Interaction of aurein 1.2 and its analogue with DPPC lipid bilayer

Antibacterial peptides have potential as novel therapeutic agents for bacterial infections. Aurein 1.2 is one of the smallest antibacterial peptides extracted from an anuran. LLAA is a more active analogue of aurein 1.2. Antibacterial peptides usually accomplish their function by interacting with bacterial membrane selectively. In this study, we tried to find the reasons for the stronger antibacterial activity of LLAA compared with aurein 1.2. For this purpose, the interaction of aurein 1.2 and LLAA with dipalmitoylphosphatidylcholine (DPPC) was investigated by molecular dynamics (MD) simulation. In addition, the structure of peptides and their antibacterial activity were investigated by circular dichroism (CD) and dilution test method, respectively. MD results showed that LLAA is more flexible compared with aurein 1.2. Furthermore, LLAA loses its structure more than aurein 1.2 in the DPPC bilayer. A higher amount of water molecules penetrate into bilayer in the presence of LLAA relative to aurein 1.2. According to the antibacterial result that indicated LLAA is remarkably more active than aurein 1.2, it can be concluded that flexibility of the peptide is a determining factor in antibacterial activity. Probably, flexibility of the peptides facilitates formation of effective pores in the lipid bilayer.     

Effect of oestrus synchronization methods on oestrus behaviour, timing of ovulation and pregnancy rate during the breeding and low breeding seasons in Nili-Ravi buffaloes

The objective of this study was to determine the effect of oestrous synchronization methods on oestrous behaviour, timing of ovulation and pregnancy rate during the breeding and low breeding seasons in Nili-Ravi buffaloes. In Experiment 1, oestrous behaviour and timing of ovulation were determined from (n=34) oestruses. The mean (+/- S.E.M.) time of ovulation after the onset of standing oestrus was greater (P<0.05) in PGF(2alpha)-induced luteolysis (30.6+/-1.5h) compared to Ovsynch buffaloes (15.0+/-0.8h). In Experiment 2, pregnancy rates were compared between two methods of synchronization (detected oestrus and Ovsynch protocol) during the breeding and low breeding seasons. Pregnancy rates of buffaloes bred at detected oestrus (62.5%) or by the Ovsynch protocol (36.3%) during the breeding season did not differ significantly (P>0.05) from those which were inseminated during the low breeding season (55.5%) and (30.4%), respectively. This study demonstrates clearly that (1) timing of ovulation in Nili-Ravi buffalo is about 30h after the onset of standing oestrus and (2) buffaloes can be successfully synchronized with optimum fertility using either PGF(2alpha) alone (detected oestrus) or using (Ovsynch protocol) during low breeding season, to calve during the period when milk availability is short.     

Coexistence of arbuscular mycorrhizae and dark septate endophytic fungi in an undisturbed and a disturbed site of an arid ecosystem

Effect of disturbance on root colonization and vertical distribution of arbuscular mycorrhizal fungi (AMF) and dark septate endophytes (DSE) was investigated at two adjacent sites of Lal Suhanra Biosphere Reserve, Pakistan. Disturbance clearly affected AMF and DSE colonization, vertical distribution of AMF and plant community structure. Mean colonization of AMF and DSE was slightly less at the disturbed site. Average spore densities, diversity and richness of AMF and DSE were higher at the undisturbed site. A study of the vertical distribution of AMF associated with the five plant species most common to each study site indicated that beside AMF and DSE colonization disturbance may affect AMF species composition. Correlation of AMF with DSE is also discussed.     

Quantification of indole-3-acetic acid from plant associated <Emphasis Type="Italic">Bacillus</Emphasis> spp. and their phytostimulatory effect on <Emphasis Type="Italic">Vigna radiata</Emphasis> (L.)

Sixteen Bacillus strains isolated from rhizosphere, histoplane and phyllosphere of different plant species were identified by 16S rDNA gene sequencing and evaluated for in vitro auxin production as well as growth stimulation of Vigna radiata (L.) Wilczek. Auxin production by Bacillus spp. in L-broth medium supplemented with 1,000 μg ml⁻¹ L-tryptophan ranges from 0.60 to 3.0 μg IAA ml⁻¹ as revealed by gas chromatography and mass spectrometric (GC–MS) analysis. Rhizospheric isolates exhibit relatively more IAA synthesis than histoplane and phyllosphere isolates. Plant microbe interaction experiments conducted under gnotobiotic conditions recorded 55.55, 46.46 and 46.20% increase in shoot length with Bacillus megaterium MiR-4, B. pumilus NpR-1 and B. subtilis TpP-1, respectively, over control. Bacillus inoculations also increased shoot fresh weight with B. megaterium MiR-4 (60.94%) and B. pumilus NpR-1 (37.76%). Highly significant positive correlation between auxin production analyzed by GC–MS and shoot length (r = 0.687**, P = 0.01) and shoot fresh weight (r = 0.703**, P = 0.01) was noted under gnotobiotic conditions. Similarly, significant correlation was also found between auxin production by Bacillus spp. (GC–MS analysis) and different growth parameters such as shoot length (r = 0.495*, P = 0.05), number of pods (r = 0.498*, P = 0.05) and grain weight (r = 0.537*, P = 0.05) at full maturity under natural wire house conditions. Results showed that auxin production potential of plant associated Bacillus spp. can be effectively exploited to enhance the growth and yield of V. radiata.     

