Novel dual action PARP and microtubule polymerization inhibitor AMXI-5001 powerfully inhibits growth of esophageal carcinoma both alone and in combination with radiotherapy.

Esophageal cancer is one of the leading causes of cancer deaths globally with an incidence that is concentrated in specific hot spots in Eastern Asia, the Middle East, Eastern Africa, and South America. 10-year overall survival for patients treated with standard of care chemoradiation followed by surgical resection is below 40% highlighting the need for novel therapeutics to treat this disease. We assessed the effect of AMXI-5001, a novel small molecule poly ADP-Ribose polymerase (PARP) inhibitor and microtubule polymerization inhibitor on tumor growth inhibition in both in-vitro and in-vivo murine models. We found that AMXI-5001 was the most potent growth inhibitor of 8 out of 9 different esophageal carcinoma cell lines compared to other clinically available PARP inhibitors, Olaparib, Niraparib, Rucaparib, and Talazoparib. We then confirmed the previously described mechanism of action of AMXI-5001 as a PARP-inhibitor and microtubule polymerization inhibitor using both a PARP trapping assay and immunofluorescence. To further assess AMXI-5001's potential as a therapeutic for esophageal carcinoma we evaluated the effect of AMXI-5001 in combination with standard chemotherapy agents, Cisplatin and 5 Fluorouracil. We showed that AMXI-5001 synergistically inhibits growth in KYSE-70, a squamous esophageal cell line in combination with these drugs. In addition, we found that AMXI-5001 was an effective radiosensitizer, and squamous esophageal carcinoma cell lines treated 24 hours prior to external beam radiation showed significantly more growth inhibition compared to controls. Finally, we assessed the effect of AMXI-5001 monotherapy and in combination with radiotherapy in a xenograft mouse model implanted with subcutaneous KYSE-70 cells. Compared to vehicle control, and those treated with either AMXI-5001 alone or radiation alone, mice treated with both AMXI-5001 and radiation had significant tumor response. In conclusion, AMXI-5001 is an orally bioavailable dual-action PARP and microtubule polymerization inhibitor that holds promise in the treatment of esophageal carcinoma.

Extracellular sulfatases as potential blood-based biomarkers for early detection of lung cancer.

PURPOSE: Non-small lung (NSCLC) is the deadliest cancer, with survival measured in months. Earlier diagnosis using a robust biomarker would likely improve survival. This study aims to determine whether blood levels of the extracellular sulfatases (SULF1 and SULF2) and their bio-activity can serve as novel biomarkers for NSCLC early detection. MATERIALS AND METHODS: Using human plasma specimens from NSCLC patients, nonmalignant COPD patients, and healthy individuals, we determined the association between plasma SULF levels and the presence of NSCLC. We assessed the plasma SULF levels as a function of sex and age. We also evaluated the plasma levels of heparin-binding factors potentially mobilized by the SULFs. To increase test specificity of blood SULF2 as a biomarker for the early diagnosis of NSCLC, we investigated the presence of a tumor-specific SULF2 isoform released in the blood, which could be used as a biomarker alone or in multiplex assays. RESULTS: The median level of plasma SULF2 was significantly elevated in NSCLC patients than in healthy controls (∼2 fold). However, these data were confounded by age. Surprisingly, COPD patients also showed a dramatically increased SULF2 plasma level. We showed a significant increase in the median plasma levels of several HSPG-binding factors in early-stage NSCLC patients compared to controls. Furthermore, we revealed a significant positive correlation of the SULF2 protein level with the plasma levels of two HSPG-binding factors IL6 and IL8. We demonstrated that NSCLC cancer cells and tissues overexpress a SULF2 splice variant. We determined the presence of a SULF2 splice variant form in NSCLC plasma, which was not detectable in COPD and control plasmas. CONCLUSION: Our findings highlight the potential for the plasma levels of SULF2 protein and its bio-activity as novel blood biomarkers for early diagnosis of NSCLC.

Ponatinib is a potential therapeutic approach for malignant pleural mesothelioma.

