Tube Assay Research Articles

Aedes Albopictus (Diptera: Culicidae) Susceptibility Status to Agrochemical Insecticides Used in Durian Planting Systems in Southern Thailand

High rates of dengue morbidity occur in southern Thailand. The intensive application of insecticides in orchards could affect not only agricultural insect pests, but also nontarget mosquitoes or beneficial insects. In this study, the type and quantity of insecticides commonly used across durian plantations in southern Thailand were characterized, along with the population density of Aedes albopictus (Skuse) (Diptera: Culicidae). Our primary aim was to determine the susceptibility status of field-derived Ae. albopictus to typical application concentrations of four agrochemical insecticides; cypermethrin, chlorpyrifos, carbaryl, and imidacloprid. Mosquito eggs were collected from durian cultivation sites in five provinces in southern Thailand and used to generate adults for susceptibility tests. The cultivation sites were categorized into three groups based on insecticide application: intensive application of insecticides, low application of insecticides, and no application of insecticides. Twenty ovitraps were deployed for at least three consecutive days at each study site to collect mosquito eggs and to determine Ae. albopictus population density. WHO tube assays were used to determine the susceptibility of adult mosquitoes derived from field-collected eggs to selected insecticides. This represents the first report of the susceptibility status of Ae. albopictus from durian orchards in southern Thailand to agrochemical insecticides. Results showed complete susceptibility of these Ae. albopictus to chlorpyrifos, but reduced mortality following exposure to λ-cyhalothrin, carbaryl, and imidacloprid, which is suggestive of the development of resistance. These findings provide new insights into the status of insecticide susceptibility in Ae. albopictus populations, with important implications for mosquito and mosquito-borne disease control in Thailand.

Short Persistence and Vector Susceptibility to Ficam 80WP (bendiocarb active ingredient) During Pilot Application of Indoor Residual Spraying in Burkina Faso, West Africa.

Indoor residual spraying (IRS) was applied in addition to the use of long-lasting insecticidal nets in the South West in Burkina Faso, where Anopheles gambiae s.l. the major malaria vector was resistant to pyrethroids. This study was designed to evaluate the efficacy and residual life of bendiocarb (active ingredient) used for spraying on different wall surfaces (mud and cement). Cone bioassays were done monthly with the susceptible An. gambiae 'Kisumu' strain and the local wild populations to determine the duration for which insecticide was effective in killing mosquitoes. Cone bioassay data showed low efficacy and short persistence of bendiocarb applied on mud and cement walls, lasting 2 mo with the susceptible insectary strain and less than 1 mo with An. gambiae wild populations. In addition, WHO tube assays confirmed resistance of An. gambiae wild populations to 0.1% bendiocarb with mortality rates less than 80% in both study sites (sprayed and unsprayed sites). The pilot study of IRS with bendiocarb showed that the residual efficacy of bendiocarb was very short, and resistance to bendiocarb was confirmed in wild populations of An. gambiae s.l. Therefore, Ficam 80 WP was not suitable for IRS in this area due to the short residual duration related mainly to vectors resistance to bendiocarb. While waiting for innovative malaria control tool, alternative insecticide (organophosphate or neonicotinoid classes) or combinations of insecticides have to be used for insecticide resistance management in Burkina Faso.

Transcription Factor PLAGL1 Is Associated with Angiogenic Gene Expression in the Placenta.

During pregnancy, the placenta is important for transporting nutrients and waste between the maternal and fetal blood supply, secreting hormones, and serving as a protective barrier. To better understand placental development, we must understand how placental gene expression is regulated. We used RNA-seq data and ChIP-seq data for the enhancer associated mark, H3k27ac, to study gene regulation in the mouse placenta at embryonic day (e) 9.5, when the placenta is developing a complex network of blood vessels. We identified several upregulated transcription factors with enriched binding sites in e9.5-specific enhancers. The most enriched transcription factor, PLAGL1 had a predicted motif in 233 regions that were significantly associated with vasculature development and response to insulin stimulus genes. We then performed several experiments using mouse placenta and a human trophoblast cell line to understand the role of PLAGL1 in placental development. In the mouse placenta, Plagl1 is expressed in endothelial cells of the labyrinth layer and is differentially expressed in placentas from mice with gestational diabetes compared to placentas from control mice in a sex-specific manner. In human trophoblast cells, siRNA knockdown significantly decreased expression of genes associated with placental vasculature development terms. In a tube assay, decreased PLAGL1 expression led to reduced cord formation. These results suggest that Plagl1 regulates overlapping gene networks in placental trophoblast and endothelial cells, and may play a critical role in placental development in normal and complicated pregnancies.

