From data to discovery: AI-guided analysis of disease-relevant molecules in spinal muscular atrophy (SMA).

Spinal Muscular Atrophy is caused by partial loss of survival of motoneuron (SMN) protein expression. The numerous interaction partners and mechanisms influenced by SMN loss result in a complex disease. Current treatments restore SMN protein levels to a certain extent, but do not cure all symptoms. The prolonged survival of patients creates an increasing need for a better understanding of SMA. Although many SMN-protein interactions, dysregulated pathways, and organ phenotypes are known, the connections among them remain largely unexplored. Monogenic diseases are ideal examples for the exploration of cause-and-effect relationships to create a network describing the disease-context. Machine learning tools can utilize such knowledge to analyze similarities between disease-relevant molecules and molecules not described in the disease so far. We used an artificial intelligence-based algorithm to predict new genes of interest. The transcriptional regulation of 8 out of 13 molecules selected from the predicted set were successfully validated in an SMA mouse model. This bioinformatic approach, using the given experimental knowledge for relevance predictions, enhances efficient targeted research in SMA and potentially in other disease settings.

The systemic complexity of a monogenic disease: the molecular network of spinal muscular atrophy.

Monogenic diseases are well-suited paradigms for the causal analysis of disease-driving molecular patterns. Spinal Muscular Atrophy (SMA) is one such monogenic model caused by mutation or deletion of the Survival of motor neuron 1 (SMN1) gene. Although several functions of the SMN protein have been studied, single functions and pathways alone do not allow to identify critical disease-driving molecules. Here, we analyzed the systemic characteristics of SMA employing proteomics, phosphoproteomics, translatomics and interactomics from two mouse models with different disease-severities and genetics. This systems approach revealed sub-networks and proteins characterizing commonalities and differences of both models. To link the identified molecular networks with the disease-causing SMN protein, we combined SMN-interactome data with both proteomes creating a comprehensive representation of SMA. By this approach, disease hubs and bottlenecks between SMN and downstream pathways could be identified. Linking a disease-causing molecule with widespread molecular dysregulations via multiomics is a concept for analyses of monogenic diseases.

Renal pathology in a mouse model of severe Spinal Muscular Atrophy is associated with downregulation of Glial Cell-Line Derived Neurotrophic Factor (GDNF).

Spinal muscular atrophy (SMA) occurs as a result of cell-ubiquitous depletion of the essential survival motor neuron (SMN) protein. Characteristic disease pathology is driven by a particular vulnerability of the ventral motor neurons of the spinal cord to decreased SMN. Perhaps not surprisingly, many other organ systems are also impacted by SMN depletion. The normal kidney expresses very high levels of SMN protein, equivalent to those found in the nervous system and liver, and levels are dramatically lowered by ~90-95% in mouse models of SMA. Taken together, these data suggest that renal pathology may be present in SMA. We have addressed this using an established mouse model of severe SMA. Nephron number, as assessed by gold standard stereological techniques, was significantly reduced. In addition, morphological assessment showed decreased renal vasculature, particularly of the glomerular capillary knot, dysregulation of nephrin and collagen IV, and ultrastructural changes in the trilaminar filtration layers of the nephron. To explore the molecular drivers underpinning this process, we correlated these findings with quantitative PCR measurements and protein analyses of glial cell-line-derived neurotrophic factor, a crucial factor in ureteric bud branching and subsequent nephron development. Glial cell-line-derived neurotrophic factor levels were significantly reduced at early stages of disease in SMA mice. Collectively, these findings reveal significant renal pathology in a mouse model of severe SMA, further reinforcing the need to develop and administer systemic therapies for this neuromuscular disease.

[Evaluation of velopharyngeal closing pressure during trumpet play with high-resolution manometry]. / Hochauflösungsmanometrische Evaluation des velopharyngealen Verschlussdrucks beim Trompetenspiel.

