A novel fluorescence imaging technique combining deconvolution microscopy and spectral analysis for quantitative detection of opportunistic pathogens

A novel fluorescence imaging technique based on deconvolution microscopy and spectral analysis is presented here as an alternative to confocal laser scanning microscopy. It allowed rapid, specific and simultaneous identification of five major opportunistic pathogens, relevant for public health, in suspension and provided quantitative results.     

Identification of seroreactive proteins in the culture filtrate antigen of Mycobacterium avium ssp. paratuberculosis human isolates to sera from Crohn's disease patients

The etiology of Crohn's disease (CD) is unresolved, but it is likely that an interplay of host genetic factors and environmental triggers is relevant. Mycobacterium paratuberculosis (MAP) has been focused upon as one of these triggers because it causes a similar chronic inflammatory bowel disease in animals. However, the differences among MAP antigens isolated from humans (H-MAP) and cattle (B-MAP) have not been well characterized. In this study, culture filtrate (CF) proteins from MAP isolates were tested with sera from CD patients and healthy controls in enzyme-linked immunosorbent assay (ELISA). Antibody produced by seven CD patients reacted differently according to the antigen source: strong reactivity was seen to H-MAP CF, but not to B-MAP CF. Six proteins, ModD, PepA, transaldolase, EchA9, MAP2120c, and MAP2950c, in H-MAP CF reacting specifically with CD patient sera were identified by liquid chromatography-electrospray ionization-MS. Bioinformatic analysis revealed that ModD and PepA were the same proteins reacting with sera from cattle infected with MAP. The elevated antibody responses of CD patients to rModD and rPepA were confirmed by ELISA ( P <0.001). These results support previous studies showing ModD and PepA as key antigens for the diagnosis of MAP infections. The study also identified additional proteins potentially useful in the design of assays for human MAP infections.     

Antimicrobial activity of camphor tree silver nano-particles against foulbrood diseases and finding out new strain of Serratia marcescens via DGGE-PCR,as a secondary infection on honeybee larvae

American foulbrood (AFB) and European foulbrood (EFB) are the two major bacterial diseases affecting honeybees, leading to a decrease in viability of the hive, decreasing honey production, and resulting in significant economic losses to beekeepers. Due to the inefficiency and/or low efficacy of some antibiotics, researches with nanotechnology represent, possibly, new therapeutic strategies. Nanostructure drugs have presented some advantagesover the conventional medicines, such as slow, gradual and controlled release, increased bioavailability, and reduced side-effects. In this study, different infected larvae were collected from two apiaries; the combs that had symptoms of American and European foulbrood were isolated. In vitro antimicrobial activity of camphor tree silver nano-particles against foulbrood diseases were characterized using UV–Vis spectrophotometry and scanning electron microscope (SEM) that proves the formation of silver nanoparticles with size range 160–660 nm. The antimicrobial activity of the silver nanoparticles was tested using agar diffusion assay and proved their ability to effectively cease the pathogenic bacterial growth in both AFB and EFB. DGGE-PCR technique has been applied for the identification of un-common bacterial infections honeybees depending on 16S rRNA amplification from their total extracted DNA and has been identified as Serratia marcescens (TES), deposited in GenBank with a new accession number (MT240613). The results were confirmed strain has been detected by DGGE-PCR analysis causing uniquely infected brood that was attacked by the American Foulbrood It could be concluded that greenly synthesized silver nanoparticles is projected to be used as effective treatment for honeybee bacterial diseases. These material need more investigations under field conditions and study the possibility of its residues in honeybee products such as honey, and beeswax.     

Paternal bisphenol A exposure induces testis and sperm pathologies in mice offspring: Possibly due to oxidative stress?

