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Inorganic phosphate (Pi) deficiency is a main limiting factor on crops growth and to select low-Pi tolerant breeding lines is very significant for crop breeding. Here, two contrasting maize (Zea mays L.) breeding lines showed different physiological response to Pi deficiency. The low-Pi tolerant QXN233 maintained normal growth, including high fresh weight, green leaves, strong shoots, and numerous roots relative to that of the sensitive MH05-4, mainly due to QXN233’ high Pi content in shoots under Pi deficiency. Importantly, some Pi-responsive genes were detected, and among them, Pi transporters ZmPHT1;1 and ZmPHT1;9 as well as phytase gene Zmphytase 2 were expressed increasingly in QXN233 compared to MH05-4 under Pi deprivation or Pi resupply. Moreover, QXN233 had higher proline content, soluble sugar content, and SOD activity than MH05-4, related with its tolerance. Taken together, this study enriches the understanding of the mechanism of maize responding to Pi deficiency.

Eupatorium fortunei Turcz. is a widely used Chinese herbal medicine in China. In this study, a gas chromatography-triple quadrupole mass spectrometry (GC-MS/MS) method was developed and validated to simultaneously determine the contents of p-cymene, thymol, neryl acetate, and β-caryophyllene in roots, stems, and leaves of Eupatorium fortunei Turcz. harvested at different growth periods. All four constituents could be detected in leaves, three could be detected in stems except β-caryophyllene, and only thymol could be detected in roots. The order of the total contents of four constituents in different parts was leaves > stems > roots. It indicated that the leaves could be the proper medicinal parts of Eupatorium fortunei Turcz. The content of four constituents in leaves varied a lot among different growth periods and showed an M-shaped change trend with the growth of Eupatorium fortunei Turcz. The four constituents accumulated to the highest values in early July followed by mid-September. Accordingly, the best harvest time of Eupatorium fortunei Turcz. is early July and mid-September.

Introduction Prognosis for relapsed and refractory large B-cell lymphoma (r/r LBCL) is poor, and treatment remains challenging. Relmacabtagene autoleucel (relma-cel) is a CD19-directed 4-1BB/CD3z chimeric antigen receptor T cell (CAR-T) product manufactured in China. RELIANCE study is the first CAR-T study with CD19 as target that got IND approved by the authority in China. Initial findings of the pivotal Phase 2 single-arm, multicenter RELIANCE trial demonstrated high response rates and low rates of CAR-T-associated toxicity with relma-cel treatment in heavily pretreated patients (pts) with r/r LBCL (NCT04089215; Ying et al. Cancer Med 2020;10:999-1011). Long-term follow-up data for the RELIANCE study are presented. Materials & Methods Fifty-nine adults (age ≥18 years) with heavily pretreated (≥2 prior therapies), measurable and histologically confirmed r/r LBCL were randomized to receive lymphodepletion chemotherapy (LDC: fludarabine 25 mg/m 2 + cyclophosphamide 250 mg/m 2 daily×3) followed by a single infusion of autologous CAR+ T cells at a dose of 100×10 6 or 150×10 6. Pts were evaluated for efficacy using Lugano criteria and for toxicity using Common Terminology Criteria for Adverse Events v4.03 and for cytokine release syndrome (CRS) using Lee et al. (Blood 2014;124(2):188-95). Primary endpoint was 3-month objective response rate (ORR); key secondary endpoints included complete response rate (CRR) at 3 months, duration of response (DOR), progression-free survival (PFS), overall survival (OS) and treatment-emergent adverse event (TEAE) profile. The data cutoff date was December 31, 2020. Results At the time of analysis, all 59 pts (median age 56.0 years, range 18-75 years) were at least 15 months post-treatment with relma-cel (7 pts completed trial, 34 pts on study, 18 pts withdrew). Among the modified intent-to-treat (mITT) population of 58 efficacy-evaluable pts, best ORR was 77.6%, with a best CRR of 51.7%; ORR and CRR at 3-month-postdose landmark were 60.3% and 44.8%, respectively. With a median follow-up of 17.9 months (range 0.3-25.6 months), median PFS (mITT) was 7.0 months (95% CI 4.8-not reached [NR]). Median PFS was NR (95% CI 8.8 months-NR) for pts with compete response (CR) at 3 months, the 6-, 12-, 18- and 24-month PFS rates were 80.8%, 69.2%, 69.2% and 69.2%, respectively (Figure). PFS was associated with objective response or CR at 1, 3, 6, and 12 months (log-rank test, p≤0.002). Median OS was NR (95% CI NR-NR) for both the mITT population and pts with CR at 3 months (12-month OS rate, 76.8% and 92.3%, respectively). Median DOR was NR (95% CI 4.9 months-NR) for the mITT population and (95% CI 8.0 months-NR) for pts with CR at 3 months. Levels of CAR-T cells declined to below the level of quantification (BLQ) in 41 (69.5%) pts, among whom nearly 40% had sustained response. Median time from documentation of CAR-T BLQ to disease progression was 6.1 months (95% CI 1.8-NR). Treatment-related TEAEs were reported in 93.2% of pts and were primarily grade 1-2 (89.8% pts); 54.2% of pts had grade ≥3 TEAEs. Pyrexia was the most common TEAE, reported in 59.3% of pts (all grade 1-2). The most common grade ≥3 treatment-related TEAEs were neutrophil count decrease (30.5%) and white blood cell (WBC) count decrease (13.6%). Presence of cytopenia at day 29 (78.0%) was significantly associated with WBC count after LDC, change in serum albumin between days 1 and 4 and serum interleukin 8 after LDC (p Conclusions These are the first data of long-term follow-up CD19 CAR-T study with IND approval in China reported. With nearly 18 months of median follow-up, the RELIANCE pivotal registered study demonstrated durable responses, high rates of OS and PFS, and no new safety profile found in r/r LBCL pts. Based on these data, relma-cel is undergoing approval in China. Figure: Kaplan-Meier estimates of (A) PFS and (B) OS by 3-month tumor response PR, partial response Figure 1 Figure 1. Disclosures Zhang: JW Therapeutics: Current Employment. Yang: JW Therapeutics: Current Employment. Zhou: JW Therapeutics: Current Employment. Zheng: JW Therapeutics: Current Employment.

