IGF-1 Accelerates Cell Aging by Inhibiting POLD1 Expression.

Objective: The individual cascades of the insulin-like growth factor-1 (IGF-1) signaling pathway and the molecular mechanism of aging have not been fully clarified. In the current study, we explored the effect of DNA polymerase delta 1 (POLD1) on the IGF-1 signaling pathway in cell aging. Methods: First, we analyzed the relationship between IGF-1 and POLD1 expression in aging. To investigate the effect of IGF-1 on POLD1 expression and aging, the 2BS cells were incubated with young-age or old-age human serum, IGF-1 protein, or linsitinib. Next, the effect of IGF-1 on aging was examined in the 2BS cells with increased or decreased POLD1 expression to clarify the molecular mechanism. Results: In this study, we found that IGF-1 expression increased and POLD1 expression decreased with aging in human serum and hippocampal tissues of SAMP8 mice, and a negative relationship between IGF-1 and POLD1 expression was observed. Furthermore, the cells cultured with old-age human serum or IGF-1 showed lower POLD1 expression and more pronounced senescence characteristics, and the effect could be reversed by treatment with linsitinib or overexpression of POLD1, while the effect of linsitinib on cell aging could be reversed with the knockdown of POLD1. Conclusion: Taken collectively, our findings demonstrate that IGF-1 promotes aging by binding to IGF-1R and inhibiting the expression of POLD1. These findings offer a new target for anti-aging strategies.

ABCA1-Labeled Exosomes in Serum Contain Higher MicroRNA-193b Levels in Alzheimer's Disease.

OBJECTIVE: We aimed to establish a method to determine whether microRNA-193b (miR-193b) levels in ABCA1-labeled serum exosomes might serve as a marker for the diagnosis of Alzheimer's disease. METHODS: We used immunocapture methods to determine the levels of ABCA1-labeled exosomal miR-193b in cultures of white blood cells (WBCs), red blood cells (RBCs), mouse hippocampal neuron HT-22 cells, and primary mouse neuronal cells. ABCA1-labeled exosomal miR-193b levels were also evaluated in the cerebrospinal fluid (CSF) and serum of APP/PS1 double-transgenic mice, as well as control subjects (n = 60) and study participants with subjective cognitive decline (SCD, n = 89), stage and mild cognitive impairment (MCI, n = 92), and dementia of the Alzheimer type (DAT, n = 92). RESULTS: ABCA1 levels of exosomes harvested from the medium of HT-22 cells and neurons were significantly higher than those of RBCs and WBCs (P < 0.05). Exosomal ABCA1 from the CSF of APP/PS1 mice were transmitted to the serum of wild-type mice after injection, and high miR-193b levels were observed in both the serum and CSF after injection. The ABCA1-labeled exosomal miR-193b levels were higher in the CSF of MCI and DAT patients compared with the CSF of the control group (P < 0.05). The ABCA1-labeled exosomal miR-193b were also slightly higher (P > 0.05) in the serum of SCD patients and significantly higher in the serum of MCI and DAT patients compared with the serum of the control group (P < 0.05). CONCLUSION: This study provides a method to capture specific exosomes. Detection of serum exosomes labeled with ABCA1 may facilitate the early diagnosis of AD.

Severely High Lactic Acid in Severe Pneumonia Patient: a Case Report.

BACKGROUND: Severe pneumonia (SP) is a clinically critical acute disease which has a higher mortality rate among infectious diseases. In this report, a rare case of severe pneumonia with severely high lactic acid (up to 24 mmol/L) and relatively normal pH was analyzed. METHODS: The case was discussed from different angles including acid-base balance disorder, the use of extractor-poreal membrane oxygenation (ECMO), dialysis treatment, circulatory disturbance, and inspection methodology. RESULTS: Hypoxia and dissolution of muscles caused by circulatory disorders may be the cause of the abnormal increase of lactate in this case; while the relatively normal pH may be caused by the dialysis treatment. CONCLUSIONS: Such a high blood gas lactic acid value is extremely rare, and this increase is not due to the limitations of the test method. High lactic acid may not result in the significant decrease of pH when the patient receives continuous systemic treatment.

