CATCH-UP: A High-Throughput Upstream-Pipeline for Bulk ATAC-Seq and ChIP-Seq Data.

Assay for transposase-accessible chromatin (ATAC) and chromatin immunoprecipitation (ChIP), coupled with next-generation sequencing (NGS), have revolutionized the study of gene regulation. A lack of standardization in the analysis of the highly dimensional datasets generated by these techniques has made reproducibility difficult to achieve, leading to discrepancies in the published, processed data. Part of this problem is due to the diverse range of bioinformatic tools available for the analysis of these types of data. Secondly, a number of different bioinformatic tools are required sequentially to convert raw data into a fully processed and interpretable output, and these tools require varying levels of computational skills. Furthermore, there are many options for quality control that are not uniformly employed during data processing. We address these issues with a complete assay for transposase-accessible chromatin sequencing (ATAC-seq) and chromatin immunoprecipitation sequencing (ChIP-seq) upstream pipeline (CATCH-UP), an easy-to-use, Python-based pipeline for the analysis of bulk ChIP-seq and ATAC-seq datasets from raw fastq files to visualizable bigwig tracks and peaks calls. This pipeline is simple to install and run, requiring minimal computational knowledge. The pipeline is modular, scalable, and parallelizable on various computing infrastructures, allowing for easy reporting of methodology to enable reproducible analysis of novel or published datasets.

GTAC enables parallel genotyping of multiple genomic loci with chromatin accessibility profiling in single cells.

Understanding clonal evolution and cancer development requires experimental approaches for characterizing the consequences of somatic mutations on gene regulation. However, no methods currently exist that efficiently link high-content chromatin accessibility with high-confidence genotyping in single cells. To address this, we developed Genotyping with the Assay for Transposase-Accessible Chromatin (GTAC), enabling accurate mutation detection at multiple amplified loci, coupled with robust chromatin accessibility readout. We applied GTAC to primary acute myeloid leukemia, obtaining high-quality chromatin accessibility profiles and clonal identities for multiple mutations in 88% of cells. We traced chromatin variation throughout clonal evolution, showing the restriction of different clones to distinct differentiation stages. Furthermore, we identified switches in transcription factor motif accessibility associated with a specific combination of driver mutations, which biased transformed progenitors toward a leukemia stem cell-like chromatin state. GTAC is a powerful tool to study clonal heterogeneity across a wide spectrum of pre-malignant and neoplastic conditions.

The chromatin remodeller ATRX facilitates diverse nuclear processes, in a stochastic manner, in both heterochromatin and euchromatin.

The chromatin remodeller ATRX interacts with the histone chaperone DAXX to deposit the histone variant H3.3 at sites of nucleosome turnover. ATRX is known to bind repetitive, heterochromatic regions of the genome including telomeres, ribosomal DNA and pericentric repeats, many of which are putative G-quadruplex forming sequences (PQS). At these sites ATRX plays an ancillary role in a wide range of nuclear processes facilitating replication, chromatin modification and transcription. Here, using an improved protocol for chromatin immunoprecipitation, we show that ATRX also binds active regulatory elements in euchromatin. Mutations in ATRX lead to perturbation of gene expression associated with a reduction in chromatin accessibility, histone modification, transcription factor binding and deposition of H3.3 at the sequences to which it normally binds. In erythroid cells where downregulation of α-globin expression is a hallmark of ATR-X syndrome, perturbation of chromatin accessibility and gene expression occurs in only a subset of cells. The stochastic nature of this process suggests that ATRX acts as a general facilitator of cell specific transcriptional and epigenetic programmes, both in heterochromatin and euchromatin.

Lessons from a ten-year-long journey: building a student-driven computational biology society across Turkey.

The Regional Student Group Turkey (RSG-Turkey) is officially associated with the International Society for Computational Biology (ISCB) Student Council (SC). At the RSG-Turkey, we aim to contribute to the early-career researchers in computational biology and bioinformatics fields by providing opportunities for improving their academic and technical skills in the field. Over the last ten years, we have built a well-known student-driven academic society in Turkey that organizes numerous events every year and continues to grow with over 650 current members. Celebrating the 10th anniversary of RSG-Turkey, in this communication, we share our experiences, five main lessons we learned, and the steps to establish a long-standing academic community: having a clear mission, building a robust structure, effective communication, turning challenges into opportunities, and building collaborations. We believe that our experiences can help students and academics establish long-standing communities in fast-developing areas like bioinformatics.

