Engineering mammalian cell growth dynamics for biomanufacturing.

Precise control over mammalian cell growth dynamics poses a major challenge in biopharmaceutical manufacturing. Here, we present a multi-level cell engineering strategy for the tunable regulation of growth phases in mammalian cells. Initially, we engineered mammalian death phase by employing CRISPR/Cas9 to knockout pro-apoptotic proteins Bax and Bak, resulting in a substantial attenuation of apoptosis by improving cell viability and extending culture lifespan. The second phase introduced a growth acceleration system, akin to a "gas pedal", based on an abscidic acid inducible system regulating cMYC gene expression, enabling rapid cell density increase and cell cycle control. The third phase focused on a stationary phase inducing system, comparable to a "brake pedal". A tetracycline inducible genetic circuit based on BLIMP1 gene led to cell growth cessation and arrested cell cycle upon activation. Finally, we developed a dual controllable system, combining the "gas and brake pedals", enabling for dynamic and precise orchestration of mammalian cell growth dynamics. This work exemplifies the application of synthetic biology tools and combinatorial cell engineering, offering a sophisticated framework for manipulating mammalian cell growth and providing a unique paradigm for reprogramming cell behaviour for enhancing biopharmaceutical manufacturing and further biomedical applications.

Characterising the structural and cellular role of immunoglobulin C-terminal lysine in secretory pathways.

Improved understanding of expression of recombinant immunoglobulin (IgG)-based therapies can decrease manufacturing process costs and bring down costs to patients. Deletion of C-terminal Lysine (C-Lys) from IgG molecules has been shown to greatly impact yield. This study set out to characterise structural components of IgG C-terminal variants which modulate protein expression by examination of the consequences of mutations at the C-terminal of IgG on expression and by the use of fluorescent C-terminal fragment fusion proteins. Cell-based and cell-free experiments were also implemented to characterise how the C-terminal differentially engages with cellular pathways to modulate expression. IgG variants engineered by removal of the C-terminal Lys were expressed at significantly lower rates than control variants by CHO (and HEK) cells. Engineered constructs of mCherry fused with short regions of the C-terminal regions of IgG mimicked the ordering of expressability observed for IgG variants. These fluorescent C-terminal fragment fusions offered the potential to profile how sequences (and point mutations) modified expression. Via combinations of cell and cell-free systems, screening across a range of variants of IgG and mCherry reporter constructs has shown that interactions between specific C-terminal amino acid sequences and the ribosome can regulate the rate and extent of expression. This study highlights the importance of amino acid sequence regulatory events determining the efficiency of production of desirable recombinant proteins, showing that wildtype C-terminal lysine is a necessary capping molecule for IgG1 expression. From a wider perspective, these data are especially significant towards the design of novel entities. The approach has also provided information about novel short C-terminal tags which may be used to provide selective synthesis of specific subunits in the production of multisubunit products. Alternative strategies for removing C-terminal amino acid heterogeneity whilst maintaining efficient rates of expression have been provided.

Long term culture promotes changes to growth, gene expression, and metabolism in CHO cells that are independent of production stability.

Phenotypic stability of Chinese hamster ovary (CHO) cells over long term culture (LTC) presents one of the most pressing challenges in the development of therapeutic protein manufacturing processess. However, our current understanding of the consequences of LTC on recombinant (r-) CHO cell lines is still limited, particularly as clonally-derived cell lines present distinct production stability phenotypes. This study evaluated changes of culture performance, global gene expression, and cell metabolism of two clonally-derived CHO cell lines with a stable or unstable phenotype during the LTC (early [EP] vs. late [LP] culture passages). Our findings indicated that LTC altered the behavior of CHO cells in culture, in terms of growth, overall gene expression, and cell metabolism. Regardless whether cells were categorized as stable or unstable in terms of r-protein production, CHO cells at LP presented an earlier decline in cell viability and loss of any observable stationary phase. These changes were parallelled by the upregulation of genes involved in cell proliferation and survival pathways (i.e., MAPK/ERK, PI3K-Akt). Stable and unstable CHO cell lines both showed increased consumption of glucose and amino acids at LP, with a parallel accumulation of greater amounts of lactate and TCA cycle intermediates. In terms of production stability, we found that decreased r-protein production in the unstable cell line directly correlated to the loss in r-gene copy number and r-mRNA expression. Our data revealed that LTC produced ubiquitious effects on CHO cell phenotypes, changes that were rooted in alterations in cell transcriptome and metabolome. Overall, we found that CHO cells adapted their cellular function to proliferation and survival during the LTC, some of these changes may well have limited effects on overall yield or specific productivity of the desired r-product, but they may be critical toward the capacity of cells to handle r-proteins with specific molecular features.

