Search

Description of supplementary methods for some general experimental procedures used for this study

Population doublings of nontransduced (NT), CAR19 and CAR19.αPD1 T cells upon antigen-specific stimulation for 3 days (n = 3, mean {plus minus} SEM; **P

Purpose: Despite favorable responses of chimeric antigen receptor (CAR)-engineered T-cell therapy in patients with hematologic malignancies, the outcome has been far from satisfactory in the treatment of solid tumors, partially owing to the development of an immunosuppressive tumor microenvironment. To overcome this limitation, we engineered CAR T cells secreting checkpoint inhibitors (CPI) targeting PD-1 (CAR.αPD1-T) and evaluated their efficacy in a human lung carcinoma xenograft mouse model.Experimental Design: To evaluate the effector function and expansion capacity of CAR.αPD1-T cells in vitro, we measured the production of IFNγ and T-cell proliferation following antigen-specific stimulation. Furthermore, the antitumor efficacy of CAR.αPD1-T cells, CAR T cells, and CAR T cells combined with anti–PD-1 antibody was determined using a xenograft mouse model. Finally, the underlying mechanism was investigated by analyzing the expansion and functional capacity of TILs.Results: Human anti–PD-1 CPIs secreted by CAR.αPD1-T cells efficiently bound to PD-1 and reversed the inhibitory effect of PD-1/PD-L1 interaction on T-cell function. PD-1 blockade by continuously secreted anti–PD-1 attenuated the inhibitory T-cell signaling and enhanced T-cell expansion and effector function both in vitro and in vivo. In the xenograft mouse model, we demonstrated that the secretion of anti–PD-1 enhanced the antitumor activity of CAR T cells and prolonged overall survival.Conclusions: With constitutive anti–PD-1 secretion, CAR.αPD1-T cells are more functional and expandable, and more efficient at tumor eradication than parental CAR T cells. Collectively, our study presents an important and novel strategy that enables CAR T cells to achieve better antitumor immunity, especially in the treatment of solid tumors. Clin Cancer Res; 23(22); 6982–92. ©2017 AACR.

We study the impact of transparency on stock price informativeness by using an exogenous change in China: the comply-or-explain dividend regulation. Employing a difference-in-difference (DID) desig...

Background: Although there has been some success using DNA barcoding to authenticate raw natural health product (NHP) botanical ingredients, there are many gaps in our understanding of DNA degradation, which may explain low PCR and sequencing success in processed NHPs. Objective: In this study, we measured multiple DNA variables after each step in the processing of a green tea extract in order to document DNA quality and quantity. Methods: We sampled plant material after each step of green tea extract processing: five steps at a Chinese tea farm (n = 10) and five at an NHP processing facility (n = 3). We hypothesized that processing treatments degrade and remove DNA from NHPs, reflected by decreasing quantities of extractable genomic DNA (gDNA), an increasing proportion of small DNA fragments in genomic extracts, and decreasing quantitative PCR (QPCR) efficiency [higher cycle threshold (Ct) values]. DNA from end-production green tea extract was sequenced in order to try to validate material as the botanical of interest. Results: We saw a 41.1% decrease in mean extractable gDNA through farm processing (P < 0.01) and a 99.7% decrease through facility processing (P < 0.05). There was a 26.3% decrease in mean DNA fragment size through farm processing (P < 0.001) and an 82.0% decrease through facility processing (P < 0.05). QPCR efficiency was reduced through processing, marked by significant increases in Ct values with 100 base pair (bp) and 200 bp PCR targets (P < 0.05), and an inability to amplify 300 bp targets when using DNA template from end-production green tea extract. Conclusions: Although there was significant degradation and removal of DNA through processing, sufficiently intact DNA was able to be recovered from highly processed green tea extract for further sequencing and identification. Highlights: This work addresses a key gap in the understanding of DNA degradation through processing and provides useful information to consider when designing molecular diagnostic techniques for NHP identification.

Background: Although there has been some success using DNA barcoding to authenticate raw natural health product (NHP) botanical ingredients, there are many gaps in our understanding of DNA degradation, which may explain low PCR and sequencing success in processed NHPs. Objective: In this study, we measured multiple DNA variables after each step in the processing of a green tea extract in order to document DNA quality and quantity. Methods: We sampled plant material after each step of green tea extract processing: five steps at a Chinese tea farm (n = 10) and five at an NHP processing facility (n = 3). We hypothesized that processing treatments degrade and remove DNA from NHPs, reflected by decreasing quantities of extractable genomic DNA (gDNA), an increasing proportion of small DNA fragments in genomic extracts, and decreasing quantitative PCR (QPCR) efficiency [higher cycle threshold (Ct) values]. DNA from end-production green tea extract was sequenced in order to try to validate material as the botanical of interest. Results: We saw a 41.1% decrease in mean extractable gDNA through farm processing (P < 0.01) and a 99.7% decrease through facility processing (P < 0.05). There was a 26.3% decrease in mean DNA fragment size through farm processing (P < 0.001) and an 82.0% decrease through facility processing (P < 0.05). QPCR efficiency was reduced through processing, marked by significant increases in Ct values with 100 base pair (bp) and 200 bp PCR targets (P < 0.05), and an inability to amplify 300 bp targets when using DNA template from end-production green tea extract. Conclusions: Although there was significant degradation and removal of DNA through processing, sufficiently intact DNA was able to be recovered from highly processed green tea extract for further sequencing and identification. Highlights: This work addresses a key gap in the understanding of DNA degradation through processing and provides useful information to consider when designing molecular diagnostic techniques for NHP identification.

