Flap endonuclease 1 (FEN1), a member of the Rad2 nuclease family, possesses 5’ flap endonuclease (FEN), 5’ exonuclease (EXO), and gap-endonuclease (GEN) activities. The multiple, structure-specific nuclease activities of FEN1 allow it to process different intermediate DNA structures during DNA replication and repair. We previously identified a group of FEN1 mutations and single nucleotide polymorphisms that impair FEN1’s EXO and GEN activities in human cancer patients. We also established a mouse model carrying the E160D FEN1 mutation, which mimics the mutations seen in humans. FEN1 mutant mice developed spontaneous lung cancer at high frequency at their late life stages. An important unanswered question is whether individuals carrying such FEN1 mutation are more susceptible to tobacco smoke and have an earlier onset of lung cancer. Here, we report our study on E160D mutant mice exposed to benzo[α]pyrene (B[α]P), a major DNA damaging compound found in tobacco smoke. We demonstrate that FEN1 employs its GEN activity to cleave DNA bubble substrates with BP-induced lesions, but the E160D FEN1 mutation abolishes such activity. As a consequence, Mouse cells carrying the E160D mutation display defects in the repair of B[α]P adducts and accumulate DNA double-stranded breaks and chromosomal aberrations upon treatments with B[α]P. Furthermore, more E160D mice than WT mice have an early onset of B[α]P-induced lung adenocarcinoma. All together, our current study suggests that individuals carrying the GEN-deficient FEN1 mutations have high risk to develop lung cancer upon exposure to B[α]P-containing agents such as tobacco smoke.
Flap endonuclease 1 (FEN1); Benzo[α]pyrene (B[α]P); Lung cancer; Double-stranded breaks (DSBs); Near-tetraploid aneuploidy
The concept of one-protein–multiple-function, i.e. moonlighting proteins, is an ever-expanding paradigm. We obtained compelling evidence that an array of ‘cytoplasmic’ metabolic enzymes can enter the nuclei to carry out moonlighting transcription functions; this phenomenon is conserved from Drosophila to humans. Of particular interest are the classical glycolytic enzymes GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and LDH (lactate dehydrogenase), which utilize NAD(H) as coenzymes and not only moonlight (in their nuclear forms) to regulate the transcription of S-phase-specific histone genes, but also act as metabolic/redox sensors that link histone gene switching to DNA replication and S-phase progression.
chromosome; enzyme; GAPDH; histone 2B; S-phase; ATM, ataxia telangiectasia mutated; ATR, ataxia telangiectasia mutated- and Rad3-related; awd, abnormal wing disc; CBP, CREB (cAMP-response-element-binding protein)-binding protein; CDK, cyclin-dependent kinase; dm, Drosophila melanogaster; DSB, double-strand break; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; H2B, histone 2B; HAT, histone acetyl transferase; HDAC, histone deacetylase; HU, hydroxyurea; LDH, lactate dehydrogenase; MMC, mammalian metabolic cycle; nm23, non-metastasis 23; NPAT, nuclear protein, ataxia-telangiectasia locus; OCA-S, Oct-1 co-activator in the S-phase; PIKK, phosphoinositide 3-kinase-related kinase; RNAi, RNA interference; SLBP, stem–loop-binding protein; Tip60, Tat (transactivator of transcription)-interactive protein 60 kDa; YMC, yeast metabolic cycle
A simple and reliable method of high-performance liquid chromatography with photodiode array detection (HPLC-DAD) was developed to evaluate the quality of Receptaculum Nelumbinis (dried receptacle of Nelumbo nucifera) through establishing chromatographic fingerprint and simultaneous determination of five flavonol glycosides, including hyperoside, isoquercitrin, quercetin-3-O-β-d-glucuronide, isorhamnetin-3-O-β-d-galactoside and syringetin-3-O-β-d-glucoside. In quantitative analysis, the five components showed good regression (R > 0.9998) within linear ranges, and their recoveries were in the range of 98.31%–100.32%. In the chromatographic fingerprint, twelve peaks were selected as the characteristic peaks to assess the similarities of different samples collected from different origins in China according to the State Food and Drug Administration (SFDA) requirements. Furthermore, hierarchical cluster analysis (HCA) was also applied to evaluate the variation of chemical components among different sources of Receptaculum Nelumbinis in China. This study indicated that the combination of quantitative and chromatographic fingerprint analysis can be readily utilized as a quality control method for Receptaculum Nelumbinis and its related traditional Chinese medicinal preparations.
