Recombinant AcnB, NrdR and RibD of Acinetobacter baumannii and their potential interaction with DNA adenine methyltransferase AamA.

In the last decades Acinetobacter baumannii developed into an increasingly challenging nosocomial pathogen. A. baumannii ATCC 17978 harbors a DNA-(adenine N6)-methyltransferase termed AamA. Previous studies revealed a low specific activity of AamA in vitro despite proven folding, which led us to speculate about possible interaction partners assisting AamA in targeting methylation sites. Here, applying a pulldown assay with subsequent mass spectrometry we identified aconitate hydratase 2 (AcnB) as possible interaction partner. In addition, we considered the putative transcriptional regulator gene nrdR (A1S_0220) and the pyrimidine deaminase/reductase gene ribD (A1S_0221) of A. baumannii strain ATCC 17978 to encode additional potential interaction partners due to their vicinity to the aamA gene (A1S_0222). Proteins were recombinantly produced in the milligram scale, purified to near homogeneity, and interactions with AamA were studied applying blue native gel electrophoreses, electrophoretic mobility shift assay, chemical cross-linking and co-immunoprecipitation. These analyses did not provide evidence of interaction between AamA and purified proteins. Solution structures of RibD, NrdR and AcnB were studied by small-angle X-ray scattering (SAXS) alone and in combination with AamA. While in the case of RibD and AcnB no evidence of an interaction with AamA was produced, addition of AamA to NrdR resulted in dissociation of long and rod-shaped polymeric NrdR structures, implying a specific but transient interaction. Moreover, we identified a molecular crowding effect possibly impeding the DNA methyltransferase activity in vivo and a sequence-independent DNA binding activity of AamA calling for continued efforts to identify the interaction network of AamA.

Janus bifunctional periodic mesoporous organosilica.

Synthesis of a Janus periodic mesoporous organosilica material (JPMO) is presented here. In this strategy, the surface of the hollow silica material was selectively functionalized with two different bridged organic-inorganic hybrid groups. It was found that the resulting bifunctional material is able to form a stable Pickering emulsion. This new type of PMO material may be suitable for widespread applications in various fields related to material science and catalysis.

Evaluating the Role of Interdigitated Neoadjuvant Chemotherapy and Radiation in the Management of High-Grade Soft-Tissue Sarcoma: The Johns Hopkins Experience.

OBJECTIVES: High-grade soft-tissue sarcoma (STS) has a poor prognosis. The goal of this study was to review treatment outcomes of patients with high-grade STS treated with interdigitated neoadjuvant chemotherapy (CT) and radiation at our institution. MATERIALS AND METHODS: Patients with high-grade STS (1997 to 2010) were planned for treatment with 3 cycles of neoadjuvant CT, interdigitated preoperative radiation therapy (44 Gy administered in split courses with a potential 16 Gy postoperative boost), and 3 cycles of postoperative CT. Cancer control outcomes at 3 years were analyzed. RESULTS: Sixteen patients with high-grade STS were evaluated. Median age was 53 years, the median longest tumor diameter was 14.6 cm, and median follow-up was 33 months. All 16 patients received 2 or 3 cycles of neoadjuvant CT and all patients completed neoadjuvant RT. The estimated 3-year rate for local control was 100%, disease-free survival 62.5%, and overall survival 73.4%. CONCLUSIONS: Patients with high-grade STS treated with interdigitated neoadjuvant CT and radiation before surgical resection had excellent rates of local control, along with disease-free survival and overall survival similar to previously published reports. This combined-modality approach continues to have a role in the treatment of patients with high-grade STS.

Deep Digging: Far Red Imaging for the Monitoring of Transplanted Hepatocytes in Rats.

Technologies for in vivo imaging of the distribution and integration of cell transplants gain significance for the use of novel cell therapy approaches in regenerative medicine. Applied to adequate animal models, they provide information on the spatio-temporal engraftment and functional performance of the cells transplanted. This chapter includes a detailed description of the in vivo tracking of transplanted hepatocytes in rat liver including the conjugation of antibodies to fluorochromes for far red imaging using a multispectral optical imager.

Characterization of musculoskeletal lesions on 3-T proton MR spectroscopy.