Severe hypercalcaemia of primary hyperparathyroidism: Could giant adenoma be the real culprit rather than carcinoma?

INTRODUCTION: Hypercalcaemic crisis is a rare manifestation of hyperparathyroidism and occurs in 1.6-6% of patients with primary hyperparathyroidism (pHPT). Although such high serum calcium levels (>14mg/dl) are attributed to malignancy, it is also associated with benign disease of the parathyroid glands. The aim of this study was to evaluate the clinical features and treatment modalities of patients with severe hypercalcaemia who underwent surgery for pHPT. METHODS: The medical records of 537 patients who underwent parathyroidectomy in our department for pHPT between 2005 and 2019 were reviewed retrospectively. Twenty-four (4.4%) of the patients were described as having severe hypercalcaemia. RESULTS: Among 24 patients, 71% were female and the mean age was 55.7 years (range: 40-71 years). The mean serum calcium level at time of diagnosis was 15.9mg/dl (range: 14-22.7mg/dl). According to postoperative pathology reports, solitary adenoma, parathyroid cancer and parathyromatosis were diagnosed with the rates of 87.5%, 8.3% and 4.1% respectively. The mean weight of the solitary parathyroid lesions was 14.9g (standard deviation: 8.9g, range: 4-38g). The mean longest diameter was 2.87cm (standard deviation: 1.4cm, range: 1-5.5cm). Serum calcium levels were within the normal range on the first postoperative day in 75% of the cases. CONCLUSIONS: Severe hypercalcaemia is a rare but urgent condition of pHPT and requires prompt management. Accelerated surgery after adequate medical treatment should be performed. It is important to emphasise that giant adenoma, which is a benign disease, may be a more common cause of severe hypercalcaemia than carcinoma, unlike previously thought.

Diagnostic value of four-dimensional computed tomography and four-dimensional magnetic resonance imaging in primary hyperparathyroidism when first-line imaging was inadequate.

INTRODUCTION: Accurate localisation of the abnormal hyperfunctioning gland with preoperative imaging has a critical role in parathyroid surgery to obtain a successful outcome. This study aimed to evaluate the diagnostic performance of second-line imaging and their contribution to the treatment success in primary hyperparathyroidism when the first-line methods were negative or discordant. METHODS: Among the patients who underwent parathyroidectomy due to primary hyperparathyroidism, 33 who underwent four-dimensional computed tomography and/or four-dimensional magnetic resonance imaging because of negative or discordant first-line imaging results were included. Persistent and recurrent cases were excluded. RESULTS: The majority of the patients were female (84.8%) and the mean age was 59.2 years. Seventeen patients had four-dimensional computed tomography and 25 had four-dimensional magnetic resonance imaging, respectively. Four-dimensional computed tomography and four-dimensional magnetic resonance imaging localised the culprit gland successfully in 52.9% and 84%, respectively. Twenty-five patients in whom single adenoma was detected underwent focused parathyroidectomy. The culprit gland was solitary in 32 cases and one patient had double adenoma. Normocalcaemia was achieved in all cases. Among the 29 patients who completed their postoperative sixth month success rate was 100%. CONCLUSION: Four-dimensional magnetic resonance imaging had high accuracy with fast dynamic imaging in detecting parathyroid adenomas. When the first-line imaging methods were negative or inconclusive, four-dimensional magnetic resonance imaging should be considered primarily since it is cost effective in Turkey and emits no radiation.

Spindle epithelial tumour with thymus-like differentiation: report of two cases.

