Development of bioreactor scale-down model using orthogonal projections to latent structures method and CO2 supplementation.

Scale-down model qualification is an important step for developing a large-scale cell culture process to enhance process understanding and support process characterization studies. Traditionally, only harvest data are used to show consistency between small-scale and large-scale bioreactor performance, allowing attributes that are dynamic over the cell culture period to be overlooked. A novel statistical method, orthogonal projections to latent structures (OPLS) analysis, can be utilized to compare time-course cell culture data across scales. Here we describe an example where OPLS is used to identify gaps between small-scale and large-scale bioreactor performances. In this case, differences in the partial pressure of carbon dioxide (pCO2) and lactate profiles were observed between small- and large-scale bioreactors, which were linked to differences in the product-quality attributes fragments and galactosylation. An improved small-scale model was developed, leading to improved consistency in the process performance and product qualities across scales and qualification of the scale-down model for regulatory submissions. This new statistical approach can provide valuable insights into process understanding and process scale-up.

23ME-00610, a genetically informed, first-in-class antibody targeting CD200R1 to enhance antitumor T cell function.

Immune checkpoint inhibition (ICI) has revolutionized cancer treatment; however, only a subset of patients benefit long term. Therefore, methods for identification of novel checkpoint targets and development of therapeutic interventions against them remain a critical challenge. Analysis of human genetics has the potential to inform more successful drug target discovery. We used genome-wide association studies of the 23andMe genetic and health survey database to identify an immuno-oncology signature in which genetic variants are associated with opposing effects on risk for cancer and immune diseases. This signature identified multiple pathway genes mapping to the immune checkpoint comprising CD200, its receptor CD200R1, and the downstream adapter protein DOK2. We confirmed that CD200R1 is elevated on tumor-infiltrating immune cells isolated from cancer patients compared to the matching peripheral blood mononuclear cells. We developed a humanized, effectorless IgG1 antibody (23ME-00610) that bound human CD200R1 with high affinity (KD <0.1 nM), blocked CD200 binding, and inhibited recruitment of DOK2. 23ME-00610 induced T-cell cytokine production and enhanced T cell-mediated tumor cell killing in vitro. Blockade of the CD200:CD200R1 immune checkpoint inhibited tumor growth and engaged immune activation pathways in an S91 tumor cell model of melanoma in mice.

Females with Diabetes Mellitus Increased the Incidence of Premenstrual Syndrome.

Background: Premenstrual syndrome (PMS) is a multifactorial disorder caused by hormone and autonomic imbalance. In our study, hyperglycemia-induced insulin secretion increased progesterone secretion and progressive autonomic imbalance. The young patients with diabetes mellitus (DM) revealed hypo-parasympathetic function and hypersympathetic function compared with nondiabetic controls. Young female patients with DM with higher blood sugar and autonomic malfunction may be associated with PMS. However, there is a lack of evidence about DM in females related to PMS. We evaluated female patients with DM who subsequently followed PMS in a retrospective cohort study. Methods: We retrieved data from the National Health Insurance Research Database in Taiwan. Female patients with DM between 20 and 50 years old were assessed by the International Classification of Disease, 9 Revision, Clinical Modification (ICD-9-CM) disease code of 250. Patients who were DM-free females were fourfold matched to the control group by age and disease index date. The ICD-9-CM disease code of 625.4 identified the incidence of PMS followed by the index date as events. The possible risk factors associated with PMS were detected with a Cox proportional regression. Results: DM was a significant risk factor for PMS incidence with an adjusted hazard ratio of 1.683 (95% confidence interval: 1.104−2.124, p < 0.001) in females after adjusting for age, other comorbidities, season, urbanization status of patients and the hospital status of visiting. Conclusions: This study noted an association between DM and PMS in female patients. Healthcare providers and female patients with DM must be aware of possible complications of PMS, aggressive glycemic control, decreased hyperglycemia and autonomic dysfunction to prevent this bothersome disorder.

