Transformation of Escherichia coli JM109 using pUC19 by the Yoshida effect.

Transformation of non-competent Escherichia coli JM109 was accomplished using pUC19 as donor plasmid and sepiolite as the acicular material to promote cell piercing via application of friction with a polystyrene stick or a magnetic bar on the surface of a hydrogel containing agar. An automatic spreading setup was built with a conventional stirring plate and compared to manual spreading. Several parameters were optimized, namely, the agar content of the hydrogel (2%), concentration of cells (OD=1.3 corresponding to 1.4×10(9) bacterial cells/mL), concentration of sepiolite (0.01%), manual versus mechanical spreading (automatic spreading more consistent) and spreading time (30s). Efficiency values up to 4.1×10(4) CFU/µg pUC19 were obtained. The method proved to be suitable for a rapid and low cost transformation of non-competent E. coli JM109, where higher values of efficiency do not need to be attained.

Heterologous production of caffeic acid from tyrosine in Escherichia coli.

Caffeic acid is a plant secondary metabolite and its biological synthesis has attracted increased attention due to its beneficial effects on human health. In this study, Escherichia coli was engineered for the production of caffeic acid using tyrosine as the initial precursor of the pathway. The pathway design included tyrosine ammonia lyase (TAL) from Rhodotorula glutinis to convert tyrosine to p-coumaric acid and 4-coumarate 3-hydroxylase (C3H) from Saccharothrix espanaensis or cytochrome P450 CYP199A2 from Rhodopseudomonas palustris to convert p-coumaric acid to caffeic acid. The genes were codon-optimized and different combinations of plasmids were used to improve the titer of caffeic acid. TAL was able to efficiently convert 3mM of tyrosine to p-coumaric acid with the highest production obtained being 2.62mM (472mg/L). CYP199A2 exhibited higher catalytic activity towards p-coumaric acid than C3H. The highest caffeic acid production obtained using TAL and CYP199A2 and TAL and C3H was 1.56mM (280mg/L) and 1mM (180mg/L), respectively. This is the first study that shows caffeic acid production using CYP199A2 and tyrosine as the initial precursor. This study suggests the possibility of further producing more complex plant secondary metabolites like flavonoids and curcuminoids.

Heterologous production of curcuminoids.

SUMMARY: Curcuminoids, components of the rhizome of turmeric, show several beneficial biological activities, including anticarcinogenic, antioxidant, anti-inflammatory, and antitumor activities. Despite their numerous pharmaceutically important properties, the low natural abundance of curcuminoids represents a major drawback for their use as therapeutic agents. Therefore, they represent attractive targets for heterologous production and metabolic engineering. The understanding of biosynthesis of curcuminoids in turmeric made remarkable advances in the last decade, and as a result, several efforts to produce them in heterologous organisms have been reported. The artificial biosynthetic pathway (e.g., in Escherichia coli) can start with the supplementation of the amino acid tyrosine or phenylalanine or of carboxylic acids and lead to the production of several natural curcuminoids. Unnatural carboxylic acids can also be supplemented as precursors and lead to the production of unnatural compounds with possibly novel therapeutic properties. In this paper, we review the natural conversion of curcuminoids in turmeric and their production by E. coli using an artificial biosynthetic pathway. We also explore the potential of other enzymes discovered recently or already used in other similar biosynthetic pathways, such as flavonoids and stilbenoids, to increase curcuminoid yield and activity.

A bacteriophage detection tool for viability assessment of Salmonella cells.