PURPOSE: Malignant pleural mesothelioma (MPM) is a rare and deadly malignancy. Current MPM therapies remain inadequate, and outcomes are often disappointing. New meaningful therapeutic approaches are urgently needed. Accumulating evidence indicates that the cAbl pathway promotes various tumor-stimulating processes in MPM. In this study, we sought to determine ponatinib's potential utility, a clinically approved and potent cAbl inhibitor, in MPM treatment. MATERIAL AND METHODS: Four MPM lines (MSTO211H, H28, H2452, H2052) were treated with ponatinib in vitro, and their growth was assessed. Scratch wound assay was used to investigate the ponatinib effect on cell migration. The expression levels of pAbl and its downstream effectors pCrkL, pAKT, and pSTAT5 were characterized. The in vivo ponatinib effect was evaluated in human MPM cells derived tumor model. RESULTS: In all four MPM lines, significant expression levels of phosphorylated cAbl/Arg and pCrkl were observed. Differentially but strongly, ponatinib inhibited the in vitro cell growth and migration of all four MPM line. Western blot analysis showed that the activation of Abl signaling was blocked in the ponatinib-treated MMP lines. In keeping, the cellular levels of pAbl and its downstream effector pCrkL, pAKT, and pSTAT5 were markedly decrease following ponatinib treatment. Moreover, ponatinib treatment amplified the levels of γH2AX in cells denoting increased double-strand DNA breaks levels. Notably, ponatinib treatment reduced in vivo tumor growth and reduced pCrkl and pSTAT5 levels in tumor samples. CONCLUSION: Ponatinib may offer a new therapeutic strategy for MPM patients based on cAbl signaling pathway inhibition.

Development of novel monoclonal antibodies and immunoassays for sensitive and specific detection of SULF1 endosulfatase.

BACKGROUND: Cell-surface heparan sulfate proteoglycans (HSPGs) function as receptors or co-receptors for ligand binding and mediate the transmission of critical extracellular signals into cells. The complex and dynamic modifications of heparan sulfates on the core proteins are highly regulated to achieve precise signaling transduction. Extracellular endosulfatase Sulf1 catalyzes the removal of 6-O sulfation from HSPGs and thus regulates signaling mediated by 6-O sulfation on HSPGs. The expression of Sulf1 is altered in many cancers. Further studies are needed to clarify Sulf1 role in tumorigenesis, and new tools that can expand our knowledge in this field are required. METHODS: We have developed and validated novel SULF1 monoclonal antibodies (mAbs). The isotype and subclass for each of these antibodies were determined. These antibodies provide invaluable reagents to assess SULF1- tissue and blood levels by immunohistochemistry and ELISA assays, respectively. RESULTS: This study reports novel mAbs and immunoassays developed for sensitive and specific human Sulf1 protein detection. Using these SULF1 mAbs, we developed an ELISA assay to investigate whether blood-derived SULF1 may be a useful biomarker for detecting cancer early. Furthermore, we have demonstrated the utility of these antibodies for Sulf1 protein detection, localization, and quantification in biospecimens using various immunoassays. CONCLUSIONS: This study describes novel Sulf1 mAbs suitable for various immunoassays, including Western blot analysis, ELISA, and immunohistochemistry, which can help understand Sulf1 pathophysiological role. GENERAL SIGNIFICANCE: New tools to assess and clarify SULF1 role in tumorigenesis are needed. Our novel Sulf1 mAbs and immunoassays assay may have utility for such application.

AMXI-5001, a novel dual parp1/2 and microtubule polymerization inhibitor for the treatment of human cancers.

Poly (ADP-ribose) polymerase (PARP) has recently emerged as a central mediator in cancer resistance against numerous anticancer agents to include chemotherapeutic agents such as microtubule targeting agents and DNA damaging agents. Here, we describe AMXI-5001, a novel, highly potent dual PARP1/2 and microtubule polymerization inhibitor with favorable metabolic stability, oral bioavailability, and pharmacokinetic properties. The potency and selectivity of AMXI-5001 were determined by biochemical assays. Anticancer activity either as a single-agent or in combination with other antitumor agents was evaluated in vitro. In vivo antitumor activity as a single-agent was assessed in a triple-negative breast cancer (TNBC) model. AMXI-5001 demonstrates comparable IC50 inhibition against PARP and microtubule polymerization as clinical PARP inhibitors (Olaparib, Rucaparib, Niraparib, and Talazoparib) and the potent polymerization inhibitor (Vinblastine), respectively. In vitro, AMXI-5001 exhibited selective antitumor cytotoxicity across a wide variety of human cancer cells with much lower IC50s than existing clinical PARP1/2 inhibitors. AMXI-5001 is highly active in both BRCA mutated and wild type cancers. AMXI-5001 is orally bioavailable. AMXI-5001 elicited a remarkable In vivo preclinical anti-tumor activity in a BRCA mutated TNBC model. Oral administration of AMXI-5001 induced complete regression of established tumors, including exceedingly large tumors. AMXI-5001 resulted in superior anti-tumor effects compared to either single agent (PARP or microtubule) inhibitor or combination with both agents. AMXI-5001 will enter clinical trial testing soon and represents a promising, novel first in class dual PARP1/2 and microtubule polymerization inhibitor that delivers continuous and synchronous one-two punch cancer therapy with one molecule.

