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1.  Activity and phylogenetic diversity of sulfate-reducing microorganisms in low-temperature subsurface fluids within the upper oceanic crust 
The basaltic ocean crust is the largest aquifer system on Earth, yet the rates of biological activity in this environment are unknown. Low-temperature (<100°C) fluid samples were investigated from two borehole observatories in the Juan de Fuca Ridge (JFR) flank, representing a range of upper oceanic basement thermal and geochemical properties. Microbial sulfate reduction rates (SRR) were measured in laboratory incubations with 35S-sulfate over a range of temperatures and the identity of the corresponding sulfate-reducing microorganisms (SRM) was studied by analyzing the sequence diversity of the functional marker dissimilatory (bi)sulfite reductase (dsrAB) gene. We found that microbial sulfate reduction was limited by the decreasing availability of organic electron donors in higher temperature, more altered fluids. Thermodynamic calculations indicate energetic constraints for metabolism, which together with relatively higher cell-specific SRR reveal increased maintenance requirements, consistent with novel species-level dsrAB phylotypes of thermophilic SRM. Our estimates suggest that microbially-mediated sulfate reduction may account for the removal of organic matter in fluids within the upper oceanic crust and underscore the potential quantitative impact of microbial processes in deep subsurface marine crustal fluids on marine and global biogeochemical carbon cycling.
PMCID: PMC4295021  PMID: 25642212
subseafloor life; metabolic activity; functional diversity; sulfate reduction; basaltic ocean fluids
2.  A Genetic Dichotomy between Pure Sclerosing Epithelioid Fibrosarcoma (SEF) and Hybrid SEF/Low Grade Fibromyxoid Sarcoma: A Pathologic and Molecular Study of 18 cases 
Genes, chromosomes & cancer  2014;54(1):28-38.
Sclerosing epithelioid fibrosarcoma (SEF) is a rare soft tissue tumor exhibiting considerable morphologic overlap with low grade fibromyxoid sarcoma (LGFMS). Moreover, both SEF and LGFMS show MUC4 expression by immunohistochemistry. While the majority of LGFMS cases are characterized by a FUS-CREB3L1 fusion, both FUS-CREB3L2 and EWSR1-CREB3L1 fusions were recently demonstrated in a small number of LGFMS and SEF/LGFMS hybrid tumors. In contrast, recent studies pointed out that SEF harbor frequent EWSR1 rearrangements, with only a minority of cases showing FUS-CREB3L2 fusions. In an effort to further characterize the molecular characteristics of pure SEF and hybrid SEF/LGFMS lesions, we undertook a clinicopathologic, immunohistochemical and genetic analysis of a series of 10 SEF and 8 hybrid SEF/LGFMS tumors. The mortality rate was similar between the two groups, 44% within the pure SEF group and 37% in the hybrid SEF/LGFMS with a mean overall follow-up of 66 months. All but one pure SEF and all hybrid SEF/LGFMS tested cases showed MUC4 immunoreactivity. The majority (90%) of pure SEF cases showed EWSR1 gene rearrangements by FISH with only one case exhibiting FUS rearrangement. Of the 9 EWSR1 positive cases, 6 cases harbored CREB3L1 break-apart, two had CREB3L2 rearrangement (a previously unreported finding) and one lacked evidence of CREB3L1/2 abnormalities. In contrast, all hybrid SEF/LGFMS tumors exhibited FUS and CREB3L2 rearrangements. These results further demarcate a relative cytogenetic dichotomy between pure SEF, often characterized by EWSR1 rearrangements, and hybrid SEF/LGFMS, harboring FUS-CREB3L2 fusion; the latter group recapitulating the genotype of LGFMS.
PMCID: PMC4232448  PMID: 25231134
sclerosing epithelioid fibrosarcoma; fibromyxoid sarcoma; EWSR1; FUS; CREB3L1; CREB3L2; fusion
3.  Fetal acetylcholine receptor inactivation syndrome 
Transient neonatal myasthenia gravis (TNMG) affects a proportion of infants born to mothers with myasthenia gravis (MG). Symptoms usually resolve completely within the first few months of life, but persistent myopathic features have been reported in a few isolated cases.
Here we report 8 patients from 4 families born to mothers with clinically manifest MG or mothers who were asymptomatic but had elevated acetylcholine receptor (AChR) antibody levels.
Clinical features in affected infants ranged from a mild predominantly facial and bulbar myopathy to arthrogryposis multiplex congenita. Additional clinical findings included hearing impairment, pyloric stenosis, and mild CNS involvement. In all cases, antibodies against the AChR were markedly elevated, although not always specific for the fetal AChR γ subunit. There was a correlation between maternal symptoms; the timing, intensity, and frequency of maternal treatment; and neonatal outcome.
