Plasma free amino acid profiling of five types of cancer patients and its application for early detection

Background

Recently, rapid advances have been made in metabolomics-based, easy-to-use early cancer detection methods using blood samples. Among metabolites, profiling of plasma free amino acids (PFAAs) is a promising approach because PFAAs link all organ systems and have important roles in metabolism. Furthermore, PFAA profiles are known to be influenced by specific diseases, including cancers. Therefore, the purpose of the present study was to determine the characteristics of the PFAA profiles in cancer patients and the possibility of using this information for early detection.

Methods and Findings

Plasma samples were collected from approximately 200 patients from multiple institutes, each diagnosed with one of the following five types of cancer: lung, gastric, colorectal, breast, or prostate cancer. Patients were compared to gender- and age- matched controls also used in this study. The PFAA levels were measured using high-performance liquid chromatography (HPLC)–electrospray ionization (ESI)–mass spectrometry (MS). Univariate analysis revealed significant differences in the PFAA profiles between the controls and the patients with any of the five types of cancer listed above, even those with asymptomatic early-stage disease. Furthermore, multivariate analysis clearly discriminated the cancer patients from the controls in terms of the area under the receiver-operator characteristics curve (AUC of ROC >0.75 for each cancer), regardless of cancer stage. Because this study was designed as case-control study, further investigations, including model construction and validation using cohorts with larger sample sizes, are necessary to determine the usefulness of PFAA profiling.

Conclusions

These findings suggest that PFAA profiling has great potential for improving cancer screening and diagnosis and understanding disease pathogenesis. PFAA profiles can also be used to determine various disease diagnoses from a single blood sample, which involves a relatively simple plasma assay and imposes a lower physical burden on subjects when compared to existing screening methods.     

Identification of tissues showing the lowest tolerance to freezing in larvae of the rice stem borer, Chilo suppressalis

Abstract.  Even though overwintering larvae of the rice stem borer, Chilo suppressalis , are freeze-tolerant, they cannot survive below −30 °C. Furthermore, nondiapausing larvae cannot survive freezing. However, the cause of death due to freezing is unclear. To identify the cause of death by freezing in larvae, those tissues most injured by low temperatures are identified using the vital stain trypan blue. In overwintering larvae, the midgut of dead larvae stains blue, and remarkable colour density differences between dead and surviving larvae are observed in the midgut. In nondiapausing larvae incubated at −10 °C for several hours, the fat body of dead larvae is strongly stained. Furthermore, increases in mortality with treatment time correspond with increases in the area of the fat body stained. Sterile nondiapausing larvae with lower supercooling points, below −20 °C, do not freeze at −10 °C and survive the treatment. However, all the larvae die when subjected to inoculative freezing at −10 °C, and the fat body stains blue. These results suggest that the midgut in overwintering larvae and the fat body in nondiapausing larvae have the lowest tolerance to freezing.     

Comparison of cold hardiness and sugar content between diapausing and nondiapausing pupae of the cotton bollworm, Helicoverpa armigera (Lepidoptera: Noctuidae)

Abstract. To understand overwintering of the cotton boll worm Helicoverpa armigera, cold hardiness and sugar content are compared between diapausing and nondiapausing pupae. Diapausing and nondiapausing pupae reared at 20 °C under short and long photoperiods are acclimatized with a reduction of 5 °C per 5 days to 0 °C. When the acclimation temperature reaches 0 °C, the survival of diapausing pupae is assessed. The survival gradually decreases as the period of treatment progresses and approximately half survive for 112 days. However, nondiapausing pupae survive only 14 days after exposure to 0 °C. The surpercooling points of nondiapausing, diapausing and acclimatized pupae are approximately −17 °C. The major sugars contained in pupae are trehalose and glucose. Even though trehalose contents in diapausing pupae (initial level: 0.6 mg 100 mg⁻¹ fresh weight) increase significantly during cold acclimation and continue increasing until 58 days after exposure to 0 °C (maximum level: 1.8 mg 100 mg⁻¹), glucose is maintained at low levels (0.02 mg 100 mg⁻¹) for 56 days at 0 °C. However, glucose contents increase (maximum level: 0.8 mg 100 mg⁻¹) with decreasing contents of trehalose 84 days after exposure to 0 °C. Glycogen content gradually decreases during cold acclimation. When nondiapausing pupae are acclimatized with a reduction of 5 °C per 5 days to 5 °C from the beginning of pupation until the eyespots move, trehalose content increases (maximum level: 1.0 mg 100 mg⁻¹). Glucose contents in nondiapausing pupae increase before eclosion (0.09 mg 100 mg⁻¹). From these results, diapausing pupae of H. armigera can overwinter in regions where average winter temperatures are higher than 0 °C, but nondiapausing pupae cannot.     