Receptaculum Nelumbinis; flavonoids compounds; fingerprint analysis; chemometric analysis; quality control
Mutations in genes involved in DNA replication such as FEN1, can cause single-stranded DNA breaks (SSBs) and subsequent collapse of DNA replication forks leading to DNA replication stresses. Persistent replication stresses normally induce p53-mediated senescence or apoptosis to prevent tumor progression. It is unclear how some mutant cells can overcome persistent replication stresses and bypass the p53-mediated pathways to develop malignancy. Here we show that formation of polyploidy, which is often observed in human cancers, leads to overexpression of BRCA1, p19arf and other DNA repair genes in FEN1 mutant cells. This overexpression triggers SSB repair and non-homologous end joining pathways to increase DNA repair activity, but at the cost of frequent chromosomal translocations. Meanwhile, DNA methylation silences p53 target genes, to bypass the p53-mediated senescence and apoptosis. These molecular changes rewire DNA damage response and repair gene networks in polyploid tumor cells, enabling them to escape replication stress-induced senescence barriers.
Somatic alterations of cyclin-dependent kinase 2 (CDK2)-cyclin E complex have been shown to contribute to breast cancer (BC) development and progression. This study aimed to explore the effects of single nucleotide polymorphisms (SNPs) in CDK2 and CCNE1 (a gene encoding G1/S specific cyclin E1 protein, formerly called cyclin E) on BC risk, progression and survival in a Chinese Han population.
We herein genotyped 6 haplotype-tagging SNPs (htSNPs) of CCNE1 and 2 htSNPs of CDK2 in 1207 BC cases and 1207 age-matched controls among Chinese Han women, and then reconstructed haplotype blocks according to our genotyping data and linkage disequilibrium status of these htSNPs. For CCNE1, the minor allele homozygotes of three htSNPs were associated with BC risk (rs3218035: adjusted odds ratio [aOR] = 3.35, 95% confidence interval [CI] = 1.69–6.67; rs3218038: aOR = 1.81, 95% CI = 1.22–2.70; rs3218042: aOR = 2.64, 95% CI = 1.31–5.34), and these three loci showed a dose-dependent manner in increasing BC risk (Ptrend = 0.0001). Moreover, the 5-SNP haplotype CCGTC, which carried none of minor alleles of the 3 at-risk SNPs, was associated with a favorable event-free survival (hazard ratio [HR] = 0.53, 95% CI = 0.32–0.90). Stratified analysis suggested that the minor-allele homozygote carriers of rs3218038 had a worse event-free survival among patients with aggressive tumours (in tumour size>2 cm group: HR = 2.06, 95% CI = 1.06–3.99; in positive lymph node metastasis group: HR = 2.41, 95% CI = 1.15–5.03; in stage II–IV group: HR = 2.03, 95% CI = 1.09–3.79). For CDK2, no significant association was found.
This study indicates that genetic variants in CCNE1 may contribute to BC risk and survival in Chinese Han population. They may become molecular markers for individual evaluation of BC susceptibility and prognosis. Nevertheless, further validation studies are needed.
Our previous studies revealed that the staphylococcal protein Gcp is essential for bacterial growth; however, the essential function of Gcp remains undefined. In this study, we demonstrated that Gcp plays an important role in the modulation of the branched-chain amino acids biosynthesis pathway. Specifically, we identified that the depletion of Gcp dramatically elevated the production of key enzymes that are encoded in the ilv-leu operon and responsible for the biosynthesis of the branched-chain amino acids isoleucine, leucine, and valine (ILV) using proteomic approaches. Using qPCR and promoter-lux reporter fusions, we established that Gcp negatively modulates the transcription of the ilv-leu operon. Gel-shift assays revealed that Gcp lacks the capacity to bind the promoter region of ilv. Moreover, we found that the depletion of Gcp did not influence the transcription level of CodY, a known repressor of the ilv-leu operon, while induced the transcription of CcpA, a known positive regulator of the ilv-leu operon. In addition, the depletion of Gcp decreased the biosynthesis of N6-threonylcarbamoyladenosine (t6A). To elucidate whether the essentiality of Gcp is attributable to its negative modulation of ILV biosynthesis, we determined the impact of the ilv-leu operon on the requirement of Gcp for growth, and revealed that the deletion of the ilv-leu operon did not affect the essentiality of Gcp. Taken together, our results indicate that the essentiality of Gcp isn’t attributable to its negative regulation of ILV biosynthesis in S. aureus. These findings provide new insights into the biological function of the staphylococcal Gcp.
Trastuzumab is currently approved for the clinical treatment of breast and gastric cancer patients with HER-2 positive tumors, but not yet for the treatment of esophageal carcinoma patients, whose tumors typically show 5 ~ 35% HER-2 gene amplification and 0 ~ 56% HER-2 protein expression. This study aimed to investigate the therapeutic efficacy of Trastuzumab in patient-derived esophageal squamous cell carcinoma xenograft (PDECX) mouse models.
