Background: MicroRNA (miRNA) regulates post-transcriptional gene expression through binding to complementary sites of target messenger RNA, including that from oncogenes or tumor suppressor genes. This study planned to pursue the possibility that circulating miRNA could be used for the early diagnosis of diffuse large B-cell lymphoma (DLBCL). Materials and Methods: Expression levels of miRNA obtained from serum, exosome-enriched serum, and formalin-fixed paraffinembedded (FFPE) tissue were evaluated. Samples were collected from patients with newly diagnosed DLBCL (n = 33) or healthy volunteers (n = 22). Based on the results of previous reports, ten miRNAs were selected and expression levels were analyzed by the quantitative real-time PCR. Results: The expression levels of hsa-miR-15a-3p, hsa-miR-21-5p, hsa-miR-181a-5p, and hsa-miR-210-5p differed significantly between DLBCL patients and controls in serum and/or exosomeenriched serum, but not in FFPE tissue. In contrast, expression levels of hsa-miR-155-5p in FFPE tissue were significantly higher in DLBCL patients, as previously reported. Conclusion: We confirmed that some miRNAs were differentially expressed in serum from DLBCL patients as previously reported. Measurement of these miRNA in exosome-enriched serum did not improve the accuracy in the differential diagnosis of DLBCL. In addition, these miRNAs seem to be produced outside of lymphoma tissue.
Lymph node metastasis is one of the most important factors for tumor dissemination. Quantifying microRNA (miRNA) expression using real-time PCR in formalin-fixed, paraffin-embedded (FFPE) lymph node can provide valuable information regarding the biological research for cancer metastasis. However, a universal endogenous reference gene has not been identified in FFPE lymph node. This study aimed to identify suitable endogenous reference genes for miRNA expression analysis in FFPE lymph node. FFPE lymph nodes were obtained from 41 metastatic cancer and from 16 non-cancerous tissues. We selected 10 miRNAs as endogenous reference gene candidates using the global mean method. The stability of candidate genes was assessed by the following four statistical tools: BestKeeper, geNorm, NormFinder, and the comparative ΔCt method. miR-103a was the most stable gene among candidate genes. However, the use of a single miR-103a was not recommended because its stability value exceeded the reference value. Thus, we combined stable genes and investigated the stability and the effect of gene normalization. The combination of miR-24, miR-103a, and let-7a was identified as one of the most stable sets of endogenous reference genes for normalization in FFPE lymph node. This study may provide a basis for miRNA expression analysis in FFPE lymph node tissue.
The endogenous volatile organic compounds (VOCs) in exhaled breath can be promising biomarkers for various diseases including cancers. An olfactory sensor has a possibility for extracting a specific feature from collective variations of the related VOCs with a certain health condition. For this approach, it is important to establish a feasible protocol for sampling exhaled breath in practical conditions to provide reproducible signal features. Here we report a robust protocol for the breath analysis, focusing on total expiratory breath measured by a Membrane-type Surface stress Sensor (MSS), which possesses practical characteristics for artificial olfactory systems. To assess its reproducibility, 83 exhaled breath samples were collected from one subject throughout more than a year. It has been confirmed that the reduction of humidity effects on the sensing signals either by controlling the humidity of purging room air or by normalizing the signal intensities leads to reasonable reproducibility verified by statistical analyses. We have also demonstrated the applicability of the protocol for detecting a target material by discriminating exhaled breaths collected from different subjects with pre- and post-alcohol ingestion on different occasions. This simple yet reproducible protocol based on the total expiratory breath measured by the MSS olfactory sensors will contribute to exploring the possibilities of clinical applications of breath diagnostics.
The giant water bug Appasus major exhibits a peculiar reproductive behavior where females lay eggs on the backs of males. A male and female pair performs frequent repeat copulations during the oviposition behavior, and the male carries the deposited eggs until hatching. Such characteristic behaviors predict that the eggs are fertilized by the brooding males. If males carry eggs of other unrelated males, the egg carrying will drastically decrease the fitness of the carriers. In this study, we examined genetic relationships between the 489 eggs and nine males carrying them collected from the field, using microsatellite DNA markers. We revealed that in total, 28.4% of the eggs were of other male origin. This is the first report of frequent brood parasitism in an aquatic egg-carrying hemipteran insect. The brood parasitism is adaptive for the females probably because it enables them to increase the chance of oviposition, or it can equalize motility risk of the eggs of each mate.
Bone marrow microenvironment plays a crucial role for the development and progression of multiple myeloma (MM). To gain insights about the abnormality in microenvironment and to predict the prognosis, we comprehensively analyzed cytokines/chemokines in MM patients. Serum concentrations of 49 cytokines/chemokines were measured by using the suspension array system, Bio-Plex (Bio-Rad), and enzyme-linked immunosorbent assay (ELISA) kits. Serum samples were obtained from 35 MM patients including those with active disease (patients with newly diagnosed myeloma or exacerbated myeloma after more than 6 months of therapy-free period, n=26) and those with controlled disease (patients achieving ≥VGPR within 6 months of auto-PBSCT, n=9), 14 MGUS patients, and 32 healthy volunteers. Serum concentrations of 40 out of 49 assayed cytokines/chemokines were significantly increased or decreased in active disease MM patients in comparison with those of controls. As such a skewed cytokine/chemokine profile has never been observed in colorectal cancer (our unpublished data), we consider that this abnormality is a unique feature of MM. To extract cytokines/chemokines related to the disease progression, we compared serum concentrations of cytokines/chemokines between ISS stage I and II-III patients. Among the 40 cytokines/chemokines, 5 were selected as cytokines/chemokines whose concentrations became more deviated from the normal range as the disease progression. Indeed, serum concentrations of b-NGF (p=0.016), IL-2Ra (p=0.009), IL-12 (p40) (p=0.013), MIG (p=0.002), and SCF (p=0.003) increased significantly in stage II-III patients in comparison with stage I patients. Moreover, overall survival of patients with higher concentration of each of these 5 cytokines/chemokines was significantly worse in comparison with those with lower concentration (b-NGF: p=0.009; IL-2Ra: p=0.021; IL-12 (p40): p=0.007; MIG: p=0.006; and SCF: p=0.005). To our interest, concentrations of all of these 5 cytokines/chemokines were in normal range in MGUS patients. We thus consider that these 5 cytokines/chemokines are related to the development and progression of MM. We next analyzed whether such an abnormality in cytokines/chemokines is corrected by the disease control. For this purpose, we compared serum concentrations of cytokines/chemokines between MM patients with active disease and those with controlled disease. Contrary to our expectation, normalization of the serum concentration was observed only in MIG, and other 4 cytokines/chemokines remained in abnormal range even after achieving VGPR. It suggests that systemic changes of cytokine/chemokine profile in MM patients persist long even after an efficient treatment, which possibly contributes to the exacerbation. In summary, our findings suggest that extensive abnormality in internal environment does exist in MM patients and this abnormality is not corrected even after disease control. Future researches are warranted to elucidate the underlying mechanism of the abnormality in cytokines/chemokines in MM patients. Figure Figure. Disclosures No relevant conflicts of interest to declare.
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