Two Cases of Post-Traumatic Mucormycosis due to Mucor circinelloides: Salvage Therapy with a Combination of Adjunctive Therapies.

Mucormycosis is a rare, emerging angioinvasive infection caused by ubiquitous filamentous fungi. In recent decades, an increase in cutaneous or post-traumatic mucormycosis has been reported. We describe two cases of post-traumatic wound infections with Mucor circinelloides, a mucor species only rarely reported as a cause of post-traumatic mucormycosis. Often considered lethal, management required a combination of medical and surgical therapies to achieve a favorable outcome in both cases.

Effectiveness of the mHealth intervention 'MyDayPlan' to increase physical activity: an aggregated single case approach.

BACKGROUND: e- and mHealth interventions using self-regulation techniques like action and coping planning have the potential to tackle the worldwide problem of physical inactivity. However, they often use one-week self-regulation cycles, providing support toward an active lifestyle on a weekly basis. This may be too long to anticipate on certain contextual factors that may fluctuate from day to day and may influence physical activity. Consequently, the formulated action and coping plans often lack specificity and instrumentality, which may decrease effectiveness of the intervention. The aim of this study was to evaluate effectiveness of a self-regulation, app-based intervention called 'MyDayPlan'. "MyDayPlan' provides an innovative daily cycle in which users are guided towards more physical activity via self-regulation techniques such as goal setting, action planning, coping planning and self-monitoring of behaviour. METHODS: An ABAB single-case design was conducted in 35 inactive adults between 18 and 58 years (M = 40 years). The A phases (A1 and A2) were the control phases in which the 'MyDayPlan' intervention was not provided. The B phases (B1 and B2) were the intervention phases in which 'MyDayPlan' was used on a daily basis. The length of the four phases varied within and between the participants. Each phase lasted a minimum of 5 days and the total study lasted 32 days for each participant. Participants wore a Fitbit activity tracker during waking hours to assess number of daily steps as an outcome. Single cases were aggregated and data were analysed using multilevel models to test intervention effects and possible carry-over effects. RESULTS: Results showed an average intervention effect with a significant increase in number of daily steps from the control to intervention phases for each AB combination. From A1 to B1, an increase of 1424 steps (95% CI [775.42, 2072.32], t (1082) = 4.31,p < .001), and from A2 to B2, an increase of 1181 steps (95% CI [392.98, 1968.16], t (1082) = 2.94, p = .003) were found. Furthermore, the number of daily steps decreased significantly (1134 steps) when going from the first intervention phase (B1) to the second control phase (A2) (95% CI [- 1755.60, - 512.38], t (1082) = - 3.58, p < .001). We found no evidence for a difference in trend between the two control (95% CI [- 114.59, 197.99], t (1078) = .52, p = .60) and intervention phases (95% CI [- 128.79,284.22], t (1078) = .74, p = .46). This reveals, in contrast to what was hypothesized, no evidence for a carry-over effect after removing the 'MyDayPlan' app after the first intervention phase (B1). CONCLUSION: This study adds evidence that the self-regulation mHealth intervention, 'MyDayPlan' has the capacity to positively influence physical activity levels in an inactive adult population. Furthermore, this study provides evidence for the potential of interventions adopting a daily self-regulation cycle in general.

Acceptability and feasibility of the mHealth intervention 'MyDayPlan' to increase physical activity in a general adult population.