Background: A requirement of current good manufacturing practices for dietary supplements is that manufacturers must identify their dietary ingredients. DNA-based methods can provide species-level authentication that may sometimes be difficult to achieve using conventional morphological and chemical analysis methods. However, because of varying levels of DNA degradation in botanical materials, many commercial tests fail to generate consistent test results across all types of botanical materials. AOAC published guidelines for validation of botanical identification methods and proposed probability of identification (POI) as a method performance parameter. However, few DNA-based botanical authentication methods in the literature follow these guidelines and evaluate POI. Objective: To provide a targeted PCR method validation example that follows AOAC guidelines for validation of botanical identification methods. Methods: Using Matricaria chamomilla (chamomile) as an example, we performed a single-laboratory validation for a targeted PCR method that aimed to identify both raw and processed chamomile materials. The performance parameters of the test were evaluated by carrying out an inclusivity/exclusivity study and a Specified Superior Test Material/Specified Inferior Test Material study to demonstrate that the method’s POI meets industry requirements. Results: The chamomile samples were identified by the method and achieved a POI greater than 0.9 with respect to all types of chamomile botanical materials. Conclusions: The method was validated for DNA-based identification of raw and processed chamomile materials, such as sterilized powders and extracts. Highlights: This work will provide insight for laboratories and manufacturers that aim to develop and validate DNA-based botanical identification methods.

Authentication of Panax ginseng and Panax quinquefolius products is important to be able to mitigate instances of adulteration and substitution that exist within the international supply chain of ginseng. To address this issue, species-specific hydrolysis probe qPCR assays were developed and validated for both P. ginseng and P. quinquefolius herbal dietary supplements. Performance of the probe-based assays was evaluated using analytical validation criteria, which included evaluation of: (1) specificity, in selectively identifying the target species, (2) sensitivity, in detecting the lowest amount of the target material, and (3) repeatability and reproducibility of the method in detecting the target species in raw materials on a real-time PCR platform (reliability). The species-specific probes were developed and successfully passed the validation criteria with 100% specificity, 80–120% efficiency and 100% reliability. The methods developed in this study are fit for purpose, rapid, and easy to implement in quality assurance programs, authentication of ginseng herbal supplements is possible, even with extracts where DNA is fragmented and of low quality and quantity.

When testing botanical ingredients of herbal medicines and dietary supplements, the complexity of botanical matrixes often requires the use of orthogonal methods to establish identification procedures suitable for quality control purposes. Genomic-based botanical identification methods are evolving and emerging as useful quality control tools to complement traditional morphological and chemical identification methods. Species-specific polymerase chain reaction methods are being evaluated for botanical quality control and as a cost-effective approach to identify and discriminate between closely related botanical species. This paper describes orthogonal identification of Panax ginseng, P. quinquefolius, and P. notoginseng materials in commerce as an example of the development and validation of a set of species-specific polymerase chain reaction methods to establish botanical identity in ginseng roots. This work also explored the possibility of extending the application of species-specific polymerase chain reaction methods to provide species identity information for processed materials, such as steamed roots and hydroalcoholic extracts, and showed success with this approach. Finally, the paper provides recommendations for an out-of-specification investigation of samples that may pass some of the orthogonal tests and fail others.

Several botanicals have been traditionally used as protein sources, including the leguminous Pisum sativum L. and Glycine max (L.) Merr. While a rich history exists of cultivating these plants for their whole, protein-rich grain, modern use as powdered supplements present a new challenge in material authentication. The absence of clear morphological identifiers of an intact plant and the existence of long, complex supply chains behoove industry to create quick, reliable analytical tools to identify the botanical source of a protein product (many of which contain multiple sources). The utility of molecular tools for plant-based protein powder authentication is gaining traction, but few validated tools exist. Multiplex quantitative polymerase chain reaction (qPCR) can provide an economical means by which sources can be identified and relative proportions quantified. We followed established guidelines for the design, optimization, and validation of qPCR assay, and developed a triplex qPCR assay that can amplify and quantify pea and soy DNA targets, normalized by a calibrator. The assay was evaluated for analytical specificity, analytical sensitivity, efficiency, precision, dynamic range, repeatability, and reproducibility. We tested the quantitative ability of the assay using pea and soy DNA mixtures, finding exceptional quantitative linearity for both targets – 0.9983 (p < 0.0001) for soy and 0.9915 (p < 0.0001) for pea. Ratios based on mass of protein powder were also tested, resulting in non-linear patterns in data that suggested the requirement of further sample preparation optimization or algorithmic correction. Variation in fragment size within different lots of commercial protein powder samples was also analyzed, revealing low SD among lots. Ultimately, this study demonstrated the utility of qPCR in the context of protein powder mixtures and highlighted key considerations to take into account for commercial implementation.

