The impact of iron and chelators on Lake Kinneret phytoplankton

The response of natural phytoplankton populations from LakeKinneret to the addition of iron (Fe) and chelator (EDTA) wastested by following growth patterns and determining the algalcomposition initially and at the end of the experiments; algalgrowth rates and yield were measured by in vivo chlorophyllfluorescence. Although the pattern of growth response varied,usually some stimulation of phytoplankton growth rate and yieldwas observed with chelator and/or Fe addition in comparisonto unsupplemented samples. However, the major impact of Fe andEDTA on the phytoplankton appeared to be expressed as changesin the algal population composition. There were clear taxonomicdifferences in the response to Fe addition, which in nearlyall experiments stimulated outgrowth of Bacillanophyta and Chlorophyta,compared with the effect of EDTA alone or with Fe which enhancedthe development of Cyanophyta, in addition to Bacillariophyta.The availability of Fe and/or chelators therefore appears tobe important in determining the composition of the phytoplanktonpopulations in Lake Kinneret and presumably in other aquaticenvironments.     

The combination of atenolol and amlodipine is better than their monotherapy for preventing end‐organ damage in different types of hypertension in rats

Combinations therapy is often used in hypertensive patients whether combination therapy is necessary for preventing end‐organ damage is not known. The objective of this study was to determine in four different hypertensive animal models the necessity of adding the calcium channel blocker amlodipine to therapy with the ß‐blocker atenolol to modulate end‐organ damage. Spontaneously hypertensive rats, DOCA‐salt hypertensive rats, two‐kidney, one‐clip renovascular hypertensive rats and Lyon genetically hypertensive rats were used to study this objective. These animal models have different sensitivities to atenolol and amlodipine. The dosages of therapy employed were 10 mg/kg atenolol alone, 1 mg/kg amlodipine, 10 mg atenolol + 1 mg/kg amlodipine and 5 mg/kg atenolol+0.5 mg/kg amlodipine. BP was continuously recorded in all animals. After determination of baroreflex sensitivity, rats were sacrificed for end‐organ damage evaluation. The combination of amlodipine and atenolol had a synergistic inhibitory effect on blood pressure and blood pressure variability, and end‐organ damage as compared with monotherapy with atenolol or amlodipine in all animal models. Baroreflex sensitivity also improved with the combination therapy more than with monotherapy. In conclusion, atenolol and amlodipine combination exerts a superior effect on blood pressure, blood pressure variability, baroreflex sensitivity and end‐organ damage. The superior effect of the combination was observed in all four models of hypertension.     

NaxD is a deacetylase required for lipid A modification and Francisella pathogenesis

Modification of specific Gram‐negative bacterial cell envelope components, such as capsule, O‐antigen and lipid A, are often essential for the successful establishment of infection. Francisella species express lipid A molecules with unique characteristics involved in circumventing host defences, which significantly contribute to their virulence. In this study, we show that NaxD, a member of the highly conserved YdjC superfamily, is a deacetylase required for an important modification of the outer membrane component lipid A in Francisella. Mass spectrometry analysis revealed that NaxD is essential for the modification of a lipid A phosphate with galactosamine in Francisella novicida, a model organism for the study of highly virulent Francisella tularensis. Significantly, enzymatic assays confirmed that this protein is necessary for deacetylation of its substrate. In addition, NaxD was involved in resistance to the antimicrobial peptide polymyxin B and critical for replication in macrophages and in vivo virulence. Importantly, this protein is also required for lipid A modification in F. tularensis as well as Bordetella bronchiseptica. Since NaxD homologues are conserved among many Gram‐negative pathogens, this work has broad implications for our understanding of host subversion mechanisms of other virulent bacteria.     

Phylogenetic and phenotypic analyses of arsenic-reducing bacteria isolated from an old tin mine area in Thailand

An agar plate screening assay was used to determine whether 100 arsenic-resistant bacterial isolates, previously obtained from arsenic-contaminated soils, had the ability to transform arsenite and arsenate. Ninety-five percent of the isolates were capable of reducing arsenate on agar plates. The isolates also grew in the presence of high concentrations of arsenite, but none of the bacterial isolates oxidized arsenite to arsenate under the growth conditions tested. About 14 % (13 of 95) of the tested isolates transformed high levels of arsenate (33–70 μM) when tested using the molybdenum blue method. Partial sequence analysis of 16S rDNA genes indicated that the isolates belonged to two broad taxonomic groups: Firmicutes and Proteobacteria. Ten isolates were assigned to four species in the genus Bacillus, and three isolates belonged to two species in the genera Enterobacter and Ochrobactrum. Taken together these results indicate that phylogenetically diverse bacteria isolated from arsenic-contaminated soils in an old tin mine area in Thailand have the ability to transform arsenate to arsenite.     

Diagnosis of extrapulmonary tuberculosis by PCR

During the last two decades, the resurgence of tuberculosis (TB) has been documented in both developed and developing nations, and much of this increase in TB burden coincided with human immunodeficiency virus (HIV) epidemics. Since then, the disease pattern has changed with a higher incidence of extrapulmonary tuberculosis (EPTB) as well as disseminated TB. EPTB cases include TB lymphadenitis, pleural TB, TB meningitis, osteoarticular TB, genitourinary TB, abdominal TB, cutaneous TB, ocular TB, TB pericarditis and breast TB, although any organ can be involved. Diagnosis of EPTB can be baffling, compelling a high index of suspicion owing to paucibacillary load in the biological specimens. A negative smear for acid-fast bacilli, lack of granulomas on histopathology and failure to culture Mycobacterium tuberculosis do not exclude the diagnosis of EPTB. Novel diagnostic modalities such as nucleic acid amplification (NAA) can be useful in varied forms of EPTB. This review is primarily focused on the diagnosis of several clinical forms of EPTB by polymerase chain reaction (PCR) using different gene targets.     

