Environmental conditions affect the nutritive value and alkaloid profiles of Lupinus forage: Opportunities and threats for sustainable ruminant systems.

The identification of crops that simultaneously contribute to the global protein supply and mitigate the effects of climate change is an urgent matter. Lupins are well adapted to nutrient-poor or contaminated soils, tolerate various abiotic stresses, and present relevant traits for acting as ecosystem engineers. Lupins are best studied for their seeds, but their full foraging potential needs further evaluation. This study evaluated the effects of location and sowing date on forage production, proximate composition, and the detailed mineral and alkaloid profiles of three species of Lupinus (L. albus cv. Estoril, L. angustifolius cv. Tango, and L. luteus cv. Cardiga). Sowing date and location and their interaction with the plant species significantly affected the vast majority of measured parameters, emphasizing the effects of climate and soil conditions on these crops. The relatively high crude protein and in vitro digestibility support the potential of the lupin species studied as sustainable forage protein sources in diets for ruminant animals. The content of individual essential macro and trace elements was below the maximum tolerable levels for cattle and sheep. Lupanine, smipine, and sparteine were the most abundant quinolizidine alkaloids in L. albus cv. Estoril, lupanine, and sparteine in L. angustifolius cv. Tango, and lupinine, gramine, ammodendrine, and sparteine in L. luteus cv. Cardiga. Based on the maximum tolerable levels of total quinolizidine alkaloid intake, the dietary inclusion of forages of L. albus cv. Estoril and L. angustifolius cv. Tango does not pose a risk to the animals, but the high alkaloid content of L. luteus cv. Cardiga may compromise its utilization at high levels in the diet. Overall, the results reveal a high potential for lupins as protein forage sources well adapted to temperate regions and soils with lower fertility, with a relevant impact on livestock sustainability in a climate change era.

Hemolysis events in the Phase 3 PEGASUS study of pegcetacoplan in patients with paroxysmal nocturnal hemoglobinuria.

Patients with paroxysmal nocturnal hemoglobinuria (PNH) experience complement-mediated intravascular hemolysis leading to anemia, fatigue, and potentially life-threatening thrombotic complications. Pegcetacoplan, a C3 inhibitor, demonstrated sustained improvements in hematological and clinical parameters in the Phase 3 PEGASUS trial in patients with PNH who remained anemic despite C5 inhibitor therapy. The current post-hoc analysis describes 26 hemolysis adverse events (AEs) experienced in 19 patients during pegcetacoplan therapy in PEGASUS and baseline patient characteristics potentially associated with increased hemolysis risk. Lactate dehydrogenase (LDH) ≥2× the upper limit of normal (ULN) was observed in 19 events, including 2 with LDH ≥10× ULN. All patients experienced decreased hemoglobin during hemolysis (mean decrease: 3.0 g/dL). In 16 events (62%) a potential complement-amplifying condition underlying the event could be identified. Hemolysis AEs led to study discontinuation in 5 patients. However, 17 of 26 (65%) hemolysis AEs were manageable without pegcetacoplan discontinuation. A greater proportion of patients with hemolysis AEs (n=19) had key characteristics of higher disease activity at baseline compared to patients without hemolysis AEs (n=61), namely higher-than-label eculizumab dose (53% vs 23%), detectable CH50 (74% vs 54%) and ≥4 transfusions in the previous 12 months (68% vs 51%). These characteristics may be useful predictors of potential future hemolysis events. ClinicalTrials.gov identifier: NCT03500549.

Mechanism of DNA origami folding elucidated by mesoscopic simulations.

Many experimental and computational efforts have sought to understand DNA origami folding, but the time and length scales of this process pose significant challenges. Here, we present a mesoscopic model that uses a switchable force field to capture the behavior of single- and double-stranded DNA motifs and transitions between them, allowing us to simulate the folding of DNA origami up to several kilobases in size. Brownian dynamics simulations of small structures reveal a hierarchical folding process involving zipping into a partially folded precursor followed by crystallization into the final structure. We elucidate the effects of various design choices on folding order and kinetics. Larger structures are found to exhibit heterogeneous staple incorporation kinetics and frequent trapping in metastable states, as opposed to more accessible structures which exhibit first-order kinetics and virtually defect-free folding. This model opens an avenue to better understand and design DNA nanostructures for improved yield and folding performance.