PDECX models were established by implanting patient esophageal squamous cell carcinoma (ESCC) tissues into immunodeficient (SCID/nude) mice. HER-2 gene copy number (GCN) and protein expression were determined in xenograft tissues and corresponding patient EC samples by FISH and IHC analysis. Trastuzumab anti-tumor efficacy was evaluated within these PDECX models (n = 8 animals/group). Furthermore, hotspot mutations of EGFR, K-ras, B-raf and PIK3CA genes were screened for in the PDECX models and their corresponding patient’s ESCC tissues. Similarity between the PDECX models and their corresponding patient’s ESCC tissue was confirmed by histology, morphology, HER-2 GCN and mutation.
None of the PDECX models (or their corresponding patient’s ESCC tissues) harbored HER-2 gene amplification. IHC staining showed HER-2 positivity (IHC 2+) in 2 PDECX models and negativity in 3 PDECX models. Significant tumor regression was observed in the Trastuzumab-treated EC044 HER-2 positive model (IHC 2+). A second HER-2 positive (IHC 2+) model, EC039, harbored a known PIK3CA mutation and showed strong activation of the AKT signaling pathway and was insensitive to Trastuzumab treatment, but could be resensitised using a combination of Trastuzumab and AKT inhibitor AZD5363. In summary, we established 5 PDECX mouse models and demonstrated tumor regression in response to Trastuzumab treatment in a HER-2 IHC 2+ model, but resistance in a HER-2 IHC 2+/PIK3CA mutated model.
This study demonstrates Trastuzumab-induced tumor regressions in HER-2 positive tumors, and highlights PIK3CA mutation as a potential resistance mechanism to Trastuzumab treatment in pre-clinical patient-derived EC xenograft models.
Esophageal carcinoma; HER-2; Herceptin; PIK3CA mutation; Xenograft model
C-src is an evolutionarily conserved proto-oncogene that regulates cell proliferation, differentiation and apoptosis. In our previous studies, we have reported that another proto-oncogene, c-erbB2, plays an important role in primordial follicle activation and development. We also found that c-src was expressed in mammalian ovaries, but its functions in primordial follicle activation remain unclear. The objective of this study is to investigate the role and mechanism of c-src during the growth of primordial follicles.
Ovaries from 2-day-old rats were cultured in vitro for 8 days. Three c-src-targeting and one negative control siRNA were designed and used in the present study. PCR, Western blotting and primordial follicle development were assessed for the silencing efficiency of the lentivirus c-src siRNA and its effect on primordial follicle onset. The expression of c-src mRNA and protein in primordial follicle growth were examined using the PCR method and immunohistochemical staining. Furthermore, the MAPK inhibitor PD98059, the PKC inhibitor Calphostin and the PI3K inhibitor LY294002 were used to explore the possible signaling pathways of c-src in primordial folliculogenesis.
The results showed that Src protein was distributed in the ooplasmic membrane and the granulosa cell membrane in the primordial follicles, and c-src expression level increased with the growth of primordial follicle. The c-src -targeting lentivirus siRNAs had a silencing effect on c-src mRNA and protein expression. Eight days after transfection of rat ovaries with c-src siRNA, the GFP fluorescence in frozen ovarian sections was clearly discernible under a fluorescence microscope, and its relative expression level was 5-fold higher than that in the control group. Furthermore, the c-src-targeting lentivirus siRNAs lowered its relative expression level 1.96 times. We also found that the development of cultured primordial follicles was completely arrested after c-src siRNA knockdown of c-src expression. Furthermore, our studies demonstrated that folliculogenesis onset was inhibited by Calphostin, PD98059 or LY294002 treatment,but none of them down-regulated c-src expression. In contrast, the expression levels of p-PKC, p-ERK1/2 and p-PI3K in the follicles were clearly decreased by c-src siRNA transfection. Correspondingly, both Calphostin and LY294002 treatment resulted in a decrease in the p-PKC level in follicles, but no change was observed in the PD98059 group. Finally, LY294002 treatment decreased the p-PI3K expression level in the follicles, but no changes were observed in the PD98059 and Calphostin groups.
C-src plays an important role in regulating primordial follicle activation and growth via the PI3K-PKC- ERK1/2 pathway.