Underestimated Ischemic Heart Disease in Major Adverse Cardiovascular Events after Septicemia Discharge.

Background and Objectives: Sepsis increases cardiovascular disease and causes death. Ischemic heart disease (IHD) without acute myocardial infarction has been discussed less, and the relationship between risk factors and IHD in septicemia survivors within six months is worthy of in-depth study. Our study demonstrated the incidence of IHD and the possible risk factors for IHD in septicemia patients within six months. Materials and Methods: An inpatient dataset of the Taiwanese Longitudinal Health Insurance Database between 2001 and 2003 was used. The events were defined as rehospitalization of stroke and IHD after discharge or death within six months after the first septicemia hospitalization. The relative factors of major adverse cardiovascular events (MACEs) and IHD were identified by multivariate Cox proportional regression. Results: There were 4323 septicemia survivors and 404 (9.3%) IHD. New-onset atrial fibrillation had a hazard ratio (HR) of 1.705 (95% confidence interval (C.I.): 1.156-2.516) for MACEs and carried a 184% risk with HR 2.836 (95% C.I.: 1.725-4.665) for IHD by adjusted area and other risk factors. Conclusions: This study explored advanced-aged patients who experienced more severe septicemia with new-onset atrial fibrillation, which increases the incidence of IHD in MACEs within six months of septicemia. Therefore, healthcare providers must identify patients with a higher IHD risk and modify risk factors beforehand.

Computational design of a modular protein sense-response system.

Sensing and responding to signals is a fundamental ability of living systems, but despite substantial progress in the computational design of new protein structures, there is no general approach for engineering arbitrary new protein sensors. Here, we describe a generalizable computational strategy for designing sensor-actuator proteins by building binding sites de novo into heterodimeric protein-protein interfaces and coupling ligand sensing to modular actuation through split reporters. Using this approach, we designed protein sensors that respond to farnesyl pyrophosphate, a metabolic intermediate in the production of valuable compounds. The sensors are functional in vitro and in cells, and the crystal structure of the engineered binding site closely matches the design model. Our computational design strategy opens broad avenues to link biological outputs to new signals.

Activation of MET promotes resistance to sorafenib in hepatocellular carcinoma cells via the AKT/ERK1/2-EGR1 pathway.

Sorafenib is an oral multikinase inhibitor that has become an established therapeutic approach in advanced hepatocellular carcinoma (HCC). However, the benefit of sorafenib in clinical therapy is often affected by drug resistance. Therefore, it is important to explore the mechanisms underlying sorafenib resistance and to develop individualized therapeutic strategies for coping with this problem. In this study, we found that addition of HGF to sorafenib-treated HCC cells activated MET and re-stimulated the downstream AKT and ERK1/2 pathways. Thereby, restored sorafenib-treated HCC cells proliferation, migration and invasion ability, and rescued cells from apoptosis. In addition, we found that HGF treatment of HCC cells induced early growth response protein (EGR1) expression, which is involved in sorafenib resistance. Importantly, the HGF rescued effect in sorafenib-treated HCC cells could be abrogated by inhibiting MET activation with PHA-665752 or by downregulating EGR1 expression with small interfering RNA (siRNA). Therefore, inhibition of the HGF/MET pathway may improve response to sorafenib in HCC, and combination therapy should be further investigated.

Identifying the differences in mechanisms of mycophenolic acid controlling fucose content of glycoproteins expressed in different CHO cell lines.