These findings suggest that persistent myopathic features following TNMG may be more common than currently recognized. Fetal AChR inactivation syndrome should be considered in the differential diagnosis of infants presenting with unexplained myopathic features, in particular marked dysarthria and velopharyngeal incompetence. Correct diagnosis requires a high degree of suspicion if the mother is asymptomatic but is crucial considering the high recurrence risk for future pregnancies and the potentially treatable nature of this condition. Infants with a history of TNMG should be followed up for subtle myopathic signs and associated complications.
PMCID: PMC4277302  PMID: 25566546
4.  Voluntary peer review as innovative tool for quality improvement in the intensive care unit – a retrospective descriptive cohort study in German intensive care units 
GMS German Medical Science  2014;12:Doc17.
Introduction: Quality improvement and safety in intensive care are rapidly evolving topics. However, there is no gold standard for assessing quality improvement in intensive care medicine yet. In 2007 a pilot project in German intensive care units (ICUs) started using voluntary peer reviews as an innovative tool for quality assessment and improvement. We describe the method of voluntary peer review and assessed its feasibility by evaluating anonymized peer review reports and analysed the thematic clusters highlighted in these reports.
Methods: Retrospective data analysis from 22 anonymous reports of peer reviews. All ICUs – representing over 300 patient beds – had undergone voluntary peer review. Data were retrieved from reports of peers of the review teams and representatives of visited ICUs. Data were analysed with regard to number of topics addressed and results of assessment questionnaires. Reports of strengths, weaknesses, opportunities and threats (SWOT reports) of these ICUs are presented.
Results: External assessment of structure, process and outcome indicators revealed high percentages of adherence to predefined quality goals. In the SWOT reports 11 main thematic clusters were identified representative for common ICUs. 58.1% of mentioned topics covered personnel issues, team and communication issues as well as organisation and treatment standards. The most mentioned weaknesses were observed in the issues documentation/reporting, hygiene and ethics. We identified several unique patterns regarding quality in the ICU of which long-term personnel problems und lack of good reporting methods were most interesting
Conclusion: Voluntary peer review could be established as a feasible and valuable tool for quality improvement. Peer reports addressed common areas of interest in intensive care medicine in more detail compared to other methods like measurement of quality indicators.
PMCID: PMC4270273  PMID: 25587245
peer review; critical care; patient safety; quality improvement; quality management
5.  Genetic disorders of thyroid metabolism and brain development 
Normal thyroid metabolism is essential for human development, including the formation and functioning of the central and peripheral nervous system. Disorders of thyroid metabolism are increasingly recognized within the spectrum of paediatric neurological disorders. Both hypothyroid and hyperthyroid disease states (resulting from genetic and acquired aetiologies) can lead to characteristic neurological syndromes, with cognitive delay, extrapyramidal movement disorders, neuropsychiatric symptoms, and neuromuscular manifestations. In this review, the neurological manifestations of genetic disorders of thyroid metabolism are outlined, with particular focus on Allan-Herndon-Dudley syndrome and benign hereditary chorea. We report in detail the clinical features, major neurological and neuropsychiatric manifestations, molecular genetic findings, disease mechanisms, and therapeutic strategies for these emerging genetic ‘brain-thyroid’ disorders.
PMCID: PMC4231219  PMID: 24665922
6.  Nebulin (NEB) mutations in a childhood onset distal myopathy with rods and cores uncovered by next generation sequencing 
European Journal of Human Genetics  2013;21(11):1249-1252.
Recessive nebulin (NEB) mutations are a common cause of nemaline myopathy (NM), typically characterized by generalized weakness of early-onset and nemaline rods on muscle biopsy. Exceptional adult cases with additional cores and an isolated distal weakness have been reported. The large NEB gene with 183 exons has been an obstacle for the genetic work-up. Here we report a childhood-onset case with distal weakness and a core-rod myopathy, associated with recessive NEB mutations identified by next generation sequencing (NGS). This 6-year-old boy presented with a history of gross-motor difficulties following a normal early development. He had distal leg weakness with bilateral foot drop, as well as axial muscle weakness, scoliosis and spinal rigidity; additionally he required nocturnal respiratory support. Muscle magnetic resonance (MR) imaging showed distal involvement in the medial and anterior compartment of the lower leg. A muscle biopsy featured both rods and cores. Initial targeted testing identified a heterozygous Nebulin exon 55 deletion. Further analysis using NGS revealed a frameshifting 4 bp duplication, c.24372_24375dup (P.Val8126fs), on the opposite allele. This case illustrates that NEB mutations can cause childhood onset distal NM, with additional cores on muscle biopsy and proves the diagnostic utility of NGS for myopathies, particularly when large genes are implicated.
PMCID: PMC3798838  PMID: 23443021
nebulin; distal myopathy; next generation sequencing
7.  Phase I/II study of pegylated arginine deiminase (ADI-PEG 20) in patients with advanced melanoma 
Investigational new drugs  2012;31(2):425-434.
Arginine deiminase (ADI) is an enzyme that degrades arginine, an amino acid that is important for growth and development of normal and neoplastic cells. Melanoma cells are auxotrophic for arginine, because they lack argininosuccinatesynthetase (ASS), a key enzyme required for the synthesis of arginine.
