Accumulation of sesquiterpenes and polysaccharides in cells of zedoary (Curcuma zedoaria Roscoe) cultured in a 10 L bioreactor

We developed a cell suspension culture system for zedoary (Curcuma zedoaria Roscoe), using 100 g fresh weight inoculum in a batch culture. The maximum cell biomass of 68.46 g/L fresh weight was obtained after 14 days of culture in a 10 L bioreactor with a pitch-blade impeller maintained at an agitation speed of 150 rpm and an aeration rate of 2.5 L/min. The accumulation of sesquiterpenes and polysaccharide in zedoary cells from 2 to 18 days was measured by HPLC and a phenol-sulfuric acid assay, respectively. The total polysaccharide concentration increased between 2 to 10 days of culture and reached a maximum value of 6.55%. HPLC revealed several eluted peaks of sesquiterpenes, which increased in amplitude from days 2 to 10. Furthermore, our results indicated that biotransformation occurred in the cell suspension, transforming certain sesquiterpenes into other types during culture.     

A thermostable phytase from Bacillus sp. MD2: cloning, expression and high-level production in Escherichia coli

Phytase is used as a feed additive for degradation of antinutritional phytate, and the enzyme is desired to be highly thermostable for it to withstand feed formulation conditions. A Bacillus sp. MD2 showing phytase activity was isolated, and the phytase encoding gene was cloned and expressed in Escherichia coli. The recombinant phytase exhibited high stability at temperatures up to 100°C. A higher enzyme activity was obtained when the gene expression was done in the presence of calcium chloride. Production of the enzyme by batch- and fed-batch cultivation in a bioreactor was studied. In batch cultivation, maintaining dissolved oxygen at 20–30% saturation and depleting inorganic phosphate below 1 mM prior to induction by IPTG resulted in over 10 U/ml phytase activity. For fed–batch cultivation, glucose concentration was maintained at 2–3 g/l, and the phytase expression was increased to 327 U/ml. Induction using lactose during fed-batch cultivation showed a lag phase of 4 h prior to an increase in the phytase activity to 71 U/ml during the same period as IPTG-induced production. Up to 90% of the total amount of expressed phytase leaked out from the E. coli cells in both IPTG- and lactose-induced fed-batch cultivations.     

Effect of oxygen supply on cell growth and saponin production in bioreactor cultures of Panax ginseng

The effects of oxygen supply within the range 20.8–50% (using pure oxygen and air), on cell cultures of Panax ginseng were investigated in a balloon-type bubble bioreactor (5 L capacity, containing 4 L Murashige and Skoog medium, supplemented with 7.0 mg L⁻¹ indolebutyric acid, 0.5 mg L⁻¹ kinetin and 30 g L⁻¹ sucrose). A 40% oxygen supply was found to be optimal for the production of both cell mass and saponin yielding values of 12.8 g (DW) L⁻¹, 4.5 mg (g DW)⁻¹ on day 25, respectively. Low (20.8%, 30%) and high (50%) oxygen concentration supplies were unfavorable to cell growth and saponin accumulation. The results indicate that oxygen supplementation to bioreactor-based ginseng cultures was beneficial for biomass accumulation and saponin production.     

Clinical stage drugs targeting inhibitor of apoptosis proteins purge episomal Hepatitis B viral genome in preclinical models

A major unmet clinical need is a therapeutic capable of removing hepatitis B virus (HBV) genome from the liver of infected individuals to reduce their risk of developing liver cancer. A strategy to deliver such a therapy could utilize the ability to target and promote apoptosis of infected hepatocytes. Presently there is no clinically relevant strategy that has been shown to effectively remove persistent episomal covalently closed circular HBV DNA (cccDNA) from the nucleus of hepatocytes. We used linearized single genome length HBV DNA of various genotypes to establish a cccDNA-like reservoir in immunocompetent mice and showed that clinical-stage orally administered drugs that antagonize the function of cellular inhibitor of apoptosis proteins can eliminate HBV replication and episomal HBV genome in the liver. Primary human liver organoid models were used to confirm the clinical relevance of these results. This study underscores a clinically tenable strategy for the potential elimination of chronic HBV reservoirs in patients.Subject terms: Target validation, Hepatitis B, Preclinical research, Translational research     

Severe bacterial neonatal infections in Madagascar,Senegal, and Cambodia: A multicentric community-based cohort study