DNA replication and repair are critical processes for all living organisms to ensure faithful duplication and transmission of genetic information. Flap endonuclease 1 (Fen1), a structure-specific nuclease, plays an important role in multiple DNA metabolic pathways and maintenance of genome stability. Human FEN1 mutations that impair its exonuclease activity have been linked to cancer development. FEN1 interacts with multiple proteins, including proliferation cell nuclear antigen (PCNA), to form various functional complexes. Interactions with these proteins are considered to be the key molecular mechanisms mediating FEN1's key biological functions. The current challenge is to experimentally demonstrate the biological consequence of a specific interaction without compromising other functions of a desired protein. To address this issue, we established a mutant mouse model harboring a FEN1 point mutation (F343A/F344A, FFAA), which specifically abolishes the FEN1/PCNA interaction. We show that the FFAA mutation causes defects in RNA primer removal and long-patch base excision repair, even in the heterozygous state, resulting in numerous DNA breaks. These breaks activate the G2/M checkpoint protein, Chk1, and induce near-tetraploid aneuploidy, commonly observed in human cancer, consequently elevating the transformation frequency. Consistent with this, inhibition of aneuploidy formation by a Chk1 inhibitor significantly suppressed the cellular transformation. WT/FFAA FEN1 mutant mice develop aneuploidy-associated cancer at a high frequency. Thus, this study establishes an exemplary case for investigating the biological significance of protein-protein interactions by knock-in of a point mutation rather than knock-out of a whole gene.
FEN1; PCNA; Okazaki fragment maturation; long patch base excision repair; tetraploidy; aneuploidy; cancer
Testes-specific protease 50 (TSP50), a newly discovered threonine enzyme, has similar amino acid sequences and enzymatic structures to those of many serine proteases. It may be an oncogene. TSP50 is up-regulated in breast cancer epithelial cells, and ectopic expression of TSP50 in TSP50-deficient Chinese hamster ovary (CHO) cells has been found to promote cell proliferation. However, the mechanisms by which TSP50 exerts its growth-promoting effects are not yet fully understood.
To delineate whether the threonine protease activity of TSP50 is essential to its function in cell proliferation, we constructed and characterized a mutant TSP50, called TSP50 T310A, which was identified as a protease-dead mutant of TSP50. By a series of proliferation analyses, colony formation assays and apoptosis analyses, we showed that T310A mutation significantly depresses TSP50-induced cell proliferation in vitro. Next, the CHO stable cell line expressing either wild-type or T310A mutant TSP50 was injected subcutaneously into nude mice. We found that the T310A mutation could abolish the tumorigenicity of TSP50 in vivo. A mechanism investigation revealed that the T310A mutation prevented interaction between TSP50 and the NF-κBIκBα complex, which is necessary for TSP50 to perform its function in cell proliferation.
Our data highlight the importance of threonine 310, the most critical protease catalytic site in TSP50, to TSP50-induced cell proliferation and tumor formation.
Prostate cancer (PCa) patients with regional lymph node involvement at radical prostatectomy often experience disease progression to other organs, with the bone as the predominant site. The transcription factor Runx2 plays an important role in bone formation and PCa cell migration, invasion and metastasis. Here we demonstrated that the forkhead protein FOXO1, a key downstream effector of the tumor suppressor PTEN, inhibits the transcriptional activity of Runx2 in PCa cells. This inhibition was enhanced by PTEN but diminished by active Akt. FOXO1 bound to Runx2 in vitro and in vivo and suppressed Runx2’s activity independent of its transcriptional function. FOXO1 inhibited Runx2-promoted migration of PCa cells while silencing of endogenous FOXO1 enhanced PCa cell migration in a Runx2-dependent manner. Forced expression of FOXO1 also inhibited Runx2-promoted PCa cell invasion. Finally, we found that expression of PTEN and the level of FOXO1 in the nucleus is inversely correlated with expression of Runx2 in a cohort of PCa specimens from patients with lymph node and bone metastasis. These data reveal FOXO1 as a critical negative regulator of Runx2 in PCa cells. Inactivation of FOXO1 due to frequent loss of PTEN in PCa cells may leave the oncogenic activities of Runx2 unchecked, thereby driving promiscuous expression of Runx2 target genes involved in cell migration and invasion and favoring PCa progression.
The present study aimed to investigate the effect of methylprednisolone (MP) on the activities of caspase-3, -6, -8 and -9 in rabbits with acute spinal cord injury (ASCI) and to explore the mechanism underlying the antiapoptotic effect of MP on ASCI. Modified Allen’s method was employed to establish the ASCI animal model. The animals were randomly divided into a sham (S; n=12), ASCI (C; n=36) and MP group (T; n=36). At 8, 24 and 72 h and 7, 14 and 28 days after ASCI, the animals were sacrificed and the spinal cord was collected. The absorbance (A) was measured with a microplate reader and the activities of caspase-3, -6, -8 and -9 were calculated followed by comparisons among the groups. In the S group, the activities of the four caspases were low. In the C and T groups, the caspase activities increased at 8 h after injury, peaked at 24 h and remained at a high level 3 days after injury. However, the caspase activities began to decrease at 7 days after injury and were significantly reduced at 14 and 28 days after ASCI. Furthermore, the caspase activities in the T group were markedly lower than those in the C group at 8 and 24 h and 3 and 7 days after surgery (P<0.05), but significant differences were not observed at 14 and 28 days after injury (P>0.05). In conclusion, MP exerted an antiapoptotic effect via inhibition of the activities of caspase-3, -6, -8 and -9 in an animal model of ASCI.
