How do industrial land transfer modes impact carbon emissions? An intermediation perspective based on industrial structure.

Industrial land is currently the main carrier and important source of global carbon emissions, and as the world's largest developing country, China's large-scale and diversified industrial land supply has made it the world's largest carbon emitter. Therefore, researching the impact of different supply methods of industrial land on carbon emissions and its impact paths in China can help provide a reference for other countries to reduce carbon emissions from the perspective of urban industrial land management, which is of great significance for effectively promoting global carbon reduction. Based on this, this paper analyses the impact of different supply methods of industrial land on carbon emissions and its urban heterogeneity using the SYS-GMM and chain-mediated effects models for 285 cities in China from 2008 to 2020. The study found that, in general, the impact of different industrial land transfer modes on carbon emission has hysteresis and persistence. Agreement and listing transfer with government intervention can significantly exacerbate carbon emissions, while more market-based bidding and auction transfer can dampen carbon emissions. In terms of intermediary effects, the transfer of industrial land by agreement and listing will inhibit the rationalization and advancement of industrial structure, thus aggravating carbon emissions. The transfer of industrial land by bidding and auction will create barriers to entry and a crowding-out effect, promote the rationalization of industrial structure and the transformation and upgrading of industrial structure and moderate carbon emissions. In terms of city heterogeneity, there is urban heterogeneity in the impact of industrial land transfer on carbon emissions in cities with different economic types. Bidding and auction transfer for industrial land in both economically developed and less developed cities can promote carbon pollution. While the more developed urban economy makes the intermediary effect of industrial structure not significant. In the future, it is necessary to strictly control the scale of industrial land supply; the whole process supervision mechanism of industrial land allocation and differentiated industrial land supply strategies will provide useful experience for many developing countries in allocating industrial land to mitigate carbon emissions, generating effective contributions to global carbon emission reduction.

Pharmacokinetics, pharmacodynamics, and toxicity of a PD-1-targeted IL-15 in cynomolgus monkeys.

PF-07209960 is a novel bispecific fusion protein composed of an anti-PD-1 antibody and engineered IL-15 cytokine mutein with reduced binding affinity to its receptors. The pharmacokinetics (PK), pharmacodynamics (PD), and toxicity of PF-07209960 were evaluated following once every other week subcutaneous (SC) or intravenous (IV) administration to cynomolgus monkeys in a repeat-dose PKPD (0.01-0.3 mg/kg/dose) and GLP toxicity study (0.1-3 mg/kg/dose). PF-07209960 showed dose dependent pharmacokinetics with a terminal T1/2 of 8 and 13 hours following IV administration at 0.03 and 0.1 mg/kg, respectively. The clearance is faster than a typical IgG1 antibody. Slightly faster clearance was also observed following the second dose, likely due to increased target pool and formation of anti-drug antibodies (ADA). Despite a high incidence rate of ADA (92%) observed in GLP toxicity study, PD-1 receptor occupancy, IL-15 signaling (STAT5 phosphorylation) and T cell expansion were comparable following the first and second doses. Activation and proliferation of T cells were observed with largest increase in cell numbers found in gamma delta T cells, followed by CD4+ and CD8+ T cells, and then NK cells. Release of cytokines IL-6, IFNγ, and IL-10 were detected, which peaked at 72 hours postdose. There was PF-07209960-related mortality at ≥1 mg/kg. At scheduled necropsy, microscopic findings were generalized mononuclear infiltration in various tissues. Both the no observed adverse effect level (NOAEL) and the highest non severely toxic dose (HNSTD) were determined to be 0.3 mg/kg/dose, which corresponded to mean Cmax and AUC48 values of 1.15 µg/mL and 37.9 µg*h/mL, respectively.

[Modulation of autophagy in the rats with acute intracerebral hemorrhage by acupuncture based on JNK pathway].