Selection of reliable reference genes for qRT-PCR analysis in human non-cancerous gastric tissue

Real-time PCR (qRT-PCR) is the standard method for studying changes in relative gene expression in complex diseases like obesity and gastritis. However, variations in amount of starting material, enzymatic efficiency and presence of amplification inhibitors can lead to quantification errors. Hence, the need for accurate data normalization is vital. Among several known strategies for data normalization, the use of reference genes as an internal control is the most common approach. Human gastric tissue has been the least investigated for stability of reference gene expression. In this study, three popular algorithms, GeNorm, NormFinder and BestKeeper were used to evaluate the reference gene stability. Conclusion: HPRT1 and GAPDH are the best performing pair of reference genes for qRT-PCR profiling experiments involving non-malignant gastric tissue samples.     

The immune response to a vesicular stomatitis virus vaccine vector is independent of particulate antigen secretion and protein turnover rate

Vesicular stomatitis virus (VSV) is a highly cytopathic virus being developed as a vaccine vector due to its ability to induce strong protective T cell and antibody responses after a single dose. However, little is known regarding the mechanisms underlying the potent immune responses elicited by VSV. We previously generated a VSV vector expressing the hepatitis B virus middle envelope surface glycoprotein (MS) that induces strong MS-specific T cell and antibody responses in mice. After synthesis in the cytoplasm, the MS protein translocates to the endoplasmic reticulum, where it forms subviral particles that are secreted from the cell. To better understand the contributions of secreted and intracellular protein to the VSV-induced immune response, we produced a vector expressing a secretion-deficient MS mutant (MS(C69A)) and compared the immunogenicity of this vector to that of the wild-type VSV-MS vector in mice. As expected, the MS(C69A) protein was not secreted from VSV-infected cells and displayed enhanced proteasome-mediated degradation. Surprisingly, despite these differences in intracellular protein processing, the T cell and antibody responses generated to MS(C69A) were comparable to those elicited by virus expressing wild-type MS protein. Therefore, when it is expressed from VSV, the immune responses to MS are independent of particulate antigen secretion and the turnover rate of cytoplasmic protein. These results are consistent with a model in which the immune responses to VSV are strongly influenced by the replication cycle of the vector and demonstrate that characteristics of the vector have the capacity to affect vaccine efficacy more than do the properties of the antigen itself.     

A transposase strategy for creating libraries of circularly permuted proteins

A simple approach for creating libraries of circularly permuted proteins is described that is called PERMutation Using Transposase Engineering (PERMUTE). In PERMUTE, the transposase MuA is used to randomly insert a minitransposon that can function as a protein expression vector into a plasmid that contains the open reading frame (ORF) being permuted. A library of vectors that express different permuted variants of the ORF-encoded protein is created by: (i) using bacteria to select for target vectors that acquire an integrated minitransposon; (ii) excising the ensemble of ORFs that contain an integrated minitransposon from the selected vectors; and (iii) circularizing the ensemble of ORFs containing integrated minitransposons using intramolecular ligation. Construction of a Thermotoga neapolitana adenylate kinase (AK) library using PERMUTE revealed that this approach produces vectors that express circularly permuted proteins with distinct sequence diversity from existing methods. In addition, selection of this library for variants that complement the growth of Escherichia coli with a temperature-sensitive AK identified functional proteins with novel architectures, suggesting that PERMUTE will be useful for the directed evolution of proteins with new functions.     

The spectrum of 4q- syndrome illustrated by a case series

Deletions of the long arm of chromosome 4 detectable by cytogenetic analysis (standard karyotyping), fluorescent in situ hybridisation (FISH), multiplex ligation-dependent probe amplification (MLPA) or comparative genomic hybridisation (CGH) cause 4q- syndrome. Here we describe 3 cases of 4q- syndrome which demonstrate the variations in clinical presentation, diagnosis and prognosis observed in this condition. Patient 1 was a female foetus diagnosed with del(4)(q33) following chorionic villus sampling (CVS) at 14weeks, and the pregnancy was terminated at 18weeks. Patient 2 was a 5-month-old boy with del(4)(q31.3) and complex congenital heart disease. He also had a duplication of chromosome 6p and died of cardiac failure. Patient 3 is a 2-year-old girl with mild dysmorphic features and an interstitial deletion del(4)(q22.1q23). She has no major malformations and only slight developmental delay.     

Spectroscopic studies of two spectral variants of light-harvesting complex 2 (LH2) from the photosynthetic purple sulfur bacterium Allochromatium vinosum

Two spectral forms of the peripheral light-harvesting complex (LH2) from the purple sulfur photosynthetic bacterium Allochromatium vinosum were purified and their photophysical properties characterized. The complexes contain bacteriochlorophyll a (BChl a) and multiple species of carotenoids. The composition of carotenoids depends on the light conditions applied during growth of the cultures. In addition, LH2 grown under high light has a noticeable split of the B800 absorption band. The influence of the change of carotenoid distribution as well as the spectral change of the excitonic absorption of the bacteriochlorophylls on the light-harvesting ability was studied using steady-state absorption, fluorescence and femtosecond time-resolved absorption at 77K. The results demonstrate that the change of the distribution of the carotenoids when cells were grown at low light adapts the absorptive properties of the complex to the light conditions and maintains maximum photon-capture performance. In addition, an explanation for the origin of the enigmatic split of the B800 absorption band is provided. This spectral splitting is also observed in LH2 complexes from other photosynthetic sulfur purple bacterial species. According to results obtained from transient absorption spectroscopy, the B800 band split originates from two spectral forms of the associated BChl a monomeric molecules bound within the same complex.