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Several candidate HIV subunit vaccines based on recombinant envelope (Env) glycoproteins have been advanced into human clinical trials. To facilitate biopharmaceutical production, it is necessary to produce these in CHO (Chinese Hamster Ovary) cells, the cellular substrate used for the manufacturing of most recombinant protein therapeutics. However, previous studies have shown that when recombinant Env proteins from clade B viruses, the major subtype represented in North America, Europe, and other parts of the world, are expressed in CHO cells, they are proteolyzed and lack important glycan-dependent epitopes present on virions. Previously, we identified C1s, a serine protease in the complement pathway, as the endogenous CHO protease responsible for the cleavage of clade B laboratory isolates of -recombinant gp120s (rgp120s) expressed in stable CHO-S cell lines. In this paper, we describe the development of two novel CHOK1 cell lines with the C1s gene inactivated by gene editing, that are suitable for the production of any protein susceptible to C1s proteolysis. One cell line, C1s-/- CHOK1 2.E7, contains a deletion in the C1s gene. The other cell line, C1s-/- MGAT1- CHOK1 1.A1, contains a deletion in both the C1s gene and the MGAT1 gene, which limits glycosylation to mannose-5 or earlier intermediates in the N-linked glycosylation pathway. In addition, we compare the substrate specificity of C1s with thrombin on the cleavage of both rgp120 and human Factor VIII, two recombinant proteins known to undergo unintended proteolysis (clipping) when expressed in CHO cells. Finally, we demonstrate the utility and practicality of the C1s-/- MGAT1- CHOK1 1.A1 cell line for the expression of clinical isolates of clade B Envs from rare individuals that possess broadly neutralizing antibodies and are able to control virus replication without anti-retroviral drugs (elite neutralizer/controller phenotypes). The Envs represent unique HIV vaccine immunogens suitable for further immunogenicity and efficacy studies.

The electromotility of outer hair cell is considered to be based on voltage-dependent conformational changes in the motor protein prestin. The structure and function of prestin have been increasingly examined in recent years. To obtain further information on prestin, a method to stably obtain prestin as the material for this research is required. This study attempted to construct a stable expression system for prestin on Chinese hamster ovary (CHO) cells and its function was evaluated. His-tagged prestin expression vectors were transfected into CHO cells and high-expression clones were obtained by drug selection and limiting dilution method. The expression and activity of prestin were examined by Western blotting and immunofluorescent experiments and by patch clamping. Clones with high fluorescent intensity tended to exhibit bell-shaped non-linear capacitance, suggesting the active function of prestin in such clones. The stable expression and activity of prestin in CHO cells were confirmed in 7 clones out of twelve clones developed. Although expression levels were 1/60-1/30 of those in OHCs, cell line maintaining the stable expression of functional prestin with His tag will be advantageous for future analyses of its structure and function., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Motoo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The sweet taste of saccharides, such as sucrose and glucose, and other sweeteners is known to result from activation of the TAS1R2/R3 receptor expressed in taste receptor cells (TRCs) of the taste bud. Recent reports have suggested the existence of an additional sweet taste signaling pathway for metabolizable saccharides that is dependent on the activity of glucose transporters, especially SGLT1, also expressed in TRCs. We have investigated the potential contribution of SGLT1 to glucose taste signaling in humans. Concentration-response analysis of glucose mediated changes in membrane potential measured in Chinese hamster ovary (CHO) cells transiently expressing the human SGLT1 (hSGLT1) yielded an EC50 value of 452 μM. The SGLT inhibitor phlorizin inhibited the membrane potential response to 10 mM glucose with an IC50 of 3.5 μM. In contrast, EC50 values of 127 and 132 mM were obtained from concentration-response analysis of glucose taste in vehicles of water or 20 mM NaCl, respectively, by rapid throughput taste discrimination with human subjects. Lactisole, an antagonist of TAS1R2/R3, at a concentration of 1 mM completely inhibited taste responses to glucose concentrations of 250 mM and below. Phlorizin (0.2 mM) and the high potency SGLT1-selective inhibitor mizagliflozin (10 μM) failed to inhibit glucose taste detection measured at peri-threshold concentrations in the rapid throughput taste discrimination assay. A Yes/No experiment using the taste discrimination assay revealed that 0.2 mM phlorizin was discriminable from water for some subjects. Taken together the results indicate that agonist activation of TAS1R2/R3 is sufficient to account for all glucose taste without contribution by an alternative SGLT-mediated signaling pathway. Furthermore, the taste of phlorizin could be a confounding variable for studies evaluating a role for SGLTs in taste., Competing Interests: The rapid throughput taste discrimination technology used in the human taste studies also is used for commercial purposes by Opertech Bio (TāStation®). RKP is a co-founder and stockholder of Opertech Bio, which independently funded the human taste studies. RKP is named as inventor on patents for the rapid throughput taste discrimination technology. This does not alter our adherence to PLOS ONE policies on sharing data and materials., (Copyright: © 2024 Palmer et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The production of envelope glycoproteins (Envs) for use as HIV vaccines is challenging. The yield of Envs expressed in stable Chinese Hamster Ovary (CHO) cell lines is typically 10-100 fold lower than other glycoproteins of pharmaceutical interest. Moreover, Envs produced in CHO cells are typically enriched for sialic acid containing glycans compared to virus associated Envs that possess mainly high-mannose carbohydrates. This difference alters the net charge and biophysical properties of Envs and impacts their antigenic structure. Here we employ a novel robotic cell line selection strategy to address the problems of low expression. Additionally, we employed a novel gene-edited CHO cell line (MGAT1- CHO) to address the problems of high sialic acid content, and poor antigenic structure. We demonstrate that stable cell lines expressing high levels of gp120, potentially suitable for biopharmaceutical production can be created using the MGAT1- CHO cell line. Finally, we describe a MGAT1- CHO cell line expressing A244-rgp120 that exhibits improved binding of three major families of bN-mAbs compared to Envs produced in normal CHO cells. The new strategy described has the potential to eliminate the bottleneck in HIV vaccine development that has limited the field for more than 25 years.

We have reported that integrins crosstalk with growth factors through direct binding to growth factors (e.g., fibroblast growth factor-1, insulin-like growth factor 1 (IGF1), neuregulin-1, fractalkine) and subsequent ternary complex formation with cognate receptor [e.g., integrin/IGF1/IGF1 receptor (IGF1R)]. IGF1 and IGF2 are overexpressed in cancer and major therapeutic targets. We previously reported that IGF1 binds to integrins ανβ3 and α6β4, and the R36E/R37E mutant in the C-domain of IGF1 is defective integrin binding and signaling functions of IGF1, and acts as an antagonist of IGF1R. We studied if integrins play a role in the signaling functions of IGF2, another member of the IGF family. Here we describe that IGF2 specifically binds to integrins ανβ3 and α6β4, and induced proliferation of CHO cells (IGF1R+) that express ανβ3 or α6β4 (β3- or α6β4-CHO cells). Arg residues to Glu at positions 24, 34, 37 and/or 38 in or close to the C-domain of IGF2 play a critical role in binding to integrins and signaling functions. The R24E/R37E/R38E, R34E/R37E/R38E, and R24E/R34E/R37E/R38E mutants were defective in integrin binding and IGF2 signaling. These mutants suppressed proliferation induced by WT IGF2, suggesting that they are dominant-negative antagonists of IGF1R. These results suggest that IGF2 also requires integrin binding for signaling functions, and the IGF2 mutants that cannot bind to integrins act as antagonists of IGF1R. The present study defines the role of the C-domain in integrin binding and signaling.

The primate-specific chemokine CCL18 is a potent chemoattractant for T cells and is expressed at elevated levels in several inflammatory diseases. However, the cognate receptor for CCL18 remains unconfirmed. Here, we describe attempts to validate a previous report that the chemokine receptor CCR8 is the human CCL18 receptor (Islam et al. J Exp Med. 2013, 210:1889-98). Two mouse pre-B cell lines (4DE4 and L1.2) exogenously expressing CCR8 exhibited robust migration in response to the known CCR8 ligand CCL1 but not to CCL18. Similarly, CCL1 but not CCL18 induced internalization of CCR8 on 4DE4 cells. CCR8 expressed on Chinese hamster ovarian (CHO) cells mediated robust G protein activation, inhibition of cAMP synthesis and β-arrestin2 recruitment in response to CCL1 but not CCL18. Several N- and C-terminal variants of CCL18 also failed to stimulate CCR8 activation. On the other hand, and as previously reported, CCL18 inhibited CCL11-stimulated migration of 4DE4 cells expressing the receptor CCR3. These data suggest that CCR8, at least in the absence of unidentified cofactors, does not function as a high affinity receptor for CCL18., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Hussain et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a common genetic disease that leads to progressive renal cyst growth and loss of renal function, and is caused by mutations in the genes encoding polycystin-1 (PC1) and polycystin-2 (PC2), respectively. The PC1/PC2 complex localizes to primary cilia and can act as a flow-dependent calcium channel in addition to numerous other signaling functions. The exact functions of the polycystins, their regulation and the purpose of the PC1/PC2 channel are still poorly understood. PC1 is an integral membrane protein with a large extracytoplasmic N-terminal domain and a short, ~200 amino acid C-terminal cytoplasmic tail. Most proteins that interact with PC1 have been found to bind via the cytoplasmic tail. Here we report that the PC1 tail has homology to the regulatory domain of myosin heavy chain including a conserved calmodulin-binding motif. This motif binds to CaM in a calcium-dependent manner. Disruption of the CaM-binding motif in PC1 does not affect PC2 binding, cilia targeting, or signaling via heterotrimeric G-proteins or STAT3. However, disruption of CaM binding inhibits the PC1/PC2 calcium channel activity and the flow-dependent calcium response in kidney epithelial cells. Furthermore, expression of CaM-binding mutant PC1 disrupts cellular energy metabolism. These results suggest that critical functions of PC1 are regulated by its ability to sense cytosolic calcium levels via binding to CaM.

