First report of detection of IgA anti-Acanthamoeba antibodies among Saudi population and amoeba isolation from their surroundings.

Acanthamoeba is an opportunistic protozoan pathogen which is found in diverse environment worldwide. Being ubiquitous nature of this amoeba we come across it in our daily life. Acanthamoeba species are recognized as human pathogens; that may cause blinding keratitis and rare but fatal granulomatous encephalitis involving central nervous system. To date, there is not a single report in literature demonstrating anti-Acanthamoeba antibodies among the Saudi population, and thus aim of the present study. Using ELISA, we identified the antibody level in the local population. Our results represent the secretory IgA antiAcanthamoeba in mucosal secretions from 133 individuals aged 15-60 years. The antiAcanthamoeba antibody prevalence rate was > 80%, and no considerable differences were observed between prevalence in males (80.28%) and that in females (80.64%). In addition, environmental sources (soil and water) from the environment of the participants in our study were evaluated for amoeba incidence. The amoeba was identified by morphological characteristics of cysts or trophozoites on non-nutrient agar plates grown with E. coli. Overall, 58.75% of samples from water and 32.85% of those from soil were culture positive for outgrowth of amoeba on non-nutrient agar plates. Furthermore, PCR was carried out with genus-specific primers to confirm the presence of Acanthamoeba DNA. Our results revealed that about 68% of cultures from water and 43% of those from soil were successfully amplified and proved to be amoeba DNA. Interestingly, a few samples yielded more than one product, which suggests that some other amoebic species may be present in the same sample (MAC-W1 and MADW1). To the best of our knowledge, we described for the first time the amoeba isolation from the participant's close environment and antibodies level among Saudi population. Our future studies will be focused on additional molecular characterization of isolated amoeba and their pathogenic potential which could be a possible threat for the community.

Extracellular Vesicle-Mediated In Vitro Transcribed mRNA Delivery for Treatment of HER2+ Breast Cancer Xenografts in Mice by Prodrug CB1954 without General Toxicity.

Prodrugs are harmless until activated by a bacterial or viral gene product; they constitute the basis of gene-delivered prodrug therapies called GDEPT, which can kill tumors without major side effects. Previously, we utilized the prodrug CNOB (C16H7CIN2O4; not clinically tested) and enzyme HChrR6 in GDEPT to generate the drug MCHB (C16H9CIN2O2) in tumors. Extracellular vesicles (EVs) were used for directed gene delivery and HChrR6 mRNA as gene. Here, the clinical transfer of this approach is enhanced by: (i) use of CB1954 (tretazicar) for which safe human dose is established; HChrR6 can activate this prodrug. (ii) EVs delivered in vitro transcribed (IVT) HChrR6 mRNA, eliminating the potentially harmful plasmid transfection of EV producer cells we utilized previously; this has not been done before. IVT mRNA loading of EVs required several steps. Naked mRNA being unstable, we ensured its prodrug activating functionality at each step. This was not possible using tretazicar itself; we relied instead on HChrR6's ability to convert CNOB into MCHB, whose fluorescence is easily visualizable. HChrR6 mRNA-translated product's ability to generate fluorescence from CNOB vicariously indicated its competence for tretazicar activation. (iii) Systemic IVT mRNA-loaded EVs displaying an anti-HER2 single-chain variable fragment ("IVT EXO-DEPTs") and tretazicar caused growth arrest of human HER2+ breast cancer xenografts in athymic mice. As this occurred without injury to other tissues, absence of off-target mRNA delivery is strongly indicated. Many cancer sites are not amenable for direct gene injection, but current GDEPTs require this. In circumventing this need, a major advance in GDEPT applicability has been accomplished.

Anti-HER2 scFv-Directed Extracellular Vesicle-Mediated mRNA-Based Gene Delivery Inhibits Growth of HER2-Positive Human Breast Tumor Xenografts by Prodrug Activation.