Genetic Variation in Healthy Oldest-Old

Individuals who live to 85 and beyond without developing major age-related diseases may achieve this, in part, by lacking disease susceptibility factors, or by possessing resistance factors that enhance their ability to avoid disease and prolong lifespan. Healthy aging is a complex phenotype likely to be affected by both genetic and environmental factors. We sequenced 24 candidate healthy aging genes in DNA samples from 47 healthy individuals aged eighty-five years or older (the ‘oldest-old’), to characterize genetic variation that is present in this exceptional group. These healthy seniors were never diagnosed with cancer, cardiovascular disease, pulmonary disease, diabetes, or Alzheimer disease. We re-sequenced all exons, intron-exon boundaries and selected conserved non-coding sequences of candidate genes involved in aging-related processes, including dietary restriction (PPARG, PPARGC1A, SIRT1, SIRT3, UCP2, UCP3), metabolism (IGF1R, APOB, SCD), autophagy (BECN1, FRAP1), stem cell activation (NOTCH1, DLL1), tumor suppression (TP53, CDKN2A, ING1), DNA methylation (TRDMT1, DNMT3A, DNMT3B) Progeria syndromes (LMNA, ZMPSTE24, KL) and stress response (CRYAB, HSPB2). We detected 935 variants, including 848 single nucleotide polymorphisms (SNPs) and 87 insertion or deletions; 41% (385) were not recorded in dbSNP. This study is the first to present a comprehensive analysis of genetic variation in aging-related candidate genes in healthy oldest-old. These variants and especially our novel polymorphisms are valuable resources to test for genetic association in models of disease susceptibility or resistance. In addition, we propose an innovative tagSNP selection strategy that combines variants identified through gene re-sequencing- and HapMap-derived SNPs.     

Expression of DNAJB12 or DNAJB14 Causes Coordinate Invasion of the Nucleus by Membranes Associated with a Novel Nuclear Pore Structure

DNAJB12 and DNAJB14 are transmembrane proteins in the endoplasmic reticulum (ER) that serve as co-chaperones for Hsc70/Hsp70 heat shock proteins. We demonstrate that over-expression of DNAJB12 or DNAJB14 causes the formation of elaborate membranous structures within cell nuclei, which we designate DJANGOS for DNAJ-associated nuclear globular structures. DJANGOS contain DNAJB12, DNAJB14, Hsc70 and markers of the ER lumen and ER and nuclear membranes. Strikingly, they are evenly distributed underneath the nuclear envelope and are of uniform size in any one nucleus. DJANGOS are composed primarily of single-walled membrane tubes and sheets that connect to the nuclear envelope via a unique configuration of membranes, in which the nuclear pore complex appears anchored exclusively to the outer nuclear membrane, allowing both the inner and outer nuclear membranes to flow past the circumference of the nuclear pore complex into the nucleus. DJANGOS break down rapidly during cell division and reform synchronously in the daughter cell nuclei, demonstrating that they are dynamic structures that undergo coordinate formation and dissolution. Genetic studies showed that the chaperone activity of DNAJ/Hsc70 is required for the formation of DJANGOS. Further analysis of these structures will provide insight into nuclear pore formation and function, activities of molecular chaperones, and mechanisms that maintain membrane identity.     

Structural and Functional Analysis of a Platelet-Activating Lysophosphatidylcholine of Trypanosoma cruzi

Background

Trypanosoma cruzi is the causative agent of the life-threatening Chagas disease, in which increased platelet aggregation related to myocarditis is observed. Platelet-activating factor (PAF) is a potent intercellular lipid mediator and second messenger that exerts its activity through a PAF-specific receptor (PAFR). Previous data from our group suggested that T. cruzi synthesizes a phospholipid with PAF-like activity. The structure of T. cruzi PAF-like molecule, however, remains elusive.