Ferritin is an iron-storage protein composed of different ratios of 24 light (L) and heavy (H) subunits. The serum level of ferritin is a clinical marker of the body's iron level. Transferrin receptor (TFR)1 is the receptor not only for transferrin but also for H-ferritin, but how it binds two different ligands and the blood cell types that preferentially incorporate H-ferritin remain unknown. To address these questions, we investigated hematopoietic cell-specific ferritin uptake by flow cytometry. Alexa Fluor 488-labeled H-ferritin was preferentially incorporated by erythroid cells among various hematopoietic cell lines examined, and was almost exclusively incorporated by bone marrow erythroblasts among human primary hematopoietic cells of various lineages. H-ferritin uptake by erythroid cells was strongly inhibited by unlabeled H-ferritin but was only partially inhibited by a large excess of holo-transferrin. On the other hand, internalization of labeled holo-transferrin by these cells was not inhibited by H-ferritin. Chinese hamster ovary cells lacking functional endogenous TFR1 but expressing human TFR1 with a mutated RGD sequence, which is required for transferrin binding, efficiently incorporated H-ferritin, indicating that TFR1 has distinct binding sites for H-ferritin and holo-transferrin. H-ferritin uptake by these cells required a threshold level of cell surface TFR1 expression, whereas there was no threshold for holo-transferrin uptake. The requirement for a threshold level of TFR1 expression can explain why among primary human hematopoietic cells, only erythroblasts efficiently take up H-ferritin.

The chemokine domain of fractalkine (FKN-CD) binds to the classical RGD-binding site of αvβ3 and that the resulting ternary complex formation (integrin-FKN-CX3CR1) is critical for CX3CR1 signaling and FKN-induced integrin activation. However, only certain cell types express CX3CR1. Here we studied if FKN-CD can activate integrins in the absence of CX3CR1. We describe that WT FKN-CD activated recombinant soluble αvβ3 in cell-free conditions, but the integrin-binding defective mutant of FKN-CD (K36E/R37E) did not. This suggests that FKN-CD can activate αvβ3 in the absence of CX3CR1 through the direct binding of FKN-CD to αvβ3. WT FKN-CD activated αvβ3 on CX3CR1-negative cells (K562 and CHO) but K36E/R37E did not, suggesting that FKN-CD can activate integrin at the cellular levels in a manner similar to that in cell-free conditions. We hypothesized that FKN-CD enhances ligand binding to the classical RGD-binding site (site 1) through binding to a second binding site (site 2) that is distinct from site 1 in αvβ3. To identify the possible second FKN-CD binding site we performed docking simulation of αvβ3-FKN-CD interaction using αvβ3 with a closed inactive conformation as a target. The simulation predicted a potential FKN-CD-binding site in inactive αvβ3 (site 2), which is located at a crevice between αv and β3 on the opposite side of site 1 in the αvβ3 headpiece. We studied if FKN-CD really binds to site 2 using a peptide that is predicted to interact with FKN-CD in site 2. Notably the peptide specifically bound to FKN-CD and effectively suppressed integrin activation by FKN-CD. This suggests that FKN-CD actually binds to site 2, and this leads to integrin activation. We obtained very similar results in α4β1 and α5β1. The FKN binding to site 2 and resulting integrin activation may be a novel mechanism of integrin activation and of FKN signaling.

The prototypic acute phase reactant C-reactive protein (CRP) is not only a marker but also a potential contributor to inflammatory diseases. CRP exists as the circulating native, pentameric CRP (pCRP) and the monomeric isoform (mCRP), formed as a result of a dissociation process of pCRP. mCRP is highly pro-inflammatory, but pCRP is not. The mechanism of pro-inflammatory action of mCRP is unclear. We studied the role of integrins in pro-inflammatory action of mCRP. Docking simulation of interaction between mCRP and integrin αvβ3 predicted that mCRP binds to αvβ3 well. We found that mCRP actually bound to integrins αvβ3 and α4β1 well. Antagonists to αvβ3 or α4β1 effectively suppressed the interaction, suggesting that the interaction is specific. Using an integrin β1 mutant (β1-3-1) that has a small fragment from the ligand binding site of β3, we showed that mCRP bound to the classical RGD-binding site in αvβ3. We studied the role of integrins in CRP signaling in monocytic U937 cells. Integrins αvβ3 and α4β1 specifically mediated binding of mCRP to U937 cells. mCRP induced AKT phosphorylation, but not ERK1/2 phosphorylation, in U937 cells. Notably, mCRP induced robust chemotaxis in U937 cells, and antagonists to integrins αvβ3 and α4β1 and an inhibitor to phosphatidylinositide 3-kinase, but not an MEK inhibitor, effectively suppressed mCRP-induced chemotaxis in U937 cells. These results suggest that the integrin and AKT/phosphatidylinositide 3-kinase pathways play a role in pro-inflammatory action of mCRP in U937 cells. In contrast, pCRP is predicted to have a limited access to αvβ3 due to steric hindrance in the simulation. Consistent with the prediction, pCRP was much less effective in integrin binding, chemotaxis, or AKT phosphorylation. These findings suggest that the ability of CRP isoforms to bind to the integrins is related to their pro-inflammatory action.

It has been documented that variations in glycosylation on glycoprotein hormones, confer distinctly different biological features to the corresponding glycoforms when multiple in vitro biochemical readings are analyzed. We here applied next generation RNA sequencing to explore changes in the transcriptome of rat granulosa cells exposed for 0, 6, and 12 h to 100 ng/ml of four highly purified follicle-stimulating hormone (FSH) glycoforms, each exhibiting different glycosylation patterns: a. human pituitary FSH18/21 (hypo-glycosylated); b. human pituitary FSH24 (fully glycosylated); c. Equine FSH (eqFSH) (hypo-glycosylated); and d. Chinese-hamster ovary cell-derived human recombinant FSH (recFSH) (fully-glycosylated). Total RNA from triplicate incubations was prepared from FSH glycoform-exposed cultured granulosa cells obtained from DES-pretreated immature female rats, and RNA libraries were sequenced in a HighSeq 2500 sequencer (2 x 125 bp paired-end format, 10-15 x 106 reads/sample). The computational workflow focused on investigating differences among the four FSH glycoforms at three levels: gene expression, enriched biological processes, and perturbed pathways. Among the top 200 differentially expressed genes, only 4 (0.6%) were shared by all 4 glycoforms at 6 h, whereas 118 genes (40%) were shared at 12 h. Follicle-stimulating hormone glycocoforms stimulated different patterns of exclusive and associated up regulated biological processes in a glycoform and time-dependent fashion with more shared biological processes after 12 h of exposure and fewer treatment-specific ones, except for recFSH, which exhibited stronger responses with more specifically associated processes at this time. Similar results were found for down-regulated processes, with a greater number of processes at 6 h or 12 h, depending on the particular glycoform. In general, there were fewer downregulated than upregulated processes at both 6 h and 12 h, with FSH18/21 exhibiting the largest number of down-regulated associated processes at 6 h while eqFSH exhibited the greatest number at 12 h. Signaling cascades, largely linked to cAMP-PKA, MAPK, and PI3/AKT pathways were detected as differentially activated by the glycoforms, with each glycoform exhibiting its own molecular signature. These data extend previous observations demonstrating glycosylation-dependent distinctly different regulation of gene expression and intracellular signaling pathways triggered by FSH in granulosa cells. The results also suggest the importance of individual FSH glycoform glycosylation for the conformation of the ligand-receptor complex and induced signalling pathways., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Zariñán et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

All subtypes of KCNQ channel subunits (KCNQ1-5) require calmodulin as a co-factor for functional channels. It has been demonstrated that calmodulin plays a critical role in KCNQ channel trafficking as well as calcium-mediated current modulation. However, how calcium-bound calmodulin suppresses the M-current is not well understood. In this study, we investigated the molecular mechanism of KCNQ2 current suppression mediated by calcium-bound calmodulin. We show that calcium induced slow calmodulin dissociation from the KCNQ2 channel subunit. In contrast, in homomeric KCNQ3 channels, calcium facilitated calmodulin binding. We demonstrate that this difference in calmodulin binding was due to the unique cysteine residue in the KCNQ2 subunit at aa 527 in Helix B, which corresponds to an arginine residue in other KCNQ subunits including KCNQ3. In addition, a KCNQ2 channel associated protein AKAP79/150 (79 for human, 150 for rodent orthologs) also preferentially bound calcium-bound calmodulin. Therefore, the KCNQ2 channel complex was able to retain calcium-bound calmodulin either through the AKPA79/150 or KCNQ3 subunit. Functionally, increasing intracellular calcium by ionomycin suppressed currents generated by KCNQ2, KCNQ2(C527R) or heteromeric KCNQ2/KCNQ3 channels to an equivalent extent. This suggests that a change in the binding configuration, rather than dissociation of calmodulin, is responsible for KCNQ current suppression. Furthermore, we demonstrate that KCNQ current suppression was accompanied by reduced KCNQ affinity toward phosphatidylinositol 4,5-bisphosphate (PIP2) when assessed by a voltage-sensitive phosphatase, Ci-VSP. These results suggest that a rise in intracellular calcium induces a change in the configuration of CaM-KCNQ binding, which leads to the reduction of KCNQ affinity for PIP2 and subsequent current suppression.

