Molecular species profiles of plasma ceramides in different clinical types of X-linked adrenoleukodystrophy.

X-linked adrenoleukodystrophy (X-ALD) is a genetic disorder associated with peroxisomal dysfunction. Patients with this rare disease accumulate very long-chain fatty acids (VLCFAs) in their bodies because of impairment of peroxisomal VLCFA ?-oxidation. Several clinical types of X-ALD, ranging from mild (axonopathy in the spinal cord) to severe (cerebral demyelination), are known. However, the molecular basis for this phenotypic variability remains largely unknown. In this study, we determined plasma ceramide (CER) profile using liquid chromatography-tandem mass spectrometry. We characterized the molecular species profile of CER in the plasma of patients with mild (adrenomyeloneuropathy;AMN) and severe (cerebral) X-ALD. Eleven X-ALD patients (five cerebral, five AMN, and one carrier) and 10 healthy volunteers participated in this study. Elevation of C26:0 CER was found to be a common feature regardless of the clinical types. The level of C26:1 CER was significantly higher in AMN but not in cerebral type, than that in healthy controls. The C26:1 CER level in the cerebral type was significantly lower than that in the AMN type. These results suggest that a high level of C26:0 CER, along with a control level of C26:1 CER, is a characteristic feature of the cerebral type X-ALD. J. Med. Invest. 70 : 403-410, August, 2023.

Novel antimyeloma therapeutic option with inhibition of the HDAC1-IRF4 axis and PIM kinase.

Multiple myeloma (MM) preferentially expands and acquires drug resistance in the bone marrow (BM). We herein examined the role of histone deacetylase 1 (HDAC1) in the constitutive activation of the master transcription factor IRF4 and the prosurvival mediator PIM2 kinase in MM cells. The knockdown or inhibition of HDAC1 by the class I HDAC inhibitor MS-275 reduced the basal expression of IRF4 and PIM2 in MM cells. Mechanistically, the inhibition of HDAC1 decreased IRF4 transcription through histone hyperacetylation and inhibiting the recruitment of RNA polymerase II at the IRF4 locus, thereby reducing IRF4-targeting genes, including PIM2. In addition to the transcriptional regulation of PIM2 by the HDAC1-IRF4 axis, PIM2 was markedly upregulated by external stimuli from BM stromal cells and interleukin-6 (IL-6). Upregulated PIM2 contributed to the attenuation of the cytotoxic effects of MS-275. Class I HDAC and PIM kinase inhibitors cooperatively suppressed MM cell growth in the presence of IL-6 and in vivo. Therefore, the present results demonstrate the potential of the simultaneous targeting of the intrinsic HDAC1-IRF4 axis plus externally activated PIM2 as an efficient therapeutic option for MM fostered in the BM.

Only one carbon difference determines the pro-apoptotic activity of α-tocopheryl esters.

α-Tocopheryl succinate (TS), a redox-silent succinyl ester of natural α-Tocopherol, has emerged as a novel anti-cancer agent. However, the underlying mechanism is unclear. We found that the terminal dicarboxylic moiety of tocopheryl esters contributes to apoptosis induction and thus cytotoxicity. To further examine this relationship, we compared the pro-apoptotic activity of TS, which has four carbon atoms in the terminal dicarboxylic moiety, to that of a newly synthesized, tocopheryl glutarate (Tglu), which has five. Cytotoxicity assays in vitro confirmed that TS stimulated apoptosis, while Tglu was non-cytotoxic. In investigating biological mechanisms leading to these opposing effects, we found that TS caused an elevation of intracellular superoxide, but Tglu did not. TS increased intracellular Ca2+ in cultured cells, suggesting induction of endoplasmic reticulum (ER) stress; however, Tglu did not affect Ca2+ homeostasis. 1,4,5-trisphosphate (IP3 ) receptor antagonist 2-Aminoethyl diphenylborinate (2-APB) decreased TS-induced intracellular Ca2+ , restored mitochondrial activity and cell viability in TS-treated cells, establishing the ER-mitochondria relationship in apoptosis induction. Moreover, real-time PCR, immunostaining and Western blotting assays revealed that TS downregulated glucose-regulated protein 78 (GRP78), which maintains ER homeostasis and promotes cell survival. Conversely, Tglu upregulates GRP78. Taken together, our results suggest a model in which TS-mediated superoxide production and GRP78 inhibition induce ER stress, which elevates intracellular Ca2+ and depolarizes mitochondria, leading to apoptosis. Because Tglu does not affect superoxide generation and increases GRP78 expression, it inhibits ER stress and is thereby non-cytotoxic. Our research provides insight into the structure-activity relationship of tocopheryl esters regarding the induction of apoptosis.