This paper deals with specific targeting of the prodrug/enzyme regimen, CNOB/HChrR6, to treat a serious disease, namely HER2+ human breast cancer with minimal off-target toxicity. HChrR6 is an improved bacterial enzyme that converts CNOB into the cytotoxic drug MCHB. Extracellular vesicles (EV) were used for mRNA-based HchrR6 gene delivery: EVs may cause minimal immune rejection, and mRNA may be superior to DNA for gene delivery. To confine HChrR6 generation and CNOB activation to the cancer, the EVHB chimeric protein was constructed. It contains high-affinity anti-HER2 scFv antibody (ML39) and is capable of latching on to EV surface. Cells transfected with EVHB-encoding plasmid generated EVs displaying this protein ("directed EVs"). Transfection of a separate batch of cells with the new plasmid, XPort/HChrR6, generated EVs containing HChrR6 mRNA; incubation with pure EVHB enabled these to target the HER2 receptor, generating "EXO-DEPT" EVs. EXO-DEPT treatment specifically enabled HER2-overexpressing BT474 cells to convert CNOB into MCHB in actinomycin D-independent manner, showing successful and specific delivery of HChrR6 mRNA. EXO-DEPTs-but not undirected EVs-plus CNOB caused near-complete growth arrest of orthotopic BT474 xenografts in vivo, demonstrating for the first time EV-mediated delivery of functional exogenous mRNA to tumors. EXO-DEPTs may be generated from patients' own dendritic cells to evade immune rejection, and without plasmids and their potentially harmful genetic material, raising the prospect of clinical use of this regimen. This approach can be used to treat any disease overexpressing a specific marker. Mol Cancer Ther; 17(5); 1133-42. ©2018 AACR.

Quantitative real time PCR analysis of apoptotic gene expression in chronic lymphocytic leukemia patients and their relationships with chemosensitivity

Background: Apoptosis-related gene expression such as BCL2, and p53 has been suggested in predicting the patient response to chemo- or radiotherapy, as well as patient's survival. Methods: The aim of this study was to determine changes in BCL2 and p53 apoptosis related gene expressions in chronic lymphocytic leukemia (CLL) patients in response to different chemotherapy regimens and number of treatment courses. The study was conducted on 55 CLL patients (44 CLL and 11 CLL/SLL; small lymphocytic lymphoma) and 40 healthy individuals as control, over three-months period. The RNA was extracted by exploitation total RNA extraction kit, treated with DNAse, then cDNA was synthesized and qRT-PCR used to analyze antiapoptotic BCL2 and tumor suppresser p53 gene expressions. Results: CLL/SLL showed higher BCL2 and p53 gene expression than CLL. The patients with CLL showed three-fold increase in BCL2 gene expression compared to healthy controls (p < 0.05), and 50% decrease in p53 gene expressions (p < 0.05). BCL2 gene expression was higher, particularly, for those who were treated with higher range of treatment courses and combination of fludarabine, cyclophosphamide and rituximab (FCR) regimen. P53 gene expression reciprocally related with BCL2 and vice versa. Conclusions: In contrary to BCL2, p53 gene was extremely expressed in patients treated with chemotherapy agents, particularly after 24­30 months disease duration; suggesting a late expression of p53 during advanced stages of the disease. A proportional change in BCL2 and p53 gene expression was reported with different treatment regimens; Chlorambucil (Clb) decreased and FCR regimen increased BCL2 gene expression. Higher p53 gene expression reported with the Chlorambucil + (Chlorambucil + Prednisolone) regimen (AU)

Utilizing native fluorescence imaging, modeling and simulation to examine pharmacokinetics and therapeutic regimen of a novel anticancer prodrug.