OBJECTIVE: In about one-third of brass instrumentalists, there are stress-related insufficiencies of velopharyngeal closure (VPC), i. e. the intraoral pressure exceeds the barrier formed by the VPC. Here, it was the aim to measure the VPC closing pressure while playing a trumpet and to evaluate the influence of a 30 minute stress sequence on the muscular activities in the VPC. MATERIAL AND METHODS: Sample: 6 healthy volunteers; task: to play the sound h1 for 5 seconds with 85 dB(A) and with 100 dB(A). METHODOLOGY: High-resolution manometry (HRM). SAMPLING: t0: measurement without warm up phase t1 after 30 min trumpet play; practice phase with predefined pieces of music. VARIABLES: mean (pmit), minimum (pmin) and maximum pressure (pmax) in the VPA at t0 and t1. STATISTICS: testing for normal distribution, t-test. RESULTS: All measured pressures in the VPC decreased from t0 to t1 for tones produced at 85 dB(A). For 100 dB(A) tones only the pmin decreased significantly. The pressures in the VPA were higher at 100 dB(A) tones overall compared to 85 dB(A) tones, significant differences were found for pmin and pmax at t0. CONCLUSION: Tones played at louder volumes require a stronger muscular contraction in the VPC. The lower VPC pressure after the exercise phase (t1) can either result from a physiological muscular adaptation to the pressure level necessary for a sufficient VPC or already be a sign of muscular fatigue. These findings may be important to assess the work ability of wind instrumentalists by HRM. As shown for the phonation, the VPC pressure profile for the trumpet play can also be described with a three-phase model consisting of an initiation, a stable phase and a termination.

Evidence of promiscuous endothelial binding by Plasmodium falciparum-infected erythrocytes.

The adhesion of infected red blood cells (iRBCs) to human endothelium is considered a key event in the pathogenesis of cerebral malaria and other life-threatening complications caused by the most prevalent malaria parasite Plasmodium falciparum. In the past 30 years, 14 endothelial receptors for iRBCs have been identified. Exposing 10 additional surface proteins of endothelial cells to a mixture of P. falciparum isolates from three Ghanaian malaria patients, we identified seven new iRBC receptors, all expressed in brain vessels. This finding strongly suggests that endothelial binding of P. falciparum iRBCs is promiscuous and may use a combination of endothelial surface moieties.

bootGSEA: a bootstrap and rank aggregation pipeline for multi-study and multi-omics enrichment analyses.

Introduction: Gene set enrichment analysis (GSEA) subsequent to differential expression analysis is a standard step in transcriptomics and proteomics data analysis. Although many tools for this step are available, the results are often difficult to reproduce because set annotations can change in the databases, that is, new features can be added or existing features can be removed. Finally, such changes in set compositions can have an impact on biological interpretation. Methods: We present bootGSEA, a novel computational pipeline, to study the robustness of GSEA. By repeating GSEA based on bootstrap samples, the variability and robustness of results can be studied. In our pipeline, not all genes or proteins are involved in the different bootstrap replicates of the analyses. Finally, we aggregate the ranks from the bootstrap replicates to obtain a score per gene set that shows whether it gains or loses evidence compared to the ranking of the standard GSEA. Rank aggregation is also used to combine GSEA results from different omics levels or from multiple independent studies at the same omics level. Results: By applying our approach to six independent cancer transcriptomics datasets, we showed that bootstrap GSEA can aid in the selection of more robust enriched gene sets. Additionally, we applied our approach to paired transcriptomics and proteomics data obtained from a mouse model of spinal muscular atrophy (SMA), a neurodegenerative and neurodevelopmental disease associated with multi-system involvement. After obtaining a robust ranking at both omics levels, both ranking lists were combined to aggregate the findings from the transcriptomics and proteomics results. Furthermore, we constructed the new R-package "bootGSEA," which implements the proposed methods and provides graphical views of the findings. Bootstrap-based GSEA was able in the example datasets to identify gene or protein sets that were less robust when the set composition changed during bootstrap analysis. Discussion: The rank aggregation step was useful for combining bootstrap results and making them comparable to the original findings on the single-omics level or for combining findings from multiple different omics levels.