acute spinal cord injury; methylprednisolone; caspase; apoptotic factors
Potassium voltage-gated channel, KQT-like subfamily, member 1 (KCNQ1) is thought to be an important candidate gene of diabetes. Several single nucleotide polymorphisms (SNPs) in a 40-kb linkage disequilibrium (LD) block in its intron 15 have been identified to be associated with diabetes in East Asian populations in recent genome-wide association studies. The aim of this study was to investigate whether KCNQ1 polymorphisms influence the levels of the metabolic phenotypes in general Chinese populations.
We investigated the associations of two SNPs (rs2237892 and rs2237895) in the aforementioned 40-kb LD block, a missense variant rs12720449 (P448R) in exon 10, and a synonymous variant rs1057128 (S546S) in exon 13 with metabolic phenotypes in a Uyghur population (n = 478) and replicated these associations in a Han population (n = 2,485). We found that rs2237892-T allele was significantly associated with decreased triglyceride levels (pcombined = 0.001). The minor G allele of the rs12720449, with sharp difference of the allelic frequency between European and East Asian populations (0.2% versus 14%, respectively), was associated with a lower triglyceride levels than G allele in Uyghur subjects (p = 0.004), in Han subjects (p = 0.052), and in subjects of meta-analysis (pcombined = 0.001). Moreover, the minor A allele of the rs1057128 was also associated with decreased triglyceride levels in meta-analysis (pcombined = 0.010).
To the best of our knowledge, this is the first report associating a missense mutation of KCNQ1, rs12720449, with triglyceride levels. Rs2237892, representing the 40-kb LD block, is also associated with triglyceride levels in Han population. Further studies are required to replicate these findings in other East Asian populations.
Morphological and functional changes during ameloblast and odontoblast differentiation suggest that enamel and dentin formation is under circadian control. Circadian rhythms are endogenous self-sustained oscillations with periods of 24 hours that control diverse physiological and metabolic processes. Mammalian clock genes play a key role in synchronizing circadian functions in many organs. However, close to nothing is known on clock genes expression during tooth development. In this work, we investigated the expression of four clock genes during tooth development. Our results showed that circadian clock genes Bmal1, clock, per1, and per2 mRNAs were detected in teeth by RT-PCR. Immunohistochemistry showed that clock protein expression was first detected in teeth at the bell stage (E17), being expressed in EOE and dental papilla cells. At post-natal day four (PN4), all four clock proteins continued to be expressed in teeth but with different intensities, being strongly expressed within the nucleus of ameloblasts and odontoblasts and down-regulated in dental pulp cells. Interestingly, at PN21 incisor, expression of clock proteins was down-regulated in odontoblasts of the crown-analogue side but expression was persisting in root-analogue side odontoblasts. In contrast, both crown and root odontoblasts were strongly stained for all four clock proteins in first molars at PN21. Within the periodontal ligament (PDL) space, epithelial rests of Malassez (ERM) showed the strongest expression among other PDL cells. Our data suggests that clock genes might be involved in the regulation of ameloblast and odontoblast functions, such as enamel and dentin protein secretion and matrix mineralization.
Clock genes; Tooth development; Bmal1; Clock; Per1; Per2; expression pattern; immunohistochemistry
AIM: To investigate the effects of resistin-like molecule β (RELMβ) over-expression on the invasion, metastasis and angiogenesis of gastric cancer cells.
METHODS: Human RELMβ encoding expression vector was constructed and transfected into the RELMβ lowly-expressed gastric cancer cell lines SGC-7901 and MKN-45. Gene expression was measured by Western blotting, reverse transcription polymerase chain reaction (PCR) and real-time quantitative PCR. Cell proliferation was measured by 2-(4,5-dimethyltriazol-2-yl)-2,5-diphenyl tetrazolium bromide colorimetry, colony formation and 5-ethynyl-20-deoxyuridine incorporation assays. The in vitro migration, invasion and metastasis of cancer cells were measured by cell adhesion assay, scratch assay and matrigel invasion assay. The angiogenic capabilities of cancer cells were measured by tube formation of endothelial cells.
