Tube Assay Research Articles

Resistance of Anopheles gambiae s.s. against commonly used insecticides and implication of cytochrome P450 monooxygenase in resistance to pyrethroids in Lambaréné (Gabon)

BackgroundInsecticides are a crucial component of vector control. However, resistance constitute a threat on their efficacy and the gains obtained over the years through malaria vector control. In Gabon, little data on phenotypic insecticide resistance in Anopheles vectors are published, compromising the rational implementation of resistance management strategies. We assessed the susceptibility to pyrethroids, carbamates and organophosphates of Anopheles gambiae sensu lato (s.l.) and discuss the mechanisms involved in the pyrethroid resistance-phenotype.MethodsA. gambiae s.l. larvae were collected from breeding sites in Lambaréné. Emerging adults were used in WHO tube assays at an insecticide concentration that defines resistance (diagnostic concentration). Subsequently, deltamethrin and permethrin were used at 5x and 10x diagnostic concentrations and after preexposure with the cytochrome p450 (and glutathione S-transferase) inhibitor piperonyl butoxide (PBO). A subset of mosquitoes was typed by molecular methods and screened using Taqman assays for mutations conferring target site resistance at the Voltage-gated sodium channel 1014 (Vgsc-1014) locus and the acetylcholinesterase (Ace-1) gene.ResultsAll mosquitoes were A. gambiae sensu stricto (s.s.) and resistant to permethrin, deltamethrin and alphacypermethrin (mortality less than 98%). However, mosquitoes were susceptible to malathion but resistant to bendiocarb. The level of resistance was high for permethrin and at least moderate for deltamethrin. Pre-exposure to PBO significantly increased the mortality of resistant mosquitoes (P < 0.0001). They became fully susceptible to deltamethrin and permethrin-induced mortality increased 4-fold. The G119S Ace-1 resistance allele, which confers resistance to both organophosphates and carbamates, was not present. All sampled mosquitoes were either homozygous for the Vgsc-L1014F or heterozygous for Vgsc-L1014F/L1014S, a marker for resistance to pyrethroids and organochlorides.ConclusionThese findings demonstrate a role of cytochrome P450 monooxygenases in the pyrethroid-resistance of A. gambiae s.s. from Lambaréné. Combining PBO with pyrethroids, as done in second generation bednets, may be used to revert resistance. In addition, malathion could also be used in combination with pyrethroids-based methods for resistance management.

Automated analysis of flow cytometry data with minimal training files: Research evaluation of an elastic image registration algorithm for TBNK, stem cell enumeration, and lymphoid screening tube assays.

Automated analysis of flow cytometry data can improve objectivity and reduce analysis time but has generally required work by software and algorithm experts. Here, we investigated the performance of BD ElastiGate™ Software (hereafter ElastiGate), which allows users to automate gating by selecting gated training files, then uses elastic image registration to gate new files. Three assays of increasing complexity were examined: TBNK, stem cell enumeration (SCE), and lymphoid screening tube (LST). For TBNK analysis, 60 peripheral blood (PB) samples from normal, HIV+, and controls were tested with ground truth analysis by an existing automated method. For SCE, 128 samples including bone marrow (BM), cord blood (CB), and apheresis were tested with analysis by multiple manual analysts. For LST, 80 PB and 28 BM samples were tested with manual analysis. For ElastiGate, a minimal number of training files was selected. Results were compared by Bland-Altman or F1 score analysis. For TBNK, ElastiGate using three training files (1 control, 1 normal, 1 HIV+) showed mean %bias across all reported populations between -1.48% and 7.13% (average 2.08%). For SCE, ElastiGate using three BM and two CB training files showed median F1 scores >0.93 in comparison to >0.94 and >0.92 for two other manual analysts. For LST, ElastiGate using four training files for each of PB and BM showed median F1 scores >0.945 for 13 of 14 PB populations and 10 of 14 BM populations, with generally similar or better performance for normal samples compared to abnormal; populations with lower scores were often associated with lower agreement between manual analysts. Based on analysis of three assays with four sample types of increasing complexity, ElastiGate with minimal training files may perform as an automated gating assistant. The results reported here are for research use only, not for use in diagnostic or therapeutic procedures.