BACKGROUND: Electronic health (eHealth) and mobile health (mHealth) interventions have the potential to tackle the worldwide problem of physical inactivity. However, they often suffer from large attrition rates. Consequently, feasibility and acceptability of interventions have become important matters in the creation of e- and mHealth interventions. The aim of this study was to evaluate participants' opinions regarding acceptability and feasibility of a self-regulation, app-based intervention called 'MyDayPlan'. 'MyDayPlan' provides an innovative daily cycle providing several self-regulation techniques throughout the day that guide users towards an active lifestyle via various self-regulation techniques. METHODS: Semi-structured interviews were conducted with 20 adults after using the app for 2 weeks. A directed content analysis was performed using NVivo Software. RESULTS: 'MyDayPlan' was well-received and seems to be feasible and acceptable with inactive adults. The straightforward lay out and ease of use of the app were appreciated. Furthermore, the incorporation of the techniques 'action planning', and 'prompting review of behavioral goals' was positively evaluated. However, the users gave some recommendations: implementation of activity trackers to self-monitor physical activity could be of added value. Furthermore, increasing intuitiveness by minimizing text input and providing more preprogrammed options could further increase the ease of use. Finally, users indicated that they would benefit from more guidance during the "coping planning" component (barrier identification/problem solving), for example by receiving more tailored examples. CONCLUSIONS: Based on these findings, adaptations will be made to the 'MyDayPlan' app before evaluating its effectiveness. Furthermore, involving potential end users and evaluating acceptability and feasibility during the development of an e- and mHealth intervention is key. Also, creating interventions with a large ease of use and straightforward layout that provides tailored support during action and coping planning is key.

RT-qPCR gene expression analysis in zebrafish: Preanalytical precautions and use of expressed repetitive elements for normalization.

Gene expression analysis is increasingly important in many fields of biological research. Understanding patterns of expressed genes is assumed to provide insight into complex regulatory networks and can lead to the identification of genes relevant to specific biological processes, including disease. Among different techniques, reverse transcription quantitative polymerase chain reaction (RT-qPCR) is currently regarded as the gold standard for targeted quantification of RNA gene expression, especially because of its high sensitivity, specificity, accuracy, and precision, and also because of its practical simplicity and processing speed. However, different critical factors can influence the outcome of RT-qPCR studies, including isolation of RNA, reverse transcription to cDNA, and data analysis. These factors need to be addressed in order to obtain biologically meaningful results. In this chapter, we describe how RT-qPCR can be used in a reliable way to successfully study differential gene expression in zebrafish. Hereby, we especially focus on how expressed repetitive elements can be employed as reference targets in zebrafish RT-qPCR studies and how they can further improve the quality of the data.

Zebrafish Collagen Type I: Molecular and Biochemical Characterization of the Major Structural Protein in Bone and Skin.

Over the last years the zebrafish imposed itself as a powerful model to study skeletal diseases, but a limit to its use is the poor characterization of collagen type I, the most abundant protein in bone and skin. In tetrapods collagen type I is a trimer mainly composed of two α1 chains and one α2 chain, encoded by COL1A1 and COL1A2 genes, respectively. In contrast, in zebrafish three type I collagen genes exist, col1a1a, col1a1b and col1a2 coding for α1(I), α3(I) and α2(I) chains. During embryonic and larval development the three collagen type I genes showed a similar spatio-temporal expression pattern, indicating their co-regulation and interdependence at these stages. In both embryonic and adult tissues, the presence of the three α(I) chains was demonstrated, although in embryos α1(I) was present in two distinct glycosylated states, suggesting a developmental-specific collagen composition. Even though in adult bone, skin and scales equal amounts of α1(I), α3(I) and α2(I) chains are present, the presented data suggest a tissue-specific stoichiometry and/or post-translational modification status for collagen type I. In conclusion, this data will be useful to properly interpret results and insights gained from zebrafish models of skeletal diseases.

Next generation sequencing to determine the cystic fibrosis mutation spectrum in Palestinian population.

