Elucidation of an archaeal replication protein network to generate enhanced PCR enzymes.

Thermostable DNA polymerases are an important tool in molecular biology. To exploit the archaeal repertoire of proteins involved in DNA replication for use in PCR, we elucidated the network of proteins implicated in this process in Archaeoglobus fulgidus. To this end, we performed extensive yeast two-hybrid screens using putative archaeal replication factors as starting points. This approach yielded a protein network involving 30 proteins potentially implicated in archaeal DNA replication including several novel factors. Based on these results, we were able to improve PCR reactions catalyzed by archaeal DNA polymerases by supplementing the reaction with predicted polymerase co-factors. In this approach we concentrated on the archaeal proliferating cell nuclear antigen (PCNA) homologue. This protein is known to encircle DNA as a ring in eukaryotes, tethering other proteins to DNA. Indeed, addition of A. fulgidus PCNA resulted in marked stimulation of PCR product generation. The PCNA-binding domain was determined, and a hybrid DNA polymerase was constructed by grafting this domain onto the classical PCR enzyme from Thermus aquaticus, Taq DNA polymerase. Addition of PCNA to PCR reactions catalyzed by the fusion protein greatly stimulated product generation, most likely by tethering the enzyme to DNA. This sliding clamp-induced increase of PCR performance implies a promising novel micromechanical principle for the development of PCR enzymes with enhanced processivity.     

Membrane Region M2C2 in Subunit KtrB of the K+ Uptake System KtrAB from Vibrio alginolyticus Forms a Flexible Gate Controlling K+ Flux: AN ELECTRON PARAMAGNETIC RESONANCE STUDY*

Transmembrane stretch M_2C from the bacterial K⁺-translocating protein KtrB is unusually long. In its middle part, termed M_2C2, it contains several small and polar amino acids. This region is flanked by the two α-helices M_2C1 and M_2C3 and may form a flexible gate at the cytoplasmic side of the membrane controlling K⁺ translocation. In this study, we provide experimental evidence for this notion by using continuous wave and pulse EPR measurements of single and double spin-labeled cysteine variants of KtrB. Most of the spin-labeled residues in M_2C2 were shown to be immobile, pointing to a compact structure. However, the high polarity revealed for the microenvironment of residue positions 317, 318, and 327 indicated the existence of a water-accessible cavity. Upon the addition of K⁺ ions, M_2C2 residue Thr-318R1 (R1 indicates the bound spin label) moved with respect to M_2B residue Asp-222R1 and M_2C3 residue Val-331R1 but not with respect to M_2C1 residue Met-311R1. Based on distances determined between spin-labeled residues of double-labeled variants of KtrB in the presence and absence of K⁺ ions, structural models of the open and closed conformations were developed.     

Reducing acetylated tau is neuroprotective in brain injury

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Voxel-Based Morphometry in Women with Borderline Personality Disorder with and without Comorbid Posttraumatic Stress Disorder

Patients with Borderline Personality Disorder (BPD) showed reduced volume of amygdala and hippocampus, but similar findings are evident in Posttraumatic Stress Disorder (PTSD). Applying voxel-based morphometry (VBM) in a larger cohort of patients with BPD, we sought to extend earlier findings of volume abnormalities in limbic regions and to evaluate the influence of co-occurring PTSD in BPD patients. We used voxel-based morphometry to study gray matter volume (GMV) in 60 healthy controls (HC) and 60 patients with BPD. Subgroup analyses on 53 patients concerning the role of co-occurring PTSD were conducted. Additionally, regression analyses were calculated to assess the relation between borderline symptom severity as well as dissociative experiences and GMV. Differences in local GMV between patients with BPD and HC were observed in the amygdale and hippocampus as well as in the fusiform and cingulate gyrus. Co-occurring PTSD was accompanied by increased GMV in the superior temporal gyrus and dorsolateral prefrontal cortex. Independent of co-occurring PTSD, severity of BPD symptoms predicted smaller GMV in the amygdala and dorsal ACC. Dissociation was positively related to GMV in the middle temporal gyrus. We could replicate earlier findings of diminished limbic GMV in patients with BPD and additionally show that patients with co-morbid PTSD feature increased GMV in prefrontal regions associated with cognitive control.     