Background: The applications of deoxyribonucleic acid (DNA) barcoding methods have been extended from authenticating taxonomic provenance of animal products to identifying botanicals used as herbal medicine and in botanical dietary supplements. DNA barcoding methods for botanical identification must be adequately validated to meet regulatory compliance. Objective: The goal of this study is to provide a validation protocol for a two-tiered DNA barcoding method that aims to identify raw botanicals. Methods: A barcode database was computationally validated to define the barcode combinations that can unambiguously identify botanicals in the database. A maximum variation sampling technique was used to capture a wide range of perspectives relating to DNA barcode-based botanical identification, including plant parts and species distance, for the experimental validation. Twenty-two authenticated botanicals were purposively sampled from different plant parts—covering both closely related and distantly related species—to validate the two-tiered DNA barcoding method. The performance of the method was assessed on accuracy, precision, ruggedness, and uncertainty. Results: High accuracy (100%) and precision (1.0) were obtained from the validation samples. The method was also found to be rugged and have acceptable uncertainty. Conclusions: The method was validated and suitable for DNA-based identification of botanical raw materials listed in the current database. Highlights: This work will provide support guidance for manufacturers and regulatory policy makers to implement equivalent validated and compliant DNA-based testing in quality control processes to improve botanical raw material identification and authentication.

Quality control in botanical products begins with the raw material supply. Traditionally, botanical identification is performed through morphological assessment and chemical analytical methods. However, the lack of availability of botanists, especially in recent years, coupled with the need to enhance quality control to combat the stresses on the supply chain brought by increasing consumer demand and climate change, necessitates alternative approaches. The goal of this protocol is to facilitate botanical species identification using a portable qPCR system on the field or in any setting, where access to laboratory equipment and expertise is limited. Target DNA is amplified using dye-based qPCR, with DNA extracted from botanical reference materials serving as a positive control. The target DNA is identified by its specific amplification and matching its melting peak against the positive control. A detailed description of the steps and parameters, from hands-on field sample collection, to DNA extraction, PCR amplification, followed by data interpretation, has been included to ensure that readers can replicate this protocol. The results produced align with traditional laboratory botanical identification methods. The protocol is easy to perform and cost-effective, enabling quality testing on raw materials as close to the point of origin of the supply chain as possible.

Quality control in botanical products begins with the raw material supply. Traditionally, botanical identification is performed through morphological assessment and chemical analytical methods. However, the lack of availability of botanists, especially in recent years, coupled with the need to enhance quality control to combat the stresses on the supply chain brought by increasing consumer demand and climate change, necessitates alternative approaches. The goal of this protocol is to facilitate botanical species identification using a portable qPCR system on the field or in any setting, where access to laboratory equipment and expertise is limited. Target DNA is amplified using dye-based qPCR, with DNA extracted from botanical reference materials serving as a positive control. The target DNA is identified by its specific amplification and matching its melting peak against the positive control. A detailed description of the steps and parameters, from hands-on field sample collection, to DNA extraction, PCR amplification, followed by data interpretation, has been included to ensure that readers can replicate this protocol. The results produced align with traditional laboratory botanical identification methods. The protocol is easy to perform and cost-effective, enabling quality testing on raw materials as close to the point of origin of the supply chain as possible.

Parsley (Petroselinum crispum) leaf is an herb widely consumed for its health benefits. Due to similar morphological and chemical profiles, celery leaf may be a source of substitution in commercial parsley materials. In order to detect this substitution, the present work characterized parsley and celery leaf at the cultivar level by physical, chemical and DNA approaches. In contrast to the variations observed in physical appearances and chemical profiles that make verification of authenticity difficult, consistent differences observed between their DNA sequences are suitable for verifying parsley material authenticity. To identify parsley and detect celery simultaneously, a multiplex qPCR assay was developed and validated with respect to efficiency and specificity. Further testing indicated the assay can be used to detect 1% (w/w) celery in parsley materials with a probability of detection greater than 0.9. The developed method is well-suited for routine quality control to prevent parsley material misidentification in commercial trade.

