial research and developed by Inscripta (Pleasanton, CA, USA). In this study, for the first time, CRISPR-MAD7 was used for genome editing in K. phaffii with a high gene-editing rate (up to 90%), as demonstrated for the three targeted genes coding for glycerol kinase 1 (GUT1), red fluorescence protein (DsRed), and zeocin resistance gene (Sh ble). Additionally, the genome-editing efficiencies of the CRISPR-MAD7 and CRISPR-Cas9 systems were systematically compared by targeting 259 kinase genes in K. phaffii. In this broad testing, the CRISPR-Cas9 had a higher genome-editing rate of about 65%, in comparison to the applied CRISPR-MAD7 toolbox (about 23%).

Psychomotor Slowing in Psychosis and Inhibitory Repetitive Transcranial Magnetic Stimulation: A Randomized Clinical Trial.

Importance: Psychomotor slowing is a frequent symptom of psychosis, impairing gross and fine motor behavior. It is associated with poor outcomes and functioning, and no treatment is available. Objective: To investigate whether 15 sessions of inhibitory repetitive transcranial magnetic stimulation (rTMS) may reduce psychomotor slowing. Design, Setting, and Participants: This was a 4-arm, double-blind, randomized, sham-controlled trial at a university hospital in Switzerland. Enrollment took place from March 2019 to August 2022. Adults aged 18 to 60 years with schizophrenia spectrum disorders and severe psychomotor slowing were eligible. All patients continued existing medications, including antipsychotics and benzodiazepines. Those with substance misuse (other than nicotine), conditions associated with impaired or aberrant movement, convulsions, history of hearing problems, other conditions typically excluded from magnetic resonance imaging or TMS, any TMS treatment in the past 3 months, or those who were pregnant or breastfeeding were excluded. Of 615 patients screened for eligibility, 103 were randomized and 88 received at least 1 session of rTMS: 22 were assigned to 1-Hz rTMS, 22 to iTBS, 22 to sham, and 22 to the waiting group. Follow-up was conducted at 6 weeks and 24 weeks following the week 3 assessments including clinical, functional, and motor measures. Interventions: Fifteen sessions of rTMS in 3 weeks over the supplementary motor area: 1-Hz rTMS, iTBS, sham, or no treatment (waiting). After 3 weeks, the waiting group received 15 sessions of 1-Hz rTMS over the supplementary motor area. Main Outcomes and Measures: The main outcome was the proportion of responders at week 3 in the Salpêtrière Retardation Rating Scale (SRRS) defined as a 30% or greater reduction from baseline (last-observation-carried-forward). The SRRS has 15 items and a maximum total score of 60. Results: Of the 88 participants analyzed, 45 were men and 43 were women. The mean (SD) age was 36.3 (12.4) years and the mean (SD) SRRS score was 24.0 (5.9). A total of 69 participants completed the study. At week 3, response rates differed between groups: 15 of 22 (68%) in the 1-Hz rTMS group, 8 of 22 (36%) in the iTBS group, 7 of 22 (32%) in the sham group, and 4 of 22 (18%) in the waiting group (χ23 = 12.1; P = .007). The 1-Hz rTMS group had more responders than sham (odds ratio [OR], 0.13; 95% CI, 0.02-0.65; P = .03), iTBS (OR, 0.12; 95% CI, 0.02-0.61; P = .02), and waiting (OR, 0.04; 95% CI, 0.01-0.22; P = .003). In the waiting group, 10 of 16 participants (63%) responded after receiving 15 sessions of 1-Hz rTMS. No serious adverse events occurred. Conclusions and Relevance: In this study, inhibitory add-on rTMS safely alleviated psychomotor slowing in psychosis compared with iTBS, sham, and no treatment. The treatment was also effective with delayed onset. Future studies need to explore the neural changes associated with supplementary motor area rTMS in psychosis. Trial Registration: ClinicalTrials.gov Identifier: NCT03921450.

Sulfate limitation increases specific plasmid DNA yield and productivity in E. coli fed-batch processes.