An extensive molecular analysis of the CF transmembrane regulator (CFTR) gene was performed to establish the CFTR mutation spectrum and frequencies in the Palestinian population, which can be considered as an understudied population. We used a targeted Next Generation Sequencing approach to sequence the entire coding region and the adjacent sequences of the CFTR gene combined with MLPA analysis of 60 unrelated CF patients. Eighteen different CF-causing mutations, including one previously undescribed mutation p.(Gly1265Arg), were identified. The overall detection rate is up to 67%, and when we consider only CF patients with sweat chloride concentrations >70 mEq/L, we even have a pickup rate of 92%. Whereas p.(Phe508del) is the most frequent allele (35% of the positive cases), 3 other mutations c.2988+1Kbdel8.6Kb, c.1393-1G>A, and p.(Gly85Glu) showed frequencies higher than 5% and a total of 9 mutations account for 84% of the mutations. This limited spectrum of CF mutations is in agreement with the homozygous ethnic origin of the Palestinian population. The relative large portion of patients without a mutation is most likely due to clinical misdiagnosis. Our results will be important in the development of an adequate molecular diagnostic test for CF in Palestine.

Attitudes of health care professionals toward carrier screening for cystic fibrosis. A review of the literature.

Recent technological developments in molecular genetics facilitate the large-scale detection of inherited genetic disorders and allow an increasing number of genetic conditions to be screened for (American College of Medical Genetics 2012). This technological evolution creates the background which makes reflection necessary about the desirability to offer community-based (preconception) carrier screening in the healthcare system. A positive attitude of potential providers is vital to the success of a screening program. Therefore, the objective of this article is to elaborate a review of the attitudes of healthcare professionals toward carrier screening. Examination of existing carrier screening programs could provide such information. The literature review will be focused on the attitudes toward carrier screening for cystic fibrosis (CF). The databases Pubmed and Web of Science, as well as the interface Google Scholar, were searched using the keywords for the period 1990-2011. Studies were selected if they were published in a peer-reviewed journal in English and described the attitudes of potential providers toward carrier screening. Eleven studies were retrieved describing the attitudes toward carrier screening for CF. In total, seven studies reported attitudes toward the best time for carrier screening; four studies described opinions toward the best setting to offer CF carrier screening; six studies investigated the willingness to be involved in a carrier screening program, and in total 11 articles reported the concerns about offering carrier screening. Ten papers described a general attitude toward carrier screening. We can conclude that health care providers state willingness to be involved in a carrier screening program, but there is need for appropriate education as well as adequate support given the time constraints already present in consultation. The prospect of an increasing number of genetic disorders for which screening becomes possible, and the potential increasing demand for such screening in the future calls for the need for further debate on the desirability of carrier screening and relevant questions such as the conditions screened, the providers involved, the information provision, and counseling.

Unmasking the obligatory components of nociceptive event-related brain potentials.

It has been hypothesized that the human cortical responses to nociceptive and nonnociceptive somatosensory inputs differ. Supporting this view, somatosensory-evoked potentials (SEPs) elicited by thermal nociceptive stimuli have been suggested to originate from areas 1 and 2 of the contralateral primary somatosensory (S1), operculo-insular, and cingulate cortices, whereas the early components of nonnociceptive SEPs mainly originate from area 3b of S1. However, to avoid producing a burn lesion, and sensitize or fatigue nociceptors, thermonociceptive SEPs are typically obtained by delivering a small number of stimuli with a large and variable interstimulus interval (ISI). In contrast, the early components of nonnociceptive SEPs are usually obtained by applying many stimuli at a rapid rate. Hence, previously reported differences between nociceptive and nonnociceptive SEPs could be due to differences in signal-to-noise ratio and/or differences in the contribution of cognitive processes related, for example, to arousal and attention. Here, using intraepidermal electrical stimulation to selectively activate Aδ-nociceptors at a fast and constant 1-s ISI, we found that the nociceptive SEPs obtained with a long ISI are no longer identified, indicating that these responses are not obligatory for nociception. Furthermore, using a blind source separation, we found that, unlike the obligatory components of nonnociceptive SEPs, the obligatory components of nociceptive SEPs do not receive a significant contribution from a contralateral source possibly originating from S1. Instead, they were best explained by sources compatible with bilateral operculo-insular and/or cingulate locations. Taken together, our results indicate that the obligatory components of nociceptive and nonnociceptive SEPs are fundamentally different.

Pharmacogenomics in children: advantages and challenges of next generation sequencing applications.