The genetic expression of cloned fluorescent proteins coupled to time-lapse fluorescence microscopy has opened the door to the direct visualization of a wide range of molecular interactions in living cells. In particular, the dynamic translocation of proteins can now be explored in real time at the single-cell level. Here we propose a reliable, easy-to-implement, quantitative image processing method to assess protein translocation in living cells based on the computation of spatial variance maps of time-lapse images. The method is first illustrated and validated on simulated images of a fluorescently-labeled protein translocating from mitochondria to cytoplasm, and then applied to experimental data obtained with fluorescently-labeled hexokinase 2 in different cell types imaged by regular or confocal microscopy. The method was found to be robust with respect to cell morphology changes and mitochondrial dynamics (fusion, fission, movement) during the time-lapse imaging. Its ease of implementation should facilitate its application to a broad spectrum of time-lapse imaging studies.

NF-κB, a transcription factor, becomes activated during the Unfolded Protein Response (UPR), an endoplasmic reticulum (ER) stress response pathway. NF-κB is normally held inactive by its inhibitor, IκBα. Multiple cellular pathways activate IKK (IκBα Kinase) which phosphorylate IκBα leading to its degradation and NF-κB activation. Here, we find that IKK is required for maximum activation of NF-κB in response to ER stress. However, unlike canonical NFκB activation, IKK activity does not increase during ER stress, but rather the level of basal IKK activity is critical for determining the extent of NF-κB activation. Furthermore, a key UPR initiator, IRE1, acts to maintain IKK basal activity through IRE1's kinase, but not RNase, activity. Inputs from IRE1 and IKK, in combination with translation repression by PERK, another UPR initiator, lead to maximal NF-κB activation during the UPR. These interdependencies have a significant impact in cancer cells with elevated IKK/NF-κB activity such as renal cell carcinoma cells (786-0). Inhibition of IKK by an IKK inhibitor, which significantly decreases NF-κB activity, is overridden by UPR induction, arguing for the importance of considering UPR activation in cancer treatment.

Receptor-tyrosine-kinase-like orphan receptor 1 (ROR1) is expressed during embryogenesis and by certain leukemias, but not by normal adult tissues. Here we show that the neoplastic cells of many human breast cancers express the ROR1 protein and high-level expression of ROR1 in breast adenocarcinoma was associated with aggressive disease. Silencing expression of ROR1 in human breast cancer cell lines found to express this protein impaired their growth in vitro and also in immune-deficient mice. We found that ROR1 could interact with casein kinase 1 epsilon (CK1ε) to activate phosphoinositide 3-kinase-mediated AKT phosphorylation and cAMP-response-element-binding protein (CREB), which was associated with enhanced tumor-cell growth. Wnt5a, a ligand of ROR1, could induce ROR1-dependent signaling and enhance cell growth. This study demonstrates that ROR1 is expressed in human breast cancers and has biological and clinical significance, indicating that it may be a potential target for breast cancer therapy.

BackgroundThe first step in glucose metabolism is conversion of glucose to glucose 6-phosphate (G-6-P) by hexokinases (HKs), a family with 4 isoforms. The two most common isoforms, HKI and HKII, have overlapping tissue expression, but different subcellular distributions, with HKI associated mainly with mitochondria and HKII associated with both mitochondrial and cytoplasmic compartments. Here we tested the hypothesis that these different subcellular distributions are associated with different metabolic roles, with mitochondrially-bound HK's channeling G-6-P towards glycolysis (catabolic use), and cytoplasmic HKII regulating glycogen formation (anabolic use).Methodology/principal findingsTo study subcellular translocation of HKs in living cells, we expressed HKI and HKII linked to YFP in CHO cells. We concomitantly recorded the effects on glucose handling using the FRET based intracellular glucose biosensor, FLIPglu-600 mM, and glycogen formation using a glycogen-associated protein, PTG, tagged with GFP. Our results demonstrate that HKI remains strongly bound to mitochondria, whereas HKII translocates between mitochondria and the cytosol in response to glucose, G-6-P and Akt, but not ATP. Metabolic measurements suggest that HKI exclusively promotes glycolysis, whereas HKII has a more complex role, promoting glycolysis when bound to mitochondria and glycogen synthesis when located in the cytosol. Glycogen breakdown upon glucose removal leads to HKII inhibition and dissociation from mitochondria, probably mediated by increases in glycogen-derived G-6-P.Conclusions/significanceThese findings show that the catabolic versus anabolic fate of glucose is dynamically regulated by extracellular glucose via signaling molecules such as intracellular glucose, G-6-P and Akt through regulation and subcellular translocation of HKII. In contrast, HKI, which activity and regulation is much less sensitive to these factors, is mainly committed to glycolysis. This may be an important mechanism by which HK's allow cells to adapt to changing metabolic conditions to maintain energy balance and avoid injury.

Finding unique peptides to target specific biological surfaces is crucial to basic research and technology development, though methods based on biological arrays or large libraries limit the speed and ease with which these necessary compounds can be found. We reasoned that because biological surfaces, such as cell surfaces, mineralized tissues, and various extracellular matrices have unique molecular compositions, they present unique physicochemical signatures to the surrounding medium which could be probed by peptides with appropriately corresponding physicochemical properties. To test this hypothesis, a naïve pilot library of 36 peptides, varying in their hydrophobicity and charge, was arranged in a two-dimensional matrix and screened against various biological surfaces. While the number of peptides in the matrix library was very small, we obtained "hits" against all biological surfaces probed. Sequence refinement of the "hits" led to peptides with markedly higher specificity and binding activity against screened biological surfaces. Genetic studies revealed that peptide binding to bacteria was mediated, at least in some cases, by specific cell-surface molecules, while examination of human tooth sections showed that this method can be used to derive peptides with highly specific binding to human tissue.

BackgroundThe genus Cronobacter (formerly called Enterobacter sakazakii) is composed of five species; C. sakazakii, C. malonaticus, C. turicensis, C. muytjensii, and C. dublinensis. The genus includes opportunistic human pathogens, and the first three species have been associated with neonatal infections. The most severe diseases are caused in neonates and include fatal necrotizing enterocolitis and meningitis. The genetic basis of the diversity within the genus is unknown, and few virulence traits have been identified.Methodology/principal findingsWe report here the first sequence of a member of this genus, C. sakazakii strain BAA-894. The genome of Cronobacter sakazakii strain BAA-894 comprises a 4.4 Mb chromosome (57% GC content) and two plasmids; 31 kb (51% GC) and 131 kb (56% GC). The genome was used to construct a 387,000 probe oligonucleotide tiling DNA microarray covering the whole genome. Comparative genomic hybridization (CGH) was undertaken on five other C. sakazakii strains, and representatives of the four other Cronobacter species. Among 4,382 annotated genes inspected in this study, about 55% of genes were common to all C. sakazakii strains and 43% were common to all Cronobacter strains, with 10-17% absence of genes.Conclusions/significanceCGH highlighted 15 clusters of genes in C. sakazakii BAA-894 that were divergent or absent in more than half of the tested strains; six of these are of probable prophage origin. Putative virulence factors were identified in these prophage and in other variable regions. A number of genes unique to Cronobacter species associated with neonatal infections (C. sakazakii, C. malonaticus and C. turicensis) were identified. These included a copper and silver resistance system known to be linked to invasion of the blood-brain barrier by neonatal meningitic strains of Escherichia coli. In addition, genes encoding for multidrug efflux pumps and adhesins were identified that were unique to C. sakazakii strains from outbreaks in neonatal intensive care units.

