Complications During Hospitalization in Patients With SARS-CoV-2 Pneumonia in a Romanian Pulmonary Center.

Introduction The coronavirus disease (COVID-19) was declared a pandemic by the World Health Organization (WHO) on March 11, 2020. Facing a new and unknown virus, the entire medical community made considerable efforts to find a specific treatment, develop guidelines, and even create a vaccine. Besides all the measures taken, a wide range of complications associated with the disease increased the mortality and morbidity rates, adding more difficulty to the management of the patients. Study design We performed a retrospective study, including the patients with SARS-CoV-2 pneumonia who were admitted to our hospital between March 2020 and August 2021. We analyzed complications that developed during the hospitalization, such as respiratory failure or acute injury to other organs (the heart, pancreas, kidneys, and liver), and whether they were treatment- and hospitalization-related. Results One thousand eight hundred and forty-four cases were evaluated, and we analyzed the complications that developed during the hospitalization. Out of this, 1392 (75.48%) cases developed at least one complication during hospitalization, most frequently respiratory failure (71.14%), hyperglycemia (43.54%), renal injury (42.67%), or cardiovascular events (7.10%). Conclusion SARS-CoV-2 infection in hospitalized patients with pneumonia can cause injuries to any organ, making the management of those patients even more difficult.

Advanced near-zero waste treatment of food processing wastewater with water, carbon, and nutrient recovery.

A near-zero waste treatment system for food processing wastewater was developed and studied. The wastewater was treated using an anaerobic membrane bioreactor (AnMBR), polished using an outdoor photobioreactor for microalgae cultivation (three species were studied), and excess sludge was treated using hydrothermal carbonization. The study was conducted under arid climate conditions for one year (four seasons). The AnMBR reduced the total organic carbon by 97%, which was mostly recovered as methane (~57%) and hydrochar (~4%). Microalgal biomass productivity in the AnMBR effluent ranged from 0.25 to 0.8 g·L-1·day-1. Nitrogen (N) and phosphorous (P) uptake varied seasonally, from 18 to 45 mg·L-1·day-1 and up to 5 mg·L-1·day-1, respectively. N and P mass balance analysis demonstrated that the process was highly efficient in the recovery of nitrogen (~77%), and phosphorus (~91%). The performance of the microalgal culture changed among seasons because of climatic variation, as a result of variation in the wastewater chemistry, and possibly due to differences among the microalgal species. Effluent standards for irrigation use were met throughout the year and were achieved within two days in summer and 4.5 days in winter. Overall, the study demonstrated a near-zero waste discharge system capable of producing high-quality effluent, achieving nutrient and carbon recovery into microalgae biomass, and energy production as biogas and hydrochar.

Characterization and utilization of hydrothermal carbonization aqueous phase as nutrient source for microalgal growth.

This study investigated the feasibility of using hydrothermal carbonization (HTC) aqueous phase as an alternative nutrient source for microalgae cultivation, and the microalgae cultivation capability to treat this complex medium to a level enabling its reuse or discharge. HTC of activated sludge was optimized in terms of the energy content of the solid hydrochar and the nutrient content of the HTC aqueous phase adequate for microalgal growth. Growth rates of Coelastrella sp. and Chlorella sp. in the HTC aqueous phase based growth medium and a control medium (mBG11) were similar, indicating that the HTC aqueous phase does not inhibit the microalgae growth. Nitrogen and phosphorus concentrations were reduced by >90% and dissolved organic carbon by 80% after 6 days of cultivation, resulting in water quality suitable for reuse or discharge. This study confirms the microalgae high potential in a circular bio-economy to valorize wet bio-waste streams from various treatment methods.

A novel endogenous selection marker for the diatom Phaeodactylum tricornutum based on a unique mutation in phytoene desaturase 1.