Selection of endogenous control genes for normalising gene expression data derived from formalin-fixed paraffin-embedded tumour tissue

Quantitative real time polymerase chain reaction (qPCR) data are normalised using endogenous control genes. We aimed to: (1) demonstrate a pathway to identify endogenous control genes for qPCR analysis of formalin-fixed paraffin-embedded (FFPE) tissue using bladder cancer as an exemplar; and (2) examine the influence of probe length and sample age on PCR amplification and co-expression of candidate genes on apparent expression stability. RNA was extracted from prospective and retrospective samples and subject to qPCR using TaqMan human endogenous control arrays or single tube assays. Gene stability ranking was assessed using coefficient of variation (CoV), GeNorm and NormFinder. Co-expressed genes were identified from The Cancer Genome Atlas (TCGA) using the on-line gene regression analysis tool GRACE. Cycle threshold (Ct) values were lower for prospective (19.49 ± 2.53) vs retrospective (23.8 ± 3.32) tissues (p < 0.001) and shorter vs longer probes. Co-expressed genes ranked as the most stable genes in the TCGA cohort by GeNorm when analysed together but ranked lower when analysed individually omitting co-expressed genes indicating bias. Stability values were < 1.5 for the 20 candidate genes in the prospective cohort. As they consistently ranked in the top ten by CoV, GeNorm and Normfinder, UBC, RPLP0, HMBS, GUSB, and TBP are the most suitable endogenous control genes for bladder cancer qPCR.

Hypoxia alleviates dexamethasone-induced inhibition of angiogenesis in cocultures of HUVECs and rBMSCs via HIF-1\u03b1

Background and aimInadequate vascularization is a challenge in bone tissue engineering because internal cells are prone to necrosis due to a lack of nutrient supply. Rat bone marrow-derived mesenchymal stem cells (rBMSCs) and human umbilical vein endothelial cells (HUVECs) were cocultured to construct prevascularized bone tissue in osteogenic induction medium (OIM) in vitro. The angiogenic capacity of HUVECs was limited in the coculture system. In this study, the effects of the components in the medium on HUVEC angiogenesis were analyzed.MethodsThe coculture system was established in OIM. Alizarin red staining and alkaline phosphatase staining were used to assess the osteogenic ability of MSCs. A Matrigel tube assay was used to assess the angiogenic ability of HUVECs in vitro. The proliferation of HUVECs was evaluated by cell counting and CCK-8 assays, and migration was evaluated by the streaked plate assay. The expression levels of angiogenesis-associated genes and proteins in HUVECs were measured by qRT-PCR and Western blotting, respectively.ResultsDexamethasone in the OIM suppressed the proliferation and migration of HUVECs, inhibiting the formation of capillary-like structures. Our research showed that dexamethasone stimulated HUVECs to secrete tissue inhibitor of metalloproteinase (TIMP-3), which competed with vascular endothelial growth factor (VEGF-A) to bind to vascular endothelial growth factor receptor 2 (VEGFR2, KDR). This effect was related to inhibiting the phosphorylation of ERK and AKT, which are two downstream targets of KDR. However, under hypoxia, the enhanced expression of hypoxia-inducible factor-1α (HIF-1α) decreased the expression of TIMP-3 and promoted the phosphorylation of KDR, improving HUVEC angiogenesis in the coculture system.ConclusionCoculture of hypoxia-preconditioned HUVECs and MSCs showed robust angiogenesis and osteogenesis in OIM, which has important implications for prevascularization in bone tissue engineering in the future.

CD34 positive cells isolated from traumatized human skeletal muscle require the CD34 protein for multi-potential differentiation

The CD34 protein is regarded as a marker of stem cells from multiple origins. Recently a mesenchymal progenitor CD34 positive cell identified from traumatized human skeletal muscle demonstrates differentiation capability into vascular endothelial cells, osteoblasts and adipocytes. Here they were treated with a small inhibitory RNA for CD34, which significantly reduced the cellular level of the CD34 protein. These treated cells had a reduced capacity to proliferate, and migrate. They were both unable to differentiation down multiple pathways and to undergo vascular endothelial differentiation as reflected by a lack of expression of VE cadherin, Tie 2 and CD31. Additionally the cells were unable to form tube-like structures in an endothelial tube assay. These treated cells were also unable to undergo osteogenesis, as revealed by lack of alizarin red and alkaline phosphatase staining and were unable to undergo adipogenesis as revealed by lack of oil red O staining. Finally, when CD34 was expressed in cells lacking this protein, the cells were able to undergo vascular endothelial differentiation as revealed by expression of Tie2, VE-cadherin and CD31. These data indicate that in cells derived from traumatized muscle the CD34 protein is required for enhanced proliferation, migration and differentiation down multiple pathways.