We recently introduced a MαCD-based method to efficiently replace virtually the entire population of plasma membrane outer leaflet phospholipids and sphingolipids of cultured mammalian cells with exogenous lipids (Li et al, (2016) Proc. Natl. Acad. Sci USA 113:14025–14030). Here, we show if the lipid-to- MαCD ratio is too high or low, cells can round up and develop membrane leakiness. We found that this cell damage can be reversed/prevented if cells are allowed to recover from the exchange step by incubation in complete growth medium. After exchange and transfer to complete growth medium cell growth was similar to that of untreated cells. In some cases, cell damage was also prevented by carrying out exchange at close to room temperature (rather than at 37°C). Exchange with lipids that do (sphingomyelin) or do not (unsaturated phosphatidylcholine) support a high level of membrane order in lipid vesicles had the analogous effect on plasma membrane order, confirming exogenous lipid localization in the plasma membrane. Importantly, changes in lipid composition and plasma membrane properties after exchange and recovery persisted for several hours. Thus, it should be possible to use lipid exchange to investigate the effect of plasma membrane lipid composition upon several aspects of membrane structure and function. [ABSTRACT FROM AUTHOR]

GPR40/FFAR1 is a Gq protein-coupled receptor expressed in pancreatic β cells and enteroendocrine cells, and mediates insulin and incretin secretion to regulate feeding behavior. Several GPR40 full agonists have been reported to reduce food intake in rodents by regulating gut hormone secretion in addition to their potent glucose-lowering effects; however, detailed mechanisms of feeding suppression are still unknown. In the present study, we characterized T-3601386, a novel compound with potent full agonistic activity for GPR40, by using in vitro Ca2+ mobilization assay in Chinese hamster ovary (CHO) cells expressing FFAR1 and in vivo hormone secretion assay. We also evaluated feeding suppression and weight loss after the administration of T-3601386 and investigated the involvement of the vagal nerve in these effects. T-3601386, but not a partial agonist fasiglifam, increased intracellular Ca2+ levels in CHO cells with low FFAR1 expression, and single dosing of T-3601386 in diet-induced obese (DIO) rats elevated plasma incretin levels, suggesting full agonistic properties of T-3601386 against GPR40. Multiple doses of T-3601386, but not fasiglifam, in DIO rats showed dose-dependent weight loss accompanied by feeding suppression and durable glucagon-like peptide-1 elevation, all of which were completely abolished in Ffar1-/- mice. Immunohistochemical analysis in the nuclei of the solitary tract demonstrated that T-3601386 increased the number of c-Fos positive cells, which also disappeared in Ffar1-/- mice. Surgical vagotomy and drug-induced deafferentation counteracted the feeding suppression and weight loss induced by the administration of T-3601386. These results suggest that T-3601386 exerts incretin release and weight loss in a GPR40-dependent manner, and that afferent vagal nerves are important for the feeding suppression induced by GPR40 full agonism. Our novel findings raise the possibility that GPR40 full agonist can induce periphery-derived weight reduction, which may provide benefits such as less adverse effects in central nervous system compared to centrally-acting anti-obesity drugs. [ABSTRACT FROM AUTHOR]

ND7/23 cells are gaining traction as a host model to express peripheral sodium channels such as NaV1.8 and NaV1.9 that have been difficult to express in widely utilized heterologous cells, like CHO and HEK293. Use of ND7/23 as a model cell to characterize the properties of sodium channels requires clear understanding of the endogenous ion channels. To define the nature of the background sodium currents in ND7/23 cells, we aimed to comprehensively profile the voltage-gated sodium channel subunits by endpoint and quantitative reverse transcription-PCR and by whole-cell patch clamp electrophysiology. We found that untransfected ND7/23 cells express endogenous peak sodium currents that average –2.12nA (n = 15) and with kinetics typical of fast sodium currents having activation and inactivation completed within few milliseconds. Furthermore, sodium currents were reduced to virtually nil upon exposure to 100nM tetrodotoxin, indicating that ND7/23 cells have essentially null background for tetrodotoxin-resistant (TTX-R) currents. qRT-PCR profiling indicated a major expression of TTX-sensitive (TTX-S) NaV1.6 and NaV1.7 at similar levels and very low expression of TTX-R NaV1.9 transcripts. There was no expression of TTX-R NaV1.8 in ND7/23 cells. There was low expression of NaV1.1, NaV1.2, NaV1.3 and no expression of cardiac or skeletal muscle sodium channels. As for the sodium channel auxiliary subunits, β1 and β3 subunits were expressed, but not the β2 and β4 subunits that covalently associate with the α-subunits. In addition, our results also showed that only the mouse forms of NaV1.6, NaV1.7 and NaV1.9 sodium channels were expressed in ND7/23 cells that was originally generated as a hybridoma of rat embryonic DRG and mouse neuroblastoma cell-line. By molecular profiling of auxiliary β- and principal α-subunits of the voltage gated sodium channel complex, our results define the background sodium channels expressed in ND7/23 cells, and confirm their utility for detailed functional studies of emerging pain channelopathies ascribed to mutations of the TTX-R sodium channels of sensory neurons. [ABSTRACT FROM AUTHOR]

As a result of a high-throughput in situ hybridization screening for adult mouse testes, we found that the mRNA for Tmco5 is expressed in round and elongating spermatids. Tmco5 belongs to the Tmco (Transmembrane and coiled-coil domains) gene family and has a coiled-coil domain in the N-terminal and a transmembrane domain in the C-terminal region. A monoclonal antibody raised against recombinant TMCO5 revealed that the protein is expressed exclusively in the elongating spermatids of step 9 to 12 and is localized to the manchette, a transiently emerging construction, which predominantly consists of cytoskeleton microtubules and actin filaments. This structure serves in the transport of Golgi-derived non-acrosomal vesicles. Moreover, induced expression of TMCO5 in CHO cells resulted in the co-localization of TMCO5 with β-tubulin besides the reorganization of the Golgi apparatus. Judging from the results and considering the domain structure of TMCO5, we assume that Tmco5 may have a role in vesicle transport along the manchette. [ABSTRACT FROM AUTHOR]

Recombinant human follicle stimulating hormone (FSH), produced in Chinese hamster ovary (CHO) cells, is widely used for treatment of fertility disorders and is subject to biosimilars development. Cell lines with high specific productivities may simplify the FSH production process. Here, we used our previously established expression system based on vector p1.1 to create new cell lines secreting heterodimeric FSH protein. To this end, we linked open reading frames of both FSH subunits by the wild-type internal ribosome entry site from the encephalomyocarditis virus (EMCV IRES). Intact and double-negative for the dihydrofolate reductase CHO cells were stably transfected by the FSH-coding plasmids. Stably transfected intact cells showed higher level of the FSH secretion and were utilized for subsequent methotrexate-driven transgene amplification, which doubled their productivity. The excess of the free α-subunit was corrected by transfecting the cells by the additional p1.1-based plasmid encoding the β-subunit of the FSH. Clonal cell lines obtained secreted mostly the heterodimeric FSH and possessed specific productivities up to 12.3±1.7 pg/cell/day. Candidate clonal cell line C-P1.3-FSH-G4 maintained a constant specific productivity for at least 2 months of culturing without the section pressure. The resulting FSH protein conformed to the international pharmaceutical quality criteria as evidenced by the receptor binding kinetics, distribution pattern of hormone isoforms and biological activity. In conclusion, our expression system offers a simple and cost-effective approach to production of FSH. [ABSTRACT FROM AUTHOR]

Non-natural amino acids (nnAA) contain unique functional moieties that greatly expand the available tool set for protein engineering. But incorporation of nnAAs requires the function of an orthogonal aminoacyl tRNA synthetase/tRNA pair. Stable cell lines expressing these components have been shown capable of producing gram per liter levels of antibodies with nnAAs. However, little has been reported on the genetic makeup of these cells. To gain a better understanding of the minimal requirements for efficient nnAA incorporation we developed qPCR methods for the quantitation of the key components. Here we describe the development of qPCR assays for the quantification of tRNApyl and pylRS. qPCR was chosen because it provides a large dynamic range, has high specificity for its target, and is a non-radioactive method used routinely for cell line characterization. Designing assays for tRNAs present challenges due to their short length (~72 nucleotides) and high secondary structure. These tRNA assays have a ≥ 5 log dynamic range with the tRNApyl assays being able to discern the mature and unprocessed forms of the tRNApyl. Cell line analysis showed tRNApyl was expressed at higher levels than the CHO-K1 endogenous Met and Phe tRNAs and that >88% of tRNApyl was the mature form. [ABSTRACT FROM AUTHOR]