BACKGROUND: Success of cancer prodrugs relying on a foreign gene requires specific delivery of the gene to the cancer, and improvements such as higher level gene transfer and expression. Attaining these objectives will be facilitated in preclinical studies using our newly discovered CNOB-GDEPT, consisting of the produrg: 6-chloro-9-nitro-5-oxo-5H-benzo-(a)-phenoxazine (CNOB) and its activating enzyme ChrR6, which generates the cytotoxic product 9-amino-6-chloro-5H-benzo[a]phenoxazine-5-one (MCHB). MCHB is fluorescent and can be noninvasively imaged in mice, and here we investigated whether MCHB fluorescence quantitatively reflects its concentration, as this would enhance its reporter value in further development of the CNOB-GDEPT therapeutic regimen. PK parameters were estimated and used to predict more effective CNOB administration schedules. METHODS: CNOB (3.3 mg/kg) was injected iv in mice implanted with humanized ChrR6 (HChrR6)-expressing 4T1 tumors. Fluorescence was imaged in live mice using IVIS Spectrum, and quantified by Living Image 3.2 software. MCHB and CNOB were quantified also by LC/MS/MS analysis. We used non-compartmental model to estimate PK parameters. Phoenix WinNonlin software was used for simulations to predict a more effective CNOB dosage regimen. RESULTS: CNOB administration significantly prolonged mice survival. MCHB fluorescence quantitatively reflected its exposure levels to the tumor and the plasma, as verified by LC/MS/MS analysis at various time points, including at a low concentration of 2 ng/g tumor. The LC/MS/MS data were used to estimate peak plasma concentrations, exposure (AUC0-24), volume of distribution, clearance and half-life in plasma and the tumor. Simulations suggested that the CNOB-GDEPT can be a successful therapy without large increases in the prodrug dosage. CONCLUSION: MCHB fluorescence quantifies this drug, and CNOB can be effective at relatively low doses. MCHB fluorescence characteristics will expedite further development of CNOB-GDEPT by, for example, facilitating specific gene delivery to the tumor, its prolonged expression, as well as other attributes necessary for successful gene-delivered enzyme prodrug therapy.

Differential fates of biomolecules delivered to target cells via extracellular vesicles.

Extracellular vesicles (EVs), specifically exosomes and microvesicles (MVs), are presumed to play key roles in cell-cell communication via transfer of biomolecules between cells. The biogenesis of these two types of EVs differs as they originate from either the endosomal (exosomes) or plasma (MVs) membranes. To elucidate the primary means through which EVs mediate intercellular communication, we characterized their ability to encapsulate and deliver different types of macromolecules from transiently transfected cells. Both EV types encapsulated reporter proteins and mRNA but only MVs transferred the reporter function to recipient cells. De novo reporter protein expression in recipient cells resulted only from plasmid DNA (pDNA) after delivery via MVs. Reporter mRNA was delivered to recipient cells by both EV types, but was rapidly degraded without being translated. MVs also mediated delivery of functional pDNA encoding Cre recombinase in vivo to tissues in transgenic Cre-lox reporter mice. Within the parameters of this study, MVs delivered functional pDNA, but not RNA, whereas exosomes from the same source did not deliver functional nucleic acids. These results have significant implications for understanding the role of EVs in cellular communication and for development of EVs as delivery tools. Moreover, studies using EVs from transiently transfected cells may be confounded by a predominance of pDNA transfer.

Sigma S-dependent antioxidant defense protects stationary-phase Escherichia coli against the bactericidal antibiotic gentamicin.

Stationary-phase bacteria are important in disease. The σ(s)-regulated general stress response helps them become resistant to disinfectants, but the role of σ(s) in bacterial antibiotic resistance has not been elucidated. Loss of σ(s) rendered stationary-phase Escherichia coli more sensitive to the bactericidal antibiotic gentamicin (Gm), and proteomic analysis suggested involvement of a weakened antioxidant defense. Use of the psfiA genetic reporter, 3'-(p-hydroxyphenyl) fluorescein (HPF) dye, and Amplex Red showed that Gm generated more reactive oxygen species (ROS) in the mutant. HPF measurements can be distorted by cell elongation, but Gm did not affect stationary-phase cell dimensions. Coadministration of the antioxidant N-acetyl cysteine (NAC) decreased drug lethality particularly in the mutant, as did Gm treatment under anaerobic conditions that prevent ROS formation. Greater oxidative stress, due to insufficient quenching of endogenous ROS and/or respiration-linked electron leakage, therefore contributed to the greater sensitivity of the mutant; infection by a uropathogenic strain in mice showed this to be the case also in vivo. Disruption of antioxidant defense by eliminating the quencher proteins, SodA/SodB and KatE/SodA, or the pentose phosphate pathway proteins, Zwf/Gnd and TalA, which provide NADPH for ROS decomposition, also generated greater oxidative stress and killing by Gm. Thus, besides its established mode of action, Gm also kills stationary-phase bacteria by generating oxidative stress, and targeting the antioxidant defense of E. coli can enhance its efficacy. Relevant aspects of the current controversy on the role of ROS in killing by bactericidal drugs of exponential-phase bacteria, which represent a different physiological state, are discussed.