Based on the relevant theories of corporate governance and the special institutional background of Chinese state-owned enterprises (SOEs), this paper systematically reviews the literature on the independence and governance effect of SOE boards. We find that the governance effect of SOE boards is driven by the dual characteristics of SOEs: state involvement in ownership and market incentives. With the state involved in ownership, SOEs adhere to the leadership of the Communist Party of China (CPC), which results in an enhanced governance effect. Under market incentives, SOEs tend to have an optimal board structure that helps mitigate both the shareholder–management agency problem (Type I agency problem) and the controlling shareholder–minority shareholder agency problem (Type II agency problem). In terms of the governance effect of boards, directors appointed by non-controlling shareholders are effective in alleviating Type I and Type II agency problems, and this highlights the importance of mixed-ownership reforms in SOEs. Independent directors, especially those with a professional background, also play a role in improving corporate governance. However, independent directors in SOEs have relatively weak incentives to monitor, which limits their governance effect. This paper shows positive implications for promoting mixed-ownership reforms and improving board governance in SOEs.

DNA-based methods have been gaining recognition as a tool for botanical authentication in herbal medicine; however, their application in processed botanical materials is challenging due to the low quality and quantity of DNA left after extensive manufacturing processes. The low amount of DNA recovered from processed materials, especially extracts, is “invisible” by current technology, which has casted doubt on the presence of amplifiable botanical DNA. A method using adapter-ligation and PCR amplification was successfully applied to visualize the “invisible” DNA in botanical extracts. The size of the “invisible” DNA fragments in botanical extracts was around 20–220 bp compared to fragments of around 600 bp for the more easily visualized DNA in botanical powders. This technique is the first to allow characterization and visualization of small fragments of DNA in processed botanical materials and will provide key information to guide the development of appropriate DNA-based botanical authentication methods in the future.

Purpose: Despite favorable responses of chimeric antigen receptor (CAR)-engineered T-cell therapy in patients with hematologic malignancies, the outcome has been far from satisfactory in the treatment of solid tumors, partially owing to the development of an immunosuppressive tumor microenvironment. To overcome this limitation, we engineered CAR T cells secreting checkpoint inhibitors (CPI) targeting PD-1 (CAR.αPD1-T) and evaluated their efficacy in a human lung carcinoma xenograft mouse model. Experimental Design: To evaluate the effector function and expansion capacity of CAR.αPD1-T cells in vitro, we measured the production of IFNγ and T-cell proliferation following antigen-specific stimulation. Furthermore, the antitumor efficacy of CAR.αPD1-T cells, CAR T cells, and CAR T cells combined with anti–PD-1 antibody was determined using a xenograft mouse model. Finally, the underlying mechanism was investigated by analyzing the expansion and functional capacity of TILs. Results: Human anti–PD-1 CPIs secreted by CAR.αPD1-T cells efficiently bound to PD-1 and reversed the inhibitory effect of PD-1/PD-L1 interaction on T-cell function. PD-1 blockade by continuously secreted anti–PD-1 attenuated the inhibitory T-cell signaling and enhanced T-cell expansion and effector function both in vitro and in vivo. In the xenograft mouse model, we demonstrated that the secretion of anti–PD-1 enhanced the antitumor activity of CAR T cells and prolonged overall survival. Conclusions: With constitutive anti–PD-1 secretion, CAR.αPD1-T cells are more functional and expandable, and more efficient at tumor eradication than parental CAR T cells. Collectively, our study presents an important and novel strategy that enables CAR T cells to achieve better antitumor immunity, especially in the treatment of solid tumors. Clin Cancer Res; 23(22); 6982–92. ©2017 AACR.

Background: Differentiation of proteins from multiple sources provides challenges in the accuracy using multiple and often disputed protein identification methods. The U.S. Pharmacopeia Food Chemical Codex does not include monographs for many protein sources, including milk proteins and soy protein isolate. Monographs that are included for proteins do not list a single comprehensive identification method but instead rely on a combined assessment of ash (total), fat, lactose, loss on drying, and protein content. A fast, inexpensive, and accurate protein source assay is tantamount to prevention of economic adulteration in protein powders. Objective: This study describes the development of a novel method to identify and differentiate animal proteins (cow protein powders as milk protein and whey protein) and plant proteins (soy protein powders). These proteins powders are of high importance to the food and dietary supplement industries, as they encompass the highest grossing and fastest growing protein sources in the global protein powder market. Methods: The developed method uses PCR amplification and gel electrophoresis of short chain DNA fragments found in processed protein powders to identify and differentiate the source of each powder. The original development was performed using reference materials of known identity and tested against an inclusivity panel of protein powders from commercial sources. Bands were identified using the Agilent Tapestation 4200 and Tapestation Analysis Software A.02.02 (SR1) using proprietary band analysis. Results: The developed method was found to be specific for the identification of each protein source, passing a computational (National Center for Biotechnology Information Basic Local Alignment Search Tool) exclusivity panel and an experimental inclusivity panel. The method was also able to detect multiple adulterants in concentrations as low as 1% (w/w). Conclusions: The developed method is fast, inexpensive, and accurate (100%) for the supplemental identification of cow and soy proteins and able to detect adulteration as low as 1% (w/w). Highlights: A new method can identify cow and soy proteins, and detect low levels of adulteration using DM-PCR.

