Phosphate Restriction Promotes Longevity via Activation of Autophagy and the Multivesicular Body Pathway

Nutrient limitation results in an activation of autophagy in organisms ranging from yeast, nematodes and flies to mammals. Several evolutionary conserved nutrient-sensing kinases are critical for efficient adaptation of yeast cells to glucose, nitrogen or phosphate depletion, subsequent cell-cycle exit and the regulation of autophagy. Here, we demonstrate that phosphate restriction results in a prominent extension of yeast lifespan that requires the coordinated activity of autophagy and the multivesicular body pathway, enabling efficient turnover of cytoplasmic and plasma membrane cargo. While the multivesicular body pathway was essential during the early days of aging, autophagy contributed to long-term survival at later days. The cyclin-dependent kinase Pho85 was critical for phosphate restriction-induced autophagy and full lifespan extension. In contrast, when cell-cycle exit was triggered by exhaustion of glucose instead of phosphate, Pho85 and its cyclin, Pho80, functioned as negative regulators of autophagy and lifespan. The storage of phosphate in form of polyphosphate was completely dispensable to in sustaining viability under phosphate restriction. Collectively, our results identify the multifunctional, nutrient-sensing kinase Pho85 as critical modulator of longevity that differentially coordinates the autophagic response to distinct kinds of starvation.

Thorsmoerkia curvula gen. et spec. nov. (Trebouxiophyceae, Chlorophyta), a semi-terrestrial microalga from Iceland exhibits high levels of unsaturated fatty acids

A terrestrial green alga was isolated at Iceland, and the strain (SAG 2627) was described for its morphology and phylogenetic position and tested for biotechnological capabilities. Cells had a distinctly curved, crescent shape with conical poles and a single parietal chloroplast. Phylogenetic analyses of 18S rDNA and rbcL markers placed the strain into the Trebouxiophyceae (Chlorophyta). The alga turned out to belong to an independent lineage without an obvious sister group within the Trebouxiophyceae. Based on morphological and phylogenetic data, the strain was described as a new genus and species, Thorsmoerkia curvula gen. et sp. nov. Biomass was generated in column reactors and subsequently screened for promising metabolites. Growth was optimized by pH-regulated, episodic CO2 supplement during the logarithmic growth-phase, and half of the biomass was thereafter exposed to nitrogen and phosphate depletion. The biomass yield reached up to 53.5 mg L−1 day−1. Fatty acid (FA) production peaked at 24 mg L−1 day−1 and up to 83% of all FAs were unsaturated. At the end of the log phase, approximately 45% of dry mass were lipids, including eicosapentaenoic acid. Carotenoid production reached up to 2.94 mg L−1 day−1 but it was halted during the stress phase. The N-linked glycans of glycoproteins were assessed to reveal chemotaxonomic patterns. The study demonstrated that new microalgae can be found at Iceland, potentially suitable for applied purposes. The advantage of T. curvula is its robustness and that significant amounts of lipids are already accumulated during log phase, making a subsequent stress exposure dispensable.

PSEUDOHYPOPHOSPHATEMIA IN A PATIENT WITH MULTIPLE MYELOMA

Objective: To discuss the diagnosis and management of paraprotein interference in the setting of multiple myeloma (MM). Methods: We discuss the evaluation of hypophosphatemia in a patient with MM and present a review of the relevant literature. Results: Our patient, who had a history of MM, was found to have persistently undetectable serum phosphate which did not respond to aggressive phosphate replacement. His clinical condition was not consistent with severe phosphate depletion and hence paraprotein interference secondary to MM was suspected. Re-analyzation of samples on a different machine showed normal serum inorganic phosphate levels. Conclusion: Paraprotein interference from MM causing pseudohypophosphatemia can be overlooked and lead to unnecessary treatment. Recognition of this phenomenon is important to all clinicians, especially in light of potential complications of unnecessary treatment.

