METTL3 Modulates Ctsk+ Lineage Supporting Cranial Osteogenesis via Hedgehog.

N6-methyladenosine (m6A) modification, a eukaryotic messenger RNA modification catalyzed by methyltransferase-like 3 (METTL3), plays a pivotal role in stem cell fate determination. Calvarial bone development and maintenance are orchestrated by the cranial sutures. Cathepsin K (CTSK)-positive calvarial stem cells (CSCs) contribute to mice calvarial ossification. However, the role of m6A modification in regulating Ctsk+ lineage cells during calvarial development remains elusive. Here, we showed that METTL3 was colocalized with cranial nonosteoclastic Ctsk+ lineage cells, which were also associated with GLI1 expression. During neonatal development, depletion of Mettl3 in the Ctsk+ lineage cells delayed suture formation and decreased mineralization. During adulthood maintenance, loss of Mettl3 in the Ctsk+ lineage cells impaired calvarial bone formation, which was featured by the increased bone porosity, enhanced bone marrow cavity, and decreased number of osteocytes with the less-developed cellular outline. The analysis of methylated RNA immunoprecipitation sequencing and RNA sequencing data indicated that loss of METTL3 reduced Hedgehog (Hh) signaling pathway. Restoration of Hh signaling pathway by crossing Sufufl/+ alleles or by local administration of SAG21 partially rescued the abnormity. Our data indicate that METTL3 modulates Ctsk+ lineage cells supporting calvarial bone formation by regulating the Hh signaling pathway, providing new insights for clinical treatment of skull vault osseous diseases.

"Very early" intrahepatic cholangiocarcinoma (≤ 2.0 cm): MRI manifestation and prognostic potential.

AIMS: To explore the MRI characteristics and clinical outcome of the "very early" intrahepatic cholangiocarcinoma (iCCA) ≤2.0cm. MATERIALS AND METHODS: Totally 213 pathologically confirmed iCCAs (44 ≤ 2.0cm and 169 of 2.0-5.0cm) from two institutes were included. Forty-four matching non-iCCA malignancies ≤2.0cm were also enrolled. Recurrence-free survival (RFS) was estimated and compared between iCCAs ≤2.0cm and 2.0-5.0cm. MRI features were analyzed and compared between iCCAs ≤2.0cm and 2.0-5.0cm, as well as between iCCAs ≤2.0cm and non-iCCAs ≤2.0cm. Univariate and multivariate regression analyses were performed to identify independent imaging features for discrimination. An MRI-based diagnostic model for iCCA ≤2.0cm was constructed by incorporating the independent imaging features. RESULTS: ICCAs ≤2.0cm had a significantly longer RFS than those of 2.0-5.0cm (log rank P=0.014). Imaging features of homogeneous signal (odds ratio (OR) = 6.677, P<0.001) and lack of vessel invasion (OR=7.56, P<0.001) were more frequently displayed in iCCAs ≤2.0cm compared to iCCAs of 2.0-5.0cm independently. In the small lesions ≤2.0cm, imaging features of progressive or persistent enhancement pattern (OR=27.78, P=0.002) and rim diffusion restriction (OR=5.70, P=0.027) were independent imaging features suggestive of iCCA over non-iCCA malignancy; their combination yielded an area under the curve value of 0.824, with a sensitivity of 97.73%. CONCLUSION: The "very early" iCCA ≤2.0cm was associated with a favorable outcome after surgery, it displayed different and relatively atypical imaging manifestations compared with those of 2.0-5.0cm. Furthermore, in the small lesions ≤ 2.0cm, MRI can be served as a useful non-invasive diagnostic tool for iCCA in clinical screening with high sensitivity.

[Study of magnetic resonance imaging based on liver imaging reporting and data system for evaluating phosphatidylinositol proteoglycan-3 expression in hepatocellular carcinoma].