In the biopharmaceutical industry, glycosylation is a critical quality attribute that can modulate the efficacy of a therapeutic glycoprotein. Obtaining a consistent glycoform profile is desired because molecular function can be defined by its carbohydrate structures. Specifically, the fucose content of oligosaccharides in glycoproteins is one of the most important attributes that can significantly affect antibody-dependent cellular cytotoxicity (ADCC) activity. It is therefore important to understand the fucosylation pathway and be able to control fucosylation at the desired level to match predecessor materials in late stage and biosimilar programs. Several strategies were explored in this study and mycophenolic acid (MPA) was able to finely modulate the fucose content with the least undesired side effects. However, the response was significantly different between CHO cell lines of different lineages. Further experiments were then performed for a deeper understanding of the mechanism of fucosylation in different CHO cell lines. Results indicated that changes in the intracellular nucleotide involved in fucosylation pathway after MPA treatment are the main cause of the differences in fucosylation level response in different CHO cell lines. Differences in MPA metabolism in the various CHO cell lines directly resulted in different levels of afucosylation measured in antibodies produced by the CHO cell lines. Biotechnol. Bioeng. 2016;113: 2367-2376. © 2016 Wiley Periodicals, Inc.

Concentrated fed-batch cell culture increases manufacturing capacity without additional volumetric capacity.

Biomanufacturing factories of the future are transitioning from large, single-product facilities toward smaller, multi-product, flexible facilities. Flexible capacity allows companies to adapt to ever-changing pipeline and market demands. Concentrated fed-batch (CFB) cell culture enables flexible manufacturing capacity with limited volumetric capacity; it intensifies cell culture titers such that the output of a smaller facility can rival that of a larger facility. We tested this hypothesis at bench scale by developing a feeding strategy for CFB and applying it to two cell lines. CFB improved cell line A output by 105% and cell line B output by 70% compared to traditional fed-batch (TFB) processes. CFB did not greatly change cell line A product quality, but it improved cell line B charge heterogeneity, suggesting that CFB has both process and product quality benefits. We projected CFB output gains in the context of a 2000-L small-scale facility, but the output was lower than that of a 15,000-L large-scale TFB facility. CFB's high cell mass also complicated operations, eroded volumetric productivity, and showed our current processes require significant improvements in specific productivity in order to realize their full potential and savings in manufacturing. Thus, improving specific productivity can resolve CFB's cost, scale-up, and operability challenges.

Toward Computationally Designed Self-Reporting Biosensors Using Leave-One-Out Green Fluorescent Protein.

Leave-one-out green fluorescent protein (LOOn-GFP) is a circularly permuted and truncated GFP lacking the nth ß-strand element. LOO7-GFP derived from the wild-type sequence (LOO7-WT) folds and reconstitutes fluorescence upon addition of ß-strand 7 (S7) as an exogenous peptide. Computational protein design may be used to modify the sequence of LOO7-GFP to fit a different peptide sequence, while retaining the reconstitution activity. Here we present a computationally designed leave-one-out GFP in which wild-type strand 7 has been replaced by a 12-residue peptide (HA) from the H5 antigenic region of the Thailand strain of H5N1 influenza virus hemagglutinin. The DEEdesign software was used to generate a sequence library with mutations at 13 positions around the peptide, coding for approximately 3 × 10(5) sequence combinations. The library was coexpressed with the HA peptide in E. coli and colonies were screened for in vivo fluorescence. Glowing colonies were sequenced, and one (LOO7-HA4) with 7 mutations was purified and characterized. LOO7-HA4 folds, fluoresces in vivo and in vitro, and binds HA. However, binding results in a decrease in fluorescence instead of the expected increase, caused by the peptide-induced dissociation of a novel, glowing oligomeric complex instead of the reconstitution of the native structure. Efforts to improve binding and recover reconstitution using in vitro evolution produced colonies that glowed brighter and matured faster. Two of these were characterized. One lost all affinity for the HA peptide but glowed more brightly in the unbound oligomeric state. The other increased in affinity to the HA peptide but still did not reconstitute the fully folded state. Despite failing to fold completely, peptide binding by computational design was observed and was improved by directed evolution. The ratio of HA to S7 binding increased from 0.0 for the wild-type sequence (no binding) to 0.01 after computational design (weak binding) and to 0.48 (comparable binding) after in vitro evolution. The novel oligomeric state is composed of an open barrel.