Patients and methods
Patients with advanced melanoma were treated with 40, 80 or 160 IU/m2 ADI-PEG 20 i.m. weekly. Primary endpoints were toxicity and tumor response, secondary endpoints included metabolic response by 18FDG-PET, pharmacodynamic (PD) effects upon circulating arginine levels, and argininosuccinate synthetase tumor expression by immunohistochemistry.
31 previously treated patients were enrolled. The main toxicities were grade 1 and 2 adverse events including injection site pain, rash, and fatigue. No objective responses were seen. Nine patients achieved stable disease (SD), with 2 of these durable for >6 months. Four of the 9 patients with SD had uveal melanoma. PD analysis showed complete plasma arginine depletion in 30/31 patients by day 8. Mean plasma levels of ADI-PEG 20 correlated inversely with ADI-PEG 20 antibody levels. Immunohistochemical ASS expression analysis in tumor tissue was negative in 24 patients, whereas 5 patients had <5 % cells positive.
ADI-PEG 20 is well tolerated in advanced melanoma patients and leads to consistent, but transient, arginine depletion. Although no RECIST responses were observed, the encouraging rate of SD in uveal melanoma patients indicates that it may be worthwhile to evaluate ADI-PEG 20 in this melanoma subgroup.
PMCID: PMC4169197  PMID: 22864522
Melanoma; Metastatic; Auxotrophy; Arginine deiminase; Argininosuccinate synthetase
8.  Adrenocortical Carcinoma Is a Lynch Syndrome–Associated Cancer 
Journal of Clinical Oncology  2013;31(24):3012-3018.
Adrenocortical carcinoma (ACC) is an endocrine malignancy with a poor prognosis. The association of adult-onset ACC with inherited cancer predisposition syndromes is poorly understood. Our study sought to define the prevalence of Lynch syndrome (LS) among patients with ACC.
Patients and Methods
One hundred fourteen patients with ACC were evaluated in a specialized endocrine oncology clinic and were prospectively offered genetic counseling and clinical genetics risk assessment (group 1). In addition, families with known mismatch repair (MMR) gene mutations that were recorded in the University of Michigan Cancer Genetics Registry were retrospectively reviewed for the presence of ACC (group 2). ACC tumors from patients with LS were tested for microsatellite instability and immunohistochemistry (IHC) to evaluate for MMR deficiency.
Ninety-four (82.5%) of 114 patients with ACC underwent genetic counseling (group 1). Three individuals (3.2%) had family histories suggestive of LS. All three families were found to have MMR gene mutations. Retrospective review of an additional 135 MMR gene–positive probands identified two with ACC (group 2). Four ACC tumors were available (group 1, 3; group 2, 1). All four tumors were microsatellite stable; three had IHC staining patterns consistent with germline mutation status.
The prevalence of LS among patients with ACC is 3.2%, which is comparable to the prevalence of LS in colorectal and endometrial cancer. Patients with ACC and a personal or family history of LS tumors should be strongly considered for genetic risk assessment. IHC screening of all ACC tumors may be an effective strategy for identifying patients with LS.
PMCID: PMC3739861  PMID: 23752102
9.  A Case of Spontaneous Systemic Immunity to Melanoma Associated with Cure after Amputation For Extensive Regional Recurrence 
Survival after amputation for melanoma is short; however, rare long-term survivors are reported. The mechanism for durable systemic tumor control in patients with regional failure is not known. To explore whether systemic tumor immunity may be implicated, tumor and circulating immune responses were examined in a patient who survived disease-free 14 years after hip disarticulation.
A 71 year-old female with extensive regional metastases of melanoma in the left lower extremity underwent amputation for palliative reasons. Tumor was collected at surgery, and blood was collected during follow-up. Tumor sections were evaluated for lymphocytic infiltration and NY-ESO-1 expression by immunohistochemistry. Cellular immune responses to defined tumor antigens were evaluated by ELIspot assay, and antibody responses to a panel of tumor antigens were assayed by ELISA.
The patient's tumor had minimal lymphocytic infiltrate (Immunotype A). NY-ESO-1 was strongly expressed by the melanoma cells. Circulating T cell responses to NY-ESO-1 peptides were observed 6 and 12 years postoperatively, and antibodies to NY-ESO-1 were detected 2-6 years after surgery.
The patient described in this report experienced relentless regional tumor progression, with intravascular metastases, then 14-year systemic disease-free survival after palliative resection, without evidence of melanoma recurrence before death from other causes. Her immune response to NY-ESO-1 likely failed to control established regional metastases because T cells were unable to infiltrate them. It is possible, however, that among other factors, the host immune response may have contributed to systemic protection.
PMCID: PMC4082724  PMID: 23666534
Melanoma; amputation for melanoma; NY-ESO-1; tumor immunity; antibody; T-cell
10.  Promoting Homework Adherence in Cognitive-Behavioral Therapy for Adolescent Depression 
This study used prospective, observational methods to evaluate six features of therapist behavior as predictors of homework adherence in cognitive-behavioral therapy (CBT) for adolescent depression, with the goal of identifying therapist strategies with the potential to improve adolescent adherence. Therapist behaviors were expected to interact with initial levels of client resistance or adherence to predict subsequent homework completion.