Background: PCR methods are the most commonly used DNA-based identity tool in the commercial food, beverage, and natural health product markets. These methods are routinely used to identify foodborne pathogens and allergens in food. Proper validation methods for some sectors have been established, while there are none in other markets, such as botanicals. Results: A survey of the literature indicates that some validation criteria are not addressed when developing PCR tests for botanicals. Objective: We provide recommendations for qualitative real-time PCR methods for validating identity tests for botanical ingredients. Methods: These include common criteria that underpin the development and validation of rigorous tests, including (1) the aim of the validation test, (2) the applicability of different matrix variants, (3) specificity in identifying the target species ingredient, (4) sensitivity in detecting the smallest amount of the target material, (5) repeatability of methods, (6) reproducibility in detecting the target species in both raw and processed materials, (7) practicability of the test in a commercial laboratory, and (8) comparison with alternative methods. In addition, we recommend additional criteria, according to which the practicability of the test method is evaluated by transferring the method to a second laboratory and by comparison with alternative methods. Conclusions and Highlights: We hope that these recommendations encourage further publication on the validation of PCR methods for many botanical ingredients. These properly validated PCR methods can be developed on small, real-time biotechnology that can be placed directly into the supply chain ledger in support of highly transparent data systems that support QC from the farm to the fork of the consumer.

DNA ligases are attractive therapeutics because of their involvement in completing the repair of almost all types of DNA damage. A series of DNA ligase inhibitors with differing selectivity for the three human DNA ligases were identified using a structure-based approach with one of these inhibitors being used to inhibit abnormal DNA ligase IIIα-dependent repair of DNA double-strand breaks (DSB)s in breast cancer, neuroblastoma and leukemia cell lines. Raghavan and colleagues reported the characterization of a derivative of one of the previously identified DNA ligase inhibitors, which they called SCR7 (designated SCR7-R in our experiments using SCR7). SCR7 appeared to show increased selectivity for DNA ligase IV, inhibit the repair of DSBs by the DNA ligase IV-dependent non-homologous end-joining (NHEJ) pathway, reduce tumor growth, and increase the efficacy of DSB-inducing therapeutic modalities in mouse xenografts. In attempting to synthesize SCR7, we encountered problems with the synthesis procedures and discovered discrepancies in its reported structure. We determined the structure of a sample of SCR7 and a related compound, SCR7-G, that is the major product generated by the published synthesis procedure for SCR7. We also found that SCR7-G has the same structure as the compound (SCR7-X) available from a commercial vendor (XcessBio). The various SCR7 preparations had similar activity in DNA ligation assay assays, exhibiting greater activity against DNA ligases I and III than DNA ligase IV. Furthermore, SCR7-R failed to inhibit DNA ligase IV-dependent V(D)J recombination in a cell-based assay. Based on our results, we conclude that SCR7 and the SCR7 derivatives are neither selective nor potent inhibitors of DNA ligase IV.

Vertebrates developed immunoglobulin heavy chain (IgH) class switch recombination (CSR) to express different IgH constant regions. Most double-strand breaks for Ig CSR occur within the repetitive portion of the switch regions located upstream of each set of constant domain exons for the Igγ, Igα or Igε heavy chain. Unlike mammalian switch regions, Xenopus switch regions do not have a high G-density on the non-template DNA strand. In previous studies, when Xenopus Sμ DNA was moved to the genome of mice, it is able to support substantial CSR when it is used to replace the murine Sγ1 region. Here, we tested both the 2 kb repetitive portion and the 4.6 kb full-length portions of the Xenopus Sμ in both their natural (forward) orientation relative to the constant domain exons, as well as the opposite (reverse) orientation. Consistent with previous work, we find that the 4.6 kb full-length Sμ mediates similar levels of CSR in both the forward and reverse orientations. Whereas, the forward orientation of the 2 kb portion can restore the majority of the CSR level of the 4.6 kb full-length Sμ, the reverse orientation poorly supports R-looping and no CSR. The forward orientation of the 2 kb repetitive portion has more GG dinucleotides on the non-template strand than the reverse orientation. The correlation of R-loop formation with CSR efficiency, as demonstrated in the 2 kb repetitive fragment of the Xenopus switch region, confirms a role played by R-looping in CSR that appears to be conserved through evolution.