The in vitro rumen exsheathment test for studying the effect of plant extracts on the exsheathment of Haemonchus contortus infective larvae.

This study applied the in vitro rumen exsheathment test (IVRET) to evaluate the exsheathment kinetics of Haemonchus contortus infective larvae (L3) incubated in ruminal liquor (RL) containing acetone:water extracts of Acacia pennatula (AP), Gymnopodium floribundum (GF), Havardia albicans (HA) or Lysiloma latisiliquum (LL). The role of polyphenols in the biological activity of the evaluated extracts was also determined. Larvae were incubated in RL either alone or added with a different plant extract (AP, GF, HA, or LL) at 1200 µg/mL. Polyethylene glycol (PEG) was added to block polyphenols in each treatment (RL+PEG, AP+PEG, GF+PEG, HA+PEG, and LL+PEG). After incubation times of 0, 1, 3, 6, 9, and 24 h, the exsheathment process was stopped to count the number of ensheathed and exsheathed L3. A Log-Logistic model was used to determine the L3 exsheathment kinetics in the different RL treatments. The inflection point of the respective kinetic curves, which indicates the time to reach 50 % exsheathed L3 (T50), was the only parameter that differed when comparing the exsheathment models (99 % probability of difference). The T50 values obtained for GF, HA, and LL treatments (T50 = 7.11 - 7.58 h) were higher in comparison to the T50 of RL (5.72 h) (≥ 70 % probability of difference). The L3 incubated in RL added with GF, HA, and LL extracts delayed their exsheathment at 3 and 6 h of incubation (28.71 - 48.06 % exsheathment reduction) compared to the RL treatment. The T50 value for AP, AP+PEG, GF+PEG, HA+PEG, and LL+PEG were similar to RL and RL+PEG (T50 = 5.34 - 6.97 h). In conclusion, the IVRET can be used to identify plants with the potential to delay the exsheathment of H. contortus L3 in the ruminal liquor. The acetone:water extracts of G. floribundum, H. albicans, and L. latisiliquum delayed the T50 of H. contortus exsheathment, which was evident at 3 and 6 h of incubation in ruminal liquor. The observed exsheathment delay was attributed to the polyphenol content of the extracts.

Agronomic, Nutritional Traits, and Alkaloids of Lupinus albus, Lupinus angustifolius and Lupinus luteus Genotypes: Effect of Sowing Dates and Locations.

Lupins (Lupinus spp.) are legumes with high relevance for the sustainability of agricultural systems as they improve the soil quality, namely, through the fixation of atmospheric nitrogen, and have good adaptability to different climates and soil conditions. Besides, they possess high nutritive value, especially due to the high protein content of the seeds. Nevertheless, the plants' productivity and metabolism can be influenced by the genotype, the edaphoclimatic conditions, and the sowing practices. In this work, the effect of edaphoclimatic conditions and sowing dates on the productivity, nutritional factors, and alkaloids of the seeds of L. albus cv. Estoril, L. angustifolius cv. Tango, and L. luteus cv. Cardiga was evaluated. High variability in the seeds and protein productions, nutritional traits, and alkaloid content related to the species was observed, along with a significant effect of the location. Lupinus albus cv. Estoril showed a good compromise between productivity and low alkaloid content, being an interesting genotype for food and feed use in the conditions of this trial.

ANGIOTENSIN-(1-7) TREATMENT EARLY IN LIFE PREVENTS CARDIAC HYPERTROPHY IN ADULT HYPERTENSIVE RATS.