OBJECTIVE: The purpose of our study was to determine the feasibility and value of proton MR spectroscopy at 3 T for characterizing musculoskeletal tumors. SUBJECTS AND METHODS: At 3 T, 18 patients with musculoskeletal lesions (four histologically proven to be malignant, 14 proven benign histologically or at clinical follow-up) underwent 23 MR spectroscopy studies, 20 with a single-voxel technique and three with a multivoxel technique. Seventeen patients were imaged with a surface coil and six with a body coil. Choline signal (3.2 ppm) was measured in each voxel and expressed relative to background noise as signal-to-noise ratio (SNR). Choline SNRs of malignant tumors and benign lesions were compared. RESULTS: Diagnostic spectra were obtained in 20 of 23 lesions. For malignant lesions (osteosarcoma with two MR spectroscopy sites, metastasis, grade 1 sarcoma), choline SNRs were 5.2 and 4.2 (performed with body coil) and 4.8 and 18.7 (performed with surface coil), respectively. For benign lesions (neurofibroma, two stress reactions, bone cyst, hemangioma, lipoma, Baker cyst), choline SNR was 6.3 (with surface coil), 5.5 (with surface coil), and not detected for five cases. Seven postoperative patients with myocutaneous flaps showed either the typical spectrum of muscle or negligible choline. Only a water peak existed in a bone cyst and a significant lipid peak in a lipoma. Choline SNRs were different for malignant and benign lesions (11.7 vs 2.3, p = 0.04, as performed with a surface coil). CONCLUSION: At 3 T, both single-voxel and multivoxel MR spectroscopy are feasible. Proton MR spectroscopy is a potential noninvasive tool for characterizing lesion composition and malignant activity.

High WT1 expression is associated with very poor survival of patients with osteogenic sarcoma metastasis.

PURPOSE: Although metastasis is the primary determinant of poor survival of patients with osteogenic sarcoma, some patients live much longer than others, indicating metastatic heterogeneity underlying survival outcome. The purpose of the investigation was to identify genes underlying survival outcome of patients with osteogenic sarcoma metastasis. EXPERIMENTAL DESIGN: We have used microarray to first compare mRNA expression between normal bone and osteogenic sarcoma specimens, identified genes overexpressed in osteogenic sarcoma, and compared expression of the selected gene between a poorly metastatic (SAOS) and two highly metastatic cell lines (LM8 and 143B). Finally, expression of the selected gene was assessed by immunostaining of osteogenic sarcoma samples with known survival outcome. RESULTS: Microarray analysis revealed 5.3-fold more expression of WT1 mRNA in osteogenic sarcoma compared with normal bone and >2-fold overexpression in 143B and LM8 cells compared with SAOS. Furthermore, WT1 mRNA was absent in normal bone (10 of 10) by reverse transcription-PCR but present in osteogenic sarcoma-derived cell lines (5 of 8). One hundred percent (42 of 42) of low-grade osteogenic sarcoma specimens expressed no WT1 as determined by immunostaining; however, 24% (12 of 49) of the high-grade specimens showed intense staining. Mean survival of patients with high-grade metastatic osteogenic sarcoma but low WT1 staining (27 of 37) was 96.5 +/- 129.3 months, whereas mean survival of patients with high-grade metastatic osteogenic sarcoma having intense staining (10 of 37) was 18.3 +/- 12.3 months (P > 0.0143). All splice variants of WT1 mRNA, including a hitherto unknown variant (lacking exons 4 and 5), were found to be expressed in osteogenic sarcoma. CONCLUSION: WT1 seems to be associated with very poor survival of patients with osteogenic sarcoma metastasis.

Characterization of pediatric skeletal tumors and tumor-like conditions: specific cross-sectional imaging signs.

The radiograph is indispensable for characterizing pediatric skeletal lesions. However, cross-sectional imaging with CT and MRI can provide additional information and augment or confirm an impression obtained from the initial radiographic findings. This review will highlight the role of CT and MRI in characterizing pediatric skeletal tumors and tumor-like conditions. Focus will be given to the contributions of each modality to the process of characterizing skeletal lesions, recalling that cross-sectional imaging is more commonly obtained for the purpose of determining extent of disease rather than for characterization.

Musculoskeletal tumors: use of proton MR spectroscopic imaging for characterization.