Bisphenol A (BPA), an endocrine and metabolic disruptor, is widely used to manufacture polycarbonate plastics and epoxy resins. Accumulating evidence suggests that paternal BPA exposure adversely affects male germlines and results in atypical reproductive phenotypes that might persist for generations to come. Our study investigated this exposure on testicular architecture and sperm quality in mouse offspring, and characterised underlying molecular mechanism(s). A total of 18 immature male Swiss albino mice (3.5 weeks old) were randomly divided into three groups and treated as follows: Group I, no treatment (sham control); Group II, sterile corn oil only (vehicle control); Group III, BPA (400 μg/kg) in sterile corn oil. At 9.5 weeks old, F0 males were mated with unexposed females. F0 offspring (F1 generation) were monitored for postnatal development for 10 weeks. At 11.5 weeks old, the animals were sacrificed to examine testicular architecture, sperm parameters, including DNA integrity, and oxidative stress biomarkers. Results showed that BPA significantly induced changes in the body and testis weights of the F0 and F1 generation BPA lineages compared to F0 and F1 generation control lineages. A decrease in sperm count and motility with further, increased sperm abnormalities, no or few sperm DNA alterations and elevated levels of MDA, PC and NO were recorded. Similar effects were found in BPA exposed F0 males, but were more pronounced in the F0 offspring. In addition, BPA caused alterations in the testicular architecture. These pathological changes extended transgenerationally to F1 generation males’ mice, but the pathological changes were more pronounced in the F1 generation. Our findings demonstrate that the biological and health BPA impacts do not end in paternal adults, but are passed on to offspring generations. Hence, linking observed testis and sperm abnormalities in the F1 generation to BPA exposure of their parental line was evident in this work. The findings also illustrate that oxidative stress appears to be a molecular component of the testis and sperm pathologies.     

Reaction of Melon (Cucumis melo L.) Cultivars to Monosporascus cannonballus (Pollack & Uecker) and their Effect on Total Phenol,Total Protein and Peroxidase Activities

Eighteen melon cultivars were screened for resistance to Monosporascus cannonballus under greenhouse conditions. The melon cultivars were grown in pasteurized sand, which had been inoculated with a high level (60 CFUs/g of soil) of M. cannonballus mycelium from culture. Cultivars Nabijani, Sfidak khatdar, Sfidak bekhat, Ghandak, Mollamosai, Chappat, Hajmashallahi and Shadgan were moderately resistant to M. cannonballus but all other melon cultivars were moderately to highly susceptible (HS) to this pathogen. A second screening was performed for resistance to M. cannonballus under greenhouse conditions. In the second screening, cultivars Nabijani, Sfidak khatdar, Sfidak bekhat, Ghandak, Mollamosai, Chappat, Hajmashallahi and Shadgan were moderately resistant to M. cannonballus. To examine the melon resistance mechanism against M. cannonballus, the activities of total phenol, total protein and peroxidase in two melon cultivars Nabijani (as resistant) and Khaghani (as susceptible) were determined at 0, 24, 48 and 72 h after inoculation. Inoculated resistant cultivar roots had always higher content of total phenol, total protein and peroxidase than the corresponding inoculated susceptible cultivar roots. The results indicated that there was a relationship between resistance in Nabijani and accumulation of total phenol, total protein and peroxidase.     

Salicylic acid-mediated changes in photosynthesis,nutrients content and antioxidant metabolism in two mustard (<Emphasis Type="Italic">Brassica juncea</Emphasis> L.) cultivars differing in salt tolerance

Mustard (Brassica juncea L.) cultivars Alankar (salt-tolerant) and PBM16 (salt-sensitive) plants were grown with 50 mM NaCl and were sprayed with 0.1, 0.5, and 1.0 mM salicylic acid (SA) to study the physiological processes determining salt tolerance and to observe the influence of SA application on the alleviation of NaCl-induced adverse effects. The content of leaf Na⁺, Cl⁻, H₂O₂, TBARS, and electrolyte leakage and the activity of SOD were higher in PBM16 than Alankar. In contrast, nutrients content, activity of APX and GR, glutathione content, photosynthetic and growth characteristics were higher in Alankar. Treatment of 50 mM NaCl resulted in increase of Na⁺ and Cl⁻, oxidative stress, activity of antioxidant enzymes and glutathione content, while nutrients content, photosynthetic, and growth characteristics decreased in both the cultivars. Application of 0.5 mM SA alleviated the negative effects of 50 mM NaCl maximally, but 1.0 mM SA proved inhibitory. The effect of SA was more conspicuous in Alankar than PBM16. It is concluded that the higher tolerance of Alankar was due to its lower leaf Na⁺ and Cl⁻ content, higher nutrients content, and efficient antioxidant metabolism. The application of 0.5 mM SA substantially alleviated salt-induced adverse effects in Alankar.     