Plasmid DNA (pDNA) is a key biotechnological product whose importance became apparent in the last years due to its role as a raw material in the messenger ribonucleic acid (mRNA) vaccine manufacturing process. In pharmaceutical production processes, cells need to grow in the defined medium in order to guarantee the highest standards of quality and repeatability. However, often these requirements result in low product titer, productivity, and yield. In this study, we used constraint-based metabolic modeling to optimize the average volumetric productivity of pDNA production in a fed-batch process. We identified a set of 13 nutrients in the growth medium that are essential for cell growth but not for pDNA replication. When these nutrients are depleted in the medium, cell growth is stalled and pDNA production is increased, raising the specific and volumetric yield and productivity. To exploit this effect we designed a three-stage process (1. batch, 2. fed-batch with cell growth, 3. fed-batch without cell growth). The transition between stage 2 and 3 is induced by sulfate starvation. Its onset can be easily controlled via the initial concentration of sulfate in the medium. We validated the decoupling behavior of sulfate and assessed pDNA quality attributes (supercoiled pDNA content) in E. coli with lab-scale bioreactor cultivations. The results showed an increase in supercoiled pDNA to biomass yield by 33% and an increase of supercoiled pDNA volumetric productivity by 13 % upon limitation of sulfate. In conclusion, even for routinely manufactured biotechnological products such as pDNA, simple changes in the growth medium can significantly improve the yield and quality.

Neural Correlates of Formal Thought Disorder Dimensions in Psychosis.

BACKGROUND AND HYPOTHESIS: Formal thought disorder (FTD) is a core symptom of psychosis, but its neural correlates remain poorly understood. This study tested whether four FTD dimensions differ in their association with brain perfusion and brain structure. STUDY DESIGN: This cross-sectional study investigated 110 patients with schizophrenia spectrum disorders using 3T magnetic resonance imaging (MRI). The Thought and Language Disorder scale (TALD) was utilized, which comprises four subscales: Objective Positive (OP), Objective Negative (ON), Subjective Positive (SP), and Subjective Negative (SN). Resting-state cerebral blood flow (rsCBF), cortical thickness (CortTh), gray matter volume (GMV), and diffusion MRI tractography were tested for associations with TALD subscales controlling for age, medication, total intracranial volume, and for variance of the 3 other TALD subscales. STUDY RESULTS: Following Bonferroni correction, the FTD dimensions presented distinct neural correlates. OP scores were associated with increased rsCBF and increased GMV in the right cerebellum lingual gyrus. Higher SP scores were linked to increased GMV in bilateral prefrontal cortex. In contrast, ON was associated with increased GMV in the right premotor cortex. At more liberal statistical thresholds, higher SP was associated with increased CortTh in the right inferior frontal gyrus, whereas SN scores were linked to decreased GMV in the right prefrontal lobe, the left inferior temporal gyrus, and the left supplementary motor area. Unadjusted analyses mostly corroborated these findings. CONCLUSION: These findings stress the heterogeneity in FTD, suggesting distinct neural patterns for specific FTD experiences. In sum, FTD in psychosis may require distinct treatment strategies and further mechanistic investigations on single-item levels.

[Catatonia Through the Ages - from Kahlbaum to the ICD-11]. / Katatonie im Wandel der Zeit ­ von Kahlbaum bis zum ICD-11.

For decades, the psychomotor syndrome of catatonia was considered exclusively a subtype of schizophrenia. The use of antipsychotics and changes in the teaching content in the further training of psychiatrists led to the fact that catatonia was hardly recognized anymore. Yet catatonia is in principle well treatable. The new status in ICD-11 will probably allow to better teach, recognize, and treat catatonia in the future.

Accuracy of additively manufactured zirconia four-unit fixed dental prostheses fabricated by stereolithography, digital light processing and material jetting compared with subtractive manufacturing.