MicroRNA-135a in ABCA1-labeled Exosome is a Serum Biomarker Candidate for Alzheimer's Disease.

OBJECTIVE: In the present study, the ABCA1 was used as a label to capture specific exosomes, the level of ABCA1-labeled exosomal microRNA-135a (miR-135a) was evaluated for the diagnosis of Alzheimer's disease (AD), especially in patients with early stages of AD. METHODS: This is a preliminary research focused on the levels of ABCA1 in WBCs, RBCs, HT-22 cells, and neuron cells. The diagnostic value of ABCA1-labeled exosomal miR-135a was examined using the CSF and serum of APP/PS1 double transgenic mice, and 152 patients with SCD, 131 patients with MCI, 198 patients with DAT, and 30 control subjects. RESULTS: The level of ABCA1 exosomes harvested from HT-22 cells and neuron culture medium was significantly higher compared to that of RBCs and WBCs ( P < 0.05). The levels of ABCA1-labeled exosomal miR-135a increased in the CSF of MCI and DAT group compared to those of control group ( P < 0.05), slightly increased ( P > 0.05) in the serum of SCD patient group, and significantly increased in MCI and DAT patient groups compared to those of the control group ( P < 0.05). CONCLUSION: This study outlines a method to capture specific exosomes and detect them using immunological methods, which is more efficient for early diagnosis of AD.

Identification by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry and Antifungal Susceptibility Testing of Non-Aspergillus Molds.

Non-Aspergillus molds including Mucorales, Fusarium, and Scedosporium, etc. are emerging pathogens leading to higher mortality in immunocompromised patients. Fifty-two isolates of genetically confirmed non-Aspergillus molds representing 16 species from 8 genera were collected to evaluate the performance of the Bruker matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) in identification of non-Aspergillus molds. Antifungal susceptibilities were determined through the Clinical & Laboratory Standards Institute (CLSI) M38-A2 broth microdilution method and the Sensititre YeastOne colorimetric method. Bruker MALDI-TOF MS identified 57.7% (30/52) of isolates cultured in broth and 15.4% (8/52) of isolates cultured on solid agar media to the species level, respectively, according to standard interpretation criteria. Lowering the species level cut-off value (COV) from ≥2.0 to ≥1.7 could improve the MALDI-TOF MS species-level identification rate to 67.3% (38/52) for isolates cultured on solid media, with a slight increase of false identification rate of 2.6% (1/38). Amphotericin B was the most in vitro fungistatic-active agent for 98.1% (51/52) of the tested non-Aspergillus molds, with minimum inhibitory concentrations (MICs) of ≤2 µg/mL. The susceptibilities to triazoles varied, with MICs of 0.12 to >16 µg/mL among different species of non-Aspergillus molds. The correlation between the CLSI method and Sensititre YeastOne on antifungal susceptibility testing of non-Aspergillus molds was good, with essential agreement (EA) rates of >90% for triazoles and echinocandins except amphotericin B, which had a lower EA rate of 84.6%. In conclusion, a favorable performance of the Bruker MALDI-TOF MS in identification of clinical non-Aspergillus isolates directly inoculated on solid agar media could be achieved with the adoption of alternative interpretation criteria. Antifungal susceptibility testing is important for non-Aspergillus molds, especially when information on triazole susceptibility is required, and the Sensititre YeastOne is a practical and reliable method to determine antifungal susceptibilities of non-Aspergillus molds.

The Serum Exosome Derived MicroRNA-135a, -193b, and -384 Were Potential Alzheimer's Disease Biomarkers.

