Between 2% and 5% of malignant germ-cell tumors in men arise at extragonadal sites. Of extragonadal germ cell tumors, testicular carcinoma in situ (CIS) are present in 31–42% of cases, and CIS are reported to have low sensitivity to chemotherapy in spite of the various morphology and to have a high likelihood of developing into testicular tumors. A testicular biopsy may thus be highly advisable when evaluating an extragonadal germ cell tumor.A 36-year-old man was diagnosed as having an extragonadal non-seminomatous germ cell tumor, that was treated by cisplatin-based chemotherapy, leading to a complete remission. In the meantime, testicular tumors were not detected by means of ultrasonography. About 4 years later, a right testicular tumor was found, and orchiectomy was carried out. Microscopically, the tumor was composed of seminoma.We herein report a case of metachronous occurrence of an extragonadal and gonadal germ cell tumor. In the evaluation of an extragonadal germ cell tumor, a histological examination should be included since ultrasonography is not sufficient to detect CIS or minute lesions of the testis. Between 2% and 5% of malignant germ-cell tumors in men arise at extragonadal sites . Cytogenrd July. Pathology showed the resected tumor was a seminoma with CIS .2. The side effect of CIS therapy  are significant, especially concerning fertility and androgen production.3. Testicular tumors early detected by adequate surveillance respond well to treatments.4. Testicular biopsy is not entitled to detect all the CIS.The author(s) declare that they have no competing interests.IK, MU, HY, KN, TT and ND carried out clinical treatments.TM carried out histopathological studies.The pre-publication history for this paper can be accessed here:


Late relapses of testicular germ cell tumor are uncommon. We report a case of cervical mature teratoma appeared 17 years after treatment of testicular teratocarcinoma.A 20- year- old patient underwent left sided orchiectomy followed by systemic therapy and retroperitoneal residual mass resection in 1989. He remained in complete remission for 200 months. In 2005 a huge left supraclavicular neck mass with extension to anterior mediastinum appeared. Radical surgical resection of the mass was performed and pathologic examination revealed mature teratoma.This is one of the longest long-term reported intervals of a mature teratoma after treatment of a testicular nonseminoma germ cell tumor. This case emphasizes the necessity for follow up of testicular cancer throughout the patient's life. The prognosis for nonseminomatous germ cell tumor (NSGCT) of the testis has been dramatically improved by using a treatment protocol of cisplatin-based chemotherapy followed by surgical resection of residual tumor mass. The complete response rate of disseminated germ cell tumor with this protocol is between 70%–80%.Most patients who relapse do so within the first year of therapy. Late relapse is defined as recurrence after a relapse- free interval of more than two years after completion of primary treatment . The cumThe incidence of late relapse after cisplatin based chemotherapy of germ cell tumor is related to initial tumor burden and patients with bulky retroperitoneal disease appear to be at an increased risk of late relapse. As tumor markers do not rise in one quarter of late relapses, they should undergo CT scans at annual follow-up evaluations. In the remaining patients, history, physical examination, tumor markers and chest X-ray may allow to detect the majority of late asymptomatic relapses .Herein we report a case of late relapse of a bulky teratocarcinoma, 17 years after completion of treatment as a mature teratoma of the neck and upper thorax.A 36 year-old man was admitted to our hospital with chief complaint of left sided neck mass. He had a history of left orchiectomy in 1988 in another center because of testicular teratocarcinoma. At that time he was referred to our center because of huge retroperitoneal mass medial to the left kidney Figure . The othGerm cell tumours, which are in complete remission two years after treatments have a high probability of cure and reports of late relapse, are rare 1.3%–6.2%) [% [2-6]. The possible mechanisms of development of a late relapse in germ cell tumors include the followings: malignant degeneration of mature teratoma to germinal malignancy, growth of an occult testicular tumor not eliminated by chemotherapy, development of a second primary germinal malignancy, persistent microscopic viable tumor with an atypical less aggressive biologic behaviour .Patient with bulky retroperitoneal disease and patient found to have teratoma following cisplatin-based chemotherapy appear to be at an increased risk of late relapse. Of 51 patients with mature teratoma in resected retroperitoneal residual tumor masses after chemotherapy, 9 patients (17.6%) relapsed. In five patients (9.8 %) relapse resulted from growing mature teratoma [Teratoma is a chemo resistant, nonseminomatous germ cell tumor composed of somatic cell type from two or more germ layers and is derived from a toti potential, malignant precursor cell . Although teratoma is a benign tumor but its biologic potential is unpredictable and it should be resected completely because it may grow and become unresectable.Disease free survival following resection of teratoma is related to completeness of resection; therefore, there are significant advantages to surgery with low volume disease. Moreover, there is the risk of malignant transformation of teratoma to carcinoma or sarcoma , so unreHerein we report a case of late relapse in the left side of neck 17 years after treatment of left testicular teratocarcinoma. The incidence of cervical metastasis in testicular germ cell tumor is about 5% . In germWhereas chemotherapy has only minor curative potential in the treatment of late relapse, patient with localized resectable disease can be cured. Modified neck dissection has a demonstrated valuable role in the treatment of metastatic non-seminomatus germ cell tumours .Our patient has been well with no evidence of disease after resection of cervical mature teratoma until the date of last follow-up (October 2006). However, continued close follow-up of this case is necessary because the large tumor burden of teratoma is a significant adverse factor predictive for further relapse . In our This case emphasizes the necessity of annual follow-up evaluation of testicular germ cell cancer patient through their life, in order to detect the majority of late relapse at an asymptomatic phase.The author(s) declare that they have no competing interest.RO carried out the surgery and drafted the manuscript.MA assisted in operation and participated in drafting the manuscript.All authors read and approved the final manuscript.


Clear cell sarcoma of the kidney (CCSK) in adults is extremely rare. Optimal treatment of adult patients with CCSK remains unclear.A 22-year-old man presented with a 2-month history of left flank pain. A color duplex sonography revealed a hypervascular, heterogeneous renal mass. Abdominal and pelvic computerized tomography showed a heterogeneous mass originating from the lower pole of the left kidney and infiltrating to the psoas muscle. Further evaluation including bone scan did not demonstrate any evidence of metastases. A left radical nephrectomy with hilar lymphadenectomy through an intraperitoneal approach with an anterior subcostal incision was performed. The histopathological diagnosis of the mass was a clear cell sarcoma of the kidney. No lymph node metastases were found. Concomitant chemo-radiotherapy was performed. Therapy-related serious side effects were not observed. There was no evidence of local recurrence or metastases during the following twenty-four months after therapy.We believe that the combination therapy is efficacious for preventing the local recurrence and distant metastases. Accurate diagnosis is very important and therapy must also include doxorubicin regardless of the disease stage in adult patients with CCSK. Clear cell sarcoma of the kidney (CCSK) is an uncommon pediatric neoplasm distinct from Wilms tumor and also usually tends to metastasize to bones being different from Wilms tumor .CCSK in adults is extremely rare. Optimal treatment of adult patients with CCSK remains unclear. Surgery, radiotherapy and chemotherapy are combined or used separately -4. AdvanHerein, we report a 22-year-old patient with clear cell sarcoma of the left kidney, who was managed with radical nephrectomy, radiotherapy and chemotherapy. We think that our case report might provide additional information about the treatment of CCSK in adults.A 22-year-old man presented with a 2-month history of left flank pain. A color duplex sonography revealed a hypervascular, heterogeneous renal mass 125 × 156 × 195 mm. in diameter. Abdominal and pelvic computerized tomography showed a heterogeneous mass originating from the lower pole of the left kidney and infiltrating to the psoas muscle  declare that they have no competing interests.Dr.s ARK and BO have provided the patients and performed the radical nephrectomy, HO has reviewed the charts, CC has evaluated the pathology specimen, IA and FYA have performed the concomitant chemo-radiotherapy. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


We report herein a rare case of primary omental gastrointestinal stromal tumor (GIST).® and is alive well with no sign of relapse.A 65 year-old man was referred to our hospital with a huge abdominal mass occupying the entire left upper abdomen as shown by sonography. On computed tomography (CT), this appeared as a heterogeneous low-density mass with faint enhancement. Abdominal angiography revealed that the right gastroepiploic artery supplied the tumor. With such an indication of gastric GIST, liposarcoma, leiomyosarcoma or mesothelioma laparotomy was performed and revealed that this large mass measured 20 × 17 × 6 cm, arising from the greater omentum. It was completely resected. Histopathologically, it was composed of proliferating spindle and epithelioid cells with an interlacing bundle pattern. Immunohistochemically, the tumor was positive for myeloid stem cell antigen (CD34), weakly positive for c-KIT (CD117) and slightly positive for neuron-specific enolase (NSE), but negative for cytokeratin (CK), alpha-smooth muscle actin (SMA) and S-100 protein. A mutation was identified in the platelet-derived growth factor alpha (PDGFRA) juxtamembrane domain  and the tumor was diagnosed as an omental GIST. The postoperative course was uneventful. The patient is treated by Glevec561 substitution. Imatinib therapy as an adjuvant to complete resection has been carried out safely. Because of the rarity of primary omental GISTs, it is inevitable to analyze accumulating data from case reports for a better and more detailed understanding of primary omental GISTs.Our case demonstrated a weak immunohistochemical expression of c-kit (CD117) and a point mutation in PDGFRA exon 12 resulting in an Asp for Val GISTs are mesenchymal tumors, the majority of which is KIT CD117)-positive, typically arising in association with the muscularis propria of the gastrointestinal tract wall. Accordingly, they occur most frequently in the stomach (60%), jejunum and ileum (30%), and less frequently in the duodenum (5%), < 5% colorectal < 1% in the esophagus and appendix 17-positi. To the Here, we report a rare case of primary omental myxoid epithelioid GIST which we have characterized immunohistochemically and genetically. We review the English-language literature on primary omental GISTs.® 300 mg/day as an adjuvant postoperative molecular targeting chemotherapy and has been living disease-free for 6 months.A 65-year-old man was referred to our hospital in October 2006 with a huge abdominal tumor. A firm mass was palpated extending from the epigastrium to the left hypogastrium. There were no laboratory abnormalities, except a slight elevation of the total bilirubin 1.3 mg/dl) and lactate dehydrogenase (LDH: 217 IU/L) levels in the serum. The tumor markers carcinoembryonic antigen (CEA) and carbohydrate antigen (CA19-9) were within the normal range. Ultrasonography showed that the mass occupied almost the entire upper abdomen anterior to the bowel loops. On computed tomography (CT), a mass behind the left hepatic lobe showed heterogeneous low density with faint enhancement  [Because of possible relapse even after complete resection of omental GISTs -16 and tmerican) ,16. At t561 substitution. Because of the rarity of primary omental GISTs, it is difficult to assess their malignant potential and their overall prognosis. Imatinib therapy as an adjuvant to complete resection has been carried out safely and may prevent relapse to prolong long-term survival. It is essential to analyze accumulating data from case reports for a better and more detailed understanding of primary omental GISTs.Our case demonstrated a weak immunohistochemical expression of c-kit (CD117) and a point mutation in PDGFRA exon 12 resulting in an Asp for ValThe author(s) declare that they have no competing interests.TS, SS and AS had contributed as molecular tumor pathologist. TS, KF, SY, RM and TM contributed as a gastrointestinal surgeon in operative performance and postoperative treatment. NH and YT contributed as gastroenterologist and diagnostic radiologist.All authors read and approved the final manuscript.