PURPOSE: To determine the value of multivoxel proton magnetic resonance spectroscopic imaging (MRSI) in distinguishing malignant skeletal tumors from benign tumors and normal bone marrow using the metabolite choline (Cho) as a marker for malignancy. MATERIALS AND METHODS: Pathologic specimens obtained from 13 patients who had undergone wide resection for skeletal tumors underwent evaluation by MRSI at 1.5 T. Coronal T1-weighted gradient-echo sequence obtained for localization purposes (TR/TE = 250/1.8 msec, field of view [FOV] = 18 x 18), and single-slice MRSI (TR/TE = 2000/272 msec, FOV = 18 x 18, 10-mm slice-thickness) were performed. Water, lipid, and Cho images were reconstructed from MRSI data. Cho signal was measured in each specimen and expressed relative to background noise level (signal-to-noise ratio [SNR]) where noise was measured between 7.0 and 9.0 ppm. Cho SNRs were compared between areas containing malignant tumor and nonmalignant tissue (benign lesion or normal bone marrow) as determined by histopathology. RESULTS: Specimens included 13 skeletal sarcomas (seven osteosarcomas, three chondrosarcomas, one malignant fibrous histiocytoma, one fibrosarcoma, and one leiomyosarcoma). All specimens included a sample of normal bone marrow and two specimens also contained benign lesions. All sarcomas demonstrated a signal at 3.2 ppm assigned to Cho-containing metabolites in areas of malignancy. Peak Cho SNR was significantly different for areas containing histologically-proven malignancy compared to nonmalignant tissue (9.8 +/- 5.1 vs. 2.7 +/- 1.4, respectively, P < 0.002). CONCLUSION: These preliminary results indicate that MRSI at 1.5 T is a promising noninvasive method of differentiating malignant skeletal tumors from nonmalignant tissue. Using MRSI, Cho can be detected in skeletal tumors and may serve as a marker for malignancy.

En bloc resection of primary sacral tumors: classification of surgical approaches and outcome.

OBJECT: En bloc resection with adequate margins is associated with the highest probability of long-term tumor control or cure in most cases of primary sacral malignancies. The authors present their experience with a systematic approach to these lesions. They provide a novel classification of surgical techniques based on the level of nerve root sacrifice and evaluate the functional and oncological outcomes. METHODS: Seventy-eight consecutive patients underwent 94 resections of sacral neoplasms at The University of Texas M. D. Anderson Cancer Center in Houston between August 1993 and June 2002. The records of 29 consecutive patients who underwent en bloc resection of primary sacral tumors were retrospectively reviewed. The median follow-up period was 55 months (range 1-103 months). Chordoma was the most frequent tumor type (16 cases). Midline sacral amputation was performed in 25 patients (eight low, four middle, seven high, and five total sacrectomies; one hemicorporectomy). Lateral sacrectomy was undertaken in four patients (two unilateral excisions of the sacroiliac joint and two hemisacrectomies). The surgical margins were wide in 19 cases, marginal in nine, and contaminated in one. The type of sacrectomy correlated with characteristic outcomes with respect to bladder, bowel, and ambulatory functions. Duration of hospital stay was related to the extent of sacrectomy (p = 0.003, Wilcoxon signed-rank test). The median Kaplan-Meier disease-free survival for patients with chordoma was 68 months (95% confidence interval 46-90 months). CONCLUSIONS: Classification of en bloc sacral resection techniques by the level of nerve root transection is useful in predicting postoperative function and the potential for morbidity. Adequate surgical margins should not be compromised to preserve function when they are necessary to affect tumor control.

Nuclear beta-catenin expression distinguishes deep fibromatosis from other benign and malignant fibroblastic and myofibroblastic lesions.