Pharmacogenetics is considered as a prime example of how personalized medicine nowadays can be put into practice. However, genotyping to guide pharmacological treatment is relatively uncommon in the routine clinical practice. Several reasons can be found why the application of pharmacogenetics is less than initially anticipated, which include the contradictory results obtained for certain variants and the lack of guidelines for clinical implementation. However, more reproducible results are being generated, and efforts have been made to establish working groups focussing on evidence-based clinical guidelines. For another pharmacogenetic hurdle, the speed by which a pharmacogenetic profile for a certain drug can be obtained in an individual patient, there has been a revolution in molecular genetics through the introduction of next generation sequencing (NGS), making it possible to sequence a large number of genes up to the complete genome in a single reaction. Besides the enthusiasm due to the tremendous increase of our sequencing capacities, several considerations need to be made regarding quality and interpretation of the sequence data as well as ethical aspects of this technology. This paper will focus on the different NGS applications that may be useful for pharmacogenomics in children and the challenges that they bring on.

The Ehlers-Danlos syndrome, a disorder with many faces.

The Ehlers-Danlos syndromes (EDSs) comprise a heterogeneous group of diseases, characterized by fragility of the soft connective tissues and widespread manifestations in skin, ligaments, joints, blood vessels and internal organs. The clinical spectrum varies from mild skin and joint hyperlaxity to severe physical disability and life-threatening vascular complications. The current Villefranche classification recognizes six subtypes, most of which are linked to mutations in genes encoding fibrillar collagens or enzymes involved in post-translational modification of these proteins. Mutations in type V and type III collagen cause classic or vascular EDS respectively, while mutations involving the processing of type I collagen are involved in the kyphoscoliosis, arthrochalasis and dermatosparaxis type of EDS. Establishing the correct EDS subtype has important implications for genetic counseling and management and is supported by specific biochemical and molecular investigations. Over the last years, several new EDS variants have been characterized which call for a refinement of the Villefranche classification. Moreover, the study of these diseases has brought new insights into the molecular pathogenesis of EDS by implicating genetic defects in the biosynthesis of other extracellular matrix (ECM) molecules, such as proteoglycans and tenascin-X, or genetic defects in molecules involved in intracellular trafficking, secretion and assembly of ECM proteins.

Superior mesenteric artery aneurysm in a 9-year-old boy with classical Ehlers-Danlos syndrome.

A 9-year-old boy with the classical type of Ehlers-Danlos syndrome (EDS) developed a symptomatic aneurysm of the superior mesenteric artery. His EDS diagnosis had been confirmed biochemically and genetically. Vascular complications are known to be associated with the vascular type of EDS, but this is the first report of a child with classical EDS who developed a major vascular complication. Clinicians should be aware that severe vascular complications albeit rare, can also occur in classical EDS.

Evaluation of RAD51C as cancer susceptibility gene in a large breast-ovarian cancer patient population referred for genetic testing.

Despite extensive analysis of the BRCA1 and BRCA2 genes, germline mutations are detected in <20% of families with a presumed genetic predisposition for breast and ovarian cancer. Recent literature reported RAD51C as a new breast cancer susceptibility gene. In this study, we report the analysis of 410 patients from 351 unrelated pedigrees. All were referred for genetic testing and we selected families with at least one reported case of ovarian cancer in which BRCA1&2 mutations were previously ruled out. We analyzed the coding exons, intron-exons boundaries, and UTRs of RAD51C. Our mutation analysis did not reveal any unequivocal deleterious mutation. In total 12 unique sequence variations were identified of which two were novel. Our study and others suggest a low prevalence of RAD51C mutations with an exception for some founder populations. This observation is in favor of the rare allele hypothesis in the debate over the nature of the genetic contribution to individual susceptibility to breast and ovarian cancer and further genome-wide studies in high risk families are warranted.

The new Ghent criteria for Marfan syndrome: what do they change?