OBJECTIVE: To investigate the effects of acupuncture on JNK pathway and autophagy level in rats with intracerebral hemorrhage （ICH） and explore the partial mechanism of acupuncture against ICH. METHODS: SD rats were randomly divided into blank group, model group and acupuncture group. Each group was divided into Day 1, Day 3 and Day 7 subgroups respectively, with 5 rats in each group. The autologous blood injection was adopted to duplicate rat model of ICH. In the acupuncture group, the needle was inserted from "Baihui" （GV20） towards "Qubin" （GB7） on the affected side, stimulating for 30 min each time, once daily； the same acupuncture technique was opera-ted in each subgroup for 1, 3 and 7 days, separately. Using Bederson scale, the neurological deficit was evaluated in each group. Western blot was adopted to detect the protein expression levels of Beclin1, LC3Ⅰ/Ⅱ, phosphorylated c-Jun amino-terminal kinase （p-JNK） and the phosphorylated （p）-c-Jun around hematoma lesion of the brain tissue of rats in each group. RESULTS: After treatment, the neurological deficit score of rats in the model group was higher than that of the blank group at each time point （P<0.05）, and the score of the acupuncture group started declining since the 3rd day of treatment when compared with the model group （P<0.05）. At each time point, compared with the blank group, the protein expression levels of LC3Ⅰ/Ⅱ, Beclin1, p-c-Jun and p-JNK was increased （P<0.01）. Compared with the model group, the protein expression level of LC3Ⅰ/Ⅱ was reduced （P<0.05）； the protein expression levels of Beclin1, p-c-Jun and p-JNK was increased （P<0.05, P<0.01） on day 3 and 7 in the acupuncture group. CONCLUSION: Acupuncture can activate the JNK pathway in the brain tissue of rats with ICH and increase the level of autophagy, thereby improving the neurological function of the rats with ICH.

Urban land use carbon emission intensity in China under the "double carbon" targets: spatiotemporal patterns and evolution trend.

In-depth research on the spatiotemporal patterns and evolution trend of urban land use carbon emission intensity (ULUCEI) can reveal the internal relationship between urban land use and carbon emissions, which is crucial for achieving carbon emission reduction and "double carbon" targets. This paper proposed a conceptual framework of ULUCEI; the methods of kernel density estimation (KDE), exploratory spatial data analysis (ESDA), and spatial Markov chains were adopted for exploring the spatiotemporal patterns and evolution trend of China's ULUCEI from 2000 to 2017. The following conclusions are drawn through research. (1) There was an increasing trend in ULUCEI in China from 0.102 in 2000 to 0.283 in 2017. From the regional perspective, the ULUCEI in the eastern region is markedly higher than that in the central and western regions. Moreover, the results of nuclear density estimation indicate that China's ULUCEI shows an obvious upward and polarized trend. (2) China's ULUCEI shows a positive spatial autocorrelation. The types of spatial agglomeration include "high-high" agglomeration, "high-low" polarization, "low-high" collapse, and "low-low" homogeneity, and there are obvious disparities in the distribution rules of cities with different spatial agglomeration forms. (3) China's ULUCEI presents strong stability and "club convergence" trend. Moreover, spatial factors significantly affect the dynamic transition of China's ULUCEI, and its effect on the shifting upwards gradually enhances with increasing lag type. This paper therefore suggests that policymakers should formulate differentiated urban land low-carbon use models and carbon emission reduction policies to reduce ULUCEI.

Has China's Low-Carbon City Construction Enhanced the Green Utilization Efficiency of Urban Land?

China has implemented the low-carbon city pilot (LCCP) policy in the hopes of efficiently limiting carbon emission intensity to combat global warming and promote green economic growth. Urban land utilization, the second-largest source of carbon emissions, is key to the LCCP policy being able to have the desired effect, which has attracted widespread attention. Based on the panel data from prefecture-level cities in China from 2006 to 2019, this study used the propensity score matching difference-in-differences method (PSM-DID) to examine the impacts of LCCP policy on green utilization efficiency of urban land (GUEUL). The results reveal that LCCP policy has a beneficial impact on GUEUL and can effectively boost the future possibilities of green and low-carbon city development. Due to variances in regional economic and resource endowment level, the impacts of LCCP are different. The pilot has pushed GUEUL in the eastern region, western region, and growing resource-based cities, but has failed to improve GUEUL in other regions. Policymakers should adhere to the long-term sustainability of the LCCP policy and adopt differentiated action strategies to promote GUEUL when implementing it in different regions.

The Spatial and Temporal Evolution of the Coordination Degree in Regard to Farmland Transfer and Cultivated Land Green Utilization Efficiency in China.

