RNA circles with minimized immunogenicity as potent PKR inhibitors.

Exon back-splicing-generated circular RNAs, as a group, can suppress double-stranded RNA (dsRNA)-activated protein kinase R (PKR) in cells. We have sought to synthesize immunogenicity-free, short dsRNA-containing RNA circles as PKR inhibitors. Here, we report that RNA circles synthesized by permuted self-splicing thymidylate synthase (td) introns from T4 bacteriophage or by Anabaena pre-tRNA group I intron could induce an immune response. Autocatalytic splicing introduces ∼74 nt td or ∼186 nt Anabaena extraneous fragments that can distort the folding status of original circular RNAs or form structures themselves to provoke innate immune responses. In contrast, synthesized RNA circles produced by T4 RNA ligase without extraneous fragments exhibit minimized immunogenicity. Importantly, directly ligated circular RNAs that form short dsRNA regions efficiently suppress PKR activation 103- to 106-fold higher than reported chemical compounds C16 and 2-AP, highlighting the future use of circular RNAs as potent inhibitors for diseases related to PKR overreaction.

DesC1 and DesC2, Δ9 Fatty Acid Desaturases of Filamentous Cyanobacteria: Essentiality and Complementarity.

DesC1 and DesC2, which are fatty acid desaturases found in cyanobacteria, are responsible for introducing a double bond at the Δ9 position of fatty-acyl chains, which are subsequently esterified to the sn-1 and sn-2 positions of the glycerol moiety, respectively. However, since the discovery of these two desaturases in the Antarctic cyanobacterium Nostoc sp. SO-36, no further research has been reported. This study presents a comprehensive characterization of DesC1 and DesC2 through targeted mutagenesis and transformation using two cyanobacteria strains: Anabaena sp. PCC 7120, comprising both desaturases, and Synechocystis sp. PCC 6803, containing a single Δ9 desaturase (hereafter referred to as DesCs) sharing similarity with DesC1 in amino acid sequence. The results suggested that both DesC1 and DesC2 were essential in Anabaena sp. PCC 7120 and that DesC1, but not DesC2, complemented DesCs in Synechocystis sp. PCC 6803. In addition, DesC2 from Anabaena sp. PCC 7120 desaturated fatty acids esterified to the sn-2 position of the glycerol moiety in Synechocystis sp. PCC 6803.

Role of exopolysaccharides of Anabaena sp. in desiccation tolerance and biodeterioration of ancient terracotta monuments of Bishnupur.

Colonization of the cyanobacteria in the Bishnupur terracotta temples, one of the heritage sites of West Bengal, India is in an alarming state of deterioration now. Among the cyanobacteria Anabaena sp. (VBCCA 052002) has been isolated from most of the crust samples of terracotta monuments of Bishnupur. The identification was done using micromorphological characters and confirmed by 16S rRNA gene sequencing. The isolated strain produces enormous exopolysaccharides, which are extracted, hydrolyzed, and analyzed by HPLC. We have studied desiccation tolerance in this cyanobacterium and found biosynthesis of trehalose with an increase in durations of desiccation. The in vitro experiment shows that Chlorophyll-a and carotenoid content increase with fourteen days of desiccation, and cellular carbohydrates increase continuously. However, cellular protein decreases with desiccation. To gain insights into the survival strategies and biodeterioration mechanisms of Anabaena sp. in the desiccated conditions of the Bishnupur monuments, the present study focuses on the physiological aspects of the cyanobacteria under controlled in vitro conditions. Our study indicates that in desiccation conditions, trehalose biosynthesis takes place in Anabaena sp. As a result of the excessive sugar and polysaccharide produced, it adheres to the surface of the terracotta structure. The continuous contraction and expansion of these polysaccharides contribute to the biodeterioration of these monuments.

Screening high-efficiency promoter to construct trans-vp28 gene Anabaena sp. PCC7120 against white spot syndrome virus of Litopenaeus vannamei.