Phaeodactylum tricornutum is a well-developed model diatom for both marine ecology and microalgal biotechnology, which has been enabled by the sequenced genome and the availability of gene delivery tools, such as biolistic transformation and E. coli-mediated conjugation. Till now, these tools have mainly relied on two selectable markers of bacterial origin which confer resistance to antibiotics Zeocin and nourseothricin. An alternative cost-effective and preferably endogenous selectable marker would facilitate gene stacking efforts through successive transformation or conjugation. We performed UV-mutagenesis of P. tricornutum to obtain mutations in the phytoene desaturase (PDS) gene, conferring resistance to the bleaching herbicide norflurazon. Two mutants displaying high tolerance to norflurazon and carrying unique mutations in PtPDS1 (PHATRDRAFT_45735) were selected. These mutants revealed novel point mutations at a conserved residue Gly290 to Ser/Arg. Homology-based structural modeling of mutated PDS1, over a resolved crystallographic model of rice PDS1 complexed with norflurazon, suggests steric hindrance by bulkier residue substitution may confer herbicide resistance. We report the characterization of PtPDS1 mutants and the development of the first endogenous selectable marker in diatoms suitable for industrial strain development, with the added benefit of biocontainment. The plasmid carrying the mutated PDS1 as a selection marker and eGFP as a reporter was created. An optimized biolistic transformation system is reported which allowed the isolation of positive transgenic events at the rate of 96.7%. Additionally, the ease of in vivo UV-mutagenesis may be employed as a strategy to create PDS-norflurazon-based selectable markers for other diatoms.

Are Thraustochytrids algae?

Thraustochytrids, a heterotrophic fungus-like clade of Stramenopiles, are becoming an increasingly important source of polyunsaturated fatty acids (PUFAs) for biotechnological industries. PUFA rich oils from these organisms are subsequently referred to in some literature and marketing sources as being derived from 'algae', in spite of their non-photosynthetic source organism. In this review, we attempt to disentangle the evolutionary relationship of the Thraustochytrids from other Protists, demonstrating that there is no scientific basis for the aforementioned misnomer. Some research has previously suggested that the ancestor of the Stramenopiles may have been photosynthetic, and subsequently lost their plastids in multiple lineages. The placement of the Thraustochytrids within the Stramenopiles and their possible plastid loss may have been a source of obfuscation. It is becoming increasingly evident that the common ancestor of the Stramenopiles was not photosynthetic, and that only the Ochrophyte lineage later engulfed a plastid via higher order endosymbiosis. Because all basal lineages of Stramenopiles are non-plastidial heterotrophs, including the Thraustochytrids, there remains no phylogenetic, biological, or ecological justification for the term 'algae' to be applied to Thraustochytrids or their products.

Cloning, molecular characterization, and phylogeny of two evolutionary distinct glutamine synthetase isoforms in the green microalga Haematococcus pluvialis (Chlorophyceae).

Haematococcus pluvialis (Chlorophyta) is a widely used microalga of great economic potential, yet its molecular genetics and evolution are largely unknown. We present new detailed molecular and phylogenetic analysis of two glutamine synthetase (GS) enzymes and genes (gln) under the Astaxanthin-inducing conditions of light- and nitrogen-stress. Structure analysis identified key residues and confirmed two decameric GS2 holoenzymes, a cytoplasmic enzyme, termed GS2c , and a plastidic form, termed GS2p , due to chloroplast-transit peptides at its N-terminus. Gene expression analysis showed dissociation of mRNA, protein, and enzyme activity levels for both GS2 under different growth conditions, indicating the strong post-transcriptional regulation. Data-mining identified novel and specified published gln genes from Prasinophyceae, Chlorophyta, Trebouxiophyceae, Charophyceae, Bryophyta, Lycopodiophyta, Spermatophyta, and Rhodophyta. Phylogenetic analysis found homologues to the cytosolic GS2c of H. pluvialis in all other photo- and non-photosynthetic Eukaryota. The chloroplastic GS2p was restricted to Chlorophyta, Bryophyta, some Proteobacteria and Fungii; no homologues were identified in Spermatophyta or other Eukaryota. This indicates two independent prokaryotic donors for these two gln genes in H. pluvialis. Combined phylogenetic analysis of GS, chl-b synthase, elongation factor, and light harvesting complex homologues project a newly refined model of Viridiplantae evolution. Herein, a GS1 evolved into the cytosolic GS2c and was passed on to all Eukaryota. Later, the chloroplastic GS2p entered the Archaeplastida lineage via a horizontal gene transfer at the divergence of Chlorophyta and Rhodophyta lineages. GS2p persisted in Chlorophyta and Bryophyta, but was lost during Spermatophyta evolution. These data suggest the revision of GS classification and nomenclature, and extend our understanding of the photosynthetic Eukaryota evolution.

Microalgae as a Source for VLC-PUFA Production.