Development of a novel tocopheryl ester for suppression of lipid accumulation without cytotoxicity by optimization of dicarboxylic ester moiety.

Tocopheryl succinate (Tsuc) is a succinic acid ester of the well-known antioxidant α-tocopherol (T). Tsuc exhibits various biological activities, including tumor growth suppression via activation of cell signaling and prevention of lipid accumulation in mouse adipocyte 3T3-L1 cells. The latter findings suggest that Tsuc may be a drug candidate for the treatment of obesity. However, Tsuc was found to induce apoptosis of normal cells (in addition to cancer cells), demonstrating the need to reduce the cytotoxicity of Tsuc without losing the suppression effect on lipid accumulation. Based on our previous findings, we focused on the ester structure of Tsuc for controlling cytotoxicity. Herein, we examined the cytotoxicity and lipid accumulation suppression effect of various T ester derivatives. We found that the terminal carboxylic group is necessary for suppression of lipid accumulation. We synthesized tocopheryl glutarate (Tglu) and tocopheryl adipate (Tadi) by elongation of carbon atoms 1 and 2 of the dicarboxylic moiety, respectively. Tglu and Tadi did not show any cytotoxicity, and both esters suppressed lipid accumulation, although their suppression activities were weaker than that of Tsuc. Tadi showed a more potent lipid accumulation inhibitory effect than Tglu. Although Tadi inhibited lipogenesis and promoted lipolysis, lipolysis was induced at lower concentrations than inhibition of lipogenesis, suggesting that Tadi mainly affects lipolysis. Taken together, we succeeded in the reduction of cytotoxicity, without loss of the suppression effect on lipid accumulation, by elongation of the dicarboxylic moiety of Tsuc. Tadi may be a promising candidate as an anti-obesity drug.

Indocyanine green-labeled dasatinib as a new fluorescent probe for molecular imaging of gastrointestinal stromal tumors.

BACKGROUND AND AIM: It is difficult to differentiate gastrointestinal stromal tumors (GISTs) from other subepithelial lesions under gastrointestinal endoscopy. Because most GISTs express tyrosine kinase receptor c-KIT, fluorescence-labeled c-KIT-specific tyrosine kinase inhibitors seem to be useful agents for molecular imaging of GIST. We aimed to develop a near-infrared fluorescent imaging technology for GIST targeting c-KIT using the novel fluorescent probe indocyanine green-labeled dasatinib (ICG-dasatinib) and to investigate the antitumor effect of ICG-dasatinib on GIST cells. METHODS: Indocyanine green-labeled dasatinib was synthesized by labeling linker-induced dasatinib with ICG derivative 3-indocyanine-green-acyl-1,3-thiazolidine-2-thione. Human GIST cell lines GIST-T1 and GIST-882M were incubated with ICG-dasatinib and observed by fluorescent microscopy. GIST cells were incubated with ICG-dasatinib, unlabeled dasatinib, or imatinib, and cell viabilities were evaluated. Subcutaneous GIST model mice or orthotopic GIST model rats were intravenously injected with ICG-dasatinib and observed using an IVIS Spectrum. RESULTS: Strong fluorescent signals of ICG-dasatinib were observed in both GIST cell lines in vitro. IC50 values for ICG-dasatinib, unlabeled dasatinib, and imatinib were 13.9, 1.17, and 16.2 nM in GIST-T1 and 26.6, 3.63, and 47.6 nM in GIST-882M cells, respectively. ICG-dasatinib accumulated in subcutaneous xenografts in mice. Fluorescent signals were also observed in liver and gallbladder, indicating biliary excretion; however, fluorescence intensity of tumors was significantly higher than that of intestine after washing. Strong fluorescent signals were observed in orthotopic xenografts through the covering normal mucosa in rats. CONCLUSIONS: Indocyanine green-labeled dasatinib could visualize GIST cells and xenografted tumors. The antitumor effect of ICG-dasatinib was preserved to the same degree as imatinib.

