Genetic Engineering of the Rock Inhabitant Knufia petricola Provides Insight Into the Biology of Extremotolerant Black Fungi

Black microcolonial fungi (Ascomycetes from Arthonio-, Dothideo-, and Eurotiomycetes) are stress-tolerant and persistent dwellers of natural and anthropogenic extreme habitats. They exhibit slow yeast-like or meristematic growth, do not form specialized reproduction structures and accumulate the black pigment 1,8-dihydroxynaphthalene (DHN) melanin in the multilayered cell walls. To understand how black fungi live, survive, colonize mineral substrates, and interact with phototrophs genetic methods are needed to test these functions and interactions. We chose the rock inhabitant Knufia petricola of the Chaetothyriales as a model for developing methods for genetic manipulation. Here, we report on the expansion of the genetic toolkit by more efficient multiplex CRISPR/Cas9 using a plasmid-based system for expression of Cas9 and multiple sgRNAs and the implementation of the three resistance selection markers genR (geneticin/nptII), baR (glufosinate/bar), and suR (chlorimuron ethyl/sur). The targeted integration of expression constructs by replacement of essential genes for pigment synthesis allows for an additional color screening of the transformants. The black-pink screening due to the elimination of pks1 (melanin) was applied for promoter studies using GFP fluorescence as reporter. The black-white screening due to the concurrent elimination of pks1 and phs1 (carotenoids) allows to identify transformants that contain the two expression constructs for co-localization or bimolecular fluorescence complementation (BiFC) studies. The co-localization and interaction of the two K. petricola White Collar orthologs were demonstrated. Two intergenic regions (igr1, igr2) were identified in which expression constructs can be inserted without causing obvious phenotypes. Plasmids of the pNXR-XXX series and new compatible entry plasmids were used for fast and easy generation of expression constructs and are suitable for a broad implementation in other fungi. This variety of genetic tools is opening a completely new perspective for mechanistic and very detailed study of expression, functioning and regulation of the genes/proteins encoded by the genomes of black fungi

Adenosine triphosphatases of thermophilic archaeal double-stranded DNA viruses

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A Cyanine‐Bridged Somatostatin Hybrid Probe for Multimodal SSTR2 Imaging in Vitro and in Vivo: Synthesis and Evaluation

Multimodal imaging probes have attracted the interest of ongoing research, for example, for the surgical removal of tumors. Modular synthesis approaches allow the construction of hybrid probes consisting of a radiotracer, a fluorophore and a targeting unit. We present the synthesis of a new asymmetric bifunctional cyanine dye that can be used as a structural and functional linker for the construction of such hybrid probes. 68Ga‐DOTATATE, a well‐characterized radiopeptide targeting the overexpressed somatostatin receptor subtype 2 (SSTR2) in neuroendocrine tumors, was labeled with our cyanine dye, thus providing additional information along with the data obtained from the radiotracer. We tested the SSTR2‐targeting and imaging properties of the resulting probe 68Ga‐DOTA‐ICC‐TATE in vitro and in a tumor xenograft mouse model. Despite the close proximity between dye and pharmacophore, we observed a high binding affinity towards SSTR2 as well as elevated uptake in SSTR2‐overexpressing tumors in the positron emission tomography (PET) scan and histological examination

Dimensions of Researcher Vulnerability in Qualitative Health Research and Recommendations for Future Practice

