Dynamic regulation of airway surface liquid pH by TMEM16A and SLC26A4 in cystic fibrosis nasal epithelia with rare mutations.

In cystic fibrosis (CF), defects in the CF transmembrane conductance regulator (CFTR) channel lead to an acidic airway surface liquid (ASL), which compromises innate defence mechanisms, predisposing to pulmonary failure. Restoring ASL pH is a potential therapy for people with CF, particularly for those who cannot benefit from current highly effective modulator therapy. However, we lack a comprehensive understanding of the complex mechanisms underlying ASL pH regulation. The calcium-activated chloride channel, TMEM16A, and the anion exchanger, SLC26A4, have been proposed as targets for restoring ASL pH, but current results are contradictory and often utilise nonphysiological conditions. To provide better evidence for a role of these two proteins in ASL pH homeostasis, we developed an efficient CRISPR-Cas9-based approach to knock-out (KO) relevant transporters in primary airway basal cells lacking CFTR and then measured dynamic changes in ASL pH under thin-film conditions in fully differentiated airway cultures, which better simulate the in vivo situation. Unexpectantly, we found that both proteins regulated steady-state as well as agonist-stimulated ASL pH, but only under inflammatory conditions. Furthermore, we identified two Food and Drug Administration (FDA)-approved drugs which raised ASL pH by activating SLC26A4. While we identified a role for SLC26A4 in fluid absorption, KO had no effect on cyclic adenosine monophosphate (cAMP)-stimulated fluid secretion in airway organoids. Overall, we have identified a role of TMEM16A in ASL pH homeostasis and shown that both TMEM16A and SLC26A4 could be important alternative targets for ASL pH therapy in CF, particularly for those people who do not produce any functional CFTR.

Chromosome-scale assemblies of Acanthamoeba castellanii genomes provide insights into Legionella pneumophila infection-related chromatin reorganization.

The unicellular amoeba Acanthamoeba castellanii is ubiquitous in aquatic environments, where it preys on bacteria. The organism also hosts bacterial endosymbionts, some of which are parasitic, including human pathogens such as Chlamydia and Legionella spp. Here we report complete, high-quality genome sequences for two extensively studied A. castellanii strains, Neff and C3. Combining long- and short-read data with Hi-C, we generated near chromosome-level assemblies for both strains with 90% of the genome contained in 29 scaffolds for the Neff strain and 31 for the C3 strain. Comparative genomics revealed strain-specific functional enrichment, most notably genes related to signal transduction in the C3 strain and to viral replication in Neff. Furthermore, we characterized the spatial organization of the A. castellanii genome and showed that it is reorganized during infection by Legionella pneumophila Infection-dependent chromatin loops were found to be enriched in genes for signal transduction and phosphorylation processes. In genomic regions where chromatin organization changed during Legionella infection, we found functional enrichment for genes associated with metabolism, organelle assembly, and cytoskeleton organization. Given Legionella infection is known to alter its host's cell cycle, to exploit the host's organelles, and to modulate the host's metabolism in its favor, these changes in chromatin organization may partly be related to mechanisms of host control during Legionella infection.

Miniature RNAs are embedded in an exceptionally protein-rich mitoribosome via an elaborate assembly pathway.

The mitochondrial ribosome (mitoribosome) has diverged drastically from its evolutionary progenitor, the bacterial ribosome. Structural and compositional diversity is particularly striking in the phylum Euglenozoa, with an extraordinary protein gain in the mitoribosome of kinetoplastid protists. Here we report an even more complex mitoribosome in diplonemids, the sister-group of kinetoplastids. Affinity pulldown of mitoribosomal complexes from Diplonema papillatum, the diplonemid type species, demonstrates that they have a mass of > 5 MDa, contain as many as 130 integral proteins, and exhibit a protein-to-RNA ratio of 11:1. This unusual composition reflects unprecedented structural reduction of ribosomal RNAs, increased size of canonical mitoribosomal proteins, and accretion of three dozen lineage-specific components. In addition, we identified >50 candidate assembly factors, around half of which contribute to early mitoribosome maturation steps. Because little is known about early assembly stages even in model organisms, our investigation of the diplonemid mitoribosome illuminates this process. Together, our results provide a foundation for understanding how runaway evolutionary divergence shapes both biogenesis and function of a complex molecular machine.

Escherichia coli RclA is a highly active hypothiocyanite reductase.

Hypothiocyanite and hypothiocyanous acid (OSCN-/HOSCN) are pseudohypohalous acids released by the innate immune system which are capable of rapidly oxidizing sulfur-containing amino acids, causing significant protein aggregation and damage to invading bacteria. HOSCN is abundant in saliva and airway secretions and has long been considered a highly specific antimicrobial that is nearly harmless to mammalian cells. However, certain bacteria, commensal and pathogenic, are able to escape damage by HOSCN and other harmful antimicrobials during inflammation, which allows them to continue to grow and, in some cases, cause severe disease. The exact genes or mechanisms by which bacteria respond to HOSCN have not yet been elucidated. We have found, in Escherichia coli, that the flavoprotein RclA, previously implicated in reactive chlorine resistance, reduces HOSCN to thiocyanate with near-perfect catalytic efficiency and strongly protects E. coli against HOSCN toxicity. This is notable in E. coli because this species thrives in the chronically inflamed environment found in patients with inflammatory bowel disease and is able to compete with and outgrow other important commensal organisms, suggesting that HOSCN may be a relevant antimicrobial in the gut, which has not previously been explored. RclA is conserved in a variety of epithelium-colonizing bacteria, implicating its HOSCN reductase activity in a variety of host-microbe interactions. We show that an rclA mutant of the probiotic Limosilactobacillus reuteri is sensitive to HOSCN and that RclA homologs from Staphylococcus aureus, Streptococcus pneumoniae, and Bacteroides thetaiotaomicron all have potent protective activity against HOSCN when expressed in E. coli.

