CREB5 promotes the proliferation and self-renewal ability of glioma stem cells.

Glioblastoma multiforme (GBM) is the most fatal form of brain cancer in humans, with a dismal prognosis and a median overall survival rate of less than 15 months upon diagnosis. Glioma stem cells (GSCs), have recently been identified as key contributors in both tumor initiation and therapeutic resistance in GBM. Both public dataset analysis and direct differentiation experiments on GSCs have demonstrated that CREB5 is more highly expressed in undifferentiated GSCs than in differentiated GSCs. Additionally, gene silencing by short hairpin RNA (shRNA) of CREB5 has prevented the proliferation and self-renewal ability of GSCs in vitro and decreased their tumor forming ability in vivo. Meanwhile, RNA-sequencing, luciferase reporter assay, and ChIP assay have all demonstrated the closely association between CREB5 and OLIG2. These findings suggest that targeting CREB5 could be an effective approach to overcoming GSCs.

Imprecision and Preferences in Interpretation of Verbal Probabilities in Health: a Systematic Review.

INTRODUCTION: Many health providers and communicators who are concerned that patients will not understand numbers instead use verbal probabilities (e.g., terms such as "rare" or "common") to convey the gist of a health message. OBJECTIVE: To assess patient interpretation of and preferences for verbal probability information in health contexts. METHODS: We conducted a systematic review of literature published through September 2020. Original studies conducted in English with samples representative of lay populations were included if they assessed health-related information and elicited either (a) numerical estimates of verbal probability terms or (b) preferences for verbal vs. quantitative risk information. RESULTS: We identified 33 original studies that referenced 145 verbal probability terms, 45 of which were included in at least two studies and 19 in three or more. Numerical interpretations of each verbal term were extremely variable. For example, average interpretations of the term "rare" ranged from 7 to 21%, and for "common," the range was 34 to 71%. In a subset of 9 studies, lay estimates of verbal probability terms were far higher than the standard interpretations established by the European Commission for drug labels. In 10 of 12 samples where preferences were elicited, most participants preferred numerical information, alone or in combination with verbal labels. CONCLUSION: Numerical interpretation of verbal probabilities is extremely variable and does not correspond well to the numerical probabilities established by expert panels. Most patients appear to prefer quantitative risk information, alone or in combination with verbal labels. Health professionals should be aware that avoiding numeric information to describe risks may not match patient preferences, and that patients interpret verbal risk terms in a highly variable way.

High-efficiency antibody discovery achieved with multiplexed microscopy.

The analysis of secreted antibody from large and diverse populations of B cells in parallel at the clonal level can reveal desirable antibodies for diagnostic or therapeutic applications. By immobilizing B cells in microdroplets with particulate reporters, decoding and isolating them in a microscopy environment, we have recovered panels of antibodies with rare attributes to therapeutically relevant targets. The ability to screen up to 100 million cells in a single experiment can be fully leveraged by accessing primary B-cell populations from evolutionarily divergent species such as chickens.

A high-affinity native human antibody neutralizes human cytomegalovirus infection of diverse cell types.

Human cytomegalovirus (HCMV) is the most common infection causing poor outcomes among transplant recipients. Maternal infection and transplacental transmission are major causes of permanent birth defects. Although no active vaccines to prevent HCMV infection have been approved, passive immunization with HCMV-specific immunoglobulin has shown promise in the treatment of both transplant and congenital indications. Antibodies targeting the viral glycoprotein B (gB) surface protein are known to neutralize HCMV infectivity, with high-affinity binding being a desirable trait, both to compete with low-affinity antibodies that promote the transmission of virus across the placenta and to displace nonneutralizing antibodies binding nearby epitopes. Using a miniaturized screening technology to characterize secreted IgG from single human B lymphocytes, 30 antibodies directed against gB were previously cloned. The most potent clone, TRL345, is described here. Its measured affinity was 1 pM for the highly conserved site I of the AD-2 epitope of gB. Strain-independent neutralization was confirmed for 15 primary HCMV clinical isolates. TRL345 prevented HCMV infection of placental fibroblasts, smooth muscle cells, endothelial cells, and epithelial cells, and it inhibited postinfection HCMV spread in epithelial cells. The potential utility for preventing congenital transmission is supported by the blockage of HCMV infection of placental cell types central to virus transmission to the fetus, including differentiating cytotrophoblasts, trophoblast progenitor cells, and placental fibroblasts. Further, TRL345 was effective at controlling an ex vivo infection of human placental anchoring villi. TRL345 has been utilized on a commercial scale and is a candidate for clinical evaluation.

Multiplexed screening of natural humoral immunity identifies antibodies at fine specificity for complex and dynamic viral targets.