Chromosomal translocations are a hallmark of hematopoietic malignancies. CG motifs within translocation fragile zones (typically 20 to 600 bp in size) are prone to chromosomal translocation in lymphomas. Here we demonstrate that the CG motifs in human translocation fragile zones are hypomethylated relative to the adjacent DNA. Using a methyltransferase footprinting assay on isolated nuclei (in vitro), we find that the chromatin at these fragile zones is accessible. We also examined in vivo accessibility using cellular expression of a prokaryotic methylase. Based on this assay, which measures accessibility over a much longer time interval than is possible with in vitro methods, these fragile zones were found to be more accessible than the adjacent DNA. Because DNA within the fragile zones can be methylated by both cellular and exogenous methyltransferases, the fragile zones are predominantly in a duplex DNA conformation. These observations permit more-refined models for why these zones are 100- to 1,000-fold more prone to undergo chromosomal translocation than the adjacent regions.

Using a large sample of listed Chinese companies, we investigate how the equity ownership of business group insiders affects subsidiary cash holdings. We find that ownership by the largest shareholders and senior managers in the listed parent firm is negatively related to its subsidiaries’ cash holdings, whereas there is a positive relationship with minority equity in subsidiaries. We also find that the market places a more significant value discount on listed firms whose cash holdings are more located in the affiliated subsidiaries. Our evidence demonstrates how cash policy inside business groups is influenced by insider ownership, and it reveals to what extent cash allocated in subsidiaries may suffer from losses in efficiency.

This study investigates the earnings effect associated with two major types of accounting estimate changes (i.e., revision of the percentage allowance for doubtful accounts receivable and revision of the depreciation rate) made by Chinese listed firms during 2003–2011. We find that the more a firm’s accounting estimate diverges from the industry-level estimate for the same category of assets, the more likely is the firm to change the estimate in convergence with (rather than deviating further from) the industry level in the following period. Moreover, the tendency to converge with the industry-level estimate is significantly stronger for previously aggressive estimates than for previously conservative estimates. These findings are consistent with the notion that management discretion in accounting estimates is restrained by the demand for verifiability of accounting information and the firm’s underlying economic forces, thus overall decreasing the degree of earnings aggressiveness.

BCL6 translocations are common in B-cell lymphomas and frequently have chromosomal breaks in immunoglobulin heavy chain (IgH) switch regions, suggesting that they occur during class-switch recombination. We analyze 120 BCL6 translocation breakpoints clustered in a 2156-bp segment of BCL6 intron 1, including 62 breakpoints (52%) joined to IgH, 12 (10%) joined to Ig light chains, and 46 (38%) joined to non-Ig partners. The BCL6 breaks in Ig-BCL6 translocations prefer known activation-induced cytosine deaminase (AID) hotspots such as WGCW and WRC (W = A/T, R = A/G), whereas BCL6 breaks in non-Ig rearrangements occur at CpG/CGC sites in addition to WGCW. Unlike previously identified CpG breaks in pro-B/pre-B-cell translocations, the BCL6 breaks do not show evidence of recombination activating gene or terminal deoxynucleotidyl transferase activity. Both WGCW/WRC and CpG/CGC breaks at BCL6 are most likely initiated by AID in germinal center B-cells, and their differential use suggests subtle mechanistic differences between Ig-BCL6 and non-Ig-BCL6 rearrangements.

Previous studies have implicated activation-induced cytidine deaminase (AID) in B-cell translocations but have failed to identify any association between their chromosomal breakpoints and known AID target sequences. Analysis of 56 unclustered IgH-CCND1 translocations in mantle cell lymphoma across the ∼ 344-kb bcl-1 breakpoint locus demonstrates that half of the CCND1 breaks are near CpG dinucleotides. Most of these CpG breaks are at CGC motifs, and half of the remaining breaks are near WGCW, both known AID targets. These findings provide the strongest evidence to date that AID initiates chromosomal breaks in translocations that occur in human bone marrow B-cell progenitors. We also identify WGCW breaks at the MYC locus in Burkitt lymphoma translocations and murine IgH-MYC translocations, both of which arise in mature germinal center B cells. Finally, we propose a developmental model to explain the transition from CpG breaks in early human B-cell progenitors to WGCW breaks in later stage B cells.

This paper finds that compared with Chinese state-owned firms, non-state-owned firms have a greater propensity to hold significant ownership in commercial banks. These results are consistent with the notion that because non-state-owned firms are more likely to suffer bank discrimination for political reasons, they tend to address their financing disadvantages by building economic bonds with banks. We also find that among non-state-owned firms, those that hold significant bank ownership have lower interest expenses, and are less likely to increase cash holdings but more likely to obtain short-term loans when the government monetary policy is tight. These results suggest that the firms building economic bonds with banks can enjoy benefits such as lower financial expenses and better lending terms during difficult times. Finally, we find that non-state-owned firms with significant bank ownership have better operating performance. Overall, we find that firms can reduce discrimination through holding bank ownership.