Objective: To clarify the diagnostic value of magnetic resonance imaging based on liver imaging reporting and data system (LI-RADS) for phosphatidylinositol proteoglycan-3 (GPC3) expression in hepatocellular carcinoma (HCC). Methods: Clinical and pathological data of 95 HCC cases with positive GPC3 expression (+) and 40 HCC cases with negative GPC3 expression (-) were retrospectively analyzed, and their MRI image features based on the 2018 version of LI-RADS were compared. Multivariate logistic regression analysis was used to determine the main predictors of GPC3 expression. Receiver operating characteristic curve was used further to determine the diagnostic efficacy of combined clinical imaging model to predict GPC3 expression. Enumeration data were compared with χ2 test or Fisher's exact test. Measurement data were compared using independent samples t-test or Mann-Whitney U-test. Results: There were statistically significant differences between HCC in GPC3 (+) and GPC3(-) group at alpha-fetoprotein (AFP) levels (χ2=31.814, P<0.000 1), and MRI features: capsular enhacement (χ2=4.108, P=0.043), halo type enhancement (χ2=4.847, P=0.028), and lesion apparent dispersion coefficient (ADC) (t=2.552, P=0.011 8). Multivariate regression analysis showed that AFP>20 µg/L (OR=9.358, P<0.000 1) and ADC≤1.404×10-3 mm2/s (OR=1.003, P=0.017) were independent predictors for GPC3 expression in HCC. The combined model and the area under the curve value for the diagnosis of GPC3(+) in HCC was 0.810, and its diagnostic sensitivity and specificity were 76.8% and 77.5%, respectively. Conclusion: AFP>20 µg/L and ADC≤1.404×10-3 mm2/s may indicate the expression of GPC3 in HCC, and the combination of the two diagnostic indicators can provide a simple and effective non-invasive diagnostic method for clinical practice.

Single-Cell Transcriptomic Atlas of Gingival Mucosa in Type 2 Diabetes.

The oral gingival barrier is a constantly stimulated and dynamic environment where homeostasis is often disrupted, resulting in inflammatory periodontal diseases. Type 2 diabetes (T2D) has been reported to be associated with gingival barrier dysfunction, but the effect and underlying mechanism are inconclusive. Herein, we performed single-cell RNA sequencing (scRNA-seq) of gingiva from leptin receptor-deficient mice (db/db) to examine the gingival heterogeneity in the context of T2D. Periodontal health of control mice is characterized by populations of Krt14+-expressing epithelial cells and Col1a1+-fibroblasts mediating immune homeostasis primarily through the enrichment of innate lymphoid cells. The db/db gingiva exhibited decreased epithelial/stromal ratio and dysfunctional barrier. We further observed stromal, particularly fibroblast immune hyperresponsiveness, linked to the recruitment of myeloid-derived cells at the db/db gingiva. Both scRNA-seq and histological analysis suggested the inflammatory signaling between fibroblasts and neutrophils as a potential driver of diabetes-induced periodontal damage. Notably, the "immune-like" stromal cells were wired toward the induction of gingival IL-17A hyperresponsiveness in db/db mice. Our work reveals that the "immune-like" fibroblasts with transcriptional diversity are involved in the innate immune homeostasis at the diabetic gingiva. It highlights a potentially significant role of these cell types in its pathogenesis.

Tunable electronic and optical properties of a BAs/As heterostructure by vertical strain and external electric field.

Based on the first-principles method, we investigated the electronic properties of a BAs/arsenene (As) van der Waals (vdW) heterostructure and found that it has an intrinsic type-II band alignment with a direct band gap of 0.25 eV, which favors the separation of photogenerated electrons and holes. The band gap can be effectively modulated by applying vertical strain and external electric field, displaying a large alteration in the band gap via the strain and experiencing an indirect-to-direct band gap transition. Moreover, the band gap of the heterostructure varies almost linearly with external electric field, and the semiconductor-to-metal transition can be realized in the presence of a strong electric field. The calculated band alignment and optical absorption reveal that the BAs/As heterostructure could present an excellent light-harvesting performance. The absorption strength can be tuned mainly by interlayer coupling, while external electric field shows clear regulating effects on the absorption strength and absorption edge.

LepR-Expressing Stem Cells Are Essential for Alveolar Bone Regeneration.

Stem cells play a critical role in bone regeneration. Multiple populations of skeletal stem cells have been identified in long bone, while their identity and functions in alveolar bone remain unclear. Here, we identified a quiescent leptin receptor-expressing (LepR+) cell population that contributed to intramembranous bone formation. Interestingly, these LepR+ cells became activated in response to tooth extraction and generated the majority of the newly formed bone in extraction sockets. In addition, genetic ablation of LepR+ cells attenuated extraction socket healing. The parabiosis experiments revealed that the LepR+ cells in the healing sockets were derived from resident tissue rather than peripheral blood circulation. Further studies on the mechanism suggested that these LepR+ cells were responsive to parathyroid hormone/parathyroid hormone 1 receptor (PTH/PTH1R) signaling. Collectively, we demonstrate that LepR+ cells, a postnatal skeletal stem cell population, are essential for alveolar bone regeneration of extraction sockets.