In many parts of the world, the shortage of cultivated land and the food crisis are worsening on a continued basis. Hence, the central and local governments of the PRC have successively issued various related policies to encourage the practice of farmland transfer, promote the eco-friendly utilization of cultivated land, and ameliorate the efficiency of cultivated land utilization. Under the context of large-scale farmland transfer and rural revitalization strategy in China, it is significant to ensure agricultural sustainability through the coordination of farmland transfer and the amelioration of cultivated land green utilization efficiency (CLGUE). In the present study, 30 Chinese provinces were taken as the research object, with the super-efficient SBM model, the coupling coordination degree model and the spatial analysis model applied in combination. Based on the measurement of CLGUE, a thorough analysis was conducted to explore the evolution of coordination degree in regard to farmland transfer and CLGUE in China from both spatial and temporal perspectives. The conclusions drawn from this study are as follows. Firstly, the overall CLGUE exhibited an upward tendency in the PRC, from 0.440 in 2005 to 0.913 in 2019, with a yearly growth rate of 5.47% on average. However, there were significant spatial disparities in CLGUE between different regions and provinces. Secondly, there was a steady increasing trend shown by the level of coordination degree regarding farmland transfer and CLGUE across China. Further, due to the variation in natural and economic conditions, there were significant spatial-temporal disparities in the coordination degree among these 30 provinces. Lastly, there were obvious spatial aggregation patterns at the provincial level regarding the coordination degree in farmland transfer and CLGUE across China. However, there was a declining trend in the level of spatial aggregation patterns for coordination degree.

Impact of Cereal Production Displacement from Urban Expansion on Ecosystem Service Values in China: Based on Three Cropland Supplement Strategies.

The acceleration of global urban expansion constantly occupies high-quality cropland and affects regional food security. The implementation of cropland protection policies has alleviated the pressure of cropland loss worldwide, and thus keeping a dynamic balance of cereal production. Such a displacement of cereal production from the lost cropland to the supplemented cropland has resulted in the massive losses of natural habitats (such as forests, grasslands, and wetlands) as well as ecosystem service values. However, the impact of cereal production displacement caused by different cropland supplement strategies has not been concerned. Therefore, taking China (mainland) as a case, this study used the LANDSCAPE model to simulate cereal production displacement caused by urban expansion and cropland supplement between 2020 and 2040, based on three scales of the Chinese administration system (i.e., the national level, the provincial level, and the municipal level). The natural habitat loss and corresponding ecosystem service value (ESV) loss were assessed. The results show that the national-scale cereal displacement will lead to a large reclamation of cropland in North China, causing the most natural habitat loss (5090 km2), and the least ESV loss (46.53 billion yuan). Cereal production displacement at the provincial and municipal scales will lead to fewer natural habitat losses (4696 km2 and 4954 km2, respectively), but more ESV losses (54.16 billion yuan and 54.02 billion yuan, respectively). Based on the national food security and ecological conservation in China, this study discussed the reasons for the ecological effects of cereal production displacement, direct and indirect natural habitat loss of urban expansion, and cropland protection policies in China. We suggest that China's cropland protection policy should emphasize avoiding large-scale cropland displacement and occupation of natural habitat with high ESV for cropland supplement.

An Engineered IL15 Cytokine Mutein Fused to an Anti-PD1 Improves Intratumoral T-cell Function and Antitumor Immunity.

The use of cytokines for immunotherapy shows clinical efficacy but is frequently accompanied by severe adverse events caused by excessive and systemic immune activation. Here, we set out to address these challenges by engineering a fusion protein of a single, potency-reduced, IL15 mutein and a PD1-specific antibody (anti-PD1-IL15m). This immunocytokine was designed to deliver PD1-mediated, avidity-driven IL2/15 receptor stimulation to PD1+ tumor-infiltrating lymphocytes (TIL) while minimally affecting circulating peripheral natural killer (NK) cells and T cells. Treatment of tumor-bearing mice with a mouse cross-reactive fusion, anti-mPD1-IL15m, demonstrated potent antitumor efficacy without exacerbating body weight loss in B16 and MC38 syngeneic tumor models. Moreover, anti-mPD1-IL15m was more efficacious than an IL15 superagonist, an anti-mPD-1, or the combination thereof in the B16 melanoma model. Mechanistically, anti-PD1-IL15m preferentially targeted CD8+ TILs and single-cell RNA-sequencing analyses revealed that anti-mPD1-IL15m treatment induced the expansion of an exhausted CD8+ TIL cluster with high proliferative capacity and effector-like signatures. Antitumor efficacy of anti-mPD1-IL15m was dependent on CD8+ T cells, as depletion of CD8+ cells resulted in the loss of antitumor activity, whereas depletion of NK cells had little impact on efficacy. The impact of anti-hPD1-IL15m on primary human TILs from patients with cancer was also evaluated. Anti-hPD1-IL15m robustly enhanced the proliferation, activation, and cytotoxicity of CD8+ and CD4+ TILs from human primary cancers in vitro, whereas tumor-derived regulatory T cells were largely unaffected. Taken together, our findings showed that anti-PD1-IL15m exhibits a high translational promise with improved efficacy and safety of IL15 for cancer immunotherapy via targeting PD1+ TILs.See related Spotlight by Felices and Miller, p. 1110.