New Chinese Accounting Standards (CAS) released on February 15, 2006, prohibit the reversal of long-lived asset impairments, effective for reporting periods beginning January 1, 2007 and later. This development in the Chinese market provides a unique experimental setting to directly investigate how firms react to the ban on previously-allowed long-lived asset impairment reversals, especially firms that use impairment charges as “cookie jar reserves.” By contrasting write-off recognition and reversal across the pre- and post- new CAS announcement regimes, we show that firms listed on Chinese stock exchanges recognized less impairment charges during the “transition” period—after announcement of the new standard and before the effective date—than in pre-announcement periods. Meanwhile, firms with substantial previous write-downs reversed more impairment charges to achieve their earnings targets in the transition period. The new CAS also constraints “big bath” reporting by loss firms. As US GAAP prohibits these reversals and IFRS allows them, our empirical evidence depicts firms’ possible reactions in other regimes contemplating doing away with such reversals.

Based on the balanced panel data of Chinese listed firms from 1999 to 2004, this paper studies the relationship between product market competition and dynamic adjustment in capital structure. Results show that, from both static and dynamic perspectives, product market competition and its changes have significant influence on the degree of deviation of a firm’ s present capital structure from its targeted one. Specifically, the more intensive the product market competition, the smaller the capital structure deviation. Meanwhile, capital structure deviation tends to decrease in a market characterized by increasingly fierce competition. However, evidence also reveals that product market competition and the speed of the capital structure adjustment are independent from one another.

To the editor: We recently published a study showing that BCL6 translocations occur at distinct sequence motifs within intron 1 of the BCL6 gene that are recognized and deaminated by activation-induced cytidine deaminase (AID).[1][1] It has been unclear, however, why these breakpoints cluster in an

We analyze data on Chinese non-state-listed firms and find that it is easier for firms with political connections to obtain long-term loans with extended debt maturities than it is for firms without political connections. Our investigation indicates that this phenomenon is significantly less common with increased media monitoring. Houston et al. (2011) find strong evidence that the state ownership of media is associated with higher levels of bank corruption in China, but our study shows that, to a certain extent, media monitoring can curb corruption.

The t(14;18) chromosomal translocation typically involves breakage at the bcl-2 major breakpoint region (MBR) to cause human follicular lymphoma. A theory to explain the striking propensity of the MBR breaks at three CpG clusters within the 175-bp MBR region invoked activation-induced deaminase (AID). In a test of that theory, we used here minichromosomal substrates in human pre-B cell lines. Consistent with the essential elements of the theory, we found that the MBR breakage process is indeed highly dependent on DNA methylation at the CpG sites and highly dependent on the AID enzyme to create lesions at peak locations within the MBR. Interestingly, breakage of the phosphodiester bonds at the AID-initiated MBR lesions is RAG dependent, but, unexpectedly, most are also dependent on Artemis. We found that Artemis is capable of nicking small heteroduplex structures and is even able to nick single-base mismatches. This raises the possibility that activated Artemis, derived from the unjoined D to J(H) DNA ends at the IgH locus on chromosome 14, nicks AID-generated TG mismatches at methyl CpG sites, and this would explain why the breaks at the chromosome 18 MBR occur within the same time window as those on chromosome 14.

Upon transcription of some sequences by RNA polymerases in vitro or in vivo, the RNA transcript can thread back onto the template DNA strand, resulting in an R loop. Previously, we showed that initiation of R-loop formation at an R-loop initiation zone (RIZ) is favored by G clusters. Here, using a purified in vitro system with T7 RNA polymerase, we show that increased distance between the promoter and the R-loop-supporting G-rich region reduces R-loop formation. When the G-rich portion of the RNA transcript is downstream from the 5' end of the transcript, the ability of this portion of the transcript to anneal to the template DNA strand is reduced. When we nucleolytically resect the beginning of the transcript, R-loop formation increases because the G-rich portion of the RNA is now closer to the 5' end of the transcript. Short G-clustered regions can act as RIZs and reduce the distance-induced suppression of R-loop formation. Supercoiled DNA is known to favor transient separation of the two DNA strands, and we find that this favors R-loop formation even in non-G-rich regions. Most strikingly, a nick can serve as a strong RIZ, even in regions with no G richness. This has important implications for class switch recombination and somatic hypermutation and possibly for other biological processes in transcribed regions.

Apelin was recently found to be an inotropic polypeptide in isolated rat hearts, and intravenous injection of apelin can induce a transient decrease in blood pressure. To illustrate the mechanism of apelin-induced vasodilation, we observed the in vitro effects of apelin on the L-arginine (L-Arg)/nitric oxide (NO) pathway in the incubated, isolated rat aorta. Apelin stimulated vascular NO(2)(-) product and NOS activation in a concentration- and time-dependent manner. Compared with no apelin treatment, incubation with apelin (10(-9), 10(-8), and 10(-7)mol/L) increased NO(2)(-) product by 33%, 46%, and 69% (all p0.01), respectively, and Ca(2+)-dependent constitutive NOS (cNOS) activity by 200%, 460%, and 550% (all p0.01), respectively. However, Ca(2+)-independent NOS (iNOS) activity was not significantly altered (p0.05). Apelin incubation (10(-9), 10(-8), and 10(-7)mol/L) increased L-Arg uptake by 130%, 180%, and 240% (all p0.01), respectively. The mRNA level of cationic amino acid transporters, CAT-1 and CAT-2B, in rat aortic tissues treated with 10(-7)mol/L apelin was increased by 110% and 128%, respectively (both p0.01). Incubation with 10(-7)mol/L apelin elevated eNOS mRNA and protein levels, by 53% (p0.05) and 319% (p0.01), respectively. Collectively, these results demonstrate that apelin directly activated the vascular L-Arg/NOS/NO pathway, which could be one of the important mechanisms of apelin-regulated vascular function.