Evaluation of multiplex ARMS-PCR for detection of Helicobacter pylori mutations conferring resistance to clarithromycin and levofloxacin

BackgroundHelicobacter pylori bacterium is a major cause of gastritis. With increasing use of antibiotics to treat infections, mutation resistant strains have emerged in most human populations. To effectively treat patients to help resolve infections, the clinician needs information on the antibiotic susceptibility profile of the infection. Therefore, a rapid and accurate test is required to provide this information. To address this issue, we designed and validated a real time multiplex ARMS-PCR assay for rapid detection of highly prevalent H. pylori clarithromycin and levofloxacin resistance mutations. The aim of the study was to evaluate the analytical and diagnostic sensitivity and specificity of ARMS-PCR, using direct Sanger sequencing of the known resistance mutations as the gold standard.ResultsIn preliminary studies using a defined number of plasmids with clarithromycin and levofloxacin resistance mutations, the analytical sensitivity of our ARMS-PCR assay was 50 plasmid copies, equating to around 50 bacterium in a gastric biopsy sample. In terms of specificity, the assay was highly specific for the targeted resistance mutations. The assay was also able to reliably and efficiently detect heteroresistance of clarithromycin and levofloxacin mutations, even at a disproportional ratio of 1:1000. From the analysis of 192 samples with suspected H. pylori infections, the diagnostic sensitivity and specificity of the assay was very high for detection of clarithromycin resistance (100% and 100%), levofloxacin resistance (98.04% and 95.04%) and clarithromycin and levofloxacin double resistance (100% and 96.91%). Amongst the 74 patients diagnosed antibiotic resistance bacteria, 23 (31.1%) had clarithromycin resistance, 21 (28.4%) had levofloxacin resistance and 30 (40.5%) had double resistance. From sample receipt to results, our single tube assay could be routinely completed in under 2 h.ConclusionsOur assay demonstrated high diagnostic sensitivity and specificity for detection of clarithromycin and levofloxacin resistant H. pylori. Based on proven accuracy, together with high efficiency, scalability and low cost, our assay has useful clinical utility for rapid diagnosis of clarithromycin and levofloxacin resistant H. pylori infections. Our assay results will provide patients with a clear diagnosis, enabling the treating clinician to administer the most effective antibiotic regimen to help the clear the infection.

Mechanical Cyclic Stretch Regulate Angiogenic Abilities of Endothelial Progenitor cells

Background & Aim Human endothelial progenitor cells (EPCs) are adult stem cells, located in the bone marrow (BM), peripheral blood, placenta and others. EPCs have important role in tissue regeneration via angiogenesis and vasculogenesis. In response to tissue injury, BM-EPCs mobilize to injury site where various growth factors such as vascular endothelial growth factors (VEGF), stromal derived factor 1 alpha (SDF-1a) are released. At the injured site, EPCs can differentiate into mature endothelial cells (ECs) and then directly incorporate into the blood vessel through the stimulation of growth factors and change of cell to cell contact. Because of immunogenicity, autologous EPC therapy is preferable than allograft. However, the number and function of EPCs derived from old and chronic patients are insufficient for successful treatment. In order to retrieve sufficient numbers of EPC from old or chronic patients and restore its function for clinical application, suitable cell preconditioning such as cell stretch, shear stress, and mechanical pressure given by blood flow in vivo may be required before transplantation. Our previous studies have shown that exposure of ECs to mechanical stretch in vitro increased the abilities of migration, proliferation, and tube formation, which could accelerate angiogenesis in impaired tissue. Methods, Results & Conclusion In this study, we explored whether mechanical stretch can improve the survival and proliferation of EPC derived from old or chronic patients by applying cyclic stretch in the range of 5%-15% with use of computerized cell strain system Flexcell-6000. In response to cyclic strain, EPCs increased cell viability, migration and tube formation ability based on matrigel tube assay. Effect of cyclic stretch on the expression of a variety of paracrine factors and extracellular matrix and also their efficacy on vasculogenesis in vivo using limb ischemia model will be estimated.

