Phylogenetic Analysis Based on DNA Barcoding and Genetic Diversity Assessment of Morinda officinalis How in Vietnam Inferred by Microsatellites.

Morinda officinalis How is well-known as a valuable medicinal plant found in some regions of Vietnam. This species is mainly used for treating male impotence, irregular menstruation, and rheumatoid arthritis. This study aimed to identify the species of and genetic diversity in three M. officinalis populations: one each in Quang Binh (QB), Thua Thien Hue (TTH), and Quang Nam (QN). In this study, four DNA barcoding markers (ITS1, ITS2, matK, and rbcL) were used to identify the species and 22 microsatellite markers were applied for population structure and diversity analyses. The results showed that the sequences of gene regions studied in M. officinalis had a high similarity (>95%) to the ITS1, ITS2, matK, and rbcL sequences of M. officinalis on BLAST. Of the four DNA barcoding markers used, ITS1 and ITS2 showed higher efficiency in DNA amplification of M. officinalis. From this study, 27 GenBank codes were published on BLAST. The results also revealed high levels of genetic diversity in populations. The average observed and expected heterozygosity values were HO = 0.513 and HE = 0.612, respectively. The average FST value was 0.206. Analysis of molecular variance (AMOVA) showed 70% variation within populations and 30% among populations. The population structure of M. officinalis inferred in STRUCTURE revealed that the optimum number of genetic groups for the admixture model was K = 2. These findings provided vital background information for future studies in the conservation of M. officinalis in both ex situ and in situ plans.

Impact of Carbon Source Supplementations on Pseudomonas aeruginosa Physiology.

Pseudomonas aeruginosa is an opportunistic pathogen highly resistant to a wide range of antimicrobial agents, making its infections very difficult to treat. Since microorganisms need to perpetually adapt to their surrounding environment, understanding the effect of carbon sources on P. aeruginosa physiology is therefore essential to avoid increasing drug-resistance and better fight this pathogen. By a global proteomic approach and phenotypic assays, we investigated the impact of various carbon source supplementations (glucose, glutamate, succinate, and citrate) on the physiology of the P. aeruginosa PA14 strain. A total of 581 proteins were identified as differentially expressed in the 4 conditions. Most of them were more abundant in citrate supplementation and were involved in virulence, motility, biofilm development, and antibiotic resistance. Phenotypic assays were performed to check these hypotheses. By coupling all this data, we highlight the importance of the environment in which the bacterium evolves on its metabolism, and thus the necessity to better understand the metabolic pathways implied in its adaptative response according to the nutrient availability.

Antibody-Conjugated Nanocarriers for Targeted Antibiotic Delivery: Application in the Treatment of Bacterial Biofilms.

Conventional antibiotic treatment is in most cases insufficient to eradicate biofilm-related infections, resulting in high risk of treatment failure and recurrent infections. Recent studies have shown that novel methods of antibiotic delivery can improve clinical outcomes and reduce the emergence of antibiotic resistance. The objectives of this work were to develop and evaluate a targeting nanocarrier system that enables effective delivery of antimicrobial drugs to Staphylococcus aureus, a commonly virulent human pathogen. For this purpose, we first prepared a formulation of polymeric nanoparticles (NPs) suitable for encapsulation and sustained release of antibiotics. A specific antibody against S. aureus was used as a targeting ligand and was covalently immobilized onto the surface of nanoparticulate materials. It was demonstrated that the targeting NPs preferentially bound S. aureus cells and presented an elevated accumulation in the S. aureus biofilm. Compared to free-form antibiotic, the antibiotic-loaded targeting NPs significantly enhanced in vitro bactericidal activity against S. aureus both in planktonic and biofilm forms. Using a mouse infection model, we observed improved therapeutic efficacy of these antibiotic-loaded NPs after a single intravenous administration. Taken together, our studies show that the targeting nanoparticulate system could be a promising strategy to enhance the biodistribution of antibiotics and thereby improve their efficacy.