BackgroundSevere bacterial infections (SBIs) are a leading cause of neonatal deaths in low- and middle-income countries (LMICs). However, most data came from hospitals, which do not include neonates who did not seek care or were treated outside the hospital. Studies from the community are scarce, and few among those available were conducted with high-quality microbiological techniques. The burden of SBI at the community level is therefore largely unknown. We aimed here to describe the incidence, etiology, risk factors, and antibiotic resistance profiles of community-acquired neonatal SBI in 3 LMICs.Methods and findingsThe BIRDY study is a prospective multicentric community-based mother and child cohort study and was conducted in both urban and rural areas in Madagascar (2012 to 2018), Cambodia (2014 to 2018), and Senegal (2014 to 2018). All pregnant women within a geographically defined population were identified and enrolled. Their neonates were actively followed from birth to 28 days to document all episodes of SBI. A total of 3,858 pregnant women (2,273 (58.9%) in Madagascar, 814 (21.1%) in Cambodia, and 771 (20.0%) in Senegal) were enrolled in the study, and, of these, 31.2% were primigravidae. Women enrolled in the urban sites represented 39.6% (900/2,273), 45.5% (370/814), and 61.9% (477/771), and those enrolled in the rural sites represented 60.4% (1,373/2,273), 54.5% (444/814), and 38.1% (294/771) of the total in Madagascar, Cambodia, and Senegal, respectively. Among the 3,688 recruited newborns, 49.6% were male and 8.7% were low birth weight (LBW). The incidence of possible severe bacterial infection (pSBI; clinical diagnosis based on WHO guidelines of the Integrated Management of Childhood Illness) was 196.3 [95% confidence interval (CI) 176.5 to 218.2], 110.1 [88.3 to 137.3], and 78.3 [59.5 to 103] per 1,000 live births in Madagascar, Cambodia, and Senegal, respectively. The incidence of pSBI differed between urban and rural sites in all study countries. In Madagascar, we estimated an incidence of 161.0 pSBI per 1,000 live births [133.5 to 194] in the urban site and 219.0 [192.6 to 249.1] pSBI per 1,000 live births in the rural site (p = 0.008). In Cambodia, estimated incidences were 141.1 [105.4 to 189.0] and 85.3 [61.0 to 119.4] pSBI per 1,000 live births in urban and rural sites, respectively (p = 0.025), while in Senegal, we estimated 103.6 [76.0 to 141.2] pSBI and 41.5 [23.0 to 75.0] pSBI per 1,000 live births in urban and rural sites, respectively (p = 0.006). The incidences of culture-confirmed SBI were 15.2 [10.6 to 21.8], 6.5 [2.7 to 15.6], and 10.2 [4.8 to 21.3] per 1,000 live births in Madagascar, Cambodia, and Senegal, respectively, with no difference between urban and rural sites in each country. The great majority of early-onset infections occurred during the first 3 days of life (72.7%). The 3 main pathogens isolated were Klebsiella spp. (11/45, 24.4%), Escherichia coli (10/45, 22.2%), and Staphylococcus spp. (11/45, 24.4%). Among the 13 gram-positive isolates, 5 were resistant to gentamicin, and, among the 29 gram-negative isolates, 13 were resistant to gentamicin, with only 1 E. coli out of 10 sensitive to ampicillin. Almost one-third of the isolates were resistant to both first-line drugs recommended for the management of neonatal sepsis (ampicillin and gentamicin). Overall, 38 deaths occurred among neonates with SBI (possible and culture-confirmed SBI together). LBW and foul-smelling amniotic fluid at delivery were common risk factors for early pSBI in all 3 countries. A main limitation of the study was the lack of samples from a significant proportion of infants with pBSI including 35 neonatal deaths. Without these samples, bacterial infection and resistance profiles could not be confirmed.ConclusionsIn this study, we observed a high incidence of neonatal SBI, particularly in the first 3 days of life, in the community of 3 LMICs. The current treatment for the management of neonatal infection is hindered by antimicrobial resistance. Our findings suggest that microbiological diagnosis of SBI remains a challenge in these settings and support more research on causes of neonatal death and the implementation of early interventions (e.g., follow-up of at-risk newborns during the first days of life) to decrease the burden of neonatal SBI and associated mortality and help achieve Sustainable Development Goal 3.

In a community-based, prospective cohort study, Bich-Tram Huynh and colleagues investigate the incidence and factors associated with several bacterial infections among neonates in rural and urban areas of three low-middle income countries.     

Evaluation of a novel monoclonal-based antigen-in-stool enzyme immunoassay (Premier Platinum HpSA PLUS) for diagnosis of Helicobacter pylori infection in Vietnamese children

Background: Helicobacter pylori infection is difficult to diagnose in children, especially in developing countries where noninvasive methods such as urea breath test are often not available. We evaluate the sensitivity and specificity of a new monoclonal antibody-based antigen-in-stool enzyme immunoassay (Premier Platinum HpSA PLUS) for diagnosis of H. pylori infection in Vietnamese children.
Materials and Methods: Sensitivity of the antigen-in-stool test was evaluated in 232 children, 3–15 years of age, who were positive for H. pylori infection by culture from biopsies. For evaluation of the specificity 98 children of similar age with nongastrointestinal conditions and who were negative for H. pylori infection by serologic assays were included with blood and stool samples.
Results: Of the 232 culture-positive children, 224 were also positive by Premier Platinum HpSA PLUS. Of the 98 control children, 93 were H. pylori negative also in the stool test. The sensitivity of Premier Platinum HpSA PLUS was thus 96.6% (95% CI 93.3–98.5) and the specificity was 94.9% (95% CI 88.5–98.3).
Conclusions: The findings have demonstrated Premium Platinum HpSA PLUS to be a reliable method for detection of H. pylori infection also in children in our area.     