Late relapse of a testicular germ cell tumour is an uncommon occurrence. We report a case of osteolytic bone metastasis appearing 23 years after the initial treatment of a metastatic testicular mixed tumour . This is one of the longest periods of recurrence reported for testicular germ cell tumours.A 52-year-old Caucasian man who underwent a right inguinal orchiectomy due to testicular tumour in 1984 presented to our outpatient clinic in a generally bad condition of health and with severe pain of his right hip joint and os ischii caused by osteolytic metastasis.This case emphasizes the need for a life-long follow-up of patients with primary metastatic testicular cancer. The prognosis of malignant testicular tumour has dramatically improved due to cisplatin-based chemotherapy. When diagnosed at an early stage, the disease has a cure rate of 95% to 100%. In advanced stages, cure rates of up to 70% are still possible . HoweverThe most complete report of late relapse was published by Baniel et al. . The autWe report here a case of a late relapse of a germ cell tumour, which occurred 23 years after orchiectomy and following four cycles of chemotherapy and five years of complete remission.A 52-year-old man presented to our outpatient clinic in a bad general condition of health and with severe pain of his right hip joint and os oschii. The pain developed approximately three months prior to presentation.The patient's medical history revealed a right inguinal orchiectomy for the removal of testicular tumour performed in France in 1984. The rest of his anamnesis was clinically insignificant.In 1984, the patient's histopathology revealed a mixed tumour, a choriocarcinoma and an embryonic carcinoma, with retroperitoneal and pulmonary metastases. To treat this, an orchiectomy was performed, followed by four cycles of bleomycin, etoposide and cisplatin (BEP) chemotherapy. Follow-up assessments were initially performed at an interval of three months, and then at an interval of six months until the fifth postoperative year.When the patient presented to our clinic, he appeared to be in bad general health, with severe pain in his right hip joint. His serum level of alpha-fetoprotein was 169.48 ng/ml  and beta-human chorionic gonadotropin (ß-hCG) was 1.30 mIU/ml . The patient's lactate dehydrogenase (LDH) level was 1155 U/L  and his alkaline phosphatase level was 104 U/L .Radiological investigation, which was later confirmed through computer tomograms, demonstrated that there was an osteolytic destruction of the patient's os ischii and hip joint Figure . In addiA biopsy of the lytic bone lesion was carried out to confirm the diagnosis. Histological and immunohistochemical investigation of the biopsy cylinders showed an anaplastic seminoma Figure . ImmunohDue to strong pain, analgesia with peripheral and central analgesics was initiated. During the next three days, however, the state of our patient dramatically declined and he died a day before the results of the biopsy were known.After initial therapy, patients with germ cell tumours who have two years of complete remission have a high cure rate. The probability of a late relapse is between 1.3% and 7% -6]. L. L6]. LaLate relapse is defined as a germ cell tumour that appears more than two years after therapy and complete remission . Of the A series of 1263 patients with germ cell testicular tumour treated at the Department of Radiotherapy and Oncology in Surrey, United Kingdom showed that only 14 patients had late relapse between the 5th and 10th year after primary treatment with a calculated annual risk of 1%. In two patients, late relapse occurred later than 10 years. One of these patients presented with metastatic seminoma, while the other presented with nonseminoma during the clinical stage I, which was then followed up by a wait-and-see attitude .The possible mechanisms for the development of late relapses are identified as: 1) the presence of mature teratoma in the germ cell tumour; 2) the growth of the remaining tumour not destroyed by the chemotherapy; and 3) the development of a secondary germ cell carcinoma and the microscopic persistence of tumour cells with atypical biological behaviour .Albers et al. wrote the European Association of Urology (EAU) guidelines on testicular cancer in a limited update in March 2009. Precise recommendations for minimum follow-up schedules in advanced nonseminomatous germ cell tumours and seminoma were provided, indicating the procedures that should be performed at one, two and three to five years after initial therapy and thereafter [Late relapses respond weakly to chemotherapy. Surgical excision remains the best treatment. Baniel et al. showed that chemotherapy alone yields poor results in patients with late relapse . Tumour-Late relapse of malignant germ cell tumours are a rare occurrence. Until recently, it was generally considered sufficient to perform follow-up assessments for up to five years after initial therapy. However, metastatic nonseminomatous tumours form an exception to this general rule, and late tumour relapses do occur. It thus seems necessary that follow-up examinations be performed for longer than five years after the primary therapy. This regime is recommended in the latest 2009 update of the EAU guidelines on testicular cancer as written by Albers and his co-authors.AFP: alpha-fetoprotein; BEP: bleomycin, etoposide and cisplatin; CEA: carcinoembryonic antigen; EAU: European Association of Urology; hCG: Human chorionic gonadotropin; LDH: lactate dehydrogenase.We were unable to obtain consent for the publication of this case due to the death of the patient and despite repeated attempts we were unable to trace the next of kin. Every effort has been made to keep the patient's identity anonymous. We would not expect the patient's family to object to publication.The authors declare that they have no competing interests.GS and TG performed the bone biopsy. SE performed the histological examination. BA was a major contributor in writing the manuscript. All authors read and approved the final manuscript.


Testicular cancer is the most common cancer for males aged 15~35 years old. The initial presentation is typically an asymptomatic enlarged testicle. The retroperitoneum is the most common metastatic area. Other metastatic sites include the lung, liver, brain, adrenal glands, gastrointestinal tract and spleen. Skin metastasis is a rare event and frequently associated with poor prognosis.A 19-year old male was diagnosed testicular mixed germ cell tumor with initial presentation of cutaneous metastasis at scalp and upper abdomen. After radical orchiectomy and four courses of cisplatin-based chemotherapy, the scalp and upper abdominal lesions regressed completely. The size of lung metastases remained unchanged.For advanced stage testicular cancer, cisplatin-based chemotherapy is still effective to achieve partial response. Cutaneous manifestation of an internal malignancy is rare, with an incidence of 2.9-9%,2. The fA 19-year-old male was in good health conditions before admission. He suffered progressively enlarging upper abdominal skin lesions and scalp nodules for 3 months fig. . These nCutaneous metastases originating from a solid organ are roughly 2% . Most ofTesticular mixed germ cell tumors are common, comprising roughly 33% of all testicular tumors. The reason for such a high incidence of testicular mixed germ-cell tumors is because germ cells in the testes are totipotent and can undergo either trophoblast or somatic differentiation. Among all the subtypes of testicular germ cell tumor, choriocarcinoma is the most aggressive with highly metastatic potential .The cutaneous metastasis as the first sign of metastatic choriocarcinoma could have been either an occult or a slow growing primary testis germ cell tumor -15. CutaAccording to the International Germ Cell Consensus Prognosis for Testicular Cancer, the 5-yThe skin is an uncommon site for testicular germ cell metastasis. A cutaneous lesion can be difficult to be differentiated from a primary cutaneous neoplasm. Excisional biopsy is required for definite diagnosis. For advanced stage testicular cancer, cisplatin-based chemotherapy is still effective to achieve biochemical remission.The authors declare that they have no competing interests with people or organizations in preparation of this study.Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.KLC was the first author, responsible for the conception and design for the manuscript, the clinical work, the search for the literature, and the editing work.SHU was responsible for the histopathological work. STP, HCC, YHC and CCL helped in the clinical work as well as the literature review. STP and SKL were responsible for editorship of the manuscript. CKC is the head of the department who supervised all the steps of the work.


