Th1 Cells Alter the Inflammatory Signature of IL-6 by Channeling STAT Transcription Factors to Alu-like Retroelements.

Cytokines that signal via STAT1 and STAT3 transcription factors instruct decisions affecting tissue homeostasis, antimicrobial host defense, and inflammation-induced tissue injury. To understand the coordination of these activities, we applied RNA sequencing, chromatin immunoprecipitation sequencing, and assay for transposase-accessible chromatin with high-throughput sequencing to identify the transcriptional output of STAT1 and STAT3 in peritoneal tissues from mice during acute resolving inflammation and inflammation primed to drive fibrosis. Bioinformatics focused on the transcriptional signature of the immunomodulatory cytokine IL-6 in both settings and examined how profibrotic IFN-γ-secreting CD4+ T cells altered the interpretation of STAT1 and STAT3 cytokine cues. In resolving inflammation, STAT1 and STAT3 cooperated to drive stromal gene expression affecting antimicrobial immunity and tissue homeostasis. The introduction of IFN-γ-secreting CD4+ T cells altered this transcriptional program and channeled STAT1 and STAT3 to a previously latent IFN-γ activation site motif in Alu-like elements. STAT1 and STAT3 binding to this conserved sequence revealed evidence of reciprocal cross-regulation and gene signatures relevant to pathophysiology. Thus, we propose that effector T cells retune the transcriptional output of IL-6 by shaping a regulatory interplay between STAT1 and STAT3 in inflammation.

Modelling dynamics between free-living amoebae and bacteria.

Free-living amoebae (FLA) serve as hosts for a variety of endosymbionts, which are microorganisms that reside and multiply within the FLA. Some of these endosymbionts pose a pathogenic threat to humans, animals, or both. The symbiotic relationship with FLA not only offers these microorganisms protection but also enhances their survival outside their hosts and assists in their dispersal across diverse habitats, thereby escalating disease transmission. This review is intended to offer an exhaustive overview of the existing mathematical models that have been applied to understand the dynamics of FLA, especially concerning their interactions with bacteria. An extensive literature review was conducted across Google Scholar, PubMed, and Scopus databases to identify mathematical models that describe the dynamics of interactions between FLA and bacteria, as published in peer-reviewed scientific journals. The literature search revealed several FLA-bacteria model systems, including Pseudomonas aeruginosa, Pasteurella multocida, and Legionella spp. Although the published mathematical models account for significant system dynamics such as predator-prey relationships and non-linear growth rates, they generally overlook spatial and temporal heterogeneity in environmental conditions, such as temperature, and population diversity. Future mathematical models will need to incorporate these factors to enhance our understanding of FLA-bacteria dynamics and to provide valuable insights for future risk assessment and disease control measures.

Neutrophils cultured ex vivo from CD34+ stem cells are immature and genetically tractable.

BACKGROUND: Neutrophils are granulocytes with essential antimicrobial effector functions and short lifespans. During infection or sterile inflammation, emergency granulopoiesis leads to release of immature neutrophils from the bone marrow, serving to boost circulating neutrophil counts. Steady state and emergency granulopoiesis are incompletely understood, partly due to a lack of genetically amenable models of neutrophil development. METHODS: We optimised a method for ex vivo production of human neutrophils from CD34+ haematopoietic progenitors. Using flow cytometry, we phenotypically compared cultured neutrophils with native neutrophils from donors experiencing emergency granulopoiesis, and steady state neutrophils from non-challenged donors. We carry out functional and proteomic characterisation of cultured neutrophils and establish genome editing of progenitors. RESULTS: We obtain high yields of ex vivo cultured neutrophils, which phenotypically resemble immature neutrophils released into the circulation during emergency granulopoiesis. Cultured neutrophils have similar rates of ROS production and bacterial killing but altered degranulation, cytokine release and antifungal activity compared to mature neutrophils isolated from peripheral blood. These differences are likely due to incomplete synthesis of granule proteins, as demonstrated by proteomic analysis. CONCLUSION: Ex vivo cultured neutrophils are genetically tractable via genome editing of precursors and provide a powerful model system for investigating the properties and behaviour of immature neutrophils.