OBJECTIVE: MicroRNAs (miRs) are attractive molecules to be considered as one of the blood-based biomarkers for neurodegenerative disorders such as Alzheimer's disease (AD). The goal of this study was to explore their potential value as biomarkers for the diagnosis of AD. METHODS: The expression levels of exosomal miR-135a, -193b, and -384 in the serum from mild cognitive impairment (MCI), dementia of Alzheimer-type (DAT), Parkinson's disease with dementia (PDD), and vascular dementia (VaD) patients were measured with a real-time quantitative reverse transcriptase PCR (qRT-PCR) method. RESULTS: Both serum exosome miR-135a and miR-384 were up-regulated while miR-193b was down-regulated in serum of AD patients compared with that of normal controls. Exosome miR-384 was the best among the three miRs to discriminate AD, VaD, and PDD. Using the cut-off value could better interpret these laboratory test results than reference intervals in the AD diagnosis. ROC curve showed that the combination of miR-135a, -193b, and -384 was proved to be better than a particular one for early AD diagnosis. CONCLUSION: Our results indicated that the exosomal miRs in the serum were not only potential biomarker of AD early diagnosis, but might also provide novel insights into the screen and prevention of the disease.

[MCT4 reduces glucose metabolism and promotes apoptosis of prostate cancer cells].

OBJECTIVE: To investigate the effect of monocarboxylate transporter 4 (MCT4) on the apoptosis and glucose metabolism of prostate cancer cells. METHODS: We constructed an adenoviral vector containing shRNA-MCT4 and transfected it into the adenocarcinoma cell lines DU145 and PC-3, using the untransfected vector as the blank control and the negative vector as the negative control (shRNA-NC). We determined the MCT4 expression, lactic acid secretion, glucose consumption and apoptosis rate in different groups of cells. RESULTS: After transfection, the expression of MCT4 in the DU145 and PC-3 cells was significantly lower in the shRNA-MCT4 than in the blank control (P = 0.008 and 0.008) and shRNA-NC groups (P = 0.007 and 0.009), and so were the secretion of lactic acid (P = 0.009 and 0.009; P = 0.009 and 0.008) and single-cell glucose consumption (P = 0.007 and 0.007; P = 0.009 and 0.007). The apoptosis rate of the DU145 cells was remarkably higher in the shRNA-MCT4 than in the blank control and shRNA-NC groups (ï¼»22.11 ± 2.68ï¼½% vs ï¼»9.81 ± 1.24ï¼½% and ï¼»10.01 ± 1.46ï¼½%, P = 0.003 and 0.003), and so was that of the PC-3 cells (ï¼»23.38 ± 3.08ï¼½% vs ï¼»10.21 ± 1.58ï¼½% and ï¼»10.91 ± 1.63ï¼½%, P = 0.004 and 0.004). CONCLUSIONS: Inhibiting the expression of MCT4 can interfere with the glucose metabolism and promote the apoptosis of prostate cancer cells. The MCT4 gene is a potential therapeutic target for the treatment of prostate cancer.

[Prevalence and subtype distribution of HPV infection among women in Beijing urban area and their correlation with age].

OBJECTIVE: To investigate the prevalence and subtype distribution of human papillomavirus (HPV) infection and its correlation with age among women in Beijing urban area, and provide some epidemiological evidence for the clinical application of HPV vaccines. METHODS: We collected cervical specimens from 1999 women in the Outpatient Department of our hospital, performed genetyping of HPV-DNA, and analyzed the incidence of HPV infection in different age groups. RESULTS: HPV infection was detected in 502 (25.2%) of the 1999 women patients, with 391 (19.6%) cases of high-risk HPV, which included 326 (83.4%, 326/391) cases of single infection. HPV-16 was the most common type (21.2%, 69/326), followed by HPV-52 (19.3%, 63/326) and HPV-58 (16.0%, 52/326). The prevalence of HPV infection was the highest among the women aged 41 -50 years and the lowest among those over 60 years. CONCLUSION: The subtype- and age-specific distribution of HPV infection among women in Beijing urban area shows an obvious heterogeneity, which deserves due consideration in the clinical application of HPV vaccines.

MicroRNA-135a and -200b, potential Biomarkers for Alzheimer׳s disease, regulate ß secretase and amyloid precursor protein.