OBJECTIVE: To evaluate the manufacturing accuracy of zirconia four-unit fixed dental prostheses (FDPs) fabricated by three different additive manufacturing technologies compared with subtractive manufacturing. METHODS: A total of 79 zirconia FDPs were produced by three different manufacturing technologies, representing additive (one stereolithography [aSLA] and one material jetting [aMJ] device, two digital light processing [aDLP1/aDLP2] devices) and subtractive manufacturing (two devices [s1/s2]), the latter serving as references. After printing, additively manufactured FDPs were debound and finally sintered. Subsequently, samples were circumferentially digitized and acquired surface areas were split in three Regions Of Interest (ROIs: inner/outer shell, margin). Design and acquired data were compared for accuracy using an inspection software. Statistical evaluation was performed using the root mean square error (RMSE) and nonparametric Kruskal-Wallis method with post hoc Wilcoxon-Mann-Whitney U tests. Bonferroni correction was applied in case of multiple testing. RESULTS: Regardless the ROI, significant differences were observed between manufacturing technologies (P < 0.001). Subtractive manufacturing was the most accurate with no significant difference regarding the material/device (s1/s2, P > 0.054). Likewise, no statistical difference regarding accurary was found when comparing s2 with aMJ and aSLA in most ROIs (P > 0.085). In general, mean surface deviation was< 50 µm for s1/s2 and aMJ and< 100 µm for aSLA and aDLP2. aDLP1 showed surface deviations> 100 µm and was the least accurate compared to the other additive/subtractive technologies. SIGNIFICANCE: Additive manufacturing represents a promising set of technologies for the manufacturing of zirconia FDPs, but not yet as accurate as subtractive manufacturing. Methodological impact on accuracy within and in between different additive technologies needs to be further investigated.

Presentation and evaluation of a modern course in disaster medicine and humanitarian assistance for medical students.

BACKGROUND: Disaster medicine is a component of the German medical education since 2003. Nevertheless, studies have shown some inconsistencies within the implementation of the national curriculum, and limits in the number of students trained over the years. Recently, the SARS-CoV-2 pandemic and other disasters have called attention to the importance of training medical students in disaster medicine on a coordinated basis. The aim of this study is to present and evaluate the disaster medicine and humanitarian assistance course, which was developed in the University of Tübingen, Germany. METHODS: The University Clinic for Anesthesiology and Intensive Care Medicine in Tübingen expanded the existing curriculum of undergraduate disaster medicine training with fundamentals of humanitarian medicine, integrating distance learning, interactive teaching and simulation sessions in a 40 h course for third-, fourth- and fifth- year medical students. This prospective and cross-sectional study evaluates the Disaster Medicine and Humanitarian Assistance course carried out over five semesters during the period between 2018 and 2020. Three survey tools were used to assess participants' previous experiences and interest in the field of disaster medicine, to compare the subjective and objective level of knowledge before and after training, and to evaluate the course quality. RESULTS: The total number of medical students attending the five courses was n = 102 of which n = 60 females (59%) and n = 42 males (41%). One hundred two students entered the mandatory knowledge assessment, with the rate of correct answers passing from 73.27% in the pre-test to 95.23% in the post-test (t [101] = 18.939, p < .001, d = 1.88). To determine the subjective perception of knowledge data were collected from 107 observations. Twenty-five did not complete the both questionnaires. Out of a remaining sample of 82 observations, the subjective perception of knowledge increased after the course (t [81] = 24.426, p < .001, d = 2.69), alongside with the interest in engaging in the field of disaster medicine (t [81] = 7.031, p < .001, d = .78). The 93.46% of the medical students (n = 100) graded the training received with an excellent overall score (1.01 out of 6). CONCLUSION: The study indicates a significant increase in students' understanding of disaster medicine using both subjective and objective measurements, as well as an increase interest in the field of disaster medicine and humanitarian assistance. Whereas former studies showed insufficient objective knowledge regarding disaster medical practices as well as subjective insecurities about their skills and knowledge to deal with disaster scenarios, the presented course seems to overcome these deficiencies preparing future physicians with the fundamentals of analysis and response to disasters. The development and successful implementation of this course is a first step towards fulfilling disaster medicine education requirements, appearing to address the deficiencies documented in previous studies. A possible adaptation with virtual reality approaches could expand access to a larger audience. Further effort must be made to develop also international training programs, which should be a mandatory component of medical schools' curricula.

Hand gesture performance is impaired in major depressive disorder: A matter of working memory performance?

