Quantifying the ultrastructure changes of air-dried and irradiated human amniotic membrane using atomic force microscopy: a preliminary study

Air-dried and sterilized amnion has been widely used as a dressing to treat burn and partial thickness wounds. Sterilisation at the standard dose of 25 kGy was reported to cause changes in the morphological structure as observed under the scanning electron microscope. This study aimed to quantify the changes in the ultrastructure of the air-dried amnion after gamma-irradiated at several doses by using atomic force microscope. Human placentae were retrieved from mothers who had undergone cesarean elective surgery. Amnion separated from chorion was processed and air-dried for 16 h. It was cut into 10?×?10 mm, individually packed and exposed to gamma irradiation at 5, 15, 25 and 35 kGy. Changes in the ultrastructural images of the amnion were quantified in term of diameter of the epithelial cells, size of the intercellular gap and membrane surface roughness. The longest diameter of the amnion cells reduced significantly after radiation (p?<?0.01) however the effect was not dose dependent. No significant changes in the shortest diameter after radiation, except at 35 kGy which decreased significantly when compared to 5 kGy (p?<?0.01). The size of the irradiated air-dried amnion cells reduced in the same direction without affecting the gross ultrastructure. At 15 kGy the intercellular gap decreased significantly (p?<?0.01) with Ra and Rq, values reflecting surface roughness, were significantly the highest (p?<?0.01). Changes in the ultrastructure quantified by using atomic force microscope could complement results from other microscopic techniques.     

Iraqi propolis increases degradation of IL-1β and NLRC4 by autophagy following Pseudomonas aeruginosa infection

Autophagy is a cellular process for maintaining cellular homeostasis. This process can be induced by different factors, such as immune stimuli and pathogen-associated molecules. Autophagy has an important role in the control of IL-1β secretion by macrophages and other cell types. In present study, we describe a novel role for Iraqi propolis affecting autophagy in controlling the secretion of IL-1β in bone-marrow macrophages (BMDMs). After infection with Pseudomonas aeruginosa in the presence of propolis, the degradation of IL-1β was induced, and the activity of inflammasome was reduced. Iraqi propolis-induced autophagy in in vitro and in vivo models decreased the levels of IL-1β and caspase-1. Results indicated that IL-1β pathway production is regulated by autophagy via two different novel mechanisms, namely, regulation of the activation of NLRC4 inflammasome and IL-1β targeting for lysosomal degradation.     

Biogenic synthesis of copper oxide nanoparticles using olea europaea leaf extract and evaluation of their toxicity activities: An in vivo and in vitro study

Copper oxide nanoparticles (CUNPs) were synthesized using Olea europaea leaf extract as reducing and protecting agent. The formation of nanoparticles was observed through a color change from yellowish to brownish black. The CUNPs were confirmed with UV–Vis spectrophotometer, which revealed a peak absorbance at 289 nm. The synthesized CUNPs were characterized by XRD, FTIR, SEM, and TEM. The XRD pattern revealed that CUNPs were crystalline in nature with a diameter around 20 nm. FTIR spectral analysis showed that CUNPs were capped with plant constituents. From SEM and TEM analyses, the CUNPs were generally found to be spherical in shape, and the size range was 20–50 nm. Free radical scavenging potential of CUNPs against DPPH was confirmed by its stable antioxidant effects. In addition, the toxicity of CUNPs in mice was also assessed by body weight and weights of liver, kidneys, spleen, and thymus. The immune response in mice was signaled through an obvious change in spleen and thymus index, with a decrease of ADA enzyme activity in serum, spleen, and thymus after CUNPs treatment. The CUNPs were found to exert cell growth arrest against AMJ‐13 and SKOV‐3 cancer cells in a dose‐dependent manner and induce cell death by apoptosis. Less significant cytotoxic effect was observed in normal dermal fibroblast cells. These findings suggest that CUNPs may have the potential to be anticancer agents. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:218–230, 2018     

Identification of a critical motif responsible for gating of Kir2.3 channel by intracellular protons.

Protons are involved in gating Kir2.3. To identify the molecular motif in the Kir2.3 channel protein that is responsible for this process, experiments were performed using wild-type and mutated Kir2. 3 and Kir2.1. CO2 and low pHi strongly inhibited wild-type Kir2.3 but not Kir2.1 in whole cell voltage clamp and excised inside-out patches. This CO2/pH sensitivity was completely eliminated in a mutant Kir2.3 in which the N terminus was substituted with that in Kir2.1, whereas a similar replacement of its C terminus had no effect. Site-specific mutations of all titratable residues in the N terminus, however, did not change the CO2/pH sensitivity. Using several chimeras generated systematically in the N terminus, a 10-residue motif near the M1 region was identified in which only three amino acids are different between Kir2.3 and Kir2.1. Mutations of these residues, especially Thr53, dramatically reduced the pH sensitivity of Kir2.3. Introducing these residues or even a single threonine to the corresponding positions of Kir2.1 made the mutant channel pH-sensitive. Thus, a critical motif responsible for gating Kir2.3 by protons was identified in the N terminus, which contained about 10 residues centered by Thr53.     

Molecular characterization of Streptococcus agalactiae strains isolated from fishes in Malaysia

Aims

The aim of this study was to characterize Streptococcus agalactiae strains that were isolated from fishes in Malaysia using random amplified polymorphic DNA (RAPD) and repetitive extragenic palindromic PCR (REP‐PCR) techniques.