Electrical stimulation in treatment of pharyngolaryngeal dysfunctions.

OBJECTIVE: Neuromuscular electrical stimulation (NMES) has been proposed in the treatment of laryngopharyngeal dysfunctions (dysphonia, dyspnoea, dysphagia) for more than 40 years. Several studies have investigated possible therapeutic effects. Some researchers described favourable results, whereas others did not find relevant benefits. This article aims to review available studies to give an overview regarding the current state of knowledge. METHODS: We conducted a selective literature search using PubMed. RESULTS: In total, 356 papers were identified: 6 case reports, 11 reviews, 43 prospective clinical trials and 3 retrospective trials were found. CONCLUSION: Due to different stimulation protocols, electrode positioning and various underlying pathological conditions, summarizing the present studies appears to be difficult. However, there is evidence that NMES is a valuable adjunct in patients with dysphagia and in patients with vocal fold paresis. Nevertheless, more empirical data is needed to fully understand the benefits provided by NMES. Further research suggestions are put forward.

The Senescence-associated Secretory Phenotype Mediates Oncogene-induced Senescence in Pediatric Pilocytic Astrocytoma.

PURPOSE: Pilocytic astrocytoma is the most common childhood brain tumor, characterized by constitutive MAPK activation. MAPK signaling induces oncogene-induced senescence (OIS), which may cause unpredictable growth behavior of pilocytic astrocytomas. The senescence-associated secretory phenotype (SASP) has been shown to regulate OIS, but its role in pilocytic astrocytoma remains unknown.Experimental Design: The patient-derived pilocytic astrocytoma cell culture model, DKFZ-BT66, was used to demonstrate presence of the SASP and analyze its impact on OIS in pilocytic astrocytoma. The model allows for doxycycline-inducible switching between proliferation and OIS. Both states were studied using gene expression profiling (GEP), Western blot, ELISA, and cell viability testing. Primary pilocytic astrocytoma tumors were analyzed by GEP and multiplex assay. RESULTS: SASP factors were upregulated in primary human and murine pilocytic astrocytoma and during OIS in DKFZ-BT66 cells. Conditioned medium induced growth arrest of proliferating pilocytic astrocytoma cells. The SASP factors IL1B and IL6 were upregulated in primary pilocytic astrocytoma, and both pathways were regulated during OIS in DKFZ-BT66. Stimulation with rIL1B but not rIL6 reduced growth of DKFZ-BT66 cells and induced the SASP. Anti-inflammatory treatment with dexamethasone induced regrowth of senescent cells and inhibited the SASP. Senescent DKFZ-BT66 cells responded to senolytic BCL2 inhibitors. High IL1B and SASP expression in pilocytic astrocytoma tumors was associated with favorable progression-free survival. CONCLUSIONS: We provide evidence for the SASP regulating OIS in pediatric pilocytic astrocytoma, with IL1B as a relevant mediator. SASP expression could enable prediction of progression in patients with pilocytic astrocytoma. Further investigation of the SASP driving the unpredictable growth of pilocytic astrocytomas, and its possible therapeutic application, is warranted.

Low-Income Texas Women's Experiences Accessing Their Desired Contraceptive Method at the First Postpartum Visit.