We describe methods to improve the efficiency with which HIV-1 Envelope glycoprotein SOSIP trimer immunogens can be produced by transient transfection of ExpiCHO-S cells and then affinity purified using the trimer-specific human monoclonal antibody PGT145. The specificity of PGT145 for properly folded trimers allows for the facile, one-step, isolation of these immunogens in research laboratories. PGT145 columns are also valuable as a component of more complex purification processes in current Good Manufacturing Practice programs. However, we found that PGT145 purification was highly variable and markedly inefficient when used to process supernatants from transiently transfected ExpiCHO-S cells expressing the BG505 SOSIP.664 and other trimeric Env proteins. In contrast, no such problems arose when the same Env proteins derived from a stable CHO cell line were processed on the same PGT145 columns, or with transient transfection supernatants from 293F cells. An investigation of the ExpiCHO-S transfection system identified the presence of polyanions, including but perhaps not limited to dextran sulfate, in the Enhancer component of the transfection system. We hypothesized that these polyanions bound to the cationic PGT145 epitope on the trimers and impeded their ability to bind to the PGT145 affinity column. We found that replacing the Enhancer component with alternative culture medium supplements substantially increased the yield of PGT145-purifiable trimers, and we also confirmed that both dextran sulfate and the Enhancer component were indeed inhibitors of PGT145 binding to BG505 SOSIP.664 trimers in immunoassays. The presence of polyanions, including but not limited to nucleic acids, should be considered in other circumstances where PGT145 columns are less efficient than expected at purifying native-like trimers. [ABSTRACT FROM AUTHOR]

Although sphingosine-1-phosphate receptor 1 (S1P1) has been shown to trigger several S1P targeted functions such as immune cell trafficking, cell proliferation, migration, or angiogenesis, tools that allow the accurate detection of endogenous S1P1 localization and trafficking remain to be obtained and validated. In this study, we developed and characterized a novel monoclonal S1P1 antibody. Mice were immunized with S1P1 produced in the yeast Pichia pastoris and nine hybridoma clones producing monoclonal antibodies were created. Using different technical approaches including Western blot, immunoprecipitation and immunocytochemistry, we show that a selected clone, hereinafter referred to as 2B9, recognizes human and mouse S1P1 in various cell lineages. The interaction between 2B9 and S1P1 is specific over receptor subtypes, as the antibody does not binds to S1P2 or S1P5 receptors. Using cell-imaging methods, we demonstrate that 2B9 binds to an epitope located at the intracellular domain of S1P1; reveals cytosolic and membrane localization of the endogenous S1P1; and receptor internalization upon S1P or FTY720-P stimulation. Finally, loss of 2B9 signal upon knockdown of endogenous S1P1 by specific small interference RNAs further confirms its specificity. 2B9 was also able to detect S1P1 in human kidney and spinal cord tissue by immunohistochemistry. Altogether, our results suggest that 2B9 could be a useful tool to detect, quantify or localize low amounts of endogenous S1P1 in various physiological and pathological processes. [ABSTRACT FROM AUTHOR]

C-type natriuretic peptide (CNP) and its receptor natriuretic peptide receptor B (NPR-B) are physiological potent positive regulators of endochondral bone growth; therefore, the CNP/NPR-B signaling pathway is one of the most promising therapeutic targets for treating growth failure and dwarfism. In this article, we summarized the pharmacological properties of a novel CNP analog peptide ASB20123 as a therapeutic agent for short stature. ASB20123, one of the CNP/ghrelin chimeric peptides, is composed of CNP(1–22) and human ghrelin(12–28, E17D). Compared to CNP(1–22), ASB20123 showed similar agonist activity for NPR-B and improved biokinetics with a longer plasma half-life in rats. In addition, the distribution of ASB20123 to the cartilage was higher than that of CNP(1–22) after single subcutaneous (sc) injection to mice. These results suggested that the C-terminal part of ghrelin, which has clusters of basic amino acid residues and a BX7B motif, might contribute to the retention of ASB20123 in the extracellular matrix of the growth plate. Multiple sc doses of ASB20123 potently stimulated skeletal growth in rats in a dose-dependent manner, and sc infusion was more effective than bolus injection at the same dose. Our data indicated that high plasma levels of ASB20123 would not necessarily be required for bone growth acceleration. Thus, pharmaceutical formulation approaches for sustained-release dosage forms to allow chronic exposure to ASB20123 might be suitable to ensure drug effectiveness and safety. [ABSTRACT FROM AUTHOR]

Cyclic Adenosine 3′,5′-monophosphate (cAMP) is a key second messenger known to directly regulate not only the protein kinase A (PKA) activity but also other important molecules such as the exchange protein activated by cAMP (EPAC), which is as a guanine nucleotide exchange factor (GEF) of the low molecular weight GTPase, Rap2. Coxiella burnetii is a Gram negative bacterium that survives and grows in a large Coxiella replicative vacuole (CRV), which displays lysosomal and autophagic features. In this report, we present evidence that both, EPAC and its downstream effector Rap2b, were recruited to the CRV. The transient over-expression of the Rap2b wt protein, but not its inactive mutant Rap2b ΔAAX, markedly inhibited the development of the large CRV. Additionally, Rap2b wtinhibited the fusion of early Coxiella phagosomes with the fully developed CRV, indicating that homotypic fusion events are altered in the presence of high levels of Rap2b wt. Likewise, the fusion of endosome/lysosomal compartments (heterotypic fusions) with the large CRV was also affected by the over-expression of this GTPase. Interestingly, cell overexpression of Rap2b wt markedly decreased the levels of the v-SNARE, Vamp7, suggesting that this down-regulation impairs the homotypic and heterotypic fusions events of the Coxiella vacuole. [ABSTRACT FROM AUTHOR]

Familial Alzheimer’s disease (FAD)-associated presenilin 1 (PS1) serves as a catalytic subunit of γ-secretase complex, which mediates the proteolytic liberation of β-amyloid (Aβ) from β-amyloid precursor protein (APP). In addition to its proteolytic role, PS1 is involved in non-proteolytic functions such as protein trafficking and ion channel regulation. Furthermore, postmortem AD brains as well as AD patients showed dysregulation of cholesterol metabolism. Since cholesterol has been implicated in regulating Aβ production, we investigated whether the FAD PS1-associated cholesterol elevation could influence APP processing. We found that in CHO cells stably expressing FAD-associated PS1 ΔE9, total cholesterol levels are elevated compared to cells expressing wild-type PS1. We also found that localization of APP in cholesterol-enriched lipid rafts is substantially increased in the mutant cells. Reducing the cholesterol levels by either methyl-β-cyclodextrin or an inhibitor of CYP51, an enzyme mediating the elevated cholesterol in PS1 ΔE9-expressing cells, significantly reduced lipid raft-associated APP. In contrast, exogenous cholesterol increased lipid raft-associated APP. These data suggest that in the FAD PS1 ΔE9 cells, the elevated cellular cholesterol level contributes to the altered APP processing by increasing APP localized in lipid rafts. [ABSTRACT FROM AUTHOR]

Cell culture process optimization is a critical solution to most of the challenges faced by the pharmaceutical manufacturing. One of the major problems encountered in large-scale production of therapeutic proteins is misfolded protein production. The accumulation of misfolded therapeutic proteins is an immunogenic signal and a risk factor for immunogenicity of the final product. The aim of this study was the statistical optimization of three-phasic temperature shift and timing for enhanced production of correctly folded Fc-fusion protein. The effect of culture temperatures were investigated using the biphasic culture system. Box–Behnken design was then used to compute temperature and time of shifting optimum. Response surface methodology revealed that maximum production with low level of misfolded protein was achieved at two-step temperature shift from 37°C to 30°C during the late logarithmic phase and 30°C to 28°C in the mid-stationary phase. The optimized condition gave the best results of 1860 mg L−1 protein titer with 24.5% misfolding level. The validation experiments were carried out under optimal conditions with three replicates and the protein misfolding level was decreased by two times while productivity increased by ~ 1.3-fold. Large-scale production in 250 L bioreactor under the optimum conditions was also verified the effectiveness and the accuracy of the model. The results showed that by utilizing two-step temperature shift, productivity and the quality of target protein have been improved simultaneously. This model could be successfully applied to other products. [ABSTRACT FROM AUTHOR]

Mutations in superoxide dismutase 1 (SOD1) associated with familial amyotrophic lateral sclerosis (fALS) induce the protein to misfold and aggregate. Missense mutations at more than 80 different amino acid positions have been associated with disease. How these mutations heighten the propensity of SOD1 to misfold and aggregate is unclear. With so many mutations, it is possible that more than one mechanism of aggregation may be involved. Of many possible mechanisms to explain heightened aggregation, one that has been suggested is that mutations that eliminate charged amino acids could diminish repulsive forces that would inhibit aberrant protein:protein interactions. Mutations at twenty-one charged residues in SOD1 have been associated with fALS, but of the 11 Lys residues in the protein, only 1 has been identified as mutated in ALS patients. Here, we examined whether loss of positively charged surface Lys residues in SOD1 would induce misfolding and formation of intracellular inclusions. We mutated four different Lys residues (K30, K36, K75, K91) in SOD1 that are not particularly well conserved, and expressed these variants as fusion proteins with yellow fluorescent protein (YFP) to assess inclusion formation. We also assessed whether these mutations induced binding to a conformation-restricted SOD1 antibody, designated C4F6, which recognizes non-natively folded protein. Although we observed some mutations to cause enhanced C4F6 binding, we did not observe that mutations that reduce charge at these positions caused the protein to form intracellular inclusions. Our findings may have implications for the low frequency of mutations at Lys residues SOD1 in ALS patients. [ABSTRACT FROM AUTHOR]