Confirmation of Insertion, Deletion, and Deletion-Insertion Variants Detected by Next-Generation Sequencing.

BACKGROUND: Despite clinically demonstrated accuracy in next generation sequencing (NGS) data, many clinical laboratories continue to confirm variants with Sanger sequencing, which increases cost of testing and turnaround time. Several studies have assessed the accuracy of NGS in detecting single nucleotide variants; however, less has been reported about insertion, deletion, and deletion-insertion variants (indels). METHODS: We performed a retrospective analysis from 2015-2022 of indel results from a subset of NGS targeted gene panel tests offered through the Mayo Clinic Genomics Laboratories. We compared results from NGS and Sanger sequencing of indels observed in clinical runs and during the intra-assay validation of the tests. RESULTS: Results demonstrated 100% concordance between NGS and Sanger sequencing for over 490 indels (217 unique), ranging in size from 1 to 68 basepairs (bp). The majority of indels were deletions (77%) and 1 to 5 bp in length (90%). Variant frequencies ranged from 11.4% to 67.4% and 85.1% to 100% for heterozygous and homozygous variants, respectively, with a median depth of coverage of 2562×. A subset of indels (7%) were located in complex regions of the genome, and these were accurately detected by NGS. We also demonstrated 100% reproducibility of indel detection (n = 179) during intra-assay validation. CONCLUSIONS: Together this data demonstrates that reportable indel variants up to 68 bp can be accurately assessed using NGS, even when they occur in complex regions. Depending on the complexity of the region or variant, Sanger sequence confirmation of indels is usually not necessary if the variants meet appropriate coverage and allele frequency thresholds.

Interleukin-6 signaling drives fibrosis in unresolved inflammation.

Fibrosis in response to tissue damage or persistent inflammation is a pathological hallmark of many chronic degenerative diseases. By using a model of acute peritoneal inflammation, we have examined how repeated inflammatory activation promotes fibrotic tissue injury. In this context, fibrosis was strictly dependent on interleukin-6 (IL-6). Repeat inflammation induced IL-6-mediated T helper 1 (Th1) cell effector commitment and the emergence of STAT1 (signal transducer and activator of transcription-1) activity within the peritoneal membrane. Fibrosis was not observed in mice lacking interferon-γ (IFN-γ), STAT1, or RAG-1. Here, IFN-γ and STAT1 signaling disrupted the turnover of extracellular matrix by metalloproteases. Whereas IL-6-deficient mice resisted fibrosis, transfer of polarized Th1 cells or inhibition of MMP activity reversed this outcome. Thus, IL-6 causes compromised tissue repair by shifting acute inflammation into a more chronic profibrotic state through induction of Th1 cell responses as a consequence of recurrent inflammation.

Characterization of Glucokinases from Pathogenic Free-Living Amoebae.

Infection with pathogenic free-living amoebae, including Naegleria fowleri, Acanthamoeba spp., and Balamuthia mandrillaris, can lead to life-threatening illnesses, primarily because of catastrophic central nervous system involvement. Efficacious treatment options for these infections are lacking, and the mortality rate due to infection is high. Previously, we evaluated the N. fowleri glucokinase (NfGlck) as a potential target for therapeutic intervention, as glucose metabolism is critical for in vitro viability. Here, we extended these studies to the glucokinases from two other pathogenic free-living amoebae, including Acanthamoeba castellanii (AcGlck) and B. mandrillaris (BmGlck). While these enzymes are similar (49.3% identical at the amino acid level), they have distinct kinetic properties that distinguish them from each other. For ATP, AcGlck and BmGlck have apparent Km values of 472.5 and 41.0 µM, while Homo sapiens Glck (HsGlck) has a value of 310 µM. Both parasite enzymes also have a higher apparent affinity for glucose than the human counterpart, with apparent Km values of 45.9 µM (AcGlck) and 124 µM (BmGlck) compared to ~8 mM for HsGlck. Additionally, AcGlck and BmGlck differ from each other and other Glcks in their sensitivity to small molecule inhibitors, suggesting that inhibitors with pan-amoebic activity could be challenging to generate.

