DARPA Phoenix Payload Orbital Delivery System: Progress towards Small Satellite Access to GEO

The emerging practice of hosting payloads on commercial geosynchronous Earth orbit (GEO) satellites is gaining traction throughout the space community because of the flight opportunities and budgetary savings that it offers. Using the hosted payload model, the DARPA Phoenix Payload Orbital Delivery (POD) system is meant to enable a higher tempo to GEO for small‐mass hardware items. The POD system proposes a departure from the typical hosted payload. The POD would provide a controlled release of the hosted payload from the commercial host near GEO. The POD standard user\u27s guide developed under the Phoenix program ensures compatibility with most of the approximately 15 commercial launches to GEO each year. By hosting with a standard user’s guide, commercial satellite providers would be capable of bringing hosted payloads quite late into the typical launch integration cycle. The combination of hightempo commercial launches and late integration would create an “express delivery” capability to GEO orbit. This POD capability would continue the paradigm shift of working with the commercial satellite provider directly to leverage the efficiencies of mass to orbit, reducing interactions with the launch provider. Phoenix is completing the design and ground testing of the POD system to help make access to new orbits more affordable and more routine for small‐mass systems

TricycloDNA-modified oligo-2′-deoxyribonucleotides reduce scavenger receptor B1 mRNA in hepatic and extra-hepatic tissues—a comparative study of oligonucleotide length, design and chemistry

We report the evaluation of 20-, 18-, 16- and 14-mer phosphorothioate (PS)-modified tricycloDNA (tcDNA) gapmer antisense oligonucleotides (ASOs) in Tm, cell culture and animal experiments and compare them to their gap-matched 20-mer 2′-O-methoxyethyl (MOE) and 14-mer 2′,4′-constrained ethyl (cEt) counterparts. The sequence-matched 20-mer tcDNA and MOE ASOs showed similar Tm and activity in cell culture under free-uptake and cationic lipid-mediated transfection conditions, while the 18-, 16- and 14-mer tcDNA ASOs were moderate to significantly less active. These observations were recapitulated in the animal experiments where the 20-mer tcDNA ASO formulated in saline showed excellent activity (ED50 3.9 mg/kg) for reducing SR-B1 mRNA in liver. The tcDNA 20-mer ASO also showed better activity than the MOE 20-mer in several extra-hepatic tissues such as kidney, heart, diaphragm, lung, fat, gastrocnemius and quadriceps. Interestingly, the 14-mer cEt ASO showed the best activity in the animal experiments despite significantly lower Tm and 5-fold reduced activity in cell culture relative to the 20-mer tcDNA and MOE-modified ASOs. Our experiments establish tcDNA as a useful modification for antisense therapeutics and highlight the role of chemical modifications in influencing ASO pharmacology and pharmacokinetic properties in animal

Retroviral integrations contribute to elevated host cancer rates during germline invasion

© 2021, The Author(s). Repeated retroviral infections of vertebrate germlines have made endogenous retroviruses ubiquitous features of mammalian genomes. However, millions of years of evolution obscure many of the immediate repercussions of retroviral endogenisation on host health. Here we examine retroviral endogenisation during its earliest stages in the koala (Phascolarctos cinereus), a species undergoing germline invasion by koala retrovirus (KoRV) and affected by highcancerprevalence. We characterise KoRV integration sites (IS) in tumour and healthy tissues from 10 koalas, detecting 1002 unique IS, with hotspots of integration occurring in the vicinity of known cancer genes. We find that tumours accumulate novel IS, with proximate genes over-represented for cancer associations. We detect dysregulation of genes containing IS and identify a highly-expressed transduced oncogene. Our data provide insights into the tremendous mutational load suffered by the host during active retroviral germline invasion, a process repeatedly experienced and overcome during the evolution of vertebrate lineages

Regulation of intestinal chloride secretion by direct and indirect activation of the epidermal growth factor receptor

Intestinal fluid transport is critical for mediating proper digestion, absorption of nutrients, and clearance of waste, toxins, and pathogens, all without excessive dehydration. Movement of water is largely passive and driven by osmotic gradients established by active transport of ions. Apposite fluidity of intestinal contents is achieved through tightly regulated coordination of a complex machinery of ion transporters involved in both absorptive and secretory processes. The intestinal epithelium must respond to an ever-changing luminal environment and fluid transport is regulated in response to a variety of endogenous stimuli including neuronal, hormonal, and immune mediators, as well as exogenous environmental factors such as bacterial agents, nutrients, and toxins. The epidermal growth factor receptor (EGFr) is an important regulator of intestinal chloride secretion and can be differentially activated in response to a variety of stimuli to elicit divergent effects. Furthermore, signaling through EGFr is modified in the context of the overall physiological setting, for instance in the presence of inflammation or infection. Different components of the secretory machinery are regulated in response to differential activation of EGFr. A number of diseases and conditions exhibit dysregulation of intestinal fluid transport characterized by disturbances in absorption, secretion, or both. To fully address many of these conditions, it is important to understand how EGFr activation regulates intestinal fluid transport under various physiological conditions. To this end, we set out to identify transporter targets subject to regulation by EGFr under conditions that differentially activate the receptor. We have identified Na⁺, K⁺, 2 Cl⁻ Cotransporter 1 (NKCC1), a widely expressed transporter important to chloride secretion, as being differentially regulated through EGFr activation. When EGFr is transactivated in response to the cholinergic agonist, carbachol, NKCC1 is endocytosed from its site of activity at the basolateral plasma membrane through a mechanism that requires EGFr-dependent activation of mitogen- activated protein kinase (MAPK) pathways. However, when EGFr is activated through direct exposure to the bona fide ligand, epidermal growth factor (EGF), phosphatidylinositide-3 kinase (PI3K) is recruited rather than MAPK and NKCC1 is not endocytosed. This divergent regulation of NKCC1 through differential activation of EGFr has implications not only for the regulation of intestinal ion and fluid transport and the treatment of their dysregulation, but also for a number of other physiological processes and pathologies involving EGFr and NKCC

