Alternative splicing expands the clinical spectrum of NDUFS6-related mitochondrial disorders.

PURPOSE: We describe 3 families with Charcot-Marie-Tooth neuropathy (CMT), harboring a homozygous NDUFS6 NM_004553.6:c.309+5G>A variant previously linked to fatal Leigh syndrome. We aimed to characterize clinically and molecularly the newly identified patients and understand the mechanism underlying their milder phenotype. METHODS: The patients underwent extensive clinical examinations. Exome sequencing was done in 4 affected individuals. The functional effect of the c.309+5G>A variant was investigated in patient-derived EBV-transformed lymphoblasts at the complementary DNA, protein, and mitochondrial level. Alternative splicing was evaluated using complementary DNA long-read sequencing. RESULTS: All patients presented with early-onset, slowly progressive axonal CMT, and nystagmus; some exhibited additional central nervous system symptoms. The c.309+5G>A substitution caused the expression of aberrantly spliced transcripts and negligible levels of the canonical transcript. Immunoblotting showed reduced levels of mutant isoforms. No detectable defects in mitochondrial complex stability or bioenergetics were found. CONCLUSION: We expand the clinical spectrum of NDUFS6-related mitochondrial disorders to include axonal CMT, emphasizing the clinical and pathophysiologic overlap between these 2 clinical entities. This work demonstrates the critical role that alternative splicing may play in modulating the severity of a genetic disorder, emphasizing the need for careful consideration when interpreting splice variants and their implications on disease prognosis.

Functional Outcomes and Complication Analysis of Plate Osteosynthesis versus Hemiarthroplasty in Three-part and Four-part Proximal Humerus Fractures.

OBJECTIVE: To compare the functional results and complications of open reduction-internal fixation and hemiarthroplasty performed in Neer three-part and four-part proximal humerus fractures. METHODS: The retrospective study was conducted at the Istanbul Training and Research Hospital, Turkey, and comprised data of patients who were diagnosed with three-part or four-part fracture of the proximal humerus and underwent surgical procedure between January 2008 and April 2013. Those who had undergone open reduction-internal fixation using anatomical locking plates were placed in group A, and those who had undergone hemiarthroplasty were in group B. Constant-Murley shoulder outcome, University of California at Los Angeles shoulder and visual analogue scale scores were compared between the two groups. The degrees of forward flexion and abduction of the glenohumeral joint were recorded. Complications in both the groups were recorded. Data was analysed using SPSS 15.\. RESULTS: Of the 48 patients, 30(62.5%) were in group A; 16(53.3%) males and 14(46.7%) females, with an overall mean age of 60.0±9.4 years. The remaining 18(37.5%) were in group B; 7(39%) males and 11(61%) females, with an overall mean age of 67.3±10.1 years. The mean follow-up period was 18.7±16.4 months. The mean Constant-Murley and University of California at Los Angeles shoulder scores were not significantly different between the two groups (p>0.05). The mean visual analogue scale score was significantly higher in group A compared to group B (p=0.021). In group A, heterotropic ossification was observed in 1(3.4%) patient, avascular necrosis in 3(10%), screw penetration without avascular necrosis in 4(13.2%), and non-union in 1(3.4%), while in group B, shoulder subluxation was noted in 2(11.1%) patients and malunion of the greater tubercle in 6(33.3%). CONCLUSION: Although statistically non-significant, higher functional scores, forward flexion and abduction degrees were observed in patients who had undergone open reduction-internal fixation, while significantly lower visual analogue scale scores were recorded in the hemiarthroplasty group.

Biallelic Mutations in ADPRHL2, Encoding ADP-Ribosylhydrolase 3, Lead to a Degenerative Pediatric Stress-Induced Epileptic Ataxia Syndrome.