Salmonellosis, one of the most common food and water-borne diseases, has a major global health and economic impact. Salmonella cells present high infection rates, persistence over inauspicious conditions and the potential to preserve virulence in dormant states when cells are viable but non-culturable (VBNC). These facts are challenging for current detection methods. Culture methods lack the capacity to detect VBNC cells, while biomolecular methods (e.g. DNA- or protein-based) hardly distinguish between dead innocuous cells and their viable lethal counterparts. This work presents and validates a novel bacteriophage (phage)-based microbial detection tool to detect and assess Salmonella viability. Salmonella Enteritidis cells in a VBNC physiological state were evaluated by cell culture, flow-cytometry and epifluorescence microscopy, and further assayed with a biosensor platform. Free PVP-SE1 phages in solution showed the ability to recognize VBNC cells, with no lysis induction, in contrast to the minor recognition of heat-killed cells. This ability was confirmed for immobilized phages on gold surfaces, where the phage detection signal follows the same trend of the concentration of viable plus VBNC cells in the sample. The phage probe was then tested in a magnetoresistive biosensor platform allowing the quantitative detection and discrimination of viable and VBNC cells from dead cells, with high sensitivity. Signals arising from 3 to 4 cells per sensor were recorded. In comparison to a polyclonal antibody that does not distinguish viable from dead cells, the phage selectivity in cell recognition minimizes false-negative and false-positive results often associated with most detection methods.

Molecular characterization of the glucose isomerase from the thermophilic bacterium Fervidobacterium gondwanense.

The gene coding for xylose isomerase from the thermophilic bacterium Fervidobacterium gondwanense was cloned and overexpressed in Escherichia coli. The produced xylose isomerase (XylA), which closely resembles counterparts from Thermotoga maritima and T. neapolitana, was purified and characterized. It is optimally active at 70 degrees C, pH 7.3, with a specific activity of 15.0 U/mg for the interconversion of glucose to fructose. When compared with T. maritima XylA at 85 degrees C, a higher catalytic efficiency was observed. Divalent metal ions Co2+ and Mg2+ were found to enhance the thermostability.

Fine-needle aspiration biopsy of the liver: a multicenter study of 602 radiologically guided FNA.

With improved radiologic techniques fine-needle aspiration (FNA) is becoming a rapid, effective diagnostic method in evaluating a wide range of liver masses. Review of six hundred two radiologically guided liver aspirates performed over a ten-year period forms the basis of this report.

Fine-needle aspiration of scalp lesions.

A variety of inflammatory and neoplastic scalp lesions are encountered in surgical pathology. However, the literature on fine-needle aspirations (FNAs) of the scalp is exceedingly rare. We report on a series of 70 FNAs involving cutaneous and subcutaneous lesions on the scalp. A total of 70 fine-needle aspirations of cutaneous and subcutaneous scalp lesions was reviewed to identify patterns of metastasis to the scalp and to demonstrate the effectiveness of FNA in diagnosing these lesions. There were 42 male and 28 female patients, ranging in age from 29-91 yr (mean, approximately 61 yr). Sixty-one patients had a previous history of malignancy. Of these aspirates, 59 were neoplastic, consistent with the patient's known primary. One case was an abscess, and the remaining case was unsatisfactorvy for cytologic evaluation. Follow-up biopsy revealed granulomatous inflammation. The most common primary tumor to metastasize to the scalp was lung carcinoma, which was seen in 18 cases, followed by hematopoietic malignancies in 14 cases. Melanoma was identified in 6 cases, head and neck tumors in 5 cases, renal malignancies in 4 cases, gastrointestinal tumors in 3 cases, sarcoma in 3 cases, breast and prostate malignancy in 2 cases each, and an olfactory neuroblastoma and meningioma in 1 case each. The remaining 9 aspirates were from patients who did not have a previous history of malignancy. These included 6 benign aspirates consisting of 3 epidermal inclusion cysts, 2 lipomas, and 1 neurofibroma. Two aspirates were malignant and included 1 primary squamous-cell carcinoma and 1 metastatic adenocarcinoma of unknown origin. The remaining case was unsatisfactory for cytologic evaluation. Follow-up biopsy of this lesion showed noncaseating granulomas. Of the aspirates from patients with a previous history of malignancy, 97% were neoplastic. Lung carcinoma and hematopoietic malignancies were the most common neoplasms that metastasized to the scalp. Since the scalp is a common site for metastasis, awareness of this fact is useful to both oncologists and dermatologists. It must be understood that FNA can provide a rapid and accurate diagnosis in the evaluation of scalp masses.

Role of fine-needle aspiration in the clinical management of solid organ transplant recipients: a review.