Participants were 50 referred adolescents (33 females, 54% ethnic minority) ages 14–18 (M=15.9) meeting diagnostic criteria for a depressive disorder, and without co-morbid psychotic disorder, bipolar disorder, autism spectrum disorder, intellectual disability, or concurrent treatments. Therapist homework-related behaviors were coded from audiotapes of Sessions 1 and 2 and used to predict adolescents’ homework adherence, coded from audiotapes of Sessions 2 and 3.
Several therapist behaviors were predictive of subsequent homework adherence, particularly for initially resistant or non-adherent adolescents. Stronger homework rationale and greater time allocated to explaining homework in Session 1 predicted greater adherence at Session 2, particularly for initially resistant adolescents. Stronger rationale and eliciting reactions/troubleshooting obstacles in Session 2 predicted greater adherence at Session 3, particularly for adolescents who were less adherent to prior homework.
Strategies such as providing a strong rationale, allocating more time to assigning homework, and eliciting reactions/troubleshooting obstacles may be effective ways to bolster homework adherence among initially less engaged, depressed teens.
PMCID: PMC3644378  PMID: 23237021
Adolescent depression; CBT; homework adherence; engagement; therapist behavior
11.  The Composite of Bone Marrow Concentrate and PRP as an Alternative to Autologous Bone Grafting 
PLoS ONE  2014;9(6):e100143.
One possible alternative to the application of autologous bone grafts represents the use of autologous bone marrow concentrate (BMC). The purpose of our study was to evaluate the potency of autologous platelet-rich plasma (PRP) in combination with BMC. In 32 mini-pigs a metaphyseal critical-size defect was surgically created at the proximal tibia. The animals were allocated to four treatment groups of eight animals each (1. BMC+CPG group, 2. BMC+CPG+PRP group, 3. autograft group, 4. CPG group). In the BMC+CPG group the defect was filled with autologous BMC in combination with calcium phosphate granules (CPG), whereas in the BMC+CPG+PRP group the defect was filled with the composite of autologous BMC, CPG and autologous PRP. In the autograft group the defect was filled with autologous cancellous graft, whereas in the CPG group the defect was filled with CPG solely. After 6 weeks radiological and histomorphometrical analysis showed significantly more new bone formation in the BMC+CPG+PRP group compared to the BMC+CPG group and the CPG group. There were no significant differences between the BMC+CPG+PRP group and the autograft group. In the PRP platelets were enriched significantly about 4.7-fold compared to native blood. In BMC the count of mononuclear cells increased significantly (3.5-fold) compared to the bone marrow aspirate. This study demonstrates that the composite of BMC+CPG+PRP leads to a significantly higher bone regeneration of critical-size defects at the proximal tibia in mini-pigs than the use of BMC+CPG without PRP. Furthermore, within the limits of the present study the composite BMC+CPG+PRP represents a comparable alternative to autologous bone grafting.
PMCID: PMC4064995  PMID: 24950251
12.  Systemic capillary leak syndrome associated with a rare abdominal and four-limb compartment syndrome: a case report 
Systemic capillary leak syndrome is a rare and life threatening disease characterized by periodic episodes of hypovolemic shock due to leakage of plasma from the intravascular to the extravascular space. It is associated with hemoconcentration, hypoalbuminemia, and generalized edema. We report the case of a patient with idiopathic systemic capillary leak syndrome who developed an unexpected and potentially fatal abdominal and four-limb compartment syndrome. This was successfully treated with fasciotomies and medical treatment including terbutaline, theophylline, and corticosteroids. To the best of our knowledge this is the first report of this kind in the literature.
Case presentation
A previously healthy 54-year-old Caucasian man presented to the emergency department of our internal medicine ward with a medical history of aggravation of general health related to dizziness, weight gain, and two syncopal attacks. Due to a massive emission of fluids and proteins from the intravascular to the extracellular compartments, he developed compartment syndromes in his upper and lower limbs and the abdominal compartment. The abdomen and all four limbs required decompression by a fasciotomy of both forearms, both thighs, both lower legs, and the abdomen within 24 hours after admission. After 60 days of treatment he was dismissed from the clinic. He was able to return to his previous occupation and reached the same level of athletic activity as before the illness.
Systemic capillary leak syndrome is a very rare disease that can lead to a fatal clinical outcome. It is important to be aware of the fatal complications that can be caused by this disease. Despite the fact that systemic capillary leak syndrome represents a very rare disease it is still important to be aware of life threatening complications, like compartment syndromes, which need surgical intervention. However, early diagnosis and interdisciplinary treatment can lead to a good clinical outcome.
