The Unstirrable Water Layer Is Unstirrable because It Does Not Exist

A number of membrane‐permeation models require the incorporation of an unstirred or unstirrable water layer (UWL). An example occurs in PAMPA models when the effective permeation rate of lipophilic acids and bases, P_e, falls behind the expected permeation rate, P_m, at pH values providing a high concentration of unionized species in the donor phase. In such cases, the compound has an apparent pK_a of a weaker acid or base. The explanation is that an UWL adjacent to the membrane provides a rate‐limiting diffusion barrier for such compounds. The thickness of the UWL is correlated with the difference between the aqueous pK_a and the apparent pK_a (pK ). Here, we provide an explanation for the pK term that requires no UWL. It comes from the fact that, in the process of passing into a membrane, an ionizable compound undergoes a change in pK_a. At some point along its path into the membrane, the compound attains a maximum free energy, at which point it is as likely to continue into the membrane, as it is to return to the donor phase. This is the transition state for absorption. The pK is the pK_a of the compound at the transition state. This is a testable hypothesis (see text). The relevance of absorption to permeation depends on the rate‐limiting step of permeation.     

Very Bright Green Fluorescent Proteins from the Pontellid Copepod Pontella mimocerami

Background

Fluorescent proteins (FP) homologous to the green fluorescent protein (GFP) from the jellyfish Aequorea victoria have revolutionized biomedical research due to their usefulness as genetically encoded fluorescent labels. Fluorescent proteins from copepods are particularly promising due to their high brightness and rapid fluorescence development.

Results

Here we report two novel FPs from Pontella mimocerami (Copepoda, Calanoida, Pontellidae), which were identified via fluorescence screening of a bacterial cDNA expression library prepared from the whole-body total RNA of the animal. The proteins are very similar in sequence and spectroscopic properties. They possess high molar extinction coefficients (79,000 M⁻¹ cm⁻) and quantum yields (0.92), which make them more than two-fold brighter than the most common FP marker, EGFP. Both proteins form oligomers, which we were able to counteract to some extent by mutagenesis of the N-terminal region; however, this particular modification resulted in substantial drop in brightness.

Conclusions

The spectroscopic characteristics of the two P. mimocerami proteins place them among the brightest green FPs ever described. These proteins may therefore become valuable additions to the in vivo imaging toolkit.     

Simple Estimation of Incident HIV Infection Rates in Notification Cohorts Based on Window Periods of Algorithms for Evaluation of Line-Immunoassay Result Patterns

Background

Tests for recent infections (TRIs) are important for HIV surveillance. We have shown that a patient''s antibody pattern in a confirmatory line immunoassay (Inno-Lia) also yields information on time since infection. We have published algorithms which, with a certain sensitivity and specificity, distinguish between incident (< = 12 months) and older infection. In order to use these algorithms like other TRIs, i.e., based on their windows, we now determined their window periods.

Methods

We classified Inno-Lia results of 527 treatment-naïve patients with HIV-1 infection < = 12 months according to incidence by 25 algorithms. The time after which all infections were ruled older, i.e. the algorithm''s window, was determined by linear regression of the proportion ruled incident in dependence of time since infection. Window-based incident infection rates (IIR) were determined utilizing the relationship ‘Prevalence  =  Incidence x Duration’ in four annual cohorts of HIV-1 notifications. Results were compared to performance-based IIR also derived from Inno-Lia results, but utilizing the relationship ‘incident  =  true incident + false incident’ and also to the IIR derived from the BED incidence assay.

Results

Window periods varied between 45.8 and 130.1 days and correlated well with the algorithms'' diagnostic sensitivity (R² = 0.962; P<0.0001). Among the 25 algorithms, the mean window-based IIR among the 748 notifications of 2005/06 was 0.457 compared to 0.453 obtained for performance-based IIR with a model not correcting for selection bias. Evaluation of BED results using a window of 153 days yielded an IIR of 0.669. Window-based IIR and performance-based IIR increased by 22.4% and respectively 30.6% in 2008, while 2009 and 2010 showed a return to baseline for both methods.

Conclusions

IIR estimations by window- and performance-based evaluations of Inno-Lia algorithm results were similar and can be used together to assess IIR changes between annual HIV notification cohorts.     