Engineering of Chinese hamster ovary cells for co-overexpressing MYC and XBP1s increased cell proliferation and recombinant EPO production.

Improving the cellular capacity of Chinese hamster ovary (CHO) cells to produce large amounts of therapeutic proteins remains a major challenge for the biopharmaceutical industry. In previous studies, we observed strong correlations between the performance of CHO cells and expression of two transcription factors (TFs), MYC and XBP1s. Here, we have evaluated the effective of overexpression of these two TFs on CHO cell productivity. To address this goal, we generated an EPO-producing cell line (CHOEPO) using a targeted integration approach, and subsequently engineered it to co-overexpress MYC and XBP1s (a cell line referred to as CHOCXEPO). Cells overexpressing MYC and XBP1s increased simultaneously viable cell densities and EPO production, leading to an enhanced overall performance in cultures. These improvements resulted from the individual effect of each TF in the cell behaviour (i.e., MYC-growth and XBP1s-productivity). An evaluation of the CHOCXEPO cells under different environmental conditions (temperature and media glucose concentration) indicated that CHOCXEPO cells increased cell productivity in high glucose concentration. This study showed the potential of combining TF-based cell engineering and process optimisation for increasing CHO cell productivity.

The secretory pathway - the key for unlocking the potential of Chinese hamster ovary cell factories for manufacturing therapeutic proteins.

Mammalian cell factories (in particular the CHO cell system) have been crucial in the rise of biopharmaceuticals. Mammalian cells have compartmentalized organelles where intricate networks of proteins manufacture highly sophisticated biopharmaceuticals in a specialized production pipeline - the secretory pathway. In the bioproduction context, the secretory pathway functioning is key for the effectiveness of cell factories to manufacture these life-changing medicines. This review describes the molecular components and events involved in the secretory pathway, and provides a comprehensive summary of the intracellular steps limiting the production of therapeutic proteins as well as the achievements in engineering CHO cell secretory machinery. We also consider antibody-producing plasma cells (so called "professional" secretory cells) to explore the mechanisms underpinning their unique secretory function/features. Such understandings offer the potential to further enhancement of the current CHO cell production platforms for manufacturing next generation of biopharmaceuticals.

Enhanced recombinant protein production in CHO cell continuous cultures under growth-inhibiting conditions is associated with an arrested cell cycle in G1/G0 phase.

Low temperature and sodium butyrate (NaBu) are two of the most used productivity-enhancing strategies in CHO cell cultures during biopharmaceutical manufacturing. While these two approaches alter the balance in the reciprocal relationship between cell growth and productivity, we do not fully understand their mechanisms of action beyond a gross cell growth inhibition. Here, we used continuous culture to evaluate the differential effect of low temperature and NaBu supplementation on CHO cell performance and gene expression profile. We found that an increase in cell-productivity under growth-inhibiting conditions was associated with the arrest of cells in the G1/G0 phase. A transcriptome analysis revealed that the molecular mechanisms by which low temperature and NaBu arrested cell cycle in G1/G0 differed from each other through the deregulation of different cell cycle checkpoints and regulators. The individual transcriptome changes in pattern observed in response to low temperature and NaBu were retained when these two strategies were combined, leading to an additive effect in arresting the cell cycle in G1/G0 phase. The findings presented here offer novel molecular insights about the cell cycle regulation during the CHO cell bioprocessing and its implications for increased recombinant protein production. This data provides a background for engineering productivity-enhanced CHO cell lines for continuous manufacturing.