ADP-ribosylation, the addition of poly-ADP ribose (PAR) onto proteins, is a response signal to cellular challenges, such as excitotoxicity or oxidative stress. This process is catalyzed by a group of enzymes referred to as poly(ADP-ribose) polymerases (PARPs). Because the accumulation of proteins with this modification results in cell death, its negative regulation restores cellular homeostasis: a process mediated by poly-ADP ribose glycohydrolases (PARGs) and ADP-ribosylhydrolase proteins (ARHs). Using linkage analysis and exome or genome sequencing, we identified recessive inactivating mutations in ADPRHL2 in six families. Affected individuals exhibited a pediatric-onset neurodegenerative disorder with progressive brain atrophy, developmental regression, and seizures in association with periods of stress, such as infections. Loss of the Drosophila paralog Parg showed lethality in response to oxidative challenge that was rescued by human ADPRHL2, suggesting functional conservation. Pharmacological inhibition of PARP also rescued the phenotype, suggesting the possibility of postnatal treatment for this genetic condition.

Enlightening the neuroprotective effect of quercetin in epilepsy: From mechanism to therapeutic opportunities.

Epilepsy is a devastating neurological disorder characterized by the repeated occurrence of epileptic seizures. Epilepsy stands as a global health concern affecting around 70 million people worldwide. The mainstream antiepileptic drugs (AEDs) only exert symptomatic relief and drug-resistant epilepsy occurs in up to 33 percent of patients. Hence, the investigation of novel therapeutic strategies against epileptic seizures that could exert disease modifying effects is of paramount importance. In this context, compounds of natural origin with potential antiepileptic properties have recently gained increasing attention. Quercetin is a plant-derived flavonoid with several pharmacological activities. Emerging evidence has demonstrated the antiepileptic potential of quercetin as well. Herein, based on the available evidence, we discuss the neuroprotective effects of quercetin against epileptic seizures and further analyze the plausible underlying molecular mechanisms. Our review suggests that quercetin might be a potential therapeutic candidate against epilepsy that deserves further investigation, and paves the way for the development of plant-derived antiepileptic treatment approaches.

Systems-level Analysis Reveals Multiple Modulators of Epithelial-mesenchymal Transition and Identifies DNAJB4 and CD81 as Novel Metastasis Inducers in Breast Cancer.

Epithelial-mesenchymal transition (EMT) is driven by complex signaling events that induce dramatic biochemical and morphological changes whereby epithelial cells are converted into cancer cells. However, the underlying molecular mechanisms remain elusive. Here, we used mass spectrometry based quantitative proteomics approach to systematically analyze the post-translational biochemical changes that drive differentiation of human mammary epithelial (HMLE) cells into mesenchymal. We identified 314 proteins out of more than 6,000 unique proteins and 871 phosphopeptides out of more than 7,000 unique phosphopeptides as differentially regulated. We found that phosphoproteome is more unstable and prone to changes during EMT compared with the proteome and multiple alterations at proteome level are not thoroughly represented by transcriptional data highlighting the necessity of proteome level analysis. We discovered cell state specific signaling pathways, such as Hippo, sphingolipid signaling, and unfolded protein response (UPR) by modeling the networks of regulated proteins and potential kinase-substrate groups. We identified two novel factors for EMT whose expression increased on EMT induction: DnaJ heat shock protein family (Hsp40) member B4 (DNAJB4) and cluster of differentiation 81 (CD81). Suppression of DNAJB4 or CD81 in mesenchymal breast cancer cells resulted in decreased cell migration in vitro and led to reduced primary tumor growth, extravasation, and lung metastasis in vivo Overall, we performed the global proteomic and phosphoproteomic analyses of EMT, identified and validated new mRNA and/or protein level modulators of EMT. This work also provides a unique platform and resource for future studies focusing on metastasis and drug resistance.

