Surface wax esters contribute to drought tolerance in Arabidopsis

Waxes are components of the cuticle covering the aerial organs of plants. Accumulation of waxes has previously been associated with protection against water loss, therefore contributing to drought tolerance. However, not much information is known about the function of individual wax components during water deficit. We studied the role of wax ester synthesis during drought. The wax ester load on Arabidopsis leaves and stems was increased during water deficiency. Expression of three genes, WSD1, WSD6 and WSD7 of the wax ester synthase/diacylglycerol acyltransferase (WS/DGAT or WSD) family was induced during drought, salt stress and abscisic acid treatment. WSD1 has previously been identified as the major wax ester synthase of stems. wsd1 mutants have shown reduced wax ester coverage on leaves and stems during normal or drought condition, while wax ester loads of wsd6, wsd7 and of the wsd6wsd7 double mutant were unchanged. The growth and relative water content of wsd1 plants were compromised during drought, while leaf water loss of wsd1 was increased. Enzyme assays with recombinant proteins expressed in insect cells revealed that WSD6 and WSD7 contain wax ester synthase activity, albeit with different substrate specificity compared with WSD1. WSD6 and WSD7 localize to the endoplasmic reticulum (ER)/Golgi. These results demonstrated that WSD1 is involved in the accumulation of wax esters during drought, while WSD6 and WSD7 might play other specific roles in wax ester metabolism during stress.     

C-Photosynthate Partitioning and Translocation in Soybeans during Reproductive Development

Partitioning and translocation of ¹⁴C-photosynthates were examined during flowering and seed maturation in soybean (Glycine max [L.]Merr.) plants to quantify allocation to sugars, amino acids, organic acids, and starch and to study transport of C and N from leaves to reproductive sinks. The trifoliolate leaf at the eighth node was exposed to steady state levels of ¹⁴CO₂ for 2 hours, followed by immediate extraction and identification of radioactive assimilates in the fed leaf blade, tissues of the transport path (e.g. petiole and stem), and fruits if they were present. About one-third of the total ¹⁴C recovered from the leaf blades was in starch until late pod-filling, after which the proportion dropped to 16%. Sugars comprised 70% to 86% of the recovered ¹⁴C from soluble assimilates of the source leaf, with highest proportions occurring during late flowering and early pod-filling. Amino acids accounted for 8% to 17% of the ¹⁴C recovered from the soluble fraction, and were most evident during early flowering and mid to late pod-filling. The ¹⁴C-organic acids comprised from 3% to 14% of the soluble ¹⁴C-assimilates in leaves. Petioles consistently contained a higher percentage of recovered radioactivity in sugars (87-97%) and a lower percentage in amino acids (3-12%) than did leaf blades. ¹⁴C-Amino acids in petioles attained their highest levels during mid and late pod-filling, while ¹⁴C-organic acids comprised 2% or less of the recovered radioactivity after pod initiation. The distribution of ¹⁴C-assimilates in the internode below the source leaf was similar to that found in petioles. A comparison of the above data to calculated C and N requirements for seed development suggests that ¹⁴C-amino acids derived from current photosynthesis and translocated from source leaves supply at least 12% to 48% of the seed N depending on the stage of pod-filling.     

Validation of Next-Generation Sequencing of Entire Mitochondrial Genomes and the Diversity of Mitochondrial DNA Mutations in Oral Squamous Cell Carcinoma

Background

Oral squamous cell carcinoma (OSCC) is mainly caused by smoking and alcohol abuse and shows a five-year survival rate of ~50%. We aimed to explore the variation of somatic mitochondrial DNA (mtDNA) mutations in primary oral tumors, recurrences and metastases.

Methods

We performed an in-depth validation of mtDNA next-generation sequencing (NGS) on an Illumina HiSeq 2500 platform for its application to cancer tissues, with the goal to detect low-level heteroplasmies and to avoid artifacts. Therefore we genotyped the mitochondrial genome (16.6 kb) from 85 tissue samples (tumors, recurrences, resection edges, metastases and blood) collected from 28 prospectively recruited OSCC patients applying both Sanger sequencing and high-coverage NGS (~35,000 reads per base).

Results

We observed a strong correlation between Sanger sequencing and NGS in estimating the mixture ratio of heteroplasmies (r = 0.99; p<0.001). Non-synonymous heteroplasmic variants were enriched among cancerous tissues. The proportions of somatic and inherited variants in a given gene region were strongly correlated (r = 0.85; p<0.001). Half of the patients shared mutations between benign and cancerous tissue samples. Low level heteroplasmies (<10%) were more frequent in benign samples compared to tumor samples, where heteroplasmies >10% were predominant. Four out of six patients who developed a local tumor recurrence showed mutations in the recurrence that had also been observed in the primary tumor. Three out of five patients, who had tumor metastases in the lymph nodes of their necks, shared mtDNA mutations between primary tumors and lymph node metastases. The percentage of mutation heteroplasmy increased from the primary tumor to lymph node metastases.

Conclusions

We conclude that Sanger sequencing is valid for heteroplasmy quantification for heteroplasmies ≥10% and that NGS is capable of reliably detecting and quantifying heteroplasmies down to the 1%-level. The finding of shared mutations between primary tumors, recurrences and metastasis indicates a clonal origin of malignant cells in oral cancer.     