More than 70 human adenovirus types were identified divided into 7 different species (A-G). Diseases caused by human adenoviruses are type-dependent and can range from mild to severe respiratory infections, gastrointestinal infections or eye infections such as epidemic keratoconjunctivitis. Unfortunately there is no specific anti-adenovirus therapy available. Here we addressed the question whether treatment with cold atmospheric plasma (CAP) for anti-adenoviral therapy such as virus-mediated ulcerations may be feasible. CAP has already been explored for the treatment of dermatological diseases such as chronic wounds. To investigate whether CAP is an effective antiviral tool, purified human adenovirus types derived from different human adenovirus species (HAdV -4, -5, -20, -35, -37, -50) tagged with luciferase were treated with defined dosages of plasma. The CAP treatment was varied by incrementally increasing the time span of CAP treatment. After CAP treatment, the virus containing solution was added to eukaryotic cells and the viral load was determined by measurement of luciferase expression levels. Through the plasma treatment the adenovirus driven luciferase expression directly correlating with adenovirus transduction efficiencies could be reduced for HAdV-5 and HAdV-37. Plasma treatment had no influence on adenovirus derived luciferase expression levels for HAdV-4 and HAdV-50 and it even had a positive effect on luciferase expression levels for HAdV-20 and HAdV-35. These results suggest that CAP has a type dependent effect on adenoviruses and that infectivity can be even increased for certain adenovirus types. Further studies should address the mechanisms behind this phenomenon. In summary we demonstrate that CAP may represent an interesting option for antiviral treatment in a virus type dependent manner. [ABSTRACT FROM AUTHOR]

A series of peptide NK2 receptor agonists was evaluated for affinity, potency, efficacy, and selectivity at human recombinant NK2 and NK1 receptors expressed in CHO cells to identify compounds with the greatest separation between NK2 and NK1 receptor agonist activity. Binding studies were performed using displacement of [125I]-NKA binding to NK2 receptors and displacement of [3H]-Septide binding to NK1 receptors expressed in CHO cells. Functional studies examining the increase in intracellular calcium levels and cyclic AMP stimulation were performed using the same cell lines. A correlation was demonstrated between binding affinities (Ki) and potency to increase intracellular calcium (EC50) for NK2 and NK1 receptors. Ranking compounds by their relative affinity (Ki) or potency (EC50) at NK2 or NK1 receptors indicated that the most selective NK2 agonists tested were [Lys5,MeLeu9,Nle10]-NKA(4-10) (NK1/NK2 Ki ratio = 674; NK1/NK2 EC50 ratio = 105) and [Arg5,MeLeu9,Nle10]-NKA(4-10) (NK1/NK2 Ki ratio = 561; NK1/NK2 EC50 ratio = 70). The endogenous peptide, NKA, lacked selectivity with an NK1/NK2 Ki ratio = 20 and NK1/NK2 EC50 ratio = 1. Of the compounds selected for evaluation in cyclic AMP stimulation assays, [β-Ala8]-NKA(4–10) had the greatest selectivity for activation of NK2 over NK1 receptors (NK1/NK2 EC50 ratio = 244), followed by [Lys5,MeLeu9,Nle10]-NKA(4-10) (ratio = 74), and NKA exhibited marginal selectivity (ratio = 2.8). [ABSTRACT FROM AUTHOR]

The Nociceptin/Orphanin FQ (N/OFQ) peptide NOP receptor is coupled to pertussis toxin (PTX)-sensitive G proteins (Gi/o) whose activation leads to the inhibition of both cAMP production and calcium channel activity, and to the stimulation of potassium currents. The label free dynamic mass redistribution (DMR) approach has been demonstrated useful for investigating the pharmacological profile of G protein-coupled receptors. Herein, we employ DMR technology to systematically characterize the pharmacology of a large panel of NOP receptor ligands. These are of peptide and non-peptide nature and display varying degrees of receptor efficacy, ranging from full agonism to pure antagonism. Using Chinese hamster ovary (CHO) cells expressing the human NOP receptor we provide rank orders of potency for full and partial agonists as well as apparent affinities for selective antagonists. We find the pharmacological profile of NOP receptor ligands to be similar but not identical to values reported in the literature using canonical assays for Gi/o-coupled receptors. Our data demonstrate that holistic label-free DMR detection can be successfully used to investigate the pharmacology of the NOP receptor and to characterize the cellular effects of novel NOP receptor ligands. [ABSTRACT FROM AUTHOR]

In the biopharmaceutical sector, Chinese hamster ovary (CHO) cells have become the host of choice to produce recombinant proteins (r-proteins) due to their capacity for correct protein folding, assembly, and posttranslational modification. However, the production of therapeutic r-proteins in CHO cells is expensive and presents insufficient production yields for certain proteins. Effective culture strategies to increase productivity (qp) include a high glucose concentration in the medium and mild hypothermia (28–34 °C), but these changes lead to a reduced specific growth rate. To study the individual and combined impacts of glucose concentration, specific growth rate and mild hypothermia on culture performance and cell metabolism, we analyzed chemostat cultures of recombinant human tissue plasminogen activator (rh-tPA)-producing CHO cell lines fed with three glucose concentrations in feeding media (20, 30 and 40 mM), at two dilution rates (0.01 and 0.018 1/h) and two temperatures (33 and 37 °C). The results indicated significant changes in cell growth, cell cycle distribution, metabolism, and rh-tPA productivity in response to the varying environmental culture conditions. High glucose feed led to constrained cell growth, increased specific rh-tPA productivity and a higher number of cells in the G2/M phase. Low specific growth rate and temperature (33 °C) reduced glucose consumption and lactate production rates. Our findings indicated that a reduced specific growth rate coupled with high feed glucose significantly improves r-protein productivity in CHO cells. We also observed that low temperature significantly reduced qp, but not cell growth when dilution rate was manipulated, regardless of the glucose concentration or dilution rate. In contrast, we determined that feed glucose concentration and consumption rate were the dominant aspects of the growth and productivity in CHO cells by using multivariate analysis. [ABSTRACT FROM AUTHOR]

Plasmodium falciparum invasion into red blood cells (RBCs) is a complex process engaging proteins on the merozoite surface and those contained and sequentially released from the apical organelles (micronemes and rhoptries). Fundamental to invasion is the formation of a moving junction (MJ), a region of close apposition of the merozoite and the RBC plasma membranes, through which the merozoite draws itself before settling into a newly formed parasitophorous vacuole (PV). SURFIN4.2 was identified at the surface of the parasitized RBCs (pRBCs) but was also found apically associated with the merozoite. Using antibodies against the N-terminus of the protein we show the presence of SURFIN4.2 in the neck of the rhoptries, its secretion into the PV and shedding into the culture supernatant upon schizont rupture. Using immunoprecipitation followed by mass spectrometry we describe here a novel protein complex we have named SURGE where SURFIN4.2 forms interacts with the rhoptry neck protein 4 (RON4) and the Glutamate Rich Protein (GLURP). The N-terminal cysteine-rich–domain (CRD) of SURFIN4.2 mediates binding to the RBC membrane and its interaction with RON4 suggests its involvement in the contact between the merozoite apex and the RBC at the MJ. Supporting this suggestion, we also found that polyclonal antibodies to the extracellular domain (including the CRD) of SURFIN4.2 partially inhibit merozoite invasion. We propose that the formation of the SURGE complex participates in the establishment of parasite infection within the PV and the RBCs. [ABSTRACT FROM AUTHOR]

The production of envelope glycoproteins (Envs) for use as HIV vaccines is challenging. The yield of Envs expressed in stable Chinese Hamster Ovary (CHO) cell lines is typically 10–100 fold lower than other glycoproteins of pharmaceutical interest. Moreover, Envs produced in CHO cells are typically enriched for sialic acid containing glycans compared to virus associated Envs that possess mainly high-mannose carbohydrates. This difference alters the net charge and biophysical properties of Envs and impacts their antigenic structure. Here we employ a novel robotic cell line selection strategy to address the problems of low expression. Additionally, we employed a novel gene-edited CHO cell line (MGAT1- CHO) to address the problems of high sialic acid content, and poor antigenic structure. We demonstrate that stable cell lines expressing high levels of gp120, potentially suitable for biopharmaceutical production can be created using the MGAT1- CHO cell line. Finally, we describe a MGAT1- CHO cell line expressing A244-rgp120 that exhibits improved binding of three major families of bN-mAbs compared to Envs produced in normal CHO cells. The new strategy described has the potential to eliminate the bottleneck in HIV vaccine development that has limited the field for more than 25 years. [ABSTRACT FROM AUTHOR]