Silicone elastomer gel impregnated with 20(S)-protopanaxadiol-loaded nanostructured lipid carriers for ordered diabetic ulcer recovery.

Inefficient diabetic ulcer healing and scar formation remain a challenge worldwide, owing to a series of disordered and dynamic biological events that occur during the process of healing. A functional wound dressing that is capable of promoting ordered diabetic wound recovery is eagerly anticipated. In this study, we designed a silicone elastomer with embedded 20(S)-protopanaxadiol-loaded nanostructured lipid carriers (PPD-NS) to achieve ordered recovery in scarless diabetic ulcer healing. The nanostructured lipid carriers were prepared through an emulsion evaporation-solidification method and then incorporated into a network of silicone elastomer to form a unique nanostructured lipid carrier-enriched gel formulation. Interestingly, the PPD-NS showed excellent in vitro anti-inflammatory and proangiogenic activity. Moreover, in diabetic mice with full-thickness skin excision wound, treatment with PPD-NS significantly promoted in vivo scarless wound healing through suppressing inflammatory infiltration in the inflammatory phase, promoting angiogenesis during the proliferation phase, and regulating collagen deposition in the remodeling phase. Hence, this study demonstrates that the developed PPD-NS could facilitate ordered diabetic wound recovery via multifunctional improvement during different wound-healing phases. This novel approach could be promising for scarless diabetic wound healing.

Eu2+/Eu3+-Based Smart Duplicate Responsive Stimuli and Time-gated Nanohybrid for Optical Recording and Encryption.

With the rapid development of information science, it is urgent that memory devices possessing high security, density, and desirable storage ability should be developed. In this work, a smart duplicate response of stimuli has been developed and a time-gate nanohybrid based on variable valence Eu2+/Eu3+ coencapsulated has been fabricated and acts as active material in the multilevel and multidimensional memory devices. The luminescence lifetime of Eu3+ in this nanohybrid gave a stimuli response due to which the energy level of the coordinated ligand could be modulated. Furthermore, by a simple sintering procedure, Eu3+ was partially in situ reduced to Eu2+ with a short lifetime in the system. And the in situ reduction ensured both Eu3+ and Eu2+ ions' uniform distribution in the nanohybrid and simultaneous response upon light excitation of variable valence Eu ions. Interestingly, Eu3+ revealed a prolonged lifetime because of the presence of an energy-transfer effect of Eu2+ → Eu3+. Such a nanohybrid had abundant luminescent properties, including the short lifetime of Eu2+, the energy transfer from the Eu2+ to Eu3+ ions, and the stimuli response of the Eu3+ lifetimes when exposed to acidic or basic vapor, thus giving birth to interesting recording and encryption performance in spatial-temporal dimensions. We believe that this research will point out a new direction for the future development of multilevel and multidimensional optical recording and encryption materials.

[Constraint priority list-based multi-objective optimization for intensity-modulated radiation therapy].