Funding Information: The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this paper: This work was supported by the German Federal Ministry of Education and Research (no. 01GP2191). Publisher Copyright: © The Author(s) 2023.Vulnerability has typically been addressed in the context of research ethics from the point of view of participants, with a focus on how to prevent the potential or exacerbation of existing harm caused by the power and role asymmetries between researchers and participants. However, more recent approaches to research ethics question whether researchers are, by definition, located in a privileged position during the research process and safe from any kind of vulnerability. In line with this, we reflect on the dimensions of researcher vulnerability specific to studies using a qualitative methodology in health research. Our argument is that participants and researchers should be on the receiving end of efforts to implement ethical procedures and protection from harm. Based on the autoethnographic analysis of our experiences as qualitative health researchers, this paper aims to identify dimensions of researcher vulnerability, and draw out relevant recommendations for practice. The reflections upon which this paper is based emerged during a spring school focusing on research ethics in qualitative health research, during which we discussed situations from our own research experience which left us feeling vulnerable. We identify four dimensions related to the experience of vulnerability (reciprocity; emotional labor; application of ethical standards; reversed power asymmetries) and five crosscutting aspects relating to these dimensions (researching sensitive topics; researching in contexts of vulnerability, poverty and structural violence; being a novice; lacking adequate support; insufficient time and space for ethical reflexivity). Our recommendations address particular challenges for these dimensions, and center on the role of reflexivity, as one of the cornerstones for enabling ethical qualitative research practice, requiring us to acknowledge and address our own vulnerability and positionality. Autoethnographic exercises are particularly useful for zooming in on ethically important moments in research related to researcher vulnerability and fruitful for identifying resources to respond to such challenges in the future.publishersversionpublishe

Increasing molar activity by HPLC purification improves 68Ga-DOTA-NAPamide tumor accumulation in a B16/F1 melanoma xenograft model

Purpose: Melanocortin receptor 1 (MC1R) is overexpressed in melanoma and may be a molecular target for imaging and peptide receptor radionuclide therapy. 68Gallium (68Ga) labeling of DOTA-conjugated peptides is an established procedure in the clinic for use in positron emission tomography (PET) imaging. Aim of this study was to compare a standard labeling protocol against the 68Ga-DOTA peptide purified from the excess of unlabeled peptide. Procedures: The MC1R ligand DOTA-NAPamide was labeled with 68Ga using a standard clinical protocol. Radioactive peptide was separated from the excess of unlabeled DOTA-NAPamide by HPLC. Immediately after the incubation of peptide and 68Ga (95˚C, 15 min), the reaction was loaded on a C18 column and separated by a water/acetonitrile gradient, allowing fractionation in less than 20 minutes. Radiolabeled products were compared in biodistribution studies and PET imaging using nude mice bearing MC1R-expressing B16/F1 xenograft tumors. Results: In biodistribution studies, non-purified 68Ga-DOTA-NAPamide did not show significant uptake in the tumor at 1 h post injection (0.78% IA/g). By the additional HPLC step, the molar activity was raised around 10,000-fold by completely removing unlabeled peptide. Application of this rapid purification strategy led to a more than 8-fold increase in tumor uptake (7.0% IA/g). The addition of various amounts of unlabeled DOTA-NAPamide to the purified product led to a blocking effect and decreased specific tumor uptake, similar to the result seen with non-purified radiopeptide. PET imaging was performed using the same tracer preparations. Purified 68Ga-DOTA-NAPamide, in comparison, showed superior tumor uptake. Conclusions: We demonstrated that chromatographic separation of radiolabeled from excess unlabeled peptide is technically feasible and beneficial, even for short-lived isotopes such as 68Ga. Unlabeled peptide molecules compete with receptor binding sites in the target tissue. Purification of the radiopeptide therefore improved tumor uptake

Reduced Time to Admit Emergency Department Patients to Inpatient Beds Using Outflow Barrier Analysis and Process Improvement

