Circulating microRNA-99 family as liquid biopsy marker in pancreatic adenocarcinoma.

PURPOSE: Recently, we identified the microRNA-99 family as unfavorable prognostic factor in patients with pancreatic ductal adenocarcinoma (PDAC). The aim of this study is to evaluate its value as circulating biomarker for PDAC. METHODS: Tissue and corresponding preoperative blood samples of 181 patients with PDAC UICC Stages I-IV (n = 90), intraductal papillary mucinous neoplasm (IPMN, n = 11), chronic pancreatitis (n = 40), pancreatic cystadenoma (n = 20), and age-matched healthy blood serum controls (n = 20) were collected between 2014 and 2017 prospectively. Expression of microRNA-21 as confirmatory marker and the microRNA-99 family, consisting of microRNA-99a, -99b, and -100, was analyzed by qRT-PCR. Target analysis of insulin-like growth factor 1 receptor (IGF1R) was performed using tissue array immunohistochemistry and Western blotting. RESULTS: Expression of microRNA-99 family members was significantly increased in macrodissected tumor tissue and corresponding blood serum samples (p < 0.05) of patients with PDAC of all stages. Correspondingly, its target protein IGF1R was upregulated (p < 0.001) in carcinoma tissue. Circulating and tissue-related microRNA-100 could well discriminate PDAC from healthy samples with area under the receiver operating characteristic (ROC) curve (AUC) values of 0.81 and 0.85, respectively. Low expression of circulating microRNA-100 was associated with significantly improved overall survival (p = 0.004) and recurrence-free survival (p = 0.03) in multivariate analyses. Circulating microRNA-21 was overexpressed in PDAC with fair discrimination between PDAC and healthy controls (AUC = 0.71) and decreased overall survival (p = 0.046) and recurrence-free survival (p = 0.03) in PDAC patients. CONCLUSIONS: Multivariate survival and ROC analyses identified circulating microRNA-100 as potential diagnostic and prognostic marker in PDAC patients.

The Rule of Histology in the Diagnosis of Periprosthetic Infection: Specific Granulocyte Counting Methods and New Immunohistologic Staining Techniques may Increase the Diagnostic Value.

OBJECTIVE: The current study investigates the diagnostic accuracy of the criteria described for frozen sections and whether modern leukocyte specific staining techniques including leukocyte peroxidase and Naphtol-AS-D-chloroacetate-esterase will improve the accuracy of the intra-operative histology. METHOD: 77 patients undergoing revision total hip and knee arthroplasty were included in this retrospective study. Patients were grouped into septic and aseptic based on intraoperative cultures. Tissue samples were analyzed utilizing the Mirra, Feldman, Lonner, Banit and Athanasou criteria. RESULTS: An experienced pathologist had a high specificity (96%), but rather low sensitivity (57%) diagnosing infection. By using the Banit-, Mirra-, or Athanasou-criteria the sensitivity is increased to 0.90. The Feldman- and Lonner-criteria have a lower sensitivity (0.48 and 0.38), however, an increased specificity of 0.96 and 0.98, respectively. The Banit cut off has the highest accuracy (86%). MPOX and NACE staining increased the sensitivity and accuracy up to 100% and 92% respectively. CONCLUSION: Banit's cut off is the most accurate histologic criteria to diagnose infection. Modern leukocyte specific staining techniques slightly improve the accuracy. The synovial fluid white blood cell count appears to be the most accurate intraoperative test.

Complex MLL rearrangement in non-infiltrated bone marrow in an infant with stage II precursor B-lymphoblastic lymphoma.