Determining mutations in G6PC and SLC37A4 genes in a sample of Brazilian patients with glycogen storage disease types Ia and Ib

Glycogen storage disease (GSD) comprises a group of autosomal recessive disorders characterized by deficiency of the enzymes that regulate the synthesis or degradation of glycogen. Types Ia and Ib are the most prevalent; while the former is caused by deficiency of glucose-6-phosphatase (G6Pase), the latter is associated with impaired glucose-6-phosphate transporter, where the catalytic unit of G6Pase is located. Over 85 mutations have been reported since the cloning of G6PC and SLC37A4 genes. In this study, twelve unrelated patients with clinical symptoms suggestive of GSDIa and Ib were investigated by using genetic sequencing of G6PC and SLC37A4 genes, being three confirmed as having GSD Ia, and two with GSD Ib. In seven of these patients no mutations were detected in any of the genes. Five changes were detected in G6PC, including three known point mutations (p.G68R, p.R83C and p.Q347X) and two neutral mutations (c.432G > A and c.1176T > C). Four changes were found in SLC37A4: a known point mutation (p.G149E), a novel frameshift insertion (c.1338_1339insT), and two neutral mutations (c.1287G > A and c.1076-28C > T). The frequency of mutations in our population was similar to that observed in the literature, in which the mutation p.R83C is also the most frequent one. Analysis of both genes should be considered in the investigation of this condition. An alternative explanation to the negative results in this molecular study is the possibility of a misdiagnosis. Even with a careful evaluation based on laboratory and clinical findings, overlap with other types of GSD is possible, and further molecular studies should be indicated.     

Dengue Fever Occurrence and Vector Detection by Larval Survey, Ovitrap and MosquiTRAP: A Space-Time Clusters Analysis

The use of vector surveillance tools for preventing dengue disease requires fine assessment of risk, in order to improve vector control activities. Nevertheless, the thresholds between vector detection and dengue fever occurrence are currently not well established. In Belo Horizonte (Minas Gerais, Brazil), dengue has been endemic for several years. From January 2007 to June 2008, the dengue vector Aedes (Stegomyia) aegypti was monitored by ovitrap, the sticky-trap MosquiTRAP™ and larval surveys in an study area in Belo Horizonte. Using a space-time scan for clusters detection implemented in SaTScan software, the vector presence recorded by the different monitoring methods was evaluated. Clusters of vectors and dengue fever were detected. It was verified that ovitrap and MosquiTRAP vector detection methods predicted dengue occurrence better than larval survey, both spatially and temporally. MosquiTRAP and ovitrap presented similar results of space-time intersections to dengue fever clusters. Nevertheless ovitrap clusters presented longer duration periods than MosquiTRAP ones, less acuratelly signalizing the dengue risk areas, since the detection of vector clusters during most of the study period was not necessarily correlated to dengue fever occurrence. It was verified that ovitrap clusters occurred more than 200 days (values ranged from 97.0±35.35 to 283.0±168.4 days) before dengue fever clusters, whereas MosquiTRAP clusters preceded dengue fever clusters by approximately 80 days (values ranged from 65.5±58.7 to 94.0±14. 3 days), the former showing to be more temporally precise. Thus, in the present cluster analysis study MosquiTRAP presented superior results for signaling dengue transmission risks both geographically and temporally. Since early detection is crucial for planning and deploying effective preventions, MosquiTRAP showed to be a reliable tool and this method provides groundwork for the development of even more precise tools.     

Replication origins are attached to the nuclear skeleton.

DNA fragments containing replication origins (oriDNA) were isolated from a chicken erythroblast cell line by a modified procedure of Zannis-Hadjopoulos et al. and studied in the renaturation reaction driven by either total or nuclear matrix DNA (nmDNA) from the same cells or from mature erythrocytes. We found that the unique sequences of nmDNA from erythroblasts (5 kb long) represented a specific subset of sequences constituting about a quarter of total DNA unique sequences, while the erythrocyte nmDNA 5 kb fragments constitute only about one tenth of total unique DNA and all are recovered among erythroblast nmDNA. Virtually all oriDNA sequences are present in the fraction of erythrocyte nmDNA. Thereafter, the putative positions of replication origins within the alpha-globine gene domain have been mapped by hybridization experiments. They were found to coincide with the previously established positions of permanent sites of DNA attachment to the nuclear matrix.     

Production of δ-Endotoxin by Bacillus thuringiensis as a Function of Glucose Concentrations

The insect pathogen Bacillus thuringiensis was cultured at different glucose concentrations. Size, protein content, and insecticidal activity of the parasporal inclusions, which are formed during the sporulation process, were measured. Increased glucose concentrations led to bigger crystalline inclusions with a higher content of protein and insecticidal activity. Maximum yields of protein and endotoxin were obtained in a semisynthetic medium that contained glucose concentrations of 6 to 8 g/liter.