Deep fibromatoses (desmoid tumors) are clonal myofibroblastic proliferations that are prone to aggressive local recurrences but that do not metastasize. They must be distinguished from a host of fibroblastic and myofibroblastic lesions as well as from smooth muscle neoplasms. Virtually all deep fibromatoses have somatic beta-catenin or adenomatous polyposis coli (APC) gene mutations leading to intranuclear accumulation of beta-catenin. Since low-grade sarcomas in general lack beta-catenin and since reactive proliferations would not be expected to have it, we predicted that nuclear beta-catenin expression would be detected in deep fibromatoses but absent in other entities in the differential diagnosis. We evaluated the role of beta-catenin to help differentiate distinguish deep fibromatoses from congeners. Formalin-fixed, paraffin-embedded sections from 21 lesions from 20 patients with deep fibromatoses were stained with monoclonal beta-catenin antibody (Transduction Laboratories) and compared with low-grade fibromyxoid sarcoma (n=12), leiomyosarcoma (n=10), various other fibrosarcoma variants (n=13, including 3 myofibrosarcomas, 3 sclerosing epithelioid fibrosarcomas, 5 low-grade fibrosarcomas, 1 classic fibrosarcoma arising in dermatofibrosarcoma protuberans, 1 inflammatory myxohyaline tumor/myxoinflammatory fibroblastic sarcoma), myofibroma/myofibromatosis (n=12), nodular fasciitis (n=11), and scars (n=9). Nuclear and cytoplasmic staining was assessed. All 21 examples of deep fibromatosis displayed nuclear beta-catenin (focal nuclear staining in one case to 90% staining). All other lesions tested (n=67) lacked nuclear labeling for beta-catenin, showing only cytoplasmic accumulation. beta-Catenin immunohistochemistry separates deep fibromatosis from entities in the differential diagnosis, a finding that can be exploited for diagnosis. Most fibromatoses have diffuse nuclear staining although occasional examples only focally label.

Radiotherapy in the management of giant cell tumor of bone.

PURPOSE: To evaluate the outcomes of patients with giant cell tumor of bone (GCTB) treated with radiotherapy (RT) with or without surgical resection. METHODS AND MATERIALS: We performed a retrospective review of the records from 25 consecutive patients with pathologically confirmed GCTB who had undergone RT between 1956 and 2000. RESULTS: Patients ranged in age from 11 to 69 years (median 32); 16 were female and 9 were male. The anatomic distribution of lesions was as follows: cervical spine, 3; temporal bone, 1; thoracic or lumbar spine, 9; sacrum, 8; ilium, 1, and humerus, radius, and thumb metacarpal, 1 each. Tumors ranged in size from 2 to 20 cm (median 9.5) at their maximal dimension. Thirteen patients had been referred for RT for primary GCTB and 12 had been referred with locally recurrent disease after having undergone one or more other treatments. Fourteen patients had undergone RT for gross disease, and the remaining 11 had been treated with RT after gross total resection. In 10 of these 11 patients, the treatment margins were positive or uncertain. Radiation doses ranged from 25 to 65 Gy (median 46). At a median follow-up of 8.8 years (range 0.67-34), 7 patients had developed isolated local recurrence, 2 had developed isolated distant recurrence, and 3 had developed both. The actuarial 5-year overall and disease-free survival rate was 91% and 58%, respectively, and the actuarial 5-year local control and distant metastasis-free survival rate was 62% and 81%, respectively. Univariate analysis suggested that treatment for recurrent disease correlated with a lower disease-free survival rate (83% vs. 33%, p = 0.06), distant metastasis-free survival rate (100% vs. 64%, p = 0.08), and local control rate (83% vs. 42%, p = 0.08) at 5 years. Of the 12 cases of recurrence, 7 were ultimately successfully treated with additional salvage therapy. In 4 of these patients, salvage therapy included interferon-alpha 2b. CONCLUSION: RT should be considered an adjuvant to surgery or as alternative therapy in cases of GCTB that are unresectable or in which excision would result in substantial functional deficits. When RT is used as primary therapy, the rate of local control seems to be satisfactory. In heavily pretreated patients, however, RT delivered as it was in this series can result in poor local control, and alternative therapies should be considered.

A role for curative surgery in the treatment of selected patients with metastatic breast cancer.