Patient-derived xenografts of triple-negative breast cancer reproduce molecular features of patient tumors and respond to mTOR inhibition.

INTRODUCTION: Triple-negative breast cancer (TNBC) is aggressive and lacks targeted therapies. Phosphatidylinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) pathways are frequently activated in TNBC patient tumors at the genome, gene expression and protein levels, and mTOR inhibitors have been shown to inhibit growth in TNBC cell lines. We describe a panel of patient-derived xenografts representing multiple TNBC subtypes and use them to test preclinical drug efficacy of two mTOR inhibitors, sirolimus (rapamycin) and temsirolimus (CCI-779). METHODS: We generated a panel of seven patient-derived orthotopic xenografts from six primary TNBC tumors and one metastasis. Patient tumors and corresponding xenografts were compared by histology, immunohistochemistry, array comparative genomic hybridization (aCGH) and phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) sequencing; TNBC subtypes were determined. Using a previously published logistic regression approach, we generated a rapamycin response signature from Connectivity Map gene expression data and used it to predict rapamycin sensitivity in 1,401 human breast cancers of different intrinsic subtypes, prompting in vivo testing of mTOR inhibitors and doxorubicin in our TNBC xenografts. RESULTS: Patient-derived xenografts recapitulated histology, biomarker expression and global genomic features of patient tumors. Two primary tumors had PIK3CA coding mutations, and five of six primary tumors showed flanking intron single nucleotide polymorphisms (SNPs) with conservation of sequence variations between primary tumors and xenografts, even on subsequent xenograft passages. Gene expression profiling showed that our models represent at least four of six TNBC subtypes. The rapamycin response signature predicted sensitivity for 94% of basal-like breast cancers in a large dataset. Drug testing of mTOR inhibitors in our xenografts showed 77 to 99% growth inhibition, significantly more than doxorubicin; protein phosphorylation studies indicated constitutive activation of the mTOR pathway that decreased with treatment. However, no tumor was completely eradicated. CONCLUSIONS: A panel of patient-derived xenograft models covering a spectrum of TNBC subtypes was generated that histologically and genomically matched original patient tumors. Consistent with in silico predictions, mTOR inhibitor testing in our TNBC xenografts showed significant tumor growth inhibition in all, suggesting that mTOR inhibitors can be effective in TNBC, but will require use with additional therapies, warranting investigation of optimal drug combinations.

Laparoscopic findings of subfertile women attended in a tertiary level care centre.

This retrospective study was done in the Obstetrics and Gynaecology Department of Bangladesh Medical College hospital during the period of July 2003 to June 2004 in the women suffering from primary and secondary subfertility, who underwent laparoscopy. The aim of this study was to see the laparoscopic findings of internal genitalia and other pelvic structures in subfertile women. The study group comprises 55 cases of which 67.37% of primary and 32.73% were of secondary subfertility. Both the ovaries were normal looking in 41.81% cases. Endometriosis was present in 5.45% of both the ovaries. Corpus luteum was seen in 20% cases on right ovary and in 27.27% cases on left ovary. Laparoscopy shows normal looking fallopian tube in 65.45% cases in right side and 61.81% cases in the left side. Right sided tubal block was in 5.46% and 9.10 % in the left side. Both the tubes were patent in 81.6% cases.

Melanotic neuroectodermal tumour of infancy.