Angiotensin (Ang)-(1-7) is a cardioprotective peptide of the renin-angiotensin system. Pre-puberty has been considered as a later susceptible window of development and stressful factors in this life phase can induce chronic diseases in adulthood. We aimed to investigate whether the treatment with Ang-(1-7) during the pre-puberty could attenuate the development of hypertension and cardiac injury in adult spontaneously hypertensive rats (SHR). SHR were treated with Ang-(1-7) (24 µg/Kg/h) from 4 to 7 weeks of age. Systolic blood pressure (SBP) was measured by tail-cuff plethysmography up to 17th of age. Thereafter, echocardiography was performed and the rats were euthanized for aorta reactivity assay and tissues and blood collections. Ang- (1-7) did not change the SBP and aortic reactivity but reduced the septal and posterior wall thickness, cardiomyocyte hypertrophy and fibrosis in SHR. Additionally, Ang-(1-7) reduced the gene expression of ANP and BNP, increased the metalloproteinase 9 expression, and reduced the ERK 1/2 phosphorylation. Ang-(1-7) also prevented the reduction of Mas receptor but did not change the protein expression of ACE2, ACE, AT1, and AT2. The treatment with Ang-(1-7) decreased the MDA levels and increased SOD-1 and catalase activity and protein expression of catalase. Our findings demonstrate that the treatment of SHR with Ang-(1-7) for three weeks early in life promotes beneficial effects in the heart later in life, even without altering blood pressure, through mechanisms involving the reduction of oxidative stress and ERK1/2 phosphorylation. Additionally, this study supports the pre-puberty as an important programming window.

Construction of Reconfigurable and Polymorphic DNA Origami Assemblies with Coiled-Coil Patches and Patterns.

DNA origami nanodevices achieve programmable structure and tunable mechanical and dynamic properties by leveraging the sequence-specific interactions of nucleic acids. Previous advances have also established DNA origami as a useful building block to make well-defined micron-scale structures through hierarchical self-assembly, but these efforts have largely leveraged the structural features of DNA origami. The tunable dynamic and mechanical properties also provide an opportunity to make assemblies with adaptive structures and properties. Here the integration of DNA origami hinge nanodevices and coiled-coil peptides are reported into hybrid reconfigurable assemblies. With the same dynamic device and peptide interaction, it is made multiple higher-order assemblies (i.e., polymorphic assembly) by organizing clusters of peptides into patches or arranging single peptides into patterns on the surfaces of DNA origami to control the relative orientation of devices. The coiled-coil interactions are used to construct circular and linear assemblies whose structure and mechanical properties can be modulated with DNA-based reconfiguration. Reconfiguration of linear assemblies leads to micron scale motions and ≈2.5-10-fold increase in bending stiffness. The results provide a foundation for stimulus-responsive hybrid assemblies that can adapt their structure and properties in response to nucleic acid, peptide, protein, or other triggers.

Oral Iptacopan Monotherapy in Paroxysmal Nocturnal Hemoglobinuria.

BACKGROUND: Persistent hemolytic anemia and a lack of oral treatments are challenges for patients with paroxysmal nocturnal hemoglobinuria who have received anti-C5 therapy or have not received complement inhibitors. Iptacopan, a first-in-class oral factor B inhibitor, has been shown to improve hemoglobin levels in these patients. METHODS: In two phase 3 trials, we assessed iptacopan monotherapy over a 24-week period in patients with hemoglobin levels of less than 10 g per deciliter. In the first, anti-C5-treated patients were randomly assigned to switch to iptacopan or to continue anti-C5 therapy. In the second, single-group trial, patients who had not received complement inhibitors and who had lactate dehydrogenase (LDH) levels more than 1.5 times the upper limit of the normal range received iptacopan. The two primary end points in the first trial were an increase in the hemoglobin level of at least 2 g per deciliter from baseline and a hemoglobin level of at least 12 g per deciliter, each without red-cell transfusion; the primary end point for the second trial was an increase in hemoglobin level of at least 2 g per deciliter from baseline without red-cell transfusion. RESULTS: In the first trial, 51 of the 60 patients who received iptacopan had an increase in the hemoglobin level of at least 2 g per deciliter from baseline, and 42 had a hemoglobin level of at least 12 g per deciliter, each without transfusion; none of the 35 anti-C5-treated patients attained the end-point levels. In the second trial, 31 of 33 patients had an increase in the hemoglobin level of at least 2 g per deciliter from baseline without red-cell transfusion. In the first trial, 59 of the 62 patients who received iptacopan and 14 of the 35 anti-C5-treated patients did not require or receive transfusion; in the second trial, no patients required or received transfusion. Treatment with iptacopan increased hemoglobin levels, reduced fatigue, reduced reticulocyte and bilirubin levels, and resulted in mean LDH levels that were less than 1.5 times the upper limit of the normal range. Headache was the most frequent adverse event with iptacopan. CONCLUSIONS: Iptacopan treatment improved hematologic and clinical outcomes in anti-C5-treated patients with persistent anemia - in whom iptacopan showed superiority to anti-C5 therapy - and in patients who had not received complement inhibitors. (Funded by Novartis; APPLY-PNH ClinicalTrials.gov number, NCT04558918; APPOINT-PNH ClinicalTrials.gov number, NCT04820530.).

