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Contributor Role Ontologies and Taxonomies (CROTs) provide a standard list of roles to specify individual contributions to research. CROTs most common application has been their inclusion alongside author bylines in scholarly publications. With the recent uptake of CROTs among publishers –particularly the Contributor Role Taxonomy (CRediT)– some have anticipated a positive impact on ethical issues regarding the attribution of credit and responsibilities, but others have voiced concerns about CROTs shortcomings and ways they could be misunderstood or have unintended consequences. Since these discussions have never been consolidated, this review collated and explored published viewpoints about the ethics of CROTs. After searching Ovid Medline, Scopus, Web of Science, and Google Scholar, 30 papers met the inclusion criteria and were analyzed. We identified eight themes and 20 specific issues related to the ethics of CROTs and provided four recommendations for CROT developers, custodians, or others seeking to use CROTs in their workflows, policy and practice: 1) Compile comprehensive instructions that explain how CROTs should be used; 2) Improve the coherence of used terms, 3) Translate roles in languages other than English, 4) Communicate a clear vision about future development plans and be transparent about CROTs’ strengths and weaknesses. We conclude that CROTs are not the panacea for unethical attributions and should be complemented with initiatives that support social and infrastructural transformation of scholarly publications.

Quantifying the marine radiocarbon reservoir effect, offsets (ΔR), and ΔR variability over time is critical to improving dating estimates of marine samples while also providing a proxy of water mass dynamics. In the northeastern Pacific, where no high-resolution time series of ΔR has yet been established, we sampled radiocarbon (14C) from exactly dated growth increments in a multicentennial chronology of the long-lived bivalve, Pacific geoduck (Paneopea generosa) at the Tree Nob site, coastal British Columbia, Canada. Samples were taken at approximately decadal time intervals from 1725 CE to 1920 CE and indicate average ΔR values of 256 ± 22 years (1σ) consistent with existing discrete estimates. Temporal variability in ΔR is small relative to analogous Atlantic records except for an unusually old-water event, 1802–1812. The correlation between ΔR and sea surface temperature (SST) reconstructed from geoduck increment width is weakly significant (r2 = .29, p = .03), indicating warm water is generally old, when the 1802–1812 interval is excluded. This interval contains the oldest (–2.1σ) anomaly, and that is coincident with the coldest (–2.7σ) anomalies of the temperature reconstruction. An additional 32 14C values spanning 1952–1980 were detrended using a northeastern Pacific bomb pulse curve. Significant positive correlations were identified between the detrended 14C data and annual El Niño Southern Oscillation (ENSO) and summer SST such that cooler conditions are associated with older water. Thus, 14C is generally relatively stable with weak, potentially inconsistent associations to climate variables, but capable of infrequent excursions as illustrated by the unusually cold, old-water 1802–1812 interval.

The purpose of this study was to assess gastric feeding intolerance for critically ill patients who received sustained neuromuscular blocker (NMB) pharmacotherapy.Adult patients (17 years of age) admitted to the trauma intensive care unit who received continuous intravenous NMB pharmacotherapy (rocuronium, cisatracurium, vecuronium, or pancuronium) for ≥48 h during continuous intragastric enteral nutrition (EN) were retrospectively evaluated. Gastric feeding intolerance was defined by initiation of a prokinetic agent (metoclopramide, erythromycin, or both) for an elevated gastric residual volume (GRV)300 ml and with distention of the abdomen by physical examination, observation of regurgitation or emesis, temporary discontinuation of EN with low intermittent gastric suctioning, or initiation of parenteral nutrition (PN). Patients were evaluated for gastric feeding intolerance for the first 3 days of combined EN and NMB pharmacotherapy. A P value 0.05 was considered statistically significant.Ten patients of the 47 patients (21%) were intolerant to EN during NMB pharmacotherapy. No statistically or clinically relevant differences in patient characteristics were found between patients who tolerated EN vs those who experienced gastric feeding intolerance, except for a higher median maximum GRV of 125 ml (28, 200) vs 300 (250, 400) ml, respectively (P 0.001). Five patients responded to prokinetic therapy and five required PN.Most patients tolerated intragastric EN during sustained NMB pharmacotherapy. Presence of NMB pharmacotherapy is not an absolute contraindication for EN.

