Mapping Transcript Cell-Specific Localization and Protein Subcellular Localization in the Adult Mosquito Aedes aegypti.

This introduction reviews techniques used to examine the distribution and expression of gene transcripts and proteins in a variety of tissues/organs in the medically important global disease vector mosquito, Aedes aegypti Specifically, these methods allow the detection of cell-specific transcript expression by fluorescent in situ hybridization; facilitate immunohistochemical mapping of a protein of interest in whole-mount small tissue/organ samples; examine the subcellular localization of proteins, such as membrane transporters, through sectioning of paraffin-embedded tissue/organ samples; and finally, enable the efficient separation of cytosolic and membrane proteins for western blot analysis without the need for specialized equipment (e.g., ultracentrifuge) in the mosquito Ae. aegypti Such techniques are useful to help answer fundamental questions in mosquito scientific research including (but not limited to) the identification of specific cells in an organ responsible for expressing a receptor of particular interest and necessary for eliciting a response to exogenous signals, including hormones. Moreover, changes in the subcellular localization of specific targets of interest can be assessed both qualitatively and quantitatively, providing insight into transient or long-term physiologically relevant regulation necessary for activity under experimental treatments or varied internal (e.g., development) or external (e.g., environmental stress) factors that might be normally experienced by the organism.

Examining Protein Localization in Aedes aegypti Cells, Tissues, and Organs: Whole-Mount Immunohistochemistry.

Immunohistochemistry (IHC) is a powerful technique used for visualizing cellular components and determining the presence and/or location of proteins or other macromolecules in tissue samples. The classical IHC process involves the detection of epitopes using a highly specific primary antibody. This is followed by a secondary antibody that is coupled to a reporter molecule or fluorophore and capable of binding to the primary antibody and allowing for protein immunodetection. Although IHC does not routinely provide quantitative results compared to an enzyme-linked immunoassay or western blotting, it permits the localization of the proteins in intact tissues. This protocol describes an IHC assay for whole-body Aedes aegypti mosquito tissues that is used to detect small proteins, specifically neuropeptide hormones. This method is useful for protein detection in whole-mount preparations; however, cross-section IHC is recommended to determine if a protein is localized in the apical versus basolateral membrane of tissues/organs or to visualize immunological distribution in larger, more complex preparations.

Examining Apical or Basolateral Protein Localization in Aedes aegypti Tissues: Cross-Section Immunohistochemistry.

Immunohistochemistry (IHC) is an important technique that permits visualization of cellular components and for determining the presence and/or distribution of proteins or other macromolecules in tissue samples. Normally, IHC involves the detection of epitopes using an antigen-specific primary antibody and a secondary antibody coupled with a reporter molecule or fluorophore that can bind to the primary antibody, allowing for the spatial distribution of a protein of interest to be detected. Although normally IHC does not provide quantitative results compared to techniques such as enzyme-linked immunoassay or western blotting, it permits the localization, expression mapping, and distribution of target proteins in intact tissues. Here, we describe an IHC protocol for examining apical versus basolateral protein staining through sectioning tissue samples from fixed, embedded tissues (e.g., IHC-paraffin) and adding primary antibodies against a target protein. This IHC protocol provides a guide for tissue fixation, sectioning, and staining of tissue samples.

Functional insight and cell-specific expression of the adipokinetic hormone/corazonin-related peptide in the human disease vector mosquito, Aedes aegypti.

The adipokinetic hormone/corazonin-related peptide (ACP) is an insect neuropeptide structurally intermediate between corazonin (CRZ) and adipokinetic hormone (AKH). Unlike the AKH and CRZ signaling systems that are widely known for their roles in the mobilization of energy substrates and stress responses, respectively, the main role of ACP and its receptor (ACPR) remains unclear in most arthropods. The current study aimed to localize the distribution of ACP in the nervous system and provide insight into its physiological roles in the disease vector mosquito, Aedes aegypti. Immunohistochemical analysis and fluorescence in situ hybridization localized the ACP peptide and transcript within a number of cells in the central nervous system, including two pairs of laterally positioned neurons in the protocerebrum of the brain and a few ventrally localized neurons within the pro- and mesothoracic regions of the fused thoracic ganglia. Further, extensive ACP-immunoreactive axonal projections with prominent blebs and varicosities were observed traversing the abdominal ganglia. Given the prominent enrichment of ACPR expression within the abdominal ganglia of adult A. aegypti mosquitoes as determined previously, the current results indicate that ACP may function as a neurotransmitter and/or neuromodulator facilitating communication between the brain and posterior regions of the nervous system. In an effort to elucidate a functional role for ACP signaling, biochemical measurement of energy substrates in female mosquitoes revealed a reduction in abdominal fat body in response to ACP that matched the actions of AKH, but interestingly, a corresponding hypertrehalosaemic effect was only found in response to AKH since ACP did not influence circulating carbohydrate levels. Comparatively, both ACP and AKH led to a significant increase in haemolymph carbohydrate levels in male mosquitoes while both peptides had no influence on their glycogen stores. Neither ACP nor AKH influenced circulating or stored lipid levels in both male and female mosquitoes. Collectively, these results reveal ACP signaling in mosquitoes may have complex sex-specific actions, and future research should aim to expand knowledge on the role of this understudied neuropeptide.