PMCID: PMC4131808  PMID: 24934689
Systemic capillary leak syndrome; Compartment syndrome; Fasciotomy; Interdisciplinary treatment
13.  Determination of the amount of leg length inequality that alters spinal posture in healthy subjects using rasterstereography 
European Spine Journal  2013;22(6):1354-1361.
Leg length inequalities (LLIs) can result in an increased energy consumption, abnormal gait or osteoarthritis of the hip. In a previous study we simulated different LLIs of up to 15 mm and evaluated their effects on the pelvic position and spinal posture. We found a correlation between LLIs and resulting changes of the pelvic position. Despite suggestions in the literature we were not able to detect significant changes of the spinal posture. Therefore, the purpose of this study was to determine the amount of LLI that would in fact alter the spinal posture.
The subjects were placed on a simulation platform, whose height could be precisely controlled by the measuring device, to simulate different LLIs of up to 20 mm. For LLIs >20 mm, additional precision-cut wooden blocks were used under one foot. After an adaptation period the resulting changes of the pelvis and spine were measured with a rasterstereographic device.
We found a significant correlation between platform height changes and changes of the pelvic position. The frontal spinal parameters surface rotation and lateral deviation changed significantly when simulating differences greater than 20 mm. No changes of the sagittal spinal curvature were measured, however, a trend to decreasing kyphotic angles was noted.
Our study has shown for the first time that LLIs >20 mm will lead to significant changes in the spinal posture of healthy test subjects. However, these changes were only found in frontal (surface rotation and lateral flexion) spinal parameters, but not in sagittal parameters. Here for the kyphotic angle only a tendency to decreasing angles was noted. We have also found a significant correlation between different leg lengths and changes of the pelvic position. Further, females and males seem to react in the same way to LLIs.
PMCID: PMC3676572  PMID: 23479027
Leg length inequalities; Rasterstereography; Posture; Pelvic obliquity; Functional scoliosis
14.  Recessive mutations in EPG5 cause Vici syndrome, a multisystem disorder with defective autophagy 
Nature genetics  2012;45(1):83-87.
Vici syndrome is a recessively inherited multisystem disorder characterized by callosal agenesis, cataracts, cardiomyopathy, combined immunodeficiency and hypopigmentation. To investigate the molecular basis of Vici syndrome, we carried out exome and Sanger sequence analysis in a cohort of 18 patients. We identified recessive mutations in EPG5 (previously KIAA1632), indicating a causative role in Vici syndrome. EPG5 is the human homologue of the metazoan-specific autophagy gene epg-5, encoding a key autophagy regulator (ectopic P-granules autophagy protein 5) implicated in the formation of autolysosomes. Further studies demonstrated a severe block of autophagosomal clearance in muscle and fibroblasts from EPG5 mutant patients, resulting in autophagic cargo accumulation in autophagosomes. These findings indicate Vici syndrome as a paradigm of a human multisystem disorder associated with defective autophagy, and suggest a fundamental role of the autophagy pathway in the anatomical and functional formation of organs such as the brain, the heart and the immune system.
PMCID: PMC4012842  PMID: 23222957
15.  The Role of Erythropoietin and Bone Marrow Concentrate in the Treatment of Osteochondral Defects in Mini-Pigs 
PLoS ONE  2014;9(3):e92766.
All available treatment options for osteochondral and chondral defects do not restore hyaline cartilage and are limited to decreasing associated pain, and maintaining or improving joint function. The purpose of this study was to evaluate the potential of erythropoietin (EPO) in combination with bone marrow aspiration concentrate (BMAC) in the treatment of osteochondral defects of mini-pigs.
14 Goettinger mini-pigs, in which a 6×10 mm osteochondral defect in the medial femoral condyle of both knee joints was created, were randomized into four groups: biphasic scaffold alone, scaffold with EPO, scaffold with BMAC and scaffold in combination with EPO and BMAC. After 26 weeks all animals were euthanized and histological slides were evaluated using a modified ÓDriscoll Score.
In the therapy groups, areas of chondrogenic tissue that contained collagen II were present. Adding EPO (p = 0.245) or BMAC (p = 0.099) alone to the scaffold led to a non-significant increase in the score compared to the control group. However, the combination of EPO and BMAC in the implanted scaffold showed a significant improvement (p = 0.02) in the histological score.
The results of our study show that in mini-pigs, the combination of EPO and BMAC leads to an enhanced osteochondral healing. However, additional research is necessary to further improve the repair tissue and to define the role of MSCs and EPO in cartilage repair.