Hypothiocyanite and host-microbe interactions.

The pseudohypohalous acid hypothiocyanite/hypothiocyanous acid (OSCN- /HOSCN) has been known to play an antimicrobial role in mammalian immunity for decades. It is a potent oxidant that kills bacteria but is non-toxic to human cells. Produced from thiocyanate (SCN- ) and hydrogen peroxide (H2 O2 ) in a variety of body sites by peroxidase enzymes, HOSCN has been explored as an agent of food preservation, pathogen killing, and even improved toothpaste. However, despite the well-recognized antibacterial role HOSCN plays in host-pathogen interactions, little is known about how bacteria sense and respond to this oxidant. In this work, we will summarize what is known and unknown about HOSCN in innate immunity and recent advances in understanding the responses that both pathogenic and non-pathogenic bacteria mount against this antimicrobial agent, highlighting studies done with three model organisms, Escherichia coli, Streptococcus spp., and Pseudomonas aeruginosa.

Genetic testing in cardiovascular disease.

Cardiovascular disease (CVD) is the leading cause of morbidity and mortality globally and is responsible for an estimated one-third of deaths as well as significant morbidity and health care utilisation. Technological and bioinformatic advances have facilitated the discovery of pathogenic germline variants for some specific CVDs, including familial hypercholesterolaemia, cardiomyopathies and arrhythmic syndromes. Use of these genetic tests for earlier disease identification is increasing due, in part, to decreasing costs, Medicare rebates, and consumer comfort with genetic testing. However, CVDs that occur more commonly, including coronary artery disease and atrial fibrillation, do not display monogenic inheritance patterns. Genetically, these diseases have generally been associated with many genetic variants each with a small effect size. This complexity can be expressed mathematically as a polygenic risk score. Genetic testing kits that provide polygenic risk scoring are becoming increasingly available directly to private-paying consumers outside the traditional clinical setting. An improved understanding of the evidence of genetics in CVD will offer clinicians new opportunities for individualised risk prediction and preventive therapy.

Recent expansion of metabolic versatility in Diplonema papillatum, the model species of a highly speciose group of marine eukaryotes.

BACKGROUND: Diplonemid flagellates are among the most abundant and species-rich of known marine microeukaryotes, colonizing all habitats, depths, and geographic regions of the world ocean. However, little is known about their genomes, biology, and ecological role. RESULTS: We present the first nuclear genome sequence from a diplonemid, the type species Diplonema papillatum. The ~ 280-Mb genome assembly contains about 32,000 protein-coding genes, likely co-transcribed in groups of up to 100. Gene clusters are separated by long repetitive regions that include numerous transposable elements, which also reside within introns. Analysis of gene-family evolution reveals that the last common diplonemid ancestor underwent considerable metabolic expansion. D. papillatum-specific gains of carbohydrate-degradation capability were apparently acquired via horizontal gene transfer. The predicted breakdown of polysaccharides including pectin and xylan is at odds with reports of peptides being the predominant carbon source of this organism. Secretome analysis together with feeding experiments suggest that D. papillatum is predatory, able to degrade cell walls of live microeukaryotes, macroalgae, and water plants, not only for protoplast feeding but also for metabolizing cell-wall carbohydrates as an energy source. The analysis of environmental barcode samples shows that D. papillatum is confined to temperate coastal waters, presumably acting in bioremediation of eutrophication. CONCLUSIONS: Nuclear genome information will allow systematic functional and cell-biology studies in D. papillatum. It will also serve as a reference for the highly diverse diplonemids and provide a point of comparison for studying gene complement evolution in the sister group of Kinetoplastida, including human-pathogenic taxa.

Pharmacological inhibitors of the cystic fibrosis transmembrane conductance regulator exert off-target effects on epithelial cation channels.

The cystic fibrosis transmembrane conductance regulator (CFTR) anion channel and the epithelial Na+ channel (ENaC) play essential roles in transepithelial ion and fluid transport in numerous epithelial tissues. Inhibitors of both channels have been important tools for defining their physiological role in vitro. However, two commonly used CFTR inhibitors, CFTRinh-172 and GlyH-101, also inhibit non-CFTR anion channels, indicating they are not CFTR specific. However, the potential off-target effects of these inhibitors on epithelial cation channels has to date not been addressed. Here, we show that both CFTR blockers, at concentrations routinely employed by many researchers, caused a significant inhibition of store-operated calcium entry (SOCE) that was time-dependent, poorly reversible and independent of CFTR. Patch clamp experiments showed that both CFTRinh-172 and GlyH-101 caused a significant block of Orai1-mediated whole cell currents, establishing that they likely reduce SOCE via modulation of this Ca2+ release-activated Ca2+ (CRAC) channel. In addition to off-target effects on calcium channels, both inhibitors significantly reduced human αßγ-ENaC-mediated currents after heterologous expression in Xenopus oocytes, but had differential effects on