Rhizosphere Bacteria with the Potential of Forming Biofilm and Plant Growth Promotion Under Salt Stress

Bacteria develop microbial communities as biofilm under different environmental stress factors like salinity, temperature, pH and antimicrobial agents and help in the adherence of bacteria to different surfaces. The growth of microorganisms is inhibited in the presence of salinity. An objective of the present research work was to check the growth response and biofilm behavior of indigenous bacteria isolated from plant rhizosphere in the presence of salt. Bacterial strains were named as TAK, TAF and SY. Biofilm formation response was also observed at different molar concentration (0M, 1M, 2M and 3M) of NaCl following test tube assay after 24, 48 and 72 hours of culture incubation. Results showed that TAK has the best biofilm forming ability on abiotic surface as compared to isolate TAF and SY, however, isolate SY showed growth and form biofilm under saline conditions. Bacterial plant growth promoting response was also determined on the basis of improvement in seed germination, shoot length and root length. In general, bacterial biofilm was best at 1M to 3M NaCl stress and 72 hours of culture incubation . Inoculation with SY improved (shoot length 3 % and root length 10.41 %) at 100 mM when compared to inoculated seedlings at 0 mM NaCl stress. It was concluded that among all three isolates, isolate SY used for broad perspective to increase soil fertility. While working to strive for the most promising isolates involved in plant growth promotion, the indigenous isolates showed the promising ability to improve germination of seeds in saline soil while helping the seeds to grow in salt stress conditions.

Impact of Insecticide Resistance on P. falciparum Vectors' Biting, Feeding, and Resting Behaviour in Selected Clusters in Teso North and South Subcounties in Busia County, Western Kenya.

Introduction Behavioural resistance to insecticides restrains the efficacy of vector control tools against mosquito-transmitted diseases. The current study is aimed at determining the impact of insecticide resistance on major malaria vectors' biting, feeding, and resting behaviour in areas with and areas without insecticide resistance in Teso North and Teso South, Busia County, Western Kenya. Methods Mosquito larvae were sampled using a dipper, reared into 3-5-day-old female mosquitoes [4944] which were exposed to 0.75% permethrin and 0.05% deltamethrin using World Health Organization tube assay method. Blood meal, species identification, and kdr Eastgene PCRs were also performed on adult mosquitoes sampled using mosquito collection methods [3448]. Biting, feeding, resting, and exiting behaviours of field-collected mosquitoes from five selected clusters were analysed. Results The lowest Kdr genotypic frequency (SS) proportion was found in female Anophelines collected in Kengatunyi at 58% while Rwatama had the highest genotypic frequency at 93%, thus susceptible and resistant clusters, respectively. The peak hour for mosquito seeking a human bite was between 0300 and 0400 hrs in the resistant cluster and 0400-0500 hrs in the susceptible cluster. The heterozygous mosquitoes maintained the known 2100-2200 hrs peak hour. There was a higher proportion of homozygous susceptible vectors (86.4%) seeking humans indoor than outdoor bitters (78.3%). Mosquito blood meals of human origin were 60% and 87% in susceptible Kengatunyi and resistant Rwatama cluster, respectively. There was significant difference between homozygous-resistant vectors feeding on human blood compared to homozygous susceptible mosquitoes (p ≤ 0.05). The proportion of bovine blood was highest in the susceptible cluster. A higher proportion of homozygous-resistant anophelines were feeding and resting indoors. No heterozygous mosquito was found resting indoor while 4.2% of the mosquitoes were caught while exiting the house through the window. Discussion. A shift in resistant Anopheles gambiae sl highest peak hour of aggressiveness from 2100-2200 hrs to 0300-0400 hrs is a key change in its biting pattern. Due to the development of resistance, mosquitoes no longer have to compete against the time the human host enters into the formerly lethal chemical and or physical barrier in the form of long-lasting insecticide-treated net. No heterozygous LS mosquito rested indoors possibly due to disadvantages of heterozygosity which could have increased their fitness costs as well as energy costs in the presence of the insecticidal agents in the treated nets. Conclusions and recommendations. Out of bed biting by female mosquitoes and partial susceptibility may contribute to residual malaria transmission. Insecticide-resistant vectors have become more endophagic and anthropophillic. Hence, insecticidal nets, zooprophylaxis, and novel repellents are still useful chemical, biological, and physical barriers against human blood questing female mosquitoes. Further studies should be done on genetic changes in mosquitoes and their effects on changing mosquito behaviour.