Preclinical Efficacy and Safety Comparison of CD3 Bispecific and ADC Modalities Targeting BCMA for the Treatment of Multiple Myeloma.

The restricted expression pattern of B-cell maturation antigen (BCMA) makes it an ideal tumor-associated antigen (TAA) for the treatment of myeloma. BCMA has been targeted by both CD3 bispecific antibody and antibody-drug conjugate (ADC) modalities, but a true comparison of modalities has yet to be performed. Here we utilized a single BCMA antibody to develop and characterize both a CD3 bispecific and 2 ADC formats (cleavable and noncleavable) and compared activity both in vitro and in vivo with the aim of generating an optimal therapeutic. Antibody affinity, but not epitope was influential in drug activity and hence a high-affinity BCMA antibody was selected. Both the bispecific and ADCs were potent in vitro and in vivo, causing dose-dependent cell killing of myeloma cell lines and tumor regression in orthotopic myeloma xenograft models. Primary patient cells were effectively lysed by both CD3 bispecific and ADCs, with the bispecific demonstrating improved potency, maximal cell killing, and consistency across patients. Safety was evaluated in cynomolgus monkey toxicity studies and both modalities were active based on on-target elimination of B lineage cells. Distinct nonclinical toxicity profiles were seen for the bispecific and ADC modalities. When taken together, results from this comparison of BCMA CD3 bispecific and ADC modalities suggest better efficacy and an improved toxicity profile might be achieved with the bispecific modality. This led to the advancement of a bispecific candidate into phase I clinical trials.

Utilization of lipopolysaccharide challenge in cynomolgus macaques to assess IL-10 receptor antagonism.

The current era of drug discovery has been marked by a significant increase in the development of immune modulating agents to address a range of diseases such as cancer, chronic inflammation, and other conditions of dysregulated immunity. Non-clinical evaluation of these agents in animal models can be challenging, as the presence of an active immune state is often required in order to detect the effects of the test agent. Modulation of interleukin (IL)-10 signaling represents this type of situation in that altering IL-10 action in vivo can be difficult to appreciate in the absence of an ongoing immune response. The study presented here reports on the use of lipopolysaccharide (LPS) challenge in cynomolgus macaques to induce predictable inflammatory cytokine responses. The results showed that IL-10 receptor (IL-10R) blockade with an antagonist monoclonal antibody (mAb) dramatically enhanced the LPS-induced cytokine response, thus demonstrating in vivo pharmacologic activity of this immunomodulatory antibody. We submit that this approach could be applied to other cases where the intent of a candidate therapeutic is to modulate components of inflammatory cytokine responses.

Enhancing antisense efficacy with multimers and multi-targeting oligonucleotides (MTOs) using cleavable linkers.

The in vivo potency of antisense oligonucleotides (ASO) has been significantly increased by reducing their length to 8-15 nucleotides and by the incorporation of high affinity RNA binders such as 2', 4'-bridged nucleic acids (also known as locked nucleic acid or LNA, and 2',4'-constrained ethyl [cET]). We now report the development of a novel ASO design in which such short ASO monomers to one or more targets are co-synthesized as homo- or heterodimers or multimers via phosphodiester linkers that are stable in plasma, but cleaved inside cells, releasing the active ASO monomers. Compared to current ASOs, these multimers and multi-targeting oligonucleotides (MTOs) provide increased plasma protein binding and biodistribution to liver, and increased in vivo efficacy against single or multiple targets with a single construct. In vivo, MTOs synthesized in both RNase H-activating and steric-blocking oligonucleotide designs provide ≈4-5-fold increased potency and ≈2-fold increased efficacy, suggesting broad therapeutic applications.

A mathematical model of the effect of immunogenicity on therapeutic protein pharmacokinetics.