Power calculation for mosquito bioassays: Quantifying variability in the WHO tube bioassay and developing sample size guidance for the PBO synergism assay using a Shiny application

Background The WHO tube bioassay is a method for exposing mosquitos to determine susceptibility to insecticides, with mortality to discriminating doses &lt;98% indicating possible resistance and &lt;90% confirming resistance. This bioassay is also used for synergism testing to assess if susceptibility is restored by pre-exposure to the synergist piperonyl butoxide. Methods Here we perform testing with pyrethroid-susceptible and pyrethroid-resistant An. gambiae to quantify the variability of the WHO tube bioassay and identify its sources. These estimates of within and between day variability are then used to evaluate the power of the bioassay to detect a mortality difference between pyrethroid-only and pyrethroid-PBO. Results We show that approximately two-thirds of variation occurs between days, with the pyrethroid-susceptible strain twice as variable as the pyrethroid-resistant strain. The total number of mosquitoes in the tube and their bodyweight contributes to approximately 10% of this variability. Changes in temperature and humidity, within a climate-controlled insectary, didn’t impact mortality. Using a simulation-based framework, we show that the current synergism guidelines, using a 4x4 design, can reliably detect a difference between 90% and 100% mortality (&gt;90% power). However, as the mortality of either group gets closer to 50%, a 10% difference between groups is more difficult to reliably detect. In the worst-case scenario where the mortality of either group is 50%, the mortality difference must be &gt;22.5% to be detected with 80% power. We provide an R shiny application to assess power for other comparisons. Conclusions Our findings indicate that detecting synergism with the WHO tube assay is more difficult than assumed by the current WHO guidelines. Additionally, we demonstrate the value of using a Shiny application to make the outputs of simulation-based power analysis readily available to end-users, allowing them to determine the number of tubes needed to detect a given mortality difference.

Projecting phytochemical bacoside A anti-mucorale agent: An in-silico and in-vitro assessment

Mucormycosis, a life-threatening fungal infection that primarily affects immunocompromised individuals.The protein family commonly observed in the fugus responsible for causing Mucormycosis. The attachment of spores to host cells surface, facilitated by a protein CotH, is a critical step for the invasion and progression of the disease. Therefore, CotH inhibitors have emerged as a promising therapeutic strategy for treating mucormycosis.This study presents a novel therapeutic target and ligand for controlling the growth of Mucorales. First, to identify potential CotH inhibitors, we surveyed a library antifungal compounds elaborated in AYUSearch database. Next, using machine learning-based algorithms we screend 20 potentials ligands, followed by structure-based molecular modelling and molecular trajectory analysis to identify the three most promising chemical constituents. In-vitro tube assays on selected Mucorales determined the minimum inhibitory concentrations (MIC) for screened chemotypes. The MIC assay revealed that Bacoside inhibits the growth and sporulation at 5 mg/ml concentrations, emerging as a probable CotH inhibitor. Further, the compound's toxicity was evaluated by adding it to the feed of C.elegans, and the finding suggests that the bacoside is reasonably safe at the studied concentration. The findings project bacoside A as a potential anti-mucorale lead compound that can be further validated with preclinical and clinical studies.

Evaluation of the Genetic Diversity, Haplotype, and Virulence of Fusarium oxysporum f. sp. vasinfectum Field Isolates from Alabama.

The United States is the third largest producer of cotton and the largest exporter of cotton globally. Fusarium wilt, caused by the soilborne fungal pathogen Fusarium oxysporum f. sp. vasinfectum (Fov), was estimated to cause a $21 million cotton yield loss in 2022. Historically, Alabama was an important producer of cotton in the Southeastern United States and was the first state in which Fusarium wilt on cotton was described. To assess the genetic diversity of Fov field isolates in Alabama, 118 field isolates were collected from six counties across the state from 2014 to 2016. Phylogenetic analysis using TEF1 and RPB2 placed the Fov field isolates into 18 haplotypes. Upon profiling the Tfo1 transposon insertion in the NAT gene, it was determined that no race 4 isolates were recovered in Alabama. Representatives of all field isolate haplotypes caused disease on Upland cotton variety Rowden in a hydroponic test tube assay. Two haplotype A isolates were the most aggressive isolates recovered, and haplotype A isolate TF1 was more aggressive than the race 4 isolate 89-1A on Upland cotton and had similar symptom severity on Pima cotton. Karyotype profiling indicted an abundance of small chromosomes characteristic of karyotypes that include accessory chromosomes, with considerable variability between isolates. Collectively, our study indicates that Fov isolates from Alabama are genetically diverse, which may have been promoted by its persistence in cotton fields.

Coexistence of β-lactamase genes and biofilm forming potential among carbapenem-resistant Acinetobacter baumannii in Lahore, Pakistan.