Clinical predictors of drug-resistant epilepsy in children

Background/aim: In up to 20% of epilepsy patients, seizures may not be controlled despite the use of antiepileptic drugs, either alone or in combination. These individuals are considered to have drug-resistant epilepsy. Drug-resistant epilepsy is usually associated with intellectual disability, psychiatric comorbidity, physical injury, sudden unexpected death, and low quality of life. Early detection and prediction of drug-resistant epilepsy are essential in determining the patient's most appropriate treatment option. This retrospective study aimed to determine the clinical, electroencephalographic, and radiological factors associated with medically intractable childhood seizures. Materials and methods: Data regarding 177 patients diagnosed with drug-resistant epilepsy were compared with 281 patients with drug-responsive epilepsy. Results: Univariate analysis showed that age at seizure onset, having mixed seizure types, history of status epilepticus, history of neonatal seizures, history of both having febrile and afebrile seizures, daily seizures at the onset, abnormality on the first electroencephalogram, generalized epileptic abnormality on electroencephalogram, abnormal neurodevelopmental status, abnormal neuroimaging, and having symptomatic etiology were significant risk factors for the development of drug-resistant epilepsy (p < 0.05). In multivariable analysis, having mixed seizure types, history of status epilepticus, having multiple seizures in a day, intellectual disability, symptomatic etiology, and neuroimaging abnormality remained significant predictors for developing drug-resistant epilepsy. Conclusions: In the course of childhood epilepsy, some clinical features may predict the outcome. Early identification of patients with high risk for drug-resistant epilepsy will help plan the appropriate treatment option. Further prospective studies should confirm these findings.

Long-Term results of total Hip Arthroplasty performed using a cementless expansive Acetabular Cup and Spotorno Femoral Stem.

OBJECTIVE: In this study we aimed to investigate the long-term clinical and radiological results, revision rates and causes, and the rate of implant survival in total hip arthroplasty performed using CLS® expansion cup and Spotorno® cementless femoral stem. METHODS: Clinical results of total hip arthroplasty performed on 131 hips of 114 patients in Istanbul Training and Research Hospital between 1993 and 2003 were retrospectively evaluated according to the Harris Hip Score. Revision rates were determined and implant survival rates were identified using the Kaplan-Meier estimator. RESULTS: Of the patients, 39 were males and 75 were females. The average age of the patients at surgery was 48.7±11.3 years. Patients were followed up for a mean period of 13.9±2.4 years. The mean Harris Hip Score was 34.35±6.09 preoperatively and 88.20±7.11 at the final follow-up (p<0.001). The Kaplan-Meier survivorship estimate for the cup at 13.9 years, taking revision for any reason as the end point was 95.6% (95% CI), while the 15th and 17th year survival rates were 90% and 85%, respectively. CONCLUSION: In total hip arthroplasty using a cementless expansive acetabular cup, a 95.6% survival rate is achieved after an average of 14 years, whereas the rate decreases to 85% after 17 years. Even if the incidence of cup breakage is reduced with proper implantation, particle disease and periacetabular osteolysis remains a problem for the long-term survival.

Comparison of open and endoscopic carpal tunnel surgery regarding clinical outcomes, complication and return to daily life: A prospective comparative study.

OBJECTIVE: This study aimed to compare the clinical results and complications as well as patient satisfaction in patients with carpal tunnel syndrome operated with open carpal tunnel release (OCTR) or endoscopic carpal tunnel release (ECTR) techniques. METHODS: This study conducted in Istanbul Training and Research Hospital between August 2016 and January 2018. A total of 54 patients were operated with the ECTR technique and 50 patients were operated with the OCTR technique after failing nonsurgical treatment. Patients functional scores are assessed with the carpal tunnel syndrome-functional status score (CTS-FSS) and carpal tunnel syndrome-symptom severity score (CTS-SSS). Operation time, incision length and complications of the two techniques were noted and compared. RESULTS: The age, sex distribution, distribution of sides, and complaint period were not significant (p > 0.05) between the groups. The preoperative or postoperative CTS-SSS and CTS-FSS values did not differ significantly (p > 0.05). Incision length, time to return to work and return to daily life in the OCTR group was significantly higher than the ECTR group (p < 0.05). CONCLUSION: ECTR has similar results in terms of symptom relief, severity, functional status, pillar pain and complication rates compared to OCTR. However, it has the advantages of early return to daily life, early return to work and less incision length.

Targeted sequencing with expanded gene profile enables high diagnostic yield in non-5q-spinal muscular atrophies.