PURPOSE: Precursor B-lymphoblastic lymphoma cells are indistinguishable by morphology, and immune phenotype from lymphoblasts in acute leukemia which in infancy is associated with MLL rearrangements and a poor prognosis. The role of MLL gene deregulation in rare cases of isolated lymphoblastic lymphoma in infants is obscure. We report the case of a 10-month-old child who presented with a cutaneous nodule on the left foot. Histological diagnosis was precursor B-lymphoblastic lymphoma. The young age of the patient motivated us to investigate the presence of an MLL rearrangement. METHODS: Cytogenetic analysis was performed by fluorescence in situ hybridization (FISH), and the genomic fusion partner of MLL was identified by long-distance inverse (LDI-)PCR and confirmed by direct PCR. RESULTS: Fluorescence in situ hybridization screening of paraffin-embedded formalin-fixed tissue indeed revealed the presence of an MLL rearrangement. The genomic fusion partner was identified as AF10 by DNA sequencing of the MLL breakpoint region. The MLL-AF10 fusion gene was further detected in cytologically normal pretreated bone marrow. Treatment was started with standard four-drug induction chemotherapy. Because of the unfavorable outcome associated with MLL rearrangements in infant leukemia, we intensified postremission treatment according to the Interfant-06 study protocol. The child is in continuous first remission 36 months after diagnosis. CONCLUSION: This is the first report of submicroscopic bone marrow involvement in MLL-rearranged isolated cutaneous B-cell precursor lymphoma in an infant. To prospectively address the role of MLL rearrangements in extramedullary B-lymphoblastic malignancies in infants, we suggest to assess both tumors and non-infiltrated bone marrow for the presence of this genetic abnormality.

Genomic EWS-FLI1 fusion sequences in Ewing sarcoma resemble breakpoint characteristics of immature lymphoid malignancies.

Chromosomal translocations between the EWS gene and members of the ETS gene family are characteristic molecular features of the Ewing sarcoma. The most common translocation t(11;22)(q24;q12) fuses the EWS gene to FLI1, and is present in 85-90% of Ewing sarcomas. In the present study, a specifically designed multiplex long-range PCR assay was applied to amplify genomic EWS-FLI1 fusion sites from as little as 100 ng template DNA. Characterization of the EWS-FLI1 fusion sites of 42 pediatric and young adult Ewing sarcoma patients and seven cell lines revealed a clustering in the 5' region of the EWS-breakpoint cluster region (BCR), in contrast to random distribution of breakpoints in the FLI1-BCR. No association of breakpoints with various recombination-inducing sequence motifs was identified. The occurrence of small deletions and duplications at the genomic junction is characteristic of involvement of the non-homologous end-joining (NHEJ) repair system, similar to findings at chromosomal breakpoints in pediatric leukemia and lymphoma.

Genome-wide analysis of histone H3 acetylation patterns in AML identifies PRDX2 as an epigenetically silenced tumor suppressor gene.

With the use of ChIP on microarray assays in primary leukemia samples, we report that acute myeloid leukemia (AML) blasts exhibit significant alterations in histone H3 acetylation (H3Ac) levels at > 1000 genomic loci compared with CD34(+) progenitor cells. Importantly, core promoter regions tended to have lower H3Ac levels in AML compared with progenitor cells, which suggested that a large number of genes are epigenetically silenced in AML. Intriguingly, we identified peroxiredoxin 2 (PRDX2) as a novel potential tumor suppressor gene in AML. H3Ac was decreased at the PRDX2 gene promoter in AML, which correlated with low mRNA and protein expression. We also observed DNA hypermethylation at the PRDX2 promoter in AML. Low protein expression of the antioxidant PRDX2 gene was clinically associated with poor prognosis in patients with AML. Functionally, PRDX2 acted as inhibitor of myeloid cell growth by reducing levels of reactive oxygen species (ROS) generated in response to cytokines. Forced PRDX2 expression inhibited c-Myc-induced leukemogenesis in vivo on BM transplantation in mice. Taken together, epigenome-wide analyses of H3Ac in AML led to the identification of PRDX2 as an epigenetically silenced growth suppressor, suggesting a possible role of ROS in the malignant phenotype in AML.

Calcitonin substitution in calcitonin deficiency reduces particle-induced osteolysis.

