Background Umbilical metastasis is one of the main characteristic signs of extensive neoplastic disease and is universally referred to as Sister Mary Joseph's nodule.
Background
Umbilical metastasis is one of the main characteristic signs of extensive neoplastic disease and is universally referred to as Sister Mary Joseph's nodule.Case presentation A 59-years-old Caucasian female underwent liver transplant for end stage liver disease due to hepatitis C with whole graft from cadaveric donor in 2003. After transplantation the patient developed multiple subcutaneous nodules in the umbilical region and bilateral inguinal lymphadenopathy. The excision biopsy of the umbilical mass showed the features of a poorly differentiated papillary serous cystadenocarcinoma. Computed tomographic scan and transvaginal ultrasonography were unable to demonstrate any primary lesion. Chemotherapy was start and the dosage of the immunosuppressive drugs was reduced. To date the patient is doing well and liver function is normal.
Case presentation
A 59-years-old Caucasian female underwent liver transplant for end stage liver disease due to hepatitis C with whole graft from cadaveric donor in 2003. After transplantation the patient developed multiple subcutaneous nodules in the umbilical region and bilateral inguinal lymphadenopathy. The excision biopsy of the umbilical mass showed the features of a poorly differentiated papillary serous cystadenocarcinoma. Computed tomographic scan and transvaginal ultrasonography were unable to demonstrate any primary lesion. Chemotherapy was start and the dosage of the immunosuppressive drugs was reduced. To date the patient is doing well and liver function is normal.Conclusions The umbilical metastasis can arise from many sites. In some cases, primary tumor may be not identified; nonetheless chemotherapy must be administrated based on patient's history, anatomical and histological findings.
Conclusions
The umbilical metastasis can arise from many sites. In some cases, primary tumor may be not identified; nonetheless chemotherapy must be administrated based on patient's history, anatomical and histological findings.

Background Metastases to the umbilicus are universally referred to as Sister Joseph's (or Sister Mary Joseph's) nodule. Etiology is related to the presence of primary malignant disease in the abdominal cavity or occasionally in the chest and/or breast [ 1 - 5 ]. Historically, Sister Mary Joseph (1856–1939) was a surgical assistant under the guidance of Dr. William Mayo. She was the first one to note the connection between the umbilical nodule and intra-abdominal cancer. The first case reporting the presence of Sister Mary Joseph's nodule was in 1864 by Storer, however; Hamilton Bailey was the first one to use the term "Sister Mary Joseph's nodule. Although skin metastasis is rare and range between 5% and 9%, it is estimated that 1% to 3% of abdomino-pelvic tumors metastasize to the umbilicus [ 2 , 3 , 5 ]. The most common primary neoplasm is adenocarcinoma (75%), more rarely squamous cell carcinoma followed by undifferentiated tumors or carcinoid can metastasize to umbilicus. In men, gastrointestinal tract (55%) is the most common location of the primary neoplasm that metastasizes to the umbilicus, followed by stomach, colon, rectum, small bowel, and pancreas in a decreasing order [ 1 - 5 ]. In women, on the other hand, gynecological neoplasms particularly ovarian cancer is the most common primary site, of which serous papillary cystadenocarcinoma (34%) is the most frequent. Further, endometrial carcinoma, cervix, vagina and vulva may also be responsible for the metastasis to the umbilicus. In addition to these the literature reports other but rare sites that could form potential grounds for metastasis to the umbilicus, namely gallbladder, liver, breast, lung, prostate, penis, peritoneum, lymphoma, bladder and kidney. In about 11% of the umbilical metastasis, the origin of the metastasis is unknown [ 4 , 5 ]. Upon recognition of the positive nodules in the umbilicus, physician must consider other potential sources such as endometriosis, melanocytic nevi, fibroma, epithelial inclusion cysts, seborrheic keratosis, pilonidal sinus, keloid, foreign body granulomas, myxoma, omphalitis, abscesses, umbilical hernia and of course primary malignant umbilical tumor (melanoma, squamous and basal cell carcinoma, sarcoma and adenocarcinoma) [ 6 ]. In many instances, Sister Joseph's nodule is the first and often the only indication of an underlying or occult cancer and/or may indicate recurrence of previously treated malignancy. The presence of umbilical metastasis usually suggests already advanced metastatic process characterized by poor prognosis [ 4 , 5 ]. Therefore, a biopsy of umbilical nodule is recommended which is safe and easy. Obtaining histological description can further direct the clinician to dictate a prompt treatment. In the case of recurrence of the neoplasm, fine needle aspiration cytology may be sufficient to provide definite diagnosis; however, sometimes clinical, cytological, histological, radiological and/or surgical investigations may not be sufficient to identify primary site of the metastasis [ 7 ]. Serous papillary cystadenocarcinoma has been documented previously as a cancer responsible for Sister Joseph's nodule because of the metastasis to the regional lymph nodes [ 6 - 9 ]. Herein, we report, to the best of our knowledge, the first case of Sister Joseph's nodule in a patient after liver transplantation complicated by serous papillary cystadenocarcinoma. In addition, we review the potential routes by which tumor spread may have occurred and the prognostic significance associated with umbilicus metastasis.
Background
Metastases to the umbilicus are universally referred to as Sister Joseph's (or Sister Mary Joseph's) nodule. Etiology is related to the presence of primary malignant disease in the abdominal cavity or occasionally in the chest and/or breast [ 1 - 5 ]. Historically, Sister Mary Joseph (1856–1939) was a surgical assistant under the guidance of Dr. William Mayo. She was the first one to note the connection between the umbilical nodule and intra-abdominal cancer. The first case reporting the presence of Sister Mary Joseph's nodule was in 1864 by Storer, however; Hamilton Bailey was the first one to use the term "Sister Mary Joseph's nodule.Although skin metastasis is rare and range between 5% and 9%, it is estimated that 1% to 3% of abdomino-pelvic tumors metastasize to the umbilicus [ 2 , 3 , 5 ]. The most common primary neoplasm is adenocarcinoma (75%), more rarely squamous cell carcinoma followed by undifferentiated tumors or carcinoid can metastasize to umbilicus. In men, gastrointestinal tract (55%) is the most common location of the primary neoplasm that metastasizes to the umbilicus, followed by stomach, colon, rectum, small bowel, and pancreas in a decreasing order [ 1 - 5 ]. In women, on the other hand, gynecological neoplasms particularly ovarian cancer is the most common primary site, of which serous papillary cystadenocarcinoma (34%) is the most frequent. Further, endometrial carcinoma, cervix, vagina and vulva may also be responsible for the metastasis to the umbilicus. In addition to these the literature reports other but rare sites that could form potential grounds for metastasis to the umbilicus, namely gallbladder, liver, breast, lung, prostate, penis, peritoneum, lymphoma, bladder and kidney. In about 11% of the umbilical metastasis, the origin of the metastasis is unknown [ 4 , 5 ]. Upon recognition of the positive nodules in the umbilicus, physician must consider other potential sources such as endometriosis, melanocytic nevi, fibroma, epithelial inclusion cysts, seborrheic keratosis, pilonidal sinus, keloid, foreign body granulomas, myxoma, omphalitis, abscesses, umbilical hernia and of course primary malignant umbilical tumor (melanoma, squamous and basal cell carcinoma, sarcoma and adenocarcinoma) [ 6 ]. In many instances, Sister Joseph's nodule is the first and often the only indication of an underlying or occult cancer and/or may indicate recurrence of previously treated malignancy. The presence of umbilical metastasis usually suggests already advanced metastatic process characterized by poor prognosis [ 4 , 5 ]. Therefore, a biopsy of umbilical nodule is recommended which is safe and easy. Obtaining histological description can further direct the clinician to dictate a prompt treatment. In the case of recurrence of the neoplasm, fine needle aspiration cytology may be sufficient to provide definite diagnosis; however, sometimes clinical, cytological, histological, radiological and/or surgical investigations may not be sufficient to identify primary site of the metastasis [ 7 ].Serous papillary cystadenocarcinoma has been documented previously as a cancer responsible for Sister Joseph's nodule because of the metastasis to the regional lymph nodes [ 6 - 9 ]. Herein, we report, to the best of our knowledge, the first case of Sister Joseph's nodule in a patient after liver transplantation complicated by serous papillary cystadenocarcinoma. In addition, we review the potential routes by which tumor spread may have occurred and the prognostic significance associated with umbilicus metastasis.Case presentation A 59-year-old Caucasian woman was admitted to our hospital for progressively enlarging but painless mass in the region of the umbilicus. The umbilical mass of approximately 5 cm in diameter and has developed two months after liver transplantation. The nodule was firm with irregular borders, wine-red shade and fixed to the surrounding tissues. The overlying skin was not ulcerated. She never smoked, consumed alcohol or used oral contraceptive pills. The gynecological history was unremarkable. The patient underwent a liver transplantation for end stage liver disease (ESLD) secondary to hepatitis C in mid of 2003. Prior to the transplant, her serum alpha-fetoprotein (alpha-FP) and CEA concentration were measured and both were within normal limits. The transplant procedure and immediate post-transplant period were unremarkable. The post-transplant immunosuppressive regimen consisted of Tacrolimus (6 mg/day) and steroids (prednisone 10 mg/day). Two months after transplantation, during routine follow-up, physical examination revealed multiple subcutaneous satellite nodules in close proximity to the umbilical region and lower abdominal wall. There was bilateral inguinal lymphadenopathy. A plain anterior/posterior chest x-ray was normal. Ultrasound of the abdominal cavity showed a 4 × 4 cm mass below the umbilicus and additional smaller nodules confined to the lower abdominal wall. Paraaortic lymph nodes were not identified. The echogenicity of the mass consisted of alternative hyper- and hypoechoic with poorly defined edges. However, the general condition of the patient was good and the laboratory test showed: hemoglobin level 9.6 g/dl, total bilirubin 0.6 mg/dl, conjugated bilirubin 0.1 mg/dl, Alkaline Phosphates 273 U/l, GGT 40 U/L, AST 36 U/L, and ALT 56 U/L. There was serological evidence of past hepatitis C virus infection. The biopsy of the umbilical mass showed features characteristic for a poorly differentiated serous papillary cystadenocarcinoma. An extended resection of the umbilical and paraumbilical metastasis was performed. In addition, extensive investigation to detect the primary neoplastic origin was initiated. Tumor marker evaluation consisted of: CEA 0.22 [normal 0–3.7 ng/ml], alpha feto protein 1.49 (normal: 0.8–9.4 UI/ml), CA125 27.73 (normal: 0–30 UI/ml), CA 19.9 was 8.61 (normal: 0–36.2 UI/ml), CA 15.3 was 22.48 (normal: 0–35 UI/ml). Multiple tumor marker measurements were obtained and each time similar results were reported. A computed tomographic (CT) scan was unable to demonstrate any lesion that could be considered a primary source of the metastasis to the umbilicus. Trans-vaginal ultrasonography showed a normal uterus and ovaries; ultrasonographic examination of thyroid and breast did not show any pathological changes as well. In spite of laborious efforts, the tumor's primary site was not found at that time. However, considering the histological results, the umbilicus metastasis could be attributed almost entirely to the ovarian cancer stage 4, since the main primary site in females are the ovaries. In order to prevent further tumor extension and expansion, tacrolimus was reduced from 6 mg/day to 1.5 mg/day. In addition, chemotherapy with Taxol was initiated (50 mg/m 2  I.V) once a week for 16 weeks. To date, after 6 months of follow-up, the patient is doing well, the liver function is normal and tacrolimus serum level is 1 ng/ml. A recent whole body CT-scan showed regression of iliac and inguinal lymph nodes involvement.