Viral entry targets with therapeutic neutralizing potential are subject to multiple escape mechanisms, including antigenic drift, immune dominance of functionally irrelevant epitopes, and subtle variations in host cell mechanisms. A surprising finding of recent years is that potent neutralizing antibodies to viral epitopes independent of strain exist, but are poorly represented across the diverse human population. Identifying these antibodies and understanding the biology mediating the specific immune response is thus difficult. An effective strategy for meeting this challenge is to incorporate multiplexed antigen screening into a high throughput survey of the memory B cell repertoire from immune individuals. We used this approach to discover suites of cross-clade antibodies directed to conformational epitopes in the stalk region of the influenza A hemagglutinin (HA) protein and to select high-affinity anti-peptide antibodies to the glycoprotein B (gB) of human cytomegalovirus. In each case, our screens revealed a restricted VH and VL germline usage, including published and previously unidentified gene families. The in vivo evolution of paratope specificity with optimal neutralizing activity was understandable after correlating biological activities with kinetic binding and epitope recognition. Iterative feedback between antigen probe design based on structure and function information with high throughput multiplexed screening demonstrated a generally applicable strategy for efficient identification of safe, native, finely tuned antibodies with the potential for high genetic barriers to viral escape.

Chk1 and Hsp90 cooperatively regulate phosphorylation of endothelial nitric oxide synthase at serine 1179.

The effects of DNA damage on NO production have not been completely elucidated. Using ultraviolet (UV) irradiation as a DNA-damaging agent, we studied its effect on NO production in bovine aortic endothelial cells (BAEC). UV irradiation acutely increased NO production, the phosphorylation of endothelial NO synthase (eNOS) at serine 1179, and eNOS activity. No alterations in eNOS expression nor phosphorylation at eNOS Thr(497) or eNOS Ser(116) were found. SB218078, a checkpoint kinase 1 (Chk1) inhibitor, inhibited UV-irradiation-stimulated eNOS-Ser(1179) phosphorylation and NO production. Similarly, ectopic expression of small interference RNA for Chk1 or a dominant-negative Chk1 repressed the UV-irradiation stimulatory effect, whereas wild-type Chk1 increased basal eNOS-Ser(1179) phosphorylation. Purified Chk1 directly phosphorylated eNOS Ser(1179) in vitro. Confocal microscopy and coimmunoprecipitation studies revealed a colocalization of eNOS and Chk1. In basal BAEC, heat shock protein 90 (Hsp90) predominantly interacted with Chk1. This interaction, which decreased significantly in response to UV irradiation, was accompanied by increased interaction of Hsp90 with eNOS. The Hsp90 inhibitor geldanamycin attenuated UV-irradiation-stimulated eNOS-Ser(1179) phosphorylation by dissociating Hsp90 from eNOS. UV irradiation and geldanamycin did not alter the interaction between eNOS and Chk1. Overall, this is the first study demonstrating that Chk1 directly phosphorylates eNOS Ser(1179) in response to UV irradiation, which is dependent on Hsp90 interaction.

Potent high-affinity antibodies for treatment and prophylaxis of respiratory syncytial virus derived from B cells of infected patients.

Native human Abs represent attractive drug candidates; however, the low frequency of B cells expressing high-quality Abs has posed a barrier to discovery. Using a novel single-cell phenotyping technology, we have overcome this barrier to discover human Abs targeting the conserved but poorly immunogenic central motif of respiratory syncytial virus (RSV) G protein. For the entire cohort of 24 subjects with recent RSV infection, B cells producing Abs meeting these stringent specificity criteria were rare, <10 per million. Several of the newly cloned Abs bind to the RSV G protein central conserved motif with very high affinity (K(d) 1-24 pM). Two of the Abs were characterized in detail and compared with palivizumab, a humanized mAb against the RSV F protein. Relative to palivizumab, the anti-G Abs showed improved viral neutralization potency in vitro and enhanced reduction of infectious virus in a prophylaxis mouse model. Furthermore, in a mouse model for postinfection treatment, both anti-G Abs were significantly more effective than palivizumab at reducing viral load. The combination of activity in mouse models for both prophylaxis and treatment makes these high-affinity human-derived Abs promising candidates for human clinical testing.

IL-18 gene expression pattern in exogenously treated AML cells.

IL-18 production may enhance immune system defense against KG-1 cells ; NB4 cells, which are associated with good prognosis, do not produce IL-18. In this study, we treated KG-1 cells with IL-18 and used microarray technology to assess subsequent effects on gene expression. In UniGene-array of 7488 human genes, expression of 57 genes, including stress related genes, increased at least 2-fold, whereas expression of 48 genes decreased at least 2-fold. Following exogenous exposure of KG-1 cells to IL-18, expression of CRYGC, NF(kappa)BIA and NACA gene were monitored. The latter is a transcriptional coactivator potentiating c-Jun-mediated transcription. NF(kappa)BIA is an inhibitor of NF(kappa)B, and affects growth regulation, apoptosis and hypoxic stress. Studies, such as this one, are beginning to clarify the differences between cells associated with good and bad cancer prognoses, which may ultimately assist in medical treatment for acute myeloid leukemia.