A burned-out seminoma of the testis is an exceptionally rare clinical entity, with few reports found in the literature.A case of burned-out tumor of the testis in a 31-year-old man is reported. The tumor presented as a retroperitoneal mass with histological characteristic of a seminoma. The testes on clinical examination were normal, and a suspicious lesion in the scrotum was only identified after ultrasound. Incision of the abdominal mass was decided, followed by orchectomy. Histological examination of the testis revealed a suspicious lesion with characteristics of spontaneous regression of germ cell tumors.We describe one of very few cases worldwide, where spontaneous regression of a primary testicular tumor occurred after demonstration of retroperitoneal lymph node metastasis, a phenomenon known as burned-out seminoma, which is hard to recognize and incompletely characterized by physicians. Seminoma is the most frequent carcinoma of the testicle in the fourth decade of life and constitutes 60% to 65% of germ cell neoplasias. Several histopathologic characteristics of the tumor have been evaluated and three types of pure seminoma have been described as follows: a) classic, b) anaplastic and c) spermatocytic.Internationally, three clinical stages for the determination of the extension of the tumor are admissible. Stage I is where the tumor is limited to the testis with or without invasion of epididymis or the spermatic cord. In Stage II the tumor has retroperitoneal lymph node metastases. Finally, in Stage III the tumor has distant metastases.The germ cell tumor often gives lymph node metastasis, except from choriocarcinoma, which in an aggressive fashion is characterized by early hematogenous spread. We describe one of the very few cases worldwide where the spontaneous regression of a primary testicular tumor occurred after demonstration of retroperitoneal lymph node metastasis, a phenomenon known as burned-out seminoma.A 31-year-old man presented with symptoms of incomplete ileus . The patient had no relevant past medical history. Abdominal CT scan showed a retroperitoneal mass of 7 × 7 × 5 cm extending along the left side of the aorta, compressing but not completely occluding the left renal vein (Figure The patient had extensive resection of the retroperitoneal mass and lymphadenectomy. Histological examination showed a mass with a thin peripheral necrotic zone and associated dystrophic calcifications and persistent fibrous and inflammatory areas. The neoplastic cells had lymphoplasmacytic and eosinophilic infiltration, which varied in intensity, and numerous nuclear mitotic actions Figure . ImmunohHistological examination of the testis after radical left orchidectomy showed a well-demarcated nodular scar. Many tubules had a Sertoli cell only pattern and others were completely hyalinized. Increased vascularity was manifested by a collection of small, atrophic, non-arborizing vessels. Testicular atrophy was demonstrated by shrunken tubules with decreased or absent spermatogenesis and thickened peritubular basement membranes peripheral to the scar Figures  and 5. TThe phenomenon of spontaneous regression of cancer has not been fully elucidated; it may possibly be defined as partial or complete disappearance of the tumor with no therapy and has been described in renal tumours, breast carcinoma, lymphoma and malignant melanoma .Burned-out seminoma is the spontaneous regression of a testicular germ cell tumor with or without metastasis. In our case report, the primary testicular tumor with histological characteristics of a seminoma regressed after the development of metastasis in the retroperitoneum. Several immunological and ischemic mechanisms have been suggested to explain this spontaneous regression , but immIn patients who present with a retroperitoneal mass, diagnosis of metastatic progression of a germ cell neoplasia should be considered. A burned-out testicular tumor shows a distinctive constellation of findings that usually permits its recognition, which is significant because pure seminomatous germ cell tumors have a long term chance of cure, irrespective of the primary tumor site.CT: computed tomography; α-FP: α-fetoprotein; β-hCG: β human chorionic gonadotrophin; LDH: lactate dehydrogenase; PLAP: placental alkaline phosphatase; SUS: scrotal ultrasonography.Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in Chief of this journal.The authors declare that they have no competing interests.All authors contributed to conceptualizing and writing this case report.


Germ-cell tumors are a high-proliferative type of cancer that may evolve to significant bulky disease. Tumor lysis syndrome is rarely reported in this setting. The reports of three patients with germ-cell tumors who developed severe acute tumor lysis syndrome following the start of their anticancer therapy are presented. All patients developed renal dysfunction and multiorgan failure. Patients with extensive germ-cell tumors should be kept on close clinical and laboratory monitoring. Physicians should be aware of this uncommon but severe complication and consider early admission to the intensive care unit for the institution of measures to prevent acute renal failure. Acute tumor lysis syndrome (ATLS) is a clinical condition that results from the massive destruction and lysis of malignant cells, and subsequent release of intracellular ions and metabolites into the bloodstream leading to hyperkalemia, hyperphosphatemia, hyperuricemia, and hypocalcemia. ATLS is accompanied by renal failure and metabolic acidosis thus increasing the risk of death. The syndrome is usually a consequence of the treatment of high-grade lymphoproliferative malignancies. It is rarely observed in patients with solid tumors and even less frequent is its spontaneous presentation.–3 Germ-cA 41-year-old male presented with left testicular mass, weight loss, dry cough followed by hemoptysis and progressive dyspnea. Computed tomography (CT) scans showed a large retroperitoneal mass and metastases to the lungs, liver, and spleen. There were high levels of human chorionic gonadotropin (HCG) (317.718 mU/ml) and of α-fetoprotein (93 ng/ml). The patient underwent a radical left orchiectomy and the hystopathological examination showed a mixed germ-cell tumor . On the first day of chemotherapy with bleomycin, etoposide, and cisplatin, he developed massive hemoptisys, acute respiratory failure, and cardiopulmonary arrest. Cardiopulmonary resuscitation was successfully performed. On day 2, he evolved with metabolic acidosis, hyperphosphatemia (8.8 mg/dl), hyperuricemia (9.4 mg/dl), hypocalcemia (6.1 mg/dl), and hyperkalemia (6.7 mEq/l). Chemotherapy was interrupted and hemodyalisis was started. However, the patient died on day 7 due to multiple organ failure.A 39-year-old male presented with weight loss, progressive dyspnea, and right testicular enlargement. CT scan showed large mediastinal, intraperitoneal, and retroperitoneal masses , 2. SeruA 30-year-old male presented with back pain, bilateral leg edema, and dyspnea because of a large retroperitoneal mass and multiple pulmonary nodules. Ultrasound examination disclosed hydronephrosis of the right kidney and deep vein thrombosis. There was no palpable testicular mass on physical examination, but a right testicular nodule was disclosed by ultrasound examination. Tumor markers showed a serum α-fetoprotein of 19.078 U/l, HCG of 5.4 U/l, and LDH of 7.150 U/l. The patient underwent right radical orchiectomy and nonseminomatous tumor was diagnosed. Etoposide and carboplatin were started. However, on day 1 of chemotherapy, he developed acute respiratory failure requiring mechanical ventilation and was transferred to the ICU. Vigorous hydration and alopurinol were started concerning the risk of tumor lysis syndrome. Nonetheless, the patient developed low urine output, metabolic acidosis, azotemia, hyperphosphatemia, hyperuricemia, hyperkalemia, and hemodynamic instability. Chemotherapy was stopped and broad-spectrum antibiotics and hemodyalisis were started. On day 6, he presented with progressive pancytopenia requiring the use of filgrastim. The patient evolved with progressive improvement of his clinical condition, mechanical ventilation was discontinued on day 22, and he was discharged to the wards on day 24. In the wards, his renal function improved allowing to the discontinuation of hemodyalisis. The patient went home on day 42 with normal renal function and lower tumor markers levels . He received three additional courses of bleomycin, etoposide, and carboplatin with no serious side effects. Two months later, he underwent an exploratory laparotomy and there was no evidence of retroperitoneal masses. However, there was disease progression characterized by the development of recurrent metastases in the central nervous system refractory to surgery, chemotherapy, and radiation therapy. The patient died 20 months after index hospital admission.The reports of three patients with extensive disseminated germ-cell tumors who developed severe ATLS following the start of chemotherapy were presented. ATLS has been occasionally described in patients with solid tumors such as melanoma, medulloblastoma, small-cell lung cancer, neuroblastoma, and germ-cell tumors.–5 The ob


