ISGylation-independent protection of cell growth by USP18 following interferon stimulation

Type 1 interferon stimulation highly up-regulates all elements of a ubiquitin-like conjugation system that leads to ISGylation of target proteins. An ISG15-specific member of the deubiquitylase family, USP18, is up-regulated in a co-ordinated manner. USP18 can also provide a negative feedback by inhibiting JAK-STAT signalling through protein interactions independently of DUB activity. Here, we provide an acute example of this phenomenon, whereby the early expression of USP18, post-interferon treatment of HCT116 colon cancer cells is sufficient to fully suppress the expression of the ISG15 E1 enzyme, UBA7. Stimulation of lung adenocarcinoma A549 cells with interferon reduces their growth rate but they remain viable. In contrast, A549 USP18 knock-out cells show similar growth characteristics under basal conditions, but upon interferon stimulation, a profound inhibition of cell growth is observed. We show that this contingency on USP18 is independent of ISGylation, suggesting non-catalytic functions are required for viability. We also demonstrate that global deISGylation kinetics are very slow compared with deubiquitylation. This is not influenced by USP18 expression, suggesting that enhanced ISGylation in USP18 KO cells reflects increased conjugating activity.</p

ISGylation-independent protection of cell growth by USP18 following interferon stimulation

Type 1 interferon stimulation highly up-regulates all elements of a ubiquitin-like conjugation system that leads to ISGylation of target proteins. An ISG15-specific member of the deubiquitylase family, USP18, is up-regulated in a co-ordinated manner. USP18 can also provide a negative feedback by inhibiting JAK-STAT signalling through protein interactions independently of DUB activity. Here, we provide an acute example of this phenomenon, whereby the early expression of USP18, post-interferon treatment of HCT116 colon cancer cells is sufficient to fully suppress the expression of the ISG15 E1 enzyme, UBA7. Stimulation of lung adenocarcinoma A549 cells with interferon reduces their growth rate but they remain viable. In contrast, A549 USP18 knock-out cells show similar growth characteristics under basal conditions, but upon interferon stimulation, a profound inhibition of cell growth is observed. We show that this contingency on USP18 is independent of ISGylation, suggesting non-catalytic functions are required for viability. We also demonstrate that global deISGylation kinetics are very slow compared with deubiquitylation. This is not influenced by USP18 expression, suggesting that enhanced ISGylation in USP18 KO cells reflects increased conjugating activity.</p

Examining relationships between parent-reported factors and recurring ear symptoms among Aboriginal and Torres Strait Islander children

Issue addressed: Aboriginal and Torres Strait Islander child ear health is complex and multiple. We examined relationships between parent-reported sociodemographic, child health, health service access factors and ear symptoms among Aboriginal and Torres Strait Islander children aged 3 to 7 years. Methods: The Longitudinal Study of Indigenous Children is a large child cohort study with annual parent-reported data collection. Generalised linear mixed model analyses examined Wave 1 (1309 children 0-5 years; 2008) predictors of being free of parent-reported ear symptoms in both Waves 2 and 3. Results: A total of 1030 (78.7%) had no reported ear symptoms in either Wave 2 or 3. In the fully adjusted model, children who had been hospitalised in the past year (aOR = 2.16; 95% CI 1.19-3.93) and those with no ear symptoms (aOR = 2.94; 95% CI, 1.59-5.46) at Wave 1 had higher odds of no ear symptoms in both the subsequent waves. There were also relationships between parent main source of income–government pension or allowance as well as parents who reported no history of their own ear symptoms and higher odds of no ear symptoms in Waves 2 and 3 after partial adjustment for sociodemographic factors. Conclusion: These findings suggest relationships between different sociodemographic and health factors and parent-reported ear symptoms among Aboriginal and Torres Strait Islander children that warrant further investigation. So what? Children with parent-reported ear symptoms during the early years need holistic support to prevent future ear symptoms that impact health, social and educational life trajectories