Application of high-throughput mini-bioreactor system for systematic scale-down modeling, process characterization, and control strategy development.

High-throughput systems and processes have typically been targeted for process development and optimization in the bioprocessing industry. For process characterization, bench scale bioreactors have been the system of choice. Due to the need for performing different process conditions for multiple process parameters, the process characterization studies typically span several months and are considered time and resource intensive. In this study, we have shown the application of a high-throughput mini-bioreactor system viz. the Advanced Microscale Bioreactor (ambr15(TM) ), to perform process characterization in less than a month and develop an input control strategy. As a pre-requisite to process characterization, a scale-down model was first developed in the ambr system (15 mL) using statistical multivariate analysis techniques that showed comparability with both manufacturing scale (15,000 L) and bench scale (5 L). Volumetric sparge rates were matched between ambr and manufacturing scale, and the ambr process matched the pCO2 profiles as well as several other process and product quality parameters. The scale-down model was used to perform the process characterization DoE study and product quality results were generated. Upon comparison with DoE data from the bench scale bioreactors, similar effects of process parameters on process yield and product quality were identified between the two systems. We used the ambr data for setting action limits for the critical controlled parameters (CCPs), which were comparable to those from bench scale bioreactor data. In other words, the current work shows that the ambr15(TM) system is capable of replacing the bench scale bioreactor system for routine process development and process characterization.

Advanced process monitoring and feedback control to enhance cell culture process production and robustness.

It is a common practice in biotherapeutic manufacturing to define a fixed-volume feed strategy for nutrient feeds, based on historical cell demand. However, once the feed volumes are defined, they are inflexible to batch-to-batch variations in cell growth and physiology and can lead to inconsistent productivity and product quality. In an effort to control critical quality attributes and to apply process analytical technology (PAT), a fully automated cell culture feedback control system has been explored in three different applications. The first study illustrates that frequent monitoring and automatically controlling the complex feed based on a surrogate (glutamate) level improved protein production. More importantly, the resulting feed strategy was translated into a manufacturing-friendly manual feed strategy without impact on product quality. The second study demonstrates the improved process robustness of an automated feed strategy based on online bio-capacitance measurements for cell growth. In the third study, glucose and lactate concentrations were measured online and were used to automatically control the glucose feed, which in turn changed lactate metabolism. These studies suggest that the auto-feedback control system has the potential to significantly increase productivity and improve robustness in manufacturing, with the goal of ensuring process performance and product quality consistency.

Perfusion seed cultures improve biopharmaceutical fed-batch production capacity and product quality.

Volumetric productivity and product quality are two key performance indicators for any biopharmaceutical cell culture process. In this work, we showed proof-of-concept for improving both through the use of alternating tangential flow perfusion seed cultures coupled with high-seed fed-batch production cultures. First, we optimized the perfusion N-1 stage, the seed train bioreactor stage immediately prior to the production bioreactor stage, to minimize the consumption of perfusion media for one CHO cell line and then successfully applied the optimized perfusion process to a different CHO cell line. Exponential growth was observed throughout the N-1 duration, reaching >40 × 10(6) vc/mL at the end of the perfusion N-1 stage. The cultures were subsequently split into high-seed (10 × 10(6) vc/mL) fed-batch production cultures. This strategy significantly shortened the culture duration. The high-seed fed-batch production processes for cell lines A and B reached 5 g/L titer in 12 days, while their respective low-seed processes reached the same titer in 17 days. The shortened production culture duration potentially generates a 30% increase in manufacturing capacity while yielding comparable product quality. When perfusion N-1 and high-seed fed-batch production were applied to cell line C, higher levels of the active protein were obtained, compared to the low-seed process. This, combined with correspondingly lower levels of the inactive species, can enhance the overall process yield for the active species. Using three different CHO cell lines, we showed that perfusion seed cultures can optimize capacity utilization and improve process efficiency by increasing volumetric productivity while maintaining or improving product quality.

