Ferredoxin5 Deletion Affects Metabolism of Algae during the Different Phases of Sulfur Deprivation.

Ferredoxin5 (FDX5), a minor ferredoxin protein in the alga Chlamydomonas (Chlamydomonas reinhardtii), helps maintain thylakoid membrane integrity in the dark. Sulfur (S) deprivation has been used to achieve prolonged hydrogen production in green algae. Here, we propose that FDX5 is involved in algal responses to S-deprivation as well as to the dark. Specifically, we tested the role of FDX5 in both the initial aerobic and subsequent anaerobic phases of S-deprivation. Under S-deprived conditions, absence of FDX5 causes a distinct delay in achieving anoxia by affecting photosynthetic O2 evolution, accompanied by reduced acetate uptake, lower starch accumulation, and delayed/lower fermentative metabolite production, including photohydrogen. We attribute these differences to transcriptional and/or posttranslational regulation of acetyl-CoA synthetase and ADP-Glc pyrophosphorylase, and increased stability of the PSII D1 protein. Interestingly, increased levels of FDX2 and FDX1 were observed in the mutant under oxic, S-replete conditions, strengthening our previously proposed hypothesis that other ferredoxins compensate in response to a lack of FDX5. Taken together, the results of our omics and pull-down experiments confirmed biochemical and physiological results, suggesting that FDX5 may have other effects on Chlamydomonas metabolism through its interaction with multiple redox partners.

Relevance of nutrient media composition for hydrogen production in Chlamydomonas.

Microalgae are capable of biological H2 photoproduction from water, solar energy, and a variety of organic substrates. Acclimation responses to different nutrient regimes finely control photosynthetic activity and can influence H2 production. Hence, nutrient stresses are an interesting scenario to study H2 production in photosynthetic organisms. In this review, we mainly focus on the H2-production mechanisms in Chlamydomonas reinhardtii and the physiological relevance of the nutrient media composition when producing H2.

Tiered regulation of sulfur deprivation responses in Chlamydomonas reinhardtii and identification of an associated regulatory factor.

During sulfur (S) deprivation, the unicellular alga Chlamydomonas reinhardtii exhibits increased expression of numerous genes. These genes encode proteins associated with sulfate (SO4(2-)) acquisition and assimilation, alterations in cellular metabolism, and internal S recycling. Administration of the cytoplasmic translational inhibitor cycloheximide prevents S deprivation-triggered accumulation of transcripts encoding arylsulfatases (ARS), an extracellular polypeptide that may be important for cell wall biosynthesis (ECP76), a light-harvesting protein (LHCBM9), the selenium-binding protein, and the haloperoxidase (HAP2). In contrast, the rapid accumulation of transcripts encoding high-affinity SO4(2-) transporters is not affected. These results suggest that there are two tiers of transcriptional regulation associated with S deprivation responses: the first is protein synthesis independent, while the second requires de novo protein synthesis. A mutant designated ars73a exhibited low ARS activity and failed to show increases in ECP76, LHCBM9, and HAP2 transcripts (among others) in response to S deprivation; increases in transcripts encoding the SO4(2-) transporters were not affected. These results suggest that the ARS73a protein, which has no known activity but might be a transcriptional regulator, is required for the expression of genes associated with the second tier of transcriptional regulation. Analysis of the ars73a strain has helped us generate a model that incorporates a number of complexities associated with S deprivation responses in C. reinhardtii.

Chlamydomonas reinhardtii and Microbacterium forte sp. nov., a mutualistic association that favors sustainable hydrogen production.