BACKGROUND: Periprosthetic osteolysis is a major cause of aseptic loosening in joint arthroplasty. This study investigates the impact of CT (calcitonin) deficiency and CT substitution under in-vivo circumstances on particle-induced osteolysis in Calca -/- mice. METHODS: We used the murine calvarial osteolysis model based on ultra-high molecular weight polyethylene (UHMWPE) particles in 10 C57BL/6J wild-type (WT) mice and twenty Calca -/- mice. The mice were divided into six groups: WT without UHMWPE particles (Group 1), WT with UHMWPE particles (Group 2), Calca -/- mice without UHMWPE particles (Group 3), Calca -/- mice with UHMWPE particles (Group 4), Calca -/- mice without UHMWPE particles and calcitonin substitution (Group 5), and Calca -/- mice with UHMWPE particle implantation and calcitonin substitution (Group 6). Analytes were extracted from serum and urine. Bone resorption was measured by bone histomorphometry. The number of osteoclasts was determined by counting the tartrate-resistant acid phosphatase (TRACP) + cells. RESULTS: Bone resorption was significantly increased in Calca -/- mice compared with their corresponding WT. The eroded surface in Calca -/- mice with particle implantation was reduced by 20.6% after CT substitution. Osteoclast numbers were significantly increased in Calca -/- mice after particle implantation. Serum OPG (osteoprotegerin) increased significantly after CT substitution. CONCLUSIONS: As anticipated, Calca -/- mice show extensive osteolysis compared with wild-type mice, and CT substitution reduces particle-induced osteolysis.

Life-threatening hypersplenism due to idiopathic portal hypertension in early childhood: case report and review of the literature.

BACKGROUND: Idiopathic portal hypertension (IPH) is a disorder of unknown etiology and is characterized clinically by portal hypertension, splenomegaly, and hypersplenism accompanied by pancytopenia. This study evaluates the pathogenic concept of the disease by a systematic review of the literature and illustrates novel pathologic and laboratory findings. CASE PRESENTATION: We report the first case of uncontrolled splenic hyperperfusion and enlargement with subsequent hypersplenism leading to life-threatening complications of IPH in infancy and emergent splenectomy. CONCLUSIONS: Our results suggest that splenic NO and VCAM-1, rather than ET-1, have a significant impact on the development of IPH, even at a very early stage of disease. The success of surgical interventions targeting the splenic hyperperfusion suggests that the primary defect in the regulation of splenic blood flow seems to be crucial for the development of IPH. Thus, beside other treatment options splenectomy needs to be considered as a prime therapeutic option for IPH.

Impact of EWS-ETS fusion type on disease progression in Ewing's sarcoma/peripheral primitive neuroectodermal tumor: prospective results from the cooperative Euro-E.W.I.N.G. 99 trial.

PURPOSE EWS-ETS fusion genes are the driving force in Ewing's sarcoma pathogenesis. Because of the variable breakpoint locations in the involved genes, there is heterogeneity in fusion RNA and protein architecture. Since previous retrospective studies suggested prognostic differences among patients expressing different EWS-FLI1 fusion types, the impact of fusion RNA architecture on disease progression and relapse was studied prospectively within the Euro-E.W.I.N.G. 99 clinical trial. PATIENTS AND METHODS Among 1,957 patients who registered before January 1, 2007, 703 primary tumors were accessible for the molecular biology study. Fusion type was assessed by polymerase chain reaction on frozen (n = 578) or paraffin-embedded materials (n = 125). The primary end point was the time to disease progression or relapse. Results After exclusion of noninformative patients, 565 patients were entered into the prognostic factor analysis comparing type 1 (n = 296), type 2 (n = 133), nontype 1/nontype 2 EWS-FLI1 (n = 91) and EWS-ERG fusions (n = 45). Median follow-up time was 4.5 years. The distribution of sex, age, tumor volume, tumor site, disease extension, or histologic response did not differ between the four fusion type groups. We did not observe any significant prognostic value of the fusion type on the risk of progression or relapse. The only slight difference was that the risk of progression or relapse associated with nontype 1/nontype 2 EWS-FLI1 fusions was 1.38 (95% CI, 0.96 to 2.0) times higher than risk associated with other fusion types, but it was not significant (P = .10). CONCLUSION In contrast to retrospective studies, the prospective evaluation did not confirm a prognostic benefit for type 1 EWS-FLI1 fusions.