Combined gene and environmental engineering offers a synergetic strategy to enhance r-protein production in Chinese hamster ovary cells.

Environmental growth-inhibition conditions (GICs) have been used extensively for increasing cell-specific productivity (qP ) of Chinese hamster ovary (CHO) cells, with the most common being temperature downshift and sodium butyrate (NaBu) treatment. B lymphocyte-induced maturation protein-1 (BLIMP1) overexpression in CHO cells can also inhibit cell growth and increase product titers and qP . Given the similar responses, this study evaluated the individual and combined effects of BLIMP1 expression, low temperature, and NaBu treatment on culture performance, cell metabolism, and recombinant protein production of CHO cells. As expected, all three interventions decreased cell growth, arrested cells in G1/G0 cell cycle phase, and increased qP . However, CHO cells presented different responses when considering cell viability, recombinant gene expression, and cell metabolism that indicated differences in the molecular loci by which BLIMP1 and GICs generated higher productivities. Combinations of BLIMP1 expression and GICs acted synergistically to inhibit cell growth and maximize r-protein production, with the BLIMP1/NaBu condition leading to the most significant improvements in product titers and qP . This latter condition also proved to substantially increase product yields (up to 9.8 g immunoglobulin G1 [IgG1]/L and 2.2 g erythropoietin-Fc [EPO-Fc]/L) and qP (up to 179 pg/cell/day [pcd] for IgG1 and 30 pcd for EPO-Fc) in high-density perfusion cultures. These findings offered mechanistic insights about the productivity-enhancing effects of BLIMP1 and GICs, as well as their complementarity for generating highly productive processes.

Reprogramming of Chinese hamster ovary cells towards enhanced protein secretion.

The deficient secretory phenotype of Chinese hamster ovary (CHO) cells is a major limitation for high-level production of biopharmaceuticals, particularly for those with complex molecular architectures and post-translational modifications. To improve CHO cell secretory capacity, we recently engineered CHO cell hosts to overexpress BLIMP1 (CHOB), in a cell engineering strategy that transformed the cellular machinery and led to significantly higher product yields and cell-specific productivities for different rproteins. Here, as a follow-up to our previous study, we developed new CHO cell hosts that co-overexpress BLIMP1 and XBP1s ( CHOBX ), two transcription factors that together drive the professional secretory function of antibody-producing plasma cells. We found that the CHOBX cells presented an improved performance over that of CHOB cells, with better product yields and cell-specific productivities for a recombinant IgG1 and a 'difficult-to-express' EPO-Fc fusion protein. These improvements in the CHOBX-derived cell lines resulted from a series of physiological and metabolic changes due to the synergetic co-expression of BLIMP1 and XBP1s. Firstly, cells presented an inhibited cell growth and arrested cell cycle in G1/G0 phase, features that were directly linked to BLIMP1 expression levels. Secondly, cells increased protein translation (both overall and recombinant protein), expanded the endoplasmic reticulum and improved their capacity to secrete protein more effectively. Lastly, cells showed a metabolic profile favouring energy production, with a pronounced lactate switch and increased consumption of amino acids. This study highlights the value of transcription factors for reprogramming CHO cells towards a desired phenotype, offering the potential to engineer cells with new functionalities for enhanced manufacturing of recombinant therapeutic proteins.

A comparative analysis of recombinant Fab and full-length antibody production in Chinese hamster ovary cells.