A naturally occurring multispecies bacterial community composed of Bacillus cereus and two novel bacteria (Microbacterium forte sp. nov. and Stenotrophomonas goyi sp. nov.) has been identified from a contaminated culture of the microalga Chlamydomonas reinhardtii. When incubated in mannitol- and yeast extract-containing medium, this bacterial community can promote and sustain algal hydrogen production up to 313 mL H2·L-1 for 17 days and 163.5 mL H2·L-1 for 25 days in high-cell (76.7 µg·mL-1 of initial chlorophyll) and low-cell density (10 µg·mL-1 of initial chlorophyll) algal cultures, respectively. In low-cell density algal cultures, hydrogen production was compatible with algal growth (reaching up to 60 µg·mL-1 of chlorophyll). Among the bacterial community, M. forte sp. nov. was the sole responsible for the improvement in hydrogen production. However, algal growth was not observed in the Chlamydomonas-M. forte sp. nov. consortium during hydrogen-producing conditions (hypoxia), suggesting that the presence of B. cereus and S. goyi sp. nov. could be crucial to support the algal growth during hypoxia. Still, under non­hydrogen producing conditions (aerobiosis) the Chlamydomonas-M. forte sp. nov. consortium allowed algal growth (up to 40 µg·mL-1 of chlorophyll) and long-term algal viability (>45 days). The genome sequence and growth tests of M. forte sp. nov. have revealed that this bacterium is auxotroph for biotin and thiamine and unable to use sulfate as sulfur source; it requires S-reduced forms such as cysteine and methionine. Cocultures of Chlamydomonas reinhardtii and M. forte sp. nov. established a mutualistic association: the alga complemented the nutrient deficiencies of the bacterium, while the bacterium released ammonium (0.19 mM·day-1) and acetic acid (0.15 mM·day-1) for the alga. This work offers a promising avenue for photohydrogen production concomitant with algal biomass generation using nutrients not suitable for mixotrophic algal growth.

Consensus Report and Recommendations on the Management of Late-stage Internal Derangement of the Temporomandibular Joint.

Introduction: This report investigates late-stage internal derangement (ID) of the temporomandibular joint (TMJ) with the aim of establishing a more effective and personalized treatment protocol to improve patients' quality of life (QoL). Material and methods: A consensus was reached among maxillofacial surgeons specializing in LSID, based on a literature research and collective expert experience following the Delphi method. Consensus was considered to be achieved when a response received at least 80% of votes. Results: Four expert groups were established, respectively, focusing on diagnosis, minimally invasive surgery (MIS), open surgery and joint replacement. A comprehensive approach to late-stage ID of the TMJ requires a consensus report. This underscores the need for a personalized treatment plan, considering the variability in clinical presentations and progression of this pathology. Our recommendations aim to optimize clinical outcomes and enhance patient QoL.

RNA-seq analysis of sulfur-deprived Chlamydomonas cells reveals aspects of acclimation critical for cell survival.

The Chlamydomonas reinhardtii transcriptome was characterized from nutrient-replete and sulfur-depleted wild-type and snrk2.1 mutant cells. This mutant is null for the regulatory Ser-Thr kinase SNRK2.1, which is required for acclimation of the alga to sulfur deprivation. The transcriptome analyses used microarray hybridization and RNA-seq technology. Quantitative RT-PCR evaluation of the results obtained by these techniques showed that RNA-seq reports a larger dynamic range of expression levels than do microarray hybridizations. Transcripts responsive to sulfur deprivation included those encoding proteins involved in sulfur acquisition and assimilation, synthesis of sulfur-containing metabolites, Cys degradation, and sulfur recycling. Furthermore, we noted potential modifications of cellular structures during sulfur deprivation, including the cell wall and complexes associated with the photosynthetic apparatus. Moreover, the data suggest that sulfur-deprived cells accumulate proteins with fewer sulfur-containing amino acids. Most of the sulfur deprivation responses are controlled by the SNRK2.1 protein kinase. The snrk2.1 mutant exhibits a set of unique responses during both sulfur-replete and sulfur-depleted conditions that are not observed in wild-type cells; the inability of this mutant to acclimate to S deprivation probably leads to elevated levels of singlet oxygen and severe oxidative stress, which ultimately causes cell death. The transcriptome results for wild-type and mutant cells strongly suggest the occurrence of massive changes in cellular physiology and metabolism as cells become depleted for sulfur and reveal aspects of acclimation that are likely critical for cell survival.