[Study on diagnostic value of extracellular volume imaging by magnetic resonance imaging for liver fibrosis of hepatitis B].

Objective: To investigate the diagnostic value of extracellular volume (ECV) imaging by magnetic resonance imaging for liver fibrosis of hepatitis B. Methods: A retrospective analysis was recruited in patients with chronic hepatitis B, who underwent liver surgery from April to October 2017 for pathological evaluation of liver tissues, and all patients underwent Gd-EOB-DTPA-enhanced T1 mapping to calculate the liver ECV score. The correlation between ECV and staging of hepatic fibrosis and inflammatory activity were compared to clarify the diagnostic value of staging of fibrosis. Results: 66 patients were enrolled in this study. Concerning the staging of liver fibrosis, there were 13, 4, 13, 10, and 26 cases with F0, F1, F2, F3 and F4 stages, respectively. ECV values had high interobserver consistency (correlation coefficient 0.860). The ECV difference between different stages of liver fibrosis was statistically significant (F = 15.02, P < 0.001). There was a significant positive correlation between ECV and fibrosis stage (r = 0.622, P < 0.001), and weak correlation with inflammatory activity (r = 0.332, P = 0.007). Fibrosis staging was an independent factor influencing ECV (P < 0.001). The area under the receiver operator characteristic curve for the diagnosis of liver fibrosis staging F≥1, F≥3 and F4 were 0.760, 0.846 and 0.873, respectively. The diagnostic sensitivity and specificity were 64.15%, 92.31%, 77.78%, 80.00% and 88.46, 72.50%, respectively. Conclusion: MRI-ECV imaging has great value for staging hepatic fibrosis of hepatitis B, and it can provide an effective method for diagnosis, staging, and evaluating the curative effect of fibrosis.

Electronic and thermal spin effect of molecular nanowires between graphene electrodes.

Based on the first-principles method, the electronic spin transport properties of terphenyl molecule bridging in zigzag graphene nanoribbon (ZGNR) electrodes with three connecting linkages were investigated, including dangling, heptagon, and pentagon-linkages. For the pentagon-linkage system, we observed a perfect spin filtering effect in the parallel (P) configuration (at almost 100% spin polarization), with the heptagon-linkages system following next (85-95% spin polarization), however, the spin filtering effect is almost negligible for the dangling-linkages system. In the antiparallel (AP) configuration, the pentagon- and heptagon-linkage systems also showed a high spin filtering effect. The terphenyl molecule was then replaced by carbon chains based on the pentagon-linkages, and these devices also show a perfect spin filtering effect (100% spin polarization). Finally, the thermally induced spin transport for the carbon chains model with pentagon-linkages was explored, and this system exhibits almost 100% thermal spin polarization.

Fluorescence-Based In Situ Quantitative Imaging for Cellular Lipids.

Membrane lipids are dynamic molecules and their local concentrations serve as regulatory signals for diverse biological processes. To achieve quantitative in situ imaging of various lipids, we developed a ratiometric analysis using fluorescence biosensors, each of which is composed of an engineered lipid-binding protein and a covalently attached solvatochromic fluorophore. To cover a wide range of lipid concentration, lipid-binding proteins are engineered to have variable dynamic ranges. These tunable sensors allow robust and sensitive in situ quantitative lipid imaging in mammalian cells, providing new insight into the spatiotemporal dynamics and fluctuation of key signaling lipids. The sensor strategy is also applicable to in situ quantification of multiple cellular lipids or a single lipid in the opposing leaflets of cell membranes.

Photoluminescence characterisations of a dynamic aging process of organic-inorganic CH3NH3PbBr3 perovskite.

After unprecedented development of organic-inorganic lead halide perovskite solar cells over the past few years, one of the biggest barriers towards their commercialization is the stability of the perovskite material. It is thus important to understand the interaction between the perovskite material and oxygen and/or humidity and the associated degradation process in order to improve device and encapsulation design for better durability. Here we characterize the dynamic aging process in vapour-assisted deposited (VASP) CH3NH3PbBr3 perovskite thin films using advanced optical techniques, such as time-resolved photoluminescence and fluorescence lifetime imaging microscopy (FLIM). Our investigation reveals that the perovskite grains grow spontaneously and the larger grains are formed at room temperature in the presence of moisture and oxygen. This crystallization process leads to a higher density of defects and a shorter carrier lifetime, specifically in the larger grains. Excitation-intensity-dependent steady-state photoluminescence shows both N2 stored and aged perovskite exhibit a super-linear increase of photoluminescence intensity with increasing excitation intensity; and the larger slope in aged sample suggests a larger density of defects is generated, consistent with time-resolved PL measurements.