Neutrophils are the most abundant white blood cells, with a vital role in innate immune defense against bacterial and fungal pathogens. Although mostly associated with pathological processes directly related to immune defense, they can also play a detrimental role in inflammatory conditions and have been found to have a pro-metastatic role in the spread of cancer cells. Here, we explore ways to temporarily suppress these detrimental activities. We first examined the possibility of using siRNA and antisense oligonucleotides (ASOs) for transient knockdown of the human and mouse C5a receptor, an important chemoattractant receptor involved in neutrophil-mediated injury that is associated with myocardial infarction, sepsis, and neurodegenerative diseases. We found that siRNAs and ASOs transfected into cultured cell lines can eliminate 70–90% of C5a receptor mRNA and protein within 72 h of administration, a clinically relevant time frame after a cardiovascular event. Targeted drug delivery to specific cells or tissues of interest in a mammalian host, however, remains a major challenge. Here, using phage display technology, we have identified peptides that bind specifically to CD177, a neutrophil-specific surface molecule. We have attached these peptides to fluorescent, lipid-based nanoparticles and confirmed targeting and delivery to cultured cells ectopically presenting either human or mouse CD177. In addition, we have shown peptide-nanoparticle binding specifically to neutrophils in human and mouse blood. We anticipate that these or related tagged nanoparticles may be therapeutically useful for delivery of siRNAs or ASOs to neutrophils for transient knockdown of pro-inflammatory proteins such as the C5a receptor. [ABSTRACT FROM AUTHOR]

Proteins were extracted from perilla (Perilla frutescens L. Britton) seed by-products and hydrolyzed with an alkaline protease. Antioxidant peptides were purified from the hydrolysate by size-exclusion chromatography and RP-HPLC. Two peptides with strong antioxidant activity were identified as Tyr-Leu (YL) and Phe-Tyr (FY) with the molecular mass of 294.33 Da and 328.33 Da, respectively. Synthesized YL and FY efficiently quenched free radicals (DPPH, ABTS and hydroxyl radicals) and showed high oxygen radical absorbance capacity. The two peptides also inhibited lipid peroxidation in the rat liver. Furthermore, YL and FY could protect HepG-2 cells against hydrogen peroxide-induced oxidative damage without cytotoxicity. Based on the structure-activity analysis, the Tyr residue was crucial for the antioxidant activity of YL and FY. The results indicate that the protein hydrolysate from perilla seed by-products possessed potent biological activity and can be utilized to develop health-related nutraceutical ingredients. [ABSTRACT FROM AUTHOR]

Splice-switching oligonucleotides (SSOs) that can modulate RNA splicing are used for the treatment of many genetic disorders. To enhance the efficacy of modulating splicing, it is important to optimize SSOs with regard to target sites, GC content, melting temperature (Tm value), chemistries, and lengths. Thus, in vitro assay systems that allow for the rapid and simple screening of SSOs are essential for optimizing SSO design. In this study, we established a novel tri-chromatic reporter cell line for SSO screening. This reporter cell line is designed to express three different fluorescent proteins (blue, green, and red) and was employed for high content screening (HCS, also known as high content analysis; HCA) for the evaluation of SSO-induced exon skipping by analyzing the expression levels of fluorescent proteins. The blue fluorescent protein is stably expressed throughout the cell and is useful for data normalization using cell numbers. Furthermore, both the green and red fluorescent proteins were used for monitoring the splicing patterns of target genes. Indeed, we demonstrated that this novel reporter cell line involving HCS leads to a more rapid and simple approach for the evaluation of exon skipping than widely used methods, such as RT-PCR, western blotting, and quantitative RT-PCR. Additionally, a brief screening of Locked nucleic acids (LNA)-based SSOs targeting exon 51 in DMD was performed using the reporter cell line. The LNA-based SSO cocktail shows high exon 51 skipping in a dose-dependent manner. Furthermore, the LNA-based SSO cocktails display high exon 51 skipping activities on endogenous DMD mRNA in human rhabdomyosarcoma cells. [ABSTRACT FROM AUTHOR]

The RV144 Phase III clinical trial with ALVAC-HIV prime and AIDSVAX B/E subtypes CRF01_AE (A244) and B (MN) gp120 boost vaccine regime in Thailand provided a foundation for the future development of improved vaccine strategies that may afford protection against the human immunodeficiency virus type 1 (HIV-1). Results from this trial showed that immune responses directed against specific regions V1V2 of the viral envelope (Env) glycoprotein gp120 of HIV-1, were inversely correlated to the risk of HIV-1 infection. Due to the low production of gp120 proteins in CHO cells (2–20 mg/L), cleavage sites in V1V2 loops (A244) and V3 loop (MN) causing heterogeneous antigen products, it was an urgent need to generate CHO cells harboring A244 gp120 with high production yields and an additional, homogenous and uncleaved subtype B gp120 protein to replace MN used in RV144 for the future clinical trials. Here we describe the generation of Chinese Hamster Ovary (CHO) cell lines stably expressing vaccine HIV-1 Env antigens for these purposes: one expressing an HIV-1 subtype CRF01_AE A244 Env gp120 protein (A244.AE) and one expressing an HIV-1 subtype B 6240 Env gp120 protein (6240.B) suitable for possible future manufacturing of Phase I clinical trial materials with cell culture expression levels of over 100 mg/L. The antigenic profiles of the molecules were elucidated by comprehensive approaches including analysis with a panel of well-characterized monoclonal antibodies recognizing critical epitopes using Biacore and ELISA, and glycosylation analysis by mass spectrometry, which confirmed previously identified glycosylation sites and revealed unknown sites of O-linked and N-linked glycosylations at non-consensus motifs. Overall, the vaccines given with MF59 adjuvant induced higher and more rapid antibody (Ab) responses as well as higher Ab avidity than groups given with aluminum hydroxide. Also, bivalent proteins (A244.AE and 6240.B) formulated with MF59 elicited distinct V2-specific Abs to the epitope previously shown to correlate with decreased risk of HIV-1 infection in the RV144 trial. All together, these results provide critical information allowing the consideration of these candidate gp120 proteins for future clinical evaluations in combination with a potent adjuvant. [ABSTRACT FROM AUTHOR]

To date, the RV144 HIV vaccine trial has been the only study to show that immunization can confer protection from HIV infection. While encouraging, the modest 31.2% (P = 0.04) efficacy achieved in this study left significant room for improvement, and created an incentive to optimize the AIDSVAX B/E vaccine immunogens to increase the level of vaccine efficacy. Since the completion of the RV144 trial, our understanding of the antigenic structure of the HIV envelope protein, gp120, and of the specificity of broadly neutralizing monoclonal antibodies (bN-mAbs) that bind to it, has significantly improved. In particular, we have learned that multiple families of bN-mAbs require specific oligomannose glycans for binding. Both of the monomeric gp120 immunogens (MN- and A244-rgp120) in the AIDSVAX B/E vaccine used in the RV144 trial were enriched for glycans containing high levels of sialic acid, and lacked critical N-linked glycosylation sites required for binding by several families of bN-mAbs. The absence of these epitopes may have contributed to the low level of efficacy achieved in this study. In this report, we describe our efforts to improve the antigenic structure of the rgp120 immunogens used in the vaccine by optimizing glycan-dependent epitopes recognized by multiple bN-mAbs. Our results demonstrated that by shifting the location of one PNGS in A244-rgp120, and by adding two PNGS to MN-rgp120, in conjunction with the production of both proteins in a cell line that favors the incorporation of oligomannose glycans, we could significantly improve the binding by three major families of bN-mAbs. The immunogens described here represent a second generation of gp120-based vaccine immunogens that exhibit potential for use in RV144 follow-up studies. [ABSTRACT FROM AUTHOR]

Chinese hamster ovary (CHO) cells are the most frequently used host for commercial production of therapeutic proteins. However, their low protein productivity in culture is the main hurdle to overcome. Mild hypothermia has been established as an effective strategy to enhance protein specific productivity, although the causes of such improvement still remain unclear. The self-regulation of global transcriptional regulatory factors, such as Myc and XBP1s, seems to be involved in increased the recombinant protein production at low temperature. This study evaluated the impact of low temperature in CHO cell cultures on myc and xbp1s expression and their effects on culture performance and cell metabolism. Two anti-TNFα producing CHO cell lines were selected considering two distinct phenotypes: i.e. maximum cell growth, (CN1) and maximum specific anti-TNFα production (CN2), and cultured at 37, 33 and 31°C in a batch system. Low temperature led to an increase in the cell viability, the expression of the recombinant anti-TNFα and the production of anti-TNFα both in CN1 and CN2. The higher production of anti-TNFα in CN2 was mainly associated with the large expression of anti-TNFα. Under mild hypothermia myc and xbp1s expression levels were directly correlated to the maximal viable cell density and the specific anti-TNFα productivity, respectively. Moreover, cells showed a simultaneous metabolic shift from production to consumption of lactate and from consumption to production of glutamine, which were exacerbated by reducing culture temperature and coincided with the increased anti-TNFα production. Our current results provide new insights of the regulation of myc and xbp1s in CHO cells at low temperature, and suggest that the presence and magnitude of the metabolic shift might be a relevant metabolic marker of productive cell line. [ABSTRACT FROM AUTHOR]