Case presentation
A 59-year-old Caucasian woman was admitted to our hospital for progressively enlarging but painless mass in the region of the umbilicus. The umbilical mass of approximately 5 cm in diameter and has developed two months after liver transplantation. The nodule was firm with irregular borders, wine-red shade and fixed to the surrounding tissues. The overlying skin was not ulcerated. She never smoked, consumed alcohol or used oral contraceptive pills. The gynecological history was unremarkable. The patient underwent a liver transplantation for end stage liver disease (ESLD) secondary to hepatitis C in mid of 2003. Prior to the transplant, her serum alpha-fetoprotein (alpha-FP) and CEA concentration were measured and both were within normal limits. The transplant procedure and immediate post-transplant period were unremarkable. The post-transplant immunosuppressive regimen consisted of Tacrolimus (6 mg/day) and steroids (prednisone 10 mg/day). Two months after transplantation, during routine follow-up, physical examination revealed multiple subcutaneous satellite nodules in close proximity to the umbilical region and lower abdominal wall. There was bilateral inguinal lymphadenopathy. A plain anterior/posterior chest x-ray was normal. Ultrasound of the abdominal cavity showed a 4 × 4 cm mass below the umbilicus and additional smaller nodules confined to the lower abdominal wall. Paraaortic lymph nodes were not identified. The echogenicity of the mass consisted of alternative hyper- and hypoechoic with poorly defined edges. However, the general condition of the patient was good and the laboratory test showed: hemoglobin level 9.6 g/dl, total bilirubin 0.6 mg/dl, conjugated bilirubin 0.1 mg/dl, Alkaline Phosphates 273 U/l, GGT 40 U/L, AST 36 U/L, and ALT 56 U/L. There was serological evidence of past hepatitis C virus infection. The biopsy of the umbilical mass showed features characteristic for a poorly differentiated serous papillary cystadenocarcinoma. An extended resection of the umbilical and paraumbilical metastasis was performed. In addition, extensive investigation to detect the primary neoplastic origin was initiated. Tumor marker evaluation consisted of: CEA 0.22 [normal 0–3.7 ng/ml], alpha feto protein 1.49 (normal: 0.8–9.4 UI/ml), CA125 27.73 (normal: 0–30 UI/ml), CA 19.9 was 8.61 (normal: 0–36.2 UI/ml), CA 15.3 was 22.48 (normal: 0–35 UI/ml). Multiple tumor marker measurements were obtained and each time similar results were reported. A computed tomographic (CT) scan was unable to demonstrate any lesion that could be considered a primary source of the metastasis to the umbilicus. Trans-vaginal ultrasonography showed a normal uterus and ovaries; ultrasonographic examination of thyroid and breast did not show any pathological changes as well. In spite of laborious efforts, the tumor's primary site was not found at that time. However, considering the histological results, the umbilicus metastasis could be attributed almost entirely to the ovarian cancer stage 4, since the main primary site in females are the ovaries. In order to prevent further tumor extension and expansion, tacrolimus was reduced from 6 mg/day to 1.5 mg/day. In addition, chemotherapy with Taxol was initiated (50 mg/m 2  I.V) once a week for 16 weeks. To date, after 6 months of follow-up, the patient is doing well, the liver function is normal and tacrolimus serum level is 1 ng/ml. A recent whole body CT-scan showed regression of iliac and inguinal lymph nodes involvement.Discussion Metastases may appear virtually anywhere in the body; however, certain sites are more common then others and umbilical metastasis are unusual sites [ 9 , 10 ]. In majority of cases, the metastatic lesions accompany the symptoms and signs of the primary tumor; however, it can be the first and often the only sign of a carcinoma, although this happens rarely [ 10 ]. A variation in vascularity and embryological development makes the umbilicus easy target for metastasis from an intra-abdominal tumor. In fact, during fetal development, the ductus venous (ductus venous Arantii) connects the umbilical portion of the left branch of the portal vein to the inferior vena cava, thus shunting oxygenated umbilical cord blood away form the liver. After birth the duct obliterates and persists as ligamentum venosum or Arantius' ligament. Because of its functional role, it is commonly believed that the ligament runs from the left branch of the portal vein to the vena cava itself. However, attention to anatomical detail demonstrates that the fibers of the ligament insert either on the left hepatic vein or at the junction between the left hepatic and the middle hepatic vein [ 1 , 11 ]. We know that in cirrhotic patients, the umbilical vein remains open due to portal hypertension. There are numerous potential routes by which a carcinoma can metastasize to the umbilicus in a liver transplant recipient. Malignant carcinomatous cells in the portal venous system may reach the umbilicus by way of a patent umbilical vein [ 12 - 15 ]. This would be more likely to occur in the presence of portal hypertension and the resulting portal-systemic shunting of blood [ 16 ]. Since our patient received liver transplant for cirrhosis secondary to hepatitis C virus infection accompanied by portal hypertension, this could be a possible mode of spread of papillary serous cystadenocarcinoma in our patient. In addition, presence of severe esophageal varicies and large umbilical vein that was viewed by angiography examination before the liver transplantation could have contributed to the spread of tumor. Therefore, in this case, malignant cells could have reached the umbilicus via the umbilical vein. In addition, a pelvic carcinoma of unknown origin may spread via lymph nodes to the umbilical region [ 9 , 17 , 18 ]. Furthermore, dermal lymphatics are a potential route of spread to the umbilicus and should be considered, since the umbilicus can be reached by retrograde lymphatic flow [ 4 ]. Nevertheless, the spread of malignant cells could have occurred via embolization thru the arterial blood supply to the umbilicus. Furthermore, direct implantation of tumor cells may have occurred via direct extension of the neoplasm from the anterior peritoneal surface or via redistribution of the peritoneal fluid flow [ 19 , 20 ]. Finally, cutaneous metastasis to the anterior abdominal wall, may perhaps cause malignant cells to spread in a retrograde direction, namely from the metastatic lesions along the subcutaneous lymphatic vessels to the umbilicus. It is known that the immunosuppression promotes tumor spreading and potentates its expansion, thus immunosuppressive regimen plays a crucial role in the tumor expansion and metastasis. Umbilical metastasis is one of many characteristic signs of extensive neoplastic disease. It suggests advanced distant metastasis and is associated with poor prognosis; mean survival is approximately 10–12 months, although long-term survival has been reported, but only in the presence of solitary metastatic umbilicus nodule [ 5 ].