CONTEXT: Early access to contraception may increase postpartum contraceptive use. However, little is known about women's experiences receiving their desired method at the first postpartum visit or how access is associated with use. METHODS: In a 2014-2016 prospective cohort study of low-income Texas women, data were collected from 685 individuals who desired a reversible contraceptive and discussed contraception with a provider at their first postpartum visit, usually within six weeks of birth. Women's experiences were captured using open- and closed-ended survey questions. Thematic and multivariate logistic regression analyses were employed to examine contraceptive access and barriers, and method use at three months postpartum. RESULTS: Twenty-three percent of women received their desired method at the first postpartum visit; 11% a prescription for their desired pill, patch or ring; 8% a method (or prescription) other than that desired; and 58% no method. Among women who did not receive their desired method, 44% reported clinic-level barriers (e.g., method unavailability or no same-day provision), 26% provider-level barriers (e.g., inaccurate contraceptive counseling) and 23% cost barriers. Women who used private practices were more likely than those who used public clinics to report availability and cost barriers (odds ratios, 6.4 and 2.7, respectively). Forty-one percent of women who did not receive their desired method, compared with 86% of those who did, were using that method at three months postpartum. CONCLUSION: Eliminating the various barriers that postpartum women face may improve their access to contraceptives. Further research is needed to improve the understanding of clinic- and provider-level barriers.

Prenatal nicotine and/or cocaine differentially alters nicotine-induced sensitization in aging offspring.

Repeated exposure to psychostimulant drugs can result in behavioral sensitization, an amplified response in locomotor activity and stereotypy, which is used to model aspects of drug addiction. The expression of behavioral sensitization, induced by i.p. injections of nicotine once daily for 5 days, was examined in 450-day-old male rats exposed prenatally on GD 8-20 to one of the following conditions: (1) low nicotine: 2.5 mg/kg/day nicotine [LN]; (2) high nicotine: 5.0 mg/kg/day nicotine [HN]; (3) low nicotine/high cocaine: 2.5 mg/kg/day nicotine plus 40 mg/kg/day cocaine [LN/HC]; (4) high nicotine/low cocaine: 5.0 mg/kg/day nicotine plus 20 mg/kg/day cocaine [HN/LC]; (5) pair-fed controls: food intake yoked to HC dams [PF]; and (6) saline controls: daily injections of 0.9% NaCl solution[SAL]. Initial injection of nicotine did not alter activity or stereotypy in comparison to saline injections, with offspring in all prenatal treatment groups showing a desensitization to nicotine. Five consecutive daily nicotine injections resulted in behavioral sensitization in HN and HN/LC prenatal drug groups. Offspring exhibited an increase in horizontal activity that was evident on day 3, and still present after a 1.0 mg/kg i.p. nicotine challenge 72 hours after the last injection (day 8). SAL offspring exhibited attenuated sensitization. In contrast, nicotine sensitization was not seen in the LN, HC/LN, and the PF offspring; activity remained at the level seen after the initial injection of nicotine. Moreover, nicotine significantly reduced total activity in the LN and PF groups in comparison with their saline-injected counterparts. These data suggest that gestational exposure to high-dose nicotine, either alone or in combination with cocaine, may carry a greater risk than low-dose nicotine exposure of stimulant abuse in later life.

Promoter polymorphism of the anion-exchange protein 1 associated with severe malarial anemia and fatality.

The anion-exchange protein 1 (AE1 or band 3) is involved in the erythrocyte invasion of the malaria parasite Plasmodium falciparum, the adhesion of infected erythrocytes to endothelial cells, and the regulation of acid-base homeostasis, which is a critical factor for human survival in severe malaria. A variant of the AE1 gene promoter 512 base pairs (bp) distant from the transcription start site and 5699 bp from the translation start codon (AE1(-5699T-->C)) has been shown to be highly frequent in a population from the Ashanti region, Ghana. In a matched-pair case-control study (736 pairs), children heterozygous for the mutation (AE1(-5699CT)) had an increased risk of severe malarial anemia (odds ratio [OR], 1.45 [95% confidence interval {CI}, 1.05-2.01]; P<.03). In children who developed this complication, carriers of the mutation AE1(-5699C) had a higher fatality rate than those with the genotype AE1(-5699TT) (relative risk, 7.1 [95% CI, 1.0-52.8]). Moreover, in children with cerebral malaria, AE1(-5699C) was positively associated with a distinctive metabolic acidosis (P<.002), and results of luciferase assays showed higher transcriptional activity of the AE1(-5699C) allele. These results demonstrate that the AE1 promoter allele might influence the infection phenotype and the risk of fatal outcome in children with severe malaria. In this regard, a crucial role of the AE1 protein in malaria is emphasized.