Unique anti-myeloma activity by thiazolidine-2,4-dione compounds with Pim inhibiting activity.

Proviral Integrations of Moloney virus 2 (PIM2) is overexpressed in multiple myeloma (MM) cells, and regarded as an important therapeutic target. Here, we aimed to validate the therapeutic efficacy of different types of PIM inhibitors against MM cells for their possible clinical application. Intriguingly, the thiazolidine-2,4-dione-family compounds SMI-16a and SMI-4a reduced PIM2 protein levels and impaired MM cell survival preferentially in acidic conditions, in contrast to other types of PIM inhibitors, including AZD1208, CX-6258 and PIM447. SMI-16a also suppressed the drug efflux function of breast cancer resistance protein, minimized the sizes of side populations and reduced in vitro colony-forming capacity and in vivo tumourigenic activity in MM cells, suggesting impairment of their clonogenic capacity. PIM2 is known to be subject to ubiquitination-independent proteasomal degradation. Consistent with this, the proteasome inhibitors bortezomib and carfilzomib increased PIM2 protein levels in MM cells without affecting its mRNA levels. However, SMI-16a mitigated the PIM2 protein increase and cooperatively enhanced anti-MM effects in combination with carfilzomib. Collectively, the thiazolidine-2,4-dione-family compounds SMI-16a and SMI-4a uniquely reduce PIM2 protein in MM cells, which may contribute to their profound efficacy in addition to their immediate kinase inhibition. Their combination with proteasome inhibitors is envisioned.

Analysis of Molecular Species Profiles of Ceramide-1-phosphate and Sphingomyelin Using MALDI-TOF Mass Spectrometry.

Ceramide-1-phosphate (C1P) is a potential signaling molecule that modulates various cellular functions in animals. It has been known that C1P with different N-acyl lengths induce biological responses differently. However, molecular species profiles of the C1P in animal tissues have not been extensively examined yet. Here, we developed a method for determination of the molecular species of a C1P using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry with Phos-tag, a phosphate capture molecule. The amounts of total C1P in skin, brain, liver, kidney and small intestine of mice were determined to be 344, 151, 198, 96 and 90 pmol/g wet weight, respectively. We found a C1P species having an α-hydroxypalmitoyl residue (h-C1P, 44 pmol/g wet weight) in mouse skin. The h-C1P was detected only in the skin, and not other tissues of mice. The same analysis was applied to sphingomyelin after conversion of sphingomyelin to C1P by Streptomyces chromofuscus phospholipase D. We found that molecular species profiles of sphingomyelin in skin, kidney and small intestine of mice were similar to those of C1P in corresponding tissues. In contrast, molecular species profiles of sphingomyelin in liver and brain were quite different from those of C1P in these tissues, indicating selective synthesis or degradation of C1P in these tissues. The method described here will be useful for detection of changes in molecular species profiles of C1P and sphingomyelin.

Reinvestigation of the synthesis of isoliquiritigenin: application of Horner-Wadsworth-Emmons reaction and Claisen-Schmidt condensation.

Isoliquiritigenin [ILG, (E)-1] was readily prepared via the Horner-Wadsworth-Emmons reactions using ß-ketophosphonates 5a, b. An improved protocol for the synthesis of (E)-1 via the Claisen-Schmidt condensation was also presented.

Evaluation of inhibitory actions of flavonols and related substances on lysophospholipase d activity of serum autotaxin by a convenient assay using a chromogenic substrate.

Overproduction of lysophosphatidic acid (LPA) by lysophospholipase D/autotaxin (lysoPLD/ATX) is postulated to be involved in the promotion of cancer and atherosclerosis. A lysoPLD inhibitor may be utilized to ameliorate the LPA-related pathological conditions. In this study, a new assay was devised to quantify p-nitrophenol from hydrolysis of chromogenic substrate by serum lysoPLD without tedious lipid extraction procedures. Flavonols, phenolic acids, free fatty acids, and N-acyltyrosines inhibited lysoPLD activity in a micromolar range. They were classified into competitive, noncompetitive, or mixed type inhibitors. The results show that the low hydrophobicity of an inhibitor is a critical factor in its preference for the binding to a noncatalytic binding site over a catalytic binding site. Considering its reported bioavailability and the low dependency of its inhibitory activity on serum dilution, flavonol is likely to be a more effective lysoPLD inhibitor in human blood circulation in vivo than the other inhibitors including LPA.