OBJECTIVE: In intensity-modulated radiation therapy (IMRT), it is time-consuming to repeatedly adjust the objectives manually to obtain the best tradeoff between the prescribed dose of the planning target volume and sparing the organs-at-risk. Here we propose a new method to realize automatic multi-objective IMRT optimization, which quantifies the clinical preferences into the constraint priority list and adjusts the dose constraints based on the list to obtain the optimal solutions under the dose constraints. This method contains automatic adjustment mechanism of the dose constraint and automatic voxel weighting factor-based FMO model. Every time the dose constraint is adjusted, the voxel weighting factor-based FMO model is launched to find a global optimal solution that satisfied the current constraints. We tested the feasibility and effectiveness of this method in 6 cases of cervical cancer with IMRT by comparing the original plan and the automatic optimization plan generated by this method. The results showed that with the same PTV coverage and uniformity, the automatic optimization plan had a better a dose sparing of the organs-at-risk and a better plan quality than the original plan, and resulted in obvious reductions of the average V45 of the rectum from (41.99∓13.31)% to (32.55∓22.27)% and of the bladder from (44.37∓4.08)% to (28.99∓15.25)%.

Efficacy and Safety of Chemotherapy Combined with Stereotactic Radiotherapy in the Treatment of Nasopharyngeal Carcinoma.

BACKGROUND The aim of this study was to investigate the efficacy and safety of chemotherapy (CT) combined with stereotactic radiotherapy (SRT) in the treatment of nasopharyngeal carcinoma (NPC). MATERIAL AND METHODS A total of 329 NPC patients without any previous treatment were included in this study between January 2009 and November 2013. These patients were divided into three groups: CT group (n=114), SRT group (n=109), and CT + SRT group (n=106). Contrast-enhanced nasopharyngeal computed tomography (CT)/magnetic resonance (MR) scan was performed on the third month after treatment. Short-term efficacy was evaluated according to the Response Evaluation Criteria in Solid Tumors (RECIST). Toxicity was graded according to the Acute Radiation Morbidity Scoring Criteria (RTOG) and the World Health Organization (WHO) toxicity grading scale. Overall survival (OS), progression free survival (PFS), and incidence rate of acute toxicity (grade ≥3) were calculated after a 24 month follow-up. RESULTS Total response rate of all patients was 85.41%. Compared with the CT group and the SRT group, the CT + SRT group showed a substantially improved efficacy in NPC treatment. The incidence rate of the acute toxicity in the CT + SRT group was slightly higher than in the CT group and the SRT group, but the difference was not statistically significant. No treatment-related deaths were observed. The CT + SRT group had the highest two-year OS and PFS, followed by the CT group and the SRT group. CONCLUSIONS It was shown that NPC patients treated with CT + SRT had better short- and long-term efficacy than those treated with CT or SRT alone.

Replacing process water and nitrogen sources with biogas slurry during cellulosic ethanol production.

BACKGROUND: Environmental issues, such as the fossil energy crisis, have resulted in increased public attention to use bioethanol as an alternative renewable energy. For ethanol production, water and nutrient consumption has become increasingly important factors being considered by the bioethanol industry as reducing the consumption of these resources would decrease the overall cost of ethanol production. Biogas slurry contains not only large amounts of wastewater, but also the nutrients required for microbial growth, e.g., nitrogen, ammonia, phosphate, and potassium. Therefore, biogas slurry is an attractive potential resource for bioethanol production that could serve as an alternative to process water and nitrogen sources. RESULTS: In this study, we propose a method that replaces the process water and nitrogen sources needed for cellulosic ethanol production by Zymomonas mobilis with biogas slurry. To test the efficacy of these methods, corn straw degradation following pretreatment with diluted NaOH and enzymatic hydrolysis in the absence of fresh water was evaluated. Then, ethanol fermentation using the ethanologenic bacterial strain Z. mobilis ZMT2 was conducted without supplementing with additional nitrogen sources. After pretreatment with 1.34% NaOH (w/v) diluted in 100% biogas slurry and continuous enzymatic hydrolysis for 144 h, 29.19 g/L glucose and 12.76 g/L xylose were generated from 30 g dry corn straw. The maximum ethanol concentration acquired was 13.75 g/L, which was a yield of 72.63% ethanol from the hydrolysate medium. Nearly 94.87% of the ammonia nitrogen was depleted and no nitrate nitrogen remained after ethanol fermentation. The use of biogas slurry as an alternative to process water and nitrogen sources may decrease the cost of cellulosic ethanol production by 10.0-20.0%. By combining pretreatment with NaOH diluted in biogas slurry, enzymatic hydrolysis, and ethanol fermentation, 56.3 kg of ethanol was produced by Z. mobilis ZMT-2 through fermentation of 1000 kg of dried corn straw. CONCLUSIONS: In this study, biogas slurry replaced process water and nitrogen sources during cellulosic ethanol production. The results suggest that biogas slurry is a potential alternative to water when pretreating corn straw and, thus, has important potential applications in cellulosic ethanol production from corn straw. This study not only provides a novel method for utilizing biogas slurry, but also demonstrates a means of reducing the overall cost of cellulosic ethanol.