Anoctamins (ANO) are activated by intracellular Ca2+ and function as phospholipid scramblases and/or Cl- channels. ANO4 is mutated in miniature schnauzers with calcium oxalate stones (CaOx), and ANO4 protein in urinary extracellular vesicles is decreased in human CaOx stone formers. In Drosophila Malpighian tubules (MT), subdued (ANO4-homolog) functions as Ca2+-activated Cl- channel, scramblase, and plays a role in host defense against gram-negative bacteria. Increasing evidence shows the participation of bacteria in urinary stone formation in humans. We used a Drosophila avatar to investigate whether subdued changes CaOx crystallization and if crystallization increases with bacterial infection.Uro:Gal4 flies were crossed with UAS:subdued-RNAi flies to knockdown (KD) subdued in MT principal cells (PC) or with w1118 flies to generate normal controls (WT). F1 generation underwent CaOx crystallization in association or not with uropathogenic E. coli (UPEC) infection. In vivo, flies were fed on diet supplemented with 20mM sodium Ox (NaOx) + UPEC:eGFP for 4 days. For ex vivo assays, dissected MTs were submerged in solution containing 10mM NaOx+ UPEC:eGFP for 90 minutes. subdued-KD in PC reduced subdued mRNA by 56% in MT. Neither crystal number nor size were changed by subdued-KD after prolonged NaOx feeding. However, when UPEC is introduced with the NaOx diet, subdued-KD flies exhibited a large bacterial presence in MT lumen and produced enlarged CaOx crystals compared to WT flies. Similar augmented presence of UPEC in subdued-KD MTs was observed during ex vivo assays. Short-term exposure to NaOx+UPEC resulted in increased number of crystals in subdued-KD MTs compared to WT MTs, but no difference was observed in crystal size.Fly cell atlas ( flycellatlas.org ) revealed that subdued is also found in MT stellate cells (SC; water and Cl- secretion). C724:Gal4>UAS:subdued-RNAi flies were used to assess the effect of subdued-KD in SC. This s ubdued-KD slightly increased bacterial infection but did change CaOx crystallization in MTs after 4 days of NaOx+UPEC feeding.The presence of subdued in PC limits CaOx crystals formation and aggregation through reducing bacterial invasion in the MT lumen. These Drosophila results suggest a role for ANO4 in bacterial-related human and dog kidney stone formation. Future experiments will determine whether other functional aspects of subdued affect CaOx crystallization. U54-DK100227, R01-DK092408, FAPESP (2022/01226-1), Mayo Foundation. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Recent evidence from well-controlled efficacy studies suggests that diffusing treatment focus across multiple concerns is associated with poorer clinical outcomes. However, research regarding treatment focus diffusion (TFD) in public mental health care (PMHC) settings, broadly or in implemented evidence-based treatments (EBT), is scarce, despite therapists in such settings often reporting more complex cases. Using multilevel modeling, this study examined TFD differences between two in-home PMHC services: (a) Multisystemic Therapy (MST; n = 911 youths, 109 therapists), an implemented EBT, and (b) a less structured service more characteristic of treatment as usual (n = 2362 youth, 457 therapists). The relationship between TFD and monthly therapeutic progress within and across these two service formats was also examined. Treatment focus diffusion occurred less in the implemented EBT. Overall, receiving services through the less structured service format and more diffused treatment focus predicted less and slower progress over the course of treatment. The relationship between TFD and less clinical progress was stronger in the MST format. These findings held when accounting for indicators of case complexity including initial level of impairment and number of diagnoses. EBTs appear to maintain a narrower treatment focus even when implemented in a public mental health system. However, even in EBTs, TFD predicts poorer clinical progress. Maintaining a narrow treatment focus, even in complex cases typical of PMHC, might improve clinical outcomes.

Adobe PDF - MCT-07-0149--Suppl_Data.pdf from Inhibition of the heat shock protein 90 molecular chaperone in vitro and in vivo by novel, synthetic, potent resorcinylic pyrazole/isoxazole amide analogues