This study aimed to select high-quality promoters to construct trans-vp28 gene Anabaena sp. PCC7120 and feed Litopenaeus vannamei to assess the effect of L.vannamei against white spot syndrome virus (WSSV). Transgenic algae were created using five plasmids containing PrbcL, Pcpc560, Ptrc, Ptac, and PpsbA. According to the gene expression efficiency and the growth index of transgenic algae, Pcpc560 was determined to be the most efficient promoter. Shrimps were continuously fed trans-vp28 gene Anabaena sp. PCC7120 for one week and then challenged with WSSV. After the challenge, the transgenic algae group (vp28-7120 group) was continuously immunized [continuous immunization for 0 days (vp28-7120-0d); continuous immunization for 2 days (vp28-7120-2d); continuous immunization for 4 days (vp28-7120-4d)]. After seven days, the daily survival rate of each experimental group was continuously tracked. Following the viral challenge, the hepatopancreas samples were assayed for their levels of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), thioredoxin peroxidase (TPX), acid phosphatase (ACP), and alkaline phosphatase (AKP) at varying time intervals. In comparison to the positive control group (challenge and no vaccination) and the wild-type group (challenge, fed wild-type Anabaena sp. PCC7120), the vp28-7120 group (challenge, fed trans-vp28 gene Anabaena sp. PCC7120) exhibited a remarkable increase in survival rates, reaching 50 % (vp28-7120-0d), 76.67 % (vp28-7120-2d), and 80 % (vp28-7120-4d). Furthermore, the vp28-7120 group consistently displayed significantly higher activities of SOD, CAT, GSH-Px, ACP, and AKP, while exhibiting notably lower TPX activity, when compared to the control group. These results indicate that the Pcpc560 promoter effectively elevated the expression level of the exogenous vp28 gene and spurred the growth of the trans-vp28 gene Anabaena sp. PCC7120. Consequently, trans-vp28 gene Anabaena sp. PCC7120 significantly bolstered the immunity of L.vannamei. Therefore, utilizing the Pcpc560 promoter to develop trans-vp28 gene Anabaena sp. PCC7120 based oral vaccine is highly beneficial for industrial-scale cultivation, advancing its commercialization prospects.

Chlorpyrifos enrichment enhances tolerance of Anabaena sp. PCC 7119 to dimethoate.

Organophosphorus (OP) insecticides are widely used for on-field pest control, constituting about 38% of global pesticide consumption. Insecticide tolerance has been recorded in microorganisms isolated from the contaminated soil. However, the cross-tolerance of laboratory-enriched cultures remains poorly understood. A chlorpyrifos tolerant (T) strain of Anabaena sp. PCC 7119 was developed through continuous enrichment of the wild strain (W). The cross-tolerance of the T strain to the OP insecticide dimethoate was assessed by measuring photosynthetic performance, key enzyme activities and degradation potential. The presence of dimethoate led to a significant reduction in the growth and pigment content of the W strain. In contrast, the T strain demonstrated improved growth and metabolic performance. Chl a and carotenoids were degraded faster than phycobiliproteins in both strains. The T strain exhibited superior photosynthetic performance, metabolic efficiency and photosystem functions, than of W strain, at both the tested dimethoate concentrations (100 and 200 µM). The treated T strain had more or less a normal OJIP fluorescence transient and bioenergetic functions, while the W strain showed a greater fluorescence rise at ≤ 300 µs indicating the inhibition of electron donation to PS II, and at 2 ms due to reduced electron release beyond QA. The T strain had significantly higher levels of esterase and phosphatases, further enhanced by insecticide treatment. Dimethoate degradation efficiency of the T strain was significantly higher than of the W strain. T strain also removed chlorpyrifos more efficiently than W strain at both the tested concentrations. The BCFs of both chlorpyrifos and dimethoate were lower in the T strain compared to the W strain. These findings suggest that the enriched strain exhibits promising results in withstanding dimethoate toxicity and could be explored for its potential as a bioremediating organism for OP degradation.