Minireview: Malassezia infections in immunocompromised patients.

Malassezia spp. form part of the normal human cutaneous flora and are implicated in several mild, but recurrent cutaneous diseases, such as pityriasis versicolor, Malassezia folliculitis, seborrhoeic dermatitis, and, with lesser frequency, a range of other dermatological disorders. Malassezia spp. have also been associated with cutaneous and systemic diseases in immunocompromised patients including folliculitis, seborrhoeic dermatitis, catheter-related fungaemia and a variety of deeply invasive infections. In this review, we provide an overview of the epidemiology, risk factors, pathogenesis, clinical manifestations, diagnosis, treatment and outcome of cutaneous and invasive Malassezia infections in immunocompromised patients.

Treatment options for recurrent giant cell tumors of bone.

BACKGROUND: Although the recurrence rate of giant cell tumors of bone (GCTB) is relatively high exact data on treatment options for the recurrent cases is lacking. The possible surgical procedures range from repeated intralesional curettage to wide resection. METHODS: Two hundred and fourteen patients with histologically certified GCTB have been treated at the authors department from 1980 to 2007. Sixty-seven patients with at least one local recurrence were included in this study. The mean follow-up was 77.3 months. The data was evaluated according the re-recurrence rate with regard to the surgical procedure for the recurrence. RESULTS: The mean time until the first local recurrence was 22.0 months; the mean number of recurrences per patient was 1.4. The recurrence occurred in 69.7% (46 out of 66 patients) within the first 2 years. If after intralesional procedures (curettage or intralesional resection) no adjunct was used the re-recurrence rate was 58.8% (10 out of 17 patients) and decreased to 21.7% (5 out of 23 patients) if a combination of all adjuncts (PMMA + burring) was used. The likelihood of re-recurrence was reduced by the factor 5.508 which was clearly significant (P = 0.016). In case of wide resection no re-recurrence occurred. Seven patients (10.5%) developed pulmonary metastases. Fourteen patients (20.9%) finally received an endoprosthesis; 12 due to tumor recurrence, 2 due to secondary arthritis. CONCLUSION: Recurrent GCTB can be treated by further curettage with additional burring and cementing with an acceptable re-recurrence rate of 21.7%. The rate of patients finally needing an endoprosthesis is 20.9%. Due to the high rate of pulmonary metastases recurrent GCTB may be considered as a severe disease.

Giant cell tumor of bone: treatment and outcome of 214 cases.

BACKGROUND: Two hundred and fourteen patients with benign giant cell tumor of bone (GCTB), treated from 1980 to 2007 at the Department of Orthopedics of the University of Muenster (Germany), were analyzed in a retrospective study. PATIENTS AND METHODS: The mean age was 33.3 years with a female-to-male ratio of 1.2 : 1. The mean follow up was 59.8 months. The recurrence rate of patients who received first treatment at our institution was 16.6%. The most common primary treatment was curettage (188 patients) usually followed by adjuvant local therapy. The effects of bone cement (PMMA), burring and hydrogen peroxide (H(2)O(2)) were statistically analyzed and the influence of a subchondral bone graft on the recurrence rate was evaluated. RESULTS: PMMA alone (n = 52) reduces the likelihood of recurrence by the factor 8.2, additional high-speed burring (n = 39) by the factor 3.9 (compared to PMMA only). H(2)O(2) (n = 42) seems to have an additional effect comparable to that of phenol although it did not reach statistical significance. CONCLUSION: The combination of all adjuncts (PMMA, burring, H(2)O(2) - n = 42) reduces the likelihood of recurrence by the factor 28.2 compared to curettage only and therefore should be recommended as a standard treatment. If the tumor reaches close to the articulating surface a subchondral bone graft (n = 42) can be performed without risking a higher recurrence rate. We add seven cases of pulmonary metastases and two cases of multicentricity to the literature. Bisphosphonates and interferon alpha may have a beneficial effect.