MacAB-TolC Contributes to the Development of Acinetobacter baumannii Biofilm at the Solid-Liquid Interface.

Acinetobacter baumannii has emerged as one of the most problematic bacterial pathogens responsible for hospital-acquired and community infections worldwide. Besides its high capacity to acquire antibiotic resistance mechanisms, it also presents high adhesion abilities on inert and living surfaces leading to biofilm development. This lifestyle confers additional protection against various treatments and allows it to persist for long periods in various hospital niches. Due to their remarkable antimicrobial tolerance, A. baumannii biofilms are difficult to control and ultimately eradicate. Further insights into the mechanism of biofilm development will help to overcome this challenge and to develop novel antibiofilm strategies. To unravel critical determinants of this sessile lifestyle, the proteomic profiles of two A. baumannii strains (ATTC17978 and SDF) grown in planktonic stationary phase or in mature solid-liquid (S-L) biofilm were compared using a semiquantitative proteomic study. Of interest, among the 69 common proteins determinants accumulated in the two strains at the S-L interface, we sorted out the MacAB-TolC system. This tripartite efflux pump played a role in A. baumannii biofilm formation as demonstrated by using ΔmacAB-tolC deletion mutant. Complementary approaches allowed us to get an overview of the impact of macAB-tolC deletion in A. baumannii physiology. Indeed, this efflux pump appeared to be involved in the envelope stress response occurring in mature biofilm. It contributes to maintain wild type (WT) membrane rigidity and provides tolerance to high osmolarity conditions. In addition, this system is probably involved in the maintenance of iron and sulfur homeostasis. MacAB-TolC might help this pathogen face and adapt to deleterious conditions occurring in mature biofilms. Increasing our knowledge of A. baumannii biofilm formation will undoubtedly help us develop new therapeutic strategies to tackle this emerging threat to human health.

Trends in antimicrobial resistance in Neisseria gonorrhoeae in Hanoi, Vietnam, 2017-2019.

BACKGROUND: Antimicrobial resistance (AMR) in Neisseria gonorrhoeae is an emerging global health threat. Surveillance of AMR in N. gonorrhoeae in the Western Pacific Region is important, as resistant strains have typically emerged from this region. There are sparse data regarding antibiotic susceptibility of N. gonorrhoeae from Vietnam. This study aimed to provide updated data on antibiotic susceptibilities in N. gonorrhoeae isolates from Hanoi, Vietnam. METHODS: From 2017 to 2019, 409 N. gonorrhoeae clinical isolates were collected at the National Hospital for Venereology and Dermatology in Hanoi, Vietnam. Antibiotic susceptibility testing was performed by disk diffusion method according to the Clinical and Laboratory Standards Institute (CLSI) protocol. The zone diameters of inhibition were recorded and interpreted according to standard CLSI criteria, except for azithromycin, due to the absence of CLSI interpretation. Categorical variables were analyzed by Chi-square and Fisher's exact tests. Linear regression was used to evaluate zones of inhibition by year. RESULTS: Among the 409 isolates, no isolates were susceptible to penicillin, 98.3% were resistant to ciprofloxacin, and all isolates were susceptible to spectinomycin. There were 122/407 (30.0%) isolates resistant to azithromycin and there was an association between resistance and year (p <  0.01), ranging from 15.3% of isolates in 2017 to 46.7% of the isolates in 2018. Resistance to cefixime was found in 13/406 (3.2%) of isolates and there was no association by year (p = 0.30). Resistance to ceftriaxone occurred in 3/408 (0.7%) of isolates. Linear regression indicated the zone of inhibition diameters decreased by 0.83 mm each year for ceftriaxone (95% CI: - 1.3, - 0.4; p <  0.01) and decreased by 0.83 mm each year (95% CI: - 1.33, - 0.33; p <  0.01) for azithromycin; the association was not significant for cefixime (p = 0.07). CONCLUSIONS: We found decreasing susceptibility of N. gonorrhoeae to ceftriaxone and azithromycin, as well as a high prevalence of resistance to azithromycin, among isolates in Hanoi, Vietnam from 2017 to 2019. The trends of decreasing susceptibility to first-line treatments are concerning and highlight the urgency of addressing antimicrobial resistance in N. gonorrhoeae. Expanded surveillance efforts within the Western Pacific Region are critical to monitoring trends and informing treatment guidelines.