The diagnosis of Marfan syndrome (MFS) is challenging and international criteria have been proposed. The 1996 Ghent criteria were adopted worldwide, but new diagnostic criteria for MFS were released in 2010, giving more weight to aortic root aneurysm and ectopia lentis. We aimed to compare the diagnosis reached by applying this new nosology vs the Ghent nosology in a well-known series of 1009 probands defined by the presence of an FBN1 mutation. A total of 842 patients could be classified as MFS according to the new nosology (83%) as compared to 894 (89%) according to the 1996 Ghent criteria. The remaining 17% would be classified as ectopia lentis syndrome (ELS), mitral valve prolapse syndrome or mitral valve, aorta, skeleton and skin (MASS) syndrome, or potential MFS in patients aged less than 20 years. Taking into account the median age at last follow-up (29 years), the possibility has to be considered that these patients would go on to develop classic MFS with time. Although the number of patients for a given diagnosis differed only slightly, the new nosology led to a different diagnosis in 15% of cases. Indeed, 10% of MFS patients were reclassified as ELS or MASS in the absence of aortic dilatation; conversely, 5% were reclassified as MFS in the presence of aortic dilatation. The nosology is easier to apply because the systemic score is helpful to reach the diagnosis of MFS only in a minority of patients. Diagnostic criteria should be a flexible and dynamic tool so that reclassification of patients with alternative diagnosis is possible, requiring regular clinical and aortic follow-up.

Ehlers-Danlos arthrochalasia type (VIIA-B)--expanding the phenotype: from prenatal life through adulthood.

The Ehlers-Danlos syndromes (EDS) form a clinically and genetically heterogeneous group of inherited connective-tissue disorders characterized by joint hypermobility, tissue fragility and skin abnormalities. Six subtypes have been well characterized based on clinical features and molecular genetic abnormalities. The arthrochalasia type EDS (formerly types VIIA and B) is characterized by severe generalized joint hypermobility with multiple dislocations including congenital bilateral dislocation of the hips, muscular hypotonia and distinct dysmorphic features. The diagnosis of the arthrochalasia type EDS is of importance in the neonatal period because of consequences of physical disability in later life. However, the differential diagnosis may be difficult because of overlap with other hypermobility syndromes. In addition, the significant hypotonia may direct the physician toward various neuromuscular diagnoses. As patients become older, the hypotonia decreases and facial features become less distinct. In this report, we describe seven patients at different ages. Timing of diagnosis varied from prenatal life to adult age. The diagnosis of EDS type VII was confirmed by biochemical studies or mutation analysis showing characteristic mutations in COL1A1 and COL1A2. These mutations result in skipping of exon 6, which leads to defective collagen synthesis. For physicians treating patients with EDS type VII, achieving mobility for the patient is the greatest challenge and it may be impossible because of recurrent dislocations of nearly all joints in severe cases.

Identification of a novel recurrent 1q42.2-1qter deletion in high risk MYCN single copy 11q deleted neuroblastomas.

Neuroblastoma is an aggressive embryonal tumor that accounts for ∼15% of childhood cancer deaths. Hitherto, despite the availability of comprehensive genomic data on DNA copy number changes in neuroblastoma, relatively little is known about the genes driving neuroblastoma tumorigenesis. In this study, high resolution array comparative genome hybridization (CGH) was performed on 188 primary neuroblastoma tumors and 33 neuroblastoma cell lines to search for previously undetected recurrent DNA copy number gains and losses. A new recurrent distal chromosome 1q deletion (del(1)(q42.2qter)) was detected in seven cases. Further analysis of available array CGH datasets revealed 13 additional similar distal 1q deletions. The majority of all detected 1q deletions was found in high risk 11q deleted tumors without MYCN amplification (Fisher exact test p = 5.61 × 10(-5) ). Using ultra-high resolution (∼115 bp resolution) custom arrays covering the breakpoints on 1q for 11 samples, clustering of nine breakpoints was observed within a 12.5-kb region, of which eight were found in a 7-kb copy number variable region, whereas the remaining two breakpoints were colocated 1.4-Mb proximal. The commonly deleted region contains one miRNA (hsa-mir-1537), four transcribed ultra conserved region elements (uc.43-uc.46) and 130 protein coding genes including at least two bona fide tumor suppressor genes, EGLN1 (or PHD2) and FH. This finding further contributes to the delineation of the genomic profile of aggressive neuroblastoma, offers perspectives for the identification of genes contributing to the disease phenotype and may be relevant in the light of assessment of response to new molecular treatments.