Melanotic neuroectodermal tumour in infancy is rare, mainly benign with little tendency to recur after excision or effective curettage. This pigmented neoplasm of neural crest origin occurring in infants before 1 year of age. The most common site of occurrence is the anterior maxillary alveolar ridge (70%), following by the skull, brain and mandible. The genital organ is the most frequent extra cranial site. We report a 6 months old male baby with a similar tumour arising from right half of cheek involving the maxilla. We diagnosed the case after histological report. We remove the tumour through a sub-labial incision. The mass was blackish in colour, and was mobilized from all side including from the maxillary sinuses. The author thought that this should be reported for improving the clinical awareness and treatment of pigmented soft tissue mass in children. Almost one year follow up of the patients showed no recurrence.

Congenital muscular torticollis: experience of 14 cases.

Congenital Muscular Torticollis (CMT) is a postural deformity of head and neck detected at birth or shortly after birth, primarily resulting from unilateral shortening of Sternocleidomastoid Muscle (SCM). In neonates and infants, patient may cure conservatively by physiotherapy but surgery is the treatment of choice for children and adolescents. There are various techniques of surgery. Here we show our experience regarding management of congenital muscular torticollis. In the present retrospective case series, fourteen patients of congenital muscular torticollis were treated. The cases were enrolled between Nov' 2005 to Oct' 2007 in Bangabandhu Sheikh Mujib Medical University, Gonosasthaya Somaj Vittik Medical College Hospital, Dhaka and different private clinics of Dhaka city of Bangladesh. Neonates and infants were treated conservatively with physiotherapy and others treated surgically by transection of both sternal and clavicular head of SCM under general anesthesia. Operated patients were released on following post operative day with advised to do physiotherapy. Patients age range from 7 days to 15 years of which ten were female and four male. SCM was shortened in all cases (8 on right side and 6 on left side). Eleven were female and three male. Of 14 patients, 2 neonates, 7 infants and 5 were more than 1 year age. There was no associated anomaly. Out of 9 neonates and infants 8 cured conservatively with physiotherapy and another one significantly improved. Six were treated surgically including one failed physiotherapy. Post operative period was uneventful and there was no complication. Results were evaluated clinically and comments of peers. Most of the patient of congenital muscular torticollis can be treated conservatively during infancy. Division of both sternal and clavicular head of SCM is easy and safe surgical technique for the treatment of CMT of older children and adolescents.

Role of nitric oxide in Salmonella typhimurium-mediated cancer cell killing.

BACKGROUND: Bacterial targeting of tumours is an important anti-cancer strategy. We previously showed that strain SL7838 of Salmonella typhimurium targets and kills cancer cells. Whether NO generation by the bacteria has a role in SL7838 lethality to cancer cells is explored. This bacterium has the mechanism for generating NO, but also for decomposing it. METHODS: Mechanism underlying Salmonella typhimurium tumour therapy was investigated through in vitro and in vivo studies. NO measurements were conducted either by chemical assays (in vitro) or using Biosensors (in vivo). Cancer cells cytotoxic assay were done by using MTS. Bacterial cell survival and tumour burden were determined using molecular imaging techniques. RESULTS: SL7838 generated nitric oxide (NO) in anaerobic cell suspensions, inside infected cancer cells in vitro and in implanted 4T1 tumours in live mice, the last, as measured using microsensors. Thus, under these conditions, the NO generating pathway is more active than the decomposition pathway. The latter was eliminated, in strain SL7842, by the deletion of hmp- and norV genes, making SL7842 more proficient at generating NO than SL7838. SL7842 killed cancer cells more effectively than SL7838 in vitro, and this was dependent on nitrate availability. This strain was also ca. 100% more effective in treating implanted 4T1 mouse tumours than SL7838. CONCLUSIONS: NO generation capability is important in the killing of cancer cells by Salmonella strains.

Visualizing implanted tumors in mice with magnetic resonance imaging using magnetotactic bacteria.