Stenotrophomonas goyi sp. nov., a novel bacterium associated with the alga Chlamydomonas reinhardtii.

Background: A culture of the green algae Chlamydomonas reinhardtii was accidentally contaminated with three different bacteria in our laboratory facilities. This contaminated alga culture showed increased algal biohydrogen production. These three bacteria were independently isolated. Methods: The chromosomic DNA of one of the isolated bacteria was extracted and sequenced using PacBio technology. Tentative genome annotation (RAST server) and phylogenetic trees analysis (TYGS server) were conducted. Diverse growth tests were assayed for the bacterium and for the alga-bacterium consortium. Results: Phylogenetic analysis indicates that the bacterium is a novel member of the Stenotrophomonas genus that has been termed in this work as S. goyi sp. nov. A fully sequenced genome (4,487,389 base pairs) and its tentative annotation (4,147 genes) are provided. The genome information suggests that S. goyi sp. nov. is unable to use sulfate and nitrate as sulfur and nitrogen sources, respectively. Growth tests have confirmed the dependence on the sulfur-containing amino acids methionine and cysteine. S. goyi sp. nov. and Chlamydomonas reinhardtii can establish a mutualistic relationship when cocultured together. Conclusions: S. goyi sp. nov. could be of interest for the design of biotechnological approaches based on the use of artificial microalgae-bacteria multispecies consortia that take advantage of the complementary metabolic capacities of their different microorganisms.

Chlamydomonas-Methylobacterium oryzae cooperation leads to increased biomass, nitrogen removal and hydrogen production.

In the context of algal wastewater bioremediation, this study has identified a novel consortium formed by the bacterium Methylobacterium oryzae and the microalga Chlamydomonas reinhardtii that greatly increase biomass generation (1.22 g L-1·d-1), inorganic nitrogen removal (>99%), and hydrogen production (33 mL·L-1) when incubated in media containing ethanol and methanol. The key metabolic aspect of this relationship relied on the bacterial oxidation of ethanol to acetate, which supported heterotrophic algal growth. However, in the bacterial monocultures the acetate accumulation inhibited bacterial growth. Moreover, in the absence of methanol, ethanol was an unsuitable carbon source and its incomplete oxidation to acetaldehyde had a toxic effect on both the alga and the bacterium. In cocultures, both alcohols were used as carbon sources by the bacteria, the inhibitory effects were overcome and both microorganisms mutually benefited. Potential biotechnological applications in wastewater treatment, biomass generation and hydrogen production are discussed.

Identification and regulation of plasma membrane sulfate transporters in Chlamydomonas.

Chlamydomonas (Chlamydomonas reinhardtii) exhibits several responses following exposure to sulfur (S)-deprivation conditions, including an increased efficiency of import and assimilation of the sulfate anion (SO(4)(2-)). Aspects of SO(4)(2-) transport during S-replete and S-depleted conditions were previously studied, although the transporters had not been functionally identified. We employed a reverse genetics approach to identify putative SO(4)(2-) transporters, examine their regulation, establish their biogenesis and subcellular locations, and explore their functionality. Upon S starvation of wild-type Chlamydomonas cells, the accumulation of transcripts encoding the putative SO(4)(2-) transporters SLT1 (for SAC1-like transporter 1), SLT2, and SULTR2 markedly increased, suggesting that these proteins function in high-affinity SO(4)(2-) transport. The Chlamydomonas sac1 and snrk2.1 mutants (defective for acclimation to S deprivation) exhibited much less of an increase in the levels of SLT1, SLT2, and SULTR2 transcripts and their encoded proteins in response to S deprivation compared with wild-type cells. All three transporters were localized to the plasma membrane, and their rates of turnover were significantly impacted by S availability; the turnover of SLT1 and SLT2 was proteasome dependent, while that of SULTR2 was proteasome independent. Finally, mutants identified for each of the S-deprivation-responsive transporters were used to establish their critical role in the transport of SO(4)(2-) into S-deprived cells.