Pre‐analytical factors affecting the establishment of a single tube assay for multiparameter liquid biopsy detection in melanoma patients

The combination of liquid biomarkers from a single blood tube can provide more comprehensive information on tumor development and progression in cancer patients compared to single analysis. Here, we evaluated whether a combined analysis of circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), and circulating cell‐free microRNA (miRNA) in total plasma and extracellular vesicles (EV) from the same blood sample is feasible and how the results are influenced by the choice of different blood tubes. Peripheral blood from 20 stage IV melanoma patients and five healthy donors (HD) was collected in EDTA, Streck, and Transfix tubes. Peripheral blood mononuclear cell fraction was used for CTC analysis, whereas plasma and EV fractions were used for ctDNA mutation and miRNA analysis. Mutations in cell‐free circulating DNA were detected in 67% of patients, with no significant difference between the tubes. CTC was detected in only EDTA blood and only in 15% of patients. miRNA NGS (next‐generation sequencing) results were highly influenced by the collection tubes and could only be performed from EDTA and Streck tubes due to hemolysis in Transfix tubes. No overlap of significantly differentially expressed miRNA (patients versus HD) could be found between the tubes in total plasma, whereas eight miRNA were commonly differentially regulated in the EV fraction. In summary, high‐quality CTCs, ctDNA, and miRNA data from a single blood tube can be obtained. However, the choice of blood collection tubes is a critical pre‐analytical variable.

Anti-CD3 Antibody Treatment Reduces Scar Formation in a Rat Model of Myocardial Infarction

Introduction: Antibody treatment with anti-thymocyte globulin (ATG) has been shown to be cardioprotective. We aimed to evaluate which single anti-T-cell epitope antibody alters chemokine expression at a level similar to ATG and identified CD3, which is a T-cell co-receptor mediating T-cell activation. Based on these results, the effects of anti-CD3 antibody treatment on angiogenesis and cardioprotection were tested in vitro and in vivo. Methods: Concentrations of IL-8 and MCP-1 in supernatants of human peripheral blood mononuclear cell (PBMC) cultures following distinct antibody treatments were evaluated by Enzyme-linked Immunosorbent Assay (ELISA). In vivo, anti-CD3 antibodies or vehicle were injected intravenously in rats subjected to acute myocardial infarction (AMI). Chemotaxis and angiogenesis were evaluated using tube and migration assays. Intracellular pathways were assessed using Western blot. Extracellular vesicles (EVs) were quantitatively evaluated using fluorescence-activated cell scanning, exoELISA, and nanoparticle tracking analysis. Also, microRNA profiles were determined by next-generation sequencing. Results: Only PBMC stimulation with anti-CD3 antibody led to IL-8 and MCP-1 changes in secretion, similar to ATG. In a rat model of AMI, systemic treatment with an anti-CD3 antibody markedly reduced infarct scar size (27.8% (Inter-quartile range; IQR 16.2–34.9) vs. 12.6% (IQR 8.3–27.2); p < 0.01). The secretomes of anti-CD3 treated PBMC neither induced cardioprotective pathways in cardiomyocytes nor pro-angiogenic mechanisms in human umbilical vein endothelial cell (HUVECs) in vitro. While EVs quantities remained unchanged, PBMC incubation with an anti-CD3 antibody led to alterations in EVs miRNA expression. Conclusion: Treatment with an anti-CD3 antibody led to decreased scar size in a rat model of AMI. Whereas cardioprotective and pro-angiogenetic pathways were unaltered by anti-CD3 treatment, qualitative changes in the EVs miRNA expression could be observed, which might be causal for the observed cardioprotective phenotype. We provide evidence that EVs are a potential cardioprotective treatment target. Our findings will also provide the basis for a more detailed analysis of putatively relevant miRNA candidates.

Species Composition, Phenotypic and Genotypic Resistance Levels in Major Malaria Vectors in Teso North and Teso South Subcounties in Busia County, Western Kenya.