Currently, no CAR-T product has been approved for use in China despite having hundreds of active CAR-T trials. Relma-cel (JWCAR029) is a CAR-T product with a CAR construct comprised of a binding domain, derived from a murine CD19-specific mAb (FMC63), a CD3ζ activation domain, and a 4-1BB costimulatory domain, and manufactured in China with a commercial-ready, highly automated, single train process to select, activate, transduce and expand both CD4 and CD8 cells to a wide range of doses with consistent product attributes. Relma-cel was evaluated in the first prospective, single-arm, multi-center, pivotal study (RELIANCE Trial) of CAR-T therapy conducted under Chinese IND to support an NMPA-accepted BLA submission in r/r large B-cell lymphoma (LBCL) (NCT 04089215) and the preliminary results are presented herein. Eligible adult patients had measurable, histologically confirmed r/r LBCL, failed at least 2 prior therapies, including anthracycline and anti-CD20 agents, and not had allogeneic transplant within 90 days or primary CNS lymphoma. Patients were randomized to receive either 100×106 (low dose) or 150×106 (high dose) CAR+ T cells as a single infusion following fludarabine 25 mg/m2 & cyclophosphamide 250 mg/m2 daily×3 as lymphodepletion. Patients were evaluated for efficacy (Lugano criteria, 2014), safety (CRS by Lee 2014, and all other events by CTCAE v4.03), and PK (by qPCR and flow cytometry). The protocol pre-specified and regulatory authority-agreed primary endpoint was 3 month ORR by landmark analysis to exclude a 3-month 20% ORR (Michael, 2017). Secondary endpoints included best overall response rate (BOR), duration of response (DOR), progression free survival (PFS), overall survival (OS), frequency/severity of AEs and CAR-T cell expansion. Investigators adjudicated disease response and progression based on imaging studies, pathology and clinical data; a sensitivity analysis was also conducted using an independent review committee adjudication. Between May 2018 and Dec 2019, 69 patients were screened, and 59 enrolled and treated with relma-cel, with median age of 56.0 years (18-75), 59.3% ECOG 1-2, and included several LBCL subtypes (DLBCL NOS 69.5%, TFL 15.3%, DHL/THL 5.1%, and PMBCL 6.8%) and nearly all with poor risk disease (81.4% treatment refractory, 45.8% ≥3 lines of prior therapies, 42.4% requiring bridging therapy, 39% IPI≥3). The median doses administered in low and high dose groups were 99.7×106 (range, 80.1-101.3) and 150.0×106 (range, 120.0-156.4) CAR+ T cells, respectively. Among the 58 efficacy evaluable patients, the primary analysis demonstrated a 3 month ORR of 58.6% (95% CI, 44.9-71.4) (the excluded patient had product that did not meet viability threshold criterion, but achieved CR at D29 that is ongoing for >1 year). As of Jun 17, 2020 data cut-off, BOR was 75.9% (95% CI, 62.8-86.1) with CR rate of 51.7% (95% CI, 38.2-65.1). With a median follow-up of 8.9 months, median OS were not reached, and 6 month DOR, PFS and OS were 60.0%, 54.2% and 90.8%, respectively. No improvement in efficacy outcomes was observed in the high dose group. Of 59 treated patients, grade ≥3 AEs occurring in >5% of patients were cytokine release syndrome (CRS), febrile neutropenia and lung infection (all, 5.1%). Grade ≥3 lab abnormalities in >10% of patients were neutrophil count decreased (30.5%), white blood cell count decreased (13.6%), leukopenia (10.2%) and neutropenia (10.2%). The rates of CRS, neurotoxicity (NT), death, and the use of tocilizumab/steroids are shown in Table 1. All events of CRS and NT resolved except for 1 patient with grade 4 CRS ongoing at the time of death as the result of sepsis at day 8. As of data cut off, no cases of severe cytopenia or severe infections occurred beyond 30 days of infusion. The two dose groups did not differ significantly in PK parameters (see Table 1). The RELIANCE Trial provided the first demonstration of licensure-quality CAR-T manufacturing and clinical trial data generation in r/r patients originating in China. These results with relma-cel demonstrate similar preliminary response rates and PK profiles while providing the potential for an improved toxicity profile in heavily-pre-treated patients with r/r DLBCL having poor risk features relative to other CD19-specific CAR-Ts approved in the US and EU. Figure Disclosures Zhang: JW Therapeutics (Shanghai) Co. Ltd: Current Employment, Current equity holder in private company. Yang:JW Therapeutics (Shanghai) Co. Ltd: Current Employment, Current equity holder in private company. Zhou:JW Therapeutics (Shanghai) Co. Ltd: Current Employment, Current equity holder in private company. Zheng:JW Therapeutics (Shanghai) Co. Ltd: Current Employment, Current equity holder in private company.

