Assessing DNA for fish identifications from reference collections: the good,bad and ugly shed light on formalin fixation and sequencing approaches

Natural history collections are repositories of biodiversity and are potentially used by molecular ecologists for comparative taxonomic, phylogenetic, biogeographic and forensic purposes. Specimens in fish collections are preserved using a combination of methods with many fixed in formalin and then preserved in ethanol for long-term storage. Formalin fixation damages DNA, thereby limiting genetic analyses. In this study, the authors compared the DNA barcoding and identification success for frozen and formalin-fixed tissues obtained from specimens in the CSIRO Australian National Fish Collection. They studied 230 samples from fishes (consisting of >160 fish species). An optimized formalin-fixed, paraffin-embedded DNA extraction method resulted in usable DNA from degraded tissues. Four mini barcoding assays of the mitochondrial DNA (mtDNA) were characterized with Sanger and Illumina amplicon sequencing. In the good quality DNA (without exposure to formalin), up to 88% of the specimens were correctly matched at the species level using the cytochrome oxidase subunit 1 (COI) mini barcodes, whereas up to 58% of the specimens exposed to formalin for less than 8 weeks were correctly identified to species. In contrast, 16S primers provided higher amplification success with formalin-exposed tissues, although the COI gene was more successful for identification. Importantly, the authors found that DNA of a certain size and quality can be amplified and sequenced despite exposure to formalin, and Illumina sequencing provided them with greater power of resolution for taxa identification even when there was little DNA present. Overall, within parameter constraints, this study highlights the possibilities of recovering DNA barcodes for identification from formalin-fixed fish specimens, and the authors provide guidelines for when successful identification could be expected.     

Spatio-temporal analysis of Egyptian flower mantis Blepharopsis mendica (order: mantodea), with notes of its future status under climate change

Egyptian flower mantis Blepharopsis mendica (Order: Mantodea) is a widespread mantis species throughout the southwest Palearctic region. The ecological and geographical distribution of such interesting species is rarely known. So, through this work, habitat suitability models for its distribution through Egyptian territory were created using MaxEnt software from 90 occurrence records. One topographic (altitude) and eleven bioclimatic variables influencing the species distribution were selected to generate the models. The predicted distribution in Egypt was focused on the Delta, South Sinai, the north-eastern part of the country, and some areas in the west including Siwa Oasis. Temporal analysis between the two periods (1900–1961) and (1961–2017) show current reduction of this species distribution through Delta and its surrounding areas, may be due to urbanization. On the other hand, it increases in newly protected areas of South Sinai. Under the future climate change scenario, the MaxEnt model predicted the habitat gains for B. mendica in RCP 2.6 for 2070 and loss of habitat in RCP 8.5 for the same year. Our results can be used as a basis for conserving this species not only in Egypt, but also throughout the whole of its range, also, it show how the using of geo-information could help in studying animal ecology.     

High efficiency versus maximal performance — The cause of oxidative stress in eukaryotes: A hypothesis

Degenerative diseases are in part based on elevated production of ROS (reactive oxygen species) in mitochondria, mainly during stress and excessive work under stress (strenuous exercise). The production of ROS increases with increasing mitochondrial membrane potential (ΔΨ_m). A mechanism is described which is suggested to keep ΔΨ_m at low values under normal conditions thus preventing ROS formation, but is switched off under stress and excessive work to maximize the rate of ATP synthesis, accompanied by decreased efficiency. Low ΔΨ_m and low ROS production are suggested to occur by inhibition of respiration at high [ATP]/[ADP] ratios. The nucleotides interact with phosphorylated cytochrome c oxidase (COX), representing the step with the highest flux-control coefficient of mitochondrial respiration. At stress and excessive work neural signals are suggested to dephosphorylate the enzyme and abolish the control of COX activity (respiration) by the [ATP]/[ADP] ratio with consequent increase of ΔΨ_m and ROS production. The control of COX by the [ATP]/[ADP] ratio, in addition, is proposed to increase the efficiency of ATP production via a third proton pumping pathway, identified in eukaryotic but not in prokaryotic COX. We conclude that ‘oxidative stress’ occurs when the control of COX activity by the [ATP]/[ADP] ratio is switched off via neural signals.     