OBJECTIVE: Individuals with depression exhibit numerous interpersonal deficits. As effective use of gestures is critical for social communication, it is possible that depressed individuals' interpersonal deficits may be due to deficits in gesture performance. The present study thus compared gesture performance of depressed patients and controls and examined whether these deficits relate to cognitive and other domains of dysfunction. METHODS: Gesture performance was evaluated in 30 depressed patients and 30 controls using the Test of Upper Limb Apraxia (TULIA). Clinical rating scales were assessed to determine if gesture deficits were associated with motor, cognitive or functional outcomes. RESULTS: Compared to controls, depressed patients exhibited impaired gesture performance with 2/3 of the patients demonstrating gesture deficits. Within depressed patients, gesture performance was highly correlated with working memory abilities. In contrast, no association between gesture performance and gestural knowledge, psychomotor retardation, depression severity, or frontal dysfunction was observed in patients. LIMITATIONS: This is a cross-sectional study and a larger size would have allowed for confident detection of more subtle, but potentially relevant effects. CONCLUSION: Gesture performance is impaired in depressed patients, and appears to be related to poor working memory abilities, suggesting a disruption in the retrieval of gestural cues indicative of a distinct clinical phenomenon that might be related to social functioning.

Loss of Neurological Disease HSAN-I-Associated Gene SPTLC2 Impairs CD8+ T Cell Responses to Infection by Inhibiting T Cell Metabolic Fitness.

Patients with the neurological disorder HSAN-I suffer frequent infections, attributed to a lack of pain sensation and failure to seek care for minor injuries. Whether protective CD8+ T cells are affected in HSAN-I patients remains unknown. Here, we report that HSAN-I-associated mutations in serine palmitoyltransferase subunit SPTLC2 dampened human T cell responses. Antigen stimulation and inflammation induced SPTLC2 expression, and murine T-cell-specific ablation of Sptlc2 impaired antiviral-T-cell expansion and effector function. Sptlc2 deficiency reduced sphingolipid biosynthetic flux and led to prolonged activation of the mechanistic target of rapamycin complex 1 (mTORC1), endoplasmic reticulum (ER) stress, and CD8+ T cell death. Protective CD8+ T cell responses in HSAN-I patient PBMCs and Sptlc2-deficient mice were restored by supplementing with sphingolipids and pharmacologically inhibiting ER stress-induced cell death. Therefore, SPTLC2 underpins protective immunity by translating extracellular stimuli into intracellular anabolic signals and antagonizes ER stress to promote T cell metabolic fitness.

A two-step process for capture and purification of human basic fibroblast growth factor from E. coli homogenate: Yield versus endotoxin clearance.

A two-step purification process for human basic fibroblast growth factor (FGF-2) from clarified E. coli homogenate has been developed in which the impurity level after the second step is below the limit of quantification. Endotoxin content is cleared to 0.02 EU/µg FGF-2 and the overall yield is 67%. The performance of the cation exchanger Carboxymethyl-Sepharose Fast Flow (CM-SFF) was compared to the affinity resin Heparin-SFF regarding the impurity profile and product quality in the elution peak. The CM-SFF eluate was further purified using hydrophobic interaction resin Toyopearl-Hexyl-650C. The relative amounts of target product, host cell proteins (HCPs), dsDNA, endotoxin, monomer content, and high molecular weight impurities differed along the elution peak depending on the applied method. The bioactive monomer (>99%) was obtained with a yield of 48% for CM-SFF and 68% for Heparin-SFF. A half-load reduction in CM-SFF increased the yield up to 67% without deterioration of the impurity content. Assuming a dose of 400 µg FGF-2, endotoxin was reduced to 188 EU/dose, dsDNA <10 ng/dose, and HCP <2 ppm/dose using the cation exchanger. In the pooled eluate fractions, dsDNA was removed 4-fold (291 ng/mL) and endotoxin 14-fold (0.47 EU/µg FGF-2) more efficiently by CM-SFF than by affinity chromatography. In contrast, HCP clearance was 3-fold (13 ppm) more efficient with Heparin-SFF than CM-SFF. In contrast to process monitoring by UV280nm or SDS-PAGE, this characterization is the basis for a Process Analytical Technology attempt when correlated with online monitored signals, as it enables knowledge-based pooling according to defined quality criteria.

Thin Film Formation and Morphology of Electrosprayed Polydimethylsiloxane.

