p62-containing, proteolytically active nuclear condensates, increase the efficiency of the ubiquitin-proteasome system.

Degradation of a protein by the ubiquitin-proteasome system (UPS) is a multistep process catalyzed by sequential reactions. Initially, ubiquitin is conjugated to the substrate in a process mediated by concerted activity of three enzymes; the last of them-a ubiquitin ligase (E3)-belongs to a family of several hundred members, each recognizing a few specific substrates. This is followed by repeated addition of ubiquitin moieties to the previously conjugated one to generate a ubiquitin chain that serves as a recognition element for the proteasome, which then degrades the substrate. Ubiquitin is recycled via the activity of deubiquitinating enzymes (DUBs). It stands to reason that efficiency of such a complex process would depend on colocalization of the different components in an assembly that allows the reactions to be carried out sequentially and processively. Here we describe nuclear condensates that are dynamic in their composition. They contain p62 as an essential component. These assemblies are generated by liquid-liquid phase separation (LLPS) and also contain ubiquitinated targets, 26S proteasome, the three conjugating enzymes, and DUBs. Under basal conditions, they serve as efficient centers for proteolysis of nuclear proteins (e.g., c-Myc) and unassembled subunits of the proteasome, suggesting they are involved in cellular protein quality control. Supporting this notion is the finding that such foci are also involved in degradation of misfolded proteins induced by heat and oxidative stresses, following recruitment of heat shock proteins and their associated ubiquitin ligase CHIP.

An immune-protein score combining TRAIL, IP-10 and CRP for predicting severe COVID-19 disease.

COVID-19 patients are oftentimes over- or under-treated due to a deficit in predictive management tools. This study reports derivation of an algorithm that integrates the host levels of TRAIL, IP-10, and CRP into a single numeric score that is an early indicator of severe outcome for COVID-19 patients and can identify patients at-risk to deteriorate. 394 COVID-19 patients were eligible; 29% meeting a severe outcome (intensive care unit admission/non-invasive or invasive ventilation/death). The score's area under the receiver operating characteristic curve (AUC) was 0.86, superior to IL-6 (AUC 0.77; p = 0.033) and CRP (AUC 0.78; p < 0.001). Likelihood of severe outcome increased significantly (p < 0.001) with higher scores. The score differentiated severe patients who further deteriorated from those who improved (p = 0.004) and projected 14-day survival probabilities (p < 0.001). The score accurately predicted COVID-19 patients at-risk for severe outcome, and therefore has potential to facilitate timely care escalation and de-escalation and appropriate resource allocation.

p62- and ubiquitin-dependent stress-induced autophagy of the mammalian 26S proteasome.

The ubiquitin-proteasome system and autophagy are the two main proteolytic systems involved in, among other functions, the maintenance of cell integrity by eliminating misfolded and damaged proteins and organelles. Both systems remove their targets after their conjugation with ubiquitin. An interesting, yet incompletely understood problem relates to the fate of the components of the two systems. Here we provide evidence that amino acid starvation enhances polyubiquitination on specific sites of the proteasome, a modification essential for its targeting to the autophagic machinery. The uptake of the ubiquitinated proteasome is mediated by its interaction with the ubiquitin-associated domain of p62/SQSTM1, a process that also requires interaction with LC3. Importantly, deletion of the PB1 domain of p62, which is important for the targeting of ubiquitinated substrates to the proteasome, has no effect on stress-induced autophagy of this proteolytic machinery, suggesting that the domain of p62 that binds to the proteasome determines the function of p62 in either targeting substrates to the proteasome or targeting the proteasome to autophagy.

A point-of-need platform for rapid measurement of a host-protein score that differentiates bacterial from viral infection: Analytical evaluation.

The objective was to evaluate the analytical performance of a new point-of-need platform for rapid and accurate measurement of a host-protein score that differentiates between bacterial and viral infection. The system comprises a dedicated test cartridge (MeMed BV®) and an analyzer (MeMed Key®). In each run, three host proteins (TRAIL, IP-10 and CRP) are measured quantitatively and a combinational score (0-100) computed that indicates the likelihood of Bacterial versus Viral infection (BV score). Serum samples collected from patients with acute infection representing viral (0 ≤ score < 35), equivocal (35 ≤ score ≤ 65), or bacterial (65 < score ≤ 100) scores based on pre-defined score cutoffs were employed for the analytical evaluation studies as well as samples from healthy individuals. To assess reproducibility, triplicate runs were conducted at 3 different sites, on 2 analyzers per site over 5 non-consecutive days. Lower limit of quantitation (LLoQ) and analytical measurement range were established utilizing recombinant proteins. Sample stability was evaluated using patient samples representative of BV score range (0-100). MeMed Key® and MeMed BV® passed the acceptance criteria for each study. In the reproducibility study, TRAIL, IP-10 and CRP measurements ranged with coefficient of variation from 9.7 to 12.7%, 4.6 to 6.2% and 5.0 to 11.6%, respectively. LLoQ concentrations were established as 15 pg/mL, 100 pg/mL and 1 mg/L for TRAIL, IP-10 and CRP, respectively. In summary, the analytical performance reported here, along with diagnostic accuracy established in the Apollo clinical validation study (NCT04690569), supports that MeMed BV® run on MeMed Key® can serve as a tool to assist clinicians in differentiating between bacterial and viral infection.