Discussion
Metastases may appear virtually anywhere in the body; however, certain sites are more common then others and umbilical metastasis are unusual sites [ 9 , 10 ]. In majority of cases, the metastatic lesions accompany the symptoms and signs of the primary tumor; however, it can be the first and often the only sign of a carcinoma, although this happens rarely [ 10 ]. A variation in vascularity and embryological development makes the umbilicus easy target for metastasis from an intra-abdominal tumor. In fact, during fetal development, the ductus venous (ductus venous Arantii) connects the umbilical portion of the left branch of the portal vein to the inferior vena cava, thus shunting oxygenated umbilical cord blood away form the liver. After birth the duct obliterates and persists as ligamentum venosum or Arantius' ligament. Because of its functional role, it is commonly believed that the ligament runs from the left branch of the portal vein to the vena cava itself. However, attention to anatomical detail demonstrates that the fibers of the ligament insert either on the left hepatic vein or at the junction between the left hepatic and the middle hepatic vein [ 1 , 11 ]. We know that in cirrhotic patients, the umbilical vein remains open due to portal hypertension.There are numerous potential routes by which a carcinoma can metastasize to the umbilicus in a liver transplant recipient. Malignant carcinomatous cells in the portal venous system may reach the umbilicus by way of a patent umbilical vein [ 12 - 15 ]. This would be more likely to occur in the presence of portal hypertension and the resulting portal-systemic shunting of blood [ 16 ]. Since our patient received liver transplant for cirrhosis secondary to hepatitis C virus infection accompanied by portal hypertension, this could be a possible mode of spread of papillary serous cystadenocarcinoma in our patient. In addition, presence of severe esophageal varicies and large umbilical vein that was viewed by angiography examination before the liver transplantation could have contributed to the spread of tumor. Therefore, in this case, malignant cells could have reached the umbilicus via the umbilical vein. In addition, a pelvic carcinoma of unknown origin may spread via lymph nodes to the umbilical region [ 9 , 17 , 18 ]. Furthermore, dermal lymphatics are a potential route of spread to the umbilicus and should be considered, since the umbilicus can be reached by retrograde lymphatic flow [ 4 ]. Nevertheless, the spread of malignant cells could have occurred via embolization thru the arterial blood supply to the umbilicus. Furthermore, direct implantation of tumor cells may have occurred via direct extension of the neoplasm from the anterior peritoneal surface or via redistribution of the peritoneal fluid flow [ 19 , 20 ]. Finally, cutaneous metastasis to the anterior abdominal wall, may perhaps cause malignant cells to spread in a retrograde direction, namely from the metastatic lesions along the subcutaneous lymphatic vessels to the umbilicus. It is known that the immunosuppression promotes tumor spreading and potentates its expansion, thus immunosuppressive regimen plays a crucial role in the tumor expansion and metastasis.Umbilical metastasis is one of many characteristic signs of extensive neoplastic disease. It suggests advanced distant metastasis and is associated with poor prognosis; mean survival is approximately 10–12 months, although long-term survival has been reported, but only in the presence of solitary metastatic umbilicus nodule [ 5 ].Conclusions Presence of umbilical nodule or nodules requires extensive, meticulous and laborious evaluations since the differential diagnosis for umbilical nodule are many. In our case although, the primary tumor was not found, we treated it as ovarian tumor considering the histological findings and the iliac-inguinal nodes involvement.
Conclusions
Presence of umbilical nodule or nodules requires extensive, meticulous and laborious evaluations since the differential diagnosis for umbilical nodule are many. In our case although, the primary tumor was not found, we treated it as ovarian tumor considering the histological findings and the iliac-inguinal nodes involvement.Competing interests The author(s) declare that they have no competing interests.
Competing interests
The author(s) declare that they have no competing interests.Authors' contributions FP : conception and design, interpretation of data, drafting the article and revising EA : interpretation of data, drafting the article and revising SDD : acquisition of data and revising NM : interpretation of data, design and coordination and helped to draft the manuscript GB : collection of data, design and coordination and helped to draft the manuscript RM : collection of data, design and coordination MM : acquisition of data and drafting the article TJ : drafting the article and interpretation FR : acquisition of data and design MC : interpretation of data, drafting the article UV : drafting the article, revising and supervision Figure 1 Preoperative Sister Mary Joseph's nodule ultrasonography: 4 × 4 cm mass confined below the umbilicus (arrows). The main lesion is partly hyperechoic and partly hypoechoic with a poorly defined edge. Figure 2 Intraoperative specimen of the umbilical region: Sister Mary Joseph's nodule and umbilicus.
Authors' contributions
FP : conception and design, interpretation of data, drafting the article and revisingFPEA : interpretation of data, drafting the article and revisingEASDD : acquisition of data and revisingSDDNM : interpretation of data, design and coordination and helped to draft the manuscriptNMGB : collection of data, design and coordination and helped to draft the manuscriptGBRM : collection of data, design and coordinationRMMM : acquisition of data and drafting the articleMMTJ : drafting the article and interpretationTJFR : acquisition of data and designFRMC : interpretation of data, drafting the articleMCUV : drafting the article, revising and supervisionUV


Background Annotating genomes remains an hazardous task. Mistakes or gaps in such a complex process may occur when relevant knowledge is ignored, whether lost, forgotten or overlooked. This paper exemplifies an approach which could help to ressucitate such meaningful data.
Background
Annotating genomes remains an hazardous task. Mistakes or gaps in such a complex process may occur when relevant knowledge is ignored, whether lost, forgotten or overlooked. This paper exemplifies an approach which could help to ressucitate such meaningful data.Results We show that a set of closely related sequences which have been annotated as ornithine carbamoyltransferases are actually putrescine carbamoyltransferases. This demonstration is based on the following points : (i) use of enzymatic data which had been overlooked, (ii) rediscovery of a short NH 2 -terminal sequence allowing to reannotate a wrongly annotated ornithine carbamoyltransferase as a putrescine carbamoyltransferase, (iii) identification of conserved motifs allowing to distinguish unambiguously between the two kinds of carbamoyltransferases, and (iv) comparative study of the gene context of these different sequences.
Results
We show that a set of closely related sequences which have been annotated as ornithine carbamoyltransferases are actually putrescine carbamoyltransferases. This demonstration is based on the following points : (i) use of enzymatic data which had been overlooked, (ii) rediscovery of a short NH 2 -terminal sequence allowing to reannotate a wrongly annotated ornithine carbamoyltransferase as a putrescine carbamoyltransferase, (iii) identification of conserved motifs allowing to distinguish unambiguously between the two kinds of carbamoyltransferases, and (iv) comparative study of the gene context of these different sequences.Conclusions We explain why this specific case of misannotation had not yet been described and draw attention to the fact that analogous instances must be rather frequent. We urge to be especially cautious when high sequence similarity is coupled with an apparent lack of biochemical information. Moreover, from the point of view of genome annotation, proteins which have been studied experimentally but are not correlated with sequence data in current databases qualify as "orphans", just as unassigned genomic open reading frames do. The strategy we used in this paper to bridge such gaps in knowledge could work whenever it is possible to collect a body of facts about experimental data, homology, unnoticed sequence data, and accurate informations about gene context.
Conclusions
We explain why this specific case of misannotation had not yet been described and draw attention to the fact that analogous instances must be rather frequent. We urge to be especially cautious when high sequence similarity is coupled with an apparent lack of biochemical information. Moreover, from the point of view of genome annotation, proteins which have been studied experimentally but are not correlated with sequence data in current databases qualify as "orphans", just as unassigned genomic open reading frames do. The strategy we used in this paper to bridge such gaps in knowledge could work whenever it is possible to collect a body of facts about experimental data, homology, unnoticed sequence data, and accurate informations about gene context.

Background As a consequence of the deluge of completely sequenced genomes belonging to a large array of species, one can expect to identify many homologues of enzymes which have been previously well studied at the experimental level. This seems to be the general rule and the public sequence databanks (DDBJ/EMBL/GenBank) are now inundated by putative amino acid sequences which have been annotated uniquely by the widely used two-step process : (1) detection of a homologous relationship by a pairwise sequence similarity search at the level of primary structure and (2) inference of functional similarity from this detected homology. However, the opposite might be true. For various reasons one can either miss or misinterpret the actual function of a putative protein when annotating by homology, resulting in a wrong function transfer. Several studies have already emphasized this point (see, for example, [ 1 - 3 ]). On the other hand, beside these now well identified errors which are often due to automatic processes, more subtle mistakes may occur when some of the numerous effects of divergent evolution are overlooked. In particular, one of the insufficiently appreciated problems of functional assignment is that homologous proteins might catalyse different biochemical reactions. Here, we discuss an instance of erroneous annotation (misannotation) in genes of nitrogen metabolism which to our knowledge has not yet been brought up. We explain why this is so and draw attention to the fact that similar cases must actually be rather frequent.