Comparative Network Biology Discovers Protein Complexes That Underline Cellular Differentiation in Anabaena sp.

The filamentous cyanobacterium Anabaena sp. PCC 7120 can differentiate into heterocysts to fix atmospheric nitrogen. During cell differentiation, cellular morphology and gene expression undergo a series of significant changes. To uncover the mechanisms responsible for these alterations, we built protein-protein interaction (PPI) networks for these two cell types by cofractionation coupled with mass spectrometry. We predicted 280 and 215 protein complexes, with 6322 and 2791 high-confidence PPIs in vegetative cells and heterocysts, respectively. Most of the proteins in both types of cells presented similar elution profiles, whereas the elution peaks of 438 proteins showed significant changes. We observed that some well-known complexes recruited new members in heterocysts, such as ribosomes, diflavin flavoprotein, and cytochrome c oxidase. Photosynthetic complexes, including photosystem I, photosystem II, and phycobilisome, remained in both vegetative cells and heterocysts for electron transfer and energy generation. Besides that, PPI data also reveal new functions of proteins. For example, the hypothetical protein Alr4359 was found to interact with FraH and Alr4119 in heterocysts and was located on heterocyst poles, thereby influencing the diazotrophic growth of filaments. The overexpression of Alr4359 suspended heterocyst formation and altered the pigment composition and filament length. This work demonstrates the differences in protein assemblies and provides insight into physiological regulation during cell differentiation.

Biosorption Ability of Pharmaceutically Active Compounds by Anabaena sp. and Chroococcidiopsis thermalis.

Drug overuse harms the biosphere, leading to disturbances in ecosystems' functioning. Consequently, more and more actions are being taken to minimise the harmful impact of xenopharmaceuticals on the environment. One of the innovative solutions is using biosorbents-natural materials such as cells or biopolymers-to remove environmental pollutants; however, this focuses mainly on the removal of metal ions and colourants. Therefore, this study investigated the biosorption ability of selected pharmaceuticals-paracetamol, diclofenac, and ibuprofen-by the biomass of the cyanobacteria Anabaena sp. and Chroococcidiopsis thermalis, using the LC-MS/MS technique. The viability of the cyanobacteria was assessed by determining photosynthetic pigments in cells using a UV-VIS spectrophotometer. The results indicate that both tested species can be effective biosorbents for paracetamol and diclofenac. At the same time, the tested compounds did not have a toxic effect on the tested cyanobacterial species and, in some cases, stimulated their cell growth. Furthermore, the Anabaena sp. can effectively biotransform DCF into its dimer.

Muscle cell proliferation using water-soluble extract from nitrogen-fixing cyanobacteria Anabaena sp. PCC 7120 for sustainable cultured meat production.

Muscle cell cultivation, specifically the culture of artificial meat from livestock-derived cells in serum-free media is an emerging technology and has attracted much attention. However, till now, the high cost of production and environmental load have been significant deterrents. This study aims to provide an alternate growth-promoting substance that is free from animal derivatives and lowers nitrogen pollution. We have extracted water-soluble compounds from the filamentous nitrogen-fixing cyanobacteria Anabaena sp. PCC 7120 by the ultrasonication method. The heat-inactivated and molecular weight separation experiments were conducted to identify the bioactive compound present in the extract. Finally, the compounds soluble in water (CW) containing the water-soluble pigment protein, phycocyanin as a bioactive compound, was added as a growth supplement to cultivate muscle cells such as C2C12 muscle cells and quail muscle clone 7 (QM7) cells to analyze the effectiveness of the extract. The results indicated that CW had a positive role in muscle cell proliferation. A three-dimensional (3-D) cell-dense structure was fabricated by culturing QM7 cells using the extract. Furthermore, the nitrogen-fixing cyanobacterial extract has vast potential for cultured meat production without animal sera in the near future.