Microalgae present a huge and still insufficiently tapped resource of very long-chain omega-3 and omega-6 polyunsaturated fatty acids (VLC-PUFA) for human nutrition and medicinal applications. This chapter describes the diversity of unicellular eukaryotic microalgae in respect to VLC-PUFA biosynthesis. Then, we outline the major biosynthetic pathways mediating the formation of VLC-PUFA by sequential desaturation and elongation of C18-PUFA acyl groups. We address the aspects of spatial localization of those pathways and elaborate on the role for VLC-PUFA in microalgal cells. Recent progress in microalgal genetic transformation and molecular engineering has opened the way to increased production efficiencies for VLC-PUFA. The perspectives of photobiotechnology and metabolic engineering of microalgae for altered or enhanced VLC-PUFA production are also discussed.

The complete sequence of the chloroplast genome of the green microalga Lobosphaera (Parietochloris) incisa.

We hereby report the complete chloroplast genome sequence of the green unicellular alga Lobosphaera (Parietochloris) incisa (strain SAG 2468). The genome consists of a circular chromosome of 156,028 bp, which is 72% A-T rich and does not contain a large rRNA-encoding inverted repeat. It is predicted to encode a total of 111 genes including 78 protein-coding, three rRNA, and 30 tRNA genes. The genome sequence also carries a self-splicing group I intron and a group II intron remnant. Overall, the gene and intron content of the L. incisa chloroplast genome is highly similar to that of other species of Trebouxiophyceae. In contrast, the L. incisa chloroplast genome harbors 88 copies of various intergenic dispersed DNA repeat sequences that are all unique to L. incisa.

New methodology for quantifying the effects of perennials on their patch productivity in semi-arid environments.

The correlations between perennials and the herbaceous productivity in patches occupied by them were previously studied and several descriptive models were defined. Yet these studies focused on either single or several species without analyzing higher numbers and ranking their effects. Here we describe a handy analytical methodology which allows separating the effects of each perennial species on herbaceous productivity at its respective patches from those of the others in a given area, even in case of complex patches containing several species. The described methodology also allows analysts to correlate the effect of perennials to their patch sizes and the respective herbaceous biomass. Additional mathematical analysis presented here succeeded in differentiating between the perennial species stand-alone presence effect on the herbaceous productivity and that attributed to the canopy size. In addition, the effects of location along the slope and its rockiness outlines were studied. As a case study, we chose representative sloped shrubland with rockiness outlines, located in Yattir farm, Northern Negev, Israel. Based on the described analyses we found that the species with the highest positive effects on the herbaceous productivity were Echinops polyceras, Echium angustifolium, and Salvia lanigera. Contradictory effects were observed in case of Thymelea hirsute, Anchusa ramosus, and Noaea mucronata. Collectively, the presented methodology could be an important management tool for monitoring the herbaceous biomass amounts in a given shrubland.

Downregulation of a putative plastid PDC E1α subunit impairs photosynthetic activity and triacylglycerol accumulation in nitrogen-starved photoautotrophic Chlamydomonas reinhardtii.

The chloroplast pyruvate dehydrogenase complex (cpPDC) catalyses the oxidative decarboxylation of pyruvate forming acetyl-CoA, an immediate primer for the initial reactions of de novo fatty acid (FA) synthesis. Little is known about the source of acetyl-CoA in the chloroplasts of photosynthetic microalgae, which are capable of producing high amounts of the storage lipid triacylglycerol (TAG) under conditions of nutrient stresses. We generated Chlamydomonas reinhardtii CC-1618 mutants with decreased expression of the PDC2_E1α gene, encoding the putative chloroplast pyruvate dehydrogenase subunit E1α, using artificial microRNA. A comparative study on the effects of PDC2_E1α silencing on FAs and TAG production in C. reinhardtii, grown photoautotrophically and mixotrophically, with and without a nitrogen source in the nutrient medium, was carried out. Reduced expression of PDC2 _E1α led to a severely hampered photoautotrophic growth phenotype with drastic impairment in TAG accumulation under nitrogen deprivation. In the presence of acetate, downregulation of PDC2_E1α exerted little to no effect on TAG production and photosynthetic activity. In contrast, under photoautotrophic conditions, especially in the absence of a nitrogen source, a dramatic decline in photosynthetic oxygen evolution and photosystem II quantum yield against a background of the apparent over-reduction of the photosynthetic electron chain was recorded. Our results suggest an essential role of cpPDC in the supply of carbon precursors for de novo FA synthesis in microalgae under conditions of photoautotrophy. A shortage of this supply is detrimental to the nitrogen-starvation-induced synthesis of storage TAG, an important carbon and energy sink in stressed Chlamydomonas cells, thereby impairing the acclimation ability of the microalga.

Development of a nuclear transformation system for Oleaginous Green Alga Lobosphaera (Parietochloris) incisa and genetic complementation of a mutant strain, deficient in arachidonic acid biosynthesis.