TricycloDNA-modified oligo-2′-deoxyribonucleotides reduce scavenger receptor B1 mRNA in hepatic and extra-hepatic tissues - a comparative study of oligonucleotide length, design and chemistry

We report the evaluation of 20-, 18-, 16- and 14-mer phosphorothioate (PS)-modified tricycloDNA (tcDNA) gapmer antisense oligonucleotides (ASOs) in T(m), cell culture and animal experiments and compare them to their gap-matched 20-mer 2′-O-methoxyethyl (MOE) and 14-mer 2′,4′-constrained ethyl (cEt) counterparts. The sequence-matched 20-mer tcDNA and MOE ASOs showed similar T(m) and activity in cell culture under free-uptake and cationic lipid-mediated transfection conditions, while the 18-, 16- and 14-mer tcDNA ASOs were moderate to significantly less active. These observations were recapitulated in the animal experiments where the 20-mer tcDNA ASO formulated in saline showed excellent activity (ED(50) 3.9 mg/kg) for reducing SR-B1 mRNA in liver. The tcDNA 20-mer ASO also showed better activity than the MOE 20-mer in several extra-hepatic tissues such as kidney, heart, diaphragm, lung, fat, gastrocnemius and quadriceps. Interestingly, the 14-mer cEt ASO showed the best activity in the animal experiments despite significantly lower T(m) and 5-fold reduced activity in cell culture relative to the 20-mer tcDNA and MOE-modified ASOs. Our experiments establish tcDNA as a useful modification for antisense therapeutics and highlight the role of chemical modifications in influencing ASO pharmacology and pharmacokinetic properties in animals

AMPK mediates inhibition of electrolyte transport and NKCC1 activity by reactive oxygen species.

Reactive oxygen species such as H2O2 are believed to play a prominent role in the injury and loss of transport function that affect the intestinal epithelium in inflammatory conditions such as inflammatory bowel diseases. Defects in intestinal epithelial ion transport regulation contribute to dysbiosis and inflammatory phenotypes. We previously showed that H2O2 inhibits Ca2+-dependent Cl- secretion across intestinal epithelial cells (IECs) via a phosphatidylinositol 3-kinase (PI3K)- and extracellular signal-regulated kinase (ERK)-dependent mechanism that occurs, at least in part, through inhibition of the basolateral Na+-K+-2Cl- cotransporter NKCC1. NKCC1 governs Cl- entry into crypt IECs and thus plays a critical role in maintaining the driving force for Cl- secretion. Electrolyte transport consumes large amounts of cellular energy, and direct pharmacological activation of the cellular energy sensor AMP-activated protein kinase (AMPK) has been shown to inhibit a number of ion transport proteins. Here, we show that H2O2 activates AMPK in human IEC lines and ex vivo human colon. Moreover, we demonstrate that the inhibitory effect of H2O2 on Ca2+-dependent Cl- secretion and NKCC1 activity is AMPK-dependent. This inhibitory effect is associated with a physical interaction between AMPK and NKCC1, as well as increased phosphorylation (Thr212,217) of NKCC1, without causing NKCC1 internalization. These data identify a key role for AMPK-NKCC1 interaction as a point of convergence for suppression of colonic epithelial ion transport by inflammatory reactive oxygen species.NEW & NOTEWORTHY H2O2 inhibition of intestinal epithelial Ca2+-dependent Cl- secretion involves recruitment of AMP-activated protein kinase (AMPK) downstream of ERK and phosphatidylinositol 3-kinase signaling pathways, physical interaction of AMPK with the Na+-K+-2Cl- cotransporter NKCC1, and AMPK-dependent suppression of NKCC1-mediated electrolyte influx without causing NKCC1 internalization. It is intriguing that, in human intestinal epithelial cell lines and human colon, H2O2 activation of AMPK increased phosphorylation of NKCC1 residues required for promoting, not inhibiting, NKCC1 activity. These data identify an elevated complexity of AMPK regulation of NKCC1 in the setting of an inflammatory stimulus