A huge retroperitoneal tumor with a right orbital mass was detected and proved to be an extragonadal nonseminomatous germ cell tumor on biopsy. BEP chemotherapy caused some regression in orbital mass however no change in retroperitoneal tumor size as well as serum tumor marker levels occurred. Herein, we present a rarely seen entity of extragonadal retroperitoneal nonseminomatous germ cell tumor with synchronous orbital metastases and discuss its diagnosis and management. Extragonadal germ cell tumors (EGGCT) accountfor approximately 2–5% of all germcell tumors. They are mostly seen in the mediastinum and retroperitoneum. However,it can also be seen in less frequent locations such as sacrococygeal region,pineal region, prostate, orbita, liver, vagina, and gastrointestinal tract , 2.These lesions may grow up to extensivedimensions without any significant symptoms. The diagnosis is usually made in thethird decade. Advanced local disease and distant metastases might exist at thetime of diagnosis in the majority of patients .β HCG) level (37.8 mIU/mL) and an increased lactate dehydrogenase (LDH)level (800 IU/L). Serum alpha fetoprotein (AFP) level was within normal limits.A 31-year-old man complaining of bilaterallumber pain, fatigue was diagnosed with right hydronephrosis on ultrasonography (US), and a double J stent was placed into the right ureter at another hospital amonth ago. He then was referred to our institution for further evaluation. Hispast medical history revealed bilateral nephrolithotomy in 1998 and rightnephrolithotomy in 1999. Physical examination of urogenital system was withinnormal limits including the scrotal examination. On admission, there was proptosis(eccentric) present displacing his right eye infero-nasally. His blood hemoglobulinand leukocyte counts were 7.81 g/dL and 21 500, respectively. Blood chemistrywas within normal limits except elevated urea (49 mg/dL) and creatinine (2.1 mg/dL) levels. Serum tumor marker studies revealed an increased beta-humanchorionic gonadotropin  germ cell tumor (EGGCT) with orbital metastasis. Chemotherapy including cisplatin, etoposide, and bleomycine (BEP) wasadministered. After completion of first course chemotherapy, symptoms subsidedwith a significant reduction in proptosis. However, serum tumor marker levelsdid not change. Afterwards, a very rapid progression of the disease has occurredfollowing initial regression of his symptoms. Dimensions of orbital lesion increasedsignificantly with concomitant increase in serum tumor marker levels including AFPand EGGCTs are rarely seen tumors with specificbiological and clinical characteristics. Symptoms depend on the location of thetumor such as presence of a palpable mass, abdominal or back pain, dysphagia, andedema in the limbs when the tumor is located in the retroperitoneum. Constitutionalsymptoms such as fever and weight loss might accompany the disease. Thediagnosis is made histopathologically when seminomatous and nonseminomatouselements of the tumor are seen on biopsy. Tumor markers are also expected toincrease if nonseminomatous elements are present , 2.In our patient, main symptoms were abdominaland low back pain. Immunohistochemical evaluation of the biopsy for AFP and HCGstaining were positive therefore, the patient was diagnosed as having extragonadalnonseminomatous germ cell tumor.Metastases to other tissues depend on thelocalization and histological type of the primary tumor . Lung meScrotal US could easily differentiate aretroperitoneal EGGCT from primary testicular tumor metastases without routinelyperforming testicular biopsy for differential diagnosis .The prognosis is excellent in cases withseminomatous histology regardless of the localization of the EGGCT either in mediastinumor in retroperitoneum. However, prognosis of nonseminomatous EGGCT is worsethan that of the seminomatous variant. Five-year-survival rates have beenreported to be 45% for mediastinal and 62% for retroperitoneal nonseminomatoustumors [Management of patients with EGGCT is accomplishedaccording to the prognostic classification of International Germ Cell Cancer Collaboration Group . StandarSecondary surgery is an integral part of thetreatment strategy in patients with EGGCT and is mandatory in cases withresidual mass, which is true for about 50% of the patient population .We intended to treat our patient with 4 coursesof BEP chemotherapy; however, we were able to administer only two courses. Unfortunately,we lost the patient despite partial response with some regression in thediameter of the orbital mass without any change in serum tumor marker levels.In our case, a right orbital mass accompanyingthe retroperitoneal tumor which we think was metastasis rather than anotherprimary focus regressed after the administration of first course of BEPchemotherapy. Because the volume of the orbital tumor mass was smaller than thevolume of the retroperitoneal tumor, we think that orbital tumor regressed tosome degree while the retroperitoneal mass remained unchanged without anychange in serum tumor marker levels.


We report a case of primary mediastinal seminoma, which presented initially with shortness of breath and a swelling in upper part of anterior chest wall. The diagnosis of primary mediastinal seminoma was established on the basis of histologic findings and was confirmed by immunohistochemical analysis. Abdominal, pelvis and cerebral CT scan, testicular ultrasound and TC-99 MDP bone scintigraphy were negative. Chemotherapy was initiated with B.E.P. protocol ; the patient received four cycles of chemotherapy. After 8 months, the patient was seen in the clinic; he was well. Primary seminomas of the mediastinum are unusual neoplasms that are morphologically indistinguishable from their gonadal counterparts but may have different biologic behavior because they arise at this particular location. The anterior mediastinum is the most common primary extragonadal site for seminoma. There are very few published reports of mediastinal seminomas located in middle and posterior mediastinum.The case presented in this report highlights the importance of considering primary mediastinal seminoma in the differential diagnosis of anterior, middle and posterior mediastinal mass even if no abnormality of the testicle is observed.A 32-year-old male patient was admitted to the hospital with a 6-month history of retrosternal chest pain which was described as compressive in character, localized, associated with mild shortness of breath and painful swelling in upper part of anterior chest wall. On direct questioning, he mentioned weight loss. Other medical history was unremarkable. On examination, the only abnormal physical sign was the presence of fixed tender swelling over manubrium sterni (2 × 3 cm).Initial investigations revealed WBC 6,400/μL, hemoglobin 13.2 g/dl, platelets 324,000/μL, erythrocyte sedimentation rate 66 mm/hour. Blood chemistry, liver profile, arterial blood gas (ABG) analysis at room air and coagulations studies were within normal limits. A tuberculin skin test was negative. Chest X ray showed large mediastinal mass  and CT cFine needle aspiration of mediastinal mass was done and cytological diagnosis was noninformative. Bone marrow aspiration and trephine biopsy showed normal study. CT-guided biopsy was done, but unfortunately the specimen was crushed and full with artifacts. Open biopsy for mediastinal mass done by cardiothoracic surgeon and histological examination of the tissue obtained showed a malignant tumor that had two cell populations; one consisted of lobules, sheets, trabeculae and clusters of large polygonal cells with nuclei having vesicular chromatin, prominent nucleoli and several mitotic figures. The cytoplasm of those cells was moderately abundant and clear. The other cell population was clusters of small mature lymphocytes, which were scattered within the fibrous septa separating the lobules of the large polygonal tumor cells . ImmunopAbdominal, pelvis and cerebral CT scan; testicular ultrasound and TC-99 MDP bone scintigraphy were negative. Tumor markers: α fetoprotein was 2.3 IU/ml  and β-HCG was < 5 IU/L .Chemotherapy was initiated with B.E.P. protocol ; the patient received four cycles of the drugs. Two weeks after the first cycle, tumor size was reduced markedly. After 8 months, the patient was seen in the clinic; he was well.Germ cell tumors occur most commonly in gonads but infrequently appear in other locations such as mediastinum, retroperitoneum, pineal gland and sacral area; 2-5% of germ cell tumors are of extragonadal origin. It has b2The primary mediastinal seminoma has an estimated incidence of 25% of that of primary mediastinal germ cell tumors and is usually located in the anterior mediastinum,5 with th68The origin of extragonadal germ cell tumors (EGCTs) is still uncertain. The cytogenetic findings on chromosome 12q appear to suggest that extragonadal tumors would be of gonadal origin.Primary seminomas of the mediastinum are unusual neoplasms that are morphologically indistinguishable from their gonadal counterparts but may have different biologic behavior because they arise at this particular location.Histopathologically, germ cell tumors are classified as follows: teratomas (including mature and immature groups), germinomas (seminomas), embryonal carcinoma, choriocarcinoma, yolk sac tumors and mixed germ cell tumors. AlthoughExtragonadal germ cell tumors grow slowly and initially produce few symptoms . Around 30% of patients with mediastinal seminoma were asymptomatic and the mass was discovered incidentally during routine chest X ray.According to the results from an international analysis with 635 cases with extragonadal germ cell tumors, 83% (524) were nonseminomatous and 16% (104) seminomatous germ cell tumors.14In this patient the differential diagnosis of mediastinal seminoma includes teratoma, primary large B cell lymphoma and thymoma. Some features commonly associated with seminoma, such as epitheloid granuloma, cystic changes and lymphocytic infiltration, were not prominent, but the application of immunohistochemical studies showed cytoplasmic staining with placental-like alkaline phosphatase. The application of immunohistochemical studies for placental-like alkaline phosphatase may be of discriminatory value in equivocal case.It is accepted that routine biopsy or orchidectomy is not needed in EGCTs if no evidence exists for retroperitoneal involvement and no abnormality of the testicle is observed.15In our patient a careful clinical and ultrasound examination showed normal testis. Abdominal and pelvis CT showed no evidence of retroperitoneal involvement while brain CT scan was normal.With regard to the treatment of mediastinal malignant GCTs, the current consensus is that initial systemic chemotherapy should be followed by aggressive complete resection of all macroscopic residual tumor when necessary.17Prognosis for patients with mediastinal seminoma is fairly good, with a 5-year survival rate of around 90%. Conversely, nonseminomas have a worse prognosis than seminoma, with a 5-year survival rate of around 45%.14 PatienThus, mediastinal seminoma should be considered in the differential diagnosis of middle, posterior and anterior superior mediastinal mass even in the absence of testicle lesions.


Patient.  A 65-year-old man was admitted with a large primary retroperitoneal            tumor and an increased β-human chorionic gonadotropin (β-HCG) serum level. A germ            cell tumor was suspected; however, a computed tomography-guided biopsy failed to enable            tumor classification. After two courses of chemotherapy, the β-HCG serum level had returned            to the normal level and a diagnostic laparotomy with incisional biopsy was performed.            The immunohistochemical examination of the specimen identified the tumor as a            retroperitoneal pleomorphic leiomyosarcoma.            Discussion. Tumor markers play only a marginal role in the work-up            of patients with soft tissue sarcomas. In men with suspected retroperitoneal sarcomas,            however, the determination of germ cell tumor markers occasionally enables a preoperative          distinguishing of primary retroperitoneal germ cell tumors with considerable consequences          for management. In this setting, a retroperitoneal tumor associated with a moderately          elevated β-HCG is a diagnostic dilemma, and surgeons should be aware of the pitfall          of a β-HCG-producing leiomyosarcoma in the differential diagnosis.