Spindle epithelial tumour with thymus-like differentiation (SETTLE) is a very rare neck malignancy arising from thymic tissue or branchial pouch, mostly seen in the early decades of life. SETTLE is thought to be a low-grade neoplasm but distant metastases have been reported. Fewer than 50 cases have been reported in the PubMed database. We report on the four-year follow-up for a 13-year-old boy, with no signs of recurrence or distant metastasis. In addition, a 45-year-old woman was admitted to hospital with neck mass. Fine-needle aspiration biopsy was compatible with medullary thyroid carcinoma but serum levels of calcitonin were normal. Total thyroidectomy and central compartment lymph node dissection was planned with an initial diagnosis of medullary thyroid carcinoma. At surgery, frozen section analysis converted our diagnosis to SETTLE and the intraoperative strategy was changed. SETTLE should be considered in a patient with normal serum calcitonin levels in the presence of suspicious medullary thyroid carcinoma at fine-needle aspiration biopsy. Frozen section analysis may help to manage the intraoperative strategy. This report describes the first case of SETTLE successfully diagnosed by frozen section analysis.

Parathyromatosis: a very rare cause of recurrent primary hyperparathyroidism - case report and review of the literature.

Parathyromatosis is a rare entity and usually appears as a consequence of the seeding on previous parathyroid surgery which was applied for the secondary hyperparathyroidism. A 63-year-old woman presented with a history of subtotal thyroidectomy 20 years ago and parathyroidectomy due to primary hyperparathyroidism (PHPT) four years ago. Imaging methods revealed multiple parathyromatosis foci on subcutaneous tissue of the neck. En-bloc resection was performed and pathological examination confirmed the diagnosis of parathyromatosis. After an uneventful 10 months, biochemical and radiological tests revealed recurrence on bilateral thyroid lodges. En-bloc resection was performed. The patient has remained well for 24 months after the second operation and has been followed-up with normal parathormone and serum calcium values. To the best of our knowledge, this report describes the twenty-first case of parathyromatosis in PHPT setting in the literature. It should be kept in mind that parathyromatosis may recur at different sites in the neck even in patients with PHPT.

SMPDL3b modulates insulin receptor signaling in diabetic kidney disease.

Sphingomyelin phosphodiesterase acid-like 3b (SMPDL3b) is a lipid raft enzyme that regulates plasma membrane (PM) fluidity. Here we report that SMPDL3b excess, as observed in podocytes in diabetic kidney disease (DKD), impairs insulin receptor isoform B-dependent pro-survival insulin signaling by interfering with insulin receptor isoforms binding to caveolin-1 in the PM. SMPDL3b excess affects the production of active sphingolipids resulting in decreased ceramide-1-phosphate (C1P) content as observed in human podocytes in vitro and in kidney cortexes of diabetic db/db mice in vivo. Podocyte-specific Smpdl3b deficiency in db/db mice is sufficient to restore kidney cortex C1P content and to protect from DKD. Exogenous administration of C1P restores IR signaling in vitro and prevents established DKD progression in vivo. Taken together, we identify SMPDL3b as a modulator of insulin signaling and demonstrate that supplementation with exogenous C1P may represent a lipid therapeutic strategy to treat diabetic complications such as DKD.

Intraoperative nerve monitoring during thyroidectomy: evaluation of signal loss, prognostic value and surgical strategy.

INTRODUCTION: Intraoperative neural monitoring of the recurrent laryngeal nerve has been widely used to avoid nerve injury during thyroidectomy. We discuss the results of the change in surgical strategy after unilateral signal loss surgeries using intermittent intraoperative neural monitoring in a high-volume referral centre. MATERIALS AND METHODS: Details of consecutive patients who underwent thyroidectomy with intermittent intraoperative neural monitoring between January 2014 and December 2017 were prospectively recorded and retrospectively reviewed. Loss of signal was defined as recurrent laryngeal nerve amplitude level lower than 100 µV during surgery. The rate of loss of signal and change in surgical strategy during the operation were evaluated. RESULTS: Loss of signal was detected in 25 (5.4%) of 456 patients for whom intermittent intraoperative neural monitoring was performed. Four patients had anatomic nerve disruption and surgery was completed by an experienced endocrine surgeon making use of intraoperative neural monitoring with continuous vagal stimulation. Staged thyroidectomy was performed on 16 patients with unilateral loss of signal in whom the nerves were intact visually. Postoperative vocal cord paralysis was encountered in 18 of 21 (85.7%) patients with loss of signal, and 16 of 18 (88.8%) were improved during the follow-up period. Patients' voices were subjectively normal to the surgeon postoperatively in 9 of 21 (42.8%) patients who were found to have loss of signal with intact nerves. CONCLUSIONS: Intraoperative neural monitoring can be used safely in thyroid surgery to avoid recurrent laryngeal nerve injury. It enables the surgeon to diagnose recurrent laryngeal nerve injury intraoperatively to estimate the postoperative nerve function and to modify the surgical strategy to avoid bilateral vocal cord paralysis.