Although the heat shock protein 90 (HSP90) inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) shows clinical promise, potential limitations encourage development of alternative chemotypes. We discovered the 3,4-diarylpyrazole resorcinol CCT018159 by high-throughput screening and used structure-based design to generate more potent pyrazole amide analogues, exemplified by VER-49009. Here, we describe the detailed biological properties of VER-49009 and the corresponding isoxazole VER-50589. X-ray crystallography showed a virtually identical HSP90 binding mode. However, the dissociation constant (Kd) of VER-50589 was 4.5 ± 2.2 nmol/L compared with 78.0 ± 10.4 nmol/L for VER-49009, attributable to higher enthalpy for VER-50589 binding. A competitive binding assay gave a lower IC50 of 21 ± 4 nmol/L for VER-50589 compared with 47 ± 9 nmol/L for VER-49009. Cellular uptake of VER-50589 was 4-fold greater than for VER-49009. Mean cellular antiproliferative GI50 values for VER-50589 and VER-49009 for a human cancer cell line panel were 78 ± 15 and 685 ± 119 nmol/L, respectively, showing a 9-fold potency gain for the isoxazole. Unlike 17-AAG, but as with CCT018159, cellular potency of these analogues was independent of NAD(P)H:quinone oxidoreductase 1/DT-diaphorase and P-glycoprotein expression. Consistent with HSP90 inhibition, VER-50589 and VER-49009 caused induction of HSP72 and HSP27 alongside depletion of client proteins, including C-RAF, B-RAF, and survivin, and the protein arginine methyltransferase PRMT5. Both caused cell cycle arrest and apoptosis. Extent and duration of pharmacodynamic changes in an orthotopic human ovarian carcinoma model confirmed the superiority of VER-50589 over VER-49009. VER-50589 accumulated in HCT116 human colon cancer xenografts at levels above the cellular GI50 for 24 h, resulting in 30% growth inhibition. The results indicate the therapeutic potential of the resorcinylic pyrazole/isoxazole amide analogues as HSP90 inhibitors. [Mol Cancer Ther 2007;6(4):1198–211]

AHA1 (activator of HSP90 ATPase) is a cochaperone of the ATP-dependent molecular chaperone, HSP90, which is involved in the maturation, stabilization/degradation, and function of oncogenic proteins. HSP90 operates in a multimeric complex driven by the binding and hydrolysis of ATP. Treatment of cells with the HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) results in the degradation of client proteins via the ubiquitin-proteasome pathway. As AHA1 increases the ATPase activity of HSP90, we hypothesized that modulation of AHA1 expression could influence the activity of client proteins and/or the cellular response to 17-AAG. We show that the basal expression of AHA1 is different across a panel of human cancer cell lines, and that treatment with 17-AAG resulted in sustained AHA1 up-regulation. Increasing the expression of AHA1 did not affect the sensitivity to 17-AAG, but did increase C-RAF activity and the levels of phosphorylated MEK1/2 and ERK1/2 without affecting total levels of these proteins or of client proteins C-RAF, ERBB2, or CDK4. Conversely, small interfering RNA–selective knockdown of >80% of AHA1 expression decreased C-RAF activity and reduced the levels of MEK1/2 and ERK1/2 phosphorylation. Moreover, the AHA1 knockdown resulted in a significant (P < 0.05) increase in sensitivity to 17-AAG, due in part to a 2- to 3-fold increase in apoptosis. These results show that the reduction of AHA1 levels could decrease the phosphorylation of key signal transduction proteins, and for the first time, separate the activation and stabilization functions of HSP90. Furthermore, AHA1 knockdown could sensitize cancer cells to 17-AAG. We conclude that modulation of AHA1 might be a potential therapeutic strategy to increase sensitivity to HSP90 inhibitors. [Cancer Res 2008;68(4):1187–97]

Supplementary Methods, Figures 1-3 from Silencing of HSP90 Cochaperone AHA1 Expression Decreases Client Protein Activation and Increases Cellular Sensitivity to the HSP90 Inhibitor 17-Allylamino-17-Demethoxygeldanamycin