PMCID: PMC3968023  PMID: 24676029
16.  Phylogenetic diversity of microorganisms in subseafloor crustal fluids from Holes 1025C and 1026B along the Juan de Fuca Ridge flank 
To expand investigations into the phylogenetic diversity of microorganisms inhabiting the subseafloor biosphere, basalt-hosted crustal fluids were sampled from Circulation Obviation Retrofit Kits (CORKs) affixed to Holes 1025C and 1026B along the Juan de Fuca Ridge (JdFR) flank using a clean fluid pumping system. These boreholes penetrate the crustal aquifer of young ocean crust (1.24 and 3.51 million years old, respectively), but differ with respect to borehole depth and temperature at the sediment-basement interface (147 m and 39°C vs. 295 m and 64°C, respectively). Cloning and sequencing of PCR-amplified small subunit ribosomal RNA genes revealed that fluids retrieved from Hole 1025C were dominated by relatives of the genus Desulfobulbus of the Deltaproteobacteria (56% of clones) and Candidatus Desulforudis of the Firmicutes (17%). Fluids sampled from Hole 1026B also contained plausible deep subseafloor inhabitants amongst the most abundant clone lineages; however, both geochemical analysis and microbial community structure reveal the borehole to be compromised by bottom seawater intrusion. Regardless, this study provides independent support for previous observations seeking to identify phylogenetic groups of microorganisms common to the deep ocean crustal biosphere, and extends previous observations by identifying additional lineages that may be prevalent in this unique environment.
PMCID: PMC3971187  PMID: 24723917
deep subsurface; microorganisms; diversity; Juan de Fuca Ridge; SSU ribosomal RNA gene; Ocean Drilling Program
17.  NY-ESO-1 antibody as a novel tumour marker of gastric cancer 
British Journal of Cancer  2013;108(5):1119-1125.
NY-ESO-1 antibodies are specifically observed in patients with NY-ESO-1-expressing tumours. We analysed whether the NY-ESO-1 humoral immune response is a useful tumour marker of gastric cancer.
Sera from 363 gastric cancer patients were screened by enzyme-linked immunosorbent assay (ELISA) to detect NY-ESO-1 antibodies. Serial serum samples were obtained from 25 NY-ESO-1 antibody-positive patients, including 16 patients with curative resection and 9 patients who received chemotherapy alone.
NY-ESO-1 antibodies were detected in 3.4% of stage I, 4.4% of stage II, 25.3% of stage III, and 20.0% of stage IV patients. The frequency of antibody positivity increased with disease progression. When the NY-ESO-1 antibody was used in combination with carcinoembryonic antigen and CA19-9 to detect gastric cancer, information gains of 11.2% in stages III and IV, and 5.8% in all patients were observed. The NY-ESO-1 immune response levels of the patients without recurrence fell below the cutoff level after surgery. Two of the patients with recurrence displayed incomplete decreases. The nine patients who received chemotherapy alone continued to display NY-ESO-1 immune responses.
When combined with conventional tumour markers, the NY-ESO-1 humoral immune response could be a useful tumour marker for detecting advanced gastric cancer and inferring the post-treatment tumour load in seropositive patients.
PMCID: PMC3619069  PMID: 23403818
surgical treatment; detection marker; follow-up marker; recurrence; prognosis
18.  Development and validation of an in vitro model for measurements of cervical root dentine permeability 
Clinical Oral Investigations  2014;18(9):2077-2086.
The aim of this series of studies was the development and validation of a new model for evaluation of dentinal hypersensitivity (DH) therapies.
Materials and methods
Roots from extracted human teeth were sealed with a flowable composite. In the cervical area, a 3-mm-wide circular window was ground through the seal 1 mm deep into dentine. The pulp lumen was connected to a reservoir of artificial dentinal fluid (ADF) containing protein, mineral salts and methylene blue. At increased pulpal pressure, the ADF released through the said window was collected in containers each with 20 ml of physiologic saline for a consecutive series of 30-min intervals and ADF concentration (absorption) was determined photometrically. The model was verified by three experiments. In experiment 1, the lower limit of quantification (LLoQ, coefficient of variation = 20 % and difference of 5 standard deviations (SD) from blank) of ADF in physiologic saline was determined by measuring the absorption of 15 dilutions of ADF in physiologic saline (containing 0.625 ng to 12.5 μg methylene blue/ml) photometrically for ten times. In experiment 2, long-term linearity of ADF perfusion/outflow was investigated using 11 specimens. The ADF released through the window was collected in the said containers separately for each consecutive interval of 30 min for up to 240 min. Absorption was determined and analysed by linear regression over time. In experiment 3, perfusion before (2×) and after single treatment according to the following three groups was measured: BisGMA-based sealant (Seal&Protect®), an acidic fluoride solution (elmex fluid®) and control (no treatment).
In experiment 1, the LLoQ was 0.005 μg methylene blue/ml. In experiment 2, permeability was different within the specimens and decreased highly linearly with time, allowing the prediction of future values. In experiment 3, Seal&Protect® completely occluded dentinal tubules. elmex fluid® increased tubular permeability by about 30 % compared to control.
A model comprising the use of artificial dentinal fluid was developed and validated allowing screening of therapeutic agents for the treatment of DH through reliable measurement of permeability of cervical root dentine.
Clinical relevance
The described in vitro model allows evaluation of potential agents for the treatment of DH at the clinically relevant cervical region of human teeth.
