AP-1 homolog BZLF1 of Epstein-Barr virus has two essential functions dependent on the epigenetic state of the viral genome.

EBV, a member of the herpes virus family, is a paradigm for human tumor viruses and a model of viral latency amenable for study in vitro. It induces resting human B lymphocytes to proliferate indefinitely in vitro and initially establishes a strictly latent infection in these cells. BZLF1, related to the cellular activating protein 1 (AP-1) family of transcription factors, is the viral master gene essential and sufficient to mediate the switch to induce the EBV lytic phase in latently infected B cells. Enigmatically, after infection BZLF1 is expressed very early in the majority of primary B cells, but its early expression fails to induce the EBV lytic phase. We show that the early expression of BZLF1 has a critical role in driving the proliferation of quiescent naïve and memory B cells but not of activated germinal center B cells. BZLF1's initial failure to induce the EBV lytic phase relies on the viral DNA at first being unmethylated. We have found that the eventual and inevitable methylation of viral DNA is a prerequisite for productive infection in stably, latently infected B cells which then yield progeny virus lacking cytosine-phosphatidyl-guanosine (CpG) methylation. This progeny virus then can repeat EBV's epigenetically regulated, biphasic life cycle. Our data indicate that the viral BZLF1 protein is crucial both to establish latency and to escape from it. Our data also indicate that EBV has evolved to appropriate its host's mode of methylating DNA for its own epigenetic regulation.

CpG-methylation regulates a class of Epstein-Barr virus promoters.

DNA methylation is the major modification of eukaryotic genomes and plays an essential role in mammalian gene regulation. In general, cytosine-phosphatidyl-guanosine (CpG)-methylated promoters are transcriptionally repressed and nuclear proteins such as MECP2, MBD1, MBD2, and MBD4 bind CpG-methylated DNA and contribute to epigenetic silencing. Methylation of viral DNA also regulates gene expression of Epstein-Barr virus (EBV), which is a model of herpes virus latency. In latently infected human B cells, the viral DNA is CpG-methylated, the majority of viral genes is repressed and virus synthesis is therefore abrogated. EBV's BZLF1 encodes a transcription factor of the AP-1 family (Zta) and is the master gene to overcome viral gene repression. In a genome-wide screen, we now identify and characterize those viral genes, which Zta regulates. Among them are genes essential for EBV's lytic phase, which paradoxically depend on strictly CpG-methylated promoters for their Zta-induced expression. We identified novel DNA recognition motifs, termed meZRE (methyl-Zta-responsive element), which Zta selectively binds in order to 'read' DNA in a methylation- and sequence-dependent manner unlike any other known protein. Zta is a homodimer but its binding characteristics to meZREs suggest a sequential, non-palindromic and bipartite DNA recognition element, which confers superior DNA binding compared to CpG-free ZREs. Our findings indicate that Zta has evolved to transactivate cytosine-methylated, hence repressed, silent promoters as a rule to overcome epigenetic silencing.

Towards maximum acceleration of monoclonal antibody development: Leveraging transposase-mediated cell line generation to enable GMP manufacturing within 3 months using a stable pool.

In recent years, acceleration of development timelines has become a major focus within the biopharmaceutical industry to bring innovative therapies faster to patients. However, in order to address a high unmet medical need even faster further acceleration potential has to be identified to transform "speed-to-clinic" concepts into "warp-speed" development programs. Recombinant Chinese hamster ovary (CHO) cell lines are the predominant expression system for monoclonal antibodies (mAbs) and are routinely generated by random transgene integration (RTI) of the genetic information into the host cell genome. This process, however, exhibits considerable challenges such as the requirement for a time-consuming clone screening process to identify a suitable clonally derived manufacturing cell line. Hence, RTI represents an error prone and tedious method leading to long development timelines until availability of Good Manufacturing Practice (GMP)-grade drug substance (DS). Transposase-mediated semi-targeted transgene integration (STI) has been recently identified as a promising alternative to RTI as it allows for a more rapid generation of high-performing and stable production cell lines. In this report, we demonstrate how a STI technology was leveraged to develop a very robust DS manufacturing process based on a stable pool cell line at unprecedented pace. Application of the novel strategy resulted in the manufacturing of GMP-grade DS at 2,000 L scale in less than three months paving the way for a start of Phase I clinical trials only six months after transfection. Finally, using a clonally derived production cell line, which was established from the parental stable pool, we were able to successfully implement a process with an increased mAb titer of up to 5 g per liter at the envisioned commercial scale (12,000 L) within eight months.