Correlations between growth and wool quality traits of genetically divergent Australian lambs in response to canola or flaxseed oil supplementation.

The correlations between growth and wool traits in response to canola and flaxseed oil supplementation were evaluated in Australian prime lambs. Sixty dual-purpose prime lambs including purebred Merino and crossbred lambs were allocated to one of five treatments of lucerne hay basal diet supplemented with isocaloric and isonitrogenous wheat-based pellets. Treatments were: no oil inclusion (Control); 2.5% canola oil; 5% canola oil; 2.5% flaxseed oil and 5% flaxseed oil, with lamb groups balanced by breed and gender. Each lamb was daily supplemented with 1kg of pellets and had free access to lucerne hay and water throughout the 7-week feeding trial, after a 3-week adaptation. Individual animal basal and supplementary pellet feed intakes were recorded daily, while body conformation traits, body condition scores and liveweights were measured on days 0, 21, 35 and 49. The lambs were dye-banded on the mid-side and shorn before commencing the feeding trial and mid-side wool samples were collected from the same dye-banded area of each lamb at the end of the experiment. Correlations between wool quality traits and lamb performance were non-significant (P>0.05). Oil supplementation had no detrimental effect on lamb growth and wool quality traits (P > 0.05). Gender significantly affected wither height gain and fibre diameter. There were significant interactions between oil supplementation and lamb breed on chest girth. The correlations between clean fleece yield (CFY) and other wool quality traits were moderate ranging from 0.29 to 0.55. Moderate to high correlations between fibre diameter (FD) and other wool quality traits were detected (0.46-0.99) with the strongest relationship between FD and wool spinning fineness (SF). The relationship between CFY and wool comfort factor (CF) were positive, while negative relationships between CFY and the others were observed. A combination of 5% oil supplementation and genetics is an effective and strategic management tool for enhancing feed efficiency and growth performance without negative effects on wool quality in dual-purpose lamb production. This is a good outcome for dual-purpose sheep farmers. It essentially means the absorbed nutrients in supplemented lambs yielded good growth performance without any detrimental impact on wool quality; a win-win case of nutrient partitioning into the synthesis of muscle and wool without compromising either traits.

Enhancement of dairy sheep cheese eating quality with increased n-3 long-chain polyunsaturated fatty acids.

This study investigated the effect of different plant oil-infused and rumen-protected wheat-based pellets containing eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) on n-3 long-chain (≥C20) polyunsaturated fatty acid (LC-PUFA) content, fatty acid recovery, and sensory attributes of ripened cheese from dairy sheep. During a 10-wk supplementary feeding trial, 60 dairy ewes balanced by live weight, milk yield, parity, and sire breed were randomly divided into 6 groups that were (1) supplemented with on-farm existing commercial wheat-based pellets without oil inclusion (control) or supplemented with wheat-based pellets infused with 50 mL/kg dry matter of oils from (2) canola, (3) rice bran, (4) flaxseed, (5) safflower, and (6) rumen-protected EPA + DHA. Milk samples from each treatment were collected separately by sire breed during the experimental period for cheese processing at the end of the experiment. Twelve batches of cheese (2 batches per treatment) were processed and ripened for 120 d. Three cheese samples were collected and analyzed for each cheese making session (total of 36 cheese samples) at d 120 of ripening. Processed cheese of rumen-protected EPA + DHA had the most efficiency at elevating total n-3 LC-PUFA [total EPA + DHA + docosapentaenoic acid (DPA, 22:5n-3] content compared with the control (0.49 vs. 0.28%). Flaxseed elicited the greatest enhancement of α-linolenic acid (ALA, 18:3n-3), whereas safflower was the most effective diet in enhancing the level of linoleic acid (18:2n-6) in cheese (1.29 vs. 0.71% and 4.8 vs. 3.3%, respectively). Parallel recoveries of n-3 and n-6 LC-PUFA were observed across all treatments except for α-linolenic acid and EPA. Cheese eating sensory traits were also highly affected by oil supplementation, with the highest score of 7.5 in cheese from the rice bran and flaxseed treatments. These results provide new insights into the biological mechanisms and processes that determine dairy ewe milk productivity by underpinning the vital biological role of n-3 LC-PUFA in not only enhancing the healthy composition of cheese from ewes but also translating it into consumer acceptability.