Natalisins (NTLs) are conservative neuropeptides, which are only found in arthropods and are documented to regulate reproductive behaviors in insects. In our previous study, we have confirmed that NTLs regulate the reproductive process in an important agricultural pest, Bactrocera dorsalis (Hendel). Hence, in this study, to further confirm the in vivo function of NTL receptor (NTLR) and assess the potential of NTLR as an insecticide target, RNA interference targeting NTLR mRNA was performed. We found that mating frequencies of both males and females were reduced by RNAi-mediated knockdown of the NTLR transcript, while there was no effect on mating duration. Moreover, we functionally expressed the B. dorsalis NTLR in Chinese Hamster Ovary (CHO) cells and was co-transfected with an aequorin reporter to measure ligand activities. A total of 13 biostable multi-Aib analogs were tested for agonistic and antagonistic activities. While most of these NTL analogs did not show strong activity, one analog (NLFQV[Aib]DPFF[Aib]TRamide) had moderate antagonistic activity. Taken together, we provided evidence for the important roles of NTLR in regulating mating frequencies of both male and female in this fly and also provided in vitro data on mimetic analogs that serve as leading structures for the development of agonists and antagonists to disrupt the NTL signaling pathway. [ABSTRACT FROM AUTHOR]

Late-infantile neuronal ceroid lipofuscinosis is a fatal neurodegenerative disease of children caused by mutations resulting in loss of activity of the lysosomal protease, tripeptidyl peptidase 1 (TPP1). While Tpp1-targeted mouse models of LINCL exist, the goal of this study was to create a transgenic mouse with inducible TPP1 to benchmark treatment approaches, evaluate efficacy of treatment at different stages of disease, and to provide insights into the pathobiology of disease. A construct containing a loxP-flanked stop cassette inserted between the chicken-actin promoter and a sequence encoding murine TPP1 (TgLSL-TPP1) was integrated into the ROSA26 locus in mice by homologous recombination. Tested in both transfected CHO cells and in transgenic mice, the TgLSL-TPP1 did not express TPP1 until cre-mediated removal of the LSL cassette, which resulted in supraphysiological levels of TPP1 activity. We tested four cre/ERT2 transgenes to allow tamoxifen-inducible removal of the LSL cassette and subsequent TPP1 expression at any stage of disease. However, two of the cre/ERT2 driver transgenes had significant cre activity in the absence of tamoxifen, while cre-mediated recombination could not be induced by tamoxifen by two others. These results highlight potential problems with the use of cre/ERT2 transgenes in applications that are sensitive to low levels of basal cre expression. However, the germline-recombined mouse transgenic that constitutively overexpresses TPP1 will allow long-term evaluation of overexposure to the enzyme and in cell culture, the inducible transgene may be a useful tool in biomarker discovery projects. [ABSTRACT FROM AUTHOR]

Muc1 is one of the most studied tumor antigens. However, antibodies or antibody-toxin conjugates against Muc1 have not shown significant efficacy for tumors with Muc1 overexpression. In this study, we employed bispecific antibody approach to target Muc1 positive tumor cells. A novel bispecific antibody, Muc1-Bi-1, was constructed by linking single domain antibodies, anti-Muc1-VHH and anti-CD16-VHH. Muc1-Bi-2, the humanized form of Muc1-Bi-1, was also constructed by grafting. Both Muc1-Bi bispecific antibodies can be efficiently expressed and purified from bacteria. In vitro, the Muc1-Bi bispecific antibodies can recruit Natural Killer (NK) cells to drive potent and specific cell killing of Muc1-overexpressing tumor cells. In xenograft model, the Muc1-Bi bispecific antibodies can suppress tumor growth in the presence of human peripheral blood mononuclear cells (PBMC). These data suggested that the single domain based Muc1-Bi may provide a valid strategy for targeting tumors with Muc1 overexpression. [ABSTRACT FROM AUTHOR]

Recombinant protein therapeutics have become important components of the modern medicine. Majority of them are produced with mammalian cells that are cultured either through adherent culturing, in which cells are cultured on substrates, or suspension culturing, in which cells are suspended and cultured in agitated cell culture medium in a culture vessel. The adherent cell culturing method is limited by its low yield. In suspension culturing, cells need extensive genetic manipulation to grow as single cells at high density, which is time- and labor-consuming. Here, we report a new method, which utilizes a thermoreversible hydrogel as the scaffold for culturing protein-expressing cells. The hydrogel scaffolds not only provide 3D spaces for the cells, but also act as physical barriers to prevent excessive cellular agglomeration and protect cells from the hydrodynamic stresses. As a result, cells can grow at high viability, high growth rate, and extremely high yield even without genetic manipulations. The cell yield in the hydrogels is around 20 times of the suspension culturing. In addition, the protein productivity per cell per day in the hydrogel is higher than the adherent culturing method. This new method is simple, scalable and defined. It will be of great value for both the research laboratories and pharmaceutical industry for producing proteins. [ABSTRACT FROM AUTHOR]

The orexins and their cognate G-protein coupled receptors have been widely studied due to their associations with various behaviors and cellular processes. However, the detailed downstream signaling cascades that mediate these effects are not completely understood. We report the generation of a neuronal model cell line that stably expresses the OX1 orexin receptor (OX1) and an RNA-Seq analysis of changes in gene expression seen upon receptor activation. Upon treatment with orexin, several families of related transcription factors are transcriptionally regulated, including the early growth response genes (Egr), the Kruppel-like factors (Klf), and the Nr4a subgroup of nuclear hormone receptors. Furthermore, some of the transcriptional effects observed have also been seen in data from in vivo sleep deprivation microarray studies, supporting the physiological relevance of the data set. Additionally, inhibition of one of the most highly regulated genes, serum and glucocorticoid-regulated kinase 1 (Sgk1), resulted in the diminished orexin-dependent induction of a subset of genes. These results provide new insight into the molecular signaling events that occur during OX1 signaling and support a role for orexin signaling in the stimulation of wakefulness during sleep deprivation studies. [ABSTRACT FROM AUTHOR]

The utility of HepG2 cells to assess drug metabolism and toxicity induced by chemical compounds is hampered by their low cytochrome P450 (CYP) activities. To overcome this limitation, we established HepG2 cell lines expressing major CYP enzymes involved in drug metabolism (CYP2C9, CYP2C19, CYP2D6, and CYP3A4) and CYP oxidoreductase (POR) using the mammalian-derived artificial chromosome vector. Transchromosomic HepG2 (TC-HepG2) cells expressing four CYPs and POR were used to determine time- and concentration-dependent inhibition and toxicity of several compounds by luminescence detection of CYP-specific substrates and cell viability assays. Gene expression levels of all four CYPs and POR, as well as the CYP activities, were higher in TC-HepG2 clones than in parental HepG2 cells. Additionally, the activity levels of all CYPs were reduced in a concentration-dependent manner by specific CYP inhibitors. Furthermore, preincubation of TC-HepG2 cells with CYP inhibitors known as time-dependent inhibitors (TDI) prior to the addition of CYP-specific substrates determined that CYP inhibition was enhanced in the TDI group than in the non-TDI group. Finally, the IC50 of bioactivable compound aflatoxin B1 was lower in TC-HepG2 cells than in HepG2 cells. In conclusion, the TC-HepG2 cells characterized in the current study are a highly versatile model to evaluate drug-drug interactions and hepatotoxicity in initial screening of candidate drug compounds, which require a high degree of processing capacity and reliability. [ABSTRACT FROM AUTHOR]

In recent years, monoclonal antibodies (mAbs) have been developed as powerful therapeutic and diagnostic agents and Chinese hamster ovary (CHO) cells have emerged as the dominant host for the recombinant expression of these proteins. A critical step in recombinant expression is the utilization of strong promoters, such as the Chinese Hamster Elongation Factor-1α (CHEF-1) promoter. To compare the strengths of CHEF with cytomegalovirus (CMV) promoter for mAb expression in CHO cells, four bicistronic vectors bearing either internal ribosome entry site (IRES) or Furin/2A (F2A) sequences were designed. The efficiency of these promoters was evaluated by measuring level of expressed antibody in stable cell pools. Our results indicated that CHEF promoter-based expression of mAbs was 2.5 fold higher than CMV-based expression in F2A-mediated vectors. However, this difference was less significant in IRES-mediated mAb expressing cells. Studying the stability of the F2A expression system in the course of 18 weeks, we observed that the cells having CHEF promoter maintained their antibody expression at higher level than those transfected with CMV promoter. Further analyses showed that both IRES-mediated vectors, expressed mAbs with correct size, whereas in antibodies expressed via F2A system heterogeneity of light chains were detected due to incomplete furin cleavage. Our findings indicated that the CHEF promoter is a viable alternative to CMV promoter-based expression in F2A-mediated vectors by providing both higher expression and level of stability. [ABSTRACT FROM AUTHOR]