Monoclonal antibodies are the leading class of biopharmaceuticals in terms of numbers approved for therapeutic purposes. Antigen-binding fragments (Fab) are also used as biotherapeutics and used widely in research applications. The dominant expression systems for full-length antibodies are mammalian cell-based, whereas for Fab molecules the preference has been an expression in bacterial systems. However, advances in CHO and downstream technologies make mammalian systems an equally viable option for small- and large-scale Fab production. Using a panel of full-length IgG antibodies and their corresponding Fab pair with different antigen specificities, we investigated the impact of the IgG and Fab molecule format on production from Chinese hamster ovary (CHO) cells and assessed the cellular capability to process and produce these formats. The full-length antibody format resulted in the recovery of fewer mini-pools posttransfection when compared to the corresponding Fab fragment format that could be interpreted as indicative of a greater overall burden on cells. Antibody-producing cell pools that did recover were subsequently able to achieve higher volumetric protein yields (mg/L) and specific productivity than the corresponding Fab pools. Importantly, when the actual molecules produced per cell of a given format was considered (as opposed to mass), CHO cells produced a greater number of Fab molecules per cell than obtained with the corresponding IgG, suggesting that cells were more efficient at making the smaller Fab molecule. Analysis of cell pools showed that gene copy number was not correlated to the subsequent protein production. The amount of mRNA correlated with secreted Fab production but not IgG, whereby posttranscriptional processes act to limit antibody production. In summary, we provide the first comparative description of how full-length IgG and Fab antibody formats impact on the outcomes of a cell line construction process and identify potential limitations in their production that could be targeted for engineering increases in the efficiency in the manufacture of these recombinant antibody formats.

Overexpression of transcription factor BLIMP1/prdm1 leads to growth inhibition and enhanced secretory capacity in Chinese hamster ovary cells.

Chinese hamster ovary (CHO) cells present inherent limitations for processing and secretion of large amounts of recombinant proteins, especially for those requiring complex post-translational processing. To tackle these limitations, we engineered CHO host cells (CHOK1 and CHOS) to overexpress the transcription factor BLIMP1/prdm1 (a master regulator of the highly-secreting phenotype of antibody-producing plasma cells), generating novel CHO cell lines (referred to as CHOB). The CHOB cell lines exhibited decreased cell densities, prolonged stationary phase and arrested cell cycle in G1/G0 phase but simultaneously had significantly greater product titre for recombinant IgG1 (> 2-fold increase) coupled with a significantly greater cell-specific productivities (> 3-fold increase). We demonstrated that the improved productive phenotype of CHOB cells resulted from a series of changes to cell physiology and metabolism. CHOB cells showed a significantly greater ER size and increased protein synthesis and secretion capacity compared to control cells. In addition, CHOB cells presented a metabolic profile that favoured energy production to support increased recombinant protein production. This study indicated that a cell engineering approach based on BLIMP1 expression offers great potential for improving the secretory capacity of CHO cell hosts utilised for manufacture of recombinant biopharmaceuticals. Our findings also provides a greater understanding of the relationship between cell growth and productivity, valuable generic information for improving productive phenotypes for CHO cell lines during industrial cell line development.

Predictive approaches to guide the expression of recombinant vaccine targets in Escherichia coli: a case study presentation utilising Absynth Biologics Ltd. proprietary Clostridium difficile vaccine antigens.

Bacterial expression systems remain a widely used host for recombinant protein production. However, overexpression of recombinant target proteins in bacterial systems such as Escherichia coli can result in poor solubility and the formation of insoluble aggregates. As a consequence, numerous strategies or alternative engineering approaches have been employed to increase recombinant protein production. In this case study, we present the strategies used to increase the recombinant production and solubility of 'difficult-to-express' bacterial antigens, termed Ant2 and Ant3, from Absynth Biologics Ltd.'s Clostridium difficile vaccine programme. Single recombinant antigens (Ant2 and Ant3) and fusion proteins (Ant2-3 and Ant3-2) formed insoluble aggregates (inclusion bodies) when overexpressed in bacterial cells. Further, proteolytic cleavage of Ant2-3 was observed. Optimisation of culture conditions and changes to the construct design to include N-terminal solubility tags did not improve antigen solubility. However, screening of different buffer/additives showed that the addition of 1-15 mM dithiothreitol alone decreased the formation of insoluble aggregates and improved the stability of both Ant2 and Ant3. Structural models were generated for Ant2 and Ant3, and solubility-based prediction tools were employed to determine the role of hydrophobicity and charge on protein production. The results showed that a large non-polar region (containing hydrophobic amino acids) was detected on the surface of Ant2 structures, whereas positively charged regions (containing lysine and arginine amino acids) were observed for Ant3, both of which were associated with poor protein solubility. We present a guide of strategies and predictive approaches that aim to guide the construct design, prior to expression studies, to define and engineer sequences/structures that could lead to increased expression and stability of single and potentially multi-domain (or fusion) antigens in bacterial expression systems.