Linking multiple aspects of thermal performance to explore the potential for thermal resource partitioning between a native and an invasive crayfish.

Ecologists require standardized, ecologically relevant information on the thermal ecology of aquatic ectotherms to address growing concerns related to changing climates, altered habitats, and introduced species. We measured multiple thermal endpoints to investigate potential for establishment of the invasive Ringed Crayfish (Faxonius neglectus) in thermally heterogeneous habitat of the narrowly distributed endemic Coldwater Crayfish (Faxonius eupunctus). For each species, we examined the relationships between thermal endpoints at the cellular and organismal levels. We then compared results between the two species to gain insight as to the generality of linkages between cellular and organismal-level endpoints, as well as the potential for thermal niche separation between the native and potential invader. At the cellular level, we found no differences in the temperature for maximum activity of electron transport system enzymes (ETSmax) between species. At the organismal level, F. neglectus preferred significantly warmer temperatures than F. eupunctus, but this difference was small (1.3 °C) and likely to have only limited biological significance. The critical thermal maximum (CTM) did not differ between species. For both species, the thermal performance curve for ETS enzyme activity served as a useful framework to link thermal endpoints and estimate the transition from optimal to stressful temperatures - organismal thermal preference and optimal temperature estimates consistently fell below ETSmax whereas CTM estimates fell above ETSmax. Taken together, the strong similarities in thermal endpoint patterns between the two species suggest habitats thermally suitable for the native F. eupunctus will also be thermally available to expanding populations of F. neglectus, thereby increasing the opportunity for negative interactions and population effects if F. neglectus invades one of the few remaining, uninvaded, critical habitats of F. eupunctus.

Synthesis, Cytotoxicity, and Genotoxicity of 10-Aza-9-oxakalkitoxin, an N,N,O-Trisubstituted Hydroxylamine Analog, or Hydroxalog, of a Marine Natural Product.

We describe the synthesis of 10-aza-9-oxakalkitoxin, an N,N,O-trisubstituted hydroxylamine-based analog, or hydroxalog, of the cytotoxic marine natural product kalkitoxin in which the -NMe-O- moiety replaces a -CHMe-CH2- unit in the backbone of the natural product. 10-Aza-9-oxakalkitoxin displays potent and selective cytotoxicity (IC50 2.4 ng mL-1) comparable to that of kalkitoxin itself (IC50 3.2 ng mL-1) against the human hepato-carcinoma cell line HepG2 over both the human leukemia cell line CEM and the normal hematopoietic CFU-GM. Like kalkitoxin, and contrary to the common expectation for hydroxylamines, 10-aza-9-oxakalkitoxin is not mutagenic.

In Vitro Screening of the Open-Source Medicines for Malaria Venture Malaria and Pathogen Boxes To Discover Novel Compounds with Activity against Balamuthia mandrillaris.

Balamuthia mandrillaris is an under-reported, pathogenic free-living amoeba that causes Balamuthia amoebic encephalitis (BAE) and cutaneous skin infections. Although cutaneous infections are not typically lethal, BAE with or without cutaneous involvement is usually fatal. This is due to the lack of drugs that are both efficacious and can cross the blood-brain barrier. We aimed to discover new leads for drug discovery by screening the open-source Medicines for Malaria Venture (MMV) Malaria Box and MMV Pathogen Box, with 800 compounds total. From an initial single point screen at 1 and 10 µM, we identified 54 hits that significantly inhibited the growth of B. mandrillarisin vitro Hits were reconfirmed in quantitative dose-response assays and 23 compounds (42.6%) were confirmed with activity greater than miltefosine, the current standard of care.

Phenotypic Screens Reveal Posaconazole as a Rapidly Acting Amebicidal Combination Partner for Treatment of Primary Amoebic Meningoencephalitis.