Candida species were the most frequent (isolated from 75% of participants), followed by Cladosporium (65%), Aureobasidium, Saccharomycetales (50% for both), Aspergillus (35%), Fusarium (30%), and Cryptococcus (20%). Four of these predominant genera are known to be pathogenic in humans. The low-abundance genera may represent environmental fungi present in the oral cavity and could simply be spores inhaled from the air or material ingested with food. Among the culturable genera, 61 were represented by one species each, while 13 genera comprised between 2 and 6 different species; the total number of species identified were 101. The number of species in the oral cavity of each individual ranged between 9 and 23. Principal component (PCO) analysis of the obtained data set followed by sample clustering and UniFrac analysis revealed that White males and Asian males clustered differently from each other, whereas both Asian and White females clustered together. This is the first study that identified the “basal mycobiome” of healthy individuals, and provides the basis for a detailed characterization of the oral mycobiome in health and disease.The oral microbiome–organisms residing in the oral cavity and their collective genome–are critical components of health and disease. The fungal component of the oral microbiota has not been characterized. In this study, we used a novel multitag pyrosequencing approach to characterize fungi present in the oral cavity of 20 healthy individuals, using the pan-fungal internal transcribed spacer (ITS) primers. Our results revealed the “basal” oral mycobiome profile of the enrolled individuals, and showed that across all the samples studied, the oral cavity contained 74 culturable and 11 non-culturable fungal genera. Among these genera, 39 were present in only one person, 16 genera were present in two participants, and 5 genera were present in three people, while 15 genera (including non-culturable organisms) were present in ≥4 (20%) participants.  Candida species were the most frequently obtained genera, isolated from 75% of all study participants, followed by Cladosporium (65%), Aureobasidium, Saccharomycetales (50% for both), Aspergillus (35%), Fusarium (30%), and Cryptococcus (20%). The remaining fungi detected in the oral wash samples represent organisms likely originating from the environment. This is the first study that identified the “basal mycobiome” of healthy individuals, and provides the basis for a detailed characterization of the oral mycobiome in health and disease.We characterized the fungal microbiome (mycobiome) of the oral cavity in healthy individuals. Our results demonstrate that the fungal component of the oral microbiome is diverse as revealed by the presence of 74 culturable and 11 non-culturable fungal genera in the oral cavity. A total of 101 species were identified, with between 9 and 23 culturable species present in each person. Fifteen genera (which included four known pathogenic fungi and non-culturable organisms) were present in ≥20% of the tested samples;  Candida, are important components of oral microbiota and are influenced by the immune status and therapy of affected individuals, studies of oral microbiota have focused largely on the bacterial components. In the only oral microbiome study to date that included some fungal profiling, Aas et al.Candida albicans and Saccharomyces cerevisiae in the subgingival plaque microbiota of HIV-infected patients. These investigators employed a PCR-based approach using the 18S rDNA primers  to characterize the fungi present in the plaques. The approach used by this group provided only a limited snap shot of the fungal members of the microbial biome.Organisms residing in the oral cavity and their collective genome–the oral microbiome–are critical components of health and disease. Disruption of the oral microbiome has been proposed to indicate, trigger, or influence the course of oral diseases, especially among immunocompromised patients (e.g. HIV-infected or cancer patients) To obtain a more comprehensive profile of the fungal microbiome (mycobiome), in this study we utilized a novel Multitag Pyrosequencing (MTPS) approach to interrogate the fungal taxa in the oral cavity using universal internal transcribed spacer (ITS) primers, which have broad fungal specificity Written informed consent was obtained from all participants in this study. Recruitment of study participants was performed according to protocol (number 20070413) approved by the Human Subjects Institutional Review Board (IRB) of Case Western Reserve University, Cleveland, Ohio.http://www.census.gov), in which race has been classified as White , Black/African-American, Asian, Native American/Native Alaskan etc. Inclusion criteria were: >18 years of age, non-smoking, no recent antifungal use, and no clinical signs of oral mucosal disease. Exclusion criteria were: (1) a history of receiving medication or treatment with topical or systemic steroids, pregnancy, and (2) insulin-dependent diabetes mellitus (IDDM).Oral rinse samples were obtained from 20 healthy individuals after informed consent and following review of the IRB at Case Western Reserve University/University Hospitals Case Medical Center. The individuals were all from the Cleveland area and on standard Western diets. Summary demographic information of the study participants is provided in Concentrated oral rinse has been previously used to detect the presence of oral bacteria and fungi CTTGGTCATTTAGAGGAAGTAA) and unlabeled reverse primer ITS2 (GCTGCGTTCTTCATCGATGC). The ITS primers were selected in this study to detect the presence of various fungi since these primers are able to detect consensus sequences present in a broad range of fungal organisms, and have been used for the detection of yeasts (including Candida spp.), moulds and dermatophytes 2, each dNTP at a concentration of 0.25 mM, each primer at a concentration of 0.5 µM, and 0.5 U of AmpliTaq Gold DNA polymerase . Initial denaturation at 94°C for 11 min was followed by 35 cycles of denaturation for 30s each at 94°C, annealing at 50°C for 30 s, and progressive extension at 72°C for 2 min. Following the 35 cycles there was a final extension time of 30 min to minimize artifacts induced by TAQ polymerase. Fungal PCR products were separated on the SCE 9610 capillary DNA sequencer  using GenoSpectrum software to convert fluorescent output into electropherograms. Relative peak abundance of fungal amplicons was calculated by dividing individual peak heights by the total peak heights in a given electropherogram using a custom PERL script. Interleaved, normalized abundances were compared as stacked histograms using Microsoft Excel. Mean normalized abundance for each amplicon was calculated from the three PCR replicates of each sample, excluding means below 1%. Results were analyzed by visual inspection and Principal Coordinate (PCO) analysis using Multivariate Statistical Package . Normalized abundance of each peak in the electropherogram was calculated with respect to the total peak area, since it is not possible to calculate absolute abundances with either the LH-PCR or MTPS technology.The first step in the mycobiome analysis was extraction of DNA from the cell pellet  followed by PCR analysis. Samples were extracted individually using the Fast DNA Spin Kit for fungi following manufacturer's instructions . Each extraction tube was agitated three times using a Fast Prep FP120 instrument at a speed setting of 5 for 30 s. Tubes were cooled on ice between agitations. The ITS1 region from DNA sample extracts was amplified in triplicate using primers with high specificity for ascomycete fungi  technique, which can be used for detailed characterization of nucleic acids and has the advantages of accuracy, flexibility, parallel processing, and easy automation potential We developed a custom PERL script to demultiplex the MTPS data by sorting the sequences into bins based on the barcodes and the taxa in the samples, automatically blasting the pyrosequence data against Genbank . The annotations for each sequence were downloaded and a PERL script used to tabulate the taxa as a percentage of the total oral community in each sample. Fungal ITS sequences were compared with the Assembling Fungal Tree of Life (AFTOL) database using the BLAST interface of Web Accessible Sequence Analysis for Biological Inference (WASABI) as well as against the NCBI nucleotide database.Principal Coordinate (PCO) analysis has been recognized as a simple and straight-forward method to group or separate samples in a dataset, and has been used in disease-association studies http://bmf2.colorado.edu/unifrac/index.psp). The analysis was weighted using the abundance of the sequence in each cluster. The P-value generated from the UniFrac significance test describes the degree of similarity between classes. Values reported were Bonferroni corrected for the number of sequences used in comparisons.To compare the phylogenetic distribution between the various gender and race classes and confirm the results of PCO analysis, we used UniFrac significance test The demographic characteristics of the study participants were: 21–60 years of age, 8 females and 12 males, no history of smoking, insulin-dependent diabetes mellitus, or active medications; the self-reported ethnicities of study participants were White , Asian  , or African-American . All participants were from the metropolitan Cleveland Ohio area, and were Faculty, staff or students at Case Western Reserve University . Study pCandida (15/20), Cladosporium (13/20), Aureobasidium (10/20), and organisms belonging to the family Saccharomycetales (10/20). Interestingly, a large percentage (36.1%) of fungi belonged to non-culturable category. The minimum number of genera identified in a sample (sample E3) was 3 , while the maximum number of genera identified was 16 in sample A2, which included Candida (5.5%), Dothediomycete (10.7%), Fusarium (4.1%), Aspergillus (3.9%), and Xylariales (7.7%)  were longer than 100 bases and were used in the analysis. Our analyses revealed an average of 1,702 sequences per sample with an average length of 248 bases . A locals (7.7%) . The nonCandida species were the most frequently obtained genera, isolated from 75% of all study participants, followed by Cladosporium (65%), Aureobasidium and Saccharomycetales (50% for both). Other fungi that were present in the oral cavity of healthy individuals were Aspergillus (35%), Fusarium (30%), and Cryptococcus (20%). Fifty three percent (39/74) of the identified genera were observed only once in the tested samples.To determine the basal fungal distribution in healthy individuals, we identified fungi that were present in at least 20%  of the study participants. This analysis revealed that 15 genera were present in ≥20% of the tested samples , followed by Candida (5 species), Cladosporium (4 species), Fusarium (3 species), and Penicillium (3 species) , while the non-albicans Candida species indentified were: C. parapsilosis (15%), C. tropicalis (15%), C. khmerensis and C. metapsilosis (in 5% of the subjects). Two species each of Alternaria, Cryptococcus, Ophiostoma, Glomus, Phoma, Schizosaccharomyces, and Zygosaccharmoyces were identified in the participants.Analysis of the species distribution of the oral mycobiome revealed that 12 fungi were represented by two or more species in the oral rinse samples  . CandidaTo investigate whether there is an association between any of the subject demographics and changes in mycobiome, we performed PCO analysis and sample clustering followed by UniFrac analysis. Our analysis revealed that White and Asian males clustered differently from each other, whereas both Asian and White females clustered together . UniFracIn the current study, we demonstrated the presence of 74 culturable and 11 non-culturable fungal genera in the oral cavity of healthy individuals, with between 9 and 23 culturable species present in each person, representing a total of 101 species for all study participants.Candida; rather it is represented by a large number of diverse fungi. The perception that fungi in the oral cavity are limited to only few species originated from previous studies that relied upon the use of culture-based methods or species-specific targeted PCR approach. In addition to Candida, other fungi previously reported in the oral cavity include S. cerevisiae, Penicillium, Geotrichum, Aspergillus, Scopulariopsis, Hemispora, and HormodendrumOur results demonstrate that the fungal component of the oral microbiome is not limited to a few species, principally et al.Only two previous studies have investigated the profile of microbes present in the oral cavity of healthy individuals and both focused on the bacterial microbiome et al.S. cerevisiae in 4 and C. albicans in 2 patients. No other fungal species were detected in analysis of 306 18S rDNA clones. In contrast, we found that the oral cavity of healthy individuals had 101 fungal species. The reason for this difference could be attributed to differences in: (a) sampling method – oral rinse versus sub-gingival plaque; oral rinse enables the collection of organisms from the dorsum of the tongue as well as from the ever changing oral mucosal environment as compared to the sub gingival biofilm plaque, (b) detection probe – 18S rDNA probes that detected Candida and eight other genera, versus the pan-fungal ITS1/ITS2 probe that could identify all fungi, and (c) sequencing technique – cloning of rDNA fragments followed by sequencing, versus real-time pyrosequencing.While the fungal component of the oral cavity has not been investigated in healthy individuals, a previous study identified fungi present in the oral cavity of HIV-infected patients et al.We also found that the distribution of fungal species varied greatly between different individuals. Similar variation was recently reported for bacterial microbiota by Nasidje Candida (present in 75% of the cohort), Aspergillus (35%), Fusarium (30%), and Cryptococcus (20%). While the abundance of Candida in these healthy individuals was not surprising, the actual percentage was higher than reported in earlier culture-based studies, where 40 to 50% of healthy individuals have been shown to contain Candida species in their oral cavity Candida detected in this study could be attributed to the use of the more sensitive ITS/pyrosequencing approach. Another interesting finding was the different types Candida species identified in the oral cavity of healthy individuals. The most abundant Candida species in this study was found to be albicans (in 40% of the subjects), followed by C. parapsilosis (15%), C. tropicalis (15%), C. khmerensis (5%) and C. metapsilosis (5%). These results are in agreement with those reported by earlier studies using culture-based as well as PCR-based analyses The oral mycobiome of at least 20% of the enrolled individuals included the four most common pathogenic fungi – Aspergillus, Fusarium, and Cryptococcus isolates in the oral cavity of healthy individuals was unexpected, since these fungi have not been reported to be colonizers of the oral cavity. It is possible that the pathogenicity of these fungi is controlled in healthy individuals by other fungi in the oral mycobiome, as well as a functional immune system. It is possible that inter-dependent relationships may exist between components of the oral mycobiome, and need to be investigated using broader sampling and longitudinal studies.The presence of In our studies, we also identified 60 fungal genera that are ubiquitous in the environment  and not normally associated with infections. Among these genera, 39 genera occurred once among the 20 samples analyzed, 16 genera occurred with a frequency of 2, while 5 genera occurred in three individuals. Due to their ubiquitous nature, the presence of these organisms in the oral cavities of healthy individuals was not surprising, which are most likely of environmental origin, from food and mouth breathing.et al.In this study, non-culturable fungi represented a large percentage (36.1%) of the organisms identified in the oral mycobiome of healthy individuals. This is the first study demonstrating the presence of non-culturable fungal organisms in the oral cavity, which may play important role in the oral milieu. The presence of non-culturable organisms has been reported for bacterial species in the oral cavity; about half the population of oral bacteria has been reported to be non-culturable. For example, Aas http://nihroadmap.nih.gov/hmp/)”. Results from our study provides critical information that is likely to form the basis of further “hypothesis-driven” studies evaluating the oral mycobiome in terms of individual variabilities, longitudinal trends, and the effect of diet and geography, and studies focused on determining the association of oral mycobiota with health and disease.Ours is the first study that provides a snapshot of the oral mycobiome in various individuals, and addresses an important component of the Human Microbiome Project (HMP). Since this is the first study that identifies the oral mycobiota, our findings complement the HMP  and support its stated goal of “generating resources enabling comprehensive characterization of the human microbiota and analysis of its role in human health and disease  in an individual could be the first step in predisposing the host to opportunistic infections. In this regard, oral Candida colonization has been known to be a risk factor for Candida infections in immunocompromised patients The clinical relevance for the presence of a diverse population of fungal species in the oral cavity is unknown. It is possible that the presence of a given fungal isolate (e.g. Table S1Sequence data(1.15 MB ZIP)Click here for additional data file.Table S2Frequency distribution of fungi belonging to different genera present with at least one percent abundance in the oral mycobiome of healthy individuals(0.05 MB XLS)Click here for additional data file.Table S3Species distribution of fungi present in oral cavity of healthy individuals(0.05 MB XLS)Click here for additional data file.


Environ Health Perspect 122:100–106 (2014)], there was an error in Table 1. The number of women in exposure quartile 4 who had missing data for maternal smoking was given as zero instead of 3. The corrected table appears below and has also been corrected online.In the article “Exposure to Brominated Trihalomethanes in Water During Pregnancy and Micronuclei Frequency in Maternal and Cord Blood Lymphocytes” by Stayner et al. [EHP regrets the error.


Jasus edwardsii, shows clear phenotypic differences between shallow water (red coloured) and deeper water  individuals. Translocations of individuals from deeper water to shallower waters are currently being trialled as a management strategy to facilitate a phenotypic change from lower value pale colouration, common in deeper waters, to the higher value red colouration found in shallow waters. Although panmixia across the J. edwardsii range has been long assumed, it is critical to assess the genetic variability of the species to ensure that the level of population connectivity is appropriately understood and translocations do not have unintended consequences. Eight microsatellite loci were used to investigate genetic differentiation between six sites  across southern Tasmania, Australia, and one from New Zealand. Based on analyses the assumption of panmixia was rejected, revealing small levels of genetic differentiation across southern Tasmania, significant levels of differentiation between Tasmania and New Zealand, and high levels of asymmetric gene flow in an easterly direction from Tasmania into New Zealand. These results suggest that translocation among Tasmanian populations are not likely to be problematic, however, a re-consideration of panmictic stock structure for this species is necessary.The southern rock lobster,  Human-mediated movement of species, known as translocation or assisted migration, is increasing in popularity as a strategy to maintain species abundance, connectivity and diversity. Translocation has been used commonly throughout agricultural history, and it is currently also an important conservation strategy for threatened species ,2. SucceJasus edwardsii), to determine if it was possible to improve value and productivity of the Australian stock [Pilot translocations were trialled in the southern rock lobster  Iacchei et al [P. interruptus also possesses a long larval duration (240-330 days) [Phenotypic differences between shallow and deep populations of the southern rock lobster are due to differences in habitat, and are not genetic, as pilot studies have shown that translocated, pale individuals change to the more desired phenotype after a single moult ,8. Althoei et al  show sli30 days) . Indeed 30 days) -14.Like many marine species with long larval phases, the southern rock lobster has long been assumed to be panmictic throughout the range of Australasia -17. KnowThis study aims to use microsatellite markers to investigate genetic differentiation among Tasmanian populations of the southern rock lobster, where translocations are under consideration as an ongoing management strategy. Analysis of genetic structure is evaluated at different levels, including between 1) shallow and deep water populations, 2) fine scale geographic separation of Tasmanian populations and 3) the oceanic divide of Tasmania and a New Zealand site. This will help to determine if current translocation efforts stand to negatively impact the southern rock lobster, and if there is significance in the scale and directionality of connectivity for this species. Potential patterns in connectivity and source-sink recruitment relationships may be important in the appropriate management and success of translocation for this species in the future.Lobsters were sampled from six sites across the southern coast of Tasmania, and one site from New Zealand (NZ) . All hanBaited traps deployed and collected from research and commercial vessels were used to catch lobsters. All handling of Tasmanian rock lobsters in this study met the Australian Government National Health and Medical Research Council code of practice for the care and use of animals for scientific purposes. Although currently ethics approval is not required for research on invertebrates under this code of practice, the guidelines for ethical and humane treatment of animals in research were followed in all handling of lobsters. Sampling of rock lobster did not involve interactions with endangered or threatened species. Three shallow water sites sampled , were between 0 and 30 metres water depth, and comprised of lobsters with red coloured phenotypes. Three deep water sites sampled  were greater than 60 metres in depth, and