Background
As a consequence of the deluge of completely sequenced genomes belonging to a large array of species, one can expect to identify many homologues of enzymes which have been previously well studied at the experimental level. This seems to be the general rule and the public sequence databanks (DDBJ/EMBL/GenBank) are now inundated by putative amino acid sequences which have been annotated uniquely by the widely used two-step process : (1) detection of a homologous relationship by a pairwise sequence similarity search at the level of primary structure and (2) inference of functional similarity from this detected homology.However, the opposite might be true. For various reasons one can either miss or misinterpret the actual function of a putative protein when annotating by homology, resulting in a wrong function transfer. Several studies have already emphasized this point (see, for example, [ 1 - 3 ]). On the other hand, beside these now well identified errors which are often due to automatic processes, more subtle mistakes may occur when some of the numerous effects of divergent evolution are overlooked. In particular, one of the insufficiently appreciated problems of functional assignment is that homologous proteins might catalyse different biochemical reactions. Here, we discuss an instance of erroneous annotation (misannotation) in genes of nitrogen metabolism which to our knowledge has not yet been brought up. We explain why this is so and draw attention to the fact that similar cases must actually be rather frequent.Results and Discussion Annotating distant carbamoyltransferases Our group ([ 4 , 5 ]) is presently involved in deciphering the evolutionary relationships between two ubiquitous and essential proteins, aspartate carbamoyltransferase (ATCase, EC 2.1.3.2) which catalyses the first committed step of  de novo  pyrimidine biosynthesis and ornithine carbamoyltransferase (OTCase, EC 2.1.3.3) which plays a crucial role in both anabolism and catabolism of arginine. In a recent study of the phylogeny of the 245 available OTCases (paper in preparation), we confirmed the existence of two families, OTC alpha and OTC beta, previously proposed on the basis of phylogenetic studies [ 4 ]. However, the advent of many new sequences further led to a more complex topology of the distance tree schematized on Fig.  1 . It appears now that a significant number of sequences which have been annotated as OTCases are distantly related to either family as outlined on Fig.  1 . These sequences, which are forming several clusters emerging in different locations between the root and the two families OTC alpha and OTC beta, have been provisionally annotated as UTC (unknown carbamoyltransferases). Indeed, among these UTC we found two sequences which do not correspond to classical OTCases. Figure 1 A schematic evolutionary tree of carbamoyltransferases.  A distance tree of the 245 available ornithine carbamoyltransferases has been obtained using the PhyloTree programme [33]. This tree was rooted by a few paralogous aspartate carbamoyltransferases belonging to either family ATC I or ATC II ([4], [5]). The root is indicated by a closed circle. The two families OTC alpha and OTC beta are schematized as triangles. The homologous carbamoyltransferases which are branching far from these ATC or OTC families are labelled as unknown (UTC) even if some of them have been annotated as ornithine carbamoyltransferases (see text). The few UTC sequences which have been annotated as carbamoyltransferase-like are enclosed in ovals. The clade of reannotated putrescine carbamoyltransferases is framed in red. The YgeW protein encoded by  Escherichia coli  and its close homologue from  Clostridium botulinum  are both located on a long branch emerging at the basis of this OTCases tree. YgeW is annotated as belonging to the ATCase/OTCase family (see, for example, the SwissProt knowledgebase [ 6 ]). On the branch which is next to the root we find the sequence of a protein which has been reported to be essential for arginine biosynthesis in the anaerobic bacterium  Bacteroides fragilis  [ 7 ]. This protein has been crystallized and characterized as a carbamoyltransferase-like protein since it does not display OTCase activity  in vitro  [ 7 ]. Indeed, several of its residues have been substituted in sites which are viewed as crucial for OTCase activity. Moreover, Dashuang  et al . [ 7 ] indicated that a similar protein has been found in  Xylella fastidiosa . Our phylogenetic data are in agreement with this observation since the protein annotated as OTCases in two strains of  X. fastidiosa  and its close relative present in two species of the  Xanthomonas  genus are found to branch close to that of  B. fragilis . Therefore, the functional identification of these different UTC is certainly not straightforward and requires further investigations. Furthermore, it occurred to us that, more than thirty years ago, another carbamoyltransferase was discovered by Roon and Barker [ 8 ]. A putrescine carbamoyltransferase (PTCase, EC 2.1.3.6) was found to be synthesized by the Gram-positive bacterium  Streptococcus  (now  Enterococcus )  faecalis  when it was grown on agmatine but not arginine as primary energy source. This PTCase was easily separated from the OTCase synthesized by the same organism grown on arginine [ 8 ]. This putrescine carbamoyltransferase had further been studied by V. Stalon's group ([ 9 - 12 ]). Two features of this study – which had apparently been overlooked in recent genome annotations – appear now to be crucial for the interpretation of the data shown on Fig.  1 . (1) Wargnies  et al . [ 9 ] showed that the putrescine carbamoyltransferase of  E. faecalis  had a weak but unambiguous OTCase activity (7.4% in terms of Vmax, with K M  for putrescine and L-ornithine of 0.029 mM and 13.0 mM respectively); (2) A short NH 2 -terminal sequence (29 residues) was published ten years later [ 13 ]. Since the complete genome of  E. faecalis  has now been sequenced [ 14 ], we could identify that one of the three putative ornithine carbamoyltransferases encoded by this genome, the open reading frame EF0732 annotated as ArgF-2 [ 14 ], is actually the putrescine carbamoyltransferase previously studied by the group of Stalon. A family of putrescine carbamoyltransferases In a second step, we extended this reannotation of a wrong OTCase as a PTCase to six other sequences encoded by  Lactococcus lactis ,  Streptococcus mutans, Pediococcus pentosaceus, Lactobacillus brevis  (and a very close partial sequence in  Lactobacillus sakei )  , Listeria monocytogenes  and  Mycoplasma mycoides , respectively. Indeed, these eight sequences, which have been annotated as either ArgF or ArcB (Table  1 ), (i) share high identity at the level of their amino acid sequence; (ii) they form a monophyletic group (Fig.  1 ) and (iii) they match the previously published  E. faecalis  NH 2 -terminal 29 residues sequence [ 13 ]. Moreover, as shown on Fig.  2 , these sequences share several specific motifs which are not found in the homologous OTCases. These motifs, especially the five longer ones, are well conserved, even in  M. mycoides  which is however more distant. When these motifs are used together to query either the UniProt knowledgebase [ 15 ] or the nr (non-redundant) database using the PHI-Blast programme, we obtain only these putative PTCase sequences (including that of  M. mycoides ) to the exclusion of any other carbamoyltransferase. Table 1 The seven ornithine carbamoyltransferases sequences which have been reannotated as putrescine carbamoyltransferases. Species name gene name CDS Swissprot TREMBL Web link Enterococcus faecalis argF-2 EF0732 Q837U7 Lactococcus lactis argF2 LL1700 Q9CEY4 Streptococcus mutans arcB SMU.262 Q8DW19 Pediococcus pentosaceus a - Scaffold 18 Gene 459 - Lactobacillus brevis a - Scaffold 15 Gene 476 - Lactobacillus sakei b argF - Q8RPX3 Listeria monocytogenes arcB LMO0036 Q8YAS7 Mycoplasma mycoides arcB MSC_0703 CAE77322 a  : draft genome  b  : fragment Figure 2 Long motifs specific of putrescine carbamoyltransferases.  The amino acid sequence of the open reading frame EF0732 of  E. faecalis  annotated as ArgF-2 [14] but now reannotated as putrescine carbamoyltransferase is shown. The NH 2 -terminal 29 residues sequence [13] which helped to this reannotation is indicated by a red bar above the respective residues. The amino acids which are specifically conserved in putrescine carbamoyltransferases and not in ornithine carbamoyltransferases are printed in bold. Inside the long motifs (> 15 residues) found along the whole sequence and numbered as "Motif 1" to Motif 5"are indicated the few substitutions present in the other sequences (lines above the  E. faecalis  sequence) including those of  M. mycoides  which is more distant (line in italic below the  E. faecalis  sequence). The residues forming the respective binding sites of carbamoylphosphate and of either putrescine or ornithine are also in bold and framed. Gene context, another tool for gene reannotation In a third step, the reannotation of this clade of OTCase sequences as PTCases was confirmed by a comparative study of the neighbourhood of their encoding genes present in the four genomes completely sequenced and published ( E. faecalis ,  Lc. lactis ,  S. mutans  and  L. monocytogenes ). As shown on Fig.  3 , the same set of neighbouring genes were present in these four species. We have successively a transcriptional regulator, the reannotated PTCase, an amino acid permease (probably an antiporter), a conserved hypothetical protein and finally the carbamate kinase ArcC-3 (EC 2.7.2.2). In a next step, we found that the so-called conserved hypothetical protein is homologous to the agmatine deiminase (or agmatine iminohydrolase, EC 3.5.3.12) of  Bacillus cereus  [ 15 ]. Fig.  3  further shows that the gene order found in  E. faecalis , is completely conserved in  Lc. lactis  and  S. mutans  and slightly modified in  L. monocytogenes . Figure 3 Conservation of the gene context of putrescine carbamoyltransferases.  The open reading frame (ORF) EF0732 of  E. faecalis  is shown with neighbouring ORFs. Under each numbered ORF (EF0731 to EF0735) is indicated the putative function proposed by the annotators of this complete genome [14]. Below is the reannotation we are proposing. This schematic representation is repeated for the similar genomic regions present in three other completely sequenced genomes, those of  Lc. lactis ,  S. mutans  and  L. monocytogenes . The homologous sequences are indicated by using the same color : white for transcriptional regulator, yellow for PTCase, pink for permease, green for agmatine deiminase, blue for carbamate kinase, respectively. Thus, these four clusters of genes appear to encode the full set of enzymes which are expected to form the catabolic agmatine deiminase pathway [ 10 ]. Agmatine deiminase, PTCase and carbamate kinase were already known to become coinduced by agmatine in  E. faecalis  when it is used as sole energy source [ 10 ], strongly suggesting that these gene clusters are functional operons. In  Pseudomonas aeruginosa  PAO1, the homologous agmatine deiminase is encoded by the gene  aguA  belonging to an operon  aguBA  induced by agmatine and N-carbamoylputrescine ([ 16 , 17 ]) but in this species N-carbamoylputrescine is converted by a N-carbamoylputrescine amidohydrolase (EC 3.5.1.53, the  aguB  product) into putrescine and CO 2  + ammonium rather than into putrescine and carbamoylphosphate. More recently, a similar pathway for polyamine biosynthesis has been identified by homology in higher plants [ 18 ]. In the alternative pathway corresponding to the analogous sets of genes shown on Fig.  3 , it is thus a PTCase which catalyzes the second step and catabolically converts N-carbamoylputrescine to putrescine and carbamoylphosphate which can further be used to synthesize ATP via carbamate kinase [ 19 ]. Furthermore, when we compare the clusters of genes shown in Fig.  3  to those surrounding gene  argF  or  arcB , encoding the catabolic OTCase functioning in the arginine deiminase (ADI) pathway present in many microbial genomes (see [ 20 - 22 ]), we observe a very similar distribution, namely a transcriptional regulator, the arginine deiminase (EC 3.5.3.6), an arginine/ornithine antiporter and a (sometimes two) carbamate kinase. There is thus a very close analogy between the set of genes encoding the enzymes catalyzing the different steps of the agmatine deiminase pathway found in these different Firmicutes and that encoding the enzymes catalyzing the different steps of the arginine deiminase pathway.