RESULTS: Transfection of RELMβ vector into SGC-7901 and MKN-45 cells resulted in over-expression of RELMβ, which did not influence the cellular proliferation. However, over-expression of RELMβ suppressed the in vitro adhesion, invasion and metastasis of cancer cells, accompanied by decreased expression of matrix metalloproteinase-2 (MMP-2) and MMP-9. Moreover, transfection of RELMβ attenuated the expression of vascular endothelial growth factor and in vitro angiogenic capabilities of cancer cells.
CONCLUSION: Over-expression of RELMβ abolishes the invasion, metastasis and angiogenesis of gastric cancer cells in vitro, suggesting its potentials as a novel therapeutic target for gastric cancer.
Resistin-like molecule β; Gastric cancer; Invasion; Metastasis; Angio-genesis
Clinical immunity to malaria in human populations is developed after repeated exposure to malaria. Regulation and balance of host immune responses may lead to optimal immunity against malaria parasite infection. Polysaccharides (ABPS) derived from the Chinese herb ox knee Achyranthes bidentata possess immuno-modulatory functions. The aim of this study is to use the rodent malaria model Plasmodium yoelii 17XL (P. y17XL) to examine whether pretreatment with ABPS will modulate host immunity against malaria infection and improve the outcome of the disease.
To determine whether ABPS could modulate immunity against malaria, mice were pretreated with ABPS prior to blood-stage infection by P. y17XL. Host survival and parasitaemia were monitored daily. The effect of pretreatment on host immune responses was studied through the quantitation of cytokines, dendritic cell populations, and natural regulatory T cells (Treg).
Pretreatment with ABPS prior to infection significantly extended the survival time of mice after P. y17XL infection. At three and five days post-infection, ABPS pretreated mice developed stronger Th1 immune responses against malaria infection with the number of F4/80+CD36+ macrophages and levels of IFN-γ, TNF-α and nitric oxide being significantly higher than in the control group. More importantly, ABPS-treated mice developed more myeloid (CD11c+CD11b+) and plasmacytoid dendritic cells (CD11c+CD45R+/B220+) than control mice. ABPS pretreatment also resulted in modulated expression of MHC-II, CD86, and especially Toll-like receptor 9 by CD11c+ dendritic cells. In comparison, pretreatment with ABPS did not alter the number of natural Treg or the production of the anti-inflammatory cytokine IL-10.
Pretreatment with the immuno-modulatory ABPS selectively enhanced Th1 immune responses to control the proliferation of malaria parasites, and prolonged the survival of mice during subsequent malaria infection.
Achyranthes bidentata polysaccharides; Plasmodium yoelii 17XL; Immune responses; Immuno modulatory effect
Overexpression of squamous cell carcinoma antigen 1 (SCCA1) in hepatitis G2 (HepG2) and Chinese hamster ovary cells can increase hepatitis B virus (HBV) binding capacity by interacting with the preS1 domain of the HBV surface antigen. However, the magnitude of increase in binding capacity was higher by several orders in the former, indicating the existence of additional factor(s) produced by HepG2 cells, which facilitates HBV attachment. Ferritin light chain (FTL) was identified as the sole high hit candidate by screening human liver cDNA library using a bacterial two-hybrid system with either preS or SCCA1 as the bait. Subsequent in vitro protein–protein interaction assays confirmed the binding activity of FTL to both preS and SCCA1, as well as the formation of triple complex preS-FTL-SCCA1, and narrowed down the binding sites on FTL. In vitro overexpression of FTL could further enhance HBV attachment in both HepG2 and Chinese hamster ovary cells, which were already overexpressing SCCA1. Importantly, in vivo co-expression of human FTL and SCCA1 in mouse liver by means of tailvein hydrodynamic injection increased serum levels of HBV surface antigen transiently 24 hours post challenge with HBV-positive human sera, and a large amount of HBV core antigen-positive hepatocytes around blood vessels could be identified by immunohistochemical staining 48 hours post challenge. The data strongly suggest that FTL and SCCA1 may serve as coreceptors in HBV cellular attachment and virus entry into hepatocytes.
squamous cell carcinoma antigen 1; ferritin light chain; hepatitis B virus; coreceptors
Endurance exercise is an inexpensive intervention that is thought to provide substantial protection against several age-related pathologies, as well as inducing acute changes to endurance capacity and metabolism. Recently, it has been established that endurance exercise induces conserved alterations in physiological capacity in the invertebrate Drosophila model. If the genetic factors underlying these exercise-induced physiological alterations are widely conserved, then invertebrate genetic model systems will become a valuable tool for testing of genetic and pharmacological mimetics for endurance training. Here, we assess whether the Drosophila homolog of the vertebrate exercise response gene PGC-1α spargel (srl) is necessary or sufficient to induce exercise-dependent phenotypes. We find that reduction of srl expression levels acutely compromises negative geotaxis ability and reduces exercise-induced improvement in both negative geotaxis and time to exhaustion. Conversely, muscle/heart specific srl overexpression improves negative geotaxis and cardiac performance in unexercised flies. In addition, we find that srl overexpression mimics some, but not all, exercise-induced phenotypes, suggesting that other factors also act in parallel to srl to regulate exercise-induced physiological changes in muscle and heart.
