Description of local and global shape properties of protein helices

A new method, dubbed “HAXIS” is introduced to describe local and global shape properties of a protein helix via its axis. HAXIS is based on coarse-graining and spline-fitting of the helix backbone. At each Cα anchor point of the backbone, a Frenet frame is calculated, which directly provides the local vector presentation of the helix. After cubic spline-fitting of the axis line, its curvature and torsion are calculated. This makes a rapid comparison of different helix forms and the determination of helix similarity possible. Distortions of the helix caused by individual residues are projected onto the helix axis and presented either by the rise parameter per residue or by the local curvature of the axis. From a non-redundant set of 2,017 proteins, 15,068 helices were investigated in this way. Helix start and helix end as well as bending and kinking of the helix are accurately described. The global properties of the helix are assessed by a polynomial fit of the helix axis and the determination of its overall curving and twisting. Long helices are more regular shaped and linear whereas short helices are often strongly bent and twisted. The distribution of different helix forms as a function of helix length is analyzed.     

E. coli biofilm formation and its susceptibility towards T4 bacteriophages studied in a continuously operating mixing – tubular bioreactor system

A system consisting of a connected mixed and tubular bioreactor was designed to study bacterial biofilm formation and the effect of its exposure to bacteriophages under different experimental conditions. The bacterial biofilm inside silicone tubular bioreactor was formed during the continuous pumping of bacterial cells at a constant physiological state for 2 h and subsequent washing with a buffer for 24 h. Monitoring bacterial and bacteriophage concentration along the tubular bioreactor was performed via a piercing method. The presence of biofilm and planktonic cells was demonstrated by combining the piercing method, measurement of planktonic cell concentration at the tubular bioreactor outlet, and optical microscopy. The planktonic cell formation rate was found to be 8.95 × 10⁻³ h⁻¹ and increased approximately four-fold (4×) after biofilm exposure to an LB medium. Exposure of bacterial biofilm to bacteriophages in the LB medium resulted in a rapid decrease of biofilm and planktonic cell concentration, to below the detection limit within < 2 h. When bacteriophages were supplied in the buffer, only a moderate decrease in the concentration of both bacterial cell types was observed. After biofilm washing with buffer to remove unadsorbed bacteriophages, its exposure to the LB medium (without bacteriophages) resulted in a rapid decrease in bacterial concentration: again below the detection limit in < 2 h.     

A Systematic Review of the Amount of Water per Person per Day Needed to Prevent Morbidity and Mortality in (Post-)Disaster Settings

BackgroundIn order to improve the effectiveness and efficiency of humanitarian efforts, minimum standards for humanitarian assistance and key indicators, showing whether a standard has been attained, have been developed. However, many of these standards and indicators are based on a consensus on best practices and experiences in humanitarian response, because relevant evidence on the impact of humanitarian interventions is often lacking.ObjectivesOne important example of a standard in humanitarian aid in a disaster setting is “water quantity.” The accompanying indicator states how many litres of water are needed per person per day in a disaster setting. It was our objective to determine the evidence base behind this indicator, in order to improve health outcomes such as morbidity (e.g., diarrhoea) and mortality.MethodsA systematic review was performed searching The Cochrane Library, Medline and Embase. We included studies performed during disasters and in refugee camps that reported a specific water amount and health-related outcomes related to water shortages, including diarrhoea, cholera, and mortality. We used GRADE to determine the quality of evidence.ResultsOut of 3,630 articles, 111 references relevant to our question were selected. Based on our selection criteria, we finally retained 6 observational studies, including 1 study that was performed during the disaster and 5 studies in a post-disaster phase. From two studies there is conclusive evidence on the relationship between the amount of water received and diarrhoea or mortality rates in refugee camps. However, overall, these studies do not contain enough data with relevance to a specific amount of water, and the level of evidence is very low.ConclusionsMore primary research on water amounts in a disaster setting is necessary, so that the humanitarian sector can further professionalise its water-related standards, indicators and interventions.     