Molecular characterization of HEK293 cells as emerging versatile cell factories.

HEK293 cell lines are used for the production of recombinant proteins, virus-like particles and viral vectors. Recent work has generated molecular (systems level) characterisation of HEK293 variants that has enabled re-engineering of the cells towards enhanced use for manufacture-scale production of recombinant biopharmaceuticals (assessment of 'safe harbours' for gene insertion, engineering of new variants for stable, amplifiable expression). In parallel, there have been notable advances in the bioprocessing conditions (suspension adaptation, development of defined serum-free media) that offer the potential for large-scale manufacture, a feature especially important in the drive to produce viral vectors at large-scale and at commercially viable costs for gene therapy. The combination of cell-based and bioprocess-based modification of existing HEK293 cell processes, frequently informed by understandings transferred from developments with Chinese hamster ovary cell lines, seems destined to place the HEK293 cell systems firmly as a critical platform for production of future biologically based therapeutics.

Temperature Down-Shift Modifies Expression of UPR-/ERAD-Related Genes and Enhances Production of a Chimeric Fusion Protein in CHO Cells.

Low culture temperature enhances the cell-specific productivity of Chinese hamster ovary (CHO) cells expressing varied recombinant (r-) proteins, but the mechanisms remain unclear. Regulation of unfolded protein response (UPR) pathway genes, such as transcriptional regulatory factors and endoplasmic reticulum (ER)-resident proteins, appear to be involved in the improvements of r-protein production under low temperature conditions. The transcriptional regulation of UPR-specific targets is studied in response to decreased culture temperature in relation to production of a difficult-to-express protein. A clonally-derived CHO cell line expressing a chimeric fusion protein (human erythropoietin [hEPO] linked to a murine Fc region, hEPO-Fc) is evaluated in terms of growth, metabolism, r-protein production and UPR-/ER associated degradation (ERAD)-specific gene expression at standard (37 °C) and low (32 °C) temperature in batch and fed-batch systems. Low temperature decreased peak cell density, improved viability, generated cell cycle arrest in the G1 phase and enhanced hEPO-Fc expression in both batch and fed-batch cultures. A low culture temperature significantly upregulated genes encoding UPR-specific transcriptional activators (xbp1s, ddit3, and atf5) and ER-resident proteins (grp78, grp94, trib3, and ero1α), that are associated with folding and processing of proteins within the ER. Further, low culture temperature decreased expression of genes involved in ERAD (edem3, sels, herpud1, and syvn1) indicating a decreased potential for protein degradation.

Metabolic profiling of Chinese hamster ovary cell cultures at different working volumes and agitation speeds using spin tube reactors.

Culture systems based on spin tube reactors have been consolidated in the development of manufacturing processes based on Chinese hamster ovary (CHO) cells. Despite their widespread use, there is little information about the consequences of varying operational setting parameters on the culture performance of recombinant CHO cell lines. Here, we investigated the effect of varying working volumes and agitation speeds on cell growth, protein production, and cell metabolism of two clonally derived CHO cell lines (expressing an IgG1 and a "difficult-to-express" fusion protein). Interestingly, low culture volumes increased recombinant protein production and decreased cell growth, while high culture volumes had the opposite effect. Altering agitation speeds exacerbated or moderated the differences observed due to culture volume changes. Combining low agitation rates with high culture volumes suppressed growth and recombinant protein production in CHO cells. Meanwhile, high agitation rates narrowed the differences in culture performance between low and high working volumes. These differences were also reflected in cell metabolism, where low culture volumes enhanced oxidative metabolism (linked to a productive phenotype) and high culture volume generated a metabolic profile that was predominately glycolytic (linked to a proliferative phenotype). Our findings indicate that the culture volume influence on metabolism modulates the balance between cell growth and protein production, a key feature that may be useful to adjust CHO cells toward a more productive phenotype.