Endoscopic ultrasound with conventional probe and miniprobe in preoperative staging of esophageal cancer.

BACKGROUND: Using an endoscopic ultrasound (EUS) miniprobe, even highly stenotic esophageal cancers precluding the passage of a conventional probe can be examined without prior dilatation. OBJECTIVE: To assess: (1) staging accuracy of conventional EUS probe and miniprobe, (2) variables influencing staging accuracy, (3) endoscopic features predicting tumor stage. METHODS: Ninety-seven consecutive patients with esophageal cancer undergoing complete surgical resection were included. Preoperative EUS was performed using a conventional probe in nonstenotic tumors and a miniprobe in stenotic tumors. Accuracy of EUS for T and N stages was compared to pathohistological staging. RESULTS: Overall EUS staging accuracy was 73.2% for T stage and 74.2% for N stage. It was similar for the miniprobe used in stenotic tumors vs the conventional probe used in nonstenotic tumors. Based on EUS, 84.5% of the patients would have been assigned to the appropriate therapy protocol (primary surgery vs neoadjuvant therapy). Endoscopic tumor features had no influence on staging accuracy. Tumor length >5 cm predicted advanced T and nodal positive stages. CONCLUSIONS: The miniprobe allows adequate EUS staging of stenotic esophageal tumors precluding the passage of a conventional probe. Therefore, dilatation therapy of stenotic cancers to conduct conventional EUS should be avoided.

Continuous versus bolus infusion of terlipressin in ovine endotoxemia.

In patients with sepsis, hemodynamic support is often complicated by a tachyphylaxis against conventional vasopressor agents. Bolus infusion of terlipressin, a vasopressin analog, has been reported to increase mean arterial pressure in patients with catecholamine-resistant septic shock. However, bolus infusion of terlipressin may be associated with severe side effects, including pulmonary vasoconstriction and impairment of oxygen delivery. We hypothesized that continuous low-dose infusion of terlipressin may reverse sepsis-related systemic arterial hypotension with reduced side effects as compared with the traditional concept of bolus administration. Twenty-seven adult sheep were instrumented for chronic study. After a baseline measurement, Salmonella typhosa endotoxin (10 ng.kg-1.min-1) was continuously administered for the next 40 h. After 16 h of endotoxemia, the surviving sheep (n = 24) were randomly assigned to be treated with either a continuous infusion of terlipressin (2 mg for 24 h), bolus injections of terlipressin (1 mg every 6 h), or placebo (normal saline; each n = 8). Continuous infusion of terlipressin permanently reversed endotoxin-induced systemic arterial hypotension (P < 0.001) and improved left ventricular stroke work index in all sheep (P < 0.05). Intermittent bolus injections of terlipressin were linked to decreases in heart rate and cardiac index and increases in pulmonary vascular resistance index (each, P < 0.001). These unwanted side effects were prevented by continuous low-dose infusion of the drug. In conclusion, continuous infusion of terlipressin stabilized hemodynamics and improved myocardial performance in endotoxemic ewes without obvious side effects. Continuous low-dose terlipressin infusion may represent a useful alternative treatment of arterial hypotension related to sepsis and systemic inflammatory response syndrome.

Evaluation of tumour necrosis during chemotherapy with diffusion-weighted MR imaging: preliminary results in osteosarcomas.