Microalgae are considered a promising source for various high value products, such as carotenoids, ω-3 and ω-6 polyunsaturated fatty acids (PUFA). The unicellular green alga Lobosphaera (Parietochloris) incisa is an outstanding candidate for the efficient phototrophic production of arachidonic acid (AA), an essential ω-6 PUFA for infant brain development and a widely used ingredient in the baby formula industry. Although phototrophic production of such algal products has not yet been established, estimated costs are considered to be 2-5 times higher than competing heterotrophic production costs. This alga accumulates unprecedented amounts of AA within triacylglycerols and the molecular pathway of AA biosynthesis in L. incisa has been previously elucidated. Thus, progress in transformation and metabolic engineering of this high value alga could be exploited for increasing the efficient production of AA at competitive prices. We describe here the first successful transformation of L. incisa using the ble gene as a selection marker, under the control of the endogenous RBCS promoter. Furthermore, we have succeeded in the functional complementation of the L. incisa mutant strain P127, containing a mutated, inactive version of the delta-5 (Δ5) fatty acid desaturase gene. A copy of the functional Δ5 desaturase gene, linked to the ble selection marker, was transformed into the P127 mutant. The resulting transformants selected for zeocine resistant, had AA biosynthesis partially restored, indicating the functional complementation of the mutant strain with the wild-type gene. The results of this study present a platform for the successful genetic engineering of L. incisa and its long-chain PUFA metabolism.

The effects of long time conservation of heavily grazed shrubland: a case study in the Northern Negev, Israel.

One of the major reasons for desertification is unrestricted grazing leading to vegetation depletion, soil erosion and degradation, phenomena often considered irreversible in the short term. Here, we compare soil and biological parameters of degraded and conserved, recently rehabilitated arid shrubland in the Northern Negev, Israel. The study area was restored by conservation efforts including a strictly controlled grazing regime initiated in 1992. The visually recognizable improvement in the ecology of the restored shrubland is reflected in significant improvement in all examined biotic (herbaceous biomass, shrub patch density, and insect activity), and soil parameters (nutrients, organic matter content, moisture, and water infiltration). The difference is created predominantly by restoration of large biological patches composed of shrubs and other perennial plants often associated with ant or termite nests, where the most significant increases in productivity and soil quality were observed. In the conserved shrubland such patches covered 35 or 25 % of the area (in a normal and a drought year, respectively). In the degraded shrubland 5 % or less of the area was occupied by such patches that were much smaller and of lower biological complexity. With respect to plant biodiversity, six plant species were found only-and 18 others became significantly more common-in the rehabilitated area. The results of this article indicate that functional arid drylands can be restored within <16 years relying on strict conservation management with reduced grazing intensity.

PCR amplification and DNA sequence of mcyA gene: the distribution profile of a toxigenic Microcystis aeruginosa in the Hartbeespoort Dam, South Africa.

Using new polymerase chain reaction (PCR) primers, a once known to be under-transcribed microcystin synthetase A (mcyA) gene from the only known toxigenic cyanobacterium Microcystis aeruginosa dominating the Hartbeespoort Dam was consistently amplified from genomic DNA extracted from a set of algal and cell free water samples collected across this dam. In addition to this, five more mcy genes (mcyBCDEG) were also amplified during this study. The resultant mcyA PCR products (518 bp) were purified and sequenced and gave nucleotide sequence segments of 408 bp sizes. The obtained sequence was aligned to the published mcyA gene sequence available online on the NCBI database and resulted in 100% similarity to a 408 bp mcyA gene sequence segment of M. aeruginosa UWOCC RID-1. Furthermore, it was found that the above sequence segment (408 bp) spans from a common base in M. aeruginosa PCC 7806 and M. aeruginosa PCC 7820 from 141 to 548 bp in the N-methyl transferase (NMT) region signifying their closer relatedness to M. aeruginosa UWOCC strains. This study has for the first time amplified mcyA gene consistently from both intracellular and extracellular DNA extracts obtained from algal and cell free water samples, respectively. Sequence data and the amplified mcy genes showed that M. aeruginosa is widely distributed and dominant in this dam.

LIGHT-INDUCED OIL GLOBULE MIGRATION IN HAEMATOCOCCUS PLUVIALIS (CHLOROPHYCEAE).