Preferential recognition of epitopes on peroxynitrite-modified alpha-2-macroglobulin by circulating autoantibodies in rheumatoid arthritis patients

Abstract

Autoimmune responses against post-translationally modified antigens are a hallmark of several autoimmune diseases. In this work, we have studied the changes in alpha-2-macroglobulin (α₂M) upon modification by peroxynitrite. Furthermore, we have evaluated the immunogenicity of modified α₂M in experimental rabbits and rheumatoid arthritis (RA) patients. Peroxynitrite-modified α₂M showed disturbed microenvironment and altered aromatic residues under UV and fluorescence studies. Aggregation, reduction in β-sheet content, production of nitrotyrosine and shift in amide I and II bands were observed in the modified α₂M by polyacrylamide gel electrophoresis besides CD and FTIR spectroscopic analysis. The exposure of hydrophobic clusters and changes in contact positions were observed in ANS and ThT binding assays. Immunological studies using ELISA showed peroxynitrite-modified α₂M as highly immunogenic producing high titre of specific antibodies in immunized rabbits. Cross-reactivity studies revealed the polyspecificity of the elicited antibodies. Direct binding ELISA and competitive inhibition studies confirmed the presence of circulating antibodies in the sera of RA patients having high specificity towards the peroxynitrite-modified α₂M as compared to the native α₂M. Sera from healthy (normal) human subjects showed lower binding with the native and modified protein. This study confirms that peroxynitrite induces structural modifications in α₂M and makes it immunogenic. The presence of neo-antigenic determinants on modified α₂M with enhanced binding for circulating autoantibodies in RA patients could offer new possibilities for diagnosis and etiopathology of the disease.

Communicated by Ramaswamy H. Sarma     

Microbial maceration and carbonate dissolution on cold‐temperate shelves

Nummulites beaumonti d'Archiac & Haime, 1853, originally described from El Basatin, Gebel Mokattam, Greater Cairo, is redefined from a nummulitic bank at the base of the Maadi Formation from its type locality as neotype. Schaub (1981) described the species from Libya and the type example of this species is still unknown. N. discorbinus Schlotheim (1820) is described as neotype from a nummulitic bank in the Mokattam Formation, which is located in the site of the Citadel tombs, near the Salah El Din Citadel, Gebel Mokattam, Egypt. According to Schaub (1981), the type example of this species is also unknown. For this reason the establishment of the neotypes of these two widespread nummulites species: N. discorbinus (Late Lutetian) and N. beaumonti (Bartonian) is important and tackled in this paper. N. qurnensis n. sp. ( = N. sp. cf. beaumonti in Boukhary et al. 2002) is introduced as a new species from the lower Bartonian part of the Qurn Formation of Helwan. Biometrical studies are carried out on the three species to present up to date information on these Nummulites, with particular attention to their biostratigraphic correlation with the Shallow Benthic Zones (SBZ) of Serra-Kiel et al. 1998. The authors would like to stress the importance of a careful designation of a neotype to avoid the introduction of complicated and unnecessary taxonomic problems.     

Iron and nickel complexes with heterocyclic ligands: Stability,synthesis, spectral characterization,antimicrobial activity,acute and subacute toxicity

The synthesis and characterization by elemental analysis, emission atomic spectroscopy, TG measurements, magnetic measurements, FTIR, ¹H NMR, UV–visible spectra and conductivity of a series of iron (II) and nickel (II) complexes with two heterocyclic ligands (L¹(SMX): sulfamethoxazole and L²(MIZ): metronidazole) used in pharmaceutical field and with a new ligand derived benzoxazole (L³(MPBO): 2-(5-methylpyridine-2-yl)benzoxazole), were reported. The formulae obtained for the complexes are: [M(L¹)₂ Cl₂]·nH₂O, [M(L²)₂Cl₂(H₂O)₂]·H₂O and [M(L³)₂(OH)₂]·nH₂O. Stability constants of these complexes have been determined by potentiometric methods in water–ethanol (90:10, v/v) mixture at a 0.2 mol L^?1 ionic strength (NaCl) and at 25.0 ± 0.1 °C. Sirko program was used to determine the protonation constants as well as the binding constants of three species [ML₂H₂]²⁺, [ML₂] and [ML]²⁺. The antimicrobial activity of the ligands and complexes was evaluated in vitro against different human bacteria and fungi using agar diffusion method.Iron sulfamethoxazole complex showed a remarkable inhibition of bacteria growth especially on Staphylococcus aureus and P. aeruginosa. The iron metronidazole complex is active against yeasts especially on Candida tropicalis strain. Nickel complexes presented different antibacterial and antifungal behavior's against bacteria and fungal.The acute toxicity study revealed that the iron complexes are not toxic at 2000 mg/kg dose orally administrated.LD₅₀ for nickel complexes was determined using graphical method.No significant differences in the body weights between the control and the treated groups of both rat sexes in subacute toxicity study using for iron complexes. Hematological and clinical blood chemistry analysis revealed no toxicity effects of the iron complexes. Pathologically, neither gross abnormalities nor histopathological changes were observed for these complexes.     

In-vitro GIT Tolerance of Microencapsulated Bifidobacterium bifidum ATCC 35914 Using Polysaccharide-Protein Matrix

Probiotics and Antimicrobial Proteins - Longevity of probiotic is the main concern for getting maximum benefits when added in food product. Bifidobacterium, a probiotic, tends to lose its viability...