Chrysanthemum oreastrum is a perennial herb of Compositae and one of the traditional Chinese medicinal herb in China. In this study, the complete chloroplast genome of C. oreastrum was illu...

Background: JWCAR029 is a CD19-directed 4-1 BB CAR T cell product, of which CD4 and CD8 CAR T cells are produced together and transfused in non-fixed ratio. A phase I, single-arm, open label study was conducted to evaluate the safety and efficacy of JWCAR029 in patients (pts) with relapsed or refractory B-NHL. Previously, preliminary data in six pts (Yan et al, Blood 2018 132:4187) showed high response rates and favorable safety profiles of JWCAR029. Herein, we presented the data of the Phase I trial of JWCAR029 (NCT03344367 and NCT03355859) in 29 pts with pharmacokinetics (PK), pharmacodynamics (PD), and anti-therapeutic antibody (ATA) evaluations. Methods: Eligible pts received lymphodepletion, with 25mg/m2 flu and 250mg/m2 cy, followed by a single dose of JWCAR029 at one of four dose levels (DL1, 25×106 cells; DL2, 50×106 cells; DL3, 100×106 cells; DL4, 150×106 cells). Blood samples were collected and analyzed for PK, PD, and ATA at a central lab per protocol defined time points. The existence and duration of CAR T cells (PK) were measured by validated flow cytometry and qPCR assays. CD4 and CD8 subpopulation of CAR+ T cells were detected by cetuximab targeting EGFRt as a marker co-expressed with CAR transgene in fresh peripheral blood. In parallel, batched frozen blood samples collected from each pt were detected for integrated CAR transgene by qPCR at the same protocol defined time points. Plasma ATA against murine CD19 scFv (FMC63) was measured with a validated electrochemiluminescent (ECL) assay. Results: As of July 5, 2019, blood samples from 29 pts who received JWCAR029 treatment with a minimum follow-up of 6 M (median, 6 M) were evaluable in the analysis. From DL1 to DL4, median Cmax, Tmax and AUC0-28 for JWCAR029 transgene detected by qPCR did not differ among dose levels (Table 1). CD4/CD8 ratio (range, 0.23-5.50) at cryopreserved drug product of JWCAR029 was not associated with best response of CR/PR at 6 M. Greater in vivo expansion was detected by both qPCR and flow cytometry in pts with best response of CR/PR than those with SD/PD at 6 M (Table 1). Higher concentration of CD8+CAR+T cells than CD4+CAR+T cells were detected in PB by flow cytometry for all treated pts (Cmax median= 30.6 vs 5.64). At 3 M, 81.5% (22/27) and 48.2% (13/27) pts had detectable CD8+ and CD4+ CAR+ T cells, respectively. Of those pts with detectable CAR+ T cells at 3 M, 70% (14/20) and 35% (7/20) had detectable CD8+ and CD4+ CAR+ cells at 6 M, respectively. Significantly higher Cmax and AUC0-28 were observed in patients with ≥ Grade 1 CRS (Cmax median= 85004 vs 16328, P 27.5% of pts (8/29) had detectable ATA in plasma, of which 25% (2/8) pts had pre-existing antibodies before CAR T cell infusion. 6 pts developed antibodies without pre-existing antibodies and were considered treatment-induced. The median time for treatment-induced antibody development was 6 M (range, 3-12). Increasing level of antibodies were detected at median time of 6 M (range, 6-6) for pts who had pre-existing antibodies and were considered treatment-boosted. No significant differences in PK profiles of JWCAR029 transgene levels were found between ATA negative group and treatment-induced ATA positive group (Cmax median= 44497 vs 50032; AUC median= 420635 vs 313654; Fig.1). Although the sample size of the treatment-boosted subgroup was small, there was a trend for lower expansion of CAR T cells in pts who had pre-existing ATA than pts who did not develop ATA (Cmax median= 3051 vs 44497; AUC median = 16437 vs 420635;Fig.1). In ATA positive subgroup, 100% (8/8) pts responded with CR rate of 75% (6/8). 