A mathematical pharmacokinetic/anti-drug-antibody (PK/ADA) model was constructed for quantitatively assessing immunogenicity for therapeutic proteins. The model is inspired by traditional pharmacokinetic/pharmacodynamic (PK/PD) models, and is based on the observed impact of ADA on protein drug clearance. The hypothesis for this work is that altered drug PK contains information about the extent and timing of ADA generation. By fitting drug PK profiles while accounting for ADA-mediated drug clearance, the model provides an approach to characterize ADA generation during the study, including the maximum ADA response, sensitivity of ADA response to drug dose level, affinity maturation rate, time lag to observe an ADA response, and the elimination rate for ADA-drug complex. The model also provides a mean to estimate putative concentration-time profiles for ADA, ADA-drug complex, and ADA binding affinity-time profile. When simulating ADA responses to various drug dose levels, bell-shaped dose-response curves were generated. The model contains simultaneous quantitative modeling and provides estimation of the characteristics of therapeutic protein drug PK and ADA responses in vivo. With further experimental validation, the model may be applied to the simulation of ADA response to therapeutic protein drugs in silico, or be applied in subsequent PK/PD models.

Modeling, simulation, and translation framework for the preclinical development of monoclonal antibodies.

The industry-wide biopharmaceutical (i.e., biologic, biotherapeutic) pipeline has been growing at an astonishing rate over the last decade with the proportion of approved new biological entities to new chemical entities on the rise. As biopharmaceuticals appear to be growing in complexity in terms of their structure and mechanism of action, so are interpretation, analysis, and prediction of their quantitative pharmacology. We present here a modeling and simulation (M&S) framework for the successful preclinical development of monoclonal antibodies (as an illustrative example of biopharmaceuticals) and discuss M&S strategies for its implementation. Critical activities during early discovery, lead optimization, and the selection of starting doses for the first-in-human study are discussed in the context of pharmacokinetic-pharmacodynamic (PKPD) and M&S. It was shown that these stages of preclinical development are and should be reliant on M&S activities including systems biology (SB), systems pharmacology (SP), and translational pharmacology (TP). SB, SP, and TP provide an integrated and rationalized framework for decision making during the preclinical development phase. In addition, they provide increased target and systems understanding, describe and interpret data generated in vitro and in vivo, predict human PKPD, and provide a rationalized approach to designing the first-in-human study.

How current understanding of clearance mechanisms and pharmacodynamics of therapeutic proteins can be applied for evaluation of their drug-drug interaction potential.

Increasing use of therapeutic proteins (TPs) in polypharmacy settings calls for more in-depth understanding of the biological interactions that can lead to increased toxicity or loss of pharmacological effect. Factors such as patient population, medications that are likely to be coadministered in that population, clearance mechanisms of a TP, and concomitant drugs have to be taken into account to determine the potential for drug-drug interactions (DDIs). The most well documented TP DDI mechanism involves cytokine-mediated changes in drug-metabolizing enzymes. Because of the limitations of the current preclinical models for addressing this type of DDI, clinical evaluation is currently the most reliable approach. Other DDI mechanisms need to be addressed on a case-by-case basis. These include altered clearance of TPs resulting from the changes in the target protein levels by the concomitant medication, displacement of TPs from binding proteins, modulation of Fcγ receptor expression, and others. The purpose of this review is to introduce the approach used by Pfizer scientists for evaluation of the DDI potential of novel TP products during drug discovery and development.

Anti-amyloid therapy protects against retinal pigmented epithelium damage and vision loss in a model of age-related macular degeneration.

Age-related macular degeneration (AMD) is a leading cause of visual dysfunction worldwide. Amyloid ß (Aß) peptides, Aß1-40 (Aß40) and Aß1-42 (Aß42), have been implicated previously in the AMD disease process. Consistent with a pathogenic role for Aß, we show here that a mouse model of AMD that invokes multiple factors that are known to modify AMD risk (aged human apolipoprotein E 4 targeted replacement mice on a high-fat, cholesterol-enriched diet) presents with Aß-containing deposits basal to the retinal pigmented epithelium (RPE), histopathologic changes in the RPE, and a deficit in scotopic electroretinographic response, which is reflective of impaired visual function. Strikingly, these electroretinographic deficits are abrogated in a dose-dependent manner by systemic administration of an antibody targeting the C termini of Aß40 and Aß42. Concomitant reduction in the levels of Aß and activated complement components in sub-RPE deposits and structural preservation of the RPE are associated with anti-Aß40/42 antibody immunotherapy and visual protection. These observations are consistent with the reduction in amyloid plaques and improvement of cognitive function in mouse models of Alzheimer's disease treated with anti-Aß antibodies. They also implicate Aß in the pathogenesis of AMD and identify Aß as a viable therapeutic target for its treatment.