BACKGROUND: During successful chemotherapy of osteosarcomas tumour size does not diminish significantly because the therapy has limited impact on the mineralized matrix of the tumour. Treatment response is considered successful if, histologically, more than 90% of tumour cells show necrosis. OBJECTIVE: To determine if osteosarcomas change their water diffusion during preoperative chemotherapy in relation to the amount of tumour necrosis. MATERIALS AND METHODS: Eight patients (age 11-19 years) with histologically proven limb osteosarcoma underwent T1-weighted, fat-suppressed T2-weighted and contrast-enhanced T1-weighted spin-echo imaging together with diffusion-weighted EPI sequences (b = 700) at 1.5 T before and after five cycles of standard chemotherapy. Tumour volume and apparent diffusion coefficient (ADC) maps were calculated before and after chemotherapy. The degree of tumour necrosis after chemotherapy was assessed using the histological Salzer-Kuntschik classification (grades 1-6). RESULTS: During chemotherapy, the ADC values of osteosarcomas changed significantly. The ADC of untreated tumour was 2.1 +/- 0.4 x 10(-3) mm(2)/s (mean +/- SD) (95% CI 1.6-2.0). The ADC of chemotherapy-treated sarcomas was 2.5 +/- 0.4 x 10(-3) mm(2)/s (95% CI 1.8-2.2). Necrotic areas, which were confirmed by macroscopic examination, showed ADC values up to 2.7 x 10(-3) mm(2)/s. Four patients with little viable tumour tissue within the neoplasm (Salzer-Kuntschik grades 1-2) had an increase in ADC of 0.4 up to 0.7 x 10(-3) mm(2)/s. Four patients with larger areas of viable tumour (Salzer-Kuntschik grade 4) showed a lesser increase in ADC of 0.0 up to 0.3 x 10(-3) mm(2)/s. The differences in ADC values in tumour tissue before and after chemotherapy were highly significant (P = 0.01). CONCLUSION: During chemotherapy of osteosarcomas, tumour ADC changes are related to the degree of tumour necrosis.

Nitric oxide synthase is up-regulated in muscle fibers in muscular dystrophy.

Nitric oxide (NO) mediates fundamental physiological actions on skeletal muscle. The neuronal NO synthase isoform (NOS1) was reported to be located exclusively in the sarcolemma. Its loss from the sarcolemma was associated with development of Duchenne muscular dystrophy (DMD). However, new studies evidence that all three NOS isoforms-NOS1, NOS2, and NOS3-are co-expressed in the sarcoplasm both in normal and in DMD skeletal muscles. To address this controversy, we assayed NOS expression in DMD myofibers in situ cytophotometrically and found NOS expression in DMD myofibers up-regulated. These results support the hypothesis that NO deficiency with consequent muscle degeneration in DMD results from NO scavenging by superoxides rather than from reduced NOS expression.

Mechanisms of ammonium transport, accumulation, and retention in ooyctes and yeast cells expressing Arabidopsis AtAMT1;1.

Ammonium is a primary source of N for plants, so knowing how it is transported, stored, and assimilated in plant cells is important for rational approaches to optimise N-use in agriculture. Electrophysiological studies of Arabidopsis AtAMT1;1 expressed in oocytes revealed passive, Deltapsi-driven transport of NH(4)(+) through this protein. Expression of AtAMT1;1 in a novel yeast mutant defective in endogenous ammonium transport and vacuolar acidification supported the above mechanism for AtAMT1;1 and revealed a central role for acid vacuoles in storage and retention of ammonia in cells. These results highlight the mechanistic differences between plant AMT proteins and related transporters in bacteria and animal cells, and suggest novel strategies to enhance nitrogen use efficiency in agriculture.

Nitric oxide synthase in muscular dystrophies: a re-evaluation.