Vascular transcription factors guide plant epidermal responses to limiting phosphate conditions

Optimal plant growth is hampered by deficiency of the essential macronutrient phosphate in most soils. Plant roots can, however, increase their root hair density to efficiently forage the soil for this immobile nutrient. By generating and exploiting a high-resolution single-cell gene expression atlas of Arabidopsis roots, we show an enrichment of TARGET OF MONOPTEROS 5/LONESOME HIGHWAY (TMO5/LHW) target gene responses in root hair cells. The TMO5/LHW heterodimer triggers biosynthesis of mobile cytokinin in vascular cells and increases root hair density during low-phosphate conditions by modifying both the length and cell fate of epidermal cells. Moreover, root hair responses in phosphate-deprived conditions are TMO5- and cytokinin-dependent. Cytokinin signaling links root hair responses in the epidermis to perception of phosphate depletion in vascular cells.

MON-364 Severe Postoperative Encephalopathy in a Patient with Oncogenic Osteomalacia and Neurofibromatosis Type 1

Background: Acute hypophosphatemia superimposed on chronic phosphate depletion results in decline in intracellular ATP and 2,3-diphosphoglycerate levels, affecting virtually all organ systems. It can lead to metabolic encephalopathy, impaired myocardial and diaphragmatic contractility, skeletal and smooth muscle dysfunction, increase in erythrocyte rigidity leading to hemolysis, and increased risk for postoperative morbidity and mortality. Risk of symptomatic hypophosphatemia may be greater in patients with urinary phosphate wasting syndromes hospitalized for intercurrent illness. Tumor-induced (oncogenic) osteomalacia (TIO) is a rare paraneoplastic disorder associated with mesenchymal tumors caused by tumor production of fibroblast-derived growth factor-23 (FGF-23). FGF-23 impairs transport of phosphorus in renal tubules, inhibits renal 25(OH)-1-α-hydroxylase activity, causing decreased levels of calcitriol, leading to hyperphosphaturia, hypophosphatemia and osteomalacia. We present a case of a patient with underlying TIO who presented with acute hypophosphatemia following hip surgery. Case: 50-year-old woman with Neurofibromatosis type-1(NF-1) complicated by TIO, presented after a fall resulting in left femur fracture requiring surgery. During her initial management in an outside hospital for 4 days and following transfer her home calcitriol and phosphorus replacement regimen was not administered leading to treatment interruption of 7 days. On postoperative day 1 she became hypotensive, tachycardic with non-specific ST-T changes and encephalopathic requiring ICU care. At the time of endocrinology consultation, she was non-verbal, not following simple commands, had severe hypophosphatemia, transaminitis, hyperbilirubinemia, elevated LDH, anemia and elevated troponin. She required aggressive phosphate and calcitriol replacement reaching 4 doses of 20mEq IV Na- Phos daily, Na-KPhos Q6H and Calcitriol IV 0.5mcg Q8H over the course of 72 hours. After 4 days in the ICU, once phosphorous levels normalized, she had dramatic improvement in mental status and gradual resolution of laboratory abnormalities. Discussion: To our knowledge, this is the first report of a patient with TIO associated with NF-1 presenting with severe symptomatic hypophosphatemia causing metabolic encephalopathy and multiple organ dysfunction following surgery. It underscores the fact that abrupt interruption of calcitriol and phosphorus supplementation in such cases can cause acute hypophosphatemia carrying high morbidity. High phosphate replacement requirements are indicative of severity of phosphate depletion. This case illustrates importance of recognition of presence of urinary phosphate wasting syndrome in hospitalized patients in order to minimize risk of preventable complications and morbidity.

Molecular Mechanisms of Phosphate Stress Activation of Pseudomonas aeruginosa Quorum Sensing Systems

