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Biologically derived fuels are viable alternatives to traditional fossil fuels, and microalgae are a particularly promising source, but improvements are required throughout the production process to increase productivity and reduce cost. Metabolic engineering to increase yields of biofuel-relevant lipids in these organisms without compromising growth is an important aspect of advancing economic feasibility. We report that the targeted knockdown of a multifunctional lipase/phospholipase/acyltransferase increased lipid yields without affecting growth in the diatom Thalassiosira pseudonana . Antisense-expressing knockdown strains 1A6 and 1B1 exhibited wild-type–like growth and increased lipid content under both continuous light and alternating light/dark conditions. Strains 1A6 and 1B1, respectively, contained 2.4- and 3.3-fold higher lipid content than wild-type during exponential growth, and 4.1- and 3.2-fold higher lipid content than wild-type after 40 h of silicon starvation. Analyses of fatty acids, lipid classes, and membrane stability in the transgenic strains suggest a role for this enzyme in membrane lipid turnover and lipid homeostasis. These results demonstrate that targeted metabolic manipulations can be used to increase lipid accumulation in eukaryotic microalgae without compromising growth.

The silicified cell walls of diatoms have inspired the interest of researchers for several centuries, and our understanding of their properties and formation has developed in synch with the development of observational and analytical techniques. Over the past 20 years, approaches used to characterize the molecular components involved in cell wall silicification have evolved, and this has provided significant insights into fundamental aspects of silicification, and promises to continue to do so. Diatom cell wall formation is highly dynamic but, apart from microscopic investigations, most previous molecular characterizations have been on completely formed structures, and thus only provide information on a static end point in the process. However, recent studies that monitor when components are made, and how they are transported to the silica deposition vesicle (SDV), indicate that investigation into the true dynamics of the process is possible. Real-time imaging and genetic manipulation offer the promise of elucidating the spatio-temporal dynamics of, and interactions between, components, which will be essential to understand how structure formation is controlled and coordinated. This review is aimed at describing the approaches that have been used to characterize diatom silicification, integrating newer concepts based on results from diverse approaches, and raising questions that still need to be addressed, leveraging the diverse tools and techniques we now have.

Some microbial eukaryotes, such as the extremophilic red alga Galdieria sulphuraria, live in hot, toxic metal-rich, acidic environments. To elucidate the underlying molecular mechanisms of adaptation, we sequenced the 13.7-megabase genome of G. sulphuraria. This alga shows an enormous metabolic flexibility, growing either photoautotrophically or heterotrophically on more than 50 carbon sources. Environmental adaptation seems to have been facilitated by horizontal gene transfer from various bacteria and archaea, often followed by gene family expansion. At least 5% of protein-coding genes of G. sulphuraria were probably acquired horizontally. These proteins are involved in ecologically important processes ranging from heavy-metal detoxification to glycerol uptake and metabolism. Thus, our findings show that a pan-domain gene pool has facilitated environmental adaptation in this unicellular eukaryote.

Background The garden pea, Pisum sativum , is among the best-investigated legume plants and of significant agro-commercial relevance. Pisum sativum has a large and complex genome and accordingly few comprehensive genomic resources exist. Results We analyzed the pea transcriptome at the highest possible amount of accuracy by current technology. We used next generation sequencing with the Roche/454 platform and evaluated and compared a variety of approaches, including diverse tissue libraries, normalization, alternative sequencing technologies, saturation estimation and diverse assembly strategies. We generated libraries from flowers, leaves, cotyledons, epi- and hypocotyl, and etiolated and light treated etiolated seedlings, comprising a total of 450 megabases. Libraries were assembled into 324,428 unigenes in a first pass assembly. A second pass assembly reduced the amount to 81,449 unigenes but caused a significant number of chimeras. Analyses of the assemblies identified the assembly step as a major possibility for improvement. By recording frequencies of Arabidopsis orthologs hit by randomly drawn reads and fitting parameters of the saturation curve we concluded that sequencing was exhaustive. For leaf libraries we found normalization allows partial recovery of expression strength aside the desired effect of increased coverage. Based on theoretical and biological considerations we concluded that the sequence reads in the database tagged the vast majority of transcripts in the aerial tissues. A pathway representation analysis showed the merits of sampling multiple aerial tissues to increase the number of tagged genes. All results have been made available as a fully annotated database in fasta format. Conclusions We conclude that the approach taken resulted in a high quality - dataset which serves well as a first comprehensive reference set for the model legume pea. We suggest future deep sequencing transcriptome projects of species lacking a genomics backbone will need to concentrate mainly on resolving the issues of redundancy and paralogy during transcriptome assembly.