PMCID: PMC4232750  PMID: 24504375
Dentine hypersensitivity; DH; Perfusion; Tubules; Dentinal fluid; Fluoride; Protein
19.  Novel deletion of lysine 7 expands the clinical, histopathological and genetic spectrum of TPM2-related myopathies 
Brain  2013;136(2):508-521.
The β-tropomyosin gene encodes a component of the sarcomeric thin filament. Rod-shaped dimers of tropomyosin regulate actin-myosin interactions and β-tropomyosin mutations have been associated with nemaline myopathy, cap myopathy, Escobar syndrome and distal arthrogryposis types 1A and 2B. In this study, we expand the allelic spectrum of β-tropomyosin-related myopathies through the identification of a novel β-tropomyosin mutation in two clinical contexts not previously associated with β-tropomyosin. The first clinical phenotype is core-rod myopathy, with a β-tropomyosin mutation uncovered by whole exome sequencing in a family with autosomal dominant distal myopathy and muscle biopsy features of both minicores and nemaline rods. The second phenotype, observed in four unrelated families, is autosomal dominant trismus-pseudocamptodactyly syndrome (distal arthrogryposis type 7; previously associated exclusively with myosin heavy chain 8 mutations). In all four families, the mutation identified was a novel 3-bp in-frame deletion (c.20_22del) that results in deletion of a conserved lysine at the seventh amino acid position (p.K7del). This is the first mutation identified in the extreme N-terminus of β-tropomyosin. To understand the potential pathogenic mechanism(s) underlying this mutation, we performed both computational analysis and in vivo modelling. Our theoretical model predicts that the mutation disrupts the N-terminus of the α-helices of dimeric β-tropomyosin, a change predicted to alter protein–protein binding between β-tropomyosin and other molecules and to disturb head-to-tail polymerization of β-tropomyosin dimers. To create an in vivo model, we expressed wild-type or p.K7del β-tropomyosin in the developing zebrafish. p.K7del β-tropomyosin fails to localize properly within the thin filament compartment and its expression alters sarcomere length, suggesting that the mutation interferes with head-to-tail β-tropomyosin polymerization and with overall sarcomeric structure. We describe a novel β-tropomyosin mutation, two clinical-histopathological phenotypes not previously associated with β-tropomyosin and pathogenic data from the first animal model of β-tropomyosin-related myopathies.
PMCID: PMC3572924  PMID: 23413262
nemaline; myopathies; muscle and nerve pathology; mutation; neuromuscular disorders
20.  Prevalence and Type of BRCA Mutations in Hispanics Undergoing Genetic Cancer Risk Assessment in the Southwestern United States: A Report From the Clinical Cancer Genetics Community Research Network 
Journal of Clinical Oncology  2012;31(2):210-216.
To determine the prevalence and type of BRCA1 and BRCA2 (BRCA) mutations among Hispanics in the Southwestern United States and their potential impact on genetic cancer risk assessment (GCRA).
Patients and Methods
Hispanics (n = 746) with a personal or family history of breast and/or ovarian cancer were enrolled in an institutional review board–approved registry and received GCRA and BRCA testing within a consortium of 14 clinics. Population-based Hispanic breast cancer cases (n = 492) enrolled in the Northern California Breast Cancer Family Registry, negative by sequencing for BRCA mutations, were analyzed for the presence of the BRCA1 ex9-12del large rearrangement.
Deleterious BRCA mutations were detected in 189 (25%) of 746 familial clinic patients (124 BRCA1, 65 BRCA2); 21 (11%) of 189 were large rearrangement mutations, of which 62% (13 of 21) were BRCA1 ex9-12del. Nine recurrent mutations accounted for 53% of the total. Among these, BRCA1 ex9-12del seems to be a Mexican founder mutation and represents 10% to 12% of all BRCA1 mutations in clinic- and population-based cohorts in the United States.
BRCA mutations were prevalent in the largest study of Hispanic breast and/or ovarian cancer families in the United States to date, and a significant proportion were large rearrangement mutations. The high frequency of large rearrangement mutations warrants screening in every case. We document the first Mexican founder mutation (BRCA1 ex9-12del), which, along with other recurrent mutations, suggests the potential for a cost-effective panel approach to ancestry-informed GCRA.
PMCID: PMC3532393  PMID: 23233716
21.  Microbial diversity within basement fluids of the sediment-buried Juan de Fuca Ridge flank 
The ISME Journal  2012;7(1):161-172.
