An innovative cloning platform enables large-scale production and maturation of an oxygen-tolerant [NiFe]-hydrogenase from Cupriavidus necator in Escherichia coli.

Expression of multiple heterologous genes in a dedicated host is a prerequisite for approaches in synthetic biology, spanning from the production of recombinant multiprotein complexes to the transfer of tailor-made metabolic pathways. Such attempts are often exacerbated, due in most cases to a lack of proper directional, robust and readily accessible genetic tools. Here, we introduce an innovative system for cloning and expression of multiple genes in Escherichia coli BL21 (DE3). Using the novel methodology, genes are equipped with individual promoters and terminators and subsequently assembled. The resulting multiple gene cassettes may either be placed in one vector or alternatively distributed among a set of compatible plasmids. We demonstrate the effectiveness of the developed tool by production and maturation of the NAD(+)reducing soluble [NiFe]-hydrogenase (SH) from Cupriavidus necator H16 (formerly Ralstonia eutropha H16) in E. coli BL21Star™ (DE3). The SH (encoded in hoxFUYHI) was successfully matured by co-expression of a dedicated set of auxiliary genes, comprising seven hyp genes (hypC1D1E1A2B2F2X) along with hoxW, which encodes a specific endopeptidase. Deletion of genes involved in SH maturation reduced maturation efficiency substantially. Further addition of hoxN1, encoding a high-affinity nickel permease from C. necator, considerably increased maturation efficiency in E. coli. Carefully balanced growth conditions enabled hydrogenase production at high cell-densities, scoring mg·(Liter culture)(-1) yields of purified functional SH. Specific activities of up to 7.2±1.15 U·mg(-1) were obtained in cell-free extracts, which is in the range of the highest activities ever determined in C. necator extracts. The recombinant enzyme was isolated in equal purity and stability as previously achieved with the native form, yielding ultrapure preparations with anaerobic specific activities of up to 230 U·mg(-1). Owing to the combinatorial power exhibited by the presented cloning platform, the system might represent an important step towards new routes in synthetic biology.

Facile analysis of short-chain fatty acids as 4-nitrophenyl esters in complex anaerobic fermentation samples by high performance liquid chromatography.

Short-chain fatty acids are crucial intermediates in the conversion of biomass to methane. Due to the complexity of raw biomass, volatile fatty acids (including n- and branched-chain compounds) as well as arylacetic and arylpropionic acids arise from digestion of carbohydrates, proteins and lipids. The development of a simple extraction procedure in combination with internal standardization and facile 4-nitrophenyl-labelling via oxalylchloride-generated acylchlorides enabled robust separation and quantification of the target compounds in crude biological samples like raw cattle manure and biogas fermenter contents. Detection limits of <100 µM and error rates of less than 4% for the quantification of individual compounds in a concentration range up to 50 mM for non-diluted samples suggest that the novel method might be of general advantage for the routine quantification of short-chain fatty acids in complex biological samples including complex fermentation media.

Evaluation of the genotoxicity of treated urban sludge in the Tradescantia micronucleus assay.

The sludge produced in sewage treatment plants can contain toxic substances. Among these, the genotoxic substances are of great concern. The present paper aimed at evaluating the genotoxicity of treated sludge samples collected at four different sewage treatment plants (STP) located in the State of São Paulo, Brazil using the Trad-MN assay. Another objective of the study was to compare the responses of the Clone #4430 with the Tradescantia pallida. Sludge samples mixed with reference soil in concentrations of 10, 25 and 50% (v/v) were tested in experiments with 3 months exposure in the field. Negative and positive controls (arsenic trioxide) were also tested with both plants. In Clone #4430 two sludge samples induced genotoxicity while in T. pallida three were positive, although no clear dose-response were observed for both plants. Results with the negative and positive controls suggest that T. pallida presented similar results when compared to the Clone #4430. The protocol using plants chronically exposed to sludge mixed with soil seems to be a promising tool to assess the genotoxicity of sludge although time consuming.

Use of a new fluorescent probe, seminaphthofluorescein-calcein, for determination of intracellular pH by simultaneous dual-emission imaging laser scanning confocal microscopy.