Trace Element Level in Different Tissues of <Emphasis Type="Italic">Rutilus frisii kutum</Emphasis> Collected from Tajan River,Iran

Tajan River is among the most significant rivers of the Caspian Sea water basin. In this study, the concentration of Cr, Cu, Fe, Mn, Ni, Pb, Cd, and Zn were determined in brain, heart, liver, gill, bile, and muscle of Rutilus frisii kutum which has great economic value in the Mazandaran state. Trace element levels in fish samples were analyzed by means of atomic absorption spectrometry. Nearly all non-essential metals levels (Ni, Pb, Cd) detected in tissues were higher than limits for fish proposed by FAO/WHO, EU, and TFC. Generally, non-essential metals (Ni, Pb) were so much higher in muscle than the essential metals (Cu, Zn, and Mn) except Fe, which was higher than other metals in nearly all parts, except in gills. Fe distribution pattern in tissues was in order of heart > brain > liver > muscle > bile > gill. Distribution patterns of metal concentrations in the muscle of fish as a main edible part followed the sequence: Fe > Pb > Ni > Cu > Mn > Zn > Cd.     

Stem cell-conditioned medium does not protect against kidney failure

Paracrine secretion of mediators may be the main route by which stem cells protect against injuries. Stem cells commonly secrete different bioactive molecules. In this study, we examined the hypothesis that administration of conditioned media of stem cells can diminish the burden of kidney injury. A mouse model of cisplatin-induced nephropathy was developed to test the putative renoprotective effects of conditioned media of human umbilical cord blood USSCs (unrestricted somatic stem cells) as well as mouse bone marrow MSCs (mesenchymal stem cells). None of these two types of conditioned medium could protect against kidney failure in terms of serum urea and creatinine, histopathologic examinations and physical activity score. Neither MSC- nor USSC-conditioned media were effective in protecting against kidney injury in our study. Possible explanations for our observations are offered, and related literature is reviewed.     

Increased lignan biosynthesis in the suspension cultures of Linum album by fungal extracts

Linum album accumulates anti-tumor podophyllotoxin (PTOX) and its related lignans, which were originally isolated from an endangered species Podophyllum. In the present study, we examined the effects of five fungal extracts on the production of lignans in L. album cell cultures. Fusarium graminearum extract induced the highest increase of PTOX [143 μg g⁻¹ dry weight (DW) of the L. album cell culture], while Rhizopus stolonifer extract enhanced the accumulation of lariciresinol up to 364 μg g⁻¹ DW, instead of PTOX. Typical elicitors, such as chitin, chitosan, or methyl jasmonate (MeJA), were shown to be less effective in lignan production in L. album cell cultures. These results verified the advantages of fungal extracts to increase lignan production in L. album cell culture, and suggested potential on-demand metabolic engineering of lignan biosynthesis using differential fungal extracts.     

The C terminus of the anaphylatoxin C3a generated upon complement activation represents a neoantigenic determinant with diagnostic potential

Activation of the C component C3 results in generation of the anaphylatoxin C3a. The C3a polypeptide chain consists of 77 amino acids. The active site of this potent mediator, which also has immunoregulatory function resides in its C terminus. This report demonstrates that the C terminus of C3a (C3a-desArg) exposed by proteolytic cleavage from C3 represents a neoantigenic determinant. Two mAb specific for this epitope were obtained after immunization with the synthetic octapeptide (OP) Arg-Ala-Ser-His-Leu-Gly-Leu-Ala [C3a(69-76)] coupled to the carrier keyhole limpet hemocyanin (KLH). These anti-C3a(69-76) antibodies (H453 and H454) reacted in an ELISA system with C3a and KLH-OP but not with C3 or with KLH alone. Free OP efficiently blocked binding of the antibodies to C3a, whereas binding of another anti-C3a mAb (H13) remained unaffected. In immunoblotting analysis, the anti-C3a(69-76) mAb reacted with purified C3a but failed to react with the denatured, noncleaved C3. A novel quantitative C3a-ELISA was established with the anti-C3a(69-76) mAb. It had a sensitivity in the nanogram range (1 to 5 ng/ml). The C3a determination was not impaired by the presence of high concentrations of C3. Therefore, C3 removal was not required in contrast to the previously described C3a assays. This C3a ELISA might facilitate clinical C3a quantitation, e.g., in samples from patients with adult respiratory distress syndrome. In these patients, C3a determination in the early phase of the disease is of diagnostic relevance and has prognostic value.