Synthesis and reaction of 1-azabicyclo[3.1.0]hexane.

The effective formation of 1-azabicyclo[3.1.0]hexane (5) by treatment of 2-(bromomethyl)pyrrolidine hydrobromide (4) with n-BuLi was established, with the reaction occurring by a rational reaction pathway via the open chain transition state 8 based on intermolecular Br...Li(+) coordination (SN2 process). The reaction of 5 with electrophiles 13a-n gave the corresponding pyrrolidines 14a-n and piperidine 6, 15a-g, i-n. The selectivity of the products in this reaction appeared to be controlled by equilibrium.

Intramolecular nonbonded S...N interaction in rabeprazole.

Intramolecular nonbonded S...N interactions in the crystal structures of the derivatives (7a-d) of sodium rabeprazole (1) and an intermolecular nonbonded S...N interaction between ethylmethylsulfoxide and pyridine in a solution were recognized. These results made us estimate that the intramolecular nonbonded S...N interaction existed in 1 and its derivatives in a solution, and formed the 4-membered quasi-ring in 2 (Fig. 1) followed by the increase of the reactivity of 2 to give the putative spiro sulfoxide 3, which is the key intermediate in the reaction cascade of 1 (Chart 1).

Synthesis of New Quinolone Antibiotics Utilizing Azetidine Derivatives Obtained from 1-Azabicyclo[1.1.0]butane.

A series of 3-sulfenylazetidine derivatives 5a-f were synthesized via the ring-opening reactions of 1-azabicyclo[1.1.0]butane (ABB, 3) with thiols 4a-f in 50-92% yields. Treatment of ABB (3) with aromatic amines 9a-e and dibenzylamine (9f) in the presence of Mg(ClO4)2 afforded the corresponding 3-aminoazetidine derivatives 10a-f in 24-65% yields. N-Benzyl-3-bromoazetidine (13), which was obtained by the reaction of ABB (3) with benzyl bromide, gave 3-aliphatic amino-substituted azetidine derivatives 15a, b. Novel fluoroquinolones 7a-f, 11a-f, 16a, b and 25a-c were obtained by the introduction of these azetidine derivatives into the C7 position of a quinolone nucleus 6 and N1-heterocyclic quinolones 21a-c in 21-83% yields. Some of them exhibited a greater antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) in comparison with that of clinically used fluoroquinolone, levofloxacin (LVFX).

Effects of magnesium salts and amines on the stereoselectivity in the imine aldol reaction.

In the imine aldol reactions of 1 with aromatic aldehydes using magnesium salts in the presence of amines, the threo/erythro ratios of products increased in the order Mg(ClO4)2>MgI2>MgBr2>MgCl2>Mg(OTf)2 and N,N,N',N'-tetramethylethylenediamine (TMEDA)>Et3N. This increase in the threo/erythro ratios of products was estimated to be caused by a retro-imine aldol reaction under thermodynamic control.

Nonenzymatic enantioselective monoacetylation of prochiral 2-protectedamino-2-alkyl-1,3-propanediols utilizing a chiral sulfonamide-Zn complex catalyst.

[structure: see text] Treatment of a chiral sulfonamide with Et(2)Zn gave quantitatively its Zn complex and then the structure was determined by X-ray crystallographic analysis. Reaction of prochiral N-Boc-2-amino-2-alkyl-1,3-propanediols with Ac(2)O in the presence of 5 mol % of chiral sulfonamide-Zn complex catalyst afforded the corresponding chiral monoacetyl products in 70-92% yields with 70-88% ee values. The proposed mechanism for the catalytic monoacetylation of a prochiral 1,3-propanediol was presented on the basis of CSI-MS analysis.

Synthesis and antibacterial activities of new quinolone derivatives utilizing 1-azabicyclo[1.1.0]butane.