Actinomycin D downregulates Sox2 and improves survival in preclinical models of recurrent glioblastoma.

BACKGROUND: Glioblastoma (GBM) has been extensively researched over the last few decades, yet despite aggressive multimodal treatment, recurrence is inevitable and second-line treatment options are limited. Here, we demonstrate how high-throughput screening (HTS) in multicellular spheroids can generate physiologically relevant patient chemosensitivity data using patient-derived cells in a rapid and cost-effective manner. Our HTS system identified actinomycin D (ACTD) to be highly cytotoxic over a panel of 12 patient-derived glioma stemlike cell (GSC) lines. ACTD is an antineoplastic antibiotic used in the treatment of childhood cancers. Here, we validate ACTD as a potential repurposed therapeutic for GBM in 3-dimensional GSC cultures and patient-derived xenograft models of recurrent glioblastoma. METHODS: Twelve patient-derived GSC lines were screened at 10 µM, as multicellular spheroids, in a 384-well serum-free assay with 133 FDA-approved compounds. GSCs were then treated in vitro with ACTD at established half-maximal inhibitory concentrations (IC50). Downregulation of sex determining region Y-box 2 (Sox2), a stem cell transcription factor, was investigated via western blot and through immunohistological assessment of murine brain tissue. RESULTS: Treatment with ACTD was shown to significantly reduce tumor growth in 2 recurrent GBM patient-derived models and significantly increased survival. ACTD is also shown to specifically downregulate the expression of Sox2 both in vitro and in vivo. CONCLUSION: These findings indicate that, as predicted by our HTS, ACTD could deplete the cancer stem cell population within the tumor mass, ultimately leading to a delay in tumor progression. KEY POINTS: 1. High-throughput chemosensitivity data demonstrated the broad efficacy of actinomycin D, which was validated in 3 preclinical models of glioblastoma.2. Actinomycin D downregulated Sox2 in vitro and in vivo, indicating that this agent could target the stem cell population of GBM tumors.

Ligno-Cellulosic Fibre Sized with Nucleating Agents Promoting Transcrystallinity in Isotactic Polypropylene Composites.

The mechanical performance of composites made from isotactic polypropylene reinforced with natural fibres depends on the interface between fibre and matrix, as well as matrix crystallinity. Sizing the fibre surface with nucleating agents to promote transcrystallinity is a potential route to improve the mechanical properties. The sizing of thermo-mechanical pulp and regenerated cellulose (Tencel™) fibres with α- and ß-nucleating agents, to improve tensile strength and impact strength respectively, was assessed in this study. Polarised microscopy, electron microscopy and differential scanning calorimetry (DSC) showed that transcrystallinity was achieved and that the bulk crystallinity of the matrix was affected during processing (compounding and injection moulding). However, despite substantial changes in crystal structure in the final composite, the sizing method used did not lead to significant changes regarding the overall composite mechanical performance.

Childhood and adolescent idiopathic urethritis: what does the current literature say?

INTRODUCTION: Idiopathic urethritis (IU) is a known entity in the childhood and adolescent age groups and is described as blood spotting of the underwear in these populations. Typically, IU presents clinically as terminal void haematuria or blood spotting, accompanied with dysuria, occasional suprapubic pain and rarely constitutional symptoms. Sometimes IU may progress to formation of urethral strictures. It carries a high morbidity because of its chronicity and recurrent course, which often persists to puberty. In spite of no single aetiology being confirmed, several treatment approaches have shown some efficacy. Although no consensus has yet been established, the consideration of previous authors' experience and expertise may contribute to the development of a possible unified algorithm. AIM: This article considers IU, collecting pertinent data and case series from the literature. The authors attempt to describe all presumptive aetiologies, endoscopic findings with established grading classifications and finally the various treatment modalities proposed. In addition, highlights from the senior's author experience regarding this entity including endoscopic, radiographic and histological images are mentioned. METHODS: Retrospective review and analysis of all relevant articles present on PubMed, SCOPUS and Cochrane Library from the time of inception to the date of first submission. RESULTS: A total of 19 series concerning IU have been reported since year 1971 to present time (Table 1). Many aetiologies have been postulated, but some have been refuted, including infectious pathogens. Children with dysfunctional voiding and meatal stenosis are more prone to exhibit IU. A variation in the urethral milieu during the prepubertal period, secondary to systematic hormonal alteration, may lead to squamous cell metaplasia resulting in IU and possible urethral strictures. Grading classifications for IU are described. Up to 20% of IU may develop urethral strictures. Various treatment approaches are discussed. CONCLUSION: A summary of current understanding of the pathogenesis and spectrum of IU has been achieved. Successful treatment can be achieved with steroids and dysfunctional voiding protocols. It may be possible to develop a consensus unified management algorithm.