were largely populated by pale coloured lobsters. Distances between sample sites (by sea) range from 10 km (between EP and MBI) to 220 km apart (between HI and TAR). We considered the effects of geographic and oceanic distance, between shallow and deep populations and any potentially resulting genetic patterns on subsequent translocations within the stock.Samples of rock lobster in Taroona reserve were collected on 1-4 February 2012 . A clip of tissue from the pleopod was stored in 95% ethanol, and the lobster released. Pleopod tissue samples from other Tasmanian sites were collected from 15 January to 15 February 2012. In addition, tissue samples were taken from lobsters collected from Taieri Mouth, Otago Harbour and Moeraki on the south island of New Zealand  during AJ. edwardsii  for capillary separation. Results were carefully scrutinised by eye using GENEIOUS PRO version 5.6.4 , using tBinned genotypes scored were formatted in GENALEX version 6.4 . MICRO-CPairwise F-statistics (Fst's) were calculated in FSTAT between assigned groups of individuals. Fst's were tested by hierarchical comparisons between: 1) all populations, 2) shallow water and deep water groups and 3) paired groups of Tasmania and New Zealand.STRUCTURE version 2.3.4 , was useBAYESASS version 3.0.1  was usedhttp://doi.org/10.5061/dryad.656gf). A total of 460 individuals were genotyped for eight microsatellite loci. Despite numerous attempts to optimise PCR conditions for all nine microsatellite loci from Thomas and Bell , locus JA significant frequency of null alleles were detected from loci JE_01, JE_LZ, JE_17, JE_40 and JE_07 using MICRO-CHECKER , althougAllelic richness for each population was similar  . TAR, HIF-statistics were used to compare across 1) all populations, 2) solely between Tasmanian populations and 3) between red  and pale (deep) populations of Tasmania. After Bonferroni correction, the data set indicated a significant difference between NZ and the six Tasmanian populations, the largest oceanographic distance compared (Fst=0.0290-0.0342) . Fst anaWhen Tasmanian populations were combined and compared to the NZ population in a pairwise Fst test, analysis showed significant levels of differentiation (Fst=0.0305) over this large distance. When populations from Tasmania only were compared by grouping the three shallow populations and the three deep populations in a pairwise analysis, no significant difference was detected. Overall, F-statistics indicated a significant difference between Tasmanian and New Zealand individuals, and a small yet still significant level of differentiation among some populations of Tasmania, yet no consistent differences between shallow and deep populations.An analysis of clusters in STRUCTURE revealed no clear grouping of individuals sampled . GroupinDAPC was tested on all individuals with a best fit for clusters found at K = 4 . The majAnalysis using STRUCTURE and DAPC suggested some level of differentiation present between individuals from Tasmania compared to New Zealand, however, no fine scale structuring was noted amongst populations of Tasmania, which suggested a high level of admixture between populations.Evaluation of migrants or admixture between populations was analysed using BAYESASS on hierarchical levels of 1) between all populations and 2) between combined populations of Tasmania and New Zealand. BAYESASS permits migration rates to be asymmetric but they must be small, the number of migrants per generation must not exceed a third, and in scenarios with low genetic differentiation (Fst<0.02) the program will struggle to define resulting migration patterns . A pairwTo take into consideration any effects of unequal number in sample sizes, pairwise comparisons of each individual Tasmanian population were run against the New Zealand population. Results showed no differences except that New Zealand was shown to realistically contribute closer to 1-3% of migrants to Tasmania . BAYESASPilot scale translocations of lobsters from deep to shallow waters around the southern coast of Tasmania and Southern Australia were financially and biologically beneficial ,7,8,47. J. edwardsii should not be disregarded completely. Rather, more complete sampling across the Tasmanian coast and Australia in general is required for a more definitive conclusion on genetic stocks. Detailed studies of population structure have not yet investigated patterns of genetic structure across Australia. The need for further study on Tasmanian populations, and the genetic stock structure of J. edwardsii across Australia is emphasised by the recent study of New Zealand populations [J. edwardsii was not homogeneous throughout its range in New Zealand, and rejected the null hypothesis of panmixia [There is minor evidence of some population structure, with low, yet statistically significant individual pairwise comparisons between some Tasmanian sites. With Fst values <0.003, it is probable that these values are not biologically significant ,49, howeulations . Thomas panmixia , althougNevertheless, finding statistically significant differences in pairwise comparisons of populations is not sufficient enough to confidently conclude that such populations are demonstrating an important level of genetic sub-structuring . StatistJ. edwardsii that are supported by models of larval trajectories that suggest trans-Tasman dispersal from Australia to New Zealand [J. edwardsii and J. novaehollandiae (based on minor differences in morphology) [Assumptions of population panmixia between Australia and New Zealand  appear t Zealand . The undphology) , until ephology) . As somephology) ,16, the J. edwardsii across its entire range of southern Australia and New Zealand. There is evidence that gene flow between distant populations does not occur equally in both directions; with both the results of this study and those of Thomas 2012 [This study provides a detailed investigation of the populations of southern Tasmania, some of which are currently involved in translocation projects. Clearly though it represents only a preliminary study of mas 2012 , suggestGiven the significant influence ocean currents have been suggested to have on population differentiation between Tasmania and New Zealand, they may also play a significant role in determining population connectivity amongst Australian populations. Migration rates were unable to be appropriately resolved between the sampled populations across Tasmania, clearly indicating important gaps in sampling that could have led to a determination of the level of self recruitment, or source stocks for southern Tasmania. Hydrological and gene flow modelling suggests a dominant eastward flow of the transport of larvae between populations , which fThere are a number of large currents across the expanse of southern Australasia that suggest source-sink relationships and an easterly pattern of step wise recruitment driving gene flow in this species . Given tThis analysis did not identify any scale of population structure that would suggest any genetic differences between shallow (red) and deep  populations. There is a significant level of genetic differentiation between Tasmania and New Zealand, and therefore the assumption of widespread population panmixia can be rejected. Although large scale translocations are genetically viable in this region of Tasmania, it is important to understand that if the indications of asymmetric gene flow and population differentiation found are transferable across the rest of this species range, then translocations should only be undertaken on a local scale. Similarly, finding significant genetic structure in an important fishery species, where previously none had been identified, means a much more detailed assessment of lobster connectivity across the range may find more unique genetic stocks, and important source sink relationships which will have important implications for successful translocations and stock structure management schemes. Given these findings, further research in this area is essential, as current management of the southern rock lobster fishery reflects national and state boundaries rather than the species biology.Figure S1STRUCTURE assignment of individuals across all populations into clusters of K=2.Colours indicate percentage contribution of individuals to assigned clusters (y axis), individuals represented by each line (x axis), black lines separate populations from which individuals belong. TAR, Taroona Reserve; MBI, Mutton Bird Island; HI, Hobbs Island; MAT, Maatsyuker Island; CQE, Cape Queen Elizabeth; EP, East Pyramids; NZ, New Zealand.(TIF)Click here for additional data file.Table S1Estimates of null allele frequencies across all populations by loci.Significant frequencies of greater than 10 percent null alleles indicated in bold. TAR, Taroona Reserve; MBI, Mutton Bird Island; HI, Hobbs Island; MAT, Maatsyuker Island; CQE, Cape Queen Elizabeth; EP, East Pyramids; NZ, New Zealand.(DOCX)Click here for additional data file.Table S2Fst's across all populations without locus JE_07.Data set of Fst values. Bold indicates significant values of p value <0.05, * indicates significant values after Bonferroni correction of p<0.002381. TAR, Taroona Reserve; MBI, Mutton Bird Island; HI, Hobbs Island; MAT, Maatsyuker Island; CQE, Cape Queen Elizabeth; EP, East Pyramids; NZ, New Zealand.(Table S2.1) Fst's across all populations without locus JE_17.Data set of Fst values. Bold indicates significant values of p value <0.05, * indicates significant values after Bonferroni correction of p<0.002381. TAR, Taroona Reserve; MBI, Mutton Bird Island; HI, Hobbs Island; MAT, Maatsyuker Island; CQE, Cape Queen Elizabeth; EP, East Pyramids; NZ, New Zealand.(Table S2.2) Fst's across All Populations without Locus JE_01Data set of Fst values. Bold indicates significant values of p value <0.05, * indicates significant values after Bonferroni correction of p<0.002381. TAR, Taroona Reserve; MBI, Mutton Bird Island; HI, Hobbs Island; MAT, Maatsyuker Island; CQE, Cape Queen Elizabeth; EP, East Pyramids; NZ, New Zealand.(Table S2.3) Fst's across all populations without loci JE_07, _17 and _01Data set of Fst values. Bold indicates significant values of p value <0.05, * indicates significant values after Bonferroni correction of p<0.002381. TAR, Taroona Reserve; MBI, Mutton Bird Island; HI, Hobbs Island; MAT, Maatsyuker Island; CQE, Cape Queen Elizabeth; EP, East Pyramids; NZ, New Zealand.(DOCX)Click here for additional data file.Table S3Migration rates (posterior probabilities) across all populations.Bold/italicised values indicate self recruitment, left column indicates where migrants travelled to, top row indicates where migrants originated from. TAR, Taroona Reserve; MBI, Mutton Bird Island; HI, Hobbs Island; MAT, Maatsyuker Island; CQE, Cape Queen Elizabeth; EP, East Pyramids; NZ, New Zealand.(DOCX)Click here for additional data file.Table S4Migration rates (posterior probabilities) compared between each Tasmanian population and New Zealand.Bold/italicised values indicate self recruitment, values in parentheses indicate standard deviation, left column indicates where migrants travelled to, top row indicates where migrants originated from. TAR, Taroona Reserve; MBI, Mutton Bird Island; HI, Hobbs Island; MAT, Maatsyuker Island; CQE, Cape Queen Elizabeth; EP, East Pyramids; NZ, New Zealand(DOCX)Click here for additional data file.