Amyloid precursor protein (APP) and ß-site amyloid precursor protein cleaving enzyme (BACE-1) play important roles in the generation of Alzheimer׳s disease (AD), a progressive neurodegenerative disorder. In the present study, microRNA (miR) microarray was used to analyze the miR expression profiles in the hippocampi from APP/PS1 transgenic and wild type mice. The miRs with significant alteration and putative targets on APP or BACE-1 were retrieved (miR-135a, -200b and -429). The deregulations of these miRs were confirmed in mice and further verified in AD patient samples by qPCR. Primary mouse hippocampal neurons, SH-SY5Y and HEK293 cells were used to study the function of miRs on APP and BACE-1. We found that miR-135a, which was downregulated significantly in hippocampi from APP/PS1 transgenic mice compared with the wild type control, directly interacted with the 3'-UTR of BACE-1 and repressed its expression and activity. On the other hand, miR-200b and -429, which were downregulated significantly in hippocampi from APP/PS1 transgenic mice compared with the wild type control, targeted the 3'-UTR of APP and repressed its expression. Furthermore, Aß42 could downregulate miR-200b expression which may generate a vicious cycle resulted in accumulating Aß42. The levels of miR-135a and -200b in the serum of DAT group were significantly lower than that of control groups (P<0.05). The serum miR-200b level of MCI group was higher than that of DAT group (P<0.05) and lower than that of control group (P<0.05). We also found decreased miR-135a and -200b levels in the cerebrospinal fluid of DAT group compared with the control group (P<0.05). In conclusion, these findings showed that miR-135a, -200b and -429 may take part in the progress of AD; miR-200b was of great potential as noninvasive and easily detected blood-based biomarkers of MCI and DAT patients.

MicroRNA-193b is a regulator of amyloid precursor protein in the blood and cerebrospinal fluid derived exosomal microRNA-193b is a biomarker of Alzheimer's disease.

Amyloid precursor protein (APP) has an important function in the generation of Alzheimer's disease (AD). In our previous study, miR­193b was found to be downregulated in the hippocampi of 9­month­old APP/PS1 double­transgenic mice using microRNA (miR) array. In the present study, bioinformatic analyses showed that miR­193b was a miR that was predicted to potentially target the 3'­untranslated region (UTR) of APP. Subsequently, the function of miR­193b on APP was studied. The levels of miR­193b, exosomal miR­193b, Aß, tau, p­tau, HCY and APOE in samples from APP/PS1 double­transgenic mice, mild cognitive impairment (MCI) and dementia of Alzheimer­type (DAT) patients, were measured. The results indicated that overexpression of miR­193b could repress the mRNA and protein expression of APP. The miR­193b inhibitor oligonucleotide induced upregulation of APP. Binding sites of miR­193b in the 3'­UTR of APP were identified by luciferase assay. MCI and DAT patients had lower exosomal miR­193b, but not total miR­193b, in the blood as compared with the controls. DAT patients had lower exosomal miR­193b levels in blood as compared with the MCI group. A decreased exosomal miR­193b expression level was additionally observed in the cerebral spinal fluid (CSF) of DAT patients. Negative correlations were found between exosomal miR­193b and Aß42 in the CSF of DAT patients. In conclusion, these findings showed that miR­193b may function in the development of AD and exosomal miR­193b has potential as a novel, non-invasive, blood­based biomarker of MCI and DAT patients.

MicroRNA-384 regulates both amyloid precursor protein and ß-secretase expression and is a potential biomarker for Alzheimer's disease.