The ring-opening reactions of 1-azabicyclo[1.1.0]butane 3 with thiols 6a-f gave 3-sulfenylazetidine derivatives 7a-f in 50-92% yields. Treatment of 3 with aromatic amines 11a-e and dibenzylamine 11f in the presence of Mg(ClO(4))(2) afforded the corresponding 3-aminoazetidine derivatives 12a-f in 24-53% yields. These azetidine derivatives were introduced into the C7 position of a quinolone nucleus 8 to afford the corresponding fluoroquinolones 9a-f and 13a-f in 21-83% yields. Some of them exhibited superior antibacterial activity against quinolone-susceptible MRSA in comparison with clinically used fluoroquinolones, such as levofloxacin, ciprofloxacin, and gatifloxacin.

Highly stereoselective asymmetric Pummerer reactions that incorporate intermolecular and intramolecular nonbonded S...O interactions.

New chiral sulfoxides (R(S),S)-3, (S(S),S)-3, (R(S),S)-4, and (S(S),S)-4 and known chiral sulfoxides (R(S))-5, (R(S))-6, and (R(S))-7 were synthesized, and the stereochemistry of the new sulfoxides (R(S),S)-3 and (R(S),S)-4 was determined by X-ray crystallographic analysis. In their crystallographic structures, the intramolecular nonbonded S...O close contacts were recognized. Analyses of several sulfoxide complexes including rac-11 with N,N-dimethylacetamide (DMAC) or N-methyl-2-pyrrolidone (NMP) in a MeOH solution utilizing cold-spray ionization mass spectrometry provided, for the first time, direct information for intermolecular nonbonded S...O interactions between sulfoxides and amide (or lactam) in a solution. Highly diastereoselective and enantioselective Pummerer reactions based on the concept of intermolecular and intramolecular nonbonded S...O interactions were performed by treatment of several chiral sulfoxides (R(S), S)-3, (S(S), S)-3, (R(S), S)-4, (S(S), S)-4, (R(S))-5, (R(S))-6, and (R(S))-7 with acetic anhydride and trimethylsilyl triflate (TMSOTf) in DMAC, NMP, N,N-dimethylformamide, and N-formylpiperidine. Mechanistic studies on these facile stereoselective Pummerer reactions revealed the necessity for the amide/TMSOTf complex, such as 26 or 27, to be an efficient activation reagent for Ac(2)O and a trapping reagent for the released acetate ion, and that DMAC and NMP had a positive effect on this highly stereoselective chiral transfer reaction.

Principle and clinical usefulness of the infrared fluorescence endoscopy.

Since there is no infrared fluorescence materials in the living body, infrared fluorescence labeling materials are very useful for making a diagnosis of a micro cancer. We have developed an infrared fluorescence endoscope (IRFE) and indocyanin green (ICG)-derivative as infrared fluorescence labeling materials to evaluate gastrointestinal neoplastic lesions. The study aims were to apply an IRFE and to demonstrate its usefulness in detecting cancerous tissue using an antibody coupled with ICG-derivative. IRFE consisted of an infrared endoscope equipped with excitation (710-790 nm) and barrier (810-920 nm) filters and an intensified CCD camera. We have developed ICG N-hydroxy sulfo succinimide ester (ICG-sulfo-OSu) and 3-ICG-acyl-1, 3-thiazolidine-2-thione (ICG-ATT) as an infrared fluorescent-labeling reagent. ICG-derivative-labeled mouse anti-human carcinoembryonic antigen (CEA) antibody and MUC1 antibody were employed in this study. Moreover, we examined the ability of a reinforcement agent, octylglucoside, to intensity fluorescence from the labeled antibody. Biopsy specimens of gastric cancer were stained with anti-CEA antibody by the avidin-biotinylated peroxidase complex method. Among the positive specimens, freshly resected stomach from three cases were used for the infrared (IR) imaging analysis. The incubation of freshly resected stomach specimens with ICG-anti-CEA antibody-complex resulted in positive staining of the tumor sites by IRFE, and the IR fluorescent images correlated well with the tumor sites. The immunohistochemical studies suggested that the intensity of IR fluorescence of ICG-ATT-MUC1 was stronger than that of ICG-sulfo-OSu. In tumor sections, the reinforcement agent intensified fluorescence, ever at low antibody concentrations. Therefore, we conclude that an anti-CEA (and/or MUC1) antibody with affinity for cancerous lesions and labeled with ICG-derivative can be imaged with this IRFE. Specific antibodies tagged with ICG-derivative with the reinforcement agent can label cancer cells and generate a strong enough fluorescent signal to detect small cancers when examined with an IR fluorescence endoscope.