Our goal was to investigate the antimicrobial resistance due to beta-lactamase genes and virulent determinants (biofilm-forming ability) expressed by Acinetobacter collected from health settings in Pakistan. A cross-sectional study was conducted for the molecular characterization of carbapenemases and biofilm-producing strains of Acinetobacter spp. Two twenty-three imipenem-resistant Acinetobacter isolates were analyzed from 2020 to 2023.The combination disk test and modified hodge test were performed. Biofilm forming ability was determined by polystyrene tube assay. Multiplex polymerase chain reaction (PCR) for virulent and biofilm-forming genes, and 16S rRNA sequencing were performed. 118 (52.9%) carbapenem-resistant Acinetobacter (CR-AB) were isolated from wounds and pus, 121 (54.2%) from males, and 92 (41.2%) from 26-50-years-olds. More than 80% of strains produced β-lactamases and carbapenemases. Based on the PCR amplification of the ITS gene, 174 (78.0%) CR-AB strains were identified from CR-Acinetobacter non-baumannii (ANB). Most CR-AB were strong and moderate biofilm producers. Genetic analysis revealed the blaOXA-23, blaTEM, blaCTX-M blaNDM-1 and blaVIM were prevalent in CR-AB with frequencies 91 (94.8%), 68 (70.8%), 19 (19.7%), 53 (55.2%), 2 (2.0%) respectively. Among virulence genes, OmpA was dominant in CR-AB isolates from wound (83, 86.4%), csuE 63 (80.7%) from non-wound specimens and significantly correlated with blaNDM and blaOXA genes. Phylogenetic analysis revealed three different clades for strains based on specimens. CR-AB was highly prevalent in Pakistan and associated with wound infections. The genes, blaOXA-23, blaTEM, blaCTX-M, and blaNDM-1 were detected in CR-AB. Most CR-AB were strong biofilm producers with virulent genes OmpA and csuE.

Entomological surveys and insecticide susceptibility profile of Aedes aegypti during the dengue outbreak in Sao Tome and Principe in 2022.

The first dengue outbreak in Sao Tome and Principe was reported in 2022. Entomological investigations were undertaken to establish the typology of Aedes larval habitats, the distribution of Ae. aegypti and Ae. albopictus, the related entomological risk and the susceptibility profile of Ae. aegypti to insecticides, to provide evidence to inform the outbreak response. Entomological surveys were performed in all seven health districts of Sao Tome and Principe during the dry and rainy seasons in 2022. WHO tube and synergist assays using piperonyl butoxide (PBO) and diethyl maleate (DEM) were carried out, together with genotyping of F1534C/V1016I/V410L mutations in Ae. aegypti. Aedes aegypti and Ae. albopictus were found in all seven health districts of the country with high abundance of Ae. aegypti in the most urbanised district, Agua Grande. Both Aedes species bred mainly in used tyres, discarded tanks and water storage containers. In both survey periods, the Breteau (BI > 50), house (HI > 35%) and container (CI > 20%) indices were higher than the thresholds established by WHO to indicate high potential risk of dengue transmission. The Ae. aegypti sampled were susceptible to all insecticides tested except dichlorodiphenyltrichloroethane (DDT) (9.2% mortality, resistant), bendiocarb (61.4% mortality, resistant) and alpha-cypermethrin (97% mortality, probable resistant). A full recovery was observed in Ae. aegypti resistant to bendiocarb after pre-exposure to synergist PBO. Only one Ae. aegypti specimen was found carrying F1534C mutation. These findings revealed a high potential risk for dengue transmission throughout the year, with the bulk of larval breeding occurring in used tyres, water storage and discarded containers. Most of the insecticides tested remain effective to control Aedes vectors in Sao Tome, except DDT and bendiocarb. These data underline the importance of raising community awareness and implementing routine dengue vector control strategies to prevent further outbreaks in Sao Tome and Principe, and elsewhere in the subregion.

Metabolic Resistance and Not Voltage-Gated Sodium Channel Gene Mutation Is Associated with Pyrethroid Resistance of Aedes albopictus (Skuse, 1894) from Cambodia.

(1) Background: In Cambodia, Aedes albopictus is an important vector of the dengue virus. Vector control using insecticides is a major strategy implemented in managing mosquito-borne diseases. Resistance, however, threatens to undermine the use of insecticides. In this study, we present the levels of insecticide resistance of Ae. albopictus in Cambodia and the mechanisms involved. (2) Methods: Two Ae. albopictus populations were collected from the capital, Phnom Penh city, and from rural Pailin province. Adults were tested with diagnostic doses of malathion (0.8%), deltamethrin (0.03%), permethrin (0.25%), and DDT (4%) using WHO tube assays. Synergist assays using piperonyl butoxide (PBO) were implemented before the pyrethroid assays to detect the potential involvement of metabolic resistance mechanisms. Adult female mosquitoes collected from Phnom Penh and Pailin were tested for voltage-gated sodium channel (VGSC) kdr (knockdown resistance) mutations commonly found in Aedes sp.-resistant populations throughout Asia (S989P, V1016G, and F1534C), as well as for other mutations (V410L, L982W, A1007G, I1011M, T1520I, and D1763Y). (3) Results: The two populations showed resistance against all the insecticides tested (<90% mortality). The use of PBO (an inhibitor of P450s) strongly restored the efficacy of deltamethrin and permethrin against the two resistant populations. Sequences of regions of the vgsc gene showed a lack of kdr mutations known to be associated with pyrethroid resistance. However, four novel non-synonymous mutations (L412P/S, C983S, Q1554STOP, and R1718L) and twenty-nine synonymous mutations were detected. It remains to be determined whether these mutations contribute to pyrethroid resistance. (4) Conclusions: Pyrethroid resistance is occurring in two Ae. albopictus populations originating from urban and rural areas of Cambodia. The resistance is likely due to metabolic resistance specifically involving P450s monooxygenases. The levels of resistance against different insecticide classes are a cause for concern in Cambodia. Alternative tools and insecticides for controlling dengue vectors should be used to minimize disease prevalence in the country.