Results and Discussion
Annotating distant carbamoyltransferases Our group ([ 4 , 5 ]) is presently involved in deciphering the evolutionary relationships between two ubiquitous and essential proteins, aspartate carbamoyltransferase (ATCase, EC 2.1.3.2) which catalyses the first committed step of  de novo  pyrimidine biosynthesis and ornithine carbamoyltransferase (OTCase, EC 2.1.3.3) which plays a crucial role in both anabolism and catabolism of arginine. In a recent study of the phylogeny of the 245 available OTCases (paper in preparation), we confirmed the existence of two families, OTC alpha and OTC beta, previously proposed on the basis of phylogenetic studies [ 4 ]. However, the advent of many new sequences further led to a more complex topology of the distance tree schematized on Fig.  1 . It appears now that a significant number of sequences which have been annotated as OTCases are distantly related to either family as outlined on Fig.  1 . These sequences, which are forming several clusters emerging in different locations between the root and the two families OTC alpha and OTC beta, have been provisionally annotated as UTC (unknown carbamoyltransferases). Indeed, among these UTC we found two sequences which do not correspond to classical OTCases. Figure 1 A schematic evolutionary tree of carbamoyltransferases.  A distance tree of the 245 available ornithine carbamoyltransferases has been obtained using the PhyloTree programme [33]. This tree was rooted by a few paralogous aspartate carbamoyltransferases belonging to either family ATC I or ATC II ([4], [5]). The root is indicated by a closed circle. The two families OTC alpha and OTC beta are schematized as triangles. The homologous carbamoyltransferases which are branching far from these ATC or OTC families are labelled as unknown (UTC) even if some of them have been annotated as ornithine carbamoyltransferases (see text). The few UTC sequences which have been annotated as carbamoyltransferase-like are enclosed in ovals. The clade of reannotated putrescine carbamoyltransferases is framed in red. The YgeW protein encoded by  Escherichia coli  and its close homologue from  Clostridium botulinum  are both located on a long branch emerging at the basis of this OTCases tree. YgeW is annotated as belonging to the ATCase/OTCase family (see, for example, the SwissProt knowledgebase [ 6 ]). On the branch which is next to the root we find the sequence of a protein which has been reported to be essential for arginine biosynthesis in the anaerobic bacterium  Bacteroides fragilis  [ 7 ]. This protein has been crystallized and characterized as a carbamoyltransferase-like protein since it does not display OTCase activity  in vitro  [ 7 ]. Indeed, several of its residues have been substituted in sites which are viewed as crucial for OTCase activity. Moreover, Dashuang  et al . [ 7 ] indicated that a similar protein has been found in  Xylella fastidiosa . Our phylogenetic data are in agreement with this observation since the protein annotated as OTCases in two strains of  X. fastidiosa  and its close relative present in two species of the  Xanthomonas  genus are found to branch close to that of  B. fragilis . Therefore, the functional identification of these different UTC is certainly not straightforward and requires further investigations. Furthermore, it occurred to us that, more than thirty years ago, another carbamoyltransferase was discovered by Roon and Barker [ 8 ]. A putrescine carbamoyltransferase (PTCase, EC 2.1.3.6) was found to be synthesized by the Gram-positive bacterium  Streptococcus  (now  Enterococcus )  faecalis  when it was grown on agmatine but not arginine as primary energy source. This PTCase was easily separated from the OTCase synthesized by the same organism grown on arginine [ 8 ]. This putrescine carbamoyltransferase had further been studied by V. Stalon's group ([ 9 - 12 ]). Two features of this study – which had apparently been overlooked in recent genome annotations – appear now to be crucial for the interpretation of the data shown on Fig.  1 . (1) Wargnies  et al . [ 9 ] showed that the putrescine carbamoyltransferase of  E. faecalis  had a weak but unambiguous OTCase activity (7.4% in terms of Vmax, with K M  for putrescine and L-ornithine of 0.029 mM and 13.0 mM respectively); (2) A short NH 2 -terminal sequence (29 residues) was published ten years later [ 13 ]. Since the complete genome of  E. faecalis  has now been sequenced [ 14 ], we could identify that one of the three putative ornithine carbamoyltransferases encoded by this genome, the open reading frame EF0732 annotated as ArgF-2 [ 14 ], is actually the putrescine carbamoyltransferase previously studied by the group of Stalon.
Annotating distant carbamoyltransferases
Our group ([ 4 , 5 ]) is presently involved in deciphering the evolutionary relationships between two ubiquitous and essential proteins, aspartate carbamoyltransferase (ATCase, EC 2.1.3.2) which catalyses the first committed step of  de novo  pyrimidine biosynthesis and ornithine carbamoyltransferase (OTCase, EC 2.1.3.3) which plays a crucial role in both anabolism and catabolism of arginine.de novoIn a recent study of the phylogeny of the 245 available OTCases (paper in preparation), we confirmed the existence of two families, OTC alpha and OTC beta, previously proposed on the basis of phylogenetic studies [ 4 ]. However, the advent of many new sequences further led to a more complex topology of the distance tree schematized on Fig.  1 . It appears now that a significant number of sequences which have been annotated as OTCases are distantly related to either family as outlined on Fig.  1 . These sequences, which are forming several clusters emerging in different locations between the root and the two families OTC alpha and OTC beta, have been provisionally annotated as UTC (unknown carbamoyltransferases). Indeed, among these UTC we found two sequences which do not correspond to classical OTCases.The YgeW protein encoded by  Escherichia coli  and its close homologue from  Clostridium botulinum  are both located on a long branch emerging at the basis of this OTCases tree. YgeW is annotated as belonging to the ATCase/OTCase family (see, for example, the SwissProt knowledgebase [ 6 ]). On the branch which is next to the root we find the sequence of a protein which has been reported to be essential for arginine biosynthesis in the anaerobic bacterium  Bacteroides fragilis  [ 7 ]. This protein has been crystallized and characterized as a carbamoyltransferase-like protein since it does not display OTCase activity  in vitro  [ 7 ]. Indeed, several of its residues have been substituted in sites which are viewed as crucial for OTCase activity. Moreover, Dashuang  et al . [ 7 ] indicated that a similar protein has been found in  Xylella fastidiosa . Our phylogenetic data are in agreement with this observation since the protein annotated as OTCases in two strains of  X. fastidiosa  and its close relative present in two species of the  Xanthomonas  genus are found to branch close to that of  B. fragilis . Therefore, the functional identification of these different UTC is certainly not straightforward and requires further investigations.Escherichia coliClostridium botulinumBacteroides fragilisin vitroet alXylella fastidiosaX. fastidiosaXanthomonasB. fragilisFurthermore, it occurred to us that, more than thirty years ago, another carbamoyltransferase was discovered by Roon and Barker [ 8 ]. A putrescine carbamoyltransferase (PTCase, EC 2.1.3.6) was found to be synthesized by the Gram-positive bacterium  Streptococcus  (now  Enterococcus )  faecalis  when it was grown on agmatine but not arginine as primary energy source. This PTCase was easily separated from the OTCase synthesized by the same organism grown on arginine [ 8 ]. This putrescine carbamoyltransferase had further been studied by V. Stalon's group ([ 9 - 12 ]). Two features of this study – which had apparently been overlooked in recent genome annotations – appear now to be crucial for the interpretation of the data shown on Fig.  1 . (1) Wargnies  et al . [ 9 ] showed that the putrescine carbamoyltransferase of  E. faecalis  had a weak but unambiguous OTCase activity (7.4% in terms of Vmax, with K M  for putrescine and L-ornithine of 0.029 mM and 13.0 mM respectively); (2) A short NH 2 -terminal sequence (29 residues) was published ten years later [ 13 ]. Since the complete genome of  E. faecalis  has now been sequenced [ 14 ], we could identify that one of the three putative ornithine carbamoyltransferases encoded by this genome, the open reading frame EF0732 annotated as ArgF-2 [ 14 ], is actually the putrescine carbamoyltransferase previously studied by the group of Stalon.StreptococcusEnterococcusfaecaliset alE. faecalisE. faecalisA family of putrescine carbamoyltransferases In a second step, we extended this reannotation of a wrong OTCase as a PTCase to six other sequences encoded by  Lactococcus lactis ,  Streptococcus mutans, Pediococcus pentosaceus, Lactobacillus brevis  (and a very close partial sequence in  Lactobacillus sakei )  , Listeria monocytogenes  and  Mycoplasma mycoides , respectively. Indeed, these eight sequences, which have been annotated as either ArgF or ArcB (Table  1 ), (i) share high identity at the level of their amino acid sequence; (ii) they form a monophyletic group (Fig.  1 ) and (iii) they match the previously published  E. faecalis  NH 2 -terminal 29 residues sequence [ 13 ]. Moreover, as shown on Fig.  2 , these sequences share several specific motifs which are not found in the homologous OTCases. These motifs, especially the five longer ones, are well conserved, even in  M. mycoides  which is however more distant. When these motifs are used together to query either the UniProt knowledgebase [ 15 ] or the nr (non-redundant) database using the PHI-Blast programme, we obtain only these putative PTCase sequences (including that of  M. mycoides ) to the exclusion of any other carbamoyltransferase. Table 1 The seven ornithine carbamoyltransferases sequences which have been reannotated as putrescine carbamoyltransferases. Species name gene name CDS Swissprot TREMBL Web link Enterococcus faecalis argF-2 EF0732 Q837U7 Lactococcus lactis argF2 LL1700 Q9CEY4 Streptococcus mutans arcB SMU.262 Q8DW19 Pediococcus pentosaceus a - Scaffold 18 Gene 459 - Lactobacillus brevis a - Scaffold 15 Gene 476 - Lactobacillus sakei b argF - Q8RPX3 Listeria monocytogenes arcB LMO0036 Q8YAS7 Mycoplasma mycoides arcB MSC_0703 CAE77322 a  : draft genome  b  : fragment Figure 2 Long motifs specific of putrescine carbamoyltransferases.  The amino acid sequence of the open reading frame EF0732 of  E. faecalis  annotated as ArgF-2 [14] but now reannotated as putrescine carbamoyltransferase is shown. The NH 2 -terminal 29 residues sequence [13] which helped to this reannotation is indicated by a red bar above the respective residues. The amino acids which are specifically conserved in putrescine carbamoyltransferases and not in ornithine carbamoyltransferases are printed in bold. Inside the long motifs (> 15 residues) found along the whole sequence and numbered as "Motif 1" to Motif 5"are indicated the few substitutions present in the other sequences (lines above the  E. faecalis  sequence) including those of  M. mycoides  which is more distant (line in italic below the  E. faecalis  sequence). The residues forming the respective binding sites of carbamoylphosphate and of either putrescine or ornithine are also in bold and framed.