Spinal muscular atrophies (SMAs) are a heterogeneous group of disorders characterized by muscular atrophy, weakness, and hypotonia due to suspected lower motor neuron degeneration (LMND). In a large cohort of 3,465 individuals suspected with SMA submitted for SMN1 testing to our routine diagnostic laboratory, 48.8% carried a homozygous SMN1 deletion, 2.8% a subtle mutation, and an SMN1 deletion, whereas 48.4% remained undiagnosed. Recently, several other genes implicated in SMA/LMND have been reported. Despite several efforts to establish a diagnostic algorithm for non-5q-SMA (SMA without deletion or point mutations in SMN1 [5q13.2]), data from large-scale studies are not available. We tested the clinical utility of targeted sequencing in non-5q-SMA by developing two different gene panels. We first analyzed 30 individuals with a small panel including 62 genes associated with LMND using IonTorrent-AmpliSeq target enrichment. Then, additional 65 individuals were tested with a broader panel encompassing up to 479 genes implicated in neuromuscular diseases (NMDs) with Agilent-SureSelect target enrichment. The NMD panel provided a higher diagnostic yield (33%) than the restricted LMND panel (13%). Nondiagnosed cases were further subjected to exome or genome sequencing. Our experience supports the use of gene panels covering a broad disease spectrum for diseases that are highly heterogeneous and clinically difficult to differentiate.

Erythropoietin may attenuate lung inflammation in a rat model of meconium aspiration syndrome.

BACKGROUND: Inflammation is believed to play a key role in the pathophysiology of meconium aspiration syndrome (MAS). PURPOSE OF THE STUDY: The objective was to determine whether the recombinant human Erythropoietin (rhEPO) pretreatment could attenuate meconium-induced inflammation. MATERIALS AND METHODS: In this study, 24 ventilated adult male rats were studied to examine the effects of recombinant human EPO (rhEPO) on meconium-induced inflammation. Seventeen rats were instilled with human meconium (1.5 mL/kg, 65 mg/mL) intratracheally and ventilated for 3 hours. rhEPO (1000 U/kg) (n = 9) or saline (n = 8) was given to the animals. Seven rats that were ventilated and not instilled with meconium served as a sham-controlled group. Analysis of the blood gases, interleukin (IL)-1ß, IL-6, IL-8, and tumor necrosis factor (TNF)-α in blood and bronchoalveolar lavage (BAL) fluid samples, and lung tissue myeloperoxidase levels were performed. RESULTS: Intrapulmonary instillation of meconium resulted in the increase of TNF-α (p = 0.005 and p < 0.001, respectively) and IL-8 concentrations (p < 0.001 and p < 0.001, respectively) in BAL fluid in the EPO + meconium and saline + meconium groups compared with the sham-controlled group. rhEPO pretreatment prevented the increase of BAL fluid IL-1ß, IL-6, and IL-8 levels (p < 0.001, p = 0.021, and p = 0.005, respectively), and serum IL-6 levels (p = 0.036). CONCLUSION: rhEPO pretreatment is associated with improved BAL fluid and serum cytokine levels. Pretreatment with rhEPO might reduce the risk of developing of meconium-induced derangements.

Mesenchymal stem cell treatment in hyperoxia-induced lung injury in newborn rats.

BACKGROUND: The aim of this study was to evaluate the effectiveness of tracheally delivered mesenchymal stem cells (MSC) on lung pathology in a hyperoxia-induced lung injury (HILI) model in neonatal rats. METHODS: For the HILI model, rat pups were exposed to 85-95% oxygen during the first 10 days of life. Rats were divided into six groups: room-air normoxia (n = 11); room air, sham (n = 11); hyperoxia exposed with normal saline as placebo (n = 9); hyperoxia exposed with culture medium of MSC (n = 10); hyperoxia exposed with medium remaining after harvesting of MSC (n = 8); and hyperoxia exposed with MSC (n = 17). Pathologic changes, number and diameter of alveoli, α-smooth muscle actin (α-SMA) expression and localization of MSC in the lungs were assessed. RESULTS: Number of alveoli increased and alveolar diameter decreased in the mesenchymal stem cell group so that there were no differences when compared with the normoxia group (P = 0.126 and P = 0.715, respectively). Expression of α-SMA decreased significantly in the mesenchymal stem cell group compared with the placebo group (P < 0001). Green fluorescent protein-positive cells were found in lung tissue from all rats given MSC. Some green fluorescent protein-positive MSC also expressed surfactant protein-C. CONCLUSION: Mesenchymal stem cells became localized in damaged lung tissue, and recovery approximated the room air control.