6 M-response rate was 65.5% (5/8) for ATA positive subgroup and 57.1% (12/21) for ATA negative subgroup. Incidence of ≥ Grade 1 CRS or NT was indistinguishable between ATA positive and negative subgroups, 50% (4/8) in ATA positive vs 57.1% (12/21) negative. Conclusion: Preliminary data from JWCAR029 Phase I study has demonstrated that pts with best response of CR/PR at 6 months had a relatively higher CAR T cell expansion. Current data suggested that the prevalence of pre-existing ATA may compromise CAR+T PK profile. No association of the presence or boost of ATA with efficacy or safety of JWCAR029 was observed. Further exploration of ATA and clinical outcomes will be studied in the ongoing pivotal Phase 2 study in 70 pts with B-NHL. Disclosures Hao: JW therapeutics (Shanghai) Co., Ltd: Employment, Equity Ownership. Wang:JW therapeutics (Shanghai) Co., Ltd: Employment, Equity Ownership. Zhou:JW therapeutics (Shanghai) Co., Ltd: Employment, Equity Ownership. Yang:JW therapeutics (Shanghai) Co., Ltd: Employment, Equity Ownership. Wang:JW therapeutics (Shanghai) Co., Ltd: Employment, Equity Ownership. Lam:JW therapeutics (Shanghai) Co., Ltd: Employment, Equity Ownership. Li:JW therapeutics (Shanghai) Co., Ltd: Employment, Equity Ownership. Zheng:JW therapeutics (Shanghai) Co., Ltd: Employment, Equity Ownership.

To overcome bad prognosis of patients with heart failure and the lack of organ donors, cardiac tissue engineering has developed as a biomimetic approach to repair, replace, and regenerate the damaged cardiac tissue. During the past decade years, researchers are devoted to find different natural and/or synthetic materials that can build appropriate physical structures to contain and organize implanted cells. In this study, we present a new method for primary neonatal rat cardiomyocytes culture in vitro using alginate/collagen/chitosan hydrogel. To investigate the feasibility of this material as scaffold for cardiac myocytes, neonatal rat ventricular myocytes were isolated and encapsulated in alginate-based beads cross-linked with calcium ion. The growth of cells was evaluated by staining with α-Sarcomeric actin (α-SCA) and Troponin T type 2 (TNNT2), and the viability of cardiomyocytes was studied in vitro by assessing the expression levels of several cardiac ion channels, including CACNL1A1, Connexin 43 and SCN5A. The results showed a significant increase in cardiac myocytes number, and the expression levels of CACNL1A1, Connexin 43 (Cx43) were up-regulated significantly except SCN5A, as compared with two-dimensional cultures. Moreover, extracellular matrix produced by the seeded cells themselves was observed by staining with fibronectin. Taken together, these findings indicate that this alginate/collagen/chitosan hydrogel bead is suitable for supporting the growth and retaining the morphologic and electrophysiologic characteristics of primary cultured rat cardiac muscle cells.