Observational cohort study of IP-10's potential as a biomarker to aid in inflammation regulation within a clinical decision support protocol for patients with severe COVID-19.

BACKGROUND: Treatment of severely ill COVID-19 patients requires simultaneous management of oxygenation and inflammation without compromising viral clearance. While multiple tools are available to aid oxygenation, data supporting immune biomarkers for monitoring the host-pathogen interaction across disease stages and for titrating immunomodulatory therapy is lacking. METHODS: In this single-center cohort study, we used an immunoassay platform that enables rapid and quantitative measurement of interferon γ-induced protein 10 (IP-10), a host protein involved in lung injury from virus-induced hyperinflammation. A dynamic clinical decision support protocol was followed to manage patients infected with severe acute respiratory syndrome coronavirus 2 and examine the potential utility of timely and serial measurements of IP-10 as tool in regulating inflammation. RESULTS: Overall, 502 IP-10 measurements were performed on 52 patients between 7 April and 10 May 2020, with 12 patients admitted to the intensive care unit. IP-10 levels correlated with COVID-19 severity scores and admission to the intensive care unit. Among patients in the intensive care unit, the number of days with IP-10 levels exceeding 1,000 pg/mL was associated with mortality. Administration of corticosteroid immunomodulatory therapy decreased IP-10 levels significantly. Only two patients presented with subsequent IP-10 flare-ups exceeding 1,000 pg/mL and died of COVID-19-related complications. CONCLUSIONS: Serial and readily available IP-10 measurements potentially represent an actionable aid in managing inflammation in COVID-19 patients and therapeutic decision-making. TRIAL REGISTRATION: Clinicaltrials.gov, NCT04389645, retrospectively registered on May 15, 2020.

What is socio-ecological research delivering? A literature survey across 25 international LTSER platforms.

With an overarching goal of addressing global and regional sustainability challenges, Long Term Socio-Ecological Research Platforms (LTSER) aim to conduct place-based research, to collect and synthesize both environmental and socio-economic data, and to involve a broader stakeholder pool to set the research agenda. To date there have been few studies examining the output from LTSER platforms. In this study we enquire if the socio-ecological research from 25 self-selected LTSER platforms of the International Long-Term Ecological Research (ILTER) network has produced research products which fulfil the aims and ambitions of the paradigm shift from ecological to socio-ecological research envisaged at the turn of the century. In total we assessed 4983 publically available publications, of which 1112 were deemed relevant to the socio-ecological objectives of the platform. A series of 22 questions were scored for each publication, assessing relevance of responses in terms of the disciplinary focus of research, consideration of human health and well-being, degree of stakeholder engagement, and other relevant variables. The results reflected the diverse origins of the individual platforms and revealed a wide range in foci, temporal periods and quantity of output from participating platforms, supporting the premise that there is a growing trend in socio-ecological research at long-term monitoring platforms. Our review highlights the challenges of realizing the top-down goal to harmonize international network activities and objectives and the need for bottom-up, self-definition for research platforms. This provides support for increasing the consistency of LTSER research while preserving the diversity of regional experiences.

The ubiquitin-proteasome system and autophagy: Coordinated and independent activities.

The living cell is an ever changing, responsive, and adaptive environment where proteins play key roles in all processes and functions. While the scientific community focused for a long time on the decoding of the information required for protein synthesis, little attention was paid to the mechanisms by which proteins are removed from the cell. We now realize that the timely and proper activity of proteins is regulated to a large extent by their degradation; that cellular coping with different physiological cues and stress conditions depends on different catabolic pathways; and that many pathological states result from improper protein breakdown. There are two major protein degradation systems in all eukaryotic cells-the ubiquitin- proteasome and the autophagy-lysosome. The two systems are highly regulated, and-via degradation of a broad array of proteins-are responsible for maintenance of protein homeostasis and adaptation to environmental changes. Each is comprised of numerous components responsible for its coordinated function, and together they encompass a considerable fraction of the entire genome. In this review, we shall discuss the common and diverse characteristics of the ubiquitin-proteasome system (UPS) and autophagy-their substructure, mechanisms of action, function and concerted regulation under varying pathophysiological conditions.

