Metabolic Silencing via Methionine-Based Amino Acid Restriction in Head and Neck Cancer.

In recent years, various forms of caloric restriction (CR) and amino acid or protein restriction (AAR or PR) have shown not only success in preventing age-associated diseases, such as type II diabetes and cardiovascular diseases, but also potential for cancer therapy. These strategies not only reprogram metabolism to low-energy metabolism (LEM), which is disadvantageous for neoplastic cells, but also significantly inhibit proliferation. Head and neck squamous cell carcinoma (HNSCC) is one of the most common tumour types, with over 600,000 new cases diagnosed annually worldwide. With a 5-year survival rate of approximately 55%, the poor prognosis has not improved despite extensive research and new adjuvant therapies. Therefore, for the first time, we analysed the potential of methionine restriction (MetR) in selected HNSCC cell lines. We investigated the influence of MetR on cell proliferation and vitality, the compensation for MetR by homocysteine, the gene regulation of different amino acid transporters, and the influence of cisplatin on cell proliferation in different HNSCC cell lines.

Mass Spectrometric Metabolic Fingerprinting of 2-Deoxy-D-Glucose (2-DG)-Induced Inhibition of Glycolysis and Comparative Analysis of Methionine Restriction versus Glucose Restriction under Perfusion Culture in the Murine L929 Model System.

All forms of restriction, from caloric to amino acid to glucose restriction, have been established in recent years as therapeutic options for various diseases, including cancer. However, usually there is no direct comparison between the different restriction forms. Additionally, many cell culture experiments take place under static conditions. In this work, we used a closed perfusion culture in murine L929 cells over a period of 7 days to compare methionine restriction (MetR) and glucose restriction (LowCarb) in the same system and analysed the metabolome by liquid chromatography mass spectrometry (LC-MS). In addition, we analysed the inhibition of glycolysis by 2-deoxy-D-glucose (2-DG) over a period of 72 h. 2-DG induced very fast a low-energy situation by a reduced glycolysis metabolite flow rate resulting in pyruvate, lactate, and ATP depletion. Under perfusion culture, both MetR and LowCarb were established on the metabolic level. Interestingly, over the period of 7 days, the metabolome of MetR and LowCarb showed more similarities than differences. This leads to the conclusion that the conditioned medium, in addition to the different restriction forms, substantially reprogramm the cells on the metabolic level.

Cysteine Restriction in Murine L929 Fibroblasts as an Alternative Strategy to Methionine Restriction in Cancer Therapy.

Methionine restriction (MetR) is an efficient method of amino acid restriction (AR) in cells and organisms that induces low energy metabolism (LEM) similar to caloric restriction (CR). The implementation of MetR as a therapy for cancer or other diseases is not simple since the elimination of a single amino acid in the diet is difficult. However, the in vivo turnover rate of cysteine is usually higher than the rate of intake through food. For this reason, every cell can enzymatically synthesize cysteine from methionine, which enables the use of specific enzymatic inhibitors. In this work, we analysed the potential of cysteine restriction (CysR) in the murine cell line L929. This study determined metabolic fingerprints using mass spectrometry (LC/MS). The profiles were compared with profiles created in an earlier work under MetR. The study was supplemented by proliferation studies using D-amino acid analogues and inhibitors of intracellular cysteine synthesis. CysR showed a proliferation inhibition potential comparable to that of MetR. However, the metabolic footprints differed significantly and showed that CysR does not induce classic LEM at the metabolic level. Nevertheless, CysR offers great potential as an alternative for decisive interventions in general and tumour metabolism at the metabolic level.

Viruses of Apple Are Seedborne but Likely Not Vertically Transmitted.

Many viruses occur in apple (Malus domestica (Borkh.)), but no information is available on their seed transmissibility. Here, we report that six viruses infecting apple trees, namely, apple chlorotic leaf spot virus (ACLSV), apple green crinkle-associated virus (AGCaV), apple rubbery wood virus 2 (ARWV2), apple stem grooving virus (ASGV), apple stem pitting virus (ASPV), and citrus concave gum-associated virus (CCGaV) occur in seeds extracted from apple fruits produced by infected maternal trees. Reverse transcription polymerase chain reaction (RT-PCR) and quantitative RT-PCR (RT-qPCR) assays revealed the presence of these six viruses in untreated apple seeds with incidence rates ranging from 20% to 96%. Furthermore, ASPV was detected by RT-PCR in the flesh and peel of fruits produced by infected maternal trees, as well as from seeds extracted from apple fruits sold for fresh consumption. Finally, a large-scale seedling grow-out experiment failed to detect ACLSV, ASGV, or ASPV in over 1000 progeny derived from sodium hypochlorite surface sterilized seeds extracted from fruits produced by infected maternal trees, suggesting no detectable transmission via embryonic tissue. This is the first report on the seedborne nature of apple-infecting viruses.