This study investigated intracellular oxidative stress and its underlying mechanisms in a rotary cell culture system used to achieve a simulated microgravity (SMG) environment. Experiments were conducted with human breast cancer cell lines MCF-7 (an estrogen receptor (ER) α positive cell line) and MDA-MB-231 (an ERα negative cell line) encapsulated in alginate/collagen carriers. After 48 h, SMG led to oxidative stress and DNA damage in the MDA-MB-231 cells but a significant increase in mitochondrial activity and minimal DNA damage in the MCF-7 cells. The activity of superoxide dismutase (SOD) significantly increased in the MCF-7 cells and decreased in MDA-MB-231 cells in the SMG environment compared with a standard gravity control. Moreover, SMG promoted expression of ERα and protein kinase C (PKC) epsilon in MCF-7 cells treated with PKC inhibitor Go6983. Overall, exposure to SMG increased mitochondrial activity in ERα positive cells but induced cellular oxidative damage in ERα negative cells. Thus, ERα may play an important role in protecting cells from oxidative stress damage under simulated microgravity.

MARVEL domain-containing 1 (MARVELD1) is a newly identified nuclear protein; however its function has not been clear until now. Here, we report that mouse MARVELD1 (mMARVELD1), which is highly conserved between mice and humans, exhibits cell cycle-dependent cellular localization. In NIH3T3 cells, MARVELD1 was observed in the nucleus and at the perinuclear region during interphase, but was localized at the mitotic spindle and midbody at metaphase, and a significant fraction of mMARVELD1 translocated to the plasma membrane during anaphase. In addition, treatment of cells with colchicine, a microtubule-depolymerizing agent, resulted in translocation of mMARVELD1 to the plasma membrane, and association of mMARVELD1 and α-tubulin was confirmed by co-immunoprecipitation. Finally, overexpression of mMARVELD1 resulted in a remarkable inhibition of cell proliferation, G1-phase arrest, and reduced cell migration. These findings indicate that mMARVELD1 is a microtubule-associated protein that plays an important role in cell cycle progression and migration.

G protein-coupled receptors (GPCRs) are critical factors in regulating morphogenesis, mating, infection and virulence in fungi. In this study, various computational strategies were applied to identify GPCR-like proteins from the genomes of both Verticillium dahliae and Verticillium albo-atrum. The putative GPCRs were distributed over 13 classes, and significantly, three of those represented novel classes of GPCR-like proteins in fungi. The three novel GPCRs had high levels of identity to their counterparts in higher eukaryotes, including Homo sapiens. The numbers of GPCR-like proteins in the two Verticillium spp. were similar to those seen in other filamentous fungi, such as Magnaporthe grisea, Neurospora crassa and Fusarium graminearum. Additionally, the carbon/amino acid receptors were divided into three different subclasses, indicating that differences among the GPCRs existed not only among different classes but also within classes. In conclusion, the identification and classification of GPCRs and their homology to some well-studied fungi will be an important starting point for future research in Verticillium spp.

MARVELD1 (MARVEL domain-containing 1) is a member of MARVEL domain-containing proteins and located on human chromosome 10q24.2. MARVELD1 has no significant similarity with other members of MARVEL domain family at amino acid level. Gene expression arrays demonstrated that MARVELD1 is widely expressed in normal human tissues and is down-regulated in primary multiple tumors derived from ovary, vulva, uterus, cervix, breast, testis, kidney, bladder and liver. The down-regulation of MARVELD1 was further identified by real-time PCR and immunohistochemical staining in primary breast cancer. In addition, we identified the reduced expression of MARVELD1 is owing to DNA methylation and could be reversed by pharmacologic demethylation. Finally, our results showed that MARVELD1 protein is located in nucleus instead of cell membrane.