Introduction Knockdown resistance (kdr) is strongly linked to pyrethroid insecticide resistance in Anopheles gambiae in Africa, which may have vital significance to the current increased use of pyrethroid-treated bed net programmes. The study is aimed at determining species composition, levels of insecticide resistance, and knockdown patterns in Anopheles gambiae sensu lato in areas with and areas without insecticide resistance in Teso North and Teso South subcounties, Western Kenya. Materials and Methods For WHO vulnerability tests, mosquito larvae were sampled using a dipper, reared into 3-5-day-old female mosquitoes (4944 at 100 mosquitoes per insecticide) which were exposed to 0.75% permethrin, 0.05% deltamethrin, and 0.1% bendiocarb using the WHO tube assay method. Species identification and kdr East gene PCRs were also performed on randomly selected mosquitoes from the collections; including adult mosquitoes (3448) sampled using standard collection methods. Results Anopheles gambiae sensu stricto were the majority in terms of species composition at 78.9%. Bendiocarb caused 100% mortality while deltamethrin had higher insecticidal effects (77%) on female mosquitoes than permethrin (71%). Susceptible Kengatunyi cluster had higher proportion of An. arabiensis (20.9%) than resistant Rwatama (10.7%). Kengatunyi mosquitoes exposed to deltamethrin had the highest KDT50 R of 8.2. Both Anopheles gambiae sensu stricto and Anopheles arabiensis had equal S allelic frequency of 0.84. Indoor resting mosquitoes had 100% mortality rate after 24 h since exposure. Overall SS genotypic frequency in Teso North and Teso South subcounties was 79.4% against 13.7% homozygous LL genotype and 6.9% heterozygous LS genotype. There was a significant difference (ρ < 0.05) in S allele frequencies between Kengatunyi (0.61) and Rwatama (0.95). Mosquito samples collected in 2013 had the highest S allelic frequency of 0.87. Discussion. Most likely, the higher the selection pressure exerted indoors by insecticidal nets, the higher were the resistance alleles. Use of pyrethroid impregnated nets and agrochemicals may have caused female mosquitoes to select for pyrethroid resistance. Different modes of action and chemical properties in different types of pyrethroids aggravated by a variety of edaphic and climatic factors may have caused different levels of susceptibility in both indoor and outdoor vectors to pyrethroids and carbamate. Species composition and populations in each collection method may have been influenced by insecticide resistance capacity in different species. Conclusions and Recommendations. Both phenotypic and genotypic insecticide resistance levels have been confirmed in Teso North and Teso South subcounties in Western Kenya. Insecticide resistance management practices in Kenya should be fast tracked and harmonized with agricultural sector agrochemical-based activities and legislation, and possibly switch to carbamate use in order to ease selection pressure on pyrethroids which are useable in insecticidal nets and indoor residual spray due to their low human toxicity. The implication of such high resistance levels in mosquitoes collected in Teso subcounties is that resistance is likely to persist and or even increase if monomolecules of permethrin and deltamethrin or both continue to be used in all net- and nonnet-based mosquito control purposes. Usage of mutually reinforcing piperonyl butoxide (PBO) that prohibits particular enzymes vital in metabolic activities inside mosquito systems and has been integrated into pyrethroid-LLINs to create pyrethroid-PBO nets is an extremely viable option.

Hold your breath - Differential behavioral and sensory acuity of mosquitoes to acetone and carbon dioxide.

Host seeking in the yellow fever mosquito, Aedes aegypti, and the African malaria mosquito, Anopheles coluzzii, relies on specific and generic host-derived odorants. Previous analyses indicate that the behavioral response of these species depends differentially on the presence of carbon dioxide (CO2) and other constituents in human breath for activation and attraction. In this study, we use a flight tube assay and electrophysiological analysis to assess the role of acetone, a major component of exhaled human breath, in modulating the behavioral and sensory neuronal response of these mosquito species, in the presence and absence of CO2. When presented alone at ecologically relevant concentrations, acetone increases attraction in Ae. aegypti, but not in An. coluzzii. Moreover, in combination with CO2, human breath-equivalents of acetone ranging between 0.1 and 10 ppm reproduces a behavioral response similar to that observed to human breath in host-seeking Ae. aegypti, but not in An. coluzzii. Acetone does, however, reduce attraction to CO2 in An. coluzzii, when presented at a higher concentration of 10 ppm. We identify the capitate peg A neuron of the maxillary palp of both species as a dual detector of CO2 and acetone. The sensory response to acetone, or binary blends of acetone and CO2, reflects the observed behavioral output in both Ae. aegypti and An. coluzzii. We conclude that host recognition is contextual and dependent on a combination of ecologically relevant odorants at naturally occurring concentrations that are encoded, in this case, by differences in the temporal structure of the neuronal response. This information should be considered when designing synthetic blends for that optimally attract mosquitoes for monitoring and control.

Correlation of biofilm formation and antibiotic resistance among clinical and soil isolates of Acinetobacter baumannii in Iraq.

Acinetobacter baumannii is an opportunistic pathogen that is reported as a major cause of nosocomial infections. The aim of this study was to investigate the biofilm formation by A. baumannii clinical and soil isolates, to display their susceptibility to 11 antibiotics and to study a possible relationship between formation of biofilm and multidrug resistance. During 8months period, from June 2016 to January 2017, a total of 52 clinical and 22 soil isolates of A. baumannii were collected and identified through conventional phenotypic, chromo agar, biochemical tests, API 20E system, and confirmed genotypically by PCR for blaOXA-51-like gene. Antibiotic susceptibility of isolates was determined by standard disk diffusion method according to Clinical and Laboratory Standard Institute. The biofilm formation was studied using Congo red agar, test tube, and microtiter plate methods. The clinical isolates were 100% resistance to ciprofloxacin, ceftazidime, piperacillin, 96.15% to gentamicin, 96.15% to imipenem, 92.31% to meropenem, and 78.85% to amikacin. The soil A. baumannii isolates were 100% sensitive to imipenem, meropenem, and gentamicin, and 90.1% to ciprofloxacin. All A. baumannii isolates (clinical and soil) were susceptible to polymyxin B. The percentage of biofilm formation in Congo red agar, test tube, and microtiter plate assays was 10.81%, 63.51%, and 86.48%, respectively. More robust biofilm former population was mainly among non-MDR isolates. Isolates with a higher level of resistance tended to form weaker biofilms. The soil isolates exhibited less resistance to antibiotics than clinical isolates. However, the soil isolates produce stronger biofilms than clinical isolates.