Optimization of Bacillus subtilis growth parameters for biosynthesis of silver nanoparticles by using response surface methodology.

Silver nanoparticles (AgNPs) are among the most widely biosynthesized and used nanomaterials. They have different unique properties and a wide range of applications. This study is concerned with optimization of the growth conditions of Bacillus subtilis NRC1 for the biosynthesis of AgNPs using two designs of response surface methodology (RSM) statistical analysis. The data obtained from Plackett-Burman design (PBD) followed by central composite design (CCD), showed a good agreement between the experimental and predicted values of AgNPs biosynthesis. The optimum conditions were 0.7% (w/v) casein hydrolysate, 5% dextrin (w/v), pH 7.5 and 57 × 106 CFU/ml inoculum size. The model was highly valid and could be applied with a confidence factor of 99.47%. Minimal inhibitory concentration (MIC) of these AgNPs synthesized using the extracellular filtrate after growth of Bacillus subtilis NRC1 in the optimized medium was found to be 41-43µg/ml for all tested microorganisms with exception of Pseudomonas aeruginosa where MIC was 169 µg/ml.

Optimization of Bacillus subtilis NRC1 growth conditions using response surface methodology for sustainable biosynthesis of gold nanoparticles.

Gold nanoparticles (AuNPs) have different unique properties and a wide range of applications in different fields. Thereby, there is a growing urgency for the production of AuNPs using a safe and an economic method. In this study, optimization of fermentation conditions by Bacillus subtilis NRC1 for extracellular AuNPs synthesis using response surface methodology was achieved. The data obtained from Plackett-Burman design followed by Box-Behnken design indicated the accuracy and reliability of the model and it could be used to navigate the design space with a reasonable accuracy. Numerical optimization of Bacillus subtilis NRC1 active extracellular filtrate production, showed the optimum conditions of 0.74% (w/v) casein hydrolysate, 3.99% (w/v) dextrin, 47 × 106 CFU/ml inoculum size at pH 7.76 and 25 [Formula: see text]C to give the maximum AuNPs biosynthesis. The model was highly valid and the obtained data had a confidence factor of 98.48%. Statistical optimization resulted in a 2.6-fold increase in AuNPs production compared with that of the non-optimized medium.

Biosynthesis of silver nanoparticles using isolated Bacillus subtilis: characterization, antimicrobial activity, cytotoxicity, and their performance as antimicrobial agent for textile materials.

Silver nanoparticles (AgNPs) have unique properties and a large range of applications. Biosynthesis of stable AgNPs using the extracellular filtrate of Bacillus subtilis was proved by the characteristic surface plasmon resonance at about 420-430 nm. They were polycrystalline with spherical, hexagonal, and irregular shapes and they were negatively charged (-40 mV) with an average diameter of 20 nm. FTIR spectrum confirmed the presence of protein molecules coating AgNPs. The optimum conditions for the synthesis of tested AgNPs were 1:6 filtrate dilution, 1 mM AgNO3, pH 7, 30 °C , 48 h contact time under static and illuminating conditions. The synthesized AgNPs showed antibacterial activities against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus cereus, and Salmonella typhi, antifungal activity against Candida albicans and antiviral activity against rotavirus. Also, they showed potent cytotoxic effects on lung and hepatic carcinoma human cell lines. Meanwhile, the acute toxicity study against mice showed no significant changes in hematological, biochemical, and histological parameters of AgNPs treated mice. They also showed mild hepatoprotective effect in thioacetamide (TAA) - induced hepatic fibrosis in rats. AgNPs treated textiles fabrics showed high antimicrobial activities against different pathogenic microorganisms as well as UV protection adequacy.

Prevalence of multidrug-resistant Gram-negative pathogens isolated from febrile neutropenic cancer patients with bloodstream infections in Egypt and new synergistic antibiotic combinations.