Membrane-bound neuregulin1 type III actively promotes Schwann cell differentiation of multipotent Progenitor cells

Many steps of peripheral glia development appear to be regulated by neuregulin1 (NRG1) signaling but the exact roles of the different NRG1 isoforms in these processes remain to be determined. While glial growth factor 2 (GGF2), a NRG1 type II isoform, is able to induce a satellite glial fate in neural crest stem cells, targeted mutations in mice have revealed a prominent role of NRG1 type III isoforms in supporting survival of Schwann cells at early developmental stages. Here, we investigated the role of NRG1 isoforms in the differentiation of Schwann cells from neural crest-derived progenitor cells. In multipotent cells isolated from dorsal root ganglia, soluble NRG1 isoforms do not promote Schwann cell features, whereas signaling by membrane-associated NRG1 type III induces the expression of the Schwann cell markers Oct-6/SCIP and S100 in neighboring cells, independent of survival. Thus, axon-bound NRG1 might actively promote both Schwann cell survival and differentiation.     

Effects of thiol modification and Ca++ on agonist-specific state transitions of nicotinic acetylcholine receptor from Torpedo californica electroplax.

The agonist binding affinity of nicotinic acetylcholine receptor (nAChR) from $\text{Torpedo}$$\text{californica}$ electroplax, as inferred from ability of agonist to inhibit specific curaremimetic neurotoxin binding to nAChR, is sensitive to the duration of exposure to agonist. The concentration of carbachol necessary to prevent one-half of toxin binding over a 30 min incubation with nAChR (K₃₀) is 10 μM when toxin and carbachol are simultaneously added to membrane-bound nAChR, and 3 μM when nAChR are pretreated with carbachol for 30 min prior to the addition of toxin. These alterations in agonist affinity may be mimicked by modification of nAChR thiol groups. Affinity of nAChR for carbachol is decreased following treatment with dithiothreitol (DTT). Dithio-bis-nitrobenzoic acid treatment of DTT-reduced membranes yields K₃₀ values of 5 μM for carbachol, while N-ethylmaleimide treatment of DTT-reduced nAChR produces nAChR with reduced affinity for carbachol, reflected in K₃₀ values of about 400 μM. In the absence of Ca⁺⁺, K₃₀ values for carbachol binding to native and DTT-reduced nAChR are diminished 3–6 fold. These affinity alterations are not observed with d-tubocurarine (antagonist) binding to nAChR. Thus, Ca⁺⁺ and the oxidation state of nAChR thiols appear to affect the affinity of nAChR for agonists (but not antagonists), and may therefore be related to agonist-mediated events in receptor activation and/or desensitization.     

Oxidative Stress in Endurance Flight: An Unconsidered Factor in Bird Migration

Migrating birds perform extraordinary endurance flights, up to 200 h non-stop, at a very high metabolic rate and while fasting. Such an intense and prolonged physical activity is normally associated with an increased production of reactive oxygen and nitrogen species (RONS) and thus increased risk of oxidative stress. However, up to now it was unknown whether endurance flight evokes oxidative stress. We measured a marker of oxidative damage (protein carbonyls, PCs) and a marker of enzymatic antioxidant capacity (glutathione peroxidase, GPx) in the European robin (Erithacus rubecula), a nocturnal migrant, on its way to the non-breeding grounds. Both markers were significantly higher in European robins caught out of their nocturnal flight than in conspecifics caught during the day while resting. Independently of time of day, both markers showed higher concentrations in individuals with reduced flight muscles. Adults had higher GPx concentrations than first-year birds on their first migration. These results show for the first time that free-flying migrants experience oxidative stress during endurance flight and up-regulate one component of antioxidant capacity. We discuss that avoiding oxidative stress may be an overlooked factor shaping bird migration strategies, e.g. by disfavouring long non-stop flights and an extensive catabolism of the flight muscles.     

Clinical Features,Treatment and Outcome of Mucosa-Associated Lymphoid Tissue (MALT) Lymphoma of the Ocular Adnexa: Single Center Experience of 60 Patients

Background

Orbital marginal zone B-cell lymphoma (OAML) constitutes for the most frequent diagnosis in orbital lymphoma. Relatively little data, however, have been reported in larger cohorts of patients staged in a uniform way and no therapy standard exists to date.

Material and Methods

We have retrospectively analyzed 60 patients diagnosed and treated at our institution 1999–2012. Median age at diagnosis was 64 years (IQR 51–75) and follow-up time 43 months (IQR 16–92). All patients had undergone uniform extensive staging and histological diagnosis was made by a reference pathologist according to the WHO classification.

Results

The majority of patients presented with stage IE (n = 40/60, 67%), three had IIE/IIIE and the remaining 17 stage IVE. Seven patients with IVE had bilateral orbital disease whereas the others showed involvement of further organs. Treatment data were available in 58 patients. Local treatment with radiotherapy (14/58, 24%) or surgery (3/58, 5%) resulted in response in 82% of patients. A total of 26 patients (45%) received systemic treatment with a response rate of 85%. Nine patients received antibiotics as initial therapy; response rate was 38%. Watchful-waiting was the initial approach in 6/58 patients. In total 28/58 patients (48%) progressed and were given further therapy. Median time-to-progression in this cohort was 20 months (IQR 9–39). There was no difference in time-to-progression after first-line therapy between the different therapy arms (p = 0.14). Elevated beta-2-microglobulin, plasmacytic differentiation, autoimmune disorder and site of lymphoma were not associated with a higher risk for progress.

Conclusion

Our data underscore the excellent prognosis of OAML irrespective of initial therapy, as there was no significant difference in time-to-progression and response between local or systemic therapy. In the absence of randomized trials, the least toxic individual approach should be chosen for OAML.