BACKGROUND: We evaluated the clinical course of the solid-organ transplant population at our institutions to determine the role of fine-needle aspiration (FNA) in the clinical management of this subgroup of patients. METHODS: 1196 allograft recipients (522 liver, 288 cardiac, 250 renal, 131 lung, 5 heart and lung) were reviewed. A total of 62 (5.2%) (32 liver, 23 heart, 6 lung, and 1 renal) transplant patients underwent an FNA procedure. Thirty-seven males and 25 females were included, ranging in age from 18 to 71 years (mean 50 years). RESULTS: Of the 62 fine-needle aspirates, 29 (47%) were neoplastic. The most common malignancies aspirated were malignant solid tumors (15 cases)-including 8 epithelial malignancies, 5 hepatocellular carcinomas, and 2 mesenchymal neoplasms-followed by posttransplant lymphoproliferative disorders (14 cases). Thirteen (21%) aspirates were inflammatory. The remaining 20 (32%) cases were benign aspirates from various sites (9 liver, 3 breast, 2 thyroid, 2 soft tissue, 2 lung, and 2 vertebral body). Surgical and/or autopsy material was available in 34 cases (55%). There was agreement between the tissue diagnosis and FNA material in 33 cases (97%). One case (3%) was a false negative. No false-positive cases were recorded. CONCLUSIONS: This study showed that over 50% of the aspirates were benign, justifying a conservative approach in the clinical management of these patients. Histologic correlation was available in 54% of the cases with an overall specificity of 100% and a sensitivity of 97%. We conclude that FNA is a highly sensitive and specific technique in the evaluation of lesions occurring in posttransplant patients. Cancer (Cancer Cytopathol)

Utilization of fine-needle aspiration in the diagnosis of metastatic tumors to the kidney.

Renal masses secondary to metastases are not common. Few comprehensive reviews exist, which consist primarily of autopsy and radiologic reports. The purpose of this study was to review the types and incidences of various neoplasms which metastasize to the kidney and to determine the usefulness of fine-needle aspiration (FNA) in diagnosing them. Two hundred and sixty-one radiologically guided FNAs of renal lesions over a 9-yr period were reviewed. The diagnoses of the 261 renal FNAs were as follows: 136 (52%) were malignant, 111 (43%) were benign, and 14 (5%) were unsatisfactory. Of the 136 positive FNAs, 28 (21%) revealed metastatic tumors. The overall incidence of renal FNAs displaying metastatic tumors was 11%. Among the 28 patients with metastases to the kidney, 23 patients were men and 5 were women, with the mean age being 58 yr. Twenty-five patients (89%) had prior history of a primary malignancy, including lung carcinoma (11 cases, 39%), lymphoma (8 cases, 29%), hepatocellular carcinoma (3 cases, 11%), and one case each of breast, pancreatic, and cervical cancer. In the remaining 3 patients (11%), with metastatic adenocarcinoma (2 cases) and squamous-cell carcinoma (1 case), the primary tumor site remained unknown despite an extensive clinical workup. Overall survival after FNA was poor, with a mean of 9.8 mo. FNA is useful in the diagnosis of masses in the kidney secondary to metastatic disease. This information is of clinical importance, principally in the exclusion of a primary malignancy, but also to avoid unnecessary surgery and to plan for subsequent patient care.

Pancreatic masses: a multi-institutional study of 364 fine-needle aspiration biopsies with histopathologic correlation.