Duchenne and Becker muscular dystrophies (DMD and BMD) are associated with decreased total nitric oxide (NO). However, mechanisms leading to NO deficiency with consequent muscle-cell degeneration remain unknown. To address this issue, we examined skeletal muscles of DMD and BMD patients for co-expression of NO synthase (NOS) with nitrotyrosine and transcription factor CREB, as well as with enzymes engaged in NO signaling. Employing immunocytochemical labeling, Western blotting and RT-PCR, we found that, in contrast to the most commonly accepted view, neuronal NOS was not restricted to the sarcolemma and that muscles of DMD and BMD patients retained all three NOS isoforms with an up-regulation of the inducible NOS isoform, CREB and nitrotyrosine. We suggest that enhanced nitrotyrosine immunostaining in muscle fibers as well as in the vasculature of DMD and BMD specimens reflects massive oxidative stress, resulting in withdrawal of NO from its regular physiological course via the scavenging actions of superoxides.

Altered cytokine responses to cognitive stress in multiple sclerosis patients with fatigue.

This study intended to examine if the immune response to a cognitive task as a variant of psychological stress in MS patients is distinct from healthy controls. The experiment was part of a larger study on mechanisms and measurements of MS fatigue. Patients (n =23) and controls (n =25) participated in a cognitive task lasting 40 minutes, in which the heart rate was continuously monitored. Blood samples were taken at baseline and directly after the stress-inducing task Whole blood stimulated cytokine production representative of the TH-1 (i.e. IFNgamma, TNFalpha) and TH-2 paradigm (i.e. IL-10) was evaluated in relation to disability, fatigue, cognitive deficit, and anxiety. Patients scored high on a disease specific fatigue score compared to controls, whereas baseline cytokine patterns did not differ between the groups. MS patients displayed a blunted response of IFNgamma (P =0.03) whereas TNFalpha and IL-10 responses did not change. Additionally MS patients showed a significantly lower heart rate increase after the task (P <0.001). Cognitive impairment was associated with a decreased heart rate reactivity (P =0.02) while depressive symptoms correlated with stronger IL-10 responses (P =0.05). Overall, cognitive stress induces IFNgamma production in healthy controls but not in MS patients with fatigue. Furthermore, a reduced cardiac response might indicate an autonomic dysfunction in this group of patients.

Characterization of the Saccharomyces cerevisiae Fol1 protein: starvation for C1 carrier induces pseudohyphal growth.

Tetrahydrofolate (vitamin B9) and its folate derivatives are essential cofactors in one-carbon (C1) transfer reactions and absolutely required for the synthesis of a variety of different compounds including methionine and purines. Most plants, microbial eukaryotes, and prokaryotes synthesize folate de novo. We have characterized an important enzyme in this pathway, the Saccharomyces cerevisiae FOL1 gene. Expression of the budding yeast gene FOL1 in Escherichia coli identified the folate biosynthetic enzyme activities dihydroneopterin aldolase (DHNA), 7,8-dihydro-6-hydroxymethylpterin-pyrophosphokinase (HPPK), and dihydropteroate synthase (DHPS). All three enzyme activities were also detected in wild-type yeast strains, whereas fol1Delta deletion strains only showed background activities, thus demonstrating that Fol1p catalyzes three sequential steps of the tetrahydrofolate biosynthetic pathway and thus is the central enzyme of this pathway, which starting from GTP consists of seven enzymatic reactions in total. Fol1p is exclusively localized to mitochondria as shown by fluorescence microscopy and immune electronmicroscopy. FOL1 is an essential gene and the nongrowth phenotype of the fol1 deletion leads to a recessive auxotrophy for folinic acid (5'-formyltetrahydrofolate). Growth of the fol1Delta deletion strain on folinic acid-supplemented rich media induced a dimorphic switch with haploid invasive and filamentous pseudohyphal growth in the presence of glucose and ammonium, which are known suppressors of filamentous and invasive growth. The invasive growth phenotype induced by the depletion of C1 carrier is dependent on the transcription factor Ste12p and the flocullin/adhesin Flo11p, whereas the filamentation phenotype is independent of Ste12p, Tec1p, Phd1p, and Flo11p, suggesting other signaling pathways as well as other adhesion proteins.