Pyrethroid resistance and gene expression profile of a new resistant An. gambiae colony from Uganda reveals multiple resistance mechanisms and overexpression of Glutathione-S-Transferases linked to survival of PBO-pyrethroid combination.

The effectiveness of long-lasting insecticidal nets (LLINs) are being threatened by growing resistance to pyrethroids. To restore their efficacy, a synergist, piperonyl butoxide (PBO) which inhibits cytochrome P450s has been incorporated into pyrethroid treated nets. A trial of PBO-LLINs was conducted in Uganda from 2017 and we attempted to characterize mechanisms of resistance that could impact intervention efficacy. We established an Anopheles gambiae s.s colony in 2018 using female mosquitoes collected from Busia district in eastern Uganda. We first assessed the phenotypic resistance profile of this colony using WHO tube and net assays using a deltamethrin dose-response approach. The Busia colony was screened for known resistance markers and RT-qPCR targeting 15 genes previously associated with insecticide resistance was performed. The Busia colony had very high resistance to deltamethrin, permethrin and DDT. In addition, the colony had moderate resistance to alpha-cypermethrin and lambda-cyhalothrin but were fully susceptible to bendiocarb and fenitrothion. Exposure to PBO in combination with permethrin and deltamethrin resulted in higher mortality rates in both net and tube assays, with a higher mortality observed in net assays than tube assays. The kdr marker, Vgsc-995S was at very high frequency (91.7-98.9%) whilst the metabolic markers Coeae1d and Cyp4j5-L43F were at very low (1.3% - 11.5%) and moderate (39.5% - 44.7%) frequencies respectively. Our analysis showed that gene expression pattern in mosquitoes exposed to deltamethrin, permethrin or DDT only were similar in comparison to the susceptible strain and there was significant overexpression of cytochrome P450s, glutathione-s-transferases (GSTs) and carboxyl esterases (COEs). However, mosquitoes exposed to both PBO and pyrethroid strikingly and significantly only overexpressed closely related GSTs compared to unexposed mosquitoes while major cytochrome P450s were underexpressed. The high levels of pyrethroid resistance observed in Busia appears associated with a wide range of metabolic gene families.

Comparison of the solid-phase red cell adherence assay and tube method for detection and identification of red blood cell antibodies.

Identifying antibodies to red blood cell antigens is one of transfusion medicine's critical responsibilities. The International Society of Blood Transfusion recognizes 354 red blood cell antigens. Accurate identification of clinically significant alloantibodies is imperative for preventing hemolytic transfusion reactions and hemolytic disease of the fetus and newborn. We compared the performance of the tube (polyethylene glycol-indirect antiglobulin test [PEG-IAT]) and solid-phase red cell adherence assay techniques. We performed a retrospective study on all antibody screens performed between 2007 and 2021 at Stanford Transfusion Services. Initially, 631,535 antibody screens were performed using a solid-phase technique. Subsequent antibody identifications were performed using a combination of tube testing and solid-phase techniques. Antibody screening resulted in 28,316 (4.5%) positive samples. Antibody identification performed on both platforms identified 50 discordant samples. The anti-E antibody had the lowest sensitivity (98.99%) in the automated solid-phase technique, while anti-Jkb had the lowest sensitivity (98.78%) with the PEG-IAT method. To our knowledge, this is the first robust, 15-year study comparing methodologic sensitivity to detect clinically significant alloantibodies. The incidence of discordant results between PEG-IAT and the solid-phase technique was low. Among discordant samples, anti-Jka was commonly detected using the solid-phase method but not with the PEG-IAT. In contrast, anti-E was commonly detected by PEG-IAT but not by the solid-phase method.

