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Pancreatic ductal adenocarcinoma (PDA) is a heterogeneous disease comprised of a basal-like subtype with mesenchymal gene signatures, undifferentiated histopathology and worse prognosis compared to the classical subtype. Despite their prognostic and therapeutic value, the key drivers that establish and control subtype identity remain unknown. Here, we demonstrate that PDA subtypes are not permanently encoded, and identify the GLI2 transcription factor as a master regulator of subtype inter-conversion. GLI2 is elevated in basal-like PDA lines and patient specimens, and forced GLI2 activation is sufficient to convert classical PDA cells to basal-like. Mechanistically, GLI2 upregulates expression of the pro-tumorigenic secreted protein, Osteopontin (OPN), which is especially critical for metastatic growth in vivo and adaptation to oncogenic KRAS ablation. Accordingly, elevated GLI2 and OPN levels predict shortened overall survival of PDA patients. Thus, the GLI2-OPN circuit is a driver of PDA cell plasticity that establishes and maintains an aggressive variant of this disease.

As a para-retrovirus, hepatitis B virus (HBV) is an enveloped virus with a double-stranded (DS) DNA genome that is replicated by reverse transcription of an RNA intermediate, the pregenomic RNA or pgRNA. HBV assembly begins with the formation of an \"immature\" nucleocapsid (NC) incorporating pgRNA, which is converted via reverse transcription within the maturing NC to the DS DNA genome. Only the mature, DS DNA-containing NCs are enveloped and secreted as virions whereas immature NCs containing RNA or single-stranded (SS) DNA are not enveloped. The current model for selective virion morphogenesis postulates that accumulation of DS DNA within the NC induces a \"maturation signal\" that, in turn, triggers its envelopment and secretion. However, we have found, by careful quantification of viral DNA and NCs in HBV virions secreted in vitro and in vivo, that the vast majority of HBV virions (over 90%) contained no DNA at all, indicating that NCs with no genome were enveloped and secreted as empty virions (i.e., enveloped NCs with no DNA). Furthermore, viral mutants bearing mutations precluding any DNA synthesis secreted exclusively empty virions. Thus, viral DNA synthesis is not required for HBV virion morphogenesis. On the other hand, NCs containing RNA or SS DNA were excluded from virion formation. The secretion of DS DNA-containing as well as empty virions on one hand, and the lack of secretion of virions containing single-stranded (SS) DNA or RNA on the other, prompted us to propose an alternative, \"Single Strand Blocking\" model to explain selective HBV morphogenesis whereby SS nucleic acid within the NC negatively regulates NC envelopment, which is relieved upon second strand DNA synthesis.

With the increasing recognition of biofilms in human disease, the development of novel antimicrobial therapies is of critical importance. For example, in patients with cystic fibrosis (CF), the acquisition of host-adapted, chronic Pseudomonas aeruginosa infection is associated with a decline in lung function and increased mortality. Our objective was to test the in vitro efficacy of a membrane-active antimicrobial peptide we designed, termed 6K-F17 (sequence: KKKKKK-AAFAAWAAFAA-NH 2 ), against multidrug resistant P . aeruginosa biofilms. This peptide displays high antimicrobial activity against a range of pathogenic bacteria, yet is non-hemolytic to human erythrocytes and non-toxic to human bronchial epithelial cells. In the present work, P . aeruginosa strain PAO1, and four multidrug resistant (MDR) isolates from chronically infected CF individuals, were grown as 48-hour biofilms in a static biofilm slide chamber model. These biofilms were then exposed to varying concentrations of 6K-F17 alone, or in the presence of tobramycin, prior to confocal imaging. Biofilm biovolume and viability were assessed. 6K-F17 was able to kill biofilms – even in the presence of sputum – and greatly reduce biofilm biovolume in PAO1 and MDR isolates. Strikingly, when used in conjunction with tobramycin, low doses of 6K-F17 significantly potentiated tobramycin killing, leading to biofilm destruction.

Safe landings are imperative to accomplish NASA’s Artemis goal to enable human exploration on the Moon, including sample collection missions. However, a process known as plume surface interaction (PSI) presents a significant hazard to lunar landings. PSI occurs when the engine exhaust of a lander interacts with the surface ejecting large amounts of regolith particles at high velocities that can interfere with the landing, disturb the surface, and damage hardware. To better understand PSI, the particle impact event (PIE) sensor is being developed to measure the kinetic energy and the flux of ejecta during landings, to quantify the potential damage, and to quantify the ejecta displaced. Multiple parameters were estimated to define the PIE instrument requirements. These estimates demonstrate that ejecta can travel at velocities of up to 800 m/s and impact the surrounding area with energies of up to 400 µJ. A significant amount of ejecta can be deposited several 10 s of meters away from the landing site, modifying the surface and causing dust-related challenges. The PIE sensor will be launched for the first time in an upcoming lunar lander. Then, PIE measurements will be used to improve PSI prediction capabilities and develop mitigation strategies to ensure safe landings.