Low-voltage dielectric actuators (DEAs) can be fabricated using submicrometer-thin polydimethylsiloxane (PDMS) films. The two established techniques, namely spin coating and molecular beam deposition, however, are inappropriate to produce multistack DEAs in an efficient way. Therefore, we propose an alternative deposition technique, i.e., the alternating current electrospray deposition (ACESD) of 5 vol % PDMS in ethyl acetate solution and subsequent ultraviolet light curing. Atomic force microscopy makes possible the three-dimensional analysis of cured droplet-like islands. These circular islands, prepared on 2 in. Si(100) wafers from four polymers with molecular masses between 800 and 62,700 g/mol, reveal a characteristic morphology with an increasing height-to-diameter ratio. Using the 6000 g/mol polymer for ACESD, the film morphology evolution was tracked by applying conventional optical microscopy and spectroscopic ellipsometry. When the deposition was terminated after 13 s, circular islands with a mean height of 30 nm were found, while terminating the deposition after about 155 s led to a confluent layer with a mean height of 91 ± 10 nm. Potential electrostatic interactions between the droplets could not be identified through the analysis of spatial island distribution. Nevertheless, ACESD is a budget-priced and competitive deposition technique that can be employed to fabricate submicrometer-thin PDMS films with true nanometer roughness.

Vesicular Galectin-3 levels decrease with donor age and contribute to the reduced osteo-inductive potential of human plasma derived extracellular vesicles.

Aging results in a decline of physiological functions and in reduced repair capacities, in part due to impaired regenerative power of stem cells, influenced by the systemic environment. In particular osteogenic differentiation capacity (ODC) of mesenchymal stem cells (MSCs) has been shown to decrease with age, thereby contributing to reduced bone formation and an increased fracture risk. Searching for systemic factors that might contribute to this age related decline of regenerative capacity led us to investigate plasma-derived extracellular vesicles (EVs). EVs of the elderly were found to inhibit osteogenesis compared to those of young individuals. By analyzing the differences in the vesicular content Galectin-3 was shown to be reduced in elderly-derived vesicles. While overexpression of Galectin-3 resulted in an enhanced ODC of MSCs, siRNA against Galectin-3 reduced osteogenesis. Modulation of intravesicular Galectin-3 levels correlated with an altered osteo-inductive potential indicating that vesicular Galectin-3 contributes to the biological response of MSCs to EVs. By site-directed mutagenesis we identified a phosphorylation-site on Galectin-3 mediating this effect. Finally, we showed that cell penetrating peptides comprising this phosphorylation-site are sufficient to increase ODC in MSCs. Therefore, we suggest that decrease of Galectin-3 in the plasma of elderly contributes to the age-related loss of ODC.

Rapamycin-induced phosphaturia.

BACKGROUND: The mammalian target of rapamycin (mTOR) is known to stimulate a variety of transport mechanisms including the intestinal phosphate transporter NaPi-IIb. The present study was performed to elucidate whether mTOR similarly regulates the major renal tubular phosphate transporter NaPi-IIa. METHODS: To this end, NaPi-IIa was expressed in Xenopus oocytes with or without mTOR and phosphate transport estimated from phosphate-induced (1 mM) current (I(pi)). RESULTS: As a result, I(pi) was observed in NaPi-IIa-expressing but not in H(2)O-injected Xenopus oocytes. Co-expression of mTOR significantly enhanced I(pi) in NaPi-IIa-expressing Xenopus oocytes, an effect abrogated by treatment with rapamycin (50 nM for the last 24 h of incubation). In a second series of experiments, the effect of rapamycin was analysed in mice. The in vivo administration of rapamycin (3 microg/g body weight/day) for 3 days resulted in phosphaturia in mice despite a tendency of plasma phosphate concentration to decrease. CONCLUSIONS: mTOR contributes to the regulation of renal phosphate transport, and rapamycin thus influences phosphate balance.

Regulation of renal tubular glucose reabsorption by Akt2/PKBß.