Effect of the ratios of unsaturated fatty acids on the expressions of genes related to fat and protein in the bovine mammary epithelial cells.

The objective of this study was to evaluate the effects of the different ratios of unsaturated fatty acids (UFAs) (oleic acid, linoleic acid, and linolenic acid) on the cell viability and triacylglycerol (TAG) content, as well as the mRNA expression of the genes related to lipid and protein synthesis in bovine mammary epithelial cells (BMECs). Primary cells were isolated from the mammary glands of Holstein dairy cows and were passaged twice. Afterward, the cells were randomly allocated to six treatments, five UFA-treated groups, and one control group. For all of the treatments, the the fetal bovine serum in the culture solution was replaced with fatty acid-free BSA (1 g/L), and the cells were treated with different ratios of oleic, linoleic, and linolenic acids (0.75:4:1, 1.5:10:1, 2:13.3:1, 3:20:1, and 4:26.7:1) for 48 h, which were group 1 to group 5. The control culture solution contained only fatty acid-free BSA without UFAs (0 µM). The results indicated that the cell viability was not affected by adding different ratios of UFAs, but the accumulation of TAG was significantly influenced by supplementing with different ratios of UFAs. Adding different ratios of UFAs suppressed the expression of ACACA and FASN but had the opposite effect on the abundances of FABP3 and CD36 mRNA. The expression levels of PPARG, SPEBF1, CSN1S1, and CSN3 mRNA in the BMECs were affected significantly after adding different ratios of UFAs. Our results suggested that groups 1, 2, and 3 (0.75:4:1, 1.5:10:1, and 2:13.3:1) had stronger auxo-action on fat synthesis in the BMECs, where group 3 (2:13.3:1) was the best, followed by group 4 (3:20:1). However, group 5 (4:26.7:1) was the worst. Genes related to protein synthesis in the BMECs were better promoted in groups 2 and 3, and group 3 had the strongest auxo-action, whereas the present study only partly examined the regulation of protein synthesis at the transcriptional level; more studies on translation level are needed in the future. Therefore, when combining fat and protein synthesis, group 3 could be obviously fat and protein synthesis in the BMECs concurrently. However, further studies are necessary to elucidate the mechanism for regulating fat and protein synthesis in the BMECs.

Synthesis and cytotoxic activity of 3-phenyl-2-thio-quinoxaline 1,4-dioxide derivatives in hypoxia and in normoxia.

A series of novel 3-phenyl-2-ethylthio/ethylsulfinyl/ethylsulfonyl/phenylthio/ phenylsulfonyl-quinoxaline 1,4-dioxide derivatives were synthesized and screened for their cytotoxicity in vitro on human leukaemia cell line HL-60, human esophagus cancer cell line ECA-109, human prostate cancer cell line PC-3, human gastric carcinoma cell line SGC-7901, and human breast cancer cell line MCF-7 in hypoxia and in normoxia. Half of tested compounds showed higher cytotoxic activity both in hypoxia and in normoxia. The mechanism of one potent compound, 67, in hypoxia showed that the mitochondria pathway is involved in the antitumor activity of this class of compounds.

Androcam is a tissue-specific light chain for myosin VI in the Drosophila testis.

Myosin VI, a ubiquitously expressed unconventional myosin, has roles in a broad array of biological processes. Unusual for this motor family, myosin VI moves toward the minus (pointed) end of actin filaments. Myosin VI has two light chain binding sites that can both bind calmodulin (CaM). However unconventional myosins could use tissue-specific light chains to modify their activity. In the Drosophila testis, myosin VI is important for maintenance of moving actin structures, called actin cones, which mediate spermatid individualization. A CaM-related protein, Androcam (Acam), is abundantly expressed in the testis and like myosin VI, accumulates on these cones. We have investigated the possibility that Acam is a testis-specific light chain of Drosophila myosin VI. We find that Acam and myosin VI precisely colocalize at the leading edge of the actin cones and that myosin VI is necessary for this Acam localization. Further, myosin VI and Acam co-immunoprecipitate from the testis and interact in yeast two-hybrid assays. Finally Acam binds with high affinity to peptide versions of both myosin VI light chain binding sites. In contrast, although Drosophila CaM also shows high affinity interactions with these peptides, we cannot detect a CaM/myosin VI interaction in the testis. We conclude that Acam and not CaM acts as a myosin VI light chain in the Drosophila testis and hypothesize that it may alter the regulation of myosin VI in this tissue.