Management of acute breakthrough hemolysis with intensive pegcetacoplan dosing in patients with PNH.

ABSTRACT: Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by complement-mediated intravascular hemolysis leading to anemia, fatigue, and potentially life-threatening thrombotic complications. Breakthrough hemolysis (BTH) was first described in patients with PNH treated with terminal complement C5 inhibitors when intravascular hemolysis reoccurred despite treatment. Pegcetacoplan, the first proximal complement C3 inhibitor, offers broad hemolysis control in patients with PNH. While experience of managing BTH on C5 inhibitors is documented, very limited guidance exists for proximal complement inhibitors. This interim analysis assessed the effect of intensive treatment with pegcetacoplan following an acute BTH event in a subset of patients enrolled in the ongoing open-label extension study of pegcetacoplan in PNH. Thirteen patients with acute BTH included in the analysis received either a single IV dose of 1080 mg (n = 4) or 1080 mg subcutaneous (SC) dosing on 3 consecutive days (n = 9). A potential, clinically-relevant complement-amplifying condition, such as infection or vaccination, was reported in approximately half of the patients experiencing an acute BTH. Lactate dehydrogenase (LDH) levels decreased between day 1 and day 2 in 8 of 12 evaluable patients and in all 13 patients at day 7 to 12. Nine of 13 patients (69%) achieved LDH <2× the upper limit of normal by day 14 to 19. All adverse events associated with the acute BTH event were considered resolved by the investigators. Overall, intensive treatment with pegcetacoplan was safe and well tolerated. These novel data support effective management of acute BTH events in patients on pegcetacoplan with intensive IV or SC pegcetacoplan dosing. This trial was registered at www.clinicaltrials.gov as #NCT03531255.

Adapting the in vitro rumen incubation method to evaluate the effect of a plant extract on the exsheathment inhibition of Haemonchus contortus infective larvae.

This study adapted the in vitro rumen incubation (IVRI) method to evaluate the biological activity of a Gymnopodium floribundum leaves extract against the exsheathment of Haemonchus contortus infective larvae (L3), and to determine the role of plant polyphenols on the biological activity. The incubation protocol followed the IVRI method, adding polyethylene glycol (PEG) as a polyphenol-blocking agent. The L3 were incubated in ruminal liquor (RL), ruminal liquor with PEG (RL+PEG), ruminal liquor with G. floribundum extract (RLE), and ruminal liquor with G. floribundum extract and PEG (RLE+PEG). Incubation condition controls included phosphate buffered saline (PBS), PBS with PEG (PBS+PEG), incubation medium (without ruminal liquor) (IM), and incubation medium with PEG (IM+PEG). The L3 were recovered after incubation times of 0, 1, 3, 6, 9, and 24 h (39 °C). The respective L3 exsheathment kinetics were estimated for the different treatments (RL, RL+PEG, RLE, and RLE+PEG) using Log-Logistic models. The parameters of the different models were compared to determine the impact of the extract, with or without PEG, on the L3 exsheathment kinetics. The exsheathment in PBS and PBS+PEG remained < 2.71% at each incubation time. The exsheathment in IM and IM+PEG reached 13.58% and 17.18% at 24 h, respectively. The exsheathment percentages for RLE were lower than those for RL at 3, 6 and 9 h of incubation. The inflection point, indicating the time required to reach 50% of the maximal exsheathment (T50), was the only parameter that differed between the ruminal liquor models. The T50 in RLE (7.106 h) was higher than the values obtained for RL (5.385 h) and RL+PEG (4.923 h) (99.99% probability of being different). Such delay resulted in a reduction of exsheathment in RLE of 62% at 3 h, 38% at 6 h, and 12% at 9 h, relative to RL values. When PEG was added with the extract (RLE+PEG), the T50 (5.045 h) was similar to that of RL and RL+PEG. The IVRI method was adapted as an in vitro rumen exsheathment test (IVRET). The IVRET showed that H. contortus L3 exposed to G. floribundum extract delayed their exsheathment kinetics at different time points. The exsheathment delay was attributed to the polyphenol content of the extract.