Gram-negative bacteremia is a major cause of global morbidity involving three phases of pathogenesis: initial site infection, dissemination, and survival in the blood and filtering organs.Klebsiella pneumoniaeis a leading cause of bacteremia and pneumonia is often the initial infection. In the lung,K. pneumoniaerelies on many factors like capsular polysaccharide and branched chain amino acid biosynthesis for virulence and fitness. However, mechanisms directly enabling bloodstream fitness are unclear. Here, we performed transposon insertion sequencing (TnSeq) in a tail-vein injection model of bacteremia and identified 58K. pneumoniaebloodstream fitness genes. These factors are diverse and represent a variety of cellular processes.In vivovalidation revealed tissue-specific mechanisms by which distinct factors support bacteremia. ArnD, involved in Lipid A modification, was required across blood filtering organs and supported resistance to soluble splenic factors. The purine biosynthesis enzyme PurD largely enhanced liver fitness and was required for replication in serum. PdxA, a member of the endogenous vitamin B6 biosynthesis pathway, optimized replication in serum and lung fitness. The stringent response regulator SspA was required for splenic fitness yet was dispensable in the liver. In a bacteremic pneumonia model that incorporates initial site infection and dissemination, splenic fitness defects were enhanced, and DsbA, SspA, and PdxA increased fitness across bacteremia phases. SspA and PdxA enhancedK. pnuemoniaeresistance to oxidative stress. SspA specifically resists oxidative stress produced by NADPH oxidase Nox2 in the lung, spleen, and liver, as it was a fitness factor in wild-type but not Nox2-deficient (Cybb−/−) mice. These results identify site-specific fitness factors that act during the progression of Gram-negative bacteremia. DefiningK. pneumoniaefitness strategies across bacteremia phases could illuminate therapeutic targets that prevent infection and sepsis.Author SummaryGram-negative bacteremia is a deadly family of infections that initiate sepsis, a leading cause of global morbidity and mortality. Only a small number of Gram-negative species contribute to the majority of clinical bacteremia.Klebsiella pneumoniaeis the second leading cause of Gram-negative bacteremia, and the third leading cause of overall bloodstream infection.K. pneumoniaeis highly linked to hospital-associated infection with increasing antimicrobial resistance, endangering the most vulnerable patients. It is critical to understand the pathogenesis ofK. pneumoniaebacteremia to better develop targets for future therapies that can prevent these deadly infections. Here, we define over 50K. pneumoniaegenes that support bloodstream fitness. These factors are diverse, support tissue-specific fitness, and increase bacterial resistance to oxidative stress. Our study is the first to systematically defineK. pneumoniaefactors enhancing bacteremia in a mammalian system. These results illuminate host-pathogen interactions duringK. pneumoniaebacteremia that may be extended to additional Gram-negative species.

A hallmark of microbial ecology is that interactions between members of a community shape community function. This includes microbial communities in human infections, such as chronic wounds, where interactions can result in more severe diseases. Staphylococcus aureus is the most common organism isolated from human chronic wound infections and has been shown to have both cooperative and competitive interactions with Pseudomonas aeruginosa . Still, despite considerable study, most interactions between these microbes have been characterized using in vitro well-mixed systems, which do not recapitulate the infection environment. Here, we characterized interactions between S. aureus and P. aeruginosa in chronic murine wounds, focusing on the role that both macro- and micro-scale spatial structures play in disease. We discovered that S. aureus and P. aeruginosa coexist at high cell densities in murine wounds. High-resolution imaging revealed that these microbes establish a patchy distribution, only occupying 5 to 25% of the wound volume. Using a quantitative framework, we identified a precise spatial structure at both the macro (mm)- and micro (µm)-scales, which was largely mediated by P. aeruginosa production of the antimicrobial 2-heptyl-4-hydroxyquinoline N-oxide, while the antimicrobial pyocyanin had no impact. Finally, we discovered that this precise spatial structure enhances S. aureus tolerance to aminoglycoside antibiotics but not vancomycin. Our results provide mechanistic insights into the biogeography of S. aureus and P. aeruginosa coinfected wounds and implicate spatial structure as a key determinant of antimicrobial tolerance in wound infections.

Quantitative gene regulation at the cell population-level can be achieved by two fundamentally different modes of regulation at individual gene copies. A “digital” mode involves binary ON/OFF expression states, with population-level variation arising from the proportion of gene copies in each state, while an “analog” mode involves graded expression levels at each gene copy. At the Arabidopsis floral repressor FLOWERING LOCUS C (FLC), “digital” Polycomb silencing is known to facilitate quantitative epigenetic memory in response to cold. However, whether FLC regulation before cold involves analog or digital modes is unknown. Using quantitative fluorescent imaging of FLC mRNA and protein, together with mathematical modelling, we find that FLC expression before cold is regulated by both analog and digital modes. We observe a temporal separation between the two modes, with analog preceding digital. The analog mode can maintain intermediate expression levels at individual FLC gene copies, before subsequent digital silencing, consistent with the copies switching OFF stochastically and heritably without cold. This switch leads to a slow reduction in FLC expression at the cell population-level. These data present a new paradigm for gradual repression, elucidating how analog transcriptional and digital epigenetic memory pathways can be integrated.