Methodology/Principal findings

Here, we have purified and structurally characterized the putative T. cruzi PAF-like molecule by electrospray ionization-tandem mass spectrometry (ESI-MS/MS). Our ESI-MS/MS data demonstrated that the T. cruzi PAF-like molecule is actually a lysophosphatidylcholine (LPC), namely sn-1 C18:1(delta 9)-LPC. Similar to PAF, the platelet-aggregating activity of C18:1-LPC was abrogated by the PAFR antagonist, WEB 2086. Other major LPC species, i.e., C16:0-, C18:0-, and C18:2-LPC, were also characterized in all T. cruzi stages. These LPC species, however, failed to induce platelet aggregation. Quantification of T. cruzi LPC species by ESI-MS revealed that intracellular amastigote and trypomastigote forms have much higher levels of C18:1-LPC than epimastigote and metacyclic trypomastigote forms. C18:1-LPC was also found to be secreted by the parasite in extracellular vesicles (EV) and an EV-free fraction. A three-dimensional model of PAFR was constructed and a molecular docking study was performed to predict the interactions between the PAFR model and PAF, and each LPC species. Molecular docking data suggested that, contrary to other LPC species analyzed, C18:1-LPC is predicted to interact with the PAFR model in a fashion similar to PAF.

Conclusions/Significance

Taken together, our data indicate that T. cruzi synthesizes a bioactive C18:1-LPC, which aggregates platelets via PAFR. We propose that C18:1-LPC might be an important lipid mediator in the progression of Chagas disease and its biosynthesis could eventually be exploited as a potential target for new therapeutic interventions.     

Gene therapy with an improved doxycycline-regulated plasmid encoding a tumour necrosis factor-alpha inhibitor in experimental arthritis

Inhibition of tumour necrosis factor (TNF)-alpha with biological molecules has proven an effective treatment for rheumatoid arthritis, achieving a 20% improvement in American College of Rheumatology score in up to 65% of patients. The main drawback to these and many other biological treatments has been their expense, which has precluded their widespread application. Biological molecules could alternatively be delivered by gene therapy as the encoding DNA. We have developed novel plasmid vectors termed pGTLMIK and pGTTMIK, from which luciferase and a dimeric TNF receptor II (dTNFR) are respectively expressed in a doxycycline (Dox)-regulated manner. Regulated expression of luciferase from the self-contained plasmid pGTLMIK was examined in vitro in a variety of cell lines and in vivo following intramuscular delivery with electroporation in DBA/1 mice. Dox-regulated expression of luciferase from pGTLMIK of approximately 1,000-fold was demonstrated in vitro, and efficient regulation was observed in vivo. The vector pGTTMIK encoding dTNFR was delivered by the same route with and without administration of Dox to mice with collagen-induced arthritis. When pGTTMIK was delivered after the onset of arthritis, progression of the disease in terms of both paw thickness and clinical score was inhibited when Dox was also administered. Vectors with similar regulation characteristics may be suitable for clinical application.     

In vitro antibiofilm and anti‐adhesion effects of magnesium oxide nanoparticles against antibiotic resistant bacteria

The aim of the current investigation was to determine the antibacterial and antibiofilm potential of MgO nanoparticles (NPs) against antibiotic‐resistant clinical strains of bacteria. MgO NPs were synthesized by a wet chemical method and further characterized by scanning electron microscopy and energy dispersive X‐ray. Antibacterial activity was determined by broth microdilution and agar diffusion methods. The Bradford method was used to assess cellular protein leakage as a result of loss of membrane integrity. Microtiter plate assay following crystal violet staining was employed to determine the effect of MgO NPs on biofilm formation and removal of established biofilms. MIC values ranged between 125 and 500 μg/mL. Moreover, treatment with MgO NPs accelerated rate of membrane disruption, measured as a function of leakage of cellular proteins. Leakage of cellular protein content was greater among gram‐negative bacteria. Cell adherence assay indicated 25.3–49.8% inhibition of bacterial attachment to plastic surfaces. According to a static biofilm method, MgO NPs reduced biofilm formation potential from 31% to 82.9% in a time‐dependent manner. Moreover, NPs also significantly reduced the biomass of 48, 72, 96 and 120 hr old biofilms (P < 0.05). Cytotoxicity experiments using a neutral red assay revealed that MgO NPs are non‐toxic to HeLa cells at concentrations of 15–120 μg/mL. These data provide in vitro scientific evidence that MgO NPs are effective and safe antibiofilm agents that inhibit adhesion, biofilm formation and removal of established biofilms of multidrug‐resistant bacteria.