Quantification of self-renewal capacity in single hematopoietic stem cells from normal and Lnk-deficient mice

Despite being a hallmark of hematopoietic stem cells (HSCs), HSC self-renewal has never been quantitatively assessed. Establishment of a clonal and quantitative assay for HSC function permitted demonstration that adult mouse HSCs are significantly heterogeneous in degree of multilineage repopulation and that higher repopulating potential reflects higher self-renewal activity. An HSC with high repopulating potential could regenerate approximately 1000 HSCs, whereas the repopulating activity of regenerated HSCs on average was significantly reduced, indicating extensive but limited self-renewal capacity in HSCs. Comparisons of wild-type mice with mutant mice deficient in the signal adaptor molecule Lnk showed that not only HSC numbers but also the self-renewal capacity of some HSCs are markedly increased when Lnk function is lost. Lnk appears to control HSC numbers by negatively regulating HSC self-renewal signaling.     

GAT (GGA and Tom1) domain responsible for ubiquitin binding and ubiquitination

GGAs (Golgi-localizing, gamma-adaptin ear domain homology, ADP-ribosylation factor (ARF)-binding proteins) are a family of monomeric adaptor proteins involved in membrane trafficking from the trans-Golgi network to endosomes. The GAT (GGA and Tom1) domains of GGAs have previously been shown to interact with GTP-bound ARF and to be crucial for membrane recruitment of GGAs. Here we show that the C-terminal subdomain of the GAT domain, which is distinct from the N-terminal GAT subdomain responsible for ARF binding, can bind ubiquitin. The binding is mediated by interactions between residues on one side of the alpha3 helix of the GAT domain and those on the so-called Ile-44 surface patch of ubiquitin. The binding of the GAT domain to ubiquitin can be enhanced by the presence of a GTP-bound form of ARF. Furthermore, GGA itself is ubiquitinated in a manner dependent on the GAT-ubiquitin interaction. These results delineate the molecular basis for the interaction between ubiquitin and GAT and suggest that GGA-mediated trafficking is regulated by the ubiquitin system as endosomal trafficking mediated by other ubiquitin-binding proteins.     

Tollip and Tom1 form a complex and recruit ubiquitin-conjugated proteins onto early endosomes

Tom1 (target of Myb1) is a protein of unknown function. Tom1 and its relative Tom1L1 have an N-terminal VHS (Vps27p/Hrs/Stam) domain followed by a GAT (GGA and Tom1) domain, both of which are also found in the GGA (Golgi-localizing, gamma-adaptin ear domain homology, ADP-ribosylation factor-binding protein) family of proteins. Although the VHS and GAT domains of GGA proteins bind to transmembrane cargo proteins and the small GTPase ADP-ribosylation factor, respectively, the VHS and GAT domains of Tom1 are unable to interact with these proteins. In this study, we show that the GAT domains of Tom1 and Tom1L1 interact with ubiquitin and Tollip (Toll-interacting protein). Ubiquitin bound the GAT domains of Tom1, Tom1L1, and GGA proteins, whereas Tollip interacted specifically with Tom1 and Tom1L1. Ubiquitin and Tollip bound to an overlapping region of the Tom1-GAT domain in a mutually exclusive manner. Tom1 was predominantly cytosolic when expressed in cells. On the other hand, Tollip was localized on early endosomes and recruited Tom1 and ubiquitinated proteins. These observations suggest that Tollip and Tom1 form a complex and regulate endosomal trafficking of ubiquitinated proteins.