Effectively α-Terpineol Suppresses Glioblastoma Aggressive Behavior and Downregulates KDELC2 Expression

BackgroundGlioblastoma (GBM) is notorious for the aggressive behaviors and easily results in chemo-resistance. Studies have shown that the use of herbal medicines as treatments for GBM as limited by the blood-brain barrier (BBB) and glioma stem cells. PurposeThe aim of this study was to investigate the relationship between GBM suppression and α-terpineol, the monoterpenoid alcohol derived from Eucalyptus glubulus and Pinus merkusii. Study DesignUsing serial in-vitro and in-vivo studies to confirm the mechanism of α-terpineol on down-regulating GBM development. MethodsThe 3-[4,5-dimethylthiazol-2-yl)]-2,5-diphenyltetrazolium bromide (MTT) assay was performed to evaluate IC50 of α-terpineol to inhibit GBM cell survival. In order to evaluate the impact of GBM aggressive behaviors by α-terpineol, the analysis of cell migration, invasion and colony formation were implemented. In addition, the ability of tumor spheres and WB of CD44 and OCT3/4 were evaluated under the impression of α-terpineol decreased GBM stemness. The regulation of neoangiogenesis by α-terpineol via the WB of angiogenic factors and human umbilical vein endothelial cells (HUVEC) tube assay. To survey the decided factors of α-terpineol downregulating GBM chemoresistance depended on the impact of O6-methylguanine-DNA methyltransferase (MGMT) expression and autophagy-related factors activation. Additionally, WB and quantitative real-time polymerase chain reaction (qRT/PCR) of KDEL (Lys-Asp-Glu-Leu) containing 2 (KDELC2), endoplasmic reticulum (ER) stress, phosphoinositide 3-kinase (PI3k), mammalian target of rapamycin (mTOR) and mitogen-activated protein kinase (MAPK) cascade signaling factors were examined to explore the mechanism of α-terpineol inhibiting GBM viability. Finally, the orthotopic GBM mouse model was applied to prove the efficacy and toxicity of α-terpineol on regulating GBM survival. Resultsα-terpineol significantly suppressed GBM growth, migration, invasion, angiogenesis and temozolomide (TMZ) resistance. Furthermore, α-terpineol specifically targeted KDELC2 to downregulate Notch and PI3k/mTOR/MAPK signaling pathway. Finally, we also demonstrated that α-terpineol could penetrate the BBB to inhibit GBM proliferation, which resulted in reduced cytotoxicity to vital organs. ConclusionCompared to published literatures, we firstly proved α-terpineol possessed the capability to inhibit GBM through various mechanisms and potentially decreased the occurrence of chemoresistance, making it a promising alternative therapeutic option for GBM in the future.

Pyrethroid resistance and gene expression profile of a new resistant An. gambiae colony from Uganda reveals multiple resistance mechanisms and overexpression of Glutathione-S-Transferases linked to survival of PBO-pyrethroid combination

Background: The effectiveness of long-lasting insecticidal nets (LLINs) are being threatened by growing resistance to pyrethroids. To restore their efficacy, a synergist, piperonyl butoxide (PBO) which inhibits cytochrome P450s has been incorporated into pyrethroid treated nets. A trial of PBO-LLINs was conducted in Uganda from 2017 and we attempted to characterize mechanisms of resistance that could impact intervention efficacy. Methods: We established an Anopheles gambiae s.s colony in 2018 using female mosquitoes collected from Busia district in eastern Uganda. We first assessed the phenotypic resistance profile of this colony using WHO tube and net assays using a deltamethrin dose-response approach. The Busia colony was screened for known resistance markers and RT-qPCR targeting 15 genes previously associated with insecticide resistance was performed. Results: The Busia colony had very high resistance to deltamethrin, permethrin and DDT. In addition, the colony had moderate resistance to alpha-cypermethrin and lambda-cyhalothrin but were fully susceptible to bendiocarb and fenitrothion. Exposure to PBO in combination with permethrin and deltamethrin resulted in higher mortality rates in both net and tube assays, with a higher mortality observed in net assays than tube assays. The kdr marker, Vgsc-995S was at very high frequency (91.7-98.9%) whilst the metabolic markers Coeae1d and Cyp4j5-L43F were at very low (1.3% - 11.5%) and moderate (39.5% - 44.7%) frequencies respectively. Our analysis showed that gene expression pattern in mosquitoes exposed to deltamethrin, permethrin or DDT only were similar in comparison to the susceptible strain and there was significant overexpression of cytochrome P450s, glutathione-s-transferases (GSTs) and carboxyl esterases (COEs). However, mosquitoes exposed to both PBO and pyrethroid strikingly and significantly only overexpressed closely related GSTs compared to unexposed mosquitoes while major cytochrome P450s were underexpressed. Conclusions: The high levels of pyrethroid resistance observed in Busia appears associated with a wide range of metabolic gene families.