Genetic interactions between regulators of Chlamydomonas phosphorus and sulfur deprivation responses.

The Chlamydomonas reinhardtii PSR1 gene is required for proper acclimation of the cells to phosphorus (P) deficiency. P-starved psr1 mutants show signs of secondary sulfur (S) starvation, exemplified by the synthesis of extracellular arylsulfatase and the accumulation of transcripts encoding proteins involved in S scavenging and assimilation. Epistasis analysis reveals that induction of the S-starvation responses in P-limited psr1 cells requires the regulatory protein kinase SNRK2.1, but bypasses the membrane-targeted activator, SAC1. The inhibitory kinase SNRK2.2 is necessary for repression of S-starvation responses during both nutrient-replete growth and P limitation; arylsulfatase activity and S deficiency-responsive genes are partially induced in the P-deficient snrk2.2 mutants and become fully activated in the P-deficient psr1snrk2.2 double mutant. During P starvation, the sac1snrk2.2 double mutants or the psr1sac1snrk2.2 triple mutants exhibit reduced arylsulfatase activity compared to snrk2.2 or psr1snrk2.2, respectively, but the sac1 mutation has little effect on the abundance of S deficiency-responsive transcripts in these strains, suggesting a post-transcriptional role for SAC1 in elicitation of S-starvation responses. Interestingly, P-starved psr1snrk2.2 cells bleach and die more rapidly than wild-type or psr1 strains, suggesting that activation of S-starvation responses during P deprivation is deleterious to the cell. From these results we infer that (i) P-deficient growth causes some internal S limitation, but the S-deficiency responses are normally inhibited during acclimation to P deprivation; (ii) the S-deficiency responses are not completely suppressed in P-deficient psr1 cells and consequently these cells synthesize some arylsulfatase and exhibit elevated levels of transcripts for S-deprivation genes; and (iii) this increased expression is controlled by regulators that modulate transcription of S-responsive genes during S-deprivation conditions. Overall, the work strongly suggests integration of the different circuits that control nutrient-deprivation responses in Chlamydomonas.

Algae-Bacteria Consortia as a Strategy to Enhance H2 Production.

Biological hydrogen production by microalgae is a potential sustainable, renewable and clean source of energy. However, many barriers limiting photohydrogen production in these microorganisms remain unsolved. In order to explore this potential and make biohydrogen industrially affordable, the unicellular microalga Chlamydomonas reinhardtii is used as a model system to solve barriers and identify new approaches that can improve hydrogen production. Recently, Chlamydomonas-bacteria consortia have opened a new window to improve biohydrogen production. In this study, we review the different consortia that have been successfully employed and analyze the factors that could be behind the improved H2 production.

Long-Term Dislocation of the Mandible: Is there an Algorithm to Success? Intraoperative Decision and Review of Literature.

PURPOSE: Long-term TMJ dislocation is a rare condition. It occurs when an acute luxation remains untreated in time. METHODS: A 52-year-old man presented with a long-term TMJ luxation in the context of Steinert's disease. A discectomy together with condylectomy and eminectomy was performed, obtaining an adequate reduction of the luxated condyle and disc. RESULTS: Twelve months after the operation, the condition has not recurred at all. A stable and centred occlusion was checked; his MIO was over 30 mm. CONCLUSION: The combination of these three techniques could be a good option in cases of Steinert's myotonia, where the condyle luxation becomes chronic and irreducible due to the altered neuromuscular condition. There is still no consensus regarding the treatment for long-term TMJ dislocations. New and more solid studies may be needed in order to find adequate treatment protocols for this condition.

Acetic acid is key for synergetic hydrogen production in Chlamydomonas-bacteria co-cultures.