Enhanced Omega-3 Polyunsaturated Fatty Acid Contents in Muscle and Edible Organs of Australian Prime Lambs Grazing Lucerne and Cocksfoot Pastures.

The enhancement of health-beneficial omega-3 long⁻chain (≥C20) polyunsaturated fatty acid (n-3 LC-PUFA) contents in the muscle, liver, heart, and kidney of Australian prime lambs through pasture grazing and supplementation with oil infused pellets was investigated. Forty-eight first-cross prime lambs were randomly assigned into a split-plot design with pasture type as the main plot effect and pellet supplementation as a sub-plot effect in a feeding trial that lasted for nine weeks. The n-3 LC-PUFA content in Longissimus dorsi muscle of all lambs was well above the 30 mg threshold for "omega-3 source" nutrition claim under the Australian Food Standards and Guidelines. Pasture type impacted the fatty acid contents in muscle, heart, and kidney of prime lambs. Lambs grazing cocksfoot grass only had high 18:3n-3 (ALA) and n-3 LC-PUFA contents (67.1 mg/100 g and 55.2 mg/100 g, respectively) in the Longissimus dorsi muscle, which was not significantly different (p > 0.8990) from the contents of lambs grazing only lucerne. Supplementation of pellets with or without oil infusion to grazing lambs generally decreased the ALA and n-3 LC-PUFA contents and increased the n-6/n-3 ratio in the Longissimus dorsi muscle. The fatty acid content in the internal organs of grazing lambs was also affected by pellet supplementation. The liver and kidney of grazing lambs were both "good sources" (60 mg/100 g) of omega-3. The cocksfoot grass showed considerable potential for producing healthy, premium quality meat with high contents of n-3 and n-3 LC-PUFA, which may consequently enhance the omega-3 intake of Australian lamb consumers.

Supplementing Dairy Ewes Grazing Low Quality Pastures with Plant-Derived and Rumen-Protected Oils Containing Eicosapentaenoic Acid and Docosahexaenoic Acid Pellets Increases Body Condition Score and Milk, Fat, and Protein Yields.

The Australian dairy sheep industry is small and mostly based on a natural grass grazing system, which can limit productivity. The current study tested different plant oil-infused and rumen protected polyunsaturated fats and their interactions with sire breeds to improve lactation traits and body condition scores (BCS) of ewes grazing low quality pastures. It was hypothesised that supplementing lactating ewe's diets with plant-derived polyunsaturated oils would improve milk production and composition without compromising BCS. Sixty ewes (n = 10/treatment) in mid-lactation, balanced by sire breed, parity, milk yield, body condition score, and liveweight, were supplemented with: (1) control: wheat-based pellets without oil inclusion; wheat-based pellets including; (2) canola oil (CO); (3) rice bran oil (RBO); (4) flaxseed oil (FSO); (5); safflower oil (SFO); and (6) rumen protected marine oil containing eicosapentaenoic acid and docosahexaenoic acid (RPO). Except for the control group, all supplementary diets included the same level of 50 mL/kg DM of oil and all diets were isocaloric and isonitrogenous. Experimental animals were grazed in the same paddock with ad libitum access to pasture, hay, and water during the 10-week study. RPO was the most effective diet that enhanced milk, fat, and protein yields by approximately 30%, 13%, and 31%, respectively (p < 0.0001). A significant increase in milk production was also observed with CO, RBO, and SFO treatments (p < 0.0001). Breed significantly influenced animal performance with higher milk yields recorded for crossbred Awassi × East Friesian (AW × EF) (578 g/day) vs. purebred Awassi (452 g/day) (p < 0.0001). This study provides empirical evidence for the use of rumen-protected and plant-derived oil-infused pellets as supplements under low quality pasture grazing conditions to improve the production performance of purebred Awassi and crossbred AW × EF ewes.