Differential allelic expression of IL13 and CSF2 genes associated with asthma

An important area of genetic research is the identification of functional mechanisms in polymorphisms associated with diseases. A highly relevant functional mechanism is the influence of polymorphisms on gene expression levels (differential allelic expression, DAE). The coding single nucleotide polymorphisms (SNPs) CSF2^rs25882 and IL13^rs20541 have been associated with asthma. In this work, we investigated whether the mRNA expression levels of CSF2 or IL13 were correlated with these SNPs. Samples were analyzed by mass spectrometry-based quantification of gene expression. Both SNPs influenced gene expression levels (CSF2^rs25882: p_overall = 0.008 and p_{DAE samples} = 0.00006; IL13^rs20541: p_overall = 0.059 and p_{DAE samples} = 0.036). For CSF2, the expression level was increased by 27.4% (95% CI: 18.5%–35.4%) in samples with significant DAE in the presence of one copy of risk variant CSF2^rs25882-T. The average expression level of IL13 was increased by 29.8% (95% CI: 3.1%–63.4%) in samples with significant DAE in the presence of one copy of risk variant IL13^rs20541-A. Enhanced expression of CSF2 could stimulate macrophages and neutrophils during inflammation and may be related to the etiology of asthma. For IL-13, higher expression could enhance the functional activity of the asthma-associated isoform. Overall, the analysis of DAE provides an efficient approach for identifying possible functional mechanisms that link disease-associated variants with altered gene expression levels.     

Polyethylene glycol conjugates of methotrexate varying in their molecular weight from MW 750 to MW 40000: synthesis,characterization, and structure-activity relationships in vitro and in vivo

Poly(ethylene glycol)s (PEGs) are potential drug carriers for improving the therapeutic index of anticancer agents. In this work, the anticancer drug methotrexate (MTX) was activated with N,N'-dicyclohexylcarbodiimide (DCC) and coupled to amino group bearing PEGs of MW 750, 2000, 5000, 10 000, 20,000, and 40,000. First, the activation process of MTX with DCC in the presence and absence of N-hydroxysuccinimide was analyzed through HPLC. Preincubation of methotrexate with DCC alone at 0 degrees C proved to be favorable with respect to the amount of activated species and the formation of byproducts. MTX-PEG conjugates were synthesized according to this procedure, isolated through size-exclusion chromatography, and characterized through analytical HPLC, MALDI-TOF spectrometry, and gel permeation chromatography. In a cell-free assay, all of the drug polymer conjugates inhibited the target enzyme of MTX, dihydrofolate reductase (DHFR), to a similar extent, but were not as active as free MTX. Additionally, incubation of the MTX-PEG40000 conjugate for 6 days at 37 degrees C in phosphate buffered saline (pH 7.4), in cell-conditioned medium, or in human serum revealed no significant release of methotrexate. These results, taken together, indicate that release of MTX from polymer conjugates is not necessary for an effective interaction with the active site of dihydrofolate reductase. Evaluation of the in vitro cytotoxicity of the MTX-PEG conjugates in two adherent and three suspension human tumor cell lines revealed that the IC(50) values of the tested compounds increased with the size of the drug-polymer conjugates. The most effective compound tested in these assays was the free drug MTX itself (IC(50) value ranging from approximately 0.01 to 0.05 microM), while the IC(50) values of the polymer conjugates were higher (IC(50) value for MTX-PEG750, 2000 and 5000: approximately 0.6-3 microM; for MTX-PEG10000 and 20000: approximately 2-7 microM; and for MTX-PEG40000: > 6 microM). Subsequently, MTX-PEG5000, MTX-PEG20000, and MTX-PEG40000 were evaluated in a human mesothelioma MSTO-211H xenograft model, and their antitumor effects were compared with free methotrexate and the albumin conjugate MTX-HSA, a conjugate that is currently in phase II clinical trials. In contrast to the in vitro results, the high molecular weight MTX-PEG conjugates exhibited the highest in vivo antitumor activity: At a dose of 40 and 80 mg/kg MTX-PEG5000 was less active than MTX at its optimal dose of 100 mg/kg; MTX-PEG20000 at a dose of 40 mg/kg showed antitumor efficacy comparable to MTX, but MTX-PEG40000 at a dose of 20 mg/kg was superior to MTX and demonstrated antitumor activity of the same order as MTX-HSA (20 mg/kg).