Is Anopheles gambiae (sensu stricto), the principal malaria vector in Africa prone to resistance development against new insecticides? Outcomes from laboratory exposure of An. gambiae (s.s.) to sub-lethal concentrations of chlorfenapyr and clothianidin

Indiscriminate use of pesticides in the public health and agriculture sectors has contributed to the development of resistance in malaria vectors following exposure to sub-lethal concentrations. To preserve the efficacy of vector control tools and prevent resistance from spreading, early resistance detection is urgently needed to inform management strategies. The introduction of new insecticides for controlling malaria vectors such as clothianidin and chlorfenapyr requires research to identify early markers of resistance which could be used in routine surveillance. This study investigated phenotypic resistance of Anopheles gambiae (sensu stricto) Muleba-Kis strain using both WHO bottle and tube assays following chlorfenapyr, clothianidin, and alpha-cypermethrin selection against larvae and adults under laboratory conditions. High mortality rates were recorded for both chlorfenapyr-selected mosquitoes that were consistently maintained for 10 generations (24-h mortality of 92–100% and 72-h mortality of 98–100% for selected larvae; and 24-h mortality of 95–100% and 72-h mortality of 98–100% for selected adults). Selection with clothianidin at larval and adult stages showed a wide range of mortality (18–91%) compared to unselected progeny where mortality was approximately 99%. On the contrary, mosquitoes selected with alpha-cypermethrin from the adult selection maintained low mortality (28% at Generation 2 and 23% at Generation 4) against discrimination concentration compared to unselected progeny where average mortality was 51%. The observed resistance in the clothianidin-selected mosquitoes needs further investigation to determine the underlying resistance mechanism against this insecticide class. Additionally, further investigation is recommended to develop molecular markers for observed clothianidin phenotypic resistance.

Selective microbial production of lacto-N-fucopentaose I in Escherichia coli using engineered α-1,2-fucosyltransferases

Lacto-N-fucopentaose I (LNFP I) is the second most abundant fucosylated human milk oligosaccharide (HMO) in breast milk after 2′-fucosyllactose (2′-FL). Studies have reported that LNFP I exhibits antimicrobial activity against group B Streptococcus and antiviral effects against Enterovirus and Norovirus. Microbial production of HMOs by engineered Escherichia coli is an attractive, low-cost process, but few studies have investigated production of long-chain HMOs, including the pentasaccharide LNFP I. LNFP I is synthesized by α1,2-fucosyltransfer reaction to the N-acetylglucosamine moiety of the lacto-N-tetraose skeleton, which is catalyzed by α1,2-fucosyltransferase (α1,2-FucT). However, α1,2-FucTs competitively transfer fucose to lactose, resulting in formation of the byproduct 2′-FL. In this study, we constructed LNFP I-producing strains of E. coli with various α1,2-fucTs, and observed undesired 2′-FL accumulation during fed-batch fermentation, although, in test tube assays, some strains produced LNFP I without 2′-FL. We hypothesized that promiscuous substrate selectivity of α1,2-FucT was responsible for 2′-FL production. Therefore, to decrease the formation of byproduct 2′-FL, we designed 15 variants of FsFucT from Francisella sp. FSC1006 by rational and semi-rational design approaches. Five of these variants of FsFucT surpassed a twofold reduction in 2′-FL production compared with wild-type FsFucT while maintaining comparable levels of LNFP I production. These designs encompassed substitutions in either a loop region of the enzyme (residues 154–171), or in specific residues (Q7, H162, and L164) that influence substrate binding either directly or indirectly. In particular, the E. coli strain that expressed FsFucT_S3 variants, with a substituted loop region (residues 154–171) forming an α-helix structure, achieved an accumulation of 19.6 g/L of LNFP I and 0.04 g/L of 2′-FL, while the E. coli strain expressing the wild-type FsFucT accumulated 12.2 g/L of LNFP I and 5.85 g/L of 2′-FL during Fed-bach fermentation. Therefore, we have successfully demonstrated the selective and efficient production of the pentasaccharide LNFP I without the byproduct 2′-FL by combining protein engineering of α1,2-FucT designed through in silico structural modeling of an α1,2-FucT and docking simulation with various ligands, with metabolic engineering of the host cell.

Anopheles gambiae s.s. resistance to pyrethroids and DDT in semi-urban and rural areas of the Moyen-Ogooué Province, Gabon

BackgroundPyrethroids are the main insecticides used in vector control for malaria. However, their extensive use in the impregnation of long-lasting insecticidal nets (LLINs) and indoor residual spraying has led to the development of resistance, threatening its success as a tool for malaria control. Baseline data prior to large scale distribution of LLINs are important for the implementation of efficient strategies. However, no data on the susceptibility of malaria vectors is available in the Moyen-Ogooué Province in Gabon. The aim of this study was to assess the susceptibility to pyrethroids and organochlorides of malaria vectors from a semi-urban and rural areas of the province and to determine the frequency of insecticide resistance genes.MethodsLarvae were collected from breeding sites in Lambaréné and Zilé and reared to adults. Three to five-day old female Anopheles gambiae sensu lato mosquitoes were used in cone tube assays following the WHO susceptibility tests protocol for adult mosquitoes. A subsample was molecularly identified using the SINE200 protocol and the frequency of Vgsc-1014 F and − 1014 S mutations were determined.ResultsAnopheles gambiae sensu stricto (s.s.) was the sole species present in both Lambaréné and Zilé. Mosquito populations from the two areas were resistant to pyrethroids and organochlorides. Resistance was more pronounced for permethrin and DDT with mortality lower than 7% for both insecticides in the two study areas. Mosquitoes were statistically more resistant (P < 0.0001) to deltamethrin in Lambaréné (51%) compared to Zilé (76%). All the mosquitoes tested were heterozygous or homozygous for the knockdown resistance (Kdr) mutations Vgsc-L1014F and Vgsc-L1014S with a higher proportion of Vgsc-L1014F homozygous in Lambaréné (76.7%) compared to Zilé (57.1%).ConclusionThis study provides evidence of widespread resistance to pyrethroids in An. gambiae s.s., the main malaria vector in the Moyen-Ogooué Province. Further investigation of the mechanisms underlining the resistance of An. gambiae s.s. to pyrethroids is needed to implement appropriate insecticide resistance management strategies.