Fibroblast growth factor 2 improves cognitive function in neonatal rats with hypoxic ischaemic brain injury.

OBJECTIVE: To determine the effect of fibroblast growth factor 2 on cognitive function in neonatal rats with hypoxic-ischaemic brain injury. METHODS: The randomised controlled study was conducted from January to June 2011 at Mersin University, School of Medicine, Experimental Animals Research Laboratory and Physiology Behaviour Laboratory, Mersin, Turkey. It included 7-d-old male rats that were randomised into four groups: fibroblast growth factor 2-20, fibroblast growth factor 2-40, control and sham. All the rats, except those in the sham group, were kept in a hypoxia chamber containing 8% oxygen for 2 hours following ligation of the right carotid artery. After hypoxic-ischaemic brain injury was induced, 20 ng g-1 or 40 ng g-1 of fibroblast growth factor 2 was administered via the intraperitoneal route. The terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelling method was used to evaluate neuronal apoptosis. The Morris water maze (MWM) test was administered to the rats at age 14 weeks. RESULTS: Of the 78 rats on the study, 18 (23%) were in the sham group, while the other three groups had 20 (25.6%) rats each. The number of apoptotic neurons in the right hemisphere in the experimental groups was significantly lower than in the control group (p=0.004 and p<0.001). The number of apoptotic neurons in the right hemisphere in the fibroblast growth factor 2-40 group was significantly lower than in the fibroblast growth factor 2-20 group (p<0.001). Moreover, fibroblast growth factor 2improved Morris water maze test cognitive performance in a dose-dependent manner. CONCLUSIONS: Fibroblast growth factor 2 treatment reduced neuronal apoptosis and improved cognitive functioning in neonatal rats with experimentally-induced hypoxic-ischaemic brain injury.

Phosphoproteomic Analysis of Aurora Kinase Inhibition in Monopolar Cytokinesis.

Cytokinesis is the last step of the cell cycle that requires coordinated activities of the microtubule cytoskeleton, actin cytoskeleton, and membrane compartments. Aurora B kinase is one of the master regulatory kinases that orchestrate multiple events during cytokinesis. To reveal targets of the Aurora B kinase, we combined quantitative mass spectrometry with chemical genetics. Using the quantitative proteomic approach, SILAC (stable isotope labeling with amino acids in cell culture), we analyzed the phosphoproteome of monopolar cytokinesis upon VX680- or AZD1152-mediated aurora kinase inhibition. In total, our analysis quantified over 20 000 phosphopeptides in response to the Aurora-B kinase inhibition; 246 unique phosphopeptides were significantly down-regulated and 74 were up-regulated. Our data provide a broad analysis of downstream effectors of Aurora kinase and offer insights into how Aurora kinase regulates cytokinesis.

Lack of IL7Rα expression in T cells is a hallmark of T-cell immunodeficiency in Schimke immuno-osseous dysplasia (SIOD).

Schimke immuno-osseous dysplasia (SIOD) is an autosomal recessive, fatal childhood disorder associated with skeletal dysplasia, renal dysfunction, and T-cell immunodeficiency. This disease is linked to biallelic loss-of-function mutations of the SMARCAL1 gene. Although recurrent infection, due to T-cell deficiency, is a leading cause of morbidity and mortality, the etiology of the T-cell immunodeficiency is unclear. Here, we demonstrate that the T cells of SIOD patients have undetectable levels of protein and mRNA for the IL-7 receptor alpha chain (IL7Rα) and are unresponsive to stimulation with IL-7, indicating a loss of functional receptor. No pathogenic mutations were detected in the exons of IL7R in these patients; however, CpG sites in the IL7R promoter were hypermethylated in SIOD T cells. We propose therefore that the lack of IL7Rα expression, associated with hypermethylation of the IL7R promoter, in T cells and possibly their earlier progenitors, restricts T-cell development in SIOD patients.