Amyloid precursor protein (APP) and ß-site APP cleaving enzyme (BACE-1) play important roles in the pathogenesis of Alzheimer's disease (AD). In this study, using bioinformatics analysis, we demonstrate that miR-384 is a microRNA (miRNA or miR) predicted to potentially target the 3' untranslated regions (3'-UTRs) of both APP and BACE-1. SH-SY5Y cells were transfected with miR-384 mimic oligonucleotide, miR-384 inhibitor oligonucleotide, or a non-specific control siRNA. We found that the overexpression of miR-384 suppressed the mRNA and protein expression of both APP and BACE-1. The miR-384 inhibitor oligonucleotide induced the upregulation of APP and BACE-1. The activity of BACE-1 was altered following the change in its protein expression. The binding sites of miR-384 on the 3'-UTRs of APP and BACE-1 were identified by luciferase assay. Furthermore, cells were treasted with amyloid-ß (Aß)42. Aß42 downregulated miR-384 expression, leading to the continuous reduction in miR-384 expression. In addition, using a mouse model of AD, as well as patients with mild cognitive impairment (MCI) and dementia of Alzheimer's type (DAT), we examined the levels of miR-384 in cerebral spinal fluid (CSF) and serum. Patients with MCI and DAT had lower blood miR-384 levels compared with the controls. In addition, patients with DAT had lower blood miR-384 levels in blood compared with the MCI group. We also found decreased miR-384 expression in the several cerebral spinal fluid (CSF) of the patients with DAT. Negative correlations were observed between miR-384 and Aß42 in the serum and CSF from patients with AD. In conclusion, these findings demonstrate that miR-384 may plays a role in the development of AD and may be a potential non-invasive biomarker for the diagnosis of AD.

Age-dependent down-regulation of DNA polymerase δ1 in human lymphocytes.

Aging progress and degeneracy of functional activity are mainly attributed to the decreased DNA repair potential. DNA polymerase (pol) δ activity plays an essential role in genome stability by virtue of its crucial DNA replication and repair capacity. To order to clarify the role of DNA pol δ in aging progression, we firstly examined the expressions of its catalytic subunit named DNA pol δ1 in human lymphocytes at different age stages, respectively, and then observed the effect of diseases on DNA pol δ1 in vivo and of nutriture on its expressions in 2BS cells in vitro. Blood samples from the healthy subjects and patients with diabetes mellitus and coronary heart disease were collected, respectively, for analysis of transcription and protein expressions of DNA pol δ1 by RT-PCR and western blot. 2BS cells of PD30 and PD47 were incubated in both normal medium and other mediums of different nutritures for verifying the differential expressions of DNA pol δ1. Results showed that the mRNA expression of DNA pol δ1 decreased substantially with age and the protein levels were well consistent with gene levels. Furthermore, there were no significant differences in DNA pol δ1 expressions between the groups of healthy individuals and the age matched patients. In addition, DNA pol δ1 gene expression levels were not affected by nutritional status in vitro. Our findings collectively confirmed that the down-regulations of DNA pol δ1 are age-related and have little bearing on diseases and nutritures. DNA pol δ1 has great potential for a new biomarker of aging.

The effect of aging on the DNA damage and repair capacity in 2BS cells undergoing oxidative stress.

Aging is associated with a reduction in the DNA repair capacity under oxidative stress. However, whether the DNA damage and repair capacity can be a biomarker of aging remains controversial. In this study, we demonstrated two cause-and-effect relationships, the one is between the DNA damage and repair capacity and the cellular age, another is between DNA damage and repair capacity and the level of oxidative stress in human embryonic lung fibroblasts (2BS) exposed to different doses of hydrogen peroxide (H2O2). To clarify the mechanisms of the age-related reduction in DNA damage and repair capacity, we preliminarily evaluated the expressions of six kinds of pivotal enzymes involved in the two classical DNA repair pathways. The DNA repair capacity was observed in human fibroblasts cells using the comet assay; the age-related DNA repair enzymes were selected by RT-PCR and then verified by Western blot in vitro. Results showed that the DNA repair capacity was negatively and linearly correlated with (i) cumulative population doubling (PD) levels only in the group of low concentration of hydrogen peroxide treatment, (ii) with the level of oxidative stress only in the group of young PD cells. The mRNA expression of DNA polymerase δ1 decreased substantially in senescent cells and showed negative linear-correlation with PD levels; the protein expression level was well consistent with the mRNA level. Taken together, DNA damage and repair capacity can be a biomarker of aging. Reduced expression of DNA polymerase δ1 may be responsible for the decrease of DNA repair capacity in senescent cells.

Two isomers of HDTIC isolated from Astragali Radix decrease the expression of p16 in 2BS cells.