PURPOSE: To determine if magnetotactic bacteria can target tumors in mice and provide positive contrast for visualization using magnetic resonance imaging. EXPERIMENTAL DESIGN: The ability of the magnetotactic bacterium, Magnetospirillum magneticum AMB-1 (referred to from here as AMB-1), to confer positive magnetic resonance imaging contrast was determined in vitro and in vivo. For the latter studies, AMB-1 were injected either i.t. or i.v. Bacterial growth conditions were manipulated to produce small (approximately 25-nm diameter) magnetite particles, which were observed using transmission electron microscopy. Tumor targeting was confirmed using 64Cu-labeled bacteria and positron emission tomography and by determination of viable cell counts recovered from different organs and the tumor. RESULTS: We show that AMB-1 bacteria with small magnetite particles generate T1-weighted positive contrast, enhancing in vivo visualization by magnetic resonance imaging. Following i.v. injection of 64Cu-labeled AMB-1, positron emission tomography imaging revealed increasing colonization of tumors and decreasing infection of organs after 4 hours. Viable cell counts showed that, by day 6, the bacteria had colonized tumors but were cleared completely from other organs. Magnetic resonance imaging showed a 1.22-fold (P = 0.003) increased positive contrast in tumors on day 2 and a 1.39-fold increase (P = 0.0007) on day 6. CONCLUSION: Magnetotactic bacteria can produce positive magnetic resonance imaging contrast and colonize mouse tumor xenografts, providing a potential tool for improved magnetic resonance imaging visualization in preclinical and translational studies to track cancer.

CNOB/ChrR6, a new prodrug enzyme cancer chemotherapy.

We report the discovery of a new prodrug, 6-chloro-9-nitro-5-oxo-5H-benzo(a)phenoxazine (CNOB). This prodrug is efficiently activated by ChrR6, the highly active prodrug activating bacterial enzyme we have previously developed. The CNOB/ChrR6 therapy was effective in killing several cancer cell lines in vitro. It also efficiently treated tumors in mice with up to 40% complete remission. 9-Amino-6-chloro-5H-benzo(a)phenoxazine-5-one (MCHB) was the only product of CNOB reduction by ChrR6. MCHB binds DNA; at nonlethal concentration, it causes cell accumulation in the S phase, and at lethal dose, it induces cell surface Annexin V and caspase-3 and caspase-9 activities. Further, MCHB colocalizes with mitochondria and disrupts their electrochemical potential. Thus, killing by CNOB involves MCHB, which likely induces apoptosis through the mitochondrial pathway. An attractive feature of the CNOB/ChrR6 regimen is that its toxic product, MCHB, is fluorescent. This feature proved helpful in in vitro studies because simple fluorescence measurements provided information on the kinetics of CNOB activation within the cells, MCHB killing mechanism, its generally efficient bystander effect in cells and cell spheroids, and its biodistribution. The emission wavelength of MCHB also permitted its visualization in live animals, allowing noninvasive qualitative imaging of MCHB in mice and the tumor microenvironment. This feature may simplify exploration of barriers to the penetration of MCHB in tumors and their amelioration.

Demonstration and partial characterization of ecto-ATPase in Balamuthia mandrillaris and its possible role in the host-cell interactions.

AIMS: To investigate the presence and partial characterization of ecto-ATPase in Balamuthia mandrillaris. METHODS AND RESULTS: In vitro assays were used to demonstrate that live B. mandrillaris hydrolyses extracellular AtP. Using nondenaturing polyacrylamide gel electrophoresis, B. mandrillaris exhibited a single ecto-ATPase band of molecular mass of more than 545 kDa. This ecto-ATPase was insensitive to ouabain, levamisole, sodium azide and sodium orthovanadate but stimulated by MgCl2. The ecto-ATPase was heat stable, but labile to detergent, sodium dodecyl sulphate. Suramin, an antagonist of P2 purinoreceptors and an inhibitor of some ecto-ATPases, inhibited B. mandrillaris binding to and cytotoxicity of HBMEC (human brain microvascular endothelial cello), in vitro. CONCLUSIONS: For the first time, we describe that live B. mandrillaris hydrolyses extracellular ATP and exhibits a > 545kDa ecto-ATPase. SIGNIFICANCE AND IMPACT OF THE STUDY: This surface enzyme may play a role in the salvage of purines from the extracellular medium and may be important for the pathogenesis of B. mandrillaris.

What leads from dead-end?