Functional characterization of two WD40 family proteins, Alr0671 and All2352, from Anabaena PCC 7120 and deciphering their role in abiotic stress management.

WD40 domain-containing proteins are one of the eukaryotes' most ancient and ubiquitous protein families. Little is known about the presence and function of these proteins in cyanobacteria in general and Anabaena in particular. In silico analysis confirmed the presence of WD40 repeats. Gene expression analysis indicated that the transcript levels of both the target proteins were up-regulated up to 4 fold in Cd and drought and 2-3 fold in heat, salt, and UV-B stress. Using a fluorescent oxidative stress indicator, we showed that the recombinant proteins were scavenging reactive oxygen species (ROS) (4-5 fold) more efficiently than empty vectors. Chromatin immunoprecipitation analysis (ChIP) and electrophoretic mobility shift assay (EMSA) revealed that the target proteins function as transcription factors after binding to the promoter sequences. The presence of kinase activity (2-4 fold) in the selected proteins indicated that these proteins could modulate the functions of other cellular proteins under stress conditions by inducing phosphorylation of specific amino acids. The chosen proteins also demonstrated interaction with Zn, Cd, and Cu (1.4-2.5 fold), which might stabilize the proteins' structure and biophysical functions under multiple abiotic stresses. The functionally characterized Alr0671 and All2352 proteins act as transcription factors and offer tolerance to agriculturally relevant abiotic stresses.

Alr0671 and All2352 are novel WD40 proteins of Anabaena capable of regulating biochemical functions and abiotic stress tolerance by acting as a transcription factor and mediating DNA-protein interaction.

Differential catalase activity and tolerance to hydrogen peroxide in the filamentous cyanobacteria Nostoc punctiforme ATCC 29133 and Anabaena sp. PCC 7120.

Photoautotrophic cyanobacteria often confront hydrogen peroxide (H2O2), a reactive oxygen species potentially toxic to cells when present in sufficiently high concentrations. In this study, H2O2 tolerance ability of filamentous cyanobacteria Nostoc punctiforme ATCC 29133 (Nostoc 29133) and Anabaena sp. PCC 7120 (Anabaena 7120) was investigated at increasing concentrations of H2O2 (0-0.5 mM). In Nostoc 29133, 0.25 and 0.5 mM H2O2 caused a reduction in chlorophyll a content by 12 and 20%, respectively, whereas with similar treatments, a total loss of chlorophyll a was detected in Anabaena 7120. Further, Nostoc 29133 was able to maintain its photosystem II performance in the presence of H2O2 up to a concentration of 0.5 mM, whereas in Anabaena 7120, 0.25 mM H2O2 caused a complete reduction of photosystem II performance. The intracellular hydroperoxide level (indicator of oxidative status) did not increase to the same high level in Nostoc 29133, as compared to in Anabaena 7120 after H2O2 treatment. This might be explained by that Nostoc 29133 showed a 20-fold higher intrinsic constitutive catalase activity than Anabaena 7120, thus indicating that the superior tolerance of Nostoc 29133 to H2O2 stems from its higher ability to decompose H2O2. It is suggested that difference in H2O2 tolerance between closely related filamentous cyanobacteria, as revealed in this study, may be taken into account for judicious selection and effective use of strains in biotechnological applications.

In Vivo Temperature Dependency of Molybdenum and Vanadium Nitrogenase Activity in the Heterocystous Cyanobacteria Anabaena variabilis.