A family of putrescine carbamoyltransferases
In a second step, we extended this reannotation of a wrong OTCase as a PTCase to six other sequences encoded by  Lactococcus lactis ,  Streptococcus mutans, Pediococcus pentosaceus, Lactobacillus brevis  (and a very close partial sequence in  Lactobacillus sakei )  , Listeria monocytogenes  and  Mycoplasma mycoides , respectively. Indeed, these eight sequences, which have been annotated as either ArgF or ArcB (Table  1 ), (i) share high identity at the level of their amino acid sequence; (ii) they form a monophyletic group (Fig.  1 ) and (iii) they match the previously published  E. faecalis  NH 2 -terminal 29 residues sequence [ 13 ]. Moreover, as shown on Fig.  2 , these sequences share several specific motifs which are not found in the homologous OTCases. These motifs, especially the five longer ones, are well conserved, even in  M. mycoides  which is however more distant. When these motifs are used together to query either the UniProt knowledgebase [ 15 ] or the nr (non-redundant) database using the PHI-Blast programme, we obtain only these putative PTCase sequences (including that of  M. mycoides ) to the exclusion of any other carbamoyltransferase.Lactococcus lactisStreptococcus mutans, Pediococcus pentosaceus, Lactobacillus brevisLactobacillus sakei, Listeria monocytogenesMycoplasma mycoidesE. faecalisM. mycoidesM. mycoidesThe seven ornithine carbamoyltransferases sequences which have been reannotated as putrescine carbamoyltransferases.Enterococcus faecalisargF-2Lactococcus lactisargF2Streptococcus mutansarcBPediococcus pentosaceusLactobacillus brevisLactobacillus sakeiargFListeria monocytogenesarcBMycoplasma mycoidesarcBa  : draft genome  b  : fragmentGene context, another tool for gene reannotation In a third step, the reannotation of this clade of OTCase sequences as PTCases was confirmed by a comparative study of the neighbourhood of their encoding genes present in the four genomes completely sequenced and published ( E. faecalis ,  Lc. lactis ,  S. mutans  and  L. monocytogenes ). As shown on Fig.  3 , the same set of neighbouring genes were present in these four species. We have successively a transcriptional regulator, the reannotated PTCase, an amino acid permease (probably an antiporter), a conserved hypothetical protein and finally the carbamate kinase ArcC-3 (EC 2.7.2.2). In a next step, we found that the so-called conserved hypothetical protein is homologous to the agmatine deiminase (or agmatine iminohydrolase, EC 3.5.3.12) of  Bacillus cereus  [ 15 ]. Fig.  3  further shows that the gene order found in  E. faecalis , is completely conserved in  Lc. lactis  and  S. mutans  and slightly modified in  L. monocytogenes . Figure 3 Conservation of the gene context of putrescine carbamoyltransferases.  The open reading frame (ORF) EF0732 of  E. faecalis  is shown with neighbouring ORFs. Under each numbered ORF (EF0731 to EF0735) is indicated the putative function proposed by the annotators of this complete genome [14]. Below is the reannotation we are proposing. This schematic representation is repeated for the similar genomic regions present in three other completely sequenced genomes, those of  Lc. lactis ,  S. mutans  and  L. monocytogenes . The homologous sequences are indicated by using the same color : white for transcriptional regulator, yellow for PTCase, pink for permease, green for agmatine deiminase, blue for carbamate kinase, respectively. Thus, these four clusters of genes appear to encode the full set of enzymes which are expected to form the catabolic agmatine deiminase pathway [ 10 ]. Agmatine deiminase, PTCase and carbamate kinase were already known to become coinduced by agmatine in  E. faecalis  when it is used as sole energy source [ 10 ], strongly suggesting that these gene clusters are functional operons. In  Pseudomonas aeruginosa  PAO1, the homologous agmatine deiminase is encoded by the gene  aguA  belonging to an operon  aguBA  induced by agmatine and N-carbamoylputrescine ([ 16 , 17 ]) but in this species N-carbamoylputrescine is converted by a N-carbamoylputrescine amidohydrolase (EC 3.5.1.53, the  aguB  product) into putrescine and CO 2  + ammonium rather than into putrescine and carbamoylphosphate. More recently, a similar pathway for polyamine biosynthesis has been identified by homology in higher plants [ 18 ]. In the alternative pathway corresponding to the analogous sets of genes shown on Fig.  3 , it is thus a PTCase which catalyzes the second step and catabolically converts N-carbamoylputrescine to putrescine and carbamoylphosphate which can further be used to synthesize ATP via carbamate kinase [ 19 ]. Furthermore, when we compare the clusters of genes shown in Fig.  3  to those surrounding gene  argF  or  arcB , encoding the catabolic OTCase functioning in the arginine deiminase (ADI) pathway present in many microbial genomes (see [ 20 - 22 ]), we observe a very similar distribution, namely a transcriptional regulator, the arginine deiminase (EC 3.5.3.6), an arginine/ornithine antiporter and a (sometimes two) carbamate kinase. There is thus a very close analogy between the set of genes encoding the enzymes catalyzing the different steps of the agmatine deiminase pathway found in these different Firmicutes and that encoding the enzymes catalyzing the different steps of the arginine deiminase pathway.