Numb Gene Enhances Radiation Sensitivity of Nonsmall Cell Lung Cancer Stem Cells.

OBJECTIVE: To study the effects of Numb gene expression on radiation sensitivity of nonsmall cell lung cancer (NSCLC) stem cells. MATERIALS AND METHODS: The side population (SP) cells A549-SP were transfected with pcDNA3.1 (pcDNA3.1 group), pcDNA-Numb (pcDNA-Numb group) and shRNA-Numb (shRNA-Numb group). Real-time quantitative polymerase chain reaction and Western blot were performed to determine Numb expression; MTT method was used to measure the proliferation activity change of the NSCLC stem cells both before and after irradiation with different doses of 60Coγ ray; Hoechst staining and Annexin V-FITC/PI were used to detect the apoptosis of the NSCLC stem cells; and colony-forming assay was used to determine the effect of Numb expression on radiation sensitivity of the NSCLC stem cells. RESULTS: Increased mRNA and protein expressions of the A549-SP cells were found in the pcDNA-Numb group, and decreased mRNA and protein expressions were found in the shRNA-Numb group. The optical density value of the cells decreased in the pcDNA-Numb group but increased in the shRNA-Numb group. The cells with over-expressed Numb showed obvious nuclear condensation and fragmentation; the apoptosis rate increased significantly. The cells with knockdown Numb showed less nuclear damage; the apoptosis rate significantly decreased. After irradiation, the cells in the pcDNA-Numb group showed decreased survival rate, clonality, and the values of D0, Dq, N, and SF2; whereas the cells in the shRNA-Numb group showed the opposite trend. CONCLUSIONS: Radiation sensitivity of NSCLC stem cells was enhanced with the increase of Numb expression. Determination of Numb expression helped to evaluate the response of lung cancer to radiotherapy, which was important for guiding tumor treatment clinically.

SULF2 Expression Is a Potential Diagnostic and Prognostic Marker in Lung Cancer.

AIMS: Lung cancer is one of the most deadly cancers; median survival from diagnosis is less than one year in those with advanced disease. Novel lung cancer biomarkers are desperately needed. In this study, we evaluated SULF2 expression by immunohistochemistry and its association with overall survival in a cohort of patients with non-small cell lung cancer (NSCLC). We also looked for the presence of SULF2 protein in plasma to evaluate its potential as an early detection biomarker for NSCLC. METHODS: We identified patients who underwent surgical resection for pulmonary adenocarcinoma or squamous cell carcinoma at our institution. A section from each paraffin-embedded specimen was stained with a SULF2 antibody. A pathologist determined the percentage and intensity of tumor cell staining. Survival analysis was performed using a multivariate Cox proportional hazards model. Using a novel SULF2 ELISA assay, we analyzed plasma levels of SULF2 in a small cohort of healthy donors and patients with early stage NSCLC. RESULTS: SULF2 staining was present in 82% of the lung cancer samples. Squamous cell carcinomas had a higher mean percentage of staining than adenocarcinomas (100% vs. 60%; p<0.0005). After adjusting for age, sex, race, histologic type, stage, and neoadjuvant therapy, there was a non-significant (31%; p = 0.65) increase in the risk of death for patients with adenocarcinoma with SULF2 staining in tumor cells. In contrast, there was a significant decrease in the risk of death (89%; p = 0.02) for patients with squamous cell carcinoma with SULF2 staining in tumor cells. SULF2 protein was present in plasma of patients with early stage NSCLC, and soluble SULF2 levels increased with age. Finally, plasma SULF2 levels were significantly elevated in early stage NSCLC patients, compared to healthy controls. CONCLUSIONS: Tumor expression of SULF2 may affect prognosis in NSCLC, while blood SULF2 levels may have a significant role in the diagnosis of this fatal disease.

Lung cancer: Biology and treatment options.