The Potential Use of Resveratrol for Cancer Prevention.

In addition to the traditional treatments of cancer and cancer prevention, the use of natural compounds, especially those found in food, should be considered. To clarify if resveratrol has the potential for cancer prevention and the possibility of use in therapy, the following must be taken into account: data from epidemiology, clinical protocol (case and control), preclinical studies (lab animals), use of established cell lines as models of cancer cells, test tube assays (enzymes activities), and requirements of nanotechnologies in order to discover new drugs to fight cancer. From this perspective and future expected advances, more information is needed such as improved efficacy, methods of application, and the synergistic sensitization of resveratrol as an adjuvant. In addition, resveratrol nanoformulation is considered to overcome its weak bioavailability.

Next generation bioformulation prepared by amalgamating Bradyrhizobium, cell free culture supernatant, and exopolysaccharides enhances the indigenous rhizospheric rhizobial population, nodulation, and productivity of pigeon pea

Addition of living cells for plant growth promotion has several constraints including erratic and inconsistent field performance, poor shelf life, and reduced population due to tough competition with resident microbes. Due to the focus on employing cell-based formulation, bacterial cell free culture supernatant (CFCS), and metabolites produced by them, have been largely ignored. Hence, present study aimed to enhance productivity and nodulation of Cajanus cajan (pigeon pea) by using diverse formulations developed from Bradyrhizobium strain IC-4059 (cell based formulation), its CFCS (supernatant based), and exopolysaccharides (EPS) (metabolite based), blended formulation (cells with CFCS; cells with metabolite; metabolite with CFCS), and combinational formulation (IC-4059+EPS + CFCS). Efficacy of developed formulations was monitored initially in tube assay, followed by field experiment with Pusa 991 pigeon pea cultivar in randomized block design. Growth promoting metabolites present in CFCS and EPS were analyzed by metabolomic approach using UPLC–MS and HPLC, respectively. In both tube assay and field experiment, seeds receiving treatment with combination of IC-4059 cells plus CFCS and EPS performed best, followed by blend IC-4059 CFCS + EPS, and IC-4059 cells + EPS, in terms of plant parameters. During field experiment seeds treated with combination showed significant enhancement in shoot length, root length, dry weight, protein content, and nodule number by 1.2, 1.3, 1.2, 1.1 and 2.0-fold, respectively, in comparison with rhizobia-based formulation. The prominent compounds identified by UPLC–MS were phosphatidylserine, phosphatidylcholine, FMC-5, pantoic acid, ascorbic acid, benzoic acid, 4-pentyl-4-formylphenyl ester, benzimidazole, and phosphatidylethanolamine. The study disseminates the use of combinational formulation developed from Bradyrhizobium sp. IC-4059, its CFCS, and its EPS for augmenting nodulation and growth of pigeon pea crop in field in comparison with application of rhizobial cell alone, as next generation of bioformulations.

Hyssopus officinalis subsp. aristatus: An unexploited wild-growing crop for new disclosed bioactives

The aerial parts of rock hyssop (Hyssopus officinalis subsp. aristatus (Godr.) Nyman), well-known for bearing essential oil-producing glands, are also rich in phenol and polyphenol compounds. These latter, still rarely studied in rock hyssop and other hyssop species, markedly differ based on the plant collection site. In fact, their biosynthesis, maximized in the wild, is finely regulated by multiple abiotic and biotic factors. In this context, to reach an accurate and deep understanding of the polyphenol constitution of a homemade-like preparation (HOEE) from rock hyssop growing wild in Southern Italy, analyses, based on ultra-high pressure liquid chromatography coupled with high-resolution mass spectrometry (UHPLC HRMS), were carried out. HOEE mainly consisted of hydroxycinnamoyl derivatives (57.2%), among which some different and never reported coumarin and lignan compounds. Dicaffeoyl quinic acids were the main chlorogenic compounds, whereas flavonoid glycosides and methoxyflavonoids were the less abundant compounds. The preliminary assessment of HOEE antiradical properties through differen t-test tube assays provided a solid basis for originally exploring its antioxidant activity towards differentiated Caco-2 cells. Experimental evidence on genoprotective activity of hyssop aerial parts, herein testified for the first time, raised the potential of their using as a functional food for oxidative stress-induced diseases prevention.