Error and Performance Analysis of MEMS-based Inertial Sensors with a Low-cost GPS Receiver.

Global Navigation Satellite Systems (GNSS), such as the Global Positioning System (GPS), have been widely utilized and their applications are becoming popular, not only in military or commercial applications, but also for everyday life. Although GPS measurements are the essential information for currently developed land vehicle navigation systems (LVNS), GPS signals are often unavailable or unreliable due to signal blockages under certain environments such as urban canyons. This situation must be compensated in order to provide continuous navigation solutions. To overcome the problems of unavailability and unreliability using GPS and to be cost and size effective as well, Micro Electro Mechanical Systems (MEMS) based inertial sensor technology has been pushing for the development of low-cost integrated navigation systems for land vehicle navigation and guidance applications. This paper will analyze the characterization of MEMS based inertial sensors and the performance of an integrated system prototype of MEMS based inertial sensors, a low-cost GPS receiver and a digital compass. The influence of the stochastic variation of sensors will be assessed and modeled by two different methods, namely Gauss-Markov (GM) and AutoRegressive (AR) models, with GPS signal blockage of different lengths. Numerical results from kinematic testing have been used to assess the performance of different modeling schemes.

Characterization of humanized antibodies secreted by Aspergillus niger.

Two different humanized immunoglobulin G1(kappa) antibodies and an Fab' fragment were produced by Aspergillus niger. The antibodies were secreted into the culture supernatant. Both light and heavy chains were initially synthesized as fusion proteins with native glucoamylase. After antibody assembly, cleavage by A. niger KexB protease allowed the release of free antibody. Purification by hydrophobic charge induction chromatography proved effective at removing any antibody to which glucoamylase remained attached. Glycosylation at N297 in the Fc region of the heavy chain was observed, but this site was unoccupied on approximately 50% of the heavy chains. The glycan was of the high-mannose type, with some galactose present, and the size ranged from Hex(6)GlcNAc(2) to Hex(15)GlcNAc(2). An aglycosyl mutant form of antibody was also produced. No significant difference between the glycosylated antibody produced by Aspergillus and that produced by mammalian cell cultures was observed in tests for affinity, avidity, pharmacokinetics, or antibody-dependent cellular cytotoxicity function.

Humanization of a chicken anti-IL-12 monoclonal antibody.

Chicken anti-IL-12 monoclonal antibodies were isolated by phage display using spleen cells from a chicken immunized with human and mouse IL-12 as a source for library construction. One of the chicken monoclonal antibodies, DD2, exhibited binding to both human and mouse IL-12 in the single-chain Fv form and also after conversion to chicken-human chimeric IgG1/lambda antibody. The chicken DD2 variable regions were humanized by transferring their CDRs and several framework amino acids onto human acceptor variable regions. In the Vlambda, six chicken framework amino acids were identified to be important for the conformation of the CDR structure by computer modeling and therefore were retained in the humanized form; likewise, five chicken amino acids in the VH framework regions were retained in the humanized VH. The affinities of humanized DD2 IgG1/lambda to human and mouse IL-12 measured by competitive binding were nearly identical to those of chicken-human chimeric DD2 IgG1/lambda. This work demonstrates that humanization of chicken monoclonal antibodies assisted by computer modeling is possible, leading to a new way to generate therapeutic humanized antibodies against antigens to which the rodent immune system may fail to efficiently raise high affinity antibodies.

Cell surface expression of C1qRP/CD93 is stabilized by O-glycosylation.

C1qRP/CD93 is a cell surface receptor predominantly expressed on monocytes, neutrophils, endothelial cells, and early stem cell precursors. In phagocytic cells, it has been characterized as contributing to the enhancement of FcR- and CR1-induced phagocytosis triggered by innate immune system defense collagens such as C1q and mannose binding lectin (MBL). Previously, we demonstrated a high level of glycosylation on C1qRP/CD93 that was predominantly O-linked. In this study, we investigate the role of glycosylation in C1qRP/CD93 stability first by inhibiting O-glycosylation by addition of benzyl 2-acetamido-2-deoxy-alpha-D-galactopyranoside (BAG) to the human histiocytic cell line U937, and secondly, by expression of C1qRP/CD93 in the CHO-derived cell line ldlD which has a reversible defect in protein glycosylation. In both U937 cells and in ldlD cells transfected to express C1qRP/CD93, glycosylation deficiency caused cell surface expression levels of C1qRP/CD93 to decrease, concomitant with the detection of C1qRP/CD93 reactivity in the culture media. Metabolic labeling studies show that when glycosylation is absent, C1qRP/CD93 is synthesized and rapidly released into the culture supernatant or degraded. These studies demonstrate that O-glycosylation is important in the stable cell surface expression of C1qRP/CD93 .