Production of no-carrier-added 186Re via deuteron induced reactions on isotopically enriched 186W.

Rhenium-186 was produced from the reaction induced by 16 MeV deuterons on isotopically enriched 186W metal powder target. Following its separation from 187W, 183Ta and the bulk of target materials 186W through an acid alumina column, the 186Re was converted to HNO3 solution through an anion exchange column, or to ammonia solution by extracting with N-235-dimethylbenzene, and finally the no-carrier-added 186Re saline solution was obtained. The radionuclidic purity of 186Re was >99.9% and the isotopic impurities were mainly 183Re (5.0 x 10(-3)%) and 184gRe (4.6 x 10(-2)%). The experimental thick-target yield of 186Re was determined to be approximately 529 microCi/microA h and the overall chemical recovery yield was > 80%.

[Ultrastructure changes of the olfactory epithelium of the patients suffering from dysosmia caused by the chronic sinusitis].

OBJECTIVE: To observe the ultrastructural changes of olfactory epithelium (OE) in patients suffering from dysosmia caused by chronic sinusitis. METHODS: The specimens of olfactory epithelium were obtained from 35 patients operated for chronic sinusitis accompanied by dysosmia. According to the results of light microscope (LM) examination, the OE was divided into three groups by the types of pathological changes: normal, atrophic and respiratory epithelium metaplasia(REM). Transmission electron microscope was used to observe the ultrastructural changes of each group. RESULTS: Under the LM, the surface ultrastructure of the OE showed some abnormal changes: (1) surface microvillus of the supporting cells disappeared; (2) olfactory vesicle changed their shape due to vacuolization; (3) disappearance of canaliculus structure in the olfactory vesicle; (4) the olfactory cilia changed the shape or reduced; some of the reduced cilia underwent metaplasia. The ultrastructural changes of atrophic OE included: (1) minor and moderate atrophy: the organelles and the membrane-limited electron dense vesicles on the upper section of the supporting cells obviously decreased or disappeared, even underwent vacuolization. The basic cell degenerated; (2) serious atrophy: the turbidity of the cell structure, even double cell structure, the nuclei of the cell aggregated as the plaque and vesiculose change or karyopyknosis. As for the cytoplasm, there were the dilation of the ERs, turgidity of the mitochondrion, the disarrangement, diminution and vacuolization. Fasciculate cilia were distributed separately in the REM group. CONCLUSION: There is a positive relationship between the atrophy degree and the degree of the abnormal ultrastructural changes of the OE. The ultrastructural changes of OE in patients suffering from dysosmia caused by chronic sinusitis may provide reference for assessment of the treatment of dysosmia.

[The expression and clinical significance of proliferative antigen Ki-67 and apoptosis-antagonizing antigen Bcl-2 in non-Hodgkin's lymphoma].

OBJECTIVE: To evaluate the expression of proliferative antigen Ki-67 and apoptosis-antagonizing protein Bcl-2 as well as their clinical significance in non-Hodgkin's lymphoma (NHL). METHODS: Ki-67 antigen and Bcl-2 protein were measured by immunohistochemistry on paraffin -embedded slices in 35 cases of NHL. RESULTS: (1) The level of Ki-67 expression in low grade NHL was lower than that in high grade NHL (P < 0.01),while Bcl-2 expression in lower grade NHL was higher than that in high grade (P < 0.01). (2) The level of Bcl-2 expression in B cell NHL was higher than that in T cell NHL (P < 0.02), but there was no difference in the expression of Ki-67. (3) The level of Bcl-2 expression in remission group was lower than that in non-remission group after the first course of chemotherapy, but there was no significant difference in Ki-67 expression. (4) The survival time was longer in the group with Ki-67 < or = 20% than that in the group with Ki-67 > 20%, but there was no significant difference of survival time between the high and low expression group of Bcl-2. CONCLUSIONS: The level of Ki-67 and Bcl-2 expression was closely related with the grade of malignant and the prognosis of NHL. They were a pair of useful markers for understanding the generation and prognosis of NHL.