ABSTRACT The hierarchical quorum sensing (QS) systems of Pseudomonas aeruginosa, consisting of las, pqs, and rhl, coordinate the expression of bacterial virulence genes. Previous studies showed that under phosphate deficiency conditions, two-component regulatory system PhoRB could activate various genes involved in cytotoxicity through modulation of QS systems, but the mechanism by which PhoR/PhoB influences QS remains largely unknown. Here, we provide evidence that among the key QS regulatory genes in P. aeruginosa, rhlR, pqsA, mvfR, and lasI were activated by the response regulator PhoB under phosphate-depleted conditions. We show that PhoB is a strong competitor against LasR and RsaL for binding to the promoter of lasI and induces significant expression of lasI, rhlR, and mvfR. However, expression of lasI, encoding the signal 3-oxo-C12-HSL, was increased only marginally under the same phosphate-depleted conditions. This seeming inconsistency was attributed to the induction of pvdQ, which encodes an enzyme for degradation of 3-oxo-C12-HSL signal molecules. Taken together, the results from this study demonstrate that through the two-component regulatory system PhoR/PhoB, phosphate depletion stress could influence the QS network by modulating several key regulators, including lasI, rhlR, mvfR, and pvdQ. The findings highlight not only the potency of the PhoR/PhoB-mediated bacterial stress response mechanism but also the plasticity of the P. aeruginosa QS systems in coping with the changed environmental conditions. IMPORTANCE It is not fully understood how phosphate deficiency could influence the virulence of Pseudomonas aeruginosa through modulation of the bacterial QS systems. This report presents a systemic investigation on the impact of phosphate depletion on the hierarchy of quorum sensing systems of P. aeruginosa. The results showed that phosphate stress could have an extensive impact on the QS networks of this bacterial pathogen. Among the 7 QS regulatory genes representing the 3 sets of QS systems tested, 4 were significantly upregulated by phosphate depletion stress through the PhoR/PhoB two-component regulatory system, especially the upstream QS regulatory gene lasI. We also present evidence that the response regulator PhoB was a strong competitor against the las regulators LasR and RsaL for the lasI promoter, unveiling the mechanistic basis of the process by which phosphate stress could modulate the bacterial QS systems.

Comparison of growth and toxicity responses between non-toxic and toxic strains of Prorocentrum hoffmannianum

We aimed to study the growth and toxicity responses of non-toxic (CCMP683) and toxic (CCMP2804) strains of Prorocentrum hoffmannianum under various nitrate and phosphate concentrations. The 2 strains were cultured in L1-Si medium with standard, depleted or 10-fold repleted nitrate or phosphate. CCMP683 cultured in standard L1-Si medium exhibited delayed growth. Nitrate or phosphate depletion decreased the cell density of both strains. Repletion of nitrate slightly increased the cell density of both strains. Repletion of phosphate also slightly increased the cell density of CCMP2804 but surprisingly decreased the cell density of CCMP683. Okadaic acid (OA) and its derivatives were not detected in CCMP683. OA was detected only in CCMP2804. Depletion of nitrate or phosphate increased the cellular concentration of OA, and repletion of nitrate or phosphate had no effect on the cellular concentration of OA. Correlation analysis indicated that the cellular concentration of OA was negatively correlated with cell density. Differences in the growth response to phosphate repletion and in the ability to produce OA suggest that the 2 strains may be good candidates for comparative studies related to phosphate metabolism and OA toxicity.

Improved, two-stage protein expression and purification via autoinduction of both autolysis and auto DNA/RNA hydrolysis conferred by phage lysozyme and DNA/RNA endonuclease

We report improved release of recombinant proteins in E. coli, which relies on combined cellular autolysis and DNA/RNA autohydrolysis, conferred by the tightly controlled autoinduction of both phage lysozyme and the non specific DNA/RNA endonuclease from S. marcescens. Autoinduction occurs in a two-stage process wherein heterologous protein expression and autolysis enzymes are induced upon entry into stationary phase by phosphate depletion. Cytoplasmic lysozyme and periplasmic endonuclease are kept from inducing lysis until membrane integrity is disrupted. Post cell harvest, the addition of detergent (0.1% Triton-X100) and a single 30 minutes freezer thaw cycle results in > 90% release of protein (GFP). This cellular lysis is accompanied by complete oligonucleotide hydrolysis. The approach has been validated for shake flask cultures, high throughput cultivation in microtiter plates and larger scale stirred-tank bioreactors. This tightly controlled system enables robust growth and resistance to lysis in routine media when cells are propagated and autolysis/hydrolysis genes are only induced upon phosphate depletion.HighlightsAutoinduction of both cell lysis and nucleotide hydrolysis>90 % lysis and DNA degradationStrains are stable to lysis in the absence of phosphate depletion.