Methods and Results

A total of 181 strains of Strep. agalactiae isolated from red hybrid tilapia (Oreochromis sp.) and golden pompano (Trachinotus blochii) were characterized using RAPD and REP‐PCR techniques. Both the fingerprinting techniques generated reproducible band patterns, differing in the number and molecular mass amplicons. The RAPD technique displayed greater discriminatory power by its production of more complex binding pattern and divided all the strains into 13 groups, compared to 9 by REP‐PCR technique. Both techniques showed the availability to differentiate the genetic profiles of the strains according to their geographical location of origin. Three strains of Strep. agalactiae that were recovered from golden pompano showed a genetic dissimilarity from the strains isolated from red hybrid tilapia, while the strain of ATCC 27956 that recovered from bovine displayed a unique profile for both methods.

Conclusions

Both techniques possess excellent discriminative capabilities and can be used as a rapid means of comparing Strep. agalactiae strains for future epidemiological investigation.

Significance and Impact of the Study

Framework as the guideline in traceability of this disease and in the search for potential local vaccine candidates for streptococcosis in this country.     

The effect of crystallinity of cellulose on the rate of reducing sugars production by heterogeneous enzymatic hydrolysis

A kinetic model is devised, from the reaction mechanism steps, to predict the rate of reducing sugar production by hydrolysis of two types of cellulose, namely, amorphous carboxymethylcellulose (CMC) and highly crystalline wood shavings, using Aspergillus niger cellulase. Experimental results in a stirred batch reactor at 40 degrees C show that the production of reducing sugar reduced at much shorter times for wood shavings in comparison to CMC at the same initial substrate concentration. The experimental results are used to determine the kinetic parameters of the model equations. The significance of crystallinity was determined using inert fraction coefficient, which is assumed to be constant and equals 0.05 and 0.98 for CMC and wood shavings, respectively. It is shown there is a good agreement between the experimental results and proposed kinetic model predictions. The effect of the inert fraction coefficient on the production of reducing sugar by the enzymatic hydrolysis of cellulose is also determined. It is found that the cellulase used extracted from A. niger is much more sensitive towards the substrate structure in comparison to that extracted from Trichoderma reesei.     

Evaluation of affymetrix severe acute respiratory syndrome resequencing GeneChips in characterization of the genomes of two strains of coronavirus infecting humans

Severe acute respiratory syndrome (SARS) was discovered during a recent global outbreak of atypical pneumonia. A number of immunologic and molecular studies of the clinical samples led to the conclusion that a novel coronavirus (SARS-CoV) was associated with the outbreak. Later, a SARS resequencing GeneChip was developed by Affymetrix to characterize the complete genome of SARS-CoV on a single GeneChip. The present study was carried out to evaluate the performance of SARS resequencing GeneChips. Two human SARS-CoV strains (CDC#200301157 and Urbani) were resequenced by the SARS GeneChips. Five overlapping PCR amplicons were generated for each strain and hybridized with these GeneChips. The successfully hybridized GeneChips generated nucleotide sequences of nearly complete genomes for the two SARS-CoV strains with an average call rate of 94.6%. Multiple alignments of nucleotide sequences obtained from SARS GeneChips and conventional sequencing revealed full concordance. Furthermore, the GeneChip-based analysis revealed no additional polymorphic sites. The results of this study suggest that GeneChip-based genome characterization is fast and reproducible. Thus, SARS resequencing GeneChips may be employed as an alternate tool to obtain genome sequences of SARS-CoV strains pathogenic for humans in order to further understand the transmission dynamics of these viruses.     

Distribution of Giardia duodenalis genotypes and subgenotypes in raw urban wastewater in Milwaukee, Wisconsin

Giardia cysts in 131 raw wastewater samples from Milwaukee, Wis., were genotyped by sequence analysis of the triosephosphate isomerase gene which showed the presence of two distinct genotypes (assemblages A and B) of Giardia duodenalis. Of the 131 samples, 111 belonged to assemblage A, and the remaining samples belonged to assemblage B. A high degree of genetic polymorphism was evident within the assemblage B cluster, with 10 distinct subgenotypes identified, eight of which have not been reported before.     

Construction costs and physico-chemical properties of the assimilatory organs of Nepenthes species in Northern Borneo

BACKGROUND AND AIMS: Species of the Nepenthaceae family are under-represented in studies of leaf traits and the consequent view of mineral nutrition and limitation in carnivorous plants. This study is aimed to complement existing data on leaf traits of carnivorous plants. METHODS: Physico-chemical properties, including construction costs (CC), of the assimilatory organs (leaf and pitcher) of a guild of lowland Nepenthes species inhabiting heath and/or peat swamp forests of Brunei, Northern Borneo were determined. KEY RESULTS: Stoichiometry analyses indicate that Nepenthes species are nitrogen limited. Most traits vary appreciably across species, but greater variations exist between the assimilatory organs. Organ mass per unit area, dry matter tissue concentration (density), nitrogen (N), phosphorus (P), carbon, heat of combustion (H(c)) and CC values were higher in the leaf relative to the pitcher, while organ thickness, potassium (K) and ash showed the opposite trend. Cross-species correlations indicate that joint rather than individual consideration of the leaf and the pitcher give better predictive relationships between variables, signalling tight coupling and functional interdependence of the two assimilatory organs. Across species, mass-based CC did not vary with N or P, but increases significantly with tissue density, carbon and H(c), and decreases with K and ash contents. Area-based CC gave the same trends (though weaker in strength) in addition to a significant positive correlation with tissue mass per unit area. CONCLUSIONS: The lower CC value for the pitcher is in agreement with the concept of low marginal cost for carnivory relative to conventional autotrophy. The poor explanatory power of N, P or N : P ratio with CC suggests that factors other than production of expensive photosynthetic machinery (which calls for a high N input), including concentrations of lignin, wax/lipids or osmoregulatory ions like K(+), may give a better explanation of the CC variation across Nepenthes species.