Limited communication resources have gradually become a critical factor toward efficiency of decentralized large scale multi-agent coordination when both system scales up and tasks become more complex. In current researches, due to the agent’s limited communication and observational capability, an agent in a decentralized setting can only choose a part of channels to access, but cannot perceive or share global information. Each agent’s cooperative decision is based on the partial observation of the system state, and as such, uncertainty in the communication network is unavoidable. In this situation, it is a major challenge working out cooperative decision-making under uncertainty with only a partial observation of the environment. In this paper, we propose a decentralized approach that allows agents cooperatively search and independently choose channels. The key to our design is to build an up-to-date observation for each agent’s view so that a local decision model is achievable in a large scale team coordination. We simplify the Dec-POMDP model problem, and each agent can jointly work out its communication policy in order to improve its local decision utilities for the choice of communication resources. Finally, we discuss an implicate resource competition game, and show that, there exists an approximate resources access tradeoff balance between agents. Based on this discovery, the tradeoff between real-time decision-making and the efficiency of cooperation using these channels can be well improved. Communication resources always play a latent role in networked large-scale agent team coordination applications, such as multi-robots system, mobile sensor system, etc. With the expansion of the system, communication resources exert a momentous impact on the cooperative efficiency , and numAs a main technical part of our research. In this paper, we model the decentralized multi-agent multi-channel access problem as a Decentralized Partially Observable Markov Decision Process (Dec-POMDP) problem. We use a continuous time Markov model to simulate the usage of channels while the constant slotted opportunity is used to support agents’ interaction. In addition, we use a sample-based Partially Observable Markov Decision Process (POMDP) to simplify the model. Finally, based on game theory, we model and analyze implicit resource competition between agents, and prove the existence of equilibrium in an ideal state.Even though centralized channel resource allocation methods can provide some sort of optimal solutions, they are less effective in situations where the central point fails. For instance, typical auction-based algorithms generally have low communication requirements , and theOpportunistic Spectrum Access (OSA)  and OppoGame Theory provides another approach to OSA. Stochastic game  as an exMany existing works assume that the observation information obtained from an agent’s neighbors is highly correlated. It can improve the efficiency of multi-agent coordination. In this case, exchange of local observations becomes important in coordination. From this view, we present a decentralized cooperative game model in which agents can iteratively adapt their strategies in terms of reduced competition or conflict, and can meet the minimum communication requirements for each agent timely. This presents a novel approach addressing the gaps in the aforementioned works.In this section, we follow the basic idea of continuous time Markov model to define the basic model of a multi-Channel access problem, and then describe the specific functional definition of each variable and the decision model.N distributed agents. Although the multi-hops information sharing method can make each agent finally gain full knowledge of the global state, this consumes a lot of communication resources and also deteriorates the system’s performance. Therefore, an agent makes decisions based on its limited observations, and the entire system would still be partially observed. The network consists of a set of contiguous, orthogonal (non-interfering) and homogeneous channels , denoted by CH = {c1, c2, …, cK}. The available channels are also numbered from 1 to K, and we assume that N > K agents are seeking channel opportunities in these K channels.We consider a multi-agent Ad-hoc network as being created by agents themselves in an open environment, with set ri can sense a set of channels and a subset of sensed channels to access. Limited by its hardware constraints, ri can sense {C1} channels,  channels and access {C2} channels,  channels. State statistics of the K channels follows a discrete-time Markov process with 2K states, where state is either idle or occupied. The channel sensing and access decisions are made to maximize agents reward by fully exploiting the sensing of vacant opportunities and the history statistics.We should recognize that agents can only access channels if the sensed channels are idle. As shown in For some reasons, some agents change the status of stable channel access, and other agents need to adjust the channel access based on their limited observation so as to ensure the global rational use of resources and QoS. Therefore, the multi-agent multi-channel access problem can be described as cooperative searching for available resources in a partially observable multi-channel network. As such, this can be modeled as a Dec-POMDP problem in terms of interdependence. A finite-horizon Dec-POMDP can be defined as a tuple S = {s0, s1, …sn} denotes the finite set of network states.Ai = {a1, a2, …, am} denotes ri’s available actions set. At each time step, all the agents in T denotes Markovian state transition function. P denotes the probability that doing action Λt and being in state s then going to state s′.ri at time t.O denotes observation function, which specifies the probability of joint observation O.t in state s.p0 = {B0, s0} is the initial belief and state distribution.a is determined by the policy π: b→a, which is the function that maps a belief state to the action that an agent should execute. i ≤ t−11 ≤ {ωt−1} denotes the known network states.Action Qi to denote the possible policy space for agent ri, and Qi− denotes the sets of policy trees for all agents except ri. With a programming approach, it is required that we generate incrementally the sets of useful policies for each agent. Thus, a joint policy Π = ×i ∈ N{πi} is a vector of policy trees. Evaluating a joint policy can then use the following formulation:ω) is the joint policy of subtree selected after observation ω. So we get the utility function as:Formally, most policies can be represented as decision trees. We use n policies to maximize a total reward function from finite horizon T under initial belief state p0, and the expected joint reward is given by Therefore, the essence of this framework is to find a set of no-information-sharing.In this section, we demonstrate an agent’s decision-making process based on current observation and resource perception, and analyze the computational complexity under the instincts of Shannon’s theory .We can use  use Eqs  and 6)  pi,jp anesign in :U=ri cannot get a full view of the state of the system, since it can only use its observation to update its actions. The goal of this problem’s model is to come up with a joint policy Π* = ri can only infer what action its neighbors may take, but the inference or conflict is inevitable. At each time slot, we can compute the expected value of a policy as follows:Based on the model described above, agent tatistic , and canT policy tree searching process.Solutions to a finite-horizon POMDP can be represented as a decision tree, where nodes denote the actions and arcs denote the observations. Similarly, solving a finite-horizon Dec-POMDP with known state space can be formulated as a multiple vector of horizon Algorithm 1: Resource aware policy search for agent ri.Require:g0 = 0; ℜ0 = {Φ}; ℜ ∈ Qi; Set Ensure: foreachrido1: randomselectploicycandidatesset {ηi}from ℜ;2:  gi(t) = 3:  forallπi ∈ ηido4:  excuteπitoobtainωt;5:   computegi(t);6:   ifthen7:   π* = πi;8:    else9:   i and get newprune π10:    end if11:   argmax {Π′ ∈ ℜ|V(η′) > R(η)};12:   end for13:  returnπ* → Π*;14:  end for15: Qi denotes the random initialized policy space with completely unspecified candidate policies. gi = η0 from ℜ, if gi’s value is bigger than πi to πi and search new lower bound for an optimal joint policy, which can be used to prune the search space. If ri has the minimum gi value in one round, then it will get priority to access its {C2}. Other agents are constantly updated to the new strategy, and after finite times evaluation and exploration that they can get all the apposite policies to fix the gi value. In a limited belief space, by retrieving the limited policies space, and the state transition probability approaching the optimal values, similarly, the decision can approach the optimal policy gi performs a summation over all possible network states and observations, and so the time complexity of this algorithm is O((|S|⋅|Qi|)T). The value of a policy is highly dependent on the other agents’ beliefs and the current system status, whereas, without sharing, the policy regeneration can only be derived on the basis of the reckoned joint policies. We define the policy update function as:Ξπ represents the conditional expectation given that policy πi is employed, and B0 is the initial belief, which can be the stationary distribution of the network state. ωt and the known status ri is As shown in Algorithm 1, ℜ ∈ ri can get a proximate full local observation. Consequently, we refer to the design in . X-axis represents the agents’ number. As in In this simulation, we test the average available channel resources for the new accessed agents under different interaction frequencies of the other agents in the network. We set 5 channels and 100 per slot new accessed agents, which are uniformly distributed in these 5 channels, the max agents number is 1000. The interaction frequency of the other agents was set to According to the experiment’s results, we can make a bold hypothesis that while the number of agents and the resource relatively balance, there should be a suitable interaction frequency that makes each agent obtain available resources to maximize its utility.In this simulation, we discuss the relationship between different team sizes when agents access channels under different assignment. We divide 100 agents into different team sizes, and allow them to access 5 channels. Simulation results are shown in Obviously, we can see that when the combination in each channel distribution is more uniform, the greater resource available, as assignment  and . In an extreme access situation with , all agents are in one channel. When communication demand escalates, all agents almost have no chance to obtain available resources. From above analysis, we can conclude that agents will gain more available resources when they distribute more uniformly.It can be seen that, with 500 tests for the same channel (distribution within the circles), agents can obtain the actual state of the channel. The red trail denotes the search result by POMDP, and black trail denotes the RANDOM method. It is obvious that the randomness and divergence of RANDOM far outweigh that of POMDP.We assumed in this paper that channel state transition probabilities can be entirely perceived, but in practice, this may not be available. The problem then becomes a decision model with unknown transition probabilities, but such mode is beyond the scope of this paper. In our design, we reduce a Dec-POMDP model to a simplified one by separating the problem into single-agent decision coordination, which may result in a low-complexity but potentially suboptimal design. In practical applications, systems Dynamics making use of pure policy space searching to solve all the problems become impractical, and need to be adjusted according to the actual situation and dynamics, and add more factors. In our future work, we will pursue the optimal joint design of the tradeoff between complexity and optimality, and will apply reinforcement learning theory on real multi-robots platform.S1 Table(XLS)Click here for additional data file.S2 Table(XLS)Click here for additional data file.S3 Table(XLS)Click here for additional data file.S4 Table(XLS)Click here for additional data file.S5 Table(XLS)Click here for additional data file.S1 File(DOC)Click here for additional data file.