Endothelin-1 stimulates resistin gene expression.

Resistin and endothelin (ET)-1 have been reported to inhibit adipogenesis and regulate adipocyte insulin resistance, respectively. Although both hormones interact with each other, the exact signaling pathway of ET-1 to act on resistin gene expression is still unknown. Using 3T3-L1 adipocytes, we investigated the signaling pathways involved in ET-1-stimulated resistin gene expression. The up-regulation of resistin mRNA expression by ET-1 depends on concentration and timing. The concentration of ET-1 that increased resistin mRNA levels by 100%-250% was approximately 100 nM for a range of 0.25-12 hours of treatment. Treatment with actinomycin D blocked ET-1-increased resistin mRNA levels, suggesting that the effect of ET-1 requires new mRNA synthesis. Treatment with an inhibitor of the ET type-A receptor, such as N-[1-Formyl-N-[N-[(hexahydro-1H-azepin-1-yl)carbonyl]-L-leucyl]-D-tryptophyl]-D-tryptophan (BQ610), but not with the ET type-B receptor antagonist N-[(cis-2,6-Dimethyl-1-piperidinyl)carbonyl]-4-methyl-L-leucyl-1-(methoxycarbonyl)-D-tryptophyl-D-norleucine (BQ788), blocked ET-1, increased the levels of resistin mRNA, and phosphorylated levels of downstream signaling molecules, such as ERK1/2, c-Jun N-terminal kinases (JNKs), protein kinase B (AKT), and signal transducer and activator of transcription 3 (STAT3). Moreover, pretreatment of specific inhibitors of either ERK1/2 (1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene [U0126] and 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one [PD98059], two inhibitors of MEK1), JNKs (SP600125), phosphatidylinositol 3-kinase/AKT (LY294002 and Wortmannin), or Janus kinase 2 (JAK2)/STAT3 ((E)-2-Cyano-3-(3,4-dihydrophenyl)-N-(phenylmethyl)-2-propenamide, AG490) prevented ET-1-increased levels of resistin mRNA and reduced the ET-1-stimulated phosphorylation of ERK1/2, JNKs, AKT, and STAT3, respectively. However, the p38 kinase antagonist 4-[5-(4-Fluorophenyl)-2-[4-(methylsulfonyl)phenyl]-1H-imidazol-4-yl]pyridine (SB203580) did not alter the effect of ET-1. These results imply that ET type-A receptor, ERK1/2, JNKs, AKT, and JAK2, but not ET type-B receptor or p38, are necessary for the ET-1 stimulation of resistin gene expression. In vivo observations that ET-1 increased resistin mRNA and protein levels in sc and epididymal adipose tissues support the in vitro findings.

Addition of valproic acid to CHO cell fed-batch cultures improves monoclonal antibody titers.

Improving the productivity of a biopharmaceutical Chinese hamster ovary (CHO) fed-batch cell culture can enable cost savings and more efficient manufacturing capacity utilization. One method for increasing CHO cell productivity is the addition of histone deacetylase (HDAC) inhibitors to the cell culture process. In this study, we examined the effect of valproic acid (VPA, 2-propylpentanoic acid), a branched-chain carboxylic acid HDAC inhibitor, on the productivity of three of our CHO cell lines that stably express monoclonal antibodies. Fed-batch shake flask VPA titrations on the three different CHO cell lines yielded cell line-specific results. Cell line A responded highly positively, cell line B responded mildly positively, and cell line C did not respond. We then performed factorial experiments to identify the optimal VPA concentration and day of addition for cell line A. After identifying the optimal conditions for cell line A, we performed verification experiments in fed-batch bioreactors for cell lines A and B. These experiments confirmed that a high dose of VPA late in the culture can increase harvest titer >20 % without greatly changing antibody aggregation, charge heterogeneity, and N-linked glycosylation profiles. Our results suggest that VPA is an attractive and viable small molecule enhancer of protein production for biopharmaceutical CHO cell culture processes.