Congenital laryngomucocele: a rare cause of airway obstruction in a newborn.

Congenital laryngoceles are defined as cystic dilatation of laryngeal saccules and are an extremely rare cause of newborn respiratory distress. A laryngomucocele occurs when the neck of the laryngocele gets obstructed and fills with the mucoid secretions of the saccule. It may cause stridor, respiratory distress, and severe airway obstruction in the narrow airway of a newborn and necessitates urgent surgical intervention. There is only 1 case of congenital laryngomucocele reported in an autopsy examination in the English literature, and here we report the first living congenital laryngomucocele case and discuss the clinical approach.

Towards single-cell LC-MS phosphoproteomics.

Protein phosphorylation is a ubiquitous posttranslational modification, which is heavily involved in signal transduction. Misregulation of protein phosphorylation is often associated with a decrease in cell viability and complex diseases such as cancer. The dynamic and low abundant nature of phosphorylated proteins makes studying phosphoproteome a challenging task. In this review, we summarize state of the art proteomic techniques to study and quantify peptide phosphorylation in biological systems and discuss their limitations. Due to its short-lived nature, the phosphorylation event cannot be precisely traced in a heterogonous cell population, which highlights the importance of analyzing phosphorylation events at the single cell level. Mainly, we focus on the methodical and instrumental developments in proteomics and nanotechnology, which will help to build more accurate and robust systems for the feasibility of phosphorylation analysis at the single cell level. We propose that an automated and miniaturized construction of analytical systems holds the key to the future of phosphoproteomics; therefore, we highlight the benchmark studies in this direction. Having advanced and automated microfluidic chip LC systems will allow us to analyze single-cell phosphoproteomics and quantitatively compare it with others. The progress in the microfluidic chip LC systems and feasibility of the single-cell phosphoproteomics will be beneficial for early diagnosis and detection of the treatment response of many crucial diseases.

Neuroprotective effect of levetiracetam on hypoxic ischemic brain injury in neonatal rats.

PURPOSE: Hypoxic-ischemic brain injury that occurs in the perinatal period is one of the leading causes of mental retardation, visual and auditory impairment, motor defects, epilepsy, cerebral palsy, and death in neonates. The severity of apoptosis that develops after ischemic hypoxia and reperfusion is an indication of brain injury. Thus, it may be possible to prevent or reduce injury with treatments that can be given before the reperfusion period following hypoxia and ischemia. Levetiracetam is a new-generation antiepileptic drug that has begun to be used in the treatment of epilepsy. METHODS: The present study investigated the effects of levetiracetam on neuronal apoptosis with histopathological and biochemical tests in the early period and behavioral experiments in the late period. RESULTS: This study showed histopathologically that levetiracetam reduces the number of apoptotic neurons and has a neuroprotective effect in a neonatal rat model of hypoxic-ischemic brain injury in the early period. On the other hand, we demonstrated that levetiracetam dose dependently improves behavioral performance in the late period. CONCLUSIONS: Based on these results, we believe that one mechanism of levetiracetam's neuroprotective effects is due to increases in glutathione peroxidase and superoxide dismutase enzyme levels. To the best of our knowledge, this study is the first to show the neuroprotective effects of levetiracetam in a neonatal rat model of hypoxic-ischemic brain injury using histopathological, biochemical, and late-period behavioral experiments within the same experimental group.

Two rare entities in the same palate lesion: hyalinizing-type clear cell carcinoma and necrotizing sialometaplasia.

Hyalinizing clear cell carcinoma is a low-grade malignant epithelial neoplasm of the salivary glands. The tumor has epithelial cells and lacks myoepithelial cells. Necrotizing sialometaplasia is a benign, self-limiting lesion of the salivary glands. The clinical and histologic features mimic those of mucoepidermoid carcinoma or squamous cell carcinoma. The importance of these entities are the rarity of both of them and their potential to be misdiagnosed as other lesions. Pathologists and clinicians should be aware of these entities to prevent misdiagnosis. This is the first clinical report of 2 rare and consecutive different entities of the same location on the hard palate to our knowledge.