The reduction of atmospheric dinitrogen by nitrogenase is a key component of terrestrial nitrogen cycling. Nitrogenases exist in several isoforms named after the metal present within their active center: the molybdenum (Mo), the vanadium (V), and the iron (Fe)-only nitrogenase. While earlier in vitro studies hint that the relative contribution of V nitrogenase to total BNF could be temperature-dependent, the effect of temperature on in vivo activity remains to be investigated. In this study, we characterize the in vivo effect of temperature (3-42 °C) on the activities of Mo nitrogenase and V nitrogenase in the heterocystous cyanobacteria Anabaena variabilis ATTC 29413 using the acetylene reduction assay by cavity ring-down absorption spectroscopy. We demonstrate that V nitrogenase becomes as efficient as Mo nitrogenase at temperatures below 10-15 °C. At temperatures above 22 °C, BNF seems to be limited by O2 availability to respiration in both enzymes. Furthermore, Anabaena variabilis cultures grown in Mo or V media achieved similar growth rates at temperatures below 20 °C. Considering the average temperature on earth is 15 °C, our findings further support the role of V nitrogenase as a viable backup enzymatic system for BNF in natural ecosystems.

A Glimpse at Siderophores Production by Anabaena flos-aquae UTEX 1444.

In this study, a strain of Anabaena flos-aquae UTEX 1444 was cultivated in six different concentrations of iron (III). Cultures were extracted with organic solvents and analyzed using our dereplication strategy, based on the combined use of high-resolution tandem mass spectrometry and molecular networking. The analysis showed the presence of the siderophores' family, named synechobactins, only in the zero iron (III) treatment culture. Seven unknown synechobactin variants were present in the extract, and their structures have been determined by a careful HRMS/MS analysis. This study unveils the capability of Anabaena flos-aquae UTEX 1444 to produce a large array of siderophores and may be a suitable model organism for a sustainable scale-up exploitation of such bioactive molecules, for the bioremediation of contaminated ecosystems, as well as in drug discovery.

Quantitative Proteomics Reveals the Protein Regulatory Network of Anabaena sp. PCC 7120 under Nitrogen Deficiency.

Anabaena sp. PCC 7120 (Anabaena 7120) is a photoautotrophic filamentous cyanobacterium capable of fixing atmospheric nitrogen. It is a model organism used for studying cell differentiation and nitrogen fixation. Under nitrogen deficiency, Anabaena 7120 forms specialized heterocysts capable of nitrogen fixation. However, the molecular mechanisms involved in the cyanobacterial adaptation to nitrogen deficiency are not well understood. Here, we employed a label-free quantitative proteomic strategy to systematically investigate the nitrogen deficiency response of Anabaena 7120 at different time points. In total, 363, 603, and 669 proteins showed significant changes in protein abundance under nitrogen deficiency for 3, 12, and 24 h, respectively. With mapping onto metabolic pathways, we revealed proteomic perturbation and regulation of carbon and nitrogen metabolism in response to nitrogen deficiency. Functional analysis confirmed the involvement of nitrogen stress-responsive proteins in biological processes, including nitrogen fixation, photosynthesis, energy and carbon metabolism, and heterocyst development. The expression of 10 proteins at different time points was further validated by using multiple reaction monitoring assays. In particular, many dysregulated proteins were found to be time-specific and involved in heterocyst development, providing new candidates for future functional studies in this model cyanobacterium. These results provide novel insights into the molecular mechanisms of nitrogen stress responses and heterocyst development in Anabaena 7120.

The CRP-family transcriptional regulator DevH regulates expression of heterocyst-specific genes at the later stage of differentiation in the cyanobacterium Anabaena sp. strain PCC 7120.

Heterocysts are terminally differentiated cells of filamentous cyanobacteria, which are specialized for nitrogen fixation. Because nitrogenase is easily inactivated by oxygen, the intracellular environment of heterocysts is kept microoxic. In heterocysts, the oxygen-evolving photosystem II is inactivated, a heterocyst-specific envelope with an outer polysaccharide layer and an inner glycolipid layer is formed to limit oxygen entry, and oxygen consumption is activated. Heterocyst differentiation, which is accompanied by drastic morphological and physiological changes, requires strictly controlled gene expression systems. Here, we investigated the functions of a CRP-family transcriptional regulator, DevH, in the process of heterocyst differentiation. A devH-knockdown strain, devH-kd, was created by replacing the original promoter with the gifA promoter, which is repressed during heterocyst differentiation. Although devH-kd formed morphologically distinct cells with the heterocyst envelope polysaccharide layer, it was unable to grow diazotrophically. Genes involved in construction of the microoxic environment, such as cox operons and the hgl island, were not upregulated in devH-kd. Moreover, expression of the nif gene cluster was completely abolished. Although CnfR was expressed in devH-kd, the nif gene cluster was not induced even under microoxic conditions. Thus, DevH is necessary for the establishment of a microoxic environment and induction of nitrogenase in heterocysts.