5. NOVEL APPROACH ENABLING THE SIMULTANEOUS DETECTION OF SNV AND CNV FOR PGT-M AND PGT-A USING A SINGLE-TUBE ASSAY

Introduction Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator gene, CFTR. An individual must inherit two defective cystic fibrosis alleles, one from each parent, to have the disease. Worldwide, over 70,000 individuals are affected by this monogenic disease. Preimplantation genetic testing (PGT) aims at selecting healthy embryos by testing for chromosome abnormalities and monogenic diseases. Often a limited number of cells or genetic material from the embryo is available to perform the detection of both aneuploidies and monogenic diseases with accuracy. To overcome this challenge, we developed a novel single tube assay enabling the simultaneous detection of both copy number variations (CNV) across the whole genome and single nucleotide variants (SNV) or small insertion/deletion (indels) located in the CFTR gene target region. Material and Methods The assay was performed using a modified SMARTer® PicoPLEX® Gold Single Cell DNA-Seq kit (Takara Bio USA, R300669). More specifically, the assay included simultaneous whole genome amplification (WGA) and targeted enrichment of selected regions of the CFTR gene. All 23 mutations of CFTR recommended to be tested by the American College of Medical Genetics (ACMG) were covered. As proof of principle, we prepared libraries from either single cells or five cells using model cell lines purchased from the Coriell Institute that contain different known CFTR variants, such as delF508, or aneuploidies of different sizes (2.5MB to 25 MB). In addition, libraries were made from trophectoderm biopsies isolated in the clinic from human blastocysts containing known CFTR mutations. The resulting libraries were sequenced on a MiSEQ to achieve 1M reads per sample (2 × 75 cycles). Results The percentage of sequencing reads allocated to both applications (CNV and SNV detections) was optimized to maximize the performance of the assay. To detect copy number variations, a shallow and even coverage of the genome is sufficient (∼0.025x). However, to detect variants, SNPs or small indels, the targeted regions of interest required a robust and deeper coverage (500x). At a sequencing depth of 1M reads, the coverage of the genome was ensured by 95% of the reads and the targeted regions of CFTR were covered by ∼5% of the reads. When using the five-cell model system, the 2.5 and 25MB chromosomal losses and the characterized heterozygous variants of CFTR were detected virtually 100% of the time (n of 18). When using single cells, we consistently detected the 25MB deletion (n of 18). Also, the expected heterozygous CFTR variants were detected over 80% of the time. Conclusion The sequencing reads allocated to both applications was optimized to maximize the performance of both CNV and SNV detection. Over 20 samples were successfully processed on a single MiSeq run. From 5 sorted cells our new approach provided representative and uniform coverage of the genome, suitable for detection of copy number variations (CNV). From the same assay, mutations contained in the CFTR gene were also accurately reported. Studies with TE biopsies with known CFTR mutations are now in progress and will be reported at the meeting.

Controlled Oil/Water Partitioning of Hydrophobic Substrates Extending the Bioanalytical Applications of Droplet-Based Microfluidics.

Functional annotation of novel proteins lags behind the number of sequences discovered by the next-generation sequencing. The throughput of conventional testing methods is far too low compared to sequencing; thus, experimental alternatives are needed. Microfluidics offer high throughput and reduced sample consumption as a tool to keep up with a sequence-based exploration of protein diversity. The most promising droplet-based systems have a significant limitation: leakage of hydrophobic compounds from water compartments to the carrier prevents their use with hydrophilic reagents. Here, we present a novel approach of substrate delivery into microfluidic droplets and apply it to high-throughput functional characterization of enzymes that convert hydrophobic substrates. Substrate delivery is based on the partitioning of hydrophobic chemicals between the oil and water phases. We applied a controlled distribution of 27 hydrophobic haloalkanes from oil to reaction water droplets to perform substrate specificity screening of eight model enzymes from the haloalkane dehalogenase family. This droplet-on-demand microfluidic system reduces the reaction volume 65 000-times and increases the analysis speed almost 100-fold compared to the classical test tube assay. Additionally, the microfluidic setup enables a convenient analysis of dependences of activity on the temperature in a range of 5 to 90 °C for a set of mesophilic and hyperstable enzyme variants. A high correlation between the microfluidic and test tube data supports the approach robustness. The precision is coupled to a considerable throughput of >20 000 reactions per day and will be especially useful for extending the scope of microfluidic applications for high-throughput analysis of reactions including compounds with limited water solubility.