Naegleria fowleri is the causative agent of primary amoebic meningoencephalitis (PAM), which is fatal in >97% of cases. In this study, we aimed to identify new, rapidly acting drugs to increase survival rates. We conducted phenotypic screens of libraries of Food and Drug Administration-approved compounds and the Medicines for Malaria Venture Pathogen Box and validated 14 hits (defined as a 50% inhibitory concentration of <1 µM). The hits were then prioritized by assessing the rate of action and efficacy in combination with current drugs used to treat PAM. Posaconazole was found to inhibit amoeba growth within the first 12 hours of exposure, which was faster than any currently used drug. In addition, posaconazole cured 33% of N. fowleri-infected mice at a dose of 20 mg/kg and, in combination with azithromycin, increased survival by an additional 20%. Fluconazole, which is currently used for PAM therapy, was ineffective in vitro and vivo. Our results suggest posaconazole could replace fluconazole in the treatment of PAM.

Digital holography efficiency measurements with excess noise.

In this paper, we use digital holography (DH) in the off-axis image plane recording geometry with a 532 nm continuous-wave laser to measure the system efficiencies (multiplicative losses) associated with a closed-form expression for the signal-to-noise ratio (SNR). Measurements of the mixing efficiency (36.8%) and the reference noise efficiency (74.5%) provide an expected total system efficiency of 22.7%±6.5% and a measured total system efficiency of 21.1%±6.3%. These total noise efficiencies do not include our measurements of the signal noise efficiency (3%-100%), which are highly dependent on the signal strength and become significant for SNRs>100. Thus, the results confirm that the mixing efficiency is generally the dominant multiplicative loss with respect to the DH system under test; however, excess reference and signal noise are significant multiplicative losses as well. Previous results also agree with these experimental findings.

Orthopaedic injuries from snowmobile accidents: a multi-centre analysis of demographics, injury patterns, and outcomes.

PURPOSE: More than 2 million people in North America use snowmobiles, resulting in an estimated 200 fatalities and 14,000 injuries annually. The purpose of this study is to document the demographics, orthopaedic injury patterns, and short-term outcomes of patients with snowmobile-related injuries. MATERIALS AND METHODS: A retrospective review was performed at two regional trauma centres in a region where snowmobile use is prevalent. Patients who sustained snowmobile-related injuries over a 12-year period were identified from the hospitals' trauma registries using E-codes (E820-E820.9). Patient demographics were recorded, as were injury characteristics including rates of substance use, open fractures, Injury Severity Score (ISS), Abbreviated Injury Score (AIS) for the extremities, and mortality. Rates of inpatient surgery, as well as hospital and ICU length of stay (LOS), were also recorded. RESULTS: We identified 528 patients with snowmobile-related injuries. Average age was 37 years, and 418 patients (79%) were male. Eighty-eight per cent of all patients with snowmobile injuries were admitted to the hospital with an average LOS of 5.7 days. Among those admitted to the hospital, average ISS was 12.3, and 28% of these patients had ISS > 15. A total of 261 patients (56%) suffered extremity injuries (including 163 upper and 173 lower extremity fractures) with an average extremity AIS of 2.4. There were 700 total fractures (1.5 per patient), and 9% of all fractures were open. A total of 208 patients (45%) suffered head injuries, and 132 patients (28%) sustained vertebral column fractures. A total of 201 patients (43%) required inpatient surgery, and eight patients (1.7%) sustained fatal injuries. CONCLUSIONS: We present a detailed multi-centre analysis of orthopaedic injury patterns and outcomes resulting from snowmobile-related injuries. Patients injured while snowmobiling share similar injury patterns with patients injured in motorcycle and other high-energy motor vehicle accidents.

Keikipukalides, Furanocembrane Diterpenes from the Antarctic Deep Sea Octocoral Plumarella delicatissima.