Novel molecular aspects of the CRISPR backbone protein 'Cas7' from cyanobacteria.

The cyanobacterium Anabaena PCC 7120 shows the presence of Type I-D CRISPR system that can potentially confer adaptive immunity. The Cas7 protein (Alr1562), which forms the backbone of the type I-D surveillance complex, was characterized from Anabaena. Alr1562, showed the presence of the non-canonical RNA recognition motif and two intrinsically disordered regions (IDRs). When overexpressed in E. coli, the Alr1562 protein was soluble and could be purified by affinity chromatography, however, deletion of IDRs rendered Alr1562 completely insoluble. The purified Alr1562 was present in the dimeric or a RNA-associated higher oligomeric form, which appeared as spiral structures under electron microscope. With RNaseA and NaCl treatment, the higher oligomeric form converted to the lower oligomeric form, indicating that oligomerization occurred due to the association of Alr1562 with RNA. The secondary structure of both these forms was largely similar, resembling that of a partially folded protein. The dimeric Alr1562 was more prone to temperature-dependent aggregation than the higher oligomeric form. In vitro, the Alr1562 bound more specifically to a minimal CRISPR unit than to the non-specific RNA. Residues required for binding of Alr1562 to RNA, identified by protein modeling-based approaches, were mutated for functional validation. Interestingly, these mutant proteins, showing reduced ability to bind RNA were predominantly present in dimeric form. Alr1562 was detected with specific antiserum in Anabaena, suggesting that the type I-D system is expressed and may be functional in vivo. This is the first report that describes the characterization of a Cas protein from any photosynthetic organism.

Isolation, biosynthesis and antimicrobial activity of gold nanoparticles produced with extracts of Anabaena spiroides.

Multidrug-resistant (MDR) pathogenic bacteria have become dangerous in bringing sporadic outbreaks in public health and nosocomial spreads from the addition of antibacterials/antibiotics continually. Obviously, the pharmacy world is in search of antibacterials that would be invincible by the evolved bacteria. Green synthesis of gold-nanoparticles (AuNps) was focused on the use of aqueous chloroauric acid (HAuCl4) and cell-free aqueous extract of the N2-fixing cyanobacterium (blue-green alga) Anabaena spiroides collected from a brackish-water, Bay of Bengal at Puri, Odisha; green-synthesized AuNps could be used as antibacterials against MDR bacteria. The synthesized AuNps were subjected to the following characterizations, UV-Vis spectrophotometry, SEM-EDX, XRD and ART-FTIR analysis. An absorption peak at 538 nm by UV-Vis spectrophotometry and the FTIR analysis confirmed the presence of AuNps. A. spiroides-AuNps were monitored for antibacterial activities against MDR pathogenic bacterial strains isolated from clinical samples, namely, Klebsiella oxytoca, MRSA and Streptococcus pyogenes, in vitro; the individual antibiograms of those bacteria were known. The recorded MIC dose values were 25, 20 and 30 mg A. spiroides-AuNps (As-AuNps) against K. oxytoca, MRSA and S. pyogenes, in vitro, respectively. Thus, As-AuNps bear promises as possible antibacterials, in future.

Crystal structure of Alr1298, a pentapeptide repeat protein from the cyanobacterium Nostoc sp. PCC 7120, determined at 2.1 Å resolution.