LmxPK4, a mitogen-activated protein kinase kinase homologue of Leishmania mexicana with a potential role in parasite differentiation.

Members of the mitogen-activated protein (MAP) kinase cascade are important for the establishment of a Leishmania mexicana infection and are involved in flagellar length control, although the underlying molecular mechanisms remain to be elucidated. This study reports the cloning and characterization of LmxPK4, a MAP kinase kinase homologue of L. mexicana displaying putative plant-like regulatory phosphorylation sites. The recombinant protein has autophosphorylating activity and phosphorylates myelin basic protein. An LmxPK4 gene deletion mutant showed a proliferation defect after infection of macrophages and no or delayed lesion development in mice. Irrespective of the onset of lesion development parasites showed an early and homogeneous lesion development in re-infection experiments. This is indicative for a compensation of the null mutant phenotype. Additionally, this phenotype could be reverted by reintroduction of the wild-type gene into the deletion background. Mutants expressing loss-of-function or N-terminally truncated versions of LmxPK4 retained the null mutant phenotype. LmxPK4 is stage-specifically expressed in promastigotes and during differentiation to amastigotes, but is not detectable in amastigotes isolated from the mammalian host. Moreover, its in vitro kinase activity increases with temperature rise up to 40 degrees C. Our results suggest that LmxPK4 is involved in the differentiation process and affects virulence of Leishmania mexicana.

LmxMPK9, a mitogen-activated protein kinase homologue affects flagellar length in Leishmania mexicana.

Components of mitogen-activated signal transduction pathways have been shown to be involved in flagellum biogenesis and maintenance. A mitogen-activated protein kinase homologue, designated LmxMPK9 from Leishmania mexicana, has been recently identified in a homology screen and its mRNA found to be present in all life stages. Three different splice-addition sites were used for mRNA maturation in trans-splicing in the different life stages. However, here we show that LmxMPK9 protein is exclusively found in the promastigote stage. Recombinant expression of LmxMPK9 in Escherichia coli and kinase assays revealed a temperature optimum at 27 degrees C, the optimal growth temperature for L. mexicana promastigotes, and a preference for manganese to promote substrate phosphorylation of myelin basic protein. A deletion mutant for the single-copy gene revealed significantly elongated flagella, whereas overexpression led to a subpopulation with rather short to no flagella suggesting a role for LmxMPK9 in flagellar morphogenesis.

Protein kinase involved in flagellar-length control.

During its life cycle, the parasitic protozoon Leishmania mexicana differentiates from a flagellated form, the promastigote, to an amastigote form carrying a rudimentary flagellum. Besides biochemical changes, this process involves a change in overall cell morphology including flagellar shortening. A mitogen-activated protein kinase kinase homologue designated LmxMKK was identified in a homology screening and found to be critically involved in the regulation of flagellar assembly and cell size. LmxMKK is exclusively expressed in the promastigote stage and is likely to be regulated by posttranslational mechanisms such as phosphorylation. A deletion mutant for the single-copy gene revealed motile flagella dramatically reduced in length and lacking the paraflagellar rod, a structure adjacent to the axoneme in kinetoplastid flagella. Moreover, a fraction of the cells showed perturbance of the axonemal structure. Complementation of the deletion mutant with the wild-type gene restored typical promastigote morphology. We propose that LmxMKK influences anterograde intraflagellar transport to maintain flagellar length in Leishmania promastigotes; as such, it is the first protein kinase known to be involved in organellar assembly.