Biology sequence comparison is a fundamental task in computational biology. According to the hydropathy profile of amino acids, a protein sequence is taken as a string with three letters. Three curves of the new protein sequence were defined to describe the protein sequence. A new method to analyze the similarity/dissimilarity of protein sequence was proposed based on the conditional probability of the protein sequence. Finally, the protein sequences of ND6 (NADH dehydrogenase subunit 6) protein of eight species were taken as an example to illustrate the new approach. The results demonstrated that the method is convenient and efficient.
Protein sequence; Sequence comparison; Similarity/dissimilarity; Conditional probability
DNA replication and repair are critical processes for all living organisms to ensure faithful duplication and transmission of genetic information. Flap endonuclease 1 (Fen1), a structure-specific nuclease, plays an important role in multiple DNA metabolic pathways and maintenance of genome stability. Human FEN1 mutations that impair its exonuclease activity have been linked to cancer development. FEN1 interacts with multiple proteins, including proliferation cell nuclear antigen (PCNA), to form various functional complexes. Interactions with these proteins are considered key molecular mechanisms mediating FEN1’s key biological functions. The current challenge is to experimentally demonstrate the biological consequence of a specific interaction without compromising other functions of a desired protein. To address this issue, we established a mutant mouse model harboring a FEN1 point mutation (F343A/F344A, FFAA), which specifically abolishes the FEN1/PCNA interaction. We show that the FFAA mutation causes defects in RNA primer removal and long-patch base excision repair, even in the heterozygous state, resulting in numerous DNA breaks. These breaks activate the G2/M checkpoint protein, Chk1, and induce near-tetraploid aneuploidy, commonly observed in human cancer, consequently elevating the transformation frequency. Consistent with this, inhibition of aneuploidy formation by a Chk1 inhibitor significantly suppressed the cellular transformation. WT/FFAA FEN1 mutant mice develop aneuploidy-associated cancer at a high frequency. Thus, this study establishes an exemplary case for investigating the biological significance of protein-protein interactions by knock in of a point mutation rather than knock out of a whole gene.
FEN1; PCNA; Okazaki fragment maturation; long patch base excision repair; tetraploidy; aneuploidy; cancer
Atractylodes macrocephala Koidz. (Asteraceae) is an economically important Chinese medicinal herb. In this study, 15 polymorphic microsatellite markers were developed from A. macrocephala using the compound microsatellite marker technique. Levels of polymorphism within the 15 markers were assessed using 83 individuals from two wild and two cultivated populations in China. The number of alleles per locus ranged from 2 to 20, with an average of 9.9 alleles. Observed and expected heterozygosities ranged from 0.083 to 1.000 and from 0.097 to 0.938, respectively. These markers will be valuable for germplasm classification and identification, as well as for assessing the genetic diversity and spatial genetic structure among wild and cultivated populations of A. macrocephala.
Atractylodes macrocephala; microsatellites; genetic diversity; Chinese medicinal herb
Insulin-like growth factor 1 receptor (IGF1R) is an attractive drug target for cancer therapy and research on IGF1R inhibitors has had success in clinical trials. A particular challenge in the development of specific IGF1R inhibitors is interference from insulin receptor (IR), which has a nearly identical sequence. A few potent inhibitors that are selective for IGF1R have been discovered experimentally with the aid of computational methods. However, studies on the rapid identification of IGF1R-selective inhibitors using virtual screening and confidence-level inspections of ligands that show different interactions with IGF1R and IR in docking analysis are rare. In this study, we established virtual screening and binding-mode prediction workflows based on benchmark results of IGF1R and several kinase receptors with IGF1R-like structures. We used comprehensive analysis of the known complexes of IGF1R and IR with their binding ligands to screen specific IGF1R inhibitors. Using these workflows, 17 of 139,735 compounds in the NCI (National Cancer Institute) database were identified as potential specific inhibitors of IGF1R. Calculations of the potential of mean force (PMF) with GROMACS were further conducted for three of the identified compounds to assess their binding affinity differences towards IGF1R and IR.
IGF1R; IR; virtual screening; binding mode prediction; selective inhibition
Transmission-blocking vaccines (TBVs) have been considered an important strategy for disrupting the malaria transmission cycle, especially for Plasmodium vivax malaria, which undergoes gametocytogenesis earlier during infection. Pvs25 and Pvs28 are transmission-blocking vaccine candidates for P. vivax malaria. Assessment of genetic diversity of the vaccine candidates will provide necessary information for predicting the performance of vaccines, which will guide us during the development of malaria vaccines.
