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.

A novel cell-based, high-content assay for phosphorylation of Lats2 by Aurora A.

Aurora A kinase is a key regulator of mitosis, which is upregulated in several human cancers, making it a potential target for anticancer therapeutics. Consequently, robust medium- to high-throughput cell-based assays to measure Aurora A kinase activity are critical for the development of small-molecule inhibitors. Here the authors compare measurement of the phosphorylation of two Aurora A substrates previously used in high-content screening Aurora A assays, Aurora A itself and TACC3, with a novel substrate Lats2. Using antibodies directed against phosphorylated forms of Aurora A (pThr288), P-TACC3 (pSer558), and P-Lats2 (pSer83), the authors investigate their suitability in parallel for development of a cell-based assay using several reference Aurora inhibitors: MLN8054, VX680, and AZD1152-HQPA. They validate a combined assay of target-specific phosphorylation of Lats2 at the centrosome and an increase in mitotic index as a measure of Aurora A activity. The assay is both sensitive and robust and has acceptable assay performance for high-throughput screening or potency estimation from concentration-response assays. It has the advantage that it can be carried out using a commercially available monoclonal antibody against phospho-Lats2 and the widely available Cellomics ArrayScan HCS reader and thus represents a significant addition to the tools available for the identification of Aurora A specific inhibitors.

Reduced maximal inhibition in phenotypic susceptibility assays indicates that viral strains resistant to the CCR5 antagonist maraviroc utilize inhibitor-bound receptor for entry.

Maraviroc is a CCR5 antagonist in clinical development as one of a new class of antiretrovirals targeting human immunodeficiency virus type 1 (HIV-1) coreceptor binding. We investigated the mechanism of HIV resistance to maraviroc by using in vitro sequential passage and site-directed mutagenesis. Serial passage through increasing maraviroc concentrations failed to select maraviroc-resistant variants from some laboratory-adapted and clinical isolates of HIV-1. However, high-level resistance to maraviroc was selected from three of six primary isolates passaged in peripheral blood lymphocytes (PBL). The SF162 strain acquired resistance to maraviroc in both treated and control cultures; all resistant variants were able to use CXCR4 as a coreceptor. In contrast, maraviroc-resistant virus derived from isolates CC1/85 and RU570 remained CCR5 tropic, as evidenced by susceptibility to the CCR5 antagonist SCH-C, resistance to the CXCR4 antagonist AMD3100, and an inability to replicate in CCR5 Delta32/Delta32 PBL. Strain-specific mutations were identified in the V3 loop of maraviroc-resistant CC1/85 and RU570. The envelope-encoding region of maraviroc-resistant CC1/85 was inserted into an NL4-3 background. This recombinant virus was completely resistant to maraviroc but retained susceptibility to aplaviroc. Reverse mutation of gp120 residues 316 and 323 in the V3 loop (numbering from HXB2) to their original sequence restored wild-type susceptibility to maraviroc, while reversion of either mutation resulted in a partially sensitive virus with reduced maximal inhibition (plateau). The plateaus are consistent with the virus having acquired the ability to utilize maraviroc-bound receptor for entry. This hypothesis was further corroborated by the observation that a high concentration of maraviroc blocks the activity of aplaviroc against maraviroc-resistant virus.

Low in vitro selection frequencies of enterococcal and staphylococcal mutants resistant to the oxazolidinone AZD2563.

Mutants with oxazolidinone MICs, 2- to 16-fold higher than those of parents, were selected from two of five clinical isolates of Enterococcus faecalis during exposure to AZD2563, but only at frequencies of ca. 10(-8). Resistance was not selected in Enterococcus faecium, Staphylococcus aureus or coagulase-negative staphylococci (CoNS). Mutants of one E. faecalis isolate had a G2576-->U 23S rRNA mutation; mutants derived from the second E. faecalis isolate lacked this mutation.

Detection of oxazolidinone-resistant Enterococcus faecalis and Enterococcus faecium strains by real-time PCR and PCR-restriction fragment length polymorphism analysis.

A real-time PCR assay identified linezolid-resistant Enterococcus faecalis and Enterococcus faecium isolates with a G2576U rRNA mutation. PCR-restriction fragment length polymorphism analysis of ribosomal DNA amplicons with NheI also detected this mutation. Both assays detected isolates heterozygous at this position. Recognition of isolates with what is presently the most frequent oxazolidinone resistance mutation may aid surveillance and individual case management.

Antibiotic resistance among clinical isolates of Acinetobacter in the UK, and in vitro evaluation of tigecycline (GAR-936).

A survey was conducted of the antimicrobial susceptibilities of 595 Acinetobacter spp. isolated from routine clinical specimens in 54 sentinel laboratories throughout the UK during 2000. Isolates of the Acinetobacter baumannii complex (genomic groups 2, 3 and 13TU; n = 443) were distinguished from other genomic groups (n = 152) by PCR fingerprinting of tDNA spacer regions. MICs of amikacin, cefotaxime, ceftazidime, ciprofloxacin, colistin, gentamicin, imipenem, meropenem, minocycline, piperacillin, piperacillin/tazobactam, rifampicin, sulbactam and tetracycline were determined on IsoSensitest agar and interpreted, wherever possible, using BSAC breakpoints. Tigecycline (GAR-936), a new glycylcycline, was also tested. Resistance to cephalosporins, aminoglycosides and ciprofloxacin was widespread, but carbapenems, colistin, sulbactam, minocycline and tigecycline were each active against >80% of the isolates. Isolates of A. baumannii were more often resistant to cefotaxime, ceftazidime, piperacillin, piperacillin/tazobactam, ciprofloxacin, gentamicin and tetracyclines than those belonging to other genomic groups, but were less often resistant to colistin; no significant differences between genomic groups were noted in the susceptibilities to amikacin, carbapenems, rifampicin or sulbactam. The relative activities of the tetracyclines were minocycline > tigecycline > tetracycline. Thirteen carbapenem-resistant isolates (MICs > or =8 mg/L; 2.2%) were received from six centres; four centres sent single isolates; one sent three and one sent six. An allele of bla(IMP) was detected in one of these isolates, but the other 12 isolates either had carbapenemase-independent resistance, or undetectable carbapenemase activity combined with other resistance mechanisms. In conclusion, carbapenems, colistin and minocycline retained greatest activity against the Acinetobacter isolates collected. Tigecycline was less active than minocycline, but both agents overcame most tetracycline resistance.