Nostoc sp. PCC 7120 are filamentous cyanobacteria capable of both oxygenic photosynthesis and nitrogen fixation, with the latter taking place in specialized cells known as heterocysts that terminally differentiate from vegetative cells under conditions of nitrogen starvation. Cyanobacteria have existed on earth for more than 2 billion years and are thought to be responsible for oxygenation of the earth's atmosphere. Filamentous cyanobacteria such as Nostoc sp. PCC 7120 may also represent the oldest multicellular organisms on earth that undergo cell differentiation. Pentapeptide repeat proteins (PRPs), which occur most abundantly in cyanobacteria, adopt a right-handed quadrilateral ß-helical structure, also referred to as a repeat five residue (Rfr) fold, with four-consecutive pentapeptide repeats constituting a single coil in the ß-helical structure. PRPs are predicted to exist in all compartments within cyanobacteria including the thylakoid and cell-wall membranes as well as the cytoplasm and thylakoid periplasmic space. Despite their intriguing structure and importance to understanding ancient cyanobacteria, the biochemical function of PRPs in cyanobacteria remains largely unknown. Here we report the crystal structure of Alr1298, a PRP from Nostoc sp. PCC 7120 predicted to reside in the cytoplasm. The structure displays the typical right-handed quadrilateral ß-helical structure and includes a four-α-helix cluster capping the N-terminus and a single α-helix capping the C-terminus. A gene cluster analysis indicated that Alr1298 may belong to an operon linked to cell proliferation and/or thylakoid biogenesis. Elevated alr1298 gene expression following nitrogen starvation indicates that Alr1298 may play a role in response to nitrogen starvation and/or heterocyst differentiation.

Effect of four kinds of complexing iron on the process of iron uptake by Anabaena flos-aquae.

AbstractIron (Fe), which is a necessary micronutrient for algal growth, plays an important role in the physiological metabolism and enzymatic reactions of algae. This study aimed to investigate the absorption process of four kinds of complexing iron absorbed by Anabaena flos-aquae. Results showed that the absorptive capacity of A. flos-aquae to complex iron was inversely proportional to the stability of the complex bond of complex iron. Complex iron with weak binding ability can be quickly adsorbed by A. flos-aquae. The absorptive rate was as follows: ferric humate, ferric oxalate >ammonium ferric citrate >EDTA Fe. For EDTA-Fe with a strong binding ability, a moderate iron concentration (e.g. 0.6 mg l-1) is favourable for iron uptake by A. flos-aquae. Our experiments also revealed that the process of separating iron from complex iron before entering algal cells was probably as follows: iron complexed with organic ligands were firstly adsorbed on the surface of algae cells; afterwards, iron ions were captured by organic matter on the surface of algae cells, accompanied by the rupture of the bond between Fe3+ and ligand; finally, the Fe3+ entered into the cell of algae while the organic ligands returned to the medium.

Siderophore piracy enhances Vibrio cholerae environmental survival and pathogenesis.

Vibrio cholerae, the aetiological agent of cholera, possesses multiple iron acquisition systems, including those for the transport of siderophores. How these systems benefit V. cholerae in low-iron, polymicrobial communities in environmental settings or during infection remains poorly understood. Here, we demonstrate that in iron-limiting conditions, co-culture of V. cholerae with a number of individual siderophore-producing microbes significantly promoted V. cholerae growth in vitro. We further show that in the host environment with low iron, V. cholerae colonizes better in adult mice in the presence of the siderophore-producing commensal Escherichia coli. Taken together, our results suggest that in aquatic reservoirs or during infection, V. cholerae may overcome environmental and host iron restriction by hijacking siderophores from other microbes.

Pegvaliase for the treatment of phenylketonuria: Results of the phase 2 dose-finding studies with long-term follow-up.