Rat Hindlimb Cryopreservation and Transplantation: A Step Toward "Organ Banking".

In 2016, over 5 million reconstructive procedures were performed in the United States. The recent successes of clinical vascularized composite allotransplantations, hand and face transplantations included, established the tremendous potential of these life-enhancing reconstructions. Nevertheless, due to limited availability and lifelong immunosuppression, application is limited. Long-term banking of composite transplants may increase the availability of esthetically compatible parts with partial or complete HLA matching, reducing the risk of rejection and the immunosuppressive burden. The study purpose was to develop efficient protocols for the cryopreservation and transplantation of a complete rodent limb. Directional freezing is a method in which a sample is cooled at a constant-velocity linear temperature gradient, enabling precise control of the process and ice crystal formation. Vitrification is an alternative cryopreservation method in which the sample solidifies without the formation of ice crystals. Testing both methods on a rat hindlimb composite tissue transplantation model, we found reliable, reproducible, and stable ways to preserve composite tissue. We believe that with further research and development, cryopreservation may lead to composite tissue "banks." This may lead to a paradigm shift from few and far apart emergent surgeries to wide-scale, well-planned, and better-controlled elective surgeries.

The evaluation of regret status in women following tubal ligation in Turkey.

BACKGROUND: Tubal ligation (TL) is increasingly common worldwide. Women may regret after undergoing this permanent form of contraception. This study aimed to investigate the regret rate and the causes of regret in women following TL in Turkey. Materials and Meth- ods: This questionnaire-based study included 253 women interviewed via telephone at least one year after TL. The women were grouped as regretful (n3 1), satisfied (n=170), and undecided (n=52) according to their answers to two questions. The ones who were undecided were excluded from the study. The comparison between the answers of regretful and satisfied women were performed using the Student t- test and the Chi Square test. RESULTS: The rate of regret after TL was 12-15%. The factors increasing the regret rate were TL before the age of 30, absence of spouse's support, and the thought of inability to have children in future. The percentage of patients stating that they accepted the TL procedure without comprehending its permanent nature or experienced health problems after TL was higher among less-educated women and these were the factors increasing regret rates. CONCLUSIONS: TL should be performed in women older than 30 years, after an adequate amount of information suited to the educational level of the patient and with full support of the patient's spouse.

The relationship between uterine prolapse and premalignant endometrial pathology.

OBJECTIVE: The aim of this study was to stress the importance of performing a thorough uterine assessment before selecting an organ- sparing surgery in patients presenting with uterine prolapse and no other complaints. MATERIALS AND METHODS: This study included a total of 111 participants who presented with pelvic organ prolapse and underwent hysterectomy for grades 3-4 uterine prolapse. The posthysterectomy histopathology results were classified as benign (atrophic endometrium, proliferative or secretory endometrium) or pathologic (endometrial hyperplasia, endometrial polyp, adenomyosis, myoma uteri, and endometrium carcinoma). RESULTS: Of the 111 patients enrolled in this study, 23 (20.2%) had endometrial hyperplasia, eight (7.2%) had endometrial polyps, 30 (27%) had uterine fibroids, and 20 (18%) had adenomyosis. CONCLUSION: There may be premalignant lesions of the endometrium in both premenopausal and postmenopausal women presenting with uterine prolapse and no other symptoms. A chronic inflammatory process resulting from the extra-vaginal location of the uterus may play a role in the development of these lesions. Further studies are needed on this subject.

A diagnostic dilemma for solid ovarian masses: the clinical and radiological aspects with differential diagnosis of 23 cases.