Only 15% of primary malignant tumors are confined to the gland at the time of presentation. Fine-needle aspiration (FNA) is valuable in confirming the malignant nature of these unresectable lesions. Benign pancreatic lesions and metastatic neoplasms can also be evaluated by fine-needle aspiration. We undertook a retrospective study to evaluate the efficacy of FNA in assessing pancreatic masses. Three hundred and sixty-four radiologically guided FNAs of the pancreas performed between 1986-1996 were reviewed. Surgical material was also evaluated when available and compared to the FNA material. There were 223 men and 141 women. Their ages ranged from 23-90, with a mean of 64 yr. Two hundred and twelve patients (58%) had primary malignant tumors, 183 had adenocarcinomas, 15 had mucinous cystadenocarcinomas, 12 had neuroendocrine tumors, and 2 had pleomorphic giant-cell carcinomas. Ninety-one (43%) had available surgical material which showed adenocarcinoma. Ninety-one patients (25%) had benign aspirates, including 53 showing benign ductal epithelium, 23 showing pancreatitis/inflammation. 10 showing pseudocysts, and 7 showing serous cystadenomas. Surgical material was available in 24 (26%) of these patients. Two of these showed adenocarcinoma. Sixteen aspirates (4%) were suspicious for malignancy, 13 (81%) of which showed adenocarcinoma on follow-up biopsies. Twenty-two aspirates (6%) showed metastatic neoplasms. Twenty-three (6%) had unsatisfactory specimens. Ten (43%) of these had follow-up biopsies, 3 of which were malignant. FNA of primary benign and malignant pancreatic masses is highly sensitive (98%) and specific (100%). Eighty-one percent of the suspicious lesions showed adenocarcinoma on follow-up biopsy. FNA of metastatic neoplasms to the pancreas is also very accurate. This technique can be useful in avoiding unnecessary surgery.

Malignant cutaneous and subcutaneous abdominal wall lesions: a fine-needle aspiration study.

Cutaneous and subcutaneous masses of the abdominal wall are uncommon. However, a variety of benign and neoplastic entities can be encountered in this region. We report a series of 22 fine-needle aspirations (FNA) of malignant cutaneous and subcutaneous lesions involving the abdominal wall. All of these lesions were metastatic neoplasms. There were 14 females and eight males, with an age range of 35-83 years (mean 65 years). Twenty-one had a previous history of malignancy. The mean interval between the primary diagnosis and FNA was 26 months (range 1.5-128 months). The sites of origin in order of decreasing frequency were colon (n = 4), ovary (n = 4), breast (n = 3), endometrium (n = 2), melanoma (n = 2), and one case each of cervix, urinary bladder, kidney, pancreas, gallbladder, and lymphoma. One case was a squamous-cell carcinoma of unknown origin. Fourteen of the 22 patients were dead at the end of this study, with a mean survival of 8.4 months (range 0.5-44 months) following FNA. One patient was alive with disease at 13 months, and seven patients were lost to follow-up. Based on this data and on review of the literature we conclude that the majority of malignant cutaneous and subcutaneous lesions of the abdominal wall subject to FNA biopsy are metastatic tumors which originate from intra-abdominal, pelvic, and retroperitoneal organs and that FNA is a highly useful technique in the assessment of these lesions of the abdominal wall.

Utilization of core wash material in the diagnosis of breast lesions by stereotactic needle biopsy.

BACKGROUND: Several reports have compared the results of fine-needle aspiration and stereotactic core needle biopsy in nonpalpable breast lesions. In this study the authors describe a simple method to retrieve cytologic material from a core breast biopsy sample that provides the diagnosis within 1 hour of the procedure. METHODS: Two hundred and eleven nonpalpable breast lesions were biopsied. Each core needle biopsy sample was placed in a mesh bag, and the bag and needle notch were washed in Cytolyt solution to obtain a monolayer using a commercial ThinPrep processor. The cytologic diagnoses were divided into four categories: benign, suspicious, malignant, and unsatisfactory, which then were compared with core needle biopsy results. RESULTS: Cytology reports of 211 lesions were as follows: 169 lesions (80%) were benign, 16 lesions (7.6%) were suspicious, 11 lesions (5.2%) were malignant, and 15 lesions (7.1%) were unsatisfactory. Core needle biopsy showed 165 of 169 samples (98%) to be benign and 4 to be malignant. Of the 16 suspicious smears, 10 were invasive carcinoma, 2 were in situ lesions, 3 were hyperplasias, and 1 was fibrosis. Of the 11 malignant smears, 10 were confirmed on core needle biopsy and 1 was read as atypia on the first core needle biopsy sample and malignant on a second, separate, follow-up core needle biopsy. Of the 15 unsatisfactory samples, 14 were found to be benign and 1 was found to be malignant on a separate, follow-up core needle biopsy. CONCLUSIONS: The core wash technique was 85% sensitive and 98% specific for malignancy. Only 7% of specimens were insufficient for diagnosis, and 93% of these were proven to be benign. This technique is useful for immediate (within 1 hour) diagnosis of breast lesions, alleviating patient anxiety and supplementing the diagnostic yield of the core biopsy.