A Turkish patient of typical Loeys-Dietz syndrome with a TGFBR2 mutation.

We describe a 2-year-old male patient with skeletal, neurological, cardiovascular, and connective tissue anomalies. Skeletal anomalies included pectus excavatum, hammer toes and hallux valgus and camptodactyly. The characteristic craniofacial findings of hypertelorism, down slanting palpebral fissures, strabismus, ptosis of eyelids, bifid uvula, high-arched palate and retrognathia were present. The proband has been operated on twice for bilateral inguinal hernia and several times for his foot deformities. Psychomotor development was retarded. At present, echocardiographic findings show aortic root dilation. The patient has important characteristics of Loeys-Dietz syndrome (LDS). Direct sequencing analysis of the transforming growth factor beta receptor I and II (TGFBR1 and 2) genes was performed and was demonstrated heterozygous missense mutation of the TGFBR2 gene in the patient, which confirms the diagnosis of LDS. This is the first Turkish patient with typical clinical signs of LDS. This report also illustrates that LDS and Shprintzen-Goldberg syndrome (SGS) have some common clinical characteristics.

Vascular haemostasis.

SUMMARY: While the majority of this session will deal with selected inherited vascular abnormalities that may manifest as a haemorrhagic disorder, the initial discussion by Dr Key will focus on the interplay between the vessel wall and components of the coagulation system, with a focus on haemophilia A and B. Although it is generally accepted that physiological haemostasis is triggered by contact of blood with tissue factor (TF), there remains some controversy regarding the cellular origin of TF in vivo. In addition, the initiation and propagation of thrombin generation are highly dependent on the balance of pro- and anticoagulant functions of endothelium, a profile that varies significantly throughout the vasculature. Drs De Paepe and Malfait address heritable collagen disorders such as the Ehlers-Danlos syndromes (EDS), a heterogeneous group of diseases involving the skin, ligaments and joints, blood vessels and internal organs. Most EDS subtypes are caused by mutations in genes encoding fibrillar collagens, or in genes coding for enzymes involved in posttranslational modifications of collagens. Accurate biochemical and molecular testing is now available for most EDS subtypes and can direct genetic counselling and medical management for these disorders. Dr Shovlin reviews recent developments in hereditary haemorrhagic telengiectasia (HHT), a frequently undiagnosed disorder characterized by arteriovenous malformations in multiple organs. These abnormal blood vessels are the result of mutations in one of a number of genes whose protein products influence TGF-beta signalling in vascular endothelial cells. Several HHT management guidelines have been published and are discussed.

Mutations in fibrillin-1 cause congenital scleroderma: stiff skin syndrome.

The predisposition for scleroderma, defined as fibrosis and hardening of the skin, is poorly understood. We report that stiff skin syndrome (SSS), an autosomal dominant congenital form of scleroderma, is caused by mutations in the sole Arg-Gly-Asp sequence-encoding domain of fibrillin-1 that mediates integrin binding. Ordered polymers of fibrillin-1 (termed microfibrils) initiate elastic fiber assembly and bind to and regulate the activation of the profibrotic cytokine transforming growth factor-beta (TGFbeta). Altered cell-matrix interactions in SSS accompany excessive microfibrillar deposition, impaired elastogenesis, and increased TGFbeta concentration and signaling in the dermis. The observation of similar findings in systemic sclerosis, a more common acquired form of scleroderma, suggests broad pathogenic relevance.