Novel approach to vaginal calculus in a girl with urogenital sinus anomaly.

Isolated urogenital sinus can cause distended bladder and/or vagina and may present with an abdominal mass and sepsis during infancy. Older children may present with recurrent urinary tract infections and hematocolpos. We describe a 3-year-old girl with recurrent urinary tract infections thought to be secondary to vesicoureteric reflux. On further investigation, an isolated urogenital sinus anomaly with a calculus inside one of the hemivaginae was noted. She was managed expectantly with a plan to intervene at puberty. At puberty, during removal of the stone, the hemivaginal introitus was found to be stenotic. Gradually increasing sizes of Amplatz type graduated renal dilators were introduced from the introitus of the urogenital sinus into the hemivaginal stone until a size 22F Amplatz sheath could be passed easily. Size 10F cystoscope was passed through this channel, and the stone was fragmented using electrohydraulic lithotripsy. At a later date, she underwent staged anterior sagittal transvulval mobilization of the urogenital sinus.

Systematic Evaluation of CRISPRa and CRISPRi Modalities Enables Development of a Multiplexed, Orthogonal Gene Activation and Repression System.

The ability to manipulate the expression of mammalian genes using synthetic transcription factors is highly desirable in both fields of basic research and industry for diverse applications, including stem cell reprogramming and differentiation, tissue engineering, and drug discovery. Orthogonal CRISPR systems can be used for simultaneous transcriptional upregulation of a subset of target genes while downregulating another subset, thus gaining control of gene regulatory networks, signaling pathways, and cellular processes whose activity depends on the expression of multiple genes. We have used a rapid and efficient modular cloning system to build and test in parallel diverse CRISPRa and CRISPRi systems and develop an efficient orthogonal gene regulation system for multiplexed and simultaneous up- and downregulation of endogenous target genes.

Surface patches on recombinant erythropoietin predict protein solubility: engineering proteins to minimise aggregation.

BACKGROUND: Protein solubility characteristics are important determinants of success for recombinant proteins in relation to expression, purification, storage and administration. Escherichia coli offers a cost-efficient expression system. An important limitation, whether for biophysical studies or industrial-scale production, is the formation of insoluble protein aggregates in the cytoplasm. Several strategies have been implemented to improve soluble expression, ranging from modification of culture conditions to inclusion of solubility-enhancing tags. RESULTS: Surface patch analysis has been applied to predict amino acid changes that can alter the solubility of expressed recombinant human erythropoietin (rHuEPO) in E. coli, a factor that has importance for both yield and subsequent downstream processing of recombinant proteins. A set of rHuEPO proteins (rHuEPO E13K, F48D, R150D, and F48D/R150D) was designed (from the framework of wild-type protein, rHuEPO WT, via amino acid mutations) that varied in terms of positively-charged patches. A variant predicted to promote aggregation (rHuEPO E13K) decreased solubility significantly compared to rHuEPO WT. In contrast, variants predicted to diminish aggregation (rHuEPO F48D, R150D, and F48D/R150D) increased solubility up to 60% in relation to rHuEPO WT. CONCLUSIONS: These findings are discussed in the wider context of biophysical calculations applied to the family of EPO orthologues, yielding a diverse range of calculated values. It is suggested that combining such calculations with naturally-occurring sequence variation, and 3D model generation, could lead to a valuable tool for protein solubility design.