[The diagnosis and treatment of Lyme disease: a report of six cases].

We retrospectively analyzed the clinical features, diagnosis, treatment, and prognosis of 6 cases of Lyme disease from November, 1994 to December, 1998. According to our report, doxycycline, penicillin, and ceftriaxone are effective drugs for Lyme disease. The shorter the disease course is, the better the treatment effect will be. Patients in phase III usually need repeated treatment.

[Clinical analysis of 36 cases with amebic liver abscess].

OBJECTIVE: To investigate the clinical features of amebic liver abscess, the causes of misdignosis and the effect of medical and surgical therapy on patient's prognosis. METHODS: The clinical features of 36 patients with amebic liver abscess admitted from 1982 to 1997 in our hospital were retrospectively reviewed. RESULTS: The major clinical manifestations were: abdominal pain (86.1%), fever (86.1%), hepatomegaly with tenderness (83.3%) and right intercostal tenderness (58.3%). Leukocytosis was observed in 61.1%, and increased of ESR in 88.5% (23/28). Serologies against Entamoeba histolytica were noted in 92.6%. Ultrasonography showed single lesions in 75% and right-lobe involvement in 75%. All patients were treated with metronidazole and 27 patients received treatment with needle aspiration or draining at the same time. After treatment, 10 patients were cured, 25 patients were improved significantly and effective rate was 97.2%. One patient died of hepatic failure. CONCLUSION: Medical therapy alone was excellent for small abscesses, while percutaneous needle aspiration or draining was a successful approach in patients with large abscesses.

[Study of 6 cases of AIDS with pulmonary complication].

OBJECTIVE: To improve and to increase the awareness of pulmonary complications of AIDS. METHODS: Six cases of confirmed AIDS from may 1992 to August 1997 were analyzed. RESULTS: Of the six cases, there were five complicated with pneumocysitis carinii pneumonia (PCP) (in which one had PCP as the first occurred manifestation). The clinical presentations of PCP were fever(5/5), dyspnea on exertion or at rest(4/5), and hypoxemi with mean PaO2 of 58.1 mm Hg. Chest X-ray showed bilateral diffuse interstitial or alveolar infiltrates. Pulmonary tuberculosis, tuberculous lymphadenities (fast-acid staining positive but PPD negative) and bronchial fungal infection were found in three cases. CONCLUSIONS: AIDS patients are at high risk of suffering from pulmonary complications, of which PCP is more common. If young pateints who were healthy in the past suddenly suffered from pneumonia and respiratory failure, PCP should be considered. When opportunistic pulmonary infections are diagnosed under special circumstances, one should be alert to the possibility of AIDS. In these cases serum HIV antibodies should be checked immediately.

[Investigation of the genetic instability of tumor cells by transfection of RER+ cell lines with exogenous microsatellite sequence].

OBJECTIVE: Frequent alterations of microsatellite sequence of cancer cells were found recently in a substantial fraction of human cancers including hereditary non-polyposis colon cancer. This paper aimed to investigate the genetic instability of tumor cells by using microsatellite instability(MI) as the marker in vitro. METHODS: Two RER+ cell lines (replication error phenotype), RKO and HCT116 and one RER- cell line, sw480, were used as the hosts for transfection with an episomal plasmid, pCMV-CAR, containing an exogenous (CA)14 repeat which was inserted within the coding sequence of lacZ reporter gene and thus made lacZ misreading. The transfectant clones were selected and established by hygromicin. Expression and production of lacZ reporter gene of restored reading frame were detected with X-gal staining assay. RESULTS: After hygromicin selection, stable pCMV-CAR transfectant clones were established. It was shown that mutation of deletion of insertion within(CA)14 occurred in the transfectant RER+ cells but not in the RER-cells. The mutation restored normal reading frame of lacZ gene, and resulted in expression and production of bio-active beta galactosidase which was detected with X-gal staining. This feature of the transfectant clones was maintained during culture passages. CONCLUSION: The alterations of the exogenous(CA)14 repeat in the transfectant RER+ clones revealed genetic instability and complicated mutation status of cancer cells. It is suggested that the exogenous (CA)14 in transfectant clones could be a useful target sequence for monitoring the effects of environmental agents on MI of human cancer cells.