Relevance of Individual Data When Assessing the Gastrointestinal Nematode Infection Level, Nutritional and Productive Variables in a Tropical Farm Context: The Median Isn't the Message.

We evaluated the relationship between individual and herd GIN infection level, nutrition, production performance and anemia parameters in a tropical farm context. Fifty-four female goats were monitored to assess their body condition score (BCS, nutritional status indicator), live weight (LW) and LW gain (LWG, both used as production level indicators), FAMACHA© and hematocrit (HT, both used as anemia indicators). Goats browsed for 4 h in a tropical forest and received balanced feed and chopped grass. The eggs per gram of feces (EPG) indicated the GIN burden, with fecal samples obtained at 7:00 (AM) and 15:00 h (PM.) from each goat at six sampling points during the study. The variables and their relationship with GIN burdens were analyzed using Kruskall-Wallis, ANOVA and Friedman tests and Spearman correlations. The fecal samples obtained in the AM and PM can be equally representative of parasitic burdens (similar and highly correlated). However, the EPG of individual goats from periods of 30 days apart can be considered independent. The BCS and LWG varied between sampling times (p < 0.05), whereas EPG, LW and HT did not (p > 0.05). The GIN burden was negatively correlated with HT and BCS (-0.21, p = 0.01 for each one). The individual pattern of infection demonstrates the true impact of GINs on their hosts. Additionally, feeding and nutritional status may present important variations influencing the performance of the goats more than the impact of GINs under the farm conditions of the present study. However, GIN infection contributed to the variation in goat health and productivity in this tropical farm.

Cooperative control of a DNA origami force sensor.

Biomolecular systems are dependent on a complex interplay of forces. Modern force spectroscopy techniques provide means of interrogating these forces, but they are not optimized for studies in constrained environments as they require attachment to micron-scale probes such as beads or cantilevers. Nanomechanical devices are a promising alternative, but this requires versatile designs that can be tuned to respond to a wide range of forces. We investigate the properties of a nanoscale force sensitive DNA origami device which is highly customizable in geometry, functionalization, and mechanical properties. The device, referred to as the NanoDyn, has a binary (open or closed) response to an applied force by undergoing a reversible structural transition. The transition force is tuned with minor alterations of 1 to 3 DNA oligonucleotides and spans tens of picoNewtons (pN). The DNA oligonucleotide design parameters also strongly influence the efficiency of resetting the initial state, with higher stability devices (≳10 pN) resetting more reliably during repeated force-loading cycles. Finally, we show the opening force is tunable in real time by adding a single DNA oligonucleotide. These results establish the potential of the NanoDyn as a versatile force sensor and provide fundamental insights into how design parameters modulate mechanical and dynamic properties.

Knowledge and attitudes regarding contraceptive methods and sex education in students and parents of eight Colombian schools 2020-2021: a mixed methods study.

Background: In Colombia, the average age of first sexual intercourse is between 14 and 15, and it has one of the highest fertility rates among adolescents in Latin America, which reflects poor access to health services and a lack of knowledge about contraception. Some laws support comprehensive sex education as a learning process that families and schools should provide. The objective of this work was to assess the frequency of adequate knowledge and attitudes of acceptance towards contraception in students, their associated factors, sexual behaviours in sexually active students, and experiences with sex education. Methods: Mixed methods study consisting of an analytical cross-sectional study and a qualitative hermeneutic phenomenological study. The former included students who completed a questionnaire with sociodemographic variables, surveys on knowledge and attitudes toward contraception, and questions about sexual behaviour. Focus groups were used in the qualitative study to evaluate students' and parents' sex education experiences. Findings: 827 students were surveyed, of whom 52.3% had adequate knowledge about contraceptives and 80.1% had attitudes of acceptance. Talking with parents about sex education, receiving sex education, and being in the eleventh grade increased the likelihood of having adequate knowledge. Each year of age, being male, being a victim of forced displacement, and studying in a public school were associated with lower possibilities of attitudes of acceptance. Using condoms, compared to other contraceptives, was associated with less adequate knowledge and attitudes of acceptance (p < 0.05). Experiences with sex education, contraception, and sexual and reproductive rights were three emerging categories in the focus groups. Interpretation: In a group of Colombian high school students, half of them had adequate knowledge, eight out of 10 had attitudes of acceptance about contraception, and both were associated with having received sex education. Nevertheless, different types of barriers to sexual education were identified at home and in schools. The results will allow the generation of educational policies that modify the educational model as well as new strategies by health professionals to raise awareness about responsible sexuality. Funding: This project was financed by internal research of the Fundación Universitaria de Ciencias de la Salud-FUCS (DI-I-0392-20).