OBJECTIVE: This study aimed to analyze the clinical characteristics and diagnostic features of ovarian fibromatous masses. MATERIALS AND METHODS: The authors reviewed the records of 23 women who underwent laparotomic surgeries and whose final histopathological diagnoses were ovarian fibroma, cellular fibroma, or fibrothecoma from January 2005 to January 2013. The clinical, ultrasonographic, magnetic resonance imaging, tumor marker, therapeutic, and histologic data were analyzed. RESULTS: The mean age of the patients was 50.9 years. Sixteen patients were menopausal. The preoperative ultrasonography examination incorrectly diagnosed seven lesions as uterine fibromas, and the magnetic resonance imaging examination incorrectly labeled three lesions as pedunculated subserous uterine fibromas. The cancer antigen-125 levels of 17 cases were measured, with four being abnormal. Twenty-three patients underwent a laparotomy. Twenty patients underwent a total hysterectomy with bilateral salpingo-oophorectomy, and three underwent a tumorectomy. The histological diagnosis was fibrothecoma in 21 cases, fibroma in one case, and cellular fibroma in one case. Histopathologic examination of the endometrium of seven of the 20 patients who underwent hysterectomy revealed simple endometrial hyperplasia without atypia. CONCLUSION: Ovarian fibromas and fibrothecomas are often misdiagnosed as uterine fibromas and occasionally mistaken for malignant tumors of the ovary preoperatively. As these tumors originate from ovarian stroma, they may be hormone-active tumors. Therefore, they may lead to premalignant changes in the endometrium. The preoperative evaluation of the endometrium is recommended.

NOX1-induced accumulation of reactive oxygen species in abdominal fat-derived mesenchymal stromal cells impinges on long-term proliferation.

Mesenchymal stromal cells (MSCs) are multipotent and can be derived from different adult tissues including fat. Our repeated attempts to produce long-term proliferative cultures of rat abdominal adipose stem cells (aASCs) under normal oxygen concentration (21%) were unsuccessful. We set to examine the events controlling this cytostasis of aASCs and found that it resulted from overproduction of reactive oxygen species (ROS) that led to apoptosis. ROS overproduction in aASCs was accompanied by increased expression of NOX1 but not of NOX2 or NOX4. NOX family members are an important source of intracellular ROS pointing to NOX1 involvement in ROS accumulation. This was verified when aASCs that were grown under 3% oxygen conditions expanded long term, displaying reduced NOX1 expression and decreased ROS accumulation. NOX1 involvement in aASC cytostasis was reaffirmed when cells that were expanded under normoxic conditions in the presence of a specific NOX1 inhibitor, ML171, demonstrated reduced ROS accumulation, reduced apoptosis and long-term expansion. aASC expansion arrest was accompanied also by a weak fat differentiation and migratory potential, which was enhanced by NOX1 inhibition. This suggests an inhibitory role for NOX1-induced ROS overproduction on aASCs, their fat differentiation and migratory potential. In contrast to aASCs, similar cells produced from subcutaneous fat were easily expanded in normoxic cultures, exhibiting low ROS concentrations, a low number of apoptotic cells and improved fat differentiation and migration. Taken together, our results show, for the first time, that NOX1-induced ROS accumulation halts ASC expansion and reduces their differentiation and migratory potential under normoxic conditions. Importantly, this phenotype comprises a tissue-specific signature as it was evident in aASCs but not in subcutaneous ASCs. NOX-induced ROS accumulation and cytokine production by fat are part of the metabolic syndrome. The similarity of this phenomenon to aASC phenotype may indicate that they arise from similar molecular mechanisms.

Effect of Ramadan fasting on metabolic markers, dietary intake and abdominal fat distribution in pregnancy.

BACKGROUND: The aim of this study is to evaluate the effect of Ramadan intermittent fasting on metabolic markers, dietary intake, anthropometric measurements, and abdominal visceral fat thickness (VFT) in pregnancy. METHODS: Seventy-eight healthy pregnant subjects who had fasted for at least 15 days during the month of Ramadan in 2012 and 2013 and 78 controls were included in this study. Metabolic markers, dietary intake, anthropometric measurements, and ultrasonographic VFT were calculated for each subject before and after Ramadan fasting. RESULTS: When before and after Ramadan values in the fasting group were compared, we found that daily protein intake was increased (p <0.001), but fat and carbohydrate intake remained unchanged. A significant reduction was observed in liquid consumption while the frequency of asymptomatic bacteriuria was increased. High-density lipoprotein significantly increased, and glycated hemoglobin, insulin, and homeostasis model index significantly decreased (p =0.005, p =0.01, p <0.001, and p =0.03, respectively). A significant increase in ferritin was found (p =0.02). No change was observed in subcutaneous fat thickness, while VFT significantly decreased (p =0.08, p =0.005). However, in the control group, only ferritin level increased. CONCLUSION: A combined change in the number and timing of meals and the portioning of the entire daily intake into only two meals per day may have beneficial metabolic effects and reduction in VFT during pregnancy. Hippokratia 2015; 19 (4): 298-303.