Objective: Because admitted emergency department (ED) patients waiting for an inpatient bed contribute to dangerous ED crowding, we conducted a patient flow investigation to discover and solve outflow delays. After solution implementation, we measured whether the time admitted ED patients waited to leave the ED was reduced. Methods: In June 2022, a team using Lean Healthcare methodologies identified flow delays and underlying barriers in a Midwest, mid-sized hospital. We calculated barriers’ magnitudes of burden by the frequency of involvement in delays. During October–December 2022, solutions targeting barriers were implemented. In October 2023, we tested whether waiting time, defined as daily median time in minutes from admission disposition to departure (ADtoD), declined by conducting independent sample, single-tailed t-test comparing pre- to post-intervention time periods, January 1–September 30, 2022 (273 days) to January 1–September 30, 2023 (273 days). Additionally, we regressed ADtoD onto pre-/post period while controlling for ED volume (total daily admissions and ED daily encounters) and hospital occupancy. A run chart analysis of monthly median ADtoD assessed improvement sustainability. Results: Process mapping revealed that three departments (ED, environmental services [EVS], and transport services) co-produced the outflow of admitted ED patients wherein 18 delays were identified. The EVS-clinical care collaboration failures explained 61% (11/18) of delays. Technology contributed to 78% (14/18) of delays primarily because staff’s technology did not display needed information, a condition we coined “digital blindness.” Comparing pre- and post-intervention days (3,144 patients admitted pre-intervention and 3,256 patients post), the median minutes a patient waited (ADtoD) significantly decreased (96.4 to 87.1 minutes, P = 0.04), even while daily ED encounter volume significantly increased (110.7 to 117.3 encounters per day, P < 0.001). After controlling in regression for other factors associated with waiting, the intervention reduced ADtoD by 12.7 minutes per patient (standard error 5.10, P = 0.01; 95% confidence interval −22.7, −2.7). We estimate that the intervention translated to ED staff avoiding 689 hours of admitted patient boarding over nine months (ADtoD coefficient [−12.7 minutes] multiplied by post-intervention ED admissions [3,256] and divided by 60). Run chart analysis substantiated the intervention’s sustainability over nine months. Conclusion: After systemwide patient flow investigation, solutions resolving digital blindness and environmental services-clinical care collaboration failures significantly reduced ED admitted patient boarding.&nbsp

Circulating brain‐derived neurotrophic factor concentrations and the risk of cardiovascular disease in the community

BACKGROUND: Brain‐derived neurotrophic factor (BDNF) is a pleiotropic peptide involved in maintaining endothelial integrity. It is unknown if circulating BDNF levels are associated with risk of cardiovascular disease (CVD). METHODS AND RESULTS: We prospectively investigated the association of circulating BDNF levels with cardiovascular events and mortality in 3687 participants (mean age 65 years, 2068 women) from the Framingham Heart Study (FHS). Using a common nonsynonomous single nucleotide polymorphism (SNP) in the BDNF gene (rs6265), we then performed a Mendelian randomization experiment in the CARDIoGRAM (Coronary ARtery DIsease Genome‐Wide Replication And Meta‐Analysis) consortium (>22 000 coronary artery disease [CAD] cases, >60 000 controls) to investigate whether SNP rs6265 was associated with CAD in CARDIoGRAM and, if so, whether the effect estimate differed from that predicted based on FHS data. On follow‐up (median 8.9 years), 467 individuals (261 women) in FHS experienced a CVD event, and 835 (430 women) died. In multivariable‐adjusted Cox regression, serum BDNF was associated inversely with CVD risk (hazard ratio [HR] per 1‐SD increase 0.88, 95% CI 0.80 to 0.97, P=0.01) and with mortality (HR 0.87, 95% CI 0.80 to 0.93, P=0.0002). SNP rs6265 was associated with BDNF concentrations (0.772 ng/mL increase per minor allele copy) in FHS. In CARDIoGRAM, SNP rs6265 was associated with CAD (odds ratio 0.957, 95% CI 0.923 to 0.992), a magnitude consistent with the predicted effect (HR per minor allele copy 0.99, 95% CI 0.98 to 1.0; P=0.06 for difference between predicted and observed effect). CONCLUSION: Higher serum BDNF is associated with a decreased risk of CVD and mortality. Mendelian randomization suggests a causal protective role of BDNF in the pathogenesis of CVD