The Effect of Hydraulic Loading Rate and Influent Source on the Binding Capacity of Phosphorus Filters

Sorption by active filter media can be a convenient option for phosphorus (P) removal and recovery from wastewater for on-site treatment systems. There is a need for a robust laboratory method for the investigation of filter materials to enable a reliable estimation of their longevity. The objectives of this study were to (1) investigate and (2) quantify the effect of hydraulic loading rate and influent source (secondary wastewater and synthetic phosphate solution) on P binding capacity determined in laboratory column tests and (3) to study how much time is needed for the P to react with the filter material (reaction time). To study the effects of these factors, a 2² factorial experiment with 11 filter columns was performed. The reaction time was studied in a batch experiment. Both factors significantly (α = 0.05) affected the P binding capacity negatively, but the interaction of the two factors was not significant. Increasing the loading rate from 100 to 1200 L m⁻² d⁻¹ decreased P binding capacity from 1.152 to 0.070 g kg⁻¹ for wastewater filters and from 1.382 to 0.300 g kg⁻¹ for phosphate solution filters. At a loading rate of 100 L m⁻² d⁻¹, the average P binding capacity of wastewater filters was 1.152 g kg⁻¹ as opposed to 1.382 g kg⁻¹ for phosphate solution filters. Therefore, influent source or hydraulic loading rate should be carefully controlled in the laboratory. When phosphate solution and wastewater were used, the reaction times for the filters to remove P were determined to be 5 and 15 minutes, respectively, suggesting that a short residence time is required. However, breakthrough in this study occurred unexpectedly quickly, implying that more time is needed for the P that has reacted to be physically retained in the filter.     

2′,3′-Dideoxyuridine triphosphate conjugated to SiO2 nanoparticles: Synthesis and evaluation of antiproliferative activity

A conjugate of triphosphorylated 2′,3′-dideoxyuridine (ddU) with SiO₂ nanoparticles was obtained via the CuAAC click chemistry between a γ-alkynyl ddU triphosphate and azido-modified SiO₂ nanoparticles. Assessment of cytotoxicity in human breast adenocarcinoma MCF7 cells demonstrated that ddU triphosphate conjugated to SiO₂ nanoparticles exhibited a 50% decrease in cancer cell growth at a concentration of 183?±?57?µg/mL, which corresponds to 22?±?7?µM of the parent nucleotide, whereas the parent nucleoside, nucleotide and alkynyl triphosphate precursor do not show any cytotoxicity. The data provide an example of remarkable potential of novel conjugates of SiO₂ nanoparticles with phosphorylated nucleoside analogues, even those, which have not been used previously as therapeutics, for application as new anticancer agents.     

Fish intake reflects on DHA level in breast milk among lactating women in Latvia

Background

Docosahexaenoic acid (DHA) is an essential fatty acid required for proper growth and development. DHA levels in breast milk vary worldwide. Higher levels are observed among coastal populations and are associated with marine food consumption. Latvia is located in Northern Europe, on the eastern shore of the Baltic Sea. Nevertheless, fish consumption among women of reproductive age is low. The aim of this study was to determine DHA levels in breast milk among lactating women in Latvia.

Methods

Invitation to participate in the study was posted on a social media member group for breastfeeding mothers. In total, 71 women were enrolled from November 2016 until December 2017. DHA levels (% of total fatty acids) in breast milk were measured by gas chromatography. Information about food consumed during the three consecutive days prior to the milk sampling was obtained and a food frequency questionnaire (FFQ) was completed. Information about maternal and infant characteristics, current breastfeeding pattern and milk expression method was also collected.

Results

The mean age of participants was 31?±?4 years and the mean BMI was 22.1?±?3.2. 27 participants were primiparas. The average age and birth weight for infants (34 males, 33 females) was 6?±?4 months and 3.46?±?0.55 kg, respectively. The median DHA level in breast milk (n?=?60) was 0.30?±?0.18% of total fatty acids and it was not influenced by any of the maternal or infant’s characteristics nor current breastfeeding pattern or milk expression manner (p?>?0.05). Fish intake was a positive predictor for DHA levels in breast milk (r?=?0.318, p?=?0.013). Average maternal DHA intake was 136?±?26, 137?±?33 and 178?±?49 (SEM – standard error of the mean) mg, for the third, second and last day prior to sampling day, respectively.

Conclusions

DHA levels in breast milk among lactating women in Latvia correspond to the suggested target DHA value in breast milk (0.30%). Fish consumption is a significant positive predictor for DHA levels in breast milk, however, daily DHA intake among the participants was lower than recommended (200 mg).

     

Anthropogenic disturbance equalizes diversity levels in arbuscular mycorrhizal fungal communities

The arbuscular mycorrhizal (AM) symbiosis is a key plant–microbe interaction in sustainable functioning ecosystems. Increasing anthropogenic disturbance poses a threat to AM fungal communities worldwide, but there is little empirical evidence about its potential negative consequences. In this global study, we sequenced AM fungal DNA in soil samples collected from pairs of natural (undisturbed) and anthropogenic (disturbed) plots in two ecosystem types (10 naturally wooded and six naturally unwooded ecosystems). We found that ecosystem type had stronger directional effects than anthropogenic disturbance on AM fungal alpha and beta diversity. However, disturbance increased alpha and beta diversity at sites where natural diversity was low and decreased diversity at sites where natural diversity was high. Cultured AM fungal taxa were more prevalent in anthropogenic than natural plots, probably due to their efficient colonization strategies and ability to recover from disturbance. We conclude that anthropogenic disturbance does not have a consistent directional effect on AM fungal diversity; rather, disturbance equalizes levels of diversity at large scales and causes changes in community functional structure.