Carvacrol-Induced Vacuole Dysfunction and Morphological Consequences in Nakaseomyces glabratus and Candida albicans.

With the prevalence of systemic fungal infections caused by Candida albicans and non-albicans species and their resistance to classical antifungals, there is an urgent need to explore alternatives. Herein, we evaluate the impact of the monoterpene carvacrol, a major component of oregano and thyme oils, on clinical and laboratory strains of C. albicans and Nakaseomyces glabratus. Carvacrol induces a wide range of antifungal effects, including the inhibition of growth and hyphal and biofilm formation. Using biochemical and microscopic approaches, we elucidate carvacrol-induced hyphal inhibition. The significantly reduced survival rates following exposure to carvacrol were accompanied by dose-dependent vacuolar acidification, disrupted membrane integrity, and aberrant morphology. Germ tube assays, used to elucidate the relationship between vacuolar dysfunction and hyphal inhibition, showed that carvacrol significantly reduced hyphal formation, which was accompanied by a defective C. albicans morphology. Thus, we show a link between vacuolar acidification/disrupted vacuole membrane integrity and compromised candidal morphology/morphogenesis, demonstrating that carvacrol exerts its anti-hyphal activity by altering vacuole integrity.

The performance of tongue swabs for detection of pulmonary tuberculosis.

Oral and/or tongue swabs have demonstrated ability to detect Mycobacterium tuberculosis (Mtb) in adults with pulmonary tuberculosis (TB). Swabs provide useful alternative specimens for diagnosis of TB using molecular assays however, the diagnostic pickup by culture requires further improvement and development. Several studies identified the presence of differentially culturable tubercle bacilli (DCTB) populations in a variety of clinical specimens. These organisms do not grow in routine laboratory media and require growth factors in the form of culture filtrate (CF) from logarithmic phase cultures of Mtb H37Rv. Herein, we compared the diagnostic performance of sputum and tongue swabs using Mycobacterial Growth Indicator Tube (MGIT) assays, Auramine smear, GeneXpert and DCTB assays supplemented with or without CF. From 89 eligible participants, 83 (93%), 66 (74%) and 79 (89%) were sputum positive by MGIT, smear and GeneXpert, respectively. The corresponding tongue swabs displayed a lower sensitivity with 39 (44%), 2 (2.0%) and 18 (20%) participants respectively for the same tests. We aimed to improve the diagnostic yield by utilizing DCTB assays. Sputum samples were associated with a higher positivity rate for CF-augmented DCTB at 82/89 (92%) relative to tongue swabs at 36/89 (40%). Similarly, sputum samples had a higher positivity rate for DCTB populations that were CF-independent at 64/89 (72%) relative to tongue swabs at 26/89 (29%). DCTB positivity increased significantly, relative to MGIT culture, for tongue swabs taken from HIV-positive participants. We next tested whether the use of an alternative smear stain, DMN-Trehalose, would improve diagnostic yield but noted no substantial increase. Collectively, our data show that while tongue swabs yield lower bacterial numbers for diagnostic testing, the use of growth supplementation may improve detection of TB particularly in HIV-positive people but this requires further interrogation in larger studies.

Mechanistic study on the anti-proinflammatory activity of Kunitz type inhibitor from Caesalpinia decapetala seeds.

The study reports the biochemical characterization and mechanism of action of a novel 19.6 kDa protease inhibitor (PIs) isolated from the seeds of Caesalpinia decapetala belonging to the Fabaceae family. A systematic study was performed to ascertain the purity, specificity, biochemical and structural characterization, and its potential in curbing inflammation in vitro conditions. A two-step chromatography technique was used to purify the PIs. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization time of flightwere employed to detect the molecular mass of the protein. N-terminal sequence analysis of the inhibitor showed sequence similarity with the Kunitz family PIs. The in vitro test tube assay was performed for determining the anti-inflammatory activity and the inhibitor is antiproliferative against macrophage (RAW264.7) and lung cancer cell lines (A549). An effective decrease in the release of inflammatory mediators (NO, IL-6, TNF-α) and on the activity of elastase was observed in macrophage cell lines (RAW264.7) which were treated with PIs. The purified inhibitor shows promising results against inflammation.