Accuracy of fully automated oscillometric central aortic blood pressure measurement techniques.

BACKGROUND: Central aortic blood pressure (cBP) is a valuable predictor of cardiovascular risk. The lack of fully automated measurement devices impeded an implementation in daily clinical practice so far. The present study compares two novel automated oscillometric devices with invasively measured cBP. METHODS: From March 2017 to March 2018, we enrolled consecutive patients undergoing elective coronary angiography to this cross-sectional study. Noninvasive assessment of cBP was performed by the SphygmoCor XCEL device and the Mobil-O-Graph NG device simultaneously to invasive measurement. RESULTS: Our study included 502 patients (228 women, 274 men) with a mean age of 67.9 ±â€Š11.6 years. The noninvasive measurement of cBP was successful in 498 patients (99%) with SphygmoCor XCEL device and in 441 patients (88%) with Mobil-O-Graph NG device (P = 0.451). Measurements of both devices revealed a high correlation to invasively measured systolic (SphygmoCor R 0.864, P < 0.001; Mobil-O-Graph R 0.763, P < 0.001) and diastolic (SphygmoCor R 0.772, P < 0.001; Mobil-O-Graph R 0.618, P < 0.001) cBP. Both devices slightly underestimated systolic and overestimated diastolic central blood pressure: biases were -5.0 ±â€Š7.7/0.5 ±â€Š6.2 mmHg with SphygmoCor XCEL and -6.0 ±â€Š10.4/3.6 ±â€Š8.3 mmHg with Mobil-O-Graph NG device. Correlations (R) were higher and biases were lower with the SphygmoCor device (P < 0.001 each). CONCLUSION: The present study is the largest validation study of noninvasive cBP measurement techniques so far and shows that two current automated oscillometric monitors are able to assess cBP with acceptable accuracy. Automated oscillometric devices may facilitate the implementation of cBP in daily clinical practice.

Hemodynamics of paradoxical severe aortic stenosis: insight from a pressure-volume loop analysis.

BACKGROUND: Controversy exists about the pathophysiology of different hemodynamic subgroups of AS. In particular, the mechanism of the paradoxical low-flow, low-gradient (PLFLG) AS with preserved ejection fraction (EF) is unclear. METHODS: A total of 41 patients with severe, symptomatic AS were divided into the following 4 subgroups based on the echocardiographically determined hemodynamics: (1) normal-flow, high-gradient (NFHG) AS; (2) low-flow, high-gradient AS; (3) paradoxical low-flow, low-gradient (PLFLG) AS with preserved EF and (4) low-flow, low-gradient (LFLG) AS with reduced EF. As part of the comprehensive invasive examinations, the analyses of the PV loops were performed with the IntraCardiac Analyzer (CD-Leycom, The Netherlands). RESULTS: PLFLG was characterized by small left ventricular volumes as well as a decreased cardiac index, a decreased systolic contractility and a lower stroke work, than the conventional NFHG AS. Alterations in effective arterial elastance (2.36 ± 0.67 mmHg/ml in NFHG versus 3.01 ± 0.79 mmHg/ml in PLFLG, p = 0.036) and end-systolic elastance (3.72 ± 1.84 mmHg/ml in NFHG versus 5.53 ± 2.3 mmHg/ml in PLFLG, p = 0.040) indicated impaired vascular function and increased chamber stiffness. CONCLUSIONS: The present study suggests that the hemodynamics of PLFLG AS can be explained by two mechanisms: (1) stiffness of the small left ventricle with reduced contractility, and (2) increased afterload due to the impairment of vascular function. Both mechanisms have similarities to those of heart failure with preserved EF. This type of remodeling may explain the poor prognosis of PLFLG AS.