Lung cancer remains the leading cause of cancer mortality in men and women in the U.S. and worldwide. About 90% of lung cancer cases are caused by smoking and the use of tobacco products. However, other factors such as radon gas, asbestos, air pollution exposures, and chronic infections can contribute to lung carcinogenesis. In addition, multiple inherited and acquired mechanisms of susceptibility to lung cancer have been proposed. Lung cancer is divided into two broad histologic classes, which grow and spread differently: small-cell lung carcinomas (SCLCs) and non-small cell lung carcinomas (NSCLCs). Treatment options for lung cancer include surgery, radiation therapy, chemotherapy, and targeted therapy. Therapeutic-modalities recommendations depend on several factors, including the type and stage of cancer. Despite the improvements in diagnosis and therapy made during the past 25 years, the prognosis for patients with lung cancer is still unsatisfactory. The responses to current standard therapies are poor except for the most localized cancers. However, a better understanding of the biology pertinent to these challenging malignancies, might lead to the development of more efficacious and perhaps more specific drugs. The purpose of this review is to summarize the recent developments in lung cancer biology and its therapeutic strategies, and discuss the latest treatment advances including therapies currently under clinical investigation.

Glycosylation alterations in lung and brain cancer.

Alterations in glycosylation are common in cancer and are thought to contribute to disease. Lung cancer and primary malignant brain cancer, most commonly glioblastoma, are genetically heterogeneous diseases with extremely poor prognoses. In this review, we summarize the data demonstrating that glycosylation is altered in lung and brain cancer. We then use specific examples to highlight the diverse roles of glycosylation in these two deadly diseases and illustrate shared mechanisms of oncogenesis. In addition to alterations in glycoconjugate biosynthesis, we also discuss mechanisms of postsynthetic glycan modification in cancer. We suggest that alterations in glycosylation in lung and brain cancer provide novel tumor biomarkers and therapeutic targets.

Fast incorporation of primary amine group into polylactide surface for improving C2C12 cell proliferation using nitrogen-based atmospheric-pressure plasma jets.

In this article, we report the development of the fast incorporation of primary amine functional groups into a polylactide (PLA) surface using the post-discharge jet region of an atmospheric-pressure nitrogen-based dielectric barrier discharge (DBD). Plasma treatments were carried out in two sequential steps: (1) nitrogen with 0.1% oxygen addition, and (2) nitrogen with 5% ammonia addition. The analyses show that the concentration of N/C ratio, surface energy, contact angle, and surface roughness of the treated PLA surface can reach 19.1%, 70.5 mJ/m(2), 38° and 73.22 nm, respectively. In addition, the proposed two-step plasma treatment procedure can produce a PLA surface exhibiting almost the same C2C12 cell attachment and proliferation performance as that of the conventional gelatin coating method. Most importantly, the processing/preparation time is reduced from 13-15 h (gelatin coating method) to 5-15 min (two-step plasma treatment), which is very useful in practical applications.

A high-confidence reference dataset of differentially expressed proteins in elongating cotton fiber cells.

In our previous study, we used a comparative proteomic approach based on 2DE to profile dynamic proteomes of cotton fibers and found 235 protein spots differentially expressed during the elongation process ranging from 5 to 25 days post-anthesis. Of them, only 106 differentially expressed proteins (DEPs) were identified by MS due to database limitations at the time. In the present work, we successfully identified the remaining 129 DEPs from the same experimental system using high-resolution MS with an updated database. Bioinformatic analysis revealed that proteins involved in carbohydrate and protein metabolism, transport, and redox homeostasis are the most abundant, and glycolysis was found to be the most significantly regulated process during fiber elongation. Our high-confidence reference dataset, composed of 235 DEPs, provides a valuable resource for future studies on the molecular mechanism of cotton fiber elongation.

Transferring isolated mitochondria into tissue culture cells.