The Comparative Root System Architecture of Declining and Non-Declining Trees in Two Apple Orchards in New York.

Rapid apple decline is a phenomenon characterized by a weakening of young apple trees in high density orchards, often followed by their quick collapse. The nature of this phenomenon remains unclear. In this work, we investigated the root system architecture (RSA) of declining and non-declining apple trees in two orchards in New York State. High-density orchard A consisted of 4-year-old 'Honeycrisp' on 'Malling 9 Nic29', and conventional orchard B consisted of 8-year-old 'Fuji' on 'Budagovsky 9'. In both orchards, a negative correlation (-0.4--0.6) was observed between RSA traits and decline symptoms, suggesting that declining trees have weaker root systems. Scion trunk diameter at the graft union, total root length, and the length of fine and coarse roots were significantly (p < 0.05) reduced in declining trees in both orchards. Additionally, internal trunk necrosis at, above, and below the graft union was observed in declining trees in orchard A but not in orchard B. Finally, latent viruses were not associated with decline, as their occurrence was documented in declining and non-declining trees in orchard A, but not in orchard B. Together, these results showed weakened root systems of declining trees, suggesting that these trees may experience deficiencies in water and nutrient uptake, although distinct RSA and trunk health traits between the two orchards were noticeable.

Low Energy Status under Methionine Restriction Is Essentially Independent of Proliferation or Cell Contact Inhibition.

Nonlimited proliferation is one of the most striking features of neoplastic cells. The basis of cell division is the sufficient presence of mass (amino acids) and energy (ATP and NADH). A sophisticated intracellular network permanently measures the mass and energy levels. Thus, in vivo restrictions in the form of amino acid, protein, or caloric restrictions strongly affect absolute lifespan and age-associated diseases such as cancer. The induction of permanent low energy metabolism (LEM) is essential in this process. The murine cell line L929 responds to methionine restriction (MetR) for a short time period with LEM at the metabolic level defined by a characteristic fingerprint consisting of the molecules acetoacetate, creatine, spermidine, GSSG, UDP-glucose, pantothenate, and ATP. Here, we used mass spectrometry (LC/MS) to investigate the influence of proliferation and contact inhibition on the energy status of cells. Interestingly, the energy status was essentially independent of proliferation or contact inhibition. LC/MS analyses showed that in full medium, the cells maintain active and energetic metabolism for optional proliferation. In contrast, MetR induced LEM independently of proliferation or contact inhibition. These results are important for cell behaviour under MetR and for the optional application of restrictions in cancer therapy.

Allometric relationships of ecologically important Antarctic and Arctic zooplankton and fish species.

Allometric relationships between body properties of animals are useful for a wide variety of purposes, such as estimation of biomass, growth, population structure, bioenergetic modelling and carbon flux studies. This study summarizes allometric relationships of zooplankton and nekton species that play major roles in polar marine food webs. Measurements were performed on 639 individuals of 15 species sampled during three expeditions in the Southern Ocean (winter and summer) and 2374 individuals of 14 species sampled during three expeditions in the Arctic Ocean (spring and summer). The information provided by this study fills current knowledge gaps on relationships between length and wet/dry mass of understudied animals, such as various gelatinous zooplankton, and of animals from understudied seasons and maturity stages, for example, for the krill Thysanoessa macrura and larval Euphausia superba caught in winter. Comparisons show that there is intra-specific variation in length-mass relationships of several species depending on season, e.g. for the amphipod Themisto libellula. To investigate the potential use of generalized regression models, comparisons between sexes, maturity stages or age classes were performed and are discussed, such as for the several krill species and T. libellula. Regression model comparisons on age classes of the fish E. antarctica were inconclusive about their general use. Other allometric measurements performed on carapaces, eyes, heads, telsons, tails and otoliths provided models that proved to be useful for estimating length or mass in, e.g. diet studies. In some cases, the suitability of these models may depend on species or developmental stages. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00300-021-02984-4.