Despite its immense size, logistical and methodological constraints have largely limited microbiological investigations of the subseafloor basement biosphere. In this study, a unique sampling system was used to collect fluids from the subseafloor basaltic crust via a Circulation Obviation Retrofit Kit (CORK) observatory at Integrated Ocean Drilling Program borehole 1301A, located at a depth of 2667 m in the Pacific Ocean on the eastern flank of the Juan de Fuca Ridge. Here, a fluid delivery line directly accesses a 3.5 million years old basalt-hosted basement aquifer, overlaid by 262 m of sediment, which serves as a barrier to direct exchange with bottom seawater. At an average of 1.2 × 104 cells ml−1, microorganisms in borehole fluids were nearly an order of magnitude less abundant than in surrounding bottom seawater. Ribosomal RNA genes were characterized from basement fluids, providing the first snapshots of microbial community structure using a high-integrity fluid delivery line. Interestingly, microbial communities retrieved from different CORKs (1026B and 1301A) nearly a decade apart shared major community members, consistent with hydrogeological connectivity. However, over three sampling years, the dominant gene clone lineage changed from relatives of Candidatus Desulforudis audaxviator within the bacterial phylum Firmicutes in 2008 to the Miscellaneous Crenarchaeotic Group in 2009 and a lineage within the JTB35 group of Gammaproteobacteria in 2010, and statistically significant variation in microbial community structure was observed. The enumeration of different phylogenetic groups of cells within borehole 1301A fluids supported our observation that the deep subsurface microbial community was temporally dynamic.
PMCID: PMC3526168  PMID: 22791235
deep subsurface; marine microorganisms; diversity; Juan de Fuca Ridge; SSU ribosomal RNA gene; IODP
22.  Pathogenic Mechanisms in Centronuclear Myopathies 
Centronuclear myopathies (CNMs) are a genetically heterogeneous group of inherited neuromuscular disorders characterized by clinical features of a congenital myopathy and abundant central nuclei as the most prominent histopathological feature. The most common forms of congenital myopathies with central nuclei have been attributed to X-linked recessive mutations in the MTM1 gene encoding myotubularin (“X-linked myotubular myopathy”), autosomal-dominant mutations in the DNM2 gene encoding dynamin-2 and the BIN1 gene encoding amphiphysin-2 (also named bridging integrator-1, BIN1, or SH3P9), and autosomal-recessive mutations in BIN1, the RYR1 gene encoding the skeletal muscle ryanodine receptor, and the TTN gene encoding titin. Models to study and rescue the affected cellular pathways are now available in yeast, C. elegans, drosophila, zebrafish, mouse, and dog. Defects in membrane trafficking have emerged as a key pathogenic mechanisms, with aberrant T-tubule formation, abnormalities of triadic assembly, and disturbance of the excitation–contraction machinery the main downstream effects studied to date. Abnormal autophagy has recently been recognized as another important collateral of defective membrane trafficking in different genetic forms of CNM, suggesting an intriguing link to primary disorders of defective autophagy with overlapping histopathological features. The following review will provide an overview of clinical, histopathological, and genetic aspects of the CNMs in the context of the key pathogenic mechanism, outline unresolved questions, and indicate promising future lines of enquiry.
PMCID: PMC4271577  PMID: 25566070
centronuclear myopathy; myotubular myopathy; MTM1 myotubularin gene; DNM2 dynamin-2 gene; BIN1 bridging integrator-1/amphiphysin-2 gene; RYR1 ryanodine receptor-1 gene; TTN titin gene; autophagy
23.  Treatable childhood neuronopathy caused by mutations in riboflavin transporter RFVT2 
Brain  2013;137(1):44-56.
Childhood onset motor neuron diseases or neuronopathies are a clinically heterogeneous group of disorders. A particularly severe subgroup first described in 1894, and subsequently called Brown-Vialetto-Van Laere syndrome, is characterized by progressive pontobulbar palsy, sensorineural hearing loss and respiratory insufficiency. There has been no treatment for this progressive neurodegenerative disorder, which leads to respiratory failure and usually death during childhood. We recently reported the identification of SLC52A2, encoding riboflavin transporter RFVT2, as a new causative gene for Brown-Vialetto-Van Laere syndrome. We used both exome and Sanger sequencing to identify SLC52A2 mutations in patients presenting with cranial neuropathies and sensorimotor neuropathy with or without respiratory insufficiency. We undertook clinical, neurophysiological and biochemical characterization of patients with mutations in SLC52A2, functionally analysed the most prevalent mutations and initiated a regimen of high-dose oral riboflavin. We identified 18 patients from 13 families with compound heterozygous or homozygous mutations in SLC52A2. Affected individuals share a core phenotype of rapidly progressive axonal sensorimotor neuropathy (manifesting with sensory ataxia, severe weakness of the upper limbs and axial muscles with distinctly preserved strength of the lower limbs), hearing loss, optic atrophy and respiratory insufficiency. We demonstrate that SLC52A2 mutations cause reduced riboflavin uptake and reduced riboflavin transporter protein expression, and we report the response to high-dose oral riboflavin therapy in patients with SLC52A2 mutations, including significant and sustained clinical and biochemical improvements in two patients and preliminary clinical response data in 13 patients with associated biochemical improvements in 10 patients. The clinical and biochemical responses of this SLC52A2-specific cohort suggest that riboflavin supplementation can ameliorate the progression of this neurodegenerative condition, particularly when initiated soon after the onset of symptoms.
PMCID: PMC3891447  PMID: 24253200
childhood neuronopathy; Brown-Vialetto-Van Laere syndrome; riboflavin therapy; RFVT2; SLC52A2

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