Gene context, another tool for gene reannotation
In a third step, the reannotation of this clade of OTCase sequences as PTCases was confirmed by a comparative study of the neighbourhood of their encoding genes present in the four genomes completely sequenced and published ( E. faecalis ,  Lc. lactis ,  S. mutans  and  L. monocytogenes ). As shown on Fig.  3 , the same set of neighbouring genes were present in these four species. We have successively a transcriptional regulator, the reannotated PTCase, an amino acid permease (probably an antiporter), a conserved hypothetical protein and finally the carbamate kinase ArcC-3 (EC 2.7.2.2). In a next step, we found that the so-called conserved hypothetical protein is homologous to the agmatine deiminase (or agmatine iminohydrolase, EC 3.5.3.12) of  Bacillus cereus  [ 15 ]. Fig.  3  further shows that the gene order found in  E. faecalis , is completely conserved in  Lc. lactis  and  S. mutans  and slightly modified in  L. monocytogenes .E. faecalisLc. lactisS. mutansL. monocytogenesBacillus cereusE. faecalisLc. lactisS. mutansL. monocytogenesThus, these four clusters of genes appear to encode the full set of enzymes which are expected to form the catabolic agmatine deiminase pathway [ 10 ]. Agmatine deiminase, PTCase and carbamate kinase were already known to become coinduced by agmatine in  E. faecalis  when it is used as sole energy source [ 10 ], strongly suggesting that these gene clusters are functional operons. In  Pseudomonas aeruginosa  PAO1, the homologous agmatine deiminase is encoded by the gene  aguA  belonging to an operon  aguBA  induced by agmatine and N-carbamoylputrescine ([ 16 , 17 ]) but in this species N-carbamoylputrescine is converted by a N-carbamoylputrescine amidohydrolase (EC 3.5.1.53, the  aguB  product) into putrescine and CO 2  + ammonium rather than into putrescine and carbamoylphosphate. More recently, a similar pathway for polyamine biosynthesis has been identified by homology in higher plants [ 18 ]. In the alternative pathway corresponding to the analogous sets of genes shown on Fig.  3 , it is thus a PTCase which catalyzes the second step and catabolically converts N-carbamoylputrescine to putrescine and carbamoylphosphate which can further be used to synthesize ATP via carbamate kinase [ 19 ].E. faecalisPseudomonas aeruginosaaguAaguBAaguBFurthermore, when we compare the clusters of genes shown in Fig.  3  to those surrounding gene  argF  or  arcB , encoding the catabolic OTCase functioning in the arginine deiminase (ADI) pathway present in many microbial genomes (see [ 20 - 22 ]), we observe a very similar distribution, namely a transcriptional regulator, the arginine deiminase (EC 3.5.3.6), an arginine/ornithine antiporter and a (sometimes two) carbamate kinase. There is thus a very close analogy between the set of genes encoding the enzymes catalyzing the different steps of the agmatine deiminase pathway found in these different Firmicutes and that encoding the enzymes catalyzing the different steps of the arginine deiminase pathway.argFarcBConclusions Genome annotation requires both reliable tools for identifying gene function and manual expertise. The frustration due to the high percentage of orphan genes found in all genomes is often compounded with another – more vicious – problem which may occur when a very strong sequence similarity is obscuring the actual functional identity of another kind of orphan. The analysis described in this paper illustrates the difficulty in identifying such a potential source of misannotation and delineates at least two fundamental parameters which must be considered especially when the results appear to be straightforward. First, one must keep in mind that proteins sharing a high level of identical residues may have different functions. A routine step for challenging the functional annotation of any putative coding sequence should be a phylogenetic analysis. Any CDS found to branch far from its homologues in an evolutionary tree, as observed in the case of the carbamoyltransferases (Fig.  1 ), should be examined with caution before assigning it a putative function. Another example of the usefulness of the phylogenetic approach to correct misannotations can be found in a comparative analysis of ureohydrolases [ 20 ]. The second parameter which must be considered is the striking lack of information in the various public databases. For example, in the case studied here (the putrescine carbamoyltransferase EC 2.1.3.6) it is reported that there is no sequence available in various first-rate databases specialized in enzymatic and/or metabolic data such as ENZYME [ 23 ], BRENDA [ 24 ], KEGG [ 25 ], BIOCYC [ 26 ], etc...as well as in the Gene Ontology (GO:0050231) Consortium [ 27 ]. A significant part of this deficit of information appears to be due to not correlating biochemical data [ 8 - 11 ] previously published and well recorded in BRENDA [ 24 ], for example, with the incomplete amino acid sequence which was not taken into account although it had been published by the same group [ 13 ] who studied this enzyme. The specific point we would like to stress in this paper reflects a more general gap – which is widely ignored – between the enormous quantity of information buried in the sequence data and the refined knowledge built up over several decades of studies on gene regulation and protein biochemistry (recorded in [ 23 ] to [ 27 ]). In this respect, experimentally studied proteins not correlated with sequence data also qualify as "orphans". In the present case, such a resulting gap in knowledge could be bridged only because we used the experimental approach detailed in this paper. After being alerted by the unusual topology of the phylogenetic tree (Fig.  1 ) and the rediscovery of the partial sequence [ 13 ], a confirmation of the reannotation as PTCases was obtained when considering their signature (Fig.  2 ) and the gene context (Fig.  3 ) which differentiate them clearly from their OTCase homologues. The strategy we used to identify such orphan sequences could work in any other case where it is possible to collect a body of facts about experimental data, homology, unnoticed sequence data, and accurate informations about gene context. Note that we incidentally used such a strategy to annotate the genes encoding a putative agmatine deiminase in the genomes listed in Fig.  3 . It is highly probable that this approach can be applied to many similar cases. Therefore, our community should feel encouraged to dig in old lab books, unpublished data buried in doctoral thesis and similar documents, in order to retrieve information crucial for correct genome annotation. Moreover, it becomes urgent to design new approaches in order to efficiently explore what has been called the "bibliome" [ 28 ]. This could help to bridge important gaps in knowledge – such as exemplified in this paper- which lead to numerous errors in genome annotation. Accordingly, it would become possible to (re)annotate conserved hypothetical proteins for which there is an apparent lack of information in the various public databases.
Conclusions
Genome annotation requires both reliable tools for identifying gene function and manual expertise. The frustration due to the high percentage of orphan genes found in all genomes is often compounded with another – more vicious – problem which may occur when a very strong sequence similarity is obscuring the actual functional identity of another kind of orphan. The analysis described in this paper illustrates the difficulty in identifying such a potential source of misannotation and delineates at least two fundamental parameters which must be considered especially when the results appear to be straightforward. First, one must keep in mind that proteins sharing a high level of identical residues may have different functions. A routine step for challenging the functional annotation of any putative coding sequence should be a phylogenetic analysis. Any CDS found to branch far from its homologues in an evolutionary tree, as observed in the case of the carbamoyltransferases (Fig.  1 ), should be examined with caution before assigning it a putative function. Another example of the usefulness of the phylogenetic approach to correct misannotations can be found in a comparative analysis of ureohydrolases [ 20 ].The second parameter which must be considered is the striking lack of information in the various public databases. For example, in the case studied here (the putrescine carbamoyltransferase EC 2.1.3.6) it is reported that there is no sequence available in various first-rate databases specialized in enzymatic and/or metabolic data such as ENZYME [ 23 ], BRENDA [ 24 ], KEGG [ 25 ], BIOCYC [ 26 ], etc...as well as in the Gene Ontology (GO:0050231) Consortium [ 27 ]. A significant part of this deficit of information appears to be due to not correlating biochemical data [ 8 - 11 ] previously published and well recorded in BRENDA [ 24 ], for example, with the incomplete amino acid sequence which was not taken into account although it had been published by the same group [ 13 ] who studied this enzyme.The specific point we would like to stress in this paper reflects a more general gap – which is widely ignored – between the enormous quantity of information buried in the sequence data and the refined knowledge built up over several decades of studies on gene regulation and protein biochemistry (recorded in [ 23 ] to [ 27 ]). In this respect, experimentally studied proteins not correlated with sequence data also qualify as "orphans". In the present case, such a resulting gap in knowledge could be bridged only because we used the experimental approach detailed in this paper. After being alerted by the unusual topology of the phylogenetic tree (Fig.  1 ) and the rediscovery of the partial sequence [ 13 ], a confirmation of the reannotation as PTCases was obtained when considering their signature (Fig.  2 ) and the gene context (Fig.  3 ) which differentiate them clearly from their OTCase homologues.The strategy we used to identify such orphan sequences could work in any other case where it is possible to collect a body of facts about experimental data, homology, unnoticed sequence data, and accurate informations about gene context. Note that we incidentally used such a strategy to annotate the genes encoding a putative agmatine deiminase in the genomes listed in Fig.  3 . It is highly probable that this approach can be applied to many similar cases. Therefore, our community should feel encouraged to dig in old lab books, unpublished data buried in doctoral thesis and similar documents, in order to retrieve information crucial for correct genome annotation. Moreover, it becomes urgent to design new approaches in order to efficiently explore what has been called the "bibliome" [ 28 ]. This could help to bridge important gaps in knowledge – such as exemplified in this paper- which lead to numerous errors in genome annotation. Accordingly, it would become possible to (re)annotate conserved hypothetical proteins for which there is an apparent lack of information in the various public databases.Methods Collecting sequences Near 450 carbamoyltransferases (ATCases and OTCases) sequences were collected from the public databases SwissProt, TREMBL and TREMBLNEW [ 15 ]. To facilitate the management of these data which are continuously growing up with the onset of new completely sequenced genomes, we assemble them in a relational database (available on request). Moreover, in the case of unpublished but completely sequenced genomes, it was often possible to recover  bona fide  sequences from specific sequencing groups sites (Joint Genome Institute [ 29 ] and Sanger Institute [ 30 ]) using either BlastP or tBlastN queries. We retained only unpublished sequences aligning along their whole length with  bona fide  carbamoyltransferases and sharing no less than 30% identity with it, using at least two distantly related seeds. Reconstructing phylogenetic trees Rooted phylogenetic trees were derived from multiple alignements of ATCases and OTCases using two different approaches. (1) New sequences were manually added and aligned to the previously published [ 4 ] multiple alignement using the BioEdit sequence alignment editor [ 31 ]. These additions were made effortless by introducing each new sequence near its closest partner (the first hit in a routine BlastP check). This processive approach minimized the risk of introducing any bias when adding numerous new sequences. However, the soundness of this manual alignement was routinely checked using automatic programmes (both ClustalX and DARWIN, see below) to verify that we did not miss any conserved motifs. We further ascertained this multiple alignement (especially the introduction of gaps) by using the informations available from the known 3D structures of ATCases and OTCases. Maximum parsimony and distance trees were derived from this alignment using the PROTPARS and NEIGHBOR programmes of the PHYLIP package [ 32 ], respectively. This PHYLIP package was further used to derive confidence limits for each node of either parsimony or distance trees using a bootstrap approach (programmes SEQBOOT and CONSENSE). (2) The PhyloTree programme of the DARWIN package [ 33 ] allows to build a multiple alignement and to derive a distance tree which is an approximation to maximum likelihood tree since the deduced evolutionary distances are weighted by computing their variance when reconstructing the tree.
Methods
Collecting sequences Near 450 carbamoyltransferases (ATCases and OTCases) sequences were collected from the public databases SwissProt, TREMBL and TREMBLNEW [ 15 ]. To facilitate the management of these data which are continuously growing up with the onset of new completely sequenced genomes, we assemble them in a relational database (available on request). Moreover, in the case of unpublished but completely sequenced genomes, it was often possible to recover  bona fide  sequences from specific sequencing groups sites (Joint Genome Institute [ 29 ] and Sanger Institute [ 30 ]) using either BlastP or tBlastN queries. We retained only unpublished sequences aligning along their whole length with  bona fide  carbamoyltransferases and sharing no less than 30% identity with it, using at least two distantly related seeds.