Improved therapies are needed against snakebite envenoming, which kills and permanently disables thousands of people each year. Recently developed neutralizing monoclonal antibodies against several snake toxins have shown promise in preclinical rodent models. Here, we use phage display technology to discover a human monoclonal antibody and show that this antibody causes antibody-dependent enhancement of toxicity (ADET) of myotoxin II from the venomous pit viper, Bothrops asper , in a mouse model of envenoming that mimics a snakebite. While clinical ADET related to snake venom has not yet been reported in humans, this report of ADET of a toxin from the animal kingdom highlights the necessity of assessing even well-known antibody formats in representative preclinical models to evaluate their therapeutic utility against toxins or venoms. This is essential to avoid potential deleterious effects as exemplified in the present study. The recent emergence of monoclonal antibodies able to neutralize snake toxins have revolutionized the approach of developing novel therapies to treat snakebite envenoming, at least in animal models. Here, the authors show antibody-dependent enhancement of toxicity (ADET) for a toxin derived from snake venom and highlight the importance of this phenomenon when testing therapeutic antibodies against snake venoms in animal models.

Dynamic phosphorylation and dephosphorylation of the hepadnavirus core protein C-terminal domain (CTD) are required for multiple steps of the viral life cycle. It remains unknown how the CTD phosphorylation state may modulate core protein functions but phosphorylation state-dependent viral or host interactions may play a role. In an attempt to identify host factors that may interact differentially with the core protein depending on its CTD phosphorylation state, pulldown assays were performed using the CTD of the duck hepatitis B virus (DHBV) and human hepatitis B virus (HBV) core protein, either with wild type (WT) sequences or with alanine or aspartic acid substitutions at the phosphorylation sites. Two host proteins, B23 and I2PP2A, were found to interact preferentially with the alanine-substituted CTD. Furthermore, the WT CTD became competent to interact with the host proteins upon dephosphorylation. Intriguingly, the binding site on the DHBV CTD for both B23 and I2PP2A was mapped to a region upstream of the phosphorylation sites even though B23 or I2PP2A binding to this site was clearly modulated by the phosphorylation state of the downstream and non-overlapping sequences. Together, these results demonstrate a novel mode of phosphorylation-regulated protein-protein interaction and provide new insights into virus-host interactions.

Monoclonal antibody (mAb) production in CHO cells depends, among other factors, on balanced co-expression of the heavy (HC) and light (LC) chains. Imbalances between HC and LC can reduce titer, compromise product quality, and negatively affect cell viability, which means that the optimal LC/HC expression ratio should be identified as early as possible in cell line development. However, systematically testing multiple LC/HC expression ratios for many antibody candidates using traditional workflows is slow and resource intensive. Here, we present a high-throughput screening platform, coupled with a design-of-experiments (DoE) strategy, to identify optimal LC/HC expression balance at the transient stage. The system uses single-vector constructs encoding both LC and HC under promoters of defined low, medium, or high strength, enabling combinatorial testing of LC/HC promoter pairs. We applied this workflow to three different antibodies and quantified titer, viable cell density, and viability 72 h post-transfection. The optimal LC/HC promoter ratio was antibody specific. For two antibodies, high LC combined with medium HC expression yielded the highest titer while maintaining cell viability. High LC with high HC expression (LC100–HC100) also produced high titer but caused reduced viable cell density and viability. For the third antibody, the best-performing configuration was medium LC with medium HC, with medium LC and high HC as a close second. Across all three antibodies, low-strength promoters for either chain consistently resulted in poor titer. Overall, this platform offers a rapid and scalable approach to define antibody-specific LC/HC promoter strength combinations that maximize productivity without compromising cell health, enabling more informed construct selection before committing to stable clone generation.

[...]even the tiniest problem can reduce a report-writing teacher to a ranting mess, incapable of seeing reason. The well-organised crew like to let you know a few weeks before the reporting deadline that they have finished writing their comments and entering their data. [...]the problem is that those weeks fly by and suddenly you are sitting a few days out from the deadline surrounded by piles of unmarked assessment pieces, copious cups of coffee and a fuse that resembles a ticking time bomb.

The relaxed state you have worked so hard to achieve starts to dissipate and the guaranteed restless sleep that ensues ensures that you return to school feeling disheveled, tired, resentful and confused. Term 4 at my school is often referred to as the \"party term\" in reference to the large number of social events that occur and the fact that Muck Up Day (we can't call it that, but we all know that's what it is) and graduation/ valedictory nights fall within it. [...]it is the same speech I have heard every principal at every school I've ever worked at deliver at this time of year.