Various polymorphisms of the MDR1 gene that encodes for P-glycoprotein (P-gp), a transmembrane pump, have been identified. A silent mutation C3435T in exon 26 and a G2677T mutation in exon 21 have been correlated with P-gp expression and function in humans. The objectives of this study were (a) to determine whether the MDR1 exon 21 and exon 26 polymorphisms were related to steroid weaning in a pediatric heart transplant (HTx) population, and (b) to determine whether an association exist between the MDR1 exon 21 and exon 26 polymorphisms in these patients. Sixty-nine pediatric HTx patients were studied. MDR1 genotyping was determined by polymerase chain reaction amplification, sequencing the DNA, and sequence evaluation using Polyphred software (University of Washington) to identify genotypes. The steroid dose at 1 year post-transplantation was recorded. For steroid weaning at one year post-HTx for MDR1 C3435T, 12 of 18 (67%) patients in the CC genotype were still on prednisone, whereas only 18 of 47 (38%) of the CT/TT group were still receiving prednisone (p = 0.04). Similar results were observed for the MDR1 G2677T genotyping and steroid weaning. Forty-three of 46 patients (93.5%) who have MDR1 C3435T allele also have a mutant G2677T allele (p < 0.001). We conclude that (a) a significantly larger number of MDR1 3435 CC HTx patients remain on steroids at 1 year after transplantation, and (b) the MDR1 C3435T genotype is associated with the G2677 genotype in pediatric HTx patients.

Background In the present study we identified a novel gene, Homo Sapiens Chromosome 1 ORF109 (c1orf109, GenBank ID: NM_017850.1), which encodes a substrate of CK2. We analyzed the regulation mode of the gene, the expression pattern and subcellular localization of the predicted protein in the cell, and its role involving in cell proliferation and cell cycle control. Methods Dual-luciferase reporter assay, chromatin immunoprecipitation and EMSA were used to analysis the basal transcriptional requirements of the predicted promoter regions. C1ORF109 expression was assessed by western blot analysis. The subcellular localization of C1ORF109 was detected by immunofluorescence and immune colloidal gold technique. Cell proliferation was evaluated using MTT assay and colony-forming assay. Results We found that two cis-acting elements within the crucial region of the c1orf109 promoter, one TATA box and one CAAT box, are required for maximal transcription of the c1orf109 gene. The 5′ flanking region of the c1orf109 gene could bind specific transcription factors and Sp1 may be one of them. Employing western blot analysis, we detected upregulated expression of c1orf109 in multiple cancer cell lines. The protein C1ORF109 was mainly located in the nucleus and cytoplasm. Moreover, we also found that C1ORF109 was a phosphoprotein in vivo and could be phosphorylated by the protein kinase CK2 in vitro. Exogenous expression of C1ORF109 in breast cancer Hs578T cells induced an increase in colony number and cell proliferation. A concomitant rise in levels of PCNA (proliferating cell nuclear antigen) and cyclinD1 expression was observed. Meanwhile, knockdown of c1orf109 by siRNA in breast cancer MDA-MB-231 cells confirmed the role of c1orf109 in proliferation. Conclusions Taken together, our findings suggest that C1ORF109 may be the downstream target of protein kinase CK2 and involved in the regulation of cancer cell proliferation.

Rotary cell culture systems (RCCS) have been shown to be promising for promoting three-dimensional (3D) cell growth and assembly of cells into functional tissues. In this study, 3D tissue-like spheroids of MCF-7 cells were constructed by encapsulating the cells in the collagen-alginate hydrogel, and then cultured in a RCCS to investigate the proliferation of MCF-7 cells. The results from the MTT assay showed that the proliferation rate of MCF-7 cells cultured in the RCCS was higher than that of the static culture control group, and the results from the flow cytometry revealed that the cells in S and G2/M phase were significantly increased compared to the control group. The expression of cell proliferation antigen PCNA and cyclin D1 was also examined with the results further supporting the enhanced proliferation of MCF-7 cells by the RCCS. The results from indirect immunofluorescence revealed that the rotary culture altered neither the cytoskeleton distribution nor the assembly of mitotic spindle. By examination, it was also shown that the rotary culture induced the ERK1/2-MAPK pathway. Taken together, this study demonstrated that the rotary culture could promote the proliferation of MCF-7 cells by inducing the ERK1/2 pathway.