Endometrial pathology in postmenopausal women with no bleeding.

OBJECTIVE: The aim of this study was to determine the rate of unexpected uterine pathology in postmenopausal women admitted to a gynecology clinic with symptoms other than vaginal bleeding and who were scheduled to undergo hysterectomy. MATERIALS AND METHODS: We reviewed retrospectively the medical records of 283 postmenopausal patients who had gynecological surgery between September 2007 and January 2014. We reviewed their presenting symptoms on admission, the indications for surgery, and their transvaginal ultrasonographic findings. Postoperative histopathological results based on uterine specimens were also recorded. The results were analyzed statistically. RESULTS: Of 283 patients who had surgery, 209 had no vaginal bleeding at the time of admission. From this group, 75.6% were found to have unsuspected pathology, including endometrial hyperplasia, endometrial polyps, uterine fibroids, adenomyosis, and one case of endometrial carcinoma (0.5%). The remaining 74 patients had experienced postmenopausal bleeding and in 87.8% there were pathological findings including 13 cases (17.6%) of endometrial cancer (p = 0.0001). CONCLUSION: Vaginal bleeding in postmenopausal women is indicative of a wide array of gynecological pathologies, including endometrial carcinoma. However, uterine fibroids, pelvic masses, or even endometrial cancer may develop without co-morbid vaginal bleeding. Therefore we advocate that postmenopausal women should undergo yearly screening and consultation, without waiting for an episode of vaginal bleeding.

Bilateral Krukenberg tumor in a 16-week pregnant woman.

The authors present the case of a G2P1001 who presented in 16-week gestation with bilateral Krukenberg tumor, abdominal pain, and iterative vomiting episodes. Although a few cases of Krukenberg tumor in pregnant women have been reported, no case reports asymptomatic and free of disease at 18 months were found in the English literature. Early detection followed by surgery and chemotherapy during pregnancy could possibly result in a favorable outcome in such patients.

The role of the fibula head flap for joint reconstruction after osteoarticular resections.

INTRODUCTION AND AIM: Endoprosthetic reconstruction is considered the mainstay of limb salvage in periarticular bone tumours. However, this procedure has limited durability especially when performed in young patients. The free fibula head flap including the proximal articular surface represents one option for hemiarthroplasty reconstruction. The aim of this study was to investigate the role of the fibula head flap for joint reconstruction after osteoarticular resections. PATIENTS AND METHODS: All patients who underwent hemiarthroplasty procedures between 2000 and 2006 using the free fibula head flap were included in the study. Functional assessments were performed using the American Musculoskeletal Tumor Society (AMTS) classification. RESULTS: There were five males and two females (mean age: 22.6 ± 15.9 years). Five patients underwent reconstruction following resection of malignant bone tumours and two for chronic osteomyelitis of the distal humerus. In three patients, the fibula was used for distal radius and wrist joint reconstruction, and the remaining four patients for reconstruction of the distal humerus and elbow joint. A vascularised growth plate transfer based on the lateral geniculate vessels was performed in two patients. Atechnetium-bone scan confirmed viability of all flaps 10 days after surgery, and radiographic bony union was confirmed on average 5 months following surgery. There were no complications with the recipient or donor site after a median follow-up of 71 months (range: 12 months to 10 years). All patients achieved reasonable return of function and were able to perform all activities of daily living. CONCLUSIONS: We demonstrate that the hemiarthroplasty procedure using the free fibula flap with its proximal head is a safe procedure with good functional results. Performing autologous arthroplasty using a free fibula head flap may be a promising alternative to an endoprosthesis or alloplastic reconstruction with a low risk of complications and morbidity.