We have developed a new method for introducing large numbers of isolated mitochondria into tissue culture cells. Direct microinjection of mitochondria into typical mammalian cells has been found to be impractical due to the large size of mitochondria relative to microinjection needles. To circumvent this problem, we inject isolated mitochondria through appropriately sized microinjection needles into rodent oocytes or single-cell embryos, which are much larger than tissue culture cells, and then withdraw a 'mitocytoplast' cell fragment containing the injected mitochondria using a modified holding needle. These mitocytoplasts are then fused to recipient cells through viral-mediated membrane fusion and the injected mitochondria are transferred into the cytoplasm of the tissue culture cell. Since mouse oocytes contain large numbers of mouse mitochondria that repopulate recipient mouse cells along with the injected mitochondria, we used either gerbil single-cell embryos or rat oocytes to package injected mouse mitochondria. We found that the gerbil mitochondrial DNA (mtDNA) is not maintained in recipient rho0 mouse cells and that rat mtDNA initially replicated but was soon completely replaced by the injected mouse mtDNA, and so with both procedures mouse cells homoplasmic for the mouse mtDNA in the injected mitochondria were obtained.

PCR-based cloning of the complete mouse mitochondrial genome and stable engineering in Escherichia coli.

We have devised a method for cloning an entire mammalian mitochondrial genome (mtDNA) in Escherichia coli using PCR-based amplification and sequential ligation. Here we test this approach by cloning the complete mouse mtDNA. The mtDNA was divided into four to five fragments based on unique restriction enzyme sites and amplified by high-fidelity long-range DNA polymerase. The synthesized fragments were cloned individually to test their toxicity in the E. coli host and then combined sequentially into a vector containing the E. coli R6K origin of DNA replication. The synthetic complete mouse mtDNA clones were replicated stably and faithfully in E. coli when maintained at moderately low copy numbers per cell. The sequence integrity of the synthetic mouse mtDNA clones was confirmed by nucleotide sequencing; no mutations or rearrangements in the genome were found. This approach can facilitate the cloning of entire mammalian mitochondrial genomes in E. coli and assist in the introduction of desired modifications into the mitochondrial genome.

Comparative proteomic analysis provides new insights into the fiber elongating process in cotton.

A comparative proteomic analysis was performed to explore the mechanism of cell elongation in developing cotton fibers. The temporal changes of global proteomes at five representative development stages (5-25 days post-anthesis [dpa]) were examined using 2-D electrophoresis. Among approximately 1800 stained protein spots reproducibly detected on each gel, 235 spots were differentially expressed with significant dynamics in elongating fibers. Of these, 120 spots showed a more than 2-fold change in at least one stage point, and 21 spots appeared to be specific to developmental stages. Furthermore, 106 differentially expressed proteins were identified from mass spectrometry to match 66 unique protein species. These proteins involve different cellular and metabolic processes with obvious functional tendencies toward energy/carbohydrate metabolism, protein turnover, cytoskeleton dynamics, cellular responses and redox homeostasis, indicating a good correlation between development-dependent proteins and fiber biochemical processes, as well as morphogenesis. Newly identified proteins such as phospholipase D alpha, vf14-3-3 protein, small ras-related protein, and GDP dissociation inhibitor will advance our knowledge of the complicated regulatory network. Identification of these proteins, combined with their changes in abundance, provides a global view of the development-dependent protein changes in cotton fibers, and offers a framework for further functional research of target proteins associated with fiber development.

Proteomic response of rice seedling leaves to elevated CO2 levels.

Previous investigations of plant responses to higher CO 2 levels were mostly based on physiological measurements and biochemical assays. In this study, a proteomic approach was employed to investigate plant response to higher CO 2 levels using rice as a model. Ten-day-old seedlings were progressively exposed to 760 ppm, 1140 ppm, and 1520 ppm CO 2 concentrations for 24 h each. The net photosynthesis rate ( P n), stomatal conductance ( G s), transpiration rate ( E), and intercellular to ambient CO 2 concentration ratio ( C i/ C a) were measured. P n, G s, and E showed a maximum increase at 1140 ppm CO 2, but further exposure to 1520 ppm for 24 h resulted in down regulation of these. Proteins extracted from leaves were subjected to 2-DE analysis, and 57 spots showing differential expression patterns, as detected by profile analysis, were identified by MALDI-TOF/TOF-MS. Most of the proteins belonged to photosynthesis, carbon metabolism, and energy pathways. Several molecular chaperones and ascorbate peroxidase were also found to respond to higher CO 2 levels. Concomitant with the down regulation of P n and G s, the levels of enzymes of the regeneration phase of the Calvin cycle were decreased. Correlations between the protein profiles and the photosynthetic measurements at the three CO 2 levels were explored.