Background Rab GTPases function as modulators in intracellular transport. Rab5a, a member of the Rab subfamily of small GTPases, is an important regulator of vesicle traffic from the plasma membrane to early endosomes. Recent findings have reported that Rab5a gene was involved in the progression of cancer. In the present study, we investigated the effect of Rab5a on cervical cancer invasion and metastasis and the molecular mechanism underlying the involvement of Rab5a. Methods Rab5a expression was assessed by immunohistochemical analysis on a cervical cancer tissue microarray. RNA interference (RNAi) was performed to knock down the endogenous expression of Rab5a gene in HeLa and SiHa cells. Cell motility was evaluated using invasion assay and wound migration assay in vitro. The expression levels of integrin-associated molecules were detected by Western blot and immunofluorescence. Results We found that Rab5a was expressed at a high level in cervical cancer tissues. Silencing of Rab5a expression significantly decreased cancer cell motility and invasiveness. The down-regulation of integrin-associated focal adhesion signaling molecules was further detected in Rab5a knockdown cells. Meanwhile, active GTP-bound Rac1, Cdc42, and RhoA were also down-regulated, accompanied with the reduction in the number and size of filopodia and lamellipodia. Conclusions Taken together, these data suggest that Rab5a functions in regulating the invasion phenotype, and we propose that this regulation may be via integrin-mediated signaling pathway in cervical cancer cells.

Cytotoxic T lymphocyte-associated antigen-4 (CTLA4) A49G is a polymorphism that is extensively studied in various cancers. To investigate whether it is associated with the occurrence of glioma in Chinese patients, we performed a case–control research study with 670 patients and 680 controls. In this group, we found that the genotype at this locus is significantly associated with glioma risk (GG vs. AA: P = 0.045; GG + AG vs. AA: P = 0.013). In some subgroups, G allele carriers are significantly less represented. We also observed significant correlations between the polymorphism genotype and glioma risk in patients with WHO histologic stages. We conclude that CTLA4 A49G might be a potential clinical biomarker for distinguishing persons with a high risk for developing gliomas.

Insulin-like growth factor-I modulates cell growth and survival, and is thought to be important in tumor development. A (CA)19 repeat polymorphism in the promoter region of IGF-I gene that may affect transcription activity has been implicated as a risk factor for cancer, but individual studies have been inconclusive or controversial. Therefore, we performed a meta-analysis of 17 studies with IGF-I (CA)19 repeat genotyping on 8,799 patients and 13,901 controls. There were seven studies with prostate cancer (2,307 cases; 2,622 controls), seven studies with breast cancer (3,533 cases; 7,771 controls), and three studies with colorectal cancer (2,959 cases; 3,508 controls). Overall, the random effects odds ratio (OR) for the (CA)19 versus non-(CA)19 allele was 1.03 [95% confidence interval (CI), 0.95–1.11], with some between-study heterogeneity (P < 0.0001). There was no suggestion of an overall effect either in recessive or dominant modeling of (CA)19 allele effects, and the comparison of (CA)19 homozygosity versus non-(CA)19 homozygosity also showed no differential susceptibility to cancer (OR, 0.99; 95% CI, 0.84–1.16). No effect of (CA)19 was seen in subjects of breast cancer (seven comparisons, OR = 1.03; 95% CI, 0.90–1.17, P = 0.005 for heterogeneity), prostate cancer (seven comparisons, OR = 1.08; 95% CI, 0.88–1.27; P = 0.0002 for heterogeneity) and colorectal cancer (three comparisons, OR = 0.96; 95% CI, 0.89–1.03, P = 0.36, no significant between-study heterogeneity). There was also no evidence that the (CA)19 allele associated with the risk of cancer in Caucasians and Asians. The meta-analysis shows that this (CA)19 repeat polymorphism is unlikely to be a major determinant of susceptibility to cancer on a wide population basis. However, a larger single study is required to further evaluate the association IGF-I (CA)19 polymorphisms and the cancer risk in a specific population.