The t(14;18)(q32;q21) involving the immunoglobulin heavy chain locus (IGH) and the MALT1 gene is a recurrent abnormality in mucosa-associated lymphoid tissue (MALT) lymphomas. However, the nucleotide sequence of only one t(14;18)-positive MALT lymphoma has been reported so far. We here report the molecular characterization of the IGH-MALT1 fusion products in 5 new cases of t(14;18)-positive MALT lymphomas. Similar to the IGH-associated translocations in follicular and mantle cell lymphomas, the IGH-MALT1 junctions in MALT lymphoma showed all features of a recombination signal sequence-guided V(D)J-mediated translocation at the IGH locus. Furthermore, analogous to follicular and mantle cell lymphoma, templated nucleotides (T-nucleotides) were identified at the t(14;18)/IGH-MALT1 breakpoint junctions. On chromosome 18, we identified a novel major breakpoint region in MALT1 upstream of its coding region. Moreover, the presence of duplications of MALT1 nucleotides in one case suggests an underlying staggered DNA-break process not consistent with V(D)J-mediated recombination. The molecular characteristics of the t(14;18)/IGH-MALT1 resemble those found in the t(14;18)/IGH-BCL2 in follicular lymphoma and t(11;14)/CCND1-IGH in mantle cell lymphoma, suggesting that these translocations could be generated by common pathomechanisms involving illegitimate V(D)J-mediated recombination on IGH as well as new synthesis of T-nucleotides and nonhomologous end joining (NHEJ) or alternative NHEJ repair pathways on the IGH-translocation partner.

Abstract 398 Most t(11;14)(q13;q32) breakpoints in mantle cell lymphoma (MCL) are scattered across a large genomic region centromeric to the CCND1 gene on chromosome 11q13. However, few t(11;14) breakpoints outside the major translocation cluster (MTC) have been sequenced. We report our analysis of 56 IGH-CCND1 fusion sequences from 32 non-MTC cases of MCL. Our analysis reveals remarkable breakpoint diversity at both CCND1 and IGH loci. The CCND1 breakpoints are located from 2 kb to 331 kb from the CCND1 gene, including 12 breakpoints (38%) in the 220kb region centromeric to the MTC, which is itself located 110 kb centromeric to the CCND1 gene. Twenty-one cases had a JH coding end breakpoint on the der(14) chromosome and a DH coding end breakpoint on the der(11). Two cases had der(14) and der(11) breakpoints derived from the coding and signal ends of the same JH or DH gene segment; one case had a JH/DH breakpoint on the der(14) and a VH breakpoint on the der(11); and one case had a breakpoint in the JH region located >100 bases from the nearest RSS motif, suggesting a RAG-independent break at IGH. No der(11) breakpoint could be amplified in eight cases. In sixteen cases (50%), one or both CCND1 breakpoints were within 4 bases of a CpG dinucleotide, a feature characteristic of translocation breakpoints in many human B cell lymphomas. Three CpG-associated “microclusters” were identified, i.e. breakpoints in two different tumors located at or near the same CpG site. Breakpoints in the sixteen (50%) “non-CpG” cases showed a significant association with AID hotspot motifs at the CCND1 locus. In addition, the non-CpG breakpoints were more likely to: 1) be located telomeric to the MTC, 2) involve 3' JH and 5' DH gene segments, and 3) be associated with immunoglobulin lambda light chain restriction; features that suggest occurrence at a late stage of pre-B cell maturation. In contrast, the CpG-associated breakpoints were: 1) more likely to be centromeric to the MTC, 2) not biased in their JH or DH segment usage, and 3) associated with kappa light chain restriction; features suggesting occurrence in an earlier pre-B cell or pro-B cell. Our results implicate AID in chromosomal breakage at both CpG and non-CpG sites within the CCND1 locus and suggest that AID and RAG collude to generate the chromosomal breaks underlying the t(11;14). Our findings also suggest that IGH-CCND1 rearrangements can occur at different stages of pre-B cell maturation. This study provides novel insights into the mechanism and developmental timing of the t(11;14) in human MCL, features that are likely to be relevant to a broad range of human lymphomas. Disclosures: Greisman: Signature Genomics, LLC: Patents & Royalties. Yi:Signature Genomics, LLC: Patents & Royalties.