During a 2013 cruise in the Southern Ocean we collected specimens of the octocoral Plumarella delicatissima between 800 and 950 m depth. Five new furanocembranoid diterpenes, keikipukalides A-E (1-5), the known diterpene pukalide aldehyde (6), and the known norditerpenoid ineleganolide (7) were isolated from the coral. These Plumarella terpenes lack mammalian cytotoxicity, while 2-7 display activity against Leishmania donovani between 1.9 and 12 µM. Structure elucidation was facilitated by one- and two-dimensional NMR spectroscopy and mass spectrometry, and keikipukalides A and E were confirmed by X-ray crystallography.

Exploitation of Mangrove Endophytic Fungi for Infectious Disease Drug Discovery.

There is an acute need for new and effective agents to treat infectious diseases. We conducted a screening program to assess the potential of mangrove-derived endophytic fungi as a source of new antibiotics. Fungi cultured in the presence and absence of small molecule epigenetic modulators were screened against Mycobacterium tuberculosis and the ESKAPE panel of bacterial pathogens, as well as two eukaryotic infective agents, Leishmania donovani and Naegleria fowleri. By comparison of bioactivity data among treatments and targets, trends became evident, such as the result that more than 60% of active extracts were revealed to be selective to a single target. Validating the technique of using small molecules to dysregulate secondary metabolite production pathways, nearly half (44%) of those fungi producing active extracts only did so following histone deacetylase inhibitory (HDACi) or DNA methyltransferase inhibitory (DNMTi) treatment.

The effect of sucrose addition at dosage stage on the foam attributes of a bottle-fermented English sparkling wine.

BACKGROUND: Approximately two-thirds of wine produced in the UK is bottle-fermented sparkling wine. Effervescence and foamability are key features used to assess English sparkling wine (ESW) quality. A critical, yet understudied, area of research is the potential for dosage to influence foam behaviour via associated changes in wine viscosity. RESULTS: In this study, dosage treatments of five increasing levels of sucrose (from 0 to 31 g L-1 ) were added to an ESW. After storage, the foamability attributes of the wines were analysed via an adapted Mosalux method and a novel image analysis method combined with free pour of the wine. Results indicate that increasing sucrose concentration improved foam formation, but reduced foam stability, likely due to the sucrose added causing a modification in wine viscosity. CONCLUSIONS: These results highlight the impact that dosage treatments can have on the quality of foam produced upon pouring, and therefore have the potential to inform future sparkling winemaking practices. © 2017 Society of Chemical Industry.

Home Safety Fall and Accident Risk Among Prematurely Aging, Formerly Homeless Adults.

OBJECTIVE: Homelessness prematurely ages people. A large subgroup of formerly homeless adults between ages 40 and 64 yr have health conditions similar to or worse than people categorized as elderly. Little is known about the impact of this group's chronic health conditions on their ability to safely function in supportive housing. METHOD: Home safety visits were carried out with 25 formerly homeless adults, ages 40-64 yr, now residing in supportive housing. RESULTS: Participants had physical, cognitive, and mental health problems that significantly interfered with their ability to perform daily life skills, safely function in an apartment, and manage chronic health conditions. Home safety hazards included cluttered walking paths, the presence of steps, and the lack of grab bars and nonskid flooring. CONCLUSION: The homeless population would benefit from aging specialists, such as occupational therapists, who could help people to maintain and function more safely in their homes. Without such services, this population may be at risk for home safety events leading to hospitalization and mortality.

A novel ciliopathic skull defect arising from excess neural crest.

The skull is essential for protecting the brain from damage, and birth defects involving disorganization of skull bones are common. However, the developmental trajectories and molecular etiologies by which many craniofacial phenotypes arise remain poorly understood. Here, we report a novel skull defect in ciliopathic Fuz mutant mice in which only a single bone pair encases the forebrain, instead of the usual paired frontal and parietal bones. Through genetic lineage analysis, we show that this defect stems from a massive expansion of the neural crest-derived frontal bone. This expansion occurs at the expense of the mesodermally-derived parietal bones, which are either severely reduced or absent. A similar, though less severe, phenotype was observed in Gli3 mutant mice, consistent with a role for Gli3 in cilia-mediated signaling. Excess crest has also been shown to drive defective palate morphogenesis in ciliopathic mice, and that defect is ameliorated by reduction of Fgf8 gene dosage. Strikingly, skull defects in Fuz mutant mice are also rescued by loss of one allele of fgf8, suggesting a potential route to therapy. In sum, this work is significant for revealing a novel skull defect with a previously un-described developmental etiology and for suggesting a common developmental origin for skull and palate defects in ciliopathies.