Green-lighting green fluorescent protein: faster and more efficient folding by eliminating a cis-trans peptide isomerization event.

Wild-type green fluorescent protein (GFP) folds on a time scale of minutes. The slow step in folding is a cis-trans peptide bond isomerization. The only conserved cis-peptide bond in the native GFP structure, at P89, was remodeled by the insertion of two residues, followed by iterative energy minimization and side chain design. The engineered GFP was synthesized and found to fold faster and more efficiently than its template protein, recovering 50% more of its fluorescence upon refolding. The slow phase of folding is faster and smaller in amplitude, and hysteresis in refolding has been eliminated. The elimination of a previously reported kinetically trapped state in refolding suggests that X-P89 is trans in the trapped state. A 2.55 Å resolution crystal structure revealed that the new variant contains only trans-peptide bonds, as designed. This is the first instance of a computationally remodeled fluorescent protein that folds faster and more efficiently than wild type.

Improving computational efficiency and tractability of protein design using a piecemeal approach. A strategy for parallel and distributed protein design.

MOTIVATION: Accuracy in protein design requires a fine-grained rotamer search, multiple backbone conformations, and a detailed energy function, creating a burden in runtime and memory requirements. A design task may be split into manageable pieces in both three-dimensional space and in the rotamer search space to produce small, fast jobs that are easily distributed. However, these jobs must overlap, presenting a problem in resolving conflicting solutions in the overlap regions. RESULTS: Piecemeal design, in which the design space is split into overlapping regions and rotamer search spaces, accelerates the design process whether jobs are run in series or in parallel. Large jobs that cannot fit in memory were made possible by splitting. Accepting the consensus amino acid selection in conflict regions led to non-optimal choices. Instead, conflicts were resolved using a second pass, in which the split regions were re-combined and designed as one, producing results that were closer to optimal with a minimal increase in runtime over the consensus strategy. Splitting the search space at the rotamer level instead of at the amino acid level further improved the efficiency by reducing the search space in the second pass. AVAILABILITY AND IMPLEMENTATION: Programs for splitting protein design expressions are available at www.bioinfo.rpi.edu/tools/piecemeal.html CONTACT: bystrc@rpi.edu Supplementary information: Supplementary data are available at Bioinformatics online.

Directed evolution of the quorum-sensing regulator EsaR for increased signal sensitivity.

The use of cell-cell communication or "quorum sensing (QS)" elements from Gram-negative Proteobacteria has enabled synthetic biologists to begin engineering systems composed of multiple interacting organisms. However, additional tools are necessary if we are to progress toward synthetic microbial consortia that exhibit more complex, dynamic behaviors. EsaR from Pantoea stewartii subsp. stewartii is a QS regulator that binds to DNA as an apoprotein and releases the DNA when it binds to its cognate signal molecule, 3-oxohexanoyl-homoserine lactone (3OC6HSL). In the absence of 3OC6HSL, EsaR binds to DNA and can act as either an activator or a repressor of transcription. Gene expression from P(esaR), which is repressed by wild-type EsaR, requires 100- to 1000-fold higher concentrations of signal than commonly used QS activators, such as LuxR and LasR. Here we have identified EsaR variants with increased sensitivity to 3OC6HSL using directed evolution and a dual ON/OFF screening strategy. Although we targeted EsaR-dependent derepression of P(esaR), our EsaR variants also showed increased 3OC6HSL sensitivity at a second promoter, P(esaS), which is activated by EsaR in the absence of 3OC6HSL. Here, the increase in AHL sensitivity led to gene expression being turned off at lower concentrations of 3OC6HSL. Overall, we have increased the signal sensitivity of EsaR more than 70-fold and generated a set of EsaR variants that recognize 3OC6HSL concentrations ranging over 4 orders of magnitude. QS-dependent transcriptional regulators that bind to DNA and are active in the absence of a QS signal represent a new set of tools for engineering cell-cell communication-dependent gene expression.