Therapeutic monoclonal antibody concentration monitoring: free or total?

Most therapeutic monoclonal antibodies are designed to bind a specific antigen to elicit pharmacological effects. Accurate quantification of a therapeutic monoclonal antibody in biological matrices is essential for assessing its pharmacokinetics and selecting an effective dosing regimen. Therapeutic antibodies may exist in free, partially bound and fully bound forms in the bloodstream. The choice of which form(s) to measure and how to measure them is gaining much attention with the increase in the number of soluble therapeutic targets. This article will review the bioanalytical methods used in supporting the clinical development of the US FDA-approved therapeutic monoclonal antibodies and also discuss how different factors, such as assay format, target and antibody concentrations, and sample dilutions, can have an impact on the measurement of each form of antibody. Appreciation of which form of drug is being measured and what factors may impact measurement under different conditions are important for interpretation of the pharmacokinetics of therapeutic antibodies.

CR011, a fully human monoclonal antibody-auristatin E conjugate, for the treatment of melanoma.

PURPOSE: Advanced melanoma is a highly drug-refractory neoplasm representing a significant unmet medical need. We sought to identify melanoma-associated cell surface molecules and to develop as well as preclinically test immunotherapeutic reagents designed to exploit such targets. EXPERIMENTAL DESIGN AND RESULTS: By transcript profiling, we identified glycoprotein NMB (GPNMB) as a gene that is expressed by most metastatic melanoma samples examined. GPNMB is predicted to be a transmembrane protein, thus making it a potential immunotherapeutic target in the treatment of this disease. A fully human monoclonal antibody, designated CR011, was generated to the extracellular domain of GPNMB and characterized for growth-inhibitory activity against melanoma. The CR011 monoclonal antibody showed surface staining of most melanoma cell lines by flow cytometry and reacted with a majority of metastatic melanoma specimens by immunohistochemistry. CR011 alone did not inhibit the growth of melanoma cells. However, when linked to the cytotoxic agent monomethylauristatin E (MMAE) to generate the CR011-vcMMAE antibody-drug conjugate, this reagent now potently and specifically inhibited the growth of GPNMB-positive melanoma cells in vitro. Ectopic overexpression and small interfering RNA transfection studies showed that GPNMB expression is both necessary and sufficient for sensitivity to low concentrations of CR011-vcMMAE. In a melanoma xenograft model, CR011-vcMMAE induced significant dose-proportional antitumor effects, including complete regressions, at doses as low as 1.25 mg/kg. CONCLUSION: These preclinical results support the continued evaluation of CR011-vcMMAE for the treatment of melanoma.

A Drosophila protein-interaction map centered on cell-cycle regulators.

BACKGROUND: Maps depicting binary interactions between proteins can be powerful starting points for understanding biological systems. A proven technology for generating such maps is high-throughput yeast two-hybrid screening. In the most extensive screen to date, a Gal4-based two-hybrid system was used recently to detect over 20,000 interactions among Drosophila proteins. Although these data are a valuable resource for insights into protein networks, they cover only a fraction of the expected number of interactions. RESULTS: To complement the Gal4-based interaction data, we used the same set of Drosophila open reading frames to construct arrays for a LexA-based two-hybrid system. We screened the arrays using a novel pooled mating approach, initially focusing on proteins related to cell-cycle regulators. We detected 1,814 reproducible interactions among 488 proteins. The map includes a large number of novel interactions with potential biological significance. Informative regions of the map could be highlighted by searching for paralogous interactions and by clustering proteins on the basis of their interaction profiles. Surprisingly, only 28 interactions were found in common between the LexA- and Gal4-based screens, even though they had similar rates of true positives. CONCLUSIONS: The substantial number of new interactions discovered here supports the conclusion that previous interaction mapping studies were far from complete and that many more interactions remain to be found. Our results indicate that different two-hybrid systems and screening approaches applied to the same proteome can generate more comprehensive datasets with more cross-validated interactions. The cell-cycle map provides a guide for further defining important regulatory networks in Drosophila and other organisms.