Bis-benzimidazole hits against Naegleria fowleri discovered with new high-throughput screens.

Naegleria fowleri is a pathogenic free-living amoeba (FLA) that causes an acute fatal disease known as primary amoebic meningoencephalitis (PAM). The major problem for infections with any pathogenic FLA is a lack of effective therapeutics, since PAM has a case mortality rate approaching 99%. Clearly, new drugs that are potent and have rapid onset of action are needed to enhance the treatment regimens for PAM. Diamidines have demonstrated potency against multiple pathogens, including FLA, and are known to cross the blood-brain barrier to cure other protozoan diseases of the central nervous system. Therefore, amidino derivatives serve as an important chemotype for discovery of new drugs. In this study, we validated two new in vitro assays suitable for medium- or high-throughput drug discovery and used these for N. fowleri. We next screened over 150 amidino derivatives of multiple structural classes and identified two hit series with nM potency that are suitable for further lead optimization as new drugs for this neglected disease. These include both mono- and diamidino derivatives, with the most potent compound (DB173) having a 50% inhibitory concentration (IC50) of 177 nM. Similarly, we identified 10 additional analogues with IC50s of <1 µM, with many of these having reasonable selectivity indices. The most potent hits were >500 times more potent than pentamidine. In summary, the mono- and diamidino derivatives offer potential for lead optimization to develop new drugs to treat central nervous system infections with N. fowleri.

Glucose metabolism in the pathogenic free-living amoebae: Tempting targets for treatment development.

Pathogenic free-living amoebae (pFLA) are single-celled eukaryotes responsible for causing intractable infections with high morbidity and mortality in humans and animals. Current therapeutic approaches include cocktails of antibiotic, antifungal, and antimicrobial compounds. Unfortunately, the efficacy of these can be limited, driving the need for the discovery of new treatments. Pan anti-amebic agents would be ideal; however, identifying these agents has been a challenge, likely due to the limited evolutionary relatedness of the different pFLA. Here, we discuss the potential of targeting amoebae glucose metabolic pathways as the differences between pFLA and humans suggest specific inhibitors could be developed as leads for new therapeutics.

Open-Path Atmospheric Transmission of Diode-Pumped Alkali Lasers in Maritime and Desert Environments.

A tunable diode laser absorption spectroscopy (TDLAS) device has been developed to study long-path atmospheric transmission near diode pumped alkali laser (DPAL) emission wavelengths. By employing a single aperture and retro reflector in a mono-static configuration, the noise associated with atmospheric and platform jitter were reduced by a factor of ∼30 and the open-air path length was extended to 4.4 km and over a very broad spectral range, up to 120 cm-1. Water vapor absorption lines near the rubidium (Rb) and cesium (Cs) variants of the DPAL near 795 and 894 nm, oxygen lines near the potassium (K) DPAL near 770 nm, and water vapor absorption in the vicinity of the neodymium-doped yttrium aluminum garnet (Nd:YAG) laser 1.064 µm and chemical oxygen iodine laser (COIL) 1.3 µm lines were studied. The detection limit for path absorbance increases from ΔA = 0.0017 at 100 m path length to 0.085 for the 4.4 km path. Comparison with meteorological instruments for maritime and desert environments yields agreement for the 2.032 km path to within 1.5% for temperature, 4.5% for pressure, and 5.1% for concentration, while agreements for the 4.4 km path are within 1.4% for temperature, 7.7% for pressure, and 23.5% for concentration. An intra cavity output spectroscopy (ICOS) device was also used as a spectral reference to verify location of atmospheric lines. Implications of TDLAS collection system design on signal-to-noise (S/N) are discussed as well as the effect of path turbulence on baseline noise and inform the selection of the DPAL variant least affected by molecular absorption.