SUMMARYDrastic alteration in macronutrients causes large changes in gene expression in the photosynthetic unicellular alga Chlamydomonas reinhardtii. Preliminary data suggested that cells follow a biphasic response to this change hinging on the initiation of lipid accumulation, and we hypothesized that drastic repatterning of metabolism also followed this biphasic modality. To test this hypothesis, transcriptomic, proteomic, and metabolite changes that occur under nitrogen (N) deprivation were analyzed. Eight sampling times were selected covering the progressive slowing of growth and induction of oil synthesis between 4 and 6 h after N deprivation. Results of the combined, systems-level investigation indicated that C. reinhardtii cells sense and respond on a large scale within 30 min to a switch to N-deprived conditions turning on a largely gluconeogenic metabolic state, which then transitions to a glycolytic stage between 4 and 6 h after N depletion. This nitrogen-sensing system is transduced to carbon-and nitrogen-responsive pathways, leading to down-regulation of carbon assimilation and chlorophyll biosynthesis, and an increase in nitrogen metabolism and lipid biosynthesis. For example, the expression of nearly all the enzymes for assimilating nitrogen from ammonium, nitrate, nitrite, urea, formamide/acetamide, purines, pyrimidines, polyamines, amino acids and proteins increased significantly. Although arginine biosynthesis enzymes were also rapidly up-regulated, arginine pool size changes and isotopic labeling results indicated no increased flux through this pathway.

Symbiotic nitrogen fixation (SNF) between rhizobia and legumes requires metabolic coordination within specialized root organs called nodules. Nodules formed in the symbiosis between S. medicae and barrel medic (M. truncatula) are indeterminate, cylindrical, and contain spatially distinct developmental zones. Bacteria in the infection zone II (ZII), interzone II-III (IZ), and nitrogen fixation zone III (ZIII) represent different stages in the metabolic progression from free-living bacteria into nitrogen fixing bacteroids. To better understand the coordination of plant and bacterial metabolism within the nodule, we used liquid and gas chromatography coupled to tandem mass spectrometry (MS) to observe protein and metabolite profiles representative of ZII, IZ, ZIII, whole-nodule, and primary root. Our MS-based approach confidently identified 361 S. medicae proteins and 888 M. truncatula proteins, as well as 160 metabolites from each tissue. The data are consistent with several organ- and zone-specific protein and metabolite localization patterns characterized previously. We used our comprehensive dataset to demonstrate how multiple branches of primary metabolism are coordinated between symbionts and zones, including central carbon, fatty acid, and amino acid metabolism. For example, M. truncatula glycolysis enzymes accumulate from zone I to zone III within the nodule, while equivalent S. medicae enzymes decrease in abundance. We also show the localization of S. medicae's transition to dicarboxylic acid-dependent carbon metabolism within the IZ. The spatial abundance patterns of S. medicae fatty acid (FA) biosynthesis enzymes indicate an increased demand for FA production in the IZ and ZIII as compared to ZI. These observations provide a resource for those seeking to understand coordinated physiological changes during the development of SNF.

Fungi are noted producers of a diverse array of secondary metabolites, many of which are of pharmacological importance. However, the biological roles of the vast majority of these molecules during the fungal life cycle in nature remain elusive. Solanapyrones are polyketide-derived secondary metabolites produced by diverse fungal species including the plant pathogen Ascochyta rabiei. This molecule was originally thought to function as a phytotoxin facilitating pathogenesis of A. rabiei. Chemical profiling and gene expression studies showed that solanapyrone A was specifically produced during saprobic, but not parasitic growth of A. rabiei. Expression of the gene encoding the final enzymatic step in solanapyrone biosynthesis was specifically associated with development of the asexual fruiting bodies of the fungus on certain substrates. In confrontation assays with saprobic fungi that were commonly found in chickpea debris in fields, A. rabiei effectively suppressed the growth of all competing fungi, such as Alternaria, Epicoccum and Ulocladium species. Solanapyrone A was directly detected in the inhibitory zone using a MALDI-imaging mass spectrometry, and the purified compound showed significant antifungal activities against the potential saprobic competitors. These results suggest that solanapyrone A plays an important role for competition and presumably the survival of the fungus.

Background: The study evaluated the clinical and radiographic outcomes of the modified Broström procedure (MBP) with periosteal flap augmentation after large subfibular ossicle excision for chronic lateral ankle instability (CLAI). Methods: Twenty-two CLAI cases with a large ossicle (≥10 mm) were treated consecutively using the MBP with periosteal flap augmentation after ossicle excision. The mean follow-up duration was 20 months (12-33). For clinical assessment, the visual analog scale (VAS), American Orthopaedic Foot & Ankle Society (AOFAS) score, and Karlsson-Peterson score were evaluated preoperatively and at the last follow-up. For radiographic assessment, the size of the ossicle was measured by magnetic resonance imaging, and the talar tilt angle and anterior talar displacement were measured preoperatively and at the last follow-up. Results: The VAS, AOFAS, and Karlsson-Peterson scores improved from 6.3, 68.7, and 56.9 preoperatively to 1.6, 94.5, and 92.4 at the last follow-up, respectively. The mean size of the ossicles was 14.8 mm (11-21 mm). The talar tilt angle and anterior talar displacement improved from 11.2 degrees and 8.3 mm preoperatively to 4.4 degrees and 3.9 mm at the last follow-up, respectively. Conclusion: MBP combined with periosteal flap augmentation after ossicle excision provided good clinical and radiographic outcomes in CLAI with a large ossicle. Level of Evidence: Level IV, case series.

Background: Sinus tarsi pain caused by accessory talar facet impingement (ATFI) owing to accessory anterolateral talar facet (AALTF) is not well documented. We evaluated prevalence of AALTF and differences of magnetic resonance imaging (MRI) findings between persons with and without sinus tarsi pain, and investigated the relevant association between MRI findings and sinus tarsi pain in persons with AALTF. Methods: We performed a case-control study on 120 ankles with sinus tarsi pain and 120 age- and gender-matched ankles without sinus tarsi pain. As MRI findings, bone marrow edema (BME), sinus tarsi fat obliteration (STFO), calcaneal cyst, talocalcaneal coalition, Gissane angle, talar inferolateral surface (TILS) angle, and calcaneal cortical thickness (CCT) were evaluated. The MRI findings were compared between persons with and without sinus tarsi pain, and between persons with and without AALTF. Among persons with AALTF, MRI findings were compared between those with and without sinus tarsi pain. Relevant association was evaluated between MRI findings and sinus tarsi pain in persons with AALTF. Results: Presence of AALTF, BME, and STFO were significantly higher in the group with sinus tarsi pain. The Gissane angle was significantly smaller, and the TILS angle and CCT were significantly larger in the group with sinus tarsi pain. The BME (OR 7.571, CI 1.453-39.446) and small Gissane angle (OR 0.891, CI 0.804-0.986) were significantly associated with sinus tarsi pain in persons with the AALTF. Conclusion: The study provides evidence for ATFI related to impingement of an AALTF associated with talocalcaneal BME. Level of Evidence: Level III, case-control study.

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