Thermodynamics of nucleotide and inhibitor binding to wild-type and ispinesib-resistant forms of human kinesin spindle protein.

Current antimitotic cancer chemotherapy based on vinca alkaloids and taxanes target tubulin, a protein required not only for mitotic spindle formation but also for the overall structural integrity of terminally differentiated cells. Among many innovations targeting specific mitotic events, inhibition of motor enzymes including KSP (or Eg5) has been validated as a highly productive approach. Many reported KSP inhibitors bind to an induced allosteric site near the site of ATP hydrolysis, and some have been tested in clinical trials with varying degrees of success. This allosteric site was defined in detail by X-ray crystallography of inhibitor complexes, yet complementary information on binding thermodynamics is still lacking. Using two model ATP-uncompetitive inhibitors, monastrol and ispinesib, we report here the results of thermal denaturation and isothermal titration calorimetric studies. These binding studies were conducted with the wild-type KSP motor domain as well as two ispinesib mutants (D130V and A133D) identified to confer resistance to ispinesib treatment. The thermodynamic parameters obtained were placed in the context of the available structural information and corresponding models of the two ispinesib-resistant mutants. The resulting overall information formed a strong basis for future structure-based design of inhibitors of KSP and related motor enzymes.

Structure-guided discovery of cyclin-dependent kinase inhibitors.

CDK2 inhibitors containing the related bicyclic heterocycles pyrazolopyrimidines and imidazopyrazines were discovered through high-throughput screening. Crystal structures of inhibitors with these bicyclic cores and two more related ones show that all but one have a common binding mode featuring two hydrogen bonds (H-bonds) to the backbone of the kinase hinge region. Even though ab initio computations indicated that the imidazopyrazine core would bind more tightly to the hinge, pyrazolopyrimidines gain an advantage in potency through participation of N4 in an H-bond network involving two catalytic residues and bridging water molecules. Further insight into inhibitor/CDK2 interactions was gained from analysis of additional crystal structures. Significant gains in potency were obtained by optimizing the fit of hydrophobic substituents to the gatekeeper region of the ATP binding site. The most potent inhibitors have good selectivity.

Artemisinin or chloroquine for blood stage Plasmodium vivax malaria in Vietnam.

Chloroquine-resistant Plasmodium vivax has not yet occurred in Vietnam. The efficacy of artemisinin for P. vivax was not established. We conducted a double-blind randomized study involving 240 inpatients with P. vivax malaria who received artemisinin (40 mg/kg over 3 days) plus placebo chloroquine (Art) or chloroquine (25 mg/kg over 3 days) plus placebo artemisinin (Chl). Patients were followed up with weekly blood smears for 28 days. In each group 113 cases were analysed. All patients recovered rapidly. The median (range) parasite clearance time of regimen Art was 24 h (8-72) and of Chl 24 h (8-64; P = 0.3). Parasites reappeared in two cases in each group on day 14, in eight cases in each group (7%) on day 16 and in 25 (23%) and 18 (16%) cases, respectively, at the end of 4-week follow-up (P = 0.3). The population parasite clearance curve followed a mono-exponential decline. The parasite reduction ratio per 48 h reproduction cycle was 2.3 x 104 for both regimens. We conclude that artemisinin and chloroquine are equally effective in the treatment of P. vivax infections in Vietnam. Reappearance of parasites before day 16 (7%) suggests the emergence of chloroquine resistance. Three days of artemisinin monotherapy does not prevent recrudescence.