INTRODUCTION: Bloodstream infections with multidrug-resistant (MDR) Gram-negative bacteria (GNB) are among the most frequent complications in immunocompromised cancer patients because of their considerable morbidity and mortality. Several guidelines on antimicrobial therapy have addressed empirical treatment for such serious infections; however, the emergence of microbial resistance has become a significant problem worldwide. MATERIALS AND METHODS: In this study, starting from November 2015 to October 2016, a total of 529 blood specimens were collected from febrile neutropenic cancer patients at a tertiary care cancer hospital in Egypt. RESULTS: On examination for positive bacterial growth, it was found that 334 specimens showed no growth, while 195 were positive. Out of the 195 positive culture specimens, 102 (102/195, 52.3%) were Gram-negative and 93 (93/195, 47.7%) were Gram-positive. Out of the 102 GNB, 70 (70/102, 68.6%) were MDR, including Escherichia coli (27/70, 38.6%), Klebsiella pneumoniae (24/70, 34.3%), Acinetobacter baumannii (9/70, 12.8%), Enterobacter cloacae (4/70, 5.7%), Pseudomonas aeruginosa (2/70, 2.8%), Klebsiella oxytoca (2/70, 2.8%), and Klebsiella ornithinolytica (2/70, 2.8%). All MDR GNB showed high resistance to ampicillin, cefepime, ceftriaxone, and cephradine (minimum inhibitory concentration at which 50% of the isolates were inhibited [MIC50] >512 µg/mL for each). However, they showed good susceptibility to colistin (MIC50 <1 µg/mL). The most common extended-spectrum ß-lactamases (ESBLs) genes detected were ctx-m (39/70, 55.7%), shv (31/70, 44.3%), and tem (22/70, 31.4%). The most common aminoglycoside-resistant gene detected was aac(6')-Ib (42/70, 60%) followed by the plasmid-mediated quinolone resistance determinants; qnrA (2/70, 2.8%), qnrB (9/70, 12.8%), and qnrS (19/70, 27.1%). ESBL determinants were significantly associated with resistance to ciprofloxacin, levofloxacin, amikacin, and carbapenems (P-value <0.005). The fractional inhibitory concentration index for ampicillin/sulbactam plus ceftriaxone, ampicillin/sulbactam plus amikacin, and amikacin plus levofloxacin showed synergism against 29 (29/70, 41.4%), 19 (19/70, 27.1%), and 11 (11/70, 15.7%) isolates of the tested MDR GNB isolates, respectively. CONCLUSION: Accordingly, new empirical antibiotics should be administered including the use of colistin or meropenem alone or both against the MDR GNB in neutropenic cancer patients.

CD11c+ and CD123+ dendritic cell subsets in peripheral blood of lung cancer patients.

Dendritic cells (DCs) play a central role in antitumor immune responses. Recent studies however have emphasized an immunosuppressive tumor influence on DCs in various types of cancer. We evaluated the percentages of myeloid and plasmacytoid related DCs in non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). Myeloid CD11c+ DCs (mDC) and plasmacytoid CD123+ DCs (pDC) cells were assessed by Flowcytometry in peripheral blood of twenty untreated lung cancer patients (13 NSCLC and 7 SCLC) and 15 healthy subjects. Lower percentages of pDCs and mDCs were found in patient with NSCLC and SCLC as compared to controls, with significant value only between NSCLC patients and controls (P= 0.001and P=0.000 respectively). The percentages of pDCs and mDCs subsets were significantly lower in patient with SCLC than NSCLC (P=0.013 and P=0.005 for pDCs and mDCs respectively). Our results suggest that NSCLC and SCLC might hamper the maturation of DCs, thus escaping an efficient immune response.

Interleukin 16 (IL-16) in asthma and allergic rhinitis. A comparison between upper and lower airways.

Asthma is a chronic inflammatory disease of bronchial mucosa, in which mast cells, eosinophils and activated T cells are of considerable importance. The increased chemotactic activity for T cells in patients with asthma is mainly attributable to IL-16. A strong association between asthma and allergic rhinitis exists from a clinical and epidemiologic standpoint of view. Although it is clear that the condition of the upper airways has impact on the lower airway physiology, the precise mechanisms underlying this relation are far from being resolved. This work was assessed the role of interleukin 16 (IL-16) in bronchoalveolar lavage (BAL) fluid in both diseases using quantitative sandwich enzyme immuno-assay, and the effect on ventilatory function. The results showed abnormally increased levels of IL-16 (294.4 +/- 15.24 pg/ml), serum eosinophils with absolute count (510.0 +/- 93.57, P>0.05), and total serum IgE (287.9 +/- 61.22 IU/ml) using ELFA in patients of combined asthma and rhinitis, than in each of them alone. There was reduction in FEV1 (forced expiratory volume in the first second) in the same group (81.6 +/- 2.01%). There was a negative correlation between BAL IL-16 and FEV1. In conclusion IL-16 may be considered as a marker of severity of airway inflammation.