Astaxanthin-rich oil globules in Haematococcus pluvialis display rapid light-induced peripheral migration that is unique to this organism and serves to protect the photosynthetic system from excessive light. We observed rapid light-induced peripheral migration that is associated with chlorophyll fluorescence quenching, whereas the recovery was slow. A simple assay to follow globule migration, based on chlorophyll fluorescence level has been developed. Globule migration was induced by high intensity blue light, but not by high intensity red light. The electron transport inhibitor dichlorophenyl-dimethylurea did not inhibit globule migration, whereas the quinone analog (dibromo-methyl-isopropylbenzoquinone), induced globule migration even at low light. Actin microfilament-directed toxins, such as cytochalasin B and latrunculin A, inhibited the light-induced globule migration, whereas toxins against microtubules were ineffective. Electron microscopic (EM) imaging confirmed the cytoplasmic localization and peripheral migration of globules upon exposure to very high light (VHL). Scanning EM of freeze-fractured cells also revealed globules within cytoplasmic bridges traversing the chloroplast, presumably representing the pathway of migration. Close alignments of globules with endoplasmic reticulum (ER) membranes were also observed following VHL illumination. We propose that light-induced globule migration is regulated by the redox state of the photosynthetic electron transport system. Possible mechanisms of actin-based globule migration are discussed.

Cloning and molecular characterization of a novel acyl-CoA:diacylglycerol acyltransferase 1-like gene (PtDGAT1) from the diatom Phaeodactylum tricornutum.

We have identified and isolated a cDNA encoding a novel acyl-CoA:diacylglycerol acyltransferase (DGAT)1-like protein, from the diatom microalga Phaeodactylum tricornutum (PtDGAT1). The full-length cDNA sequences of PtDGAT1 transcripts revealed that two types of mRNA, PtDGAT1short and PtDGAT1long, were transcribed from the single PtDGAT1 gene. PtDGAT1short encodes a 565 amino acid sequence that is homologous to several functionally characterized higher plant DGAT1 proteins, and 55% identical to the putative DGAT1 of the diatom Thalassiosira pseudonana, but shows little homology with other available putative and cloned algal DGAT sequences. PtDGAT1long lacks several catalytic domains, owing to a 63-bp nucleotide insertion in the mRNA containing a stop codon. Alternative splicing consisting of intron retention appears to regulate the amount of active DGAT1 produced, providing a possible molecular mechanism for increased triacylglycerol (TAG) biosynthesis in P. tricornutum under nitrogen starvation. DGAT mediates the last committed step in TAG biosynthesis, so we investigated the changes in expression levels of the two types of mRNA following nitrogen starvation inducing TAG accumulation. The abundance of both transcripts was markedly increased under nitrogen starvation, but much less so for PtDGAT1short. PtDGAT1 activity of PtDGAT1short was confirmed in a heterologous yeast transformation system by restoring DGAT activity in a Saccharomyces cerevisiae neutral lipid-deficient quadruple mutant strain (H1246), resulting in lipid body formation. Lipid body formation was only restored upon the expression of PtDGAT1short, and not of PtDGAT1long. The recombinant yeast appeared to display a preference for incorporating saturated C(16) and C(18) fatty acids into TAG.

Isolation of a novel oil globule protein from the green alga Haematococcus pluvialis (Chlorophyceae).

Cytoplasmic oil globules of Haematococcus pluvialis (Chlorophyceae) were isolated and analyzed for pigments, lipids and proteins. Astaxanthin appeared to be the only pigment deposited in the globules. Triacyglycerols were the main lipids (more than 90% of total fatty acids) in both the cell-free extract and in the oil globules. Lipid profile analysis of the oil globules showed that relative to the cell-free extract, they were enriched with extraplastidial lipids. A fatty acids profile revealed that the major fatty acids in the isolated globules were oleic acid (18:1) and linoleic acid (18:2). Protein extracts from the globules revealed seven enriched protein bands, all of which were possible globule-associated proteins. A major 33-kDa globule protein was partially sequenced by MS/MS analysis, and degenerate DNA primers were prepared and utilized to clone its encoding gene from cDNA extracted from cells grown in a nitrogen depleted medium under high light. The sequence of this 275-amino acid protein, termed the Haematococcus Oil Globule Protein (HOGP), revealed partial homology with a Chlamydomonas reinhardtii oil globule protein and with undefined proteins from other green algae. The HOGP transcript was barely detectable in vegetative cells, but its level increased by more than 100 fold within 12 h of exposure to nitrogen depletion/high light conditions, which induced oil accumulation. HOGP is the first oil-globule-associated protein to be identified in H. pluvialis, and it is a member of a novel gene family that may be unique to green microalgae.