BACKGROUND: Astragali Radix, the root of Astragalus membranceus (Fish) Bunge Var. mongholicus (Bge), is a crude drug considered as one of the effective traditional Chinese anti-ageing material. The two isomers of 4-hydroxy-5-hydroxymethyl-[1, 3] dioxolan-2, 6'-spirane-5', 6', 7', 8'-tetrahydro-indolizine-3'-carbaldehyde (HDTIC), HDTIC-1 and HDTIC-2, were first extracted from the herb in 2002. We demonstrated previously that 0.1 micromol/L HDTIC-1 or 1.0 micromol/L HDTIC-2 strongly delay replicative senescence of human fetal lung diploid fibroblasts (2BS). In this study, we chose them to investigate their effects on the expression of senescence-associated genes to explore the mechanism of how HDTIC delays replicative senescence. METHODS: The effects of HDTIC-1 and HDTIC-2 on the expression of p16 and p21 were observed in vitro by RT-PCR and Western blot. The anti-oxidative activities of the compounds were also observed by phenotype alteration after treatment with antioxidants. RESULTS: There was an obvious expression of p16 in the control senescent cells. However, in the 2BS cells, after 56 population doublings (PDs) grown from PD28 in 0.1 micromol/L HDTIC-1 or 1.0 micromol/L HDTIC-2, there was a weak mRNA expression of p16 and no protein expression of p16 was observed. The expression level of p21 increased with cell ageing. Moreover, there was no difference between the expression level of p21 in the control cells and that in the same PD cells cultured with HDTIC compounds. The results also showed that 2BS cells exposed to 100 micromol/L H2O2 for 5 minutes return to their non-senescent phenotype and continue to be confluent after incubating the damaged cells with HDTIC-1 (1.0 micromol/L ) or HDTIC-2 (10 micromol/L ) for 1 hour. CONCLUSIONS: Expression of p16 by 2BS cells was strongly inhibited by HDTIC compounds, which could contribute to their delayed replicative senescence by the way of p16(INK4a)/Rb/MAPK. The anti-oxidative activities of HDTIC-1 and HDTIC-2, described in this study for the first time, might be indirectly related to their inhibition of p16 expression.

Aminoguanidine delays the replicative senescence of human diploid fibroblasts.

BACKGROUND: The accumulation of free radicals and advanced glycation end products (AGEs) in cell plays a very important role in replicative senescence. Aminoguanidine (AG) has potential antioxidant effects and decreases AGE levels. This study aimed to investigate its effect on replicative senescence in vitro. METHODS: The effects of aminoguanidine on morphology, replicative lifespan, cell growth and proliferation, AGEs, DNA damage, DNA repair ability and telomere length were observed in human fetal lung diploid fibroblasts (2BS). RESULTS: Aminoguanidine maintained the non-senescent phenotype of 2BS cells even at late population doubling (PD) and increased cumulative population doublings by at least 17 - 21 PDs. Aminoguanidine also improved the potentials of growth and proliferation of 2BS cells as detected by the MTT assay. The AGE levels of late PD cells grown from early PD in DMEM containing aminiguanidine decreased significantly compared with those of late PD control cells and were similar to those of young control cells. In addition, the cells pretreated with aminoguanidine had a significant reduction in DNA strand breaks when they were exposed to 200 micromol/L H(2)O(2) for 5 minutes which indicated that the compound had a strong potential to protect genomic DNA against oxidative stress. And most of the cells exposed to 100 micromol/L H(2)O(2) had much shorter comet tails and smaller tail areas after incubation with aminoguanidine-supplemented DMEM, which indicated that the compound strongly improved the DNA repair abilities of 2BS cells. Moreover, PD55 cells grown from PD28 in 2 mmol/L or 4 mmol/L aminoguanidine retain telomere lengths of 7.94 kb or 8.12 kb, which was 0.83 kb or 1.11 kb longer than that of the control cells. CONCLUSION: Aminoguanidine delays replicative senescence of 2BS cells and the senescence-delaying effect of aminoguanidine appear to be due to its many biological properties including its potential for proliferation improvement, its inhibitory effect of AGE formation, antioxidant effect, improvement of DNA repair ability and the slowdown of telomere shortening.