Expanded explorations into the optimization of an energy function for protein design.

Nature possesses a secret formula for the energy as a function of the structure of a protein. In protein design, approximations are made to both the structural representation of the molecule and to the form of the energy equation, such that the existence of a general energy function for proteins is by no means guaranteed. Here, we present new insights toward the application of machine learning to the problem of finding a general energy function for protein design. Machine learning requires the definition of an objective function, which carries with it the implied definition of success in protein design. We explored four functions, consisting of two functional forms, each with two criteria for success. Optimization was carried out by a Monte Carlo search through the space of all variable parameters. Cross-validation of the optimized energy function against a test set gave significantly different results depending on the choice of objective function, pointing to relative correctness of the built-in assumptions. Novel energy cross terms correct for the observed nonadditivity of energy terms and an imbalance in the distribution of predicted amino acids. This paper expands on the work presented at the 2012 ACM-BCB.

Endothelin-1 stimulates suppressor of cytokine signaling-3 gene expression in adipocytes.

Endothelin (ET)-1 and suppressor of cytokine signaling (SOCS)-3 were respectively found to regulate energy metabolism and hormone signaling in fat cells. Although ET-1 can also regulate the expression of SOCS-3-stimulating hormones, it is still unknown whether ET-1 regulates SOCS-3 gene expression. This study investigated the pathways involved in ET-1's modulation of SOCS-3 gene expression in 3T3-L1 adipocytes. ET-1 upregulated SOCS-3 mRNA and protein expression in dose- and time-dependent manners. The concentration of ET-1 that increased SOCS-3 mRNA levels by 250-400% was ∼100nM with 2-4h of treatment. Treatment with actinomycin D prevented ET-1-stimulated SOCS-3 mRNA expression, suggesting that the effect of ET-1 requires new mRNA synthesis. Pretreatment with the ET type A receptor (ET(A)R) antagonist, BQ-610, but not the ET type B receptor (ET(B)R) antagonist, BQ-788, prevented the stimulatory effect of ET-1 on SOCS-3 gene expression. The specific inhibitors of either MEK1 (U-0126 and PD-98059), JAK (AG-490), JNK (SP-600125), or PI3K (LY-294002 and wortmannin) reduced ET-1-increased levels of SOCS-3 mRNA and respectively inhibited ET-1-stimulated activities of MEK1, JAK, JNK, and PI3K. These results imply that the ET(A)R, ERK, JAK, JNK, and PI3K are functionally necessary for ET-1's stimulation of SOCS-3 gene expression. Moreover, ET-1 was observed to upregulate expressions of SOCS-1, -2, -3, -4, -5, and -6 mRNAs, but not SOCS-7 or cytokine-inducible SH2-containing protein-1 mRNAs. This suggests that ET-1 selectively affects particular types of SOCS family members. Changes in SOCS gene expressions induced by ET-1 may help explain the mechanism by which ET-1 modulates hormone signaling of adipocytes.

Quantitative in vivo solubility and reconstitution of truncated circular permutants of green fluorescent protein.

Several versions of split green fluorescent protein (GFP) fold and reconstitute fluorescence, as do many circular permutants, but little is known about the dependence of reconstitution on circular permutation. Explored here is the capacity of GFP to fold and reconstitute fluorescence from various truncated circular permutants, herein called "leave-one-outs" using a quantitative in vivo solubility assay and in vivo reconstitution of fluorescence. Twelve leave-one-out permutants are discussed, one for each of the 12 secondary structure elements. The results expand the outlook for the use of permuted split GFPs as specific and self-reporting gene encoded affinity reagents.