The life cycle of the 26S proteasome: from birth, through regulation and function, and onto its death.

The 26S proteasome is a large, ∼2.5 MDa, multi-catalytic ATP-dependent protease complex that serves as the degrading arm of the ubiquitin system, which is the major pathway for regulated degradation of cytosolic, nuclear and membrane proteins in all eukaryotic organisms.

Optimizing desalinated sea water blending with other sources to meet magnesium requirements for potable and irrigation waters.

Sea water desalination provides fresh water that typically lacks minerals essential to human health and to agricultural productivity. Thus the rising proportion of desalinated sea water consumed by both the domestic and agricultural sectors constitutes a public health risk. Research on low-magnesium water irrigation showed that crops developed magnesium deficiency symptoms that could lead to plant death, and tomato yields were reduced by 10-15%. The World Health Organization (WHO) reported on a relationship between sudden cardiac death rates and magnesium intake deficits. An optimization model, developed and tested to provide recommendations for Water Distribution System (WDS) quality control in terms of meeting optimal water quality requirements, was run in computational experiments based on an actual regional WDS. The expected magnesium deficit due to the operation of a large Sea Water Desalination Plant (SWDP) was simulated, and an optimal operation policy, in which remineralization at the SWDP was combined with blending desalinated and natural water to achieve the required quality, was generated. The effects of remineralization costs and WDS physical layout on the optimal policy were examined by sensitivity analysis. As part of the sensitivity blending natural and desalinated water near the treatment plants will be feasible up to 16.2 US cents/m(3), considering all expenses. Additional chemical injection was used to meet quality criteria when blending was not feasible.

ß-Casein nanoparticle-based oral drug delivery system for potential treatment of gastric carcinoma: stability, target-activated release and cytotoxicity.

We studied a potential drug delivery system comprising the hydrophobic anticancer drug paclitaxel entrapped within ß-casein (ß-CN) nanoparticles and its cytotoxicity to human gastric carcinoma cells. Paclitaxel was entrapped by stirring its dimethyl sulfoxide (DMSO) solution into PBS containing ß-CN. Cryo-TEM analysis revealed drug nanocrystals, the growth of which was blocked by ß-CN. Entrapment efficiency was nearly 100%, and the nanovehicles formed were colloidally stable. Following encapsulation and simulated digestion with pepsin (2 hours at pH=2, 37 °C), paclitaxel retained its cytotoxic activity to human N-87 gastric cancer cells; the IC(50) value (32.5 ± 6.2 nM) was similar to that of non-encapsulated paclitaxel (25.4 ± 2.6 nM). Without prior simulated gastric digestion, ß-CN-paclitaxel nanoparticles were non-cytotoxic, suggesting the lack of untoward toxicity to bucal and esophageal epithelia. We conclude that ß-CN shows promise to be useful for target-activated oral delivery of hydrophobic chemotherapeutics in the treatment of gastric carcinoma, one of the leading causes of cancer mortality worldwide.

Brief posturographic test as an indicator of fatigue.

The purpose of the present paper was to analyze the efficiency of an abbreviated, albeit objective posturographic test as an indicator of fatigue. Posturography was measured in 10 healthy adults (age 18-33 years, male/female 7/3). Baseline posturographic measurements were taken for each subject. Later, a shorter (3-min) posturographic test was administered 12 times during 25 h of sleep deprivation. This was correlated with subjective assessment of fatigue using a questionnaire and cognitive performance assessed with a reaction time test (Psychomotor Vigilance Test). Although showing significant individual differences, the score of the abbreviated posturographic examination, normalized to each subject's baseline performance ('fatigue index') had a pronounced circadian pattern with a peak of instability in the early morning hours. Fatigue index was highly correlated with the cognitive test (r=0.80-0.90). A substantial, albeit weaker correlation was found between the fatigue index and the subjective fatigue scores (r=0.59-0.75). Fourier spectral analysis showed that low median sway (0.10-0.50 Hz), considered to be an expression of vestibular control, was most affected by fatigue. The study demonstrates that cognitive deterioration caused by fatigue can be objectively predicted by an abbreviated postural test of