This study is a proof of concept for the synergetic biohydrogen production in alga-bacteria co-cultures. Algal hydrogen photoproduction was obtained in sugar-containing media only when the green alga Chlamydomonas reinhardtii was co-cultured with Pseudomonas putida (40.8 ml H2·L-1), Escherichia coli (35.1 ml H2·L-1) and Rhizobium etli (16.1 ml H2·L-1). Hydrogen photo-production in these co-cultures was not only linked to the induction of hypoxia, but to the ability of the bacteria to produce acetic acid from sugars. Synergetic hydrogen production was achieved by integrating the photobiological and fermentative production in Chlamydomonas and Escherichia coli co-cultures supplemented with glucose, which resulted in 60% more H2 production than the sum of the respective monocultures. This cooperation relied on the ability of the alga to consume the excreted bacterial acetic acid, which benefited both bacterial and algal hydrogen production. This knowledge may open new possibilities for the biohydrogen production from industrial wastes.

Arginine is a component of the ammonium-CYG56 signalling cascade that represses genes of the nitrogen assimilation pathway in Chlamydomonas reinhardtii.

Nitrogen assimilation and metabolism are essential processes for all living organisms, yet there is still much to be learnt on how they are regulated. The use of Chlamydomonas reinhardtii as a model system has been instrumental not only in identifying conserved regulation mechanisms that control the nitrogen assimilation pathway, but also in understanding how the intracellular nitrogen status regulates metabolic processes of industrial interest such as the synthesis of biolipids. While the genetic regulators that control the nitrogen pathway are successfully being unravelled, other layers of regulation have received less attention. Amino acids, for example, regulate nitrogen assimilation in certain organisms, but their role in Chlamydomonas has not thoroughly been explored. Previous results had suggested that arginine might repress key genes of the nitrogen assimilation pathway by acting within the ammonium negative signalling cascade, upstream of the nitric oxide (NO) inducible guanylate cyclase CYG56. We tested this hypothesis with a combination of genetic and chemical approaches. Antagonising the effects of arginine with an arginine biosynthesis mutant or with two chemical analogues released gene expression from ammonium mediated repression. The cyg56 and related non1 mutants, which are partially insensitive to ammonium repression, were also partially insensitive to repression by arginine. Finally, we show that the addition of arginine to the medium leads to an increase in intracellular NO. Our data reveal that arginine acts as a negative signal for the assimilation of nitrogen within the ammonium-CYG56 negative signalling cascade, and provide a connection between amino acid metabolism and nitrogen assimilation in microalgae.

Insertional mutagenesis as a tool to study genes/functions in Chlamydomonas.

The unicellular alga Chlamydomonas reinhardtii has emerged during the last decades as a model system to understand gene functions, many of them shared by bacteria, fungi, plants, animals and humans. A powerful resource for the research community is the availability of complete collections of stable mutants for studying whole genome function. In the meantime other strategies might be developed; insertional mutagenesis has become currently the best strategy to disrupt and tag nuclear genes in Chlamydomonas allowing forward and reverse genetic approaches. Here, we outline the mutagenesis technique stressing the idea of generating databases for ordered mutant libraries, and also of improving efficient methods for reverse genetics to identify mutants defective in a particular gene.

H2 production pathways in nutrient-replete mixotrophic Chlamydomonas cultures under low light. Response to the commentary article "On the pathways feeding the H2 production process in nutrient-replete, hypoxic conditions," by Alberto Scoma and Szilvia Z. Tóth.