Background An inducible promoter for recombinant protein expression provides substantial benefits because under induction conditions cellular energy and metabolic capability can be directed into protein synthesis. The most widely used inducible promoter for diatoms is for nitrate reductase, however, nitrogen metabolism is tied into diverse aspects of cellular function, and the induction response is not necessarily robust. Silicon limitation offers a means to eliminate energy and metabolic flux into cell division processes, with little other detrimental effect on cellular function, and a protein expression system that works under those conditions could be advantageous. Results In this study, we evaluate a number of promoters for recombinant protein expression induced by silicon limitation and repressed by the presence of silicon in the diatoms Thalassiosira pseudonana and Cyclotella cryptica . In addition to silicon limitation, we describe additional strategies to elevate recombinant protein expression level, including inclusion of the 5′ fragment of the coding region of the native gene and reducing carbon flow into ancillary processes of pigment synthesis and formation of photosynthetic storage products. We achieved yields of eGFP to 1.8% of total soluble protein in C. cryptica , which is about 3.6-fold higher than that obtained with chloroplast expression and ninefold higher than nuclear expression in another well-established algal system. Conclusions Our studies demonstrate that the combination of inducible promoter and other strategies can result in robust expression of recombinant protein in a nuclear-based expression system in diatoms under silicon limited conditions, separating the protein expression regime from growth processes and improving overall recombinant protein yields.

Background Silicon plays important biological roles, but the mechanisms of cellular responses to silicon are poorly understood. We report the first analysis of cell cycle arrest and recovery from silicon starvation in the diatom Thalassiosira pseudonana using whole genome microarrays. Results Three known responses to silicon were examined, 1) silicified cell wall synthesis, 2) recovery from silicon starvation, and 3) co-regulation with silicon transporter (SIT) genes. In terms of diatom cell wall formation, thus far only cell surface proteins and proteins tightly associated with silica have been characterized. Our analysis has identified new genes potentially involved in silica formation, and other genes potentially involved in signaling, trafficking, protein degradation, glycosylation and transport, which provides a larger-scale picture of the processes involved. During silicon starvation, an overrepresentation of transcription and translation related genes were up-regulated, indicating that T. pseudonana is poised to rapidly recover from silicon starvation and resume cell cycle progression upon silicon replenishment. This is in contrast to other types of limitation, and provides the first molecular data explaining the well-established environmental response of diatoms to grow as blooms and to out-compete other classes of microalgae for growth. Comparison of our data with a previous diatom cell cycle analysis indicates that assignment of the cell cycle specific stage of particular cyclins and cyclin dependent kinases should be re-evaluated. Finally, genes co-varying in expression with the SITs enabled identification of a new class of diatom-specific proteins containing a unique domain, and a putative silicon efflux protein. Conclusions Analysis of the T. pseudonana microarray data has provided a wealth of new genes to investigate previously uncharacterized cellular phenomenon related to silicon metabolism, silicon’s interaction with cellular components, and environmental responses to silicon.

An inducible promoter for recombinant protein expression provides substantial benefits because under induction conditions cellular energy and metabolic capability can be directed into protein synthesis. The most widely used inducible promoter for diatoms is for nitrate reductase, however, nitrogen metabolism is tied into diverse aspects of cellular function, and the induction response is not necessarily robust. Silicon limitation offers a means to eliminate energy and metabolic flux into cell division processes, with little other detrimental effect on cellular function, and a protein expression system that works under those conditions could be advantageous. In this study, we evaluate a number of promoters for recombinant protein expression induced by silicon limitation and repressed by the presence of silicon in the diatoms Thalassiosira pseudonana and Cyclotella cryptica. In addition to silicon limitation, we describe additional strategies to elevate recombinant protein expression level, including inclusion of the 5' fragment of the coding region of the native gene and reducing carbon flow into ancillary processes of pigment synthesis and formation of photosynthetic storage products. We achieved yields of eGFP to 1.8% of total soluble protein in C. cryptica, which is about 3.6-fold higher than that obtained with chloroplast expression and ninefold higher than nuclear expression in another well-established algal system. Our studies demonstrate that the combination of inducible promoter and other strategies can result in robust expression of recombinant protein in a nuclear-based expression system in diatoms under silicon limited conditions, separating the protein expression regime from growth processes and improving overall recombinant protein yields.