Although metastatic breast cancer is widely believed to carry a grim prognosis, treatment developments over the past 25 years have greatly improved survival outcomes in these patients. In selected cases, aggressive treatment approaches may occasionally result in long-term survival of 15 years or more. This review considers the role of surgery in the treatment of single or multiple metastatic lesions restricted to one site. For each site, available literature from 1992-2002 was assessed to determine the role of surgery on survival outcomes and to determine appropriate criteria for selecting the best candidates for surgery. For lung, liver, brain, and sternum metastases, the use of surgery with or without adjuvant therapy resulted in greater median survival times and 5-year survival rates. The best candidate for surgery had no evidence of additional metastatic disease, good performance status, and a long disease-free interval after treatment of the primary tumor. Current treatment standards for breast cancer follow-up do not include imaging studies other than mammography. The addition of chest x-rays as part of routine follow-up should be considered as a cost-effective approach for early assessment of metastases to the lung or sternum that may be appropriate for surgical excision.

Long-term follow-up of patients with giant cell tumor of the sacrum treated with selective arterial embolization.

BACKGROUND: Giant cell tumors of the bone can behave as aggressive and sometimes lethal tumors. In the sacrum, the tumor can be extremely difficult to manage. Standard treatments, including surgery and radiation, are associated with significant complications and recurrence rates. The goal of this study is to evaluate the long-term outcome of selective arterial embolization as an alternative treatment modality. METHODS: From 1975 to 2001, 18 patients were treated with selective intraarterial embolization. The embolization method was a combination of Gelfoam particles and coils for peripheral and central occlusions, respectively. The number of embolizations was based on clinical symptoms, radiographic response, and the vascularity of the tumor. Nine patients received intraarterial cisplatin as part of their treatment. The median follow-up was 105 months. RESULTS: Of 18 patients, 14 responded favorably to embolization with improvement in pain and neurologic symptoms. Computed tomographic and magnetic resonance imaging scans showed reossification and stabilization of tumor size. Arteriograms showed diminished vascularity. With long-term follow-up, three patients developed late disease recurrences within the sacrum. Kaplan-Meier analysis showed that the risk of local recurrence is 31% at 10 years and 43% at 15 and 20 years. The long-term outcome was not affected by intraarterial cisplatin. There was one death that occurred 1 day after embolization. CONCLUSIONS: Most patients demonstrate an objective early radiographic response to embolization. Long-term follow-up shows that the response is durable in approximately one half of the patients. Given the potential morbidity of other treatments, embolization should be included in the armamentarium of treatment for this difficult disease. Embolization may be used alone or in conjunction with other therapy. Long-term follow-up is recommended for all patients because late disease recurrence or sarcomatous change can occur.

Surgical management of soft tissue sarcomas of the hand and foot.

BACKGROUND: Soft tissue sarcomas of the hand and foot present unique management challenges. The purpose of the current study study was to determine oncologic outcome, particularly with respect to factors affecting local recurrence, distant recurrence, and disease-specific survival. METHODS: A retrospective study was performed on 115 patients with soft tissue sarcomas of the hand or foot who were evaluated, treated, and followed at the authors' institution between 1980 and 1998. The medical records and radiographs were reviewed. Kaplan-Meier analysis was used to assess patient survival. RESULTS: Most patients (95%) were referred after previous surgery. The majority of tumors (75%) were T1 lesions (less than 5 cm), and most tumors (81%) were high grade. Patients who were treated by definitive, wide re-excision (n = 43) had a 10 year local recurrence-free survival of 88%, which was significantly better than the corresponding rate of 58% for patients who did not have re-excision (n = 40, P = 0.05). Radiation improved local control in patients who did not undergo re-excision (n = 17, P = 0.02). However, radiation did not improve local control in patients who had definitive re-excision with negative margins (n = 13, P = 0.51). The disease-specific survival at 5 and 10 years was 76% and 65%, respectively, for patients who presented with localized disease. Disease-specific patient survival was significantly worse for patients who had regional or distant metastasis. Radical amputation as initial surgical treatment did not decrease the likelihood of regional metastasis and did not improve disease-specific patient survival. The presence of distant metastasis at presentation was an independent predictor of local recurrence. CONCLUSION: Limb sparing treatment is possible in many patients with soft tissue sarcomas of the hand and foot. Re-excision to achieve microscopically negative surgical margins is an effective method of achieving a high rate of local control in appropriately selected patients who present after unplanned excision of the primary tumor. There does not appear to be a survival benefit to immediate radical amputation, which should be reserved for cases where surgical excision or re-excision with adequate margins cannot be performed without sacrifice of functionally significant neurovascular or osseous structures.