BACKGROUND: A recent Commentary article entitled "On the pathways feeding the H2 production process in nutrient-replete, hypoxic conditions" by Dr. Scoma and Dr. Tóth, Biotechnology for Biofuels (2017), opened a very interesting debate about the H2 production photosynthetic-linked pathways occurring in Chlamydomonas cultures grown in acetate-containing media and incubated under hypoxia/anoxia conditions. This Commentary article mainly focused on the results of our previous article "Low oxygen levels contribute to improve photohydrogen production in mixotrophic non-stressed Chlamydomonas cultures," by Jurado-Oller et al., Biotechnology for Biofuels (7, 2015; 8:149). MAIN BODY: Here, we review some previous knowledge about the H2 production pathways linked to photosynthesis in Chlamydomonas, especially focusing on the role of the PSII-dependent and -independent pathways in acetate-containing nutrient-replete cultures. The potential contributions of these pathways to H2 production under anoxia/hypoxia are discussed. CONCLUSION: Despite the fact that the PSII inhibitor DCMU is broadly used to discern between the two different photosynthetic pathways operating under H2 production conditions, its use may lead to distinctive conclusions depending on the growth conditions. The different potential sources of reductive power needed for the PSII-independent H2 production in mixotrophic nutrient-replete cultures are a matter of debate and conclusive evidences are still missing.

Glucose Uptake in Prochlorococcus: Diversity of Kinetics and Effects on the Metabolism.

We have previously shown that Prochlorococcus sp. SS120 strain takes up glucose by using a multiphasic transporter encoded by the Pro1404 gene. Here, we studied the glucose uptake kinetics in multiple Prochlorococcus strains from different ecotypes, observing diverse values for the Ks constants (15-126.60 nM) and the uptake rates (0.48-6.36 pmol min-1 mg prot-1). Multiphasic kinetics was observed in all studied strains, except for TAK9803-2. Pro1404 gene expression studies during the 21st Atlantic Meridional Transect cruise showed positive correlation with glucose concentrations in the ocean. This suggests that the Pro1404 transporter has been subjected to diversification along the Prochlorococcus evolution, in a process probably driven by the glucose availabilities at the different niches it inhabits. The glucose uptake mechanism seems to be a primary transporter. Glucose addition induced detectable transcriptomic and proteomic changes in Prochlorococcus SS120, but photosynthetic efficiency was unaffected. Our studies indicate that glucose is actively taken up by Prochlorococcus, but its uptake does not significantly alter the trophic ways of this cyanobacterium, which continues performing photosynthesis. Therefore Prochlorococcus seems to remain acting as a fundamentally phototrophic organism, capable of using glucose as an extra resource of carbon and energy when available in the environment.

Coristoma óseo lingual. Nuevo caso de una entidad extremadamente infrecuente / Lingual osseous choristoma. New case of an extremely uncommon entity

Los osteomas constituyen un grupo de tumores óseos benignos que se localizan habitualmente en el cráneo y los huesos de la región facial, siendo rara su presencia en los tejidos blandos de la cavidad oral. También se les conoce como coristomas óseos, ya que están compuestos por tejido óseo maduro de aspecto normal. En la cavidad oral, la localización más frecuente es el tercio posterior de la lengua y suele aparecer en mujeres con edades comprendidas entre la segunda y tercera década de vida. A pesar de que la mayoría de los pacientes presentan un curso asintomático, no es infrecuente que su motivo de consulta sea la sensación de cuerpo extraño y disfagia. Afortunadamente, los osteomas linguales tienen buen pronóstico, ya que una vez realizada la extirpación no hay descritos casos de recurrencia. Debido a su insólita presentación, con menos de 100 casos publicados en la literatura, se decide presentar este caso clínico. (AU)

Osteomas are rare benign tumors that can be located on the craniomaxillofacial skeleton, being unusual its presence in the soft tissues of the oral cavity. They are also known as osseous choristomas, as they consist of normal matured bone tissue. Osteomas of the tongue occur more frequently in women in their second or third decade and the most frequent location in the oral cavity is the posterior third of the tongue. Most patients are asymptomatic, though other forms of presentation could be complaining of the sensation of having a foreign body or, even, dysphagia. The fact that less than 100 cases have been reported in the literature, has motivated the presentation of this clinical case. (AU)