BACKGROUND: Phenylketonuria (PKU) is characterized by a deficiency in phenylalanine hydroxylase (PAH) that may lead to elevated blood phenylalanine (Phe) and significant neurocognitive and neuropsychological comorbidities. Pegvaliase (PALYNZIQ®, BioMarin Pharmaceutical Inc.) is a PEGylated recombinant Anabaena variabilis phenylalanine ammonia lyase (PAL), which converts Phe to trans-cinnamic acid and ammonia, and was approved in May 2018 in the United States and in May 2019 in the European Union for decreasing blood Phe levels in adults with PKU with blood Phe levels >600 µmol/L. The efficacy and safety of pegvaliase was assessed in two phase 2 dose-finding studies in adults with PKU (PAL-002, NCT00925054, and PAL-004, NCT01212744). Participants completing these studies could enroll in a long-term extension study (PAL-003, NCT00924703). METHODS: Participants in PAL-002 received pegvaliase 0.001, 0.003, 0.01, 0.03, or 0.1 mg/kg weekly for 8 weeks, then continued treatment for a further 8 weeks with dose and/or frequency adjusted to achieve blood Phe concentrations of 60 to 600 µmol/L. Participants in PAL-004 received pegvaliase 0.001 to 0.4 mg/kg 5 days/week for 13 weeks, with modifications made to the starting dose in response to safety and/or efficacy, followed by 3 additional weeks of follow-up assessments. The maximum allowable daily dose in both studies was 1.0 mg/kg/day (5.0 mg/kg/week). Participants who completed any of the phase 2 studies (PAL-002; PAL-004; or a third phase 2 study, 165-205) were eligible to enroll in an open-label, multicenter, long-term extension study (PAL-003, NCT00924703). RESULTS: Thirty-seven of the 40 enrolled participants completed PAL-002 and 15 of the 16 enrolled participants completed PAL-004. Mean blood Phe at baseline was 1311.0 (standard deviation [SD] 354) µmol/L in PAL-002 and 1482.1 (SD 363.5) µmol/L in PAL-004. Mean blood Phe did not substantially decrease with pegvaliase treatment in PAL-002 (-206.3 [SD 287.1] µmol/L at Week 16) or PAL-004 (-410.8 [SD 653.7] µmol/L at Week 13). In PAL-004, mean blood Phe dropped from baseline by 929.1 µmol/L (SD 691.1) by Week 2; subsequent to dose modifications and interruptions, this early decrease in mean Phe level was not sustained. With increased pegvaliase dose and duration in PAL-003, mean blood Phe levels steadily decreased from baseline, with mean reductions by Week 120 of 68.8% (SD 44.2%) in PAL-002 participants and 75.9% (SD 32.4%) in PAL-004 participants. All participants in PAL-002 and PAL-004 reported ≥1 adverse event (AE), with higher exposure-adjusted event rates in PAL-004. The majority of AEs were mild (87.2% in PAL-002, 86.7% in PAL-004) or moderate (12.4% in PAL-002, 13.3% in PAL-004). The most commonly reported AEs in PAL-002 were injection site reaction (50.0% of participants), headache (42.1%), injection site erythema (36.8%), nausea (34.2%), and arthralgia (29.0%), and in PAL-004 were arthralgia (75.0%), headache (62.5%), dizziness (56.3%), injection site erythema (56.3%), and injection site reaction (50.0%). CONCLUSIONS: In two phase 2 dose-finding studies, pegvaliase did not lead to substantial blood Phe reductions. Higher and more frequent pegvaliase dosing in PAL-004 led to a substantial initial drop in blood Phe, but an increase in the number of hypersensitivity AEs and dose reductions or interruptions. With increased dose and duration of treatment in PAL-003, mean blood Phe reduction was substantial and sustained, and the frequency of hypersensitivity AEs decreased and stabilized. Together, these studies led to the development of an induction-titration-maintenance regimen that has been approved for pegvaliase, with patients starting at a low weekly dose that gradually increases in dose and frequency until they achieve a standard non-weight-based daily maintenance dose. This regimen has been tested in a third phase 2 study, as well as in two successful phase 3 studies of pegvaliase.