We sequenced the coding regions of pvs25 and pvs28 from 30 P. vivax isolates from Yunnan Province, identifying five amino acid haplotypes of Pvs25 and seven amino acid haplotypes of Pvs28. Among a total of four mutant residues, the predominant haplotype of Pvs25 only had the I130T substitution. For Pvs28, a total of eight amino acid substitutions were identified. The predominant haplotype of Pvs28 had two substitution at positions 52 (M52L) and 140 (T140S) with 5-6 GSGGE/D tandem repeats at the end of fourth EGF-like domain. Most amino acid substitutions were common with previous reports from South Asian isolates. Although the nucleotide diversity of pvs28 (π = 0.0034 ± 0.0012) was significantly higher than pvs25 (π = 0.0013 ± 0.0009), it was still conserved when compared with the blood stage vaccine candidates.
Genetic analysis revealed limited genetic diversity of pvs25 and pvs28, suggesting antigenic diversity may not be a particular problem for Sal I based TBVs in most P. vivax-endemic areas of China.
Plasmodium vivax; TBVs; Pvs25; Pvs28; polymorphism
CD8+ effector cells often have an antitumor function in patients with cancer. However, CD8+Foxp3+ regulatory T cells (Tcregs) and interleukin (IL)-17-producing CD8+ T cells (Tc17 cells) also derive from the CD8+ T cell lineage. Their role in the antitumor response remains largely unknown. In the present study, we aimed to investigate the distribution, characterization, and generation of CD8+ Tcregs and Tc17 cells in NPC patients.
Peripheral blood and tumor biopsy tissues from 21 newly diagnosed patients with nasopharyngeal carcinoma (NPC) were collected, along with peripheral blood from 21 healthy donors. The biological characteristics of Tcregs and Tc17 cells from blood and tumor tissues were examined by intracellular staining, tetramer staining and fluorescence-activated cell sorting (FACS) analysis. The suppressive function of Tcregs was investigated using a proliferation assay that involved co-culture of sorted CD8+CD25+ T cells with naïve CD4+ T cells in vitro.
We observed an increased prevalence of Tcregs and Tc17 cells among tumor-infiltrating lymphocytes (TILs) and different distribution among peripheral blood mononuclear cells (PBMCs) in NPC patients. Cytokine profiles showed that the Tcregs expressed a high level of IL-10 and low level of transforming growth factor β, whereas Tc17 cells expressed a high level of tumor necrosis factor α. Interestingly, both subsets expressed a high level of interferon γ in TILs, and the Tcregs suppressed naïve CD4+ T cell proliferation by a cell contact-dependent mechanism in vitro. Moreover, we demonstrated the existence of Epstein-Barr virus latent membrane protein (LMP) 1 and LMP2 antigen-specific Tcregs in NPC.
Our data provide new insights into the composition and function of CD8+ T-cell subsets in NPC, which may have an important influence on NPC immunotherapy.
Nasopharyngeal carcinoma; Tumor-infiltrating lymphocytes; CD8+ regulatory T cells (Tcreg); IL-17-producing CD8+ T cells (Tc17)
AIM: To investigate potential antitumor effects of rAd-p53 by determining if it enhanced sensitivity of gastric cancer cells to chemotherapy.
METHODS: Three gastric cancer cell lines with distinct levels of differentiation were treated with various doses of rAd-p53 alone, oxaliplatin (OXA) alone, or a combination of both. Cell growth was assessed with an 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-diphenytetrazoliumromide assay and the expression levels of p53, Bax and Bcl-2 were determined by immunohistochemistry. The presence of apoptosis and the expression of caspase-3 were determined using flow cytometry.
RESULTS: Treatment with rAd-p53 or OXA alone inhibited gastric cancer cell growth in a time- and dose-dependent manner; moreover, significant synergistic effects were observed when these treatments were combined. Immunohistochemical analysis demonstrated that treatment with rAd-p53 alone, OXA alone or combined treatment led to decreased Bcl-2 expression and increased Bax expression in gastric cancer cells. Furthermore, flow cytometry showed that rAd-p53 alone, OXA alone or combination treatment induced apoptosis of gastric cancer cells, which was accompanied by increased expression of caspase-3.
CONCLUSION: rAd-p53 enhances the sensitivity of gastric cancer cells to chemotherapy by promoting apoptosis. Thus, our results suggest that p53 gene therapy combined with chemotherapy represents a novel avenue for gastric cancer treatment.
Gastric cancer; rAd-p53; Oxaliplatin; Chemosensitivity; Apoptosis