Efficacy of a ‘lethal house lure’ against Culex quinquefasciatus from Bouaké city, Côte d’Ivoire

BackgroundEave tube technology is a novel method of insecticide application that uses an electrostatic coating system to boost insecticide efficacy against resistant mosquitoes. A series of previous experiments showed encouraging insecticidal effects against malaria vectors. This study was undertaken to assess the effects of the eave tube approach on other Culicidae, in particular Culex quinquefasciatus, under laboratory and semi-field conditions.MethodsLarvae of Cx. quinquefasciatus from Bouaké were collected and reared to adult stage, and World Health Organization (WHO) cylinder tests were performed to determine their resistance status. WHO standard 3-min cone bioassays were conducted using PermaNet 2.0 netting versus eave tube-treated inserts. To assess the transient exposure effect on Cx. quinquefasciatus, eave tube assay utilizing smelly socks as attractant was performed with exposure time of 30 s, 1 min, and 2 min on 10% beta-cyfluthrin-treated inserts. Residual activity of these treated inserts was then monitored over 9 months. Field tests involving release–recapture of Cx. quinquefasciatus within enclosures around experimental huts fitted with windows and untreated or insecticide-treated eave tubes were conducted to determine house entry preference and the impact of tubes on the survival of this species.ResultsBouaké Cx. quinquefasciatus displayed high resistance to three out of four classes of insecticides currently used in public health. After 3 min of exposure in cone tests, 10% beta-cyfluthrin-treated inserts induced 100% mortality in Cx. quinquefasciatus, whereas the long-lasting insecticidal net (LLIN) only killed 4.5%. With reduced exposure time on the eave tube insert, mortality was still 100% after 2 min, 88% after 1 min, and 44% after 30 s. Mortality following 1 h exposure on 10% beta-cyfluthrin-treated insert was > 80% continuously up to 7 months post-treatment. Data suggest that Cx. quinquefasciatus have a stronger preference for entering a house through the eaves than through windows. Beta-cyfluthrin-treated inserts were able to kill 51% of resistant Cx. quinquefasciatus released within the enclosure.ConclusionsEave tubes are a novel method for delivery of insecticide to the house. They attract nuisance host-seeking Cx. quinquefasciatus mosquitoes and are as effective in controlling them as they are against pyrethroid-resistant Anopheles gambiae, despite the high level of resistance Cx. quinquefasciatus have developed.Graphical

Insecticide Resistance and Metabolic Mechanisms in Aedes aegypti from Two Agrosystems (Vegetable and Cotton Crops) in Côte d'Ivoire.

Background: The emergence of insecticide resistance in Aedes vectors mosquitoes poses a real challenge for arboviral-borne disease control. In Côte d'Ivoire, data are available on phenotypic resistance and the presence of kdr mutations in Aedes aegypti. Therefore, information on metabolic resistance in Aedes populations is very scarce. Here, we assessed the insecticide resistance status of Ae. aegypti in periurban and rural areas of Côte d'Ivoire, and we investigated the role of detoxification enzymes as possible resistance mechanisms. Materials and Methods: Aedes mosquito eggs were collected between June 2019 to April 2021 in two agricultural sites. Adults of Ae. aegypti were tested using World Health Organization tube assays, with seven insecticides belonging to pyrethroids, organochlorines, carbamates, and organophosphates classes. We determined the knockdown times (KdT50, KdT95) and resistance ratios of pyrethroids in natural populations. The synergist piperonyl butoxide (PBO) was used to investigate the role of enzymes in resistance. Biochemical assays were performed to detect potential increased activities in mixed-function oxidase levels, nonspecific esterases (NSEs), and glutathione S-transferases. Results: The results showed that Ae. aegypti populations were resistant to five insecticides with mortality of 46% and 89% for 0.75% permethrin, 68% and 92% for 0.05% deltamethrin, 57% and 89% for lambda-cyhalothrin, 41% and 47% for dichlorodiphenyltrichloroethane (DDT), 82% and 91% for chlorpyrifos-methyl in Songon-Agban and Kaforo, respectively. Susceptibility to carbamates was observed in our study sites. After exposure to PBO, the susceptibility of Ae. aegypti to pyrethroids and DDT was partially restored in Songon-Agban. Whereas in Kaforo, none increase of the mortality rates of Ae. aegypti for these four insecticides was observed after exposure to PBO. Increased activity of NSE (α-esterases) was found in Songon-Agban compared with the reference susceptible strain. Conclusion: These findings provide valuable information to support decisions for vector control strategies in Cote d'Ivoire. Also, we highlight the need for the monitoring of insecticide resistance management in Aedes vectors.