Risk of Early Postoperative Cardiovascular and Cerebrovascular Complication in Patients with Preoperative COVID-19 Undergoing Cancer Surgery.

BACKGROUND: As the COVID-19 pandemic shifts to an endemic phase, an increasing proportion of cancer patients with a preoperative history of COVID-19 will require surgery. This study aimed to assess the influence of preoperative COVID-19 on postoperative risk for major adverse cardiovascular and cerebrovascular events (MACE) among those undergoing surgical cancer resection. Secondary objectives included determining optimal time-to-surgery guidelines based on COVID-19 severity and discerning the influence of vaccination status on MACE risk. STUDY DESIGN: National COVID Cohort Collaborative (N3C) Data Enclave, a large multi-institutional dataset, was used to identify patients that underwent surgical cancer resection between January 2020 and February 2023. Multivariate regression analysis adjusting for demographics, comorbidities, and risk of surgery was performed to evaluate risk for 30-day postoperative MACE. RESULTS: Of 204,371 included patients, 21,313 (10.4%) patients had a history of preoperative COVID-19. History of COVID-19 was associated with an increased risk for postoperative composite MACE as well as 30-day mortality. Among patients with mild disease who did not require hospitalization, MACE risk was elevated for up to 4-weeks following infection. Postoperative MACE risk remained elevated 8+ weeks following infection in those with moderate disease. Vaccination did not reduce risk for postoperative MACE. CONCLUSION: Together, these data highlight that assessment of the severity of preoperative COVID-19 infection should be a routine component of both preoperative patient screening as well as surgical risk stratification. In addition, strategies beyond vaccination that increase patients' cardiovascular fitness and prevent COVID-19 infection are needed.

Localized Plasmonic Heating for Single-Molecule DNA Rupture Measurements in Optical Tweezers.

To date, studies on the thermodynamic and kinetic processes that underlie biological function and nanomachine actuation in biological- and biology-inspired molecular constructs have primarily focused on photothermal heating of ensemble systems, highlighting the need for probes that are localized within the molecular construct and capable of resolving single-molecule response. Here we present an experimental demonstration of wavelength-selective, localized heating at the single-molecule level using the surface plasmon resonance of a 15 nm gold nanoparticle (AuNP). Our approach is compatible with force-spectroscopy measurements and can be applied to studies of the single-molecule thermodynamic properties of DNA origami nanomachines as well as biomolecular complexes. We further demonstrate wavelength selectivity and establish the temperature dependence of the reaction coordinate for base-pair disruption in the shear-rupture geometry, demonstrating the utility and flexibility of this approach for both fundamental studies of local (nanometer-scale) temperature gradients and rapid and multiplexed nanomachine actuation.

Piggybacking functionalized DNA nanostructures into live cell nuclei.

DNA origami (DO) are promising tools for in vitro or in vivo applications including drug delivery; biosensing, detecting biomolecules; and probing chromatin sub-structures. Targeting these nanodevices to mammalian cell nuclei could provide impactful approaches for probing visualizing and controlling important biological processes in live cells. Here we present an approach to deliver DO strucures into live cell nuclei. We show that labelled DOs do not undergo detectable structural degradation in cell culture media or human cell extracts for 24 hr. To deliver DO platforms into the nuclei of human U2OS cells, we conjugated 30 nm long DO nanorods with an antibody raised against the largest subunit of RNA Polymerase II (Pol II), a key enzyme involved in gene transcription. We find that DOs remain structurally intact in cells for 24hr, including within the nucleus. Using fluorescence microscopy we demonstrate that the electroporated anti-Pol II antibody conjugated DOs are efficiently piggybacked into nuclei and exihibit sub-diffusive motion inside the nucleus. Our results reveal that functionalizing DOs with an antibody raised against a nuclear factor is a highly effective method for the delivery of nanodevices into live cell nuclei.