Quantitative trait loci for grain fructan concentration in wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Fructans (fructo-oligosaccharides) are prebiotics that are thought to selectively promote the growth of colonic bifidobacteria, thereby improving human gut health. Fructans are present in the grain of wheat, a staple food crop. In the research reported here, we aimed to detect and map loci affecting grain fructan concentration in wheat using a doubled-haploid population derived from a cross between a high-fructan breeding line, Berkut, and a low-fructan cultivar, Krichauff. Fructan concentration was measured in grain samples grown at two locations in Australia and one in Kazakhstan. Fructan concentration varied widely within the population, ranging from 0.6 to 2.6% of grain dry weight, and was quite repeatable, with broad-sense heritability estimated as 0.71. With a linkage map of 528 molecular markers, quantitative trait loci (QTLs) were detected on chromosomes 2B, 3B, 5A, 6D and 7A. Of these, the QTLs on chromosomes 6D and 7A had the largest effects, explaining 17 and 27% of the total phenotypic variance, respectively, both with the favourable (high-fructan concentration) alleles contributed from Berkut. These chromosome regions had similar effects in another mapping population, Sokoll/Krichauff, with the favourable alleles contributed from Sokoll. It is concluded that grain fructan concentration of wheat can be improved by breeding and that molecular markers could be used to select effectively for favourable alleles in two regions of the wheat genome.     

Chloroplast monothiol glutaredoxins as scaffold proteins for the assembly and delivery of [2Fe-2S] clusters

Glutaredoxins (Grxs) are small oxidoreductases that reduce disulphide bonds or protein-glutathione mixed disulphides. More than 30 distinct grx genes are expressed in higher plants, but little is currently known concerning their functional diversity. This study presents biochemical and spectroscopic evidence for incorporation of a [2Fe-2S] cluster in two heterologously expressed chloroplastic Grxs, GrxS14 and GrxS16, and in vitro cysteine desulphurase-mediated assembly of an identical [2Fe-2S] cluster in apo-GrxS14. These Grxs possess the same monothiol CGFS active site as yeast Grx5 and both were able to complement a yeast grx5 mutant defective in Fe-S cluster assembly. In vitro kinetic studies monitored by CD spectroscopy indicate that [2Fe-2S] clusters on GrxS14 are rapidly and quantitatively transferred to apo chloroplast ferredoxin. These data demonstrate that chloroplast CGFS Grxs have the potential to function as scaffold proteins for the assembly of [2Fe-2S] clusters that can be transferred intact to physiologically relevant acceptor proteins. Alternatively, they may function in the storage and/or delivery of preformed Fe-S clusters or in the regulation of the chloroplastic Fe-S cluster assembly machinery.     

From geochemical background determination to pollution assessment of heavy metals in sediments and soils

Establishing geochemical background concentrations to distinguish the natural background from anthropogenic concentrations of heavy metals in sediments and soils is necessary to develop guidelines for environmental legislation. Due to the fact that the background concentrations strongly depend on geological characteristics such as mineral composition, grain size distribution and organic matter content, several normalization methods have been developed. Empirical (geochemical), theoretical (statistical) and integrated methods (combining both empirical and theoretical methods) are the main approaches described in literature for determination of geochemical background concentrations. In this review paper, the different approaches as well as the main normalization methods for heavy metal concentrations in sediments and soils will be discussed. Both geochemical background concentrations and added risk level (maximum permissible addition) should be taken into account for setting up legal threshold limits. Moreover, different approaches to evaluate the pollution status of heavy metals in sediments and soils, from Sediment/Soil Quality Guidelines to quantitative indices (Geo-accumulation Index-I_geo, Enrichment Factor-EF, Pollution Load Index-PLI and Risk assessment Code-RAC) will be presented. Although guidelines to establish whether a sediment or soil is polluted or not are generally only related to total metal concentrations, the available/reactive pool i.e., availability/reactivity of metals should be taken into account for sediment/soil pollution assessment.     

Impact of Pregnancy-Associated Malaria on Infant Malaria Infection in Southern Benin

Background

Infants of mothers with placental Plasmodium falciparum infections at delivery are themselves more susceptible to malaria attacks or to infection in early life.

Methodology/ Principal Findings

To assess the impact of either the timing or the number of pregnancy-associated malaria (PAM) infections on the incidence of parasitemia or malaria attacks in infancy, we followed 218 mothers through pregnancy (monthly visits) up to delivery and their infants from birth to 12 months of age (fortnightly visits), collecting detailed clinical and parasitological data. After adjustment on location, mother’s age, birth season, bed net use, and placental malaria, infants born to a mother with PAM during the third trimester of pregnancy had a significantly increased risk of infection (OR [95% CI]: 4.2 [1.6; 10.5], p = 0.003) or of malaria attack (4.6 [1.7; 12.5], p = 0.003). PAM during the first and second trimesters had no such impact. Similarly significant results were found for the effect of the overall number of PAM episodes on the time to first parasitemia and first malaria attack (HR [95% CI]: 2.95 [1.58; 5.50], p = 0.001 and 3.19 [1.59; 6.38], p = 0.001) respectively.

Conclusions/ Significance

This study highlights the importance of protecting newborns by preventing repeated episodes of PAM in their mothers.