Collecting sequences
Near 450 carbamoyltransferases (ATCases and OTCases) sequences were collected from the public databases SwissProt, TREMBL and TREMBLNEW [ 15 ]. To facilitate the management of these data which are continuously growing up with the onset of new completely sequenced genomes, we assemble them in a relational database (available on request). Moreover, in the case of unpublished but completely sequenced genomes, it was often possible to recover  bona fide  sequences from specific sequencing groups sites (Joint Genome Institute [ 29 ] and Sanger Institute [ 30 ]) using either BlastP or tBlastN queries. We retained only unpublished sequences aligning along their whole length with  bona fide  carbamoyltransferases and sharing no less than 30% identity with it, using at least two distantly related seeds.bona fidebona fideReconstructing phylogenetic trees Rooted phylogenetic trees were derived from multiple alignements of ATCases and OTCases using two different approaches. (1) New sequences were manually added and aligned to the previously published [ 4 ] multiple alignement using the BioEdit sequence alignment editor [ 31 ]. These additions were made effortless by introducing each new sequence near its closest partner (the first hit in a routine BlastP check). This processive approach minimized the risk of introducing any bias when adding numerous new sequences. However, the soundness of this manual alignement was routinely checked using automatic programmes (both ClustalX and DARWIN, see below) to verify that we did not miss any conserved motifs. We further ascertained this multiple alignement (especially the introduction of gaps) by using the informations available from the known 3D structures of ATCases and OTCases. Maximum parsimony and distance trees were derived from this alignment using the PROTPARS and NEIGHBOR programmes of the PHYLIP package [ 32 ], respectively. This PHYLIP package was further used to derive confidence limits for each node of either parsimony or distance trees using a bootstrap approach (programmes SEQBOOT and CONSENSE). (2) The PhyloTree programme of the DARWIN package [ 33 ] allows to build a multiple alignement and to derive a distance tree which is an approximation to maximum likelihood tree since the deduced evolutionary distances are weighted by computing their variance when reconstructing the tree.
Reconstructing phylogenetic trees
Rooted phylogenetic trees were derived from multiple alignements of ATCases and OTCases using two different approaches. (1) New sequences were manually added and aligned to the previously published [ 4 ] multiple alignement using the BioEdit sequence alignment editor [ 31 ]. These additions were made effortless by introducing each new sequence near its closest partner (the first hit in a routine BlastP check). This processive approach minimized the risk of introducing any bias when adding numerous new sequences. However, the soundness of this manual alignement was routinely checked using automatic programmes (both ClustalX and DARWIN, see below) to verify that we did not miss any conserved motifs. We further ascertained this multiple alignement (especially the introduction of gaps) by using the informations available from the known 3D structures of ATCases and OTCases. Maximum parsimony and distance trees were derived from this alignment using the PROTPARS and NEIGHBOR programmes of the PHYLIP package [ 32 ], respectively. This PHYLIP package was further used to derive confidence limits for each node of either parsimony or distance trees using a bootstrap approach (programmes SEQBOOT and CONSENSE). (2) The PhyloTree programme of the DARWIN package [ 33 ] allows to build a multiple alignement and to derive a distance tree which is an approximation to maximum likelihood tree since the deduced evolutionary distances are weighted by computing their variance when reconstructing the tree.Authors' contributions NG dug up the "ancient" data on putrescine carbamoyltransferase, contributed his knowledge about carbamoyltransferases and made important additions to the manuscript. YX brought essential informations about the genetics and biochemistry of the enzymes involved in arginine metabolism and their evolution. DGN participated in the collection of new carbamoyltransferase sequences and their manual alignment and identified which sequence of  E. faecalis  is the putrescine carbamoyltransferase. BL carried out the phylogenetic analyses, the gene context study and drafted the manuscript which was further improved (and approved) by all authors.
Authors' contributions
NG dug up the "ancient" data on putrescine carbamoyltransferase, contributed his knowledge about carbamoyltransferases and made important additions to the manuscript. YX brought essential informations about the genetics and biochemistry of the enzymes involved in arginine metabolism and their evolution. DGN participated in the collection of new carbamoyltransferase sequences and their manual alignment and identified which sequence of  E. faecalis  is the putrescine carbamoyltransferase. BL carried out the phylogenetic analyses, the gene context study and drafted the manuscript which was further improved (and approved) by all authors.E. faecalis


A joint task force of the American Society of Cytopathology (ASC) and the National Cancer Institute (NCI) recently completed a 2-year effort to revise the Bethesda System "blue book" atlas and develop a complementary web-based collection of cervical cytology images. The web-based collection of images is housed on the ASC website, which went live on November 5 th , 2003; it can be directly accessed at  .

The second edition of the Bethesda 'blue book' atlas maintains an easy to read format with bulleted morphologic criteria and half-page color illustrations. The content has been divided into chapters based on the major 2001 Bethesda System interpretive categories. Highlights of the new edition include: (1) incorporation of liquid based cytomorphologic criteria and images; (2) new sections addressing ancillary testing, educational notes and recommendations, computer assisted interpretation and anal-rectal cytology; and (3) inclusion of sample reports, references, and detailed legends for images. Overall, the second edition has tripled in size from the original version, with a total of 186 images of which 90% are new images and 40% are from liquid based specimens. Some are classic examples of an entity while others have been selected to illustrate interpretive dilemmas or "borderline" cytomorphologic features that may not be interpreted in the same way by all cytologists.In parallel with production of the Bethesda book atlas, the ASC-NCI task force has also developed a Bethesda web-based collection of images. Approximately 350 images, (40% of which are from liquid based specimens) along with linked explanatory notes, including all the images in the published atlas, can be viewed on this site. The website is user friendly and has several search modalities for viewing the images including searching by Bethesda terminology, atlas chapter headings, keyword(s), or preparation type. It also allows for individual self-assessment by participating in a "self test" in which viewers can compare their interpretation to other participants' responses.The image selection process for the book atlas and website involved a multistage review:  Step 1 : individual Bethesda forum group members  (32 participants)  reviewed and selected images for their chapter from among those in the first edition of the atlas and new submissions; and  Step 2 : the images selected from Step 1 were reviewed individually ("validated") by 13 task force members and scored on a scale of 1–5 for agreement with interpretation and quality of image. In all over 1000 images were reviewed of which 186 images were selected for the atlas and an additional 163 for the website.Step 1(32 participants)Step 2A subset (n = 77) of the book atlas images were posted as "unknowns" on the Internet from mid July to mid September 2003 as part of a study – the Bethesda Interobserver Reproducibility Project (BIRP). The site was open to the cytopathology community to view the images and provide their interpretations. Immediately after submitting their response, participants were able to view a histogram of the distribution of results submitted by all prior participants for that image. Over 600 cytologists from around the world participated in BIRP. Summary histograms for each of the 77 images can be viewed on the Bethesda atlas website (select BIRP images from the left menu). Preliminary BIRP results presented at the ASC annual meeting in Orlando in November 2003 showed that Negative for Intraepithelial Lesion or Malignancy (NILM) and Low Grade Squamous Intraepithelial Lesion (LSIL) reference images attained the highest concordance scores, while glandular abnormalities demonstrated the most splay in distribution of interpretations. BIRP analyses are ongoing and further results should be available in 2004.ASC-NCI Working Group for the Second Edition Bethesda Atlas and Website ASC Bethesda 2001 Task Force: Ritu Nayar  (Chair), Diane Solomon (Co-Chair, NCI)  George Birdsong  (Adequacy),  Jamie Covell  (Glandular Lesions),  Ann Moriarty  (Endometrial cells),  Dennis O'Connor  (Educational notes and recommendations),  Marianne Prey  (Computer assisted interpretation),  Steve Raab  (Ancillary testing),  Mark Sherman  (Atypical squamous cells),  Sana Tabbara  (Other malignant neoplasms),  Tom Wright  (Squamous lesions),  Nancy Young  (Non-neoplastic findings). ASC Consultants :  ASC 2002/2003 Presidents:  Diane Davey and Dave Wilbur . Information Technology representatives :  Mike Montgomery (NCI) and Brandon Winbush (Northwestern University), Aquilent (Laurel, MD) . The second edition of the 'blue book' atlas can be ordered through Springer-Verlag (1-800-SPRINGE) for $34.95.
ASC-NCI Working Group for the Second Edition Bethesda Atlas and Website
ASC Bethesda 2001 Task Force: Ritu Nayar  (Chair), Diane Solomon (Co-Chair, NCI)  George Birdsong  (Adequacy),  Jamie Covell  (Glandular Lesions),  Ann Moriarty  (Endometrial cells),  Dennis O'Connor  (Educational notes and recommendations),  Marianne Prey  (Computer assisted interpretation),  Steve Raab  (Ancillary testing),  Mark Sherman  (Atypical squamous cells),  Sana Tabbara  (Other malignant neoplasms),  Tom Wright  (Squamous lesions),  Nancy Young  (Non-neoplastic findings).ASC Bethesda 2001 Task Force:ASC Bethesda 2001 Task Force:Ritu NayarGeorge BirdsongJamie CovellAnn MoriartyDennis O'ConnorMarianne PreySteve RaabMark ShermanSana TabbaraTom WrightNancy YoungASC Consultants :  ASC 2002/2003 Presidents:  Diane Davey and Dave Wilbur .ASC Consultants :Diane Davey and Dave WilburInformation Technology representatives :  Mike Montgomery (NCI) and Brandon Winbush (Northwestern University), Aquilent (Laurel, MD) .Information Technology representatives :Mike Montgomery (NCI) and Brandon Winbush (Northwestern University), Aquilent (Laurel, MD)The second edition of the 'blue book' atlas can be ordered through Springer-Verlag (1-800-SPRINGE) for $34.95.