The 129 mouse strain develops congenital testicular germ cell tumors (TGCTs) at a low frequency. TGCTs in mice resemble the testicular tumors (teratomas) that occur in human infants. The genes that cause these tumors in 129 have not been identified. The defect at the Ter locus increases TGCT incidence such that 94% of 129-Ter/Ter males develop TGCTs. The primary effect of the Ter mutation is progressive loss of primordial germ cells (PGCs) during embryonic development. This results in sterility in adult Ter/Ter mice on all mouse strain backgrounds. However, on the 129 background, Ter causes tumor development in addition to sterility. Therefore, Ter acts as a modifier of 129-derived TGCT susceptibility genes. Ter was identified to be a mutation that inactivates the Dead-end1 (Dnd1) gene. In this perspective, I discuss the possible areas of future investigations to elucidate the mechanism of TGCT development due to Dnd1 inactivation.

New enzyme for reductive cancer chemotherapy, YieF, and its improvement by directed evolution.

Reductive prodrugs, mitomycin C and 5-aziridinyl-2,4-dinitrobenzamide (CB 1954), are nontoxic in their native form but become highly toxic upon reduction. Their effectiveness in cancer chemotherapy can be enhanced by delivering to tumors enzymes with improved prodrug reduction kinetics. We report the discovery of a new prodrug-reducing enzyme, YieF, from Escherichia coli, and the improvement of its kinetics for reducing mitomycin C and CB 1954. A YieF-derived enzyme, Y6, killed HeLa spinner cells with >or=5-fold efficiency than the wild-type enzymes, YieF and NfsA, at a variety of drug and enzyme concentrations and incubation times. With adhered HeLa cells and Salmonella typhimurium SL 7838 bacteria as enzyme delivery vehicle, at least an order of magnitude less of Y6-producing bacteria were required to kill >90% of tumor cells compared with bacteria expressing the wild-type enzymes, which at a comparable level killed < 5% of the cells. Thus, Y6 is a promising enzyme for use in cancer chemotherapy, and Salmonella strain SL 7838, which specifically targets tumors, may be used to deliver the prodrug-activating enzymes to tumors.

Mechanism of chromate reduction by the Escherichia coli protein, NfsA, and the role of different chromate reductases in minimizing oxidative stress during chromate reduction.

Chromate [Cr(VI)] is a serious environmental pollutant, which is amenable to bacterial bioremediation. NfsA, the major oxygen-insensitive nitroreductase of Escherichia coli, is a flavoprotein that is able to reduce chromate to less soluble and less toxic Cr(III). We show that this process involves single-electron transfer, giving rise to a flavin semiquinone form of NfsA and Cr(V) as intermediates, which redox cycle, generating more reactive oxygen species (ROS) than a divalent chromate reducer, YieF. However, NfsA generates less ROS than a known one-electron chromate reducer, lipoyl dehydrogenase (LpDH), suggesting that NfsA employs a mixture of uni- and di-valent electron transfer steps. The presence of YieF, ChrR (another chromate reductase we previously characterized), or NfsA in an LpDH-catalysed chromate reduction reaction decreased ROS generation by c. 65, 40, or 20%, respectively, suggesting that these enzymes can pre-empt ROS generation by LpDH. We previously showed that ChrR protects Pseudomonas putida against chromate toxicity; here we show that NfsA or YieF overproduction can also increase the tolerance of E. coli to this compound.

Sensitized polygenic trait analysis.

Genetic variation in many biological processes and evolutionary adaptations is caused by polygenes--genes that act in combination to affect a particular trait. Despite the recent identification of several polygenes, many remain to be found, suggesting that new experimental and analytical methods are needed to facilitate their discovery. Here we discuss sensitized polygenetic trait analysis, a method that has emerged recently for simplifying the genetic analysis of polygenic traits. The method uses a known single gene mutation in linkage testing crosses to 'sensitize' the analysis. By increasing the frequency of affected individuals in segregating populations, linkages are more readily detected. This method has considerable potential, especially given the increasing variety of mutations that can be used to sensitize the genetic analysis of polygenic traits.