Akt/PKB is known to regulate the facilitative glucose carrier GLUT4. Nothing is known, however, of the role of Akt/PKB in the regulation of renal epithelial transport. To explore whether Akt2/PKBß influences the Na(+)-coupled glucose cotransporter SGLT1, human SGLT1 was expressed in Xenopus laevis oocytes with or without Akt/PKB, and electrogenic glucose transport was determined by dual-electrode voltage clamp. The coexpression of Akt/PKB in SGLT1-expressing oocytes was followed by an increase in glucose-induced currents. To study the functional significance of Akt/PKB-sensitive renal glucose transport, further experiments were performed in gene-targeted mice lacking functional Akt2/PKBß (akt2(-/-)) and in their wild-type littermates (akt2(+/+)). Plasma glucose concentration was significantly higher in akt2(-/-) mice than in akt2(+/+) mice but was virtually identical to the plasma glucose concentration in fructose-treated akt2(+/+) mice. Urinary glucose excretion was significantly higher in akt2(-/-) mice compared with akt2(+/+) mice with or without fructose treatment. Moreover, the glucose-induced depolarization of proximal tubular cells was significantly smaller in isolated, perfused renal tubules from akt2(-/-) mice than in those from akt2(+/+) mice. In conclusion, Akt2/PKBß plays a role in the regulation of renal glucose transport.

Complications of non-compaction of the left ventricular myocardium in a paediatric population: a prospective study.

AIMS: Non-compaction of the left ventricular myocardium (NCVM) is reportedly exceedingly rare and associated with a high morbidity and mortality. A different genetic background has been suggested for NCVM with [non-isolated NCVM (ni-NCVM)] and without [isolated NCVM (i-NCVM)] other congenital heart defects. We prospectively evaluated both the NCVM subgroups regarding frequency of occurrence and cardiovascular complications in a paediatric population. Results In a prospective, single-centre study, 66/5220 consecutive patients (1.26%) were diagnosed (25 i-NCVM, 41 ni-NCVM). The median age was 4 years (range 0-21), the median follow-up 12 months (range 0-51). The occurrence of congestive heart failure (CHF) at follow-up was 68.0%. CHF was as frequently seen in i-NCVM and ni-NCVM patients (77.5 vs. 62.1%, P = 0.322). The occurrence of arrhythmias (20.0%) and thrombo-embolic events (13.9%) was not different between subgroups. The cardiomyopathy related mortality was 7.1%, with three and one deaths in the i-NCVM and ni-NCVM groups, respectively (P = 0.126). CONCLUSION: When prospectively evaluated, NCVM appears to have been previously under-diagnosed. Whereas arrhythmias and thrombo-embolic events were rare, CHF was frequently found. An equally aggressive anticongestive treatment regimen would seem indicated for both the NCVM subgroups.

MR evaluation of dural ectasia in Marfan syndrome: reassessment of the established criteria in children, adolescents, and young adults.

PURPOSE: To retrospectively evaluate known criteria for assessment of dural ectasia by using magnetic resonance (MR) imaging in children, adolescents, and young adults with and those without Marfan syndrome. MATERIALS AND METHODS: Local ethics committee approval and informed consent were obtained. MR images of the lumbar spine in 28 patients with clinically proved Marfan syndrome (group A; 17 male, 11 female; age range, 4-21 years; mean, 12.1 years), seven patients with suspicion of Marfan syndrome (group B; six male, one female; age range, 6-18 years; mean, 10.4 years), and 55 patients without Marfan syndrome (group C; 26 male, 29 female; age range, 4-20 years; mean, 10.7 years) were evaluated retrospectively for dural ectasia criteria (scalloping, dural sac ratio, nerve root sleeve diameter, sagittal dural sac width at S1 greater than that at L4) and according to classifications by Ahn et al and Fattori et al. For statistical comparison of results, one-way analysis of variance with Scheffe post hoc comparisons was used, with an overall two-tailed significance at alpha = .05. RESULTS: No significant differences in scalloping and nerve root sleeve diameter were shown between groups. A significant difference was measured for dural sac ratios at L5 and S1 (F test, P = .003 and P < .001 at L5 and S1, respectively; post hoc t test for groups A vs C, P = .004 and P < .001 at L5 and S1, respectively). Significant differences were also obtained between groups A and C for sagittal dural sac width at S1 greater than that at L4 according to the calculated mean difference (for both F test and post hoc t test, P < .001 and P = .003 at S1 and L4, respectively). The Ahn et al and Fattori et al classifications were of limited value. CONCLUSION: The data suggest that only dural sac ratio at L5 and S1 and a sagittal dural sac width at S1 greater than that at L4 are statistically significant criteria for the assessment of dural ectasia in children, adolescents, and young adults.