Fine-needle aspiration of 697 palpable breast lesions with histopathologic correlation.

BACKGROUND: Fine-needle aspiration breast biopsy has been used increasingly as an alternative to excisional biopsy. The purpose of this study is to evaluate the accuracy of fine-needle aspiration with histopathologic confirmation. METHODS: A retrospective study was performed using a computer database over a 5-year period. All women who had had fine-needle aspiration breast biopsy with histopathologic confirmation of the diagnosis were included. Fine-needle aspirations were interpreted as malignant, suspicious, or benign. Histopathologic diagnosis included core-needle biopsy, open excisional biopsy, or mastectomy specimen. RESULTS: A total of 697 patients fulfilled the criteria. Only 5 (0.7%) of the specimens were inadequate for study. There were 401 total malignant fine-needle aspiration diagnoses, with only 3 false-positive specimens. All three were ductal hyperplasia, one from a previously radiated breast. There were 125 suspicious readings; 84 of these were malignant and 41 were false-suspicious specimens. Most of the false-suspicious lesions were fibrocystic disease. Of the 166 lesions interpreted as benign, there were 13 false-negative specimens. The test had a 97% sensitivity, 78% specificity, 92% positive predictive value, and 92% negative predictive value. CONCLUSIONS: Fine-needle aspiration is a sensitive test that can be useful as an adjunct in the diagnosis of breast cancer. "Malignant" and "benign" interpretations are highly predictive but must be used only in the context of other diagnostic modalities. "Suspicious" lesions require further investigation.

Importance of repeat fine-needle biopsy in the management of thyroid nodules.

Fine-needle aspiration (FNA) biopsy of a thyroid nodule was performed in 797 patients. Ninety-six patients had resection of the thyroid nodule performed subsequent to a one-time FNA biopsy. The surgical pathology of these 96 cases demonstrated a 5.8% false-negative rate and a 9.9% false-positive rate. As a consequence, we prospectively evaluated the routine practice of repeat FNA of cytologically benign thyroid nodules. Repeat FNA confirmed the original benign cytology in 183 (93%) of 196 patients. Seventeen of these 183 patients with benign FNA on both biopsies had resection of the nodule performed because of the development of suspicious clinical signs or in response to the patient's choice; 1 recurrent cyst was found to be carcinomatous. Of the 13 patients demonstrating a change in cytology on repeat FNA biopsy, 9 had a nodule that was classified as possibly malignant (suspicious); 6 of these patients underwent resection, and 1 patient was found to have a carcinomatous nodule. Four patients had nodules that were classified as probably malignant on repeat FNA biopsy; all of their nodules were resected, and three of them were found to be carcinomatous. This study demonstrates that, although one-time FNA biopsy of thyroid nodules is highly accurate, with a relatively low false-negative rate, repeat fine-needle biopsy improves on this diagnostic accuracy, thereby decreasing the risk of misdiagnosing a thyroid nodule that is malignant.

Nonpalpable breast lesions: findings of stereotaxic needle-core biopsy and fine-needle aspiration cytology.

Two hundred fifty mammographically detected nonpalpable breast lesions suspicious for malignancy in women who underwent routine screening mammography were stereotaxically localized. Fine-needle-aspiration (FNA) cytologic specimens and needle-core biopsy specimens were obtained before open biopsy in every case. Seventy-six lesions (30.4%) were malignant. Sixty-three (83%) of these 76 cancers were 1 cm long or smaller. Needle-core biopsy alone was used to diagnose conclusively 41% (n = 31) of these cancers, while FNA cytologic study alone was used to diagnose 32% (n = 24). No false-positive results occurred with either test. The same diagnosis was reached in 54% (n = 41) when the combined results of both needle tests were considered. In applying the two needle tests to 125 mammographically defined low-suspicion lesions, 85 (68%) were found to be benign by means of either one or both needle tests; there was one lobular carcinoma in situ. By applying this algorithm, 85 (34%) of 250 patients with abnormal mammograms, or one-third of all patients recommended for open biopsy, might have avoided surgery.