The genetic polymorphisms in the ABCB1 gene, which encodes for the membrane pump, P-glycoprotein, have been previously demonstrated to have an association with tacrolimus dosing in organ transplant patients. This study associated the haplotype and genotype for ABCB1 G2677T and C3435T variants with a sequential analysis of tacrolimus blood level (ng/mL) per mg/day dosage ([L/D]) administered to 91 adult lung transplant patients at 1, 3, 6, 9, and 12 months after transplantation. Haplotype 22 carriers had a significantly higher tacrolimus [L/D] value in comparison with nonhaplotype 22 carriers (P = .04) only at 1 month after transplant. Sequential analysis demonstrated that ABCB1 genotypes 00 and 01 had low tacrolimus [L/D] values at 1 and 3 months, but these values increased substantially at 6, 9, and 12 months after transplantation. This was not true of the other genotypes with the exception of genotypes 10 and 21, which had small numbers of patients but had consistently low tacrolimus [L/D]. Haplotype analysis also suggested that the homozygous for ABCB1 2677 variant allele had more of an impact on tacrolimus [L/D] in haplotype analysis than that of ABCB1 3435. In conclusion, sequential analysis of tacrolimus [L/D] with haplotypes can explain previous clinical observations of changes in tacrolimus dosage over time but suggests that this effect is limited to individual patient haplotypes. Sequential analysis of drug dosing and haplotypes relationships can provide important information about the induction or inhibition of drug-drug and disease-drug interactions among specific haplotypes.

Persistent rejection in the face of treatment and multiple episodes of rejection are associated with the development of chronic rejection and graft loss in solid organ transplantation. The factors that create an environment for rejection that persists in the face of treatment are as yet not understood. The objective of this study was to evaluate the risk factors, including human multidrug resistance gene ( MDR 1), cytochrome P4503A5 (CYP3A5) and cytokine gene polymorphisms, associated with acute persistent rejection (APR) in lung transplant patients. One hundred and twenty-five adult lung transplant patients were studied. MDR 1 G2677T, C3435T and CYP3A5 polymorphisms were assessed by direct sequencing of the polymorphic region in patient DNA. Cytokine genotyping for five cytokines was performed using the polymerase chain reaction-sequence specific primers (PCR-SSP) technique. Multivariate regression analysis was used to identify the predictors of acute persistent rejection. The dependent variable was the presence or absence of acute persistent rejection based on lung biopsies during the first postoperative year. The independent variables were MDR 1 G2677T and C3435T, CYP4503A5 and cytokine polymorphisms, survival status, age, gender, survival days and HLA mismatches. The MDR 1 C3435T polymorphism and age were independently associated with acute persistent rejection ( p =0.025, odds ratio=0.29, 95% CI 0.1–0.86 and p =0.016, odds ratio=0.94, 95% CI 0.89–0.98, respectively). For the MDR 1 C3435T polymorphism, 72% of patients with the C allele had acute persistent rejection in comparison to 52% for TT patients ( p =0.04). For age, a significant difference was found between the nonrejection group and the rejection group (mean±S.D. 52.1±11.2 vs. 44.4±12.3, p =0.01). This is the first report of the association of a drug disposition genotype with drug-resistant acute rejection in organ transplant patients. The major predictor of acute persistent rejection in the first postoperative year for lung transplant patients was the MDR 1 C3435T genotype. This association could be due to drug resistance, altered drug disposition or other immunologic effects associated with P-glycoprotein (P-gp) function. Future prospective treatment algorithms should be developed that will incorporate the knowledge of gene polymorphisms into treatment regimens to improve the outcome following lung transplantation.

Maize (Zea mays L.) is an important food and energy crop, and low phosphate (Pi) availability is one of the major constraints in maize production worldwide. Plants adapt suitably to acclimate to low Pi stress. However, the underlying molecular mechanism of Pi deficiency response is still unclear. In this study, comparative transcriptomic analyses were conducted to investigate the differences of transcriptional responses in two maize genotypes with different tolerances to low phosphorus (LP) stress. LP-tolerant genotype QXN233 maintained higher P and Pi levels in shoots than LP-sensitive genotype QXH0121 suffering from Pi deficiency at seedling stage. Moreover, the transcriptomic analysis identified a total of 1391 Pi-responsive genes differentially expressed between QXN233 and QXH0121 under LP stress. Among these genes, 468 (321 up- and 147 down-regulated) were identified in leaves, and 923 (626 up- and 297 down-regulated) were identified in roots. These Pi-responsive genes were involved in various metabolic pathways, the biosynthesis of secondary metabolites, ion transport, phytohormone regulation, and other adverse stress responses. Consistent with the differential tolerance to LP stress, five maize inorganic Pi transporter genes were more highly up-regulated in QXN233 than in QXH0121. Results provide important information to further study the changes in global gene expression between LP-tolerant and LP-sensitive maize genotypes and to understand the molecular mechanisms underlying maize's long-term response to Pi deficiency.