Optimal labeling condition of antibodies available for immunofluorescence endoscopy.

PURPOSE: In recent years, labeled antibodies have been used for diagnostic imaging in many studies. In this study, we investigated the mode of binding in antibodies labeled with ICG derivatives newly developed for the diagnosis of microcarcinomas, and evaluated the optimal binding molar ratio between the labeling compounds and antibody. METHODS: MUC 1 antibody and ICG derivatives (ICG-ATT and ICG-sulfo-OSu) were used. ICG derivatives non-covalently bound to the antibody were removed with ethyl acetate, and the ratio of ICG derivatives covalently bound to the labeled antibody was confirmed. During purification of the labeled antibody, the amount of each labeling compound reacting with 1 molecule of the antibody varied as follows: 4, 8, 16, and 32 molar equivalents. Subsequently, the intensity of fluorescence was evaluated by spectroscopy and infrared fluoroscopy. RESULTS: The ratio of residual ICG derivative labeling the antibody was 67.4% for ICG-ATT and 65.0% for ICG-sulfo-OSu. When fluorescent antibody labeled with ICG-ATT at an F/P ratio of 2.94 or 4.18 was used, specific and clear fluorescent images of the antigen were obtained. When ICG-ATT-labeled antibody at an F/P ratio of 6.50 or 6.75 was used, the fluorescence intensity decreased and the fluorescent images of antigen became unclear. CONCLUSIONS: It was found that the ICG-ATT-labeled antibody was a more specific and sensitive marker than ICG-sulfo-OSu-labeled antibody, and that lower binding molar ratios of ICG-ATT were more useful for labeling the antibody.

Expeditious syntheses of conjugated allenyl esters and oxazoles through a cascade reaction of alpha-alkynyl malonates under alkaline conditions.

Diethyl alpha-alkynyl-alpha-methoxymalonates (2a--e) were smoothly hydrolyzed and then decarboxylated under alkaline conditions employing 1 N KOH in EtOH to give conjugated allenyl esters (6a--e) in high yields, and similar alkaline treatment of diethyl alpha-alkynyl-alpha-acetylaminomalonates (5a, b, d, e) furnished unexpectedly the oxazoles (7a, b, d, e) having three substituent groups in excellent yields.

Fab fragment labeled with ICG-derivative for detecting digestive tract cancer.

BACKGROUND: In previous studies, we generated infrared ray fluorescence-labeled monoclonal antibodies and developed an infrared ray fluorescence endoscope capable of detecting the monoclonal antibodies to establish a novel diagnostic technique for gastrointestinal cancer. Although the whole IgG molecule has commonly been used for preparation of labeled antibodies, labeled IgG displays insufficient sensitivity and specificity, probably resulting from non-specific binding of the Fc fragment to target cells or interference between fluorochromes on the identical labeled antibody, which might be caused by molecular structure. In this in vitro study, we characterized an Fc-free fluorescence-labeled Fab fragment, which was expected to yield more specific binding to target cells than the whole IgG molecule. METHODS: An anti-mucin antibody and ICG-ATT, an ICG derivative, were used as the labeled antibody and labeling compound, respectively. Paraffin sections of excised gastric cancer tissues were subjected to staining. The labeled whole IgG molecule (ICG-ATT-labeled IgG) and the labeled Fab fragment (ICG-ATT-labeled Fab) were prepared according to a previous report, and the fluorescence properties, antibody activities, and features of fluorescence microscope images obtained from paraffin sections were compared. RESULTS: Both ICG-ATT-labeled Fab and ICG-ATT-labeled IgG were excited by a near infrared ray of 766nm, and maximum emission occurred at 804nm. Antibody activities of ICG-ATT-labeled Fab were shown to be similar to those of unlabeled anti-MUC1 antibody. The fluorescence intensity obtained from paraffin sections of excised gastric cancer tissues revealed a tendency to be greater with ICG-ATT-labeled Fab than with ICG-ATT-labeled IgG. CONCLUSIONS: The infrared ray fluorescence-labeled Fab fragment was likely to be more specific than the conventionally labeled antibodies. Fragmentation of antibodies is considered to contribute to improved sensitivity and specificity of labeled antibodies for detection of micro gastrointestinal cancers.