Background Poor growth in early childhood has been considered irreversible after 2–3 years of age and has been associated with morbidity and mortality over the short-term and with poor economic and cognitive outcomes over the long-term. The MAL-ED cohort study was performed in eight low-income settings with the goal of evaluating relationships between the child’s environment and experience (dietary, illness, and pathogen exposure, among others) and their growth and development. The goal of this analysis is to determine whether there are differences in the factors associated with growth from 24 to 60 months using two different metrics. Methods Across six MAL-ED sites, 942 children had anthropometry data at 24 and 60 months, as well as information about socioeconomic status, maternal height, gut permeability (lactulose-mannitol z-score (LMZ)), dietary intake from 9 to 24 months, and micronutrient status. Anthropometric changes were in height- or weight-for-age z-score (HAZ, WAZ), their absolute difference from the growth standard median (HAD (cm), WAD (kg)), as well as recovery from stunting/underweight. Outcomes were modeled using multivariate regression. Results At 24 months, almost half of the cohort was stunted (45%) and 21% were underweight. Among those who were stunted at 24 months ( n  = 426), 185 (43%) were no longer stunted at 60 months. Most children increased their HAZ from 24 to 60 months (81%), whereas fewer (33%) had positive changes in their HAD. Linear regression models indicate that girls improved less than boys from 24 to 60 months (HAZ: -0.21 (95% CI -0.27, -0.15); HAD: -0.75 (-1.07, -0.43)). Greater intestinal permeability (higher LMZ) at 0–24 months was associated with lower relative and absolute changes from 24 to 60 months (HAZ: -0.10 (-0.16, -0.04); HAD: -0.47 (-0.73, -0.21)). Maternal height (per 10 cm) was positively associated with changes (HAZ: 0.09 (0.03, 0.15); HAD: 0.45 (0.15, 0.75)). Similar relationships were identified for changes in WAZ and WAD. Conclusions The study children demonstrated improved growth from 24 to 60 months of age, but only a subset had positive changes in HAD and WAD. The same environmental factors were associated with growth from 24 to 60 months regardless of metric used (change in HAZ or HAD, or WAZ and WAD).

BackgroundConvalescent plasma therapy (CPT) and remdesivir (REM) have been approved for investigational use to treat coronavirus disease 2019 (COVID-19) in Nepal. MethodsIn this prospective, multicentered study, we evaluated the safety and outcomes of treatment with CPT and/or REM in 1315 hospitalized COVID-19 patients over 18 years in 31 hospitals across Nepal. REM was administered to patients with moderate, severe, or life-threatening infection. CPT was administered to patients with severe to life-threatening infections who were at high risk for progression or clinical worsening despite REM. Clinical findings and outcomes were recorded until discharge or death. ResultsPatients were classified as having moderate (24.2%), severe (64%), or life-threatening (11.7%) COVID-19 infection. The majority of CPT and CPT + REM recipients had severe to life-threatening infections (CPT 98.3%; CPT + REM 92.1%) and were admitted to the intensive care unit (ICU; CPT 91.8%; CPT + REM 94.6%) compared with those who received REM alone (73.3% and 57.5%, respectively). Of 1083 patients with reported outcomes, 78.4% were discharged and 21.6% died. The discharge rate was 84% for REM (n = 910), 39% for CPT (n = 59), and 54.4% for CPT + REM (n = 114) recipients. In a logistic model comparing death vs discharge and adjusted for age, gender, steroid use, and severity, the predicted margin for discharge was higher for recipients of remdesivir alone (0.82; 95% CI, 0.79–0.84) compared with CPT (0.58; 95% CI, 0.47–0.70) and CPT + REM (0.67; 95% CI, 0.60–0.74) recipients. Adverse events of remdesivir and CPT were reported in <5% of patients. ConclusionsThis study demonstrates a safe rollout of CPT and REM in a resource-limited setting. Remdesivir recipients had less severe infection and better outcomes. ClinicalTrials.gov identifier. NCT04570982.

Design and configuration for wetlands treating greywater are usually based on literature data obtained from domestic wastewater operating wetlands. It is very important to determine proper criteria for design and configuration to provide efficiency and minimum maintenance, avoiding bad odour and clogging amongst others, ensuring the acceptance of householders. The aim of this work was to design a wetland system treating greywater for a household and determine whether the chosen criteria were appropriate. Some of the criteria taken into consideration for design and configuration were: quantitative and qualitative characteristics, desired removal of biochemical oxygen demand (BOD) and suspended solids (TSS), substrate and ornamental aspect of the system. The system was composed of a grease trap (kitchen), sedimentation tank, a horizontal flow constructed wetland (HF-CW), intermittent feeding system, and a vertical flow constructed wetland (VF-CW). The results showed that the suggested design and configuration were in accordance with the expected efficiency. Being a compact system, it was susceptible to peak flows, temporarily deteriorating the performance of the HF-CW. The hybrid system, however, showed to cope well with influent fluctuations. The overall performance of the system shows that the removal of turbidity, TSS, COD and BOD were over 88%, reaching 95% removal for both BOD and turbidity.