Neofunctionalization driven by positive selection led to the retention of the loqs2 gene encoding an Aedes specific dsRNA binding protein.

BACKGROUND: Mosquito borne viruses, such as dengue, Zika, yellow fever and Chikungunya, cause millions of infections every year. These viruses are mostly transmitted by two urban-adapted mosquito species, Aedes aegypti and Aedes albopictus. Although mechanistic understanding remains largely unknown, Aedes mosquitoes may have unique adaptations that lower the impact of viral infection. Recently, we reported the identification of an Aedes specific double-stranded RNA binding protein (dsRBP), named Loqs2, that is involved in the control of infection by dengue and Zika viruses in mosquitoes. Preliminary analyses suggested that the loqs2 gene is a paralog of loquacious (loqs) and r2d2, two co-factors of the RNA interference (RNAi) pathway, a major antiviral mechanism in insects. RESULTS: Here we analyzed the origin and evolution of loqs2. Our data suggest that loqs2 originated from two independent duplications of the first double-stranded RNA binding domain of loqs that occurred before the origin of the Aedes Stegomyia subgenus, around 31 million years ago. We show that the loqs2 gene is evolving under relaxed purifying selection at a faster pace than loqs, with evidence of neofunctionalization driven by positive selection. Accordingly, we observed that Loqs2 is localized mainly in the nucleus, different from R2D2 and both isoforms of Loqs that are cytoplasmic. In contrast to r2d2 and loqs, loqs2 expression is stage- and tissue-specific, restricted mostly to reproductive tissues in adult Ae. aegypti and Ae. albopictus. Transgenic mosquitoes engineered to express loqs2 ubiquitously undergo developmental arrest at larval stages that correlates with massive dysregulation of gene expression without major effects on microRNAs or other endogenous small RNAs, classically associated with RNA interference. CONCLUSIONS: Our results uncover the peculiar origin and neofunctionalization of loqs2 driven by positive selection. This study shows an example of unique adaptations in Aedes mosquitoes that could ultimately help explain their effectiveness as virus vectors.

Curcumin-PVP improves the in vitro efficacy of ivermectin against resistant and susceptible Haemonchus contortus.

Ivermectin (IVM) resistance in parasitic nematodes such as Haemonchus contortus has spurred a search for substances that help to recover its efficacy. One potential agent is the natural product curcumin (CUR). In this study, CUR was combined with polyvinylpyrrolidone (PVP) (CUR/PVP) to improve its solubility and biological applicability. This study determined the effect of CUR preincubation on the effective concentration 50% (EC50) of IVM in three H. contortus isolates with different susceptibilities to IVM. The IVM EC50 was determined for three H. contortus isolates with different IVM susceptibilities using the larval migration inhibition (LMI) test. The three isolates were (i) PARAISO (IVM resistant), (ii) FMVZ-UADY (IVM susceptible), and (iii) CENID-SAI INIFAP (reference IVM susceptible). The L3 of each isolate were preincubated for 3 h with one of three concentrations of CUR (µg curcumin/mL): CONC-1 (3.67), CONC-2 (5.67), or CONC-3 (8.48). Corresponding controls were performed without CUR. The EC50 of IVM was determined for each isolate after they were exposed to the different CUR concentrations. The EC50 of IVM differed between the isolates PARAISO > FMVZ-UADY > CENID-SAI INIFAP (P < 0.05). The CUR preincubation at CONC-1 did not decrease the EC50 of IVM for any of the three isolates, suggesting a hormetic effect. By contrast, CUR preincubation at CONC-2 or CONC-3 decreased the IVM EC50 for the PARAISO isolate (P < 0.05) compared with the reference isolate and reduced the EC50 of IVM for the FMVZ-UADY and CENID-SAI INIFAP isolates below the EC50 for the CENID-SAI INIFAP isolate without CUR preincubation. In conclusion, preincubation of H. contortus L3 with CUR reduced the EC50 of IVM for field isolates classified as resistant and susceptible to IVM. The CUR preincubation reduced the IVM resistance factor in the different isolates tested.