A new pH indicator, seminaphthofluorescein (SNAFL)-calcein acetoxymethyl ester, was used for intracellular pH (pHi) measurement in living MDCK cells with a laser scanning confocal microscope (LSCM) equipped with an Argon/Krypton laser and dual-excitation and dual-emission (FITC/Texas Red) filter set. SNAFL-calcein excitation maxima are approximately 492/540 nm (acid/base) and emission maxima are approximately 535/625 nm (acid/base) with a pKa value at approximately 7.0. The absorption/emission spectra of SNAFL-calcein indicate that the ratio of emission intensities of its basic/acidic forms is pH dependent. With an Argon/Krypton LSCM, we were able to monitor the acidic and basic forms of this dye simultaneously using dual-excitation (488/568 nm) and dual-emission (525-614 nm/> or = 615 nm) wavelengths (lambda s). The simultaneous dual-excitation/emission LSCM system allows for efficient recording of pHi dynamics (time resolution approximately 1 sec) in living cells. We have analyzed emission stability of the dye at different temperatures (22 degrees C and 37 degrees C) and constant pH, and at the same temperature (22 degrees C) but various pHs (6.6, 7.0, and 7.4). Bleaching rate is slightly higher at 37 degree C than that at 22 degrees C. The basic form of the dye (lambda Em approximately 625 nm) has a slightly higher bleaching rate than the acidic form (lambda Em approximately 535 nm) in standard culture medium (pH 7.3) at either 22 degree C or 37 degrees C. The pHi in MDCK cells calculated from ratio images (535 nm/625 nm) was 7.19 +/- 0.03 (mean +/- SEM, n = 20). Calibration experiments show that the useful pH range of SNAFL-calcein appears to be between 6.2 and 7.8, as the dye is difficult to calibrate outside this pH range.

The development of a new family of intracellular calcium probes.

Four new potential fluorescent probes for Ca2+ using the coumarin moiety as a fluorophore have been synthesized and their spectral properties and binding affinities for Ca2+ determined. The most promising derivative for intracellular use, BTC, exhibits an excitation wavelength shift from 462 nm to 401 nm on binding Ca2+, with an emission wavelength of 530 nm. The quantum yield of this probe increases from 0.07 as the free indicator to 0.12 on binding Ca2+. BTC, loaded as its tetraacetoxymethyl ester (AM ester) into mouse myeloma P3X cells, responded only when cytoplasmic Ca2+ exceeded typical intracellular calcium concentrations. The dye, therefore, appears to be useful in excitatory cells or extracellular spaces with intracellular calcium concentrations high enough to saturate typical excitation ratio Ca2+ indicators such as Fura-2.

Development of multiple endocrine neoplasia type 2A does not involve substantial deletions of chromosome 10.

In MEN2A both familial and sporadic cases are known. The familial cases show a dominant pattern of inheritance. In these respects, MEN2A resembles other tumors in whose etiology so-called tumor suppressor genes play a decisive role. The MEN2A locus has been assigned to chromosome 10 by linkage studies. Analysis of tumor DNA from 42 patients shows that markers on chromosome 10 were lost in only one tumor. Thus, these results contrast with previous studies which show that tumor development is generally associated with the loss of the whole or substantial parts of the chromosome on which the putative tumor suppressor gene is located.

Unruptured intracranial aneurysm and transient focal cerebral ischemia: a follow-up study.

Transient focal cerebral ischemia may occasionally be due to distal embolization of a clot from an unruptured intracranial aneurysm. Follow-up data in 12 such patients revealed no ischemic strokes, subarachnoid or parenchymal hemorrhages in a mean follow-up period of 6.5 years. The aneurysms ranged in size from 2 to 12 mm in diameter, and only two were larger than 10 mm. Two patients had clip ligation of the aneurysm, five patients were given platelet antiaggregation therapy, one was given oral anticoagulants after aortic valve surgery, and four had no specific therapy. The prognosis for unruptured aneurysms presenting with transient focal ischemia was good, regardless of therapy.

Fever in the wake of a stroke.

We performed a prospective study of the frequency and cause of fever, defined as a rectal temperature of greater than or equal to 101 degrees F detected within the first 5 days, in 104 consecutive adults admitted to a community/teaching hospital because of a nontraumatic stroke. Fever was documented in 23 patients. A source for the fever was identified in 19 patients and was attributed to a pulmonary insult, either aspiration chemical pneumonitis or an infectious pneumonia, in 13 of these patients. Patients who experienced lacunar infarcts did not develop fever. Fever occurring in the wake of a stroke should not be attributed to the vascular process, but should direct attention to inflammatory disorders of the lungs.

Transient focal cerebral ischemia as a presenting manifestation of unruptured cerebral aneurysms.

Few reports have described an association between cerebral transient ischemic attacks (TIAs) and unruptured cerebral aneurysms. This study presents seven patients with TIA who had aneurysms in a vascular distribution appropriate to their clinical symptoms. In three patients, angiographic evidence of embolization was present distal to the aneurysm without apparent cardiac or extracranial arterial source for the emboli. The most reasonable pathogenesis for TIA in a patient with an associated saccular aneurysm would be thrombosis of the aneurysmal sac with subsequent embolization. However, subarachnoid blood can cause permanent focal intracranial narrowing, and this appeared to be a factor in at least one patient. The results imply that patients with symptoms of TIA should have their intracranial arterial circulation visualized as part of the diagnostic evaluation.