Ear and hearing care programs for First Nations children: a scoping review

Background: Ear and hearing care programs are critical to early detection and management of otitis media (or middle ear disease). Otitis media and associated hearing loss disproportionately impacts First Nations children. This affects speech and language development, social and cognitive development and, in turn, education and life outcomes. This scoping review aimed to better understand how ear and hearing care programs for First Nations children in high-income colonial-settler countries aimed to reduce the burden of otitis media and increase equitable access to care. Specifically, the review aimed to chart program strategies, map the focus of each program against 4 parts of a care pathway (prevention, detection, diagnosis/management, rehabilitation), and to identify the factors that indicated the longer-term sustainability and success of programs. Method: A database search was conducted in March 2021 using Medline, Embase, Global Health, APA PsycInfo, CINAHL, Web of Science Core Collection, Scopus, and Academic Search Premier. Programs were eligible or inclusion if they had either been developed or run at any time between January 2010 to March 2021. Search terms encompassed terms such as First Nations children, ear and hearing care, and health programs, initiatives, campaigns, and services. Results: Twenty-seven articles met the criteria to be included in the review and described a total of twenty-one ear and hearing care programs. Programs employed strategies to: (i) connect patients to specialist services, (ii) improve cultural safety of services, and (iii) increase access to ear and hearing care services. However, program evaluation measures were limited to outputs or the evaluation of service-level outcome, rather than patient-based outcomes. Factors which contributed to program sustainability included funding and community involvement although these were limited in many cases. Conclusion: The result of this study highlighted that programs primarily operate at two points along the care pathway—detection and diagnosis/management, presumably where the greatest need lies. Targeted strategies were used to address these, some which were limited in their approach. The success of many programs are evaluated as outputs, and many programs rely on funding sources which can potentially limit longer-term sustainability. Finally, the involvement of First Nations people and communities typically only occurred during implementation rather than across the development of the program. Future programs should be embedded within a connected system of care and tied to existing policies and funding streams to ensure long term viability. Programs should be governed and evaluated by First Nations communities to further ensure programs are sustainable and are designed to meet community needs

Political institutions and debt crises

This paper shows that political institutions matter in explaining defaults on external and domestic debt obligations. We explore a large number of political and macroeconomic variables using a non-parametric technique to predict safety from default. The advantage of this technique is that it is able to identify patterns in the data that are not captured in standard probit analysis. We find that political factors matter, and do so in different ways for democratic and non-democratic regimes, and for domestic and external debt. In democracies, a parliamentary system or sufficient checks and balances almost guarantee the absence of default on external debt when economic fundamentals or liquidity are sufficiently strong. In dictatorships, high stability and tenure play a similar role for default on domestic debt

Virtual PCR

The polymerase chain reaction (PCR) stands among the keystone technologies for analysis of biological sequence data. PCR is used to amplify DNA, to generate many copies from as little as a single template. This is essential, for example, in processing forensic DNA samples, pathogen detection in clinical or biothreat surveillance applications, and medical genotyping for diagnosis and treatment of disease. It is used in virtually every laboratory doing molecular, cellular, genetic, ecologic, forensic, or medical research. Despite its ubiquity, we lack the precise predictive capability that would enable detailed optimization of PCR reaction dynamics. In this LDRD, we proposed to develop Virtual PCR (VPCR) software, a computational method to model the kinetic, thermodynamic, and biological processes of PCR reactions. Given a successful completion, these tools will allow us to predict both the sequences and concentrations of all species that are amplified during PCR. The ability to answer the following questions will allow us both to optimize the PCR process and interpret the PCR results: What products are amplified when sequence mixtures are present, containing multiple, closely related targets and multiplexed primers, which may hybridize with sequence mismatches? What are the effects of time, temperature, and DNA concentrations on the concentrations of products? A better understanding of these issues will improve the design and interpretation of PCR reactions. The status of the VPCR project after 1.5 years of funding is consistent with the goals of the overall project which was scoped for 3 years of funding. At half way through the projected timeline of the project we have an early beta version of the VPCR code. We have begun investigating means to improve the robustness of the code, performed preliminary experiments to test the code and begun drafting manuscripts for publication. Although an experimental protocol for testing the code was developed, the preliminary experiments were tainted by contaminated products received from the manufacturer. Much knowledge has been gained in the development of the code thus far, but without final debugging, increasing its robustness and verifying it against experimental results, the papers which we have drafted to share our findings still require the final data necessary for publication. The following sections summarize our final progress on VPCR as it stands after 1.5 years of effort on an ambitious project scoped for a 3 year period. We have additional details of the methods than are provided here, but would like to have legal protection in place before releasing them. The result of this project, a suite of programs that predict PCR products as a function of reaction conditions and sequences, will be used to address outstanding questions in pathogen detection and forensics at LLNL. VPCR should enable scientists to optimize PCR protocols in terms of time, temperature, ion concentration, and primer sequences and concentrations, and to estimate products and error rates in advance of performing experiments. Our proposed capabilities are well ahead of all currently available technologies, which do not model non-equilibrium kinetics, polymerase extension, or predict multiple or undesired PCR products. We are currently seeking DHS funding to complete the project, at which time licensing opportunities will be explored, an updated patent application will be prepared, and a publication will be submitted. A provisional and a full patent application have already been filed (1)

Cellular localization, accumulation and trafficking of double-walled carbon nanotubes in human prostate cancer cells

Carbon nanotubes (CNTs) are at present being considered as potential nanovectors with the ability to deliver therapeutic cargoes into living cells. Previous studies established the ability of CNTs to enter cells and their therapeutic utility, but an appreciation of global intracellular trafficking associated with their cellular distribution has yet to be described. Despite the many aspects of the uptake mechanism of CNTs being studied, only a few studies have investigated internalization and fate of CNTs inside cells in detail. In the present study, intracellular localization and trafficking of RNA-wrapped, oxidized double-walled CNTs (oxDWNT–RNA) is presented. Fixed cells, previously exposed to oxDWNT–RNA, were subjected to immunocytochemical analysis using antibodies specific to proteins implicated in endocytosis; moreover cell compartment markers and pharmacological inhibitory conditions were also employed in this study. Our results revealed that an endocytic pathway is involved in the internalization of oxDWNT–RNA. The nanotubes were found in clathrin-coated vesicles, after which they appear to be sorted in early endosomes, followed by vesicular maturation, become located in lysosomes. Furthermore, we observed co-localization of oxDWNT–RNA with the small GTP-binding protein (Rab 11), involved in their recycling back to the plasma membrane via endosomes from the trans-golgi network

Network development in biological gels: role in lymphatic vessel development

In this paper, we present a model that explains the prepatterning of lymphatic vessel morphology in collagen gels. This model is derived using the theory of two phase rubber material due to Flory and coworkers and it consists of two coupled fourth order partial differential equations describing the evolution of the collagen volume fraction, and the evolution of the proton concentration in a collagen implant; as described in experiments of Boardman and Swartz (Circ. Res. 92, 801–808, 2003). Using linear stability analysis, we find that above a critical level of proton concentration, spatial patterns form due to small perturbations in the initially uniform steady state. Using a long wavelength reduction, we can reduce the two coupled partial differential equations to one fourth order equation that is very similar to the Cahn–Hilliard equation; however, it has more complex nonlinearities and degeneracies. We present the results of numerical simulations and discuss the biological implications of our model

ISGylation-independent protection of cell growth by USP18 following interferon stimulation.

Type 1 interferon stimulation highly up-regulates all elements of a ubiquitin-like conjugation system that leads to ISGylation of target proteins. An ISG15-specific member of the deubiquitylase family, USP18, is up-regulated in a co-ordinated manner. USP18 can also provide a negative feedback by inhibiting JAK-STAT signalling through protein interactions independently of DUB activity. Here, we provide an acute example of this phenomenon, whereby the early expression of USP18, post-interferon treatment of HCT116 colon cancer cells is sufficient to fully suppress the expression of the ISG15 E1 enzyme, UBA7. Stimulation of lung adenocarcinoma A549 cells with interferon reduces their growth rate but they remain viable. In contrast, A549 USP18 knock-out cells show similar growth characteristics under basal conditions, but upon interferon stimulation, a profound inhibition of cell growth is observed. We show that this contingency on USP18 is independent of ISGylation, suggesting non-catalytic functions are required for viability. We also demonstrate that global deISGylation kinetics are very slow compared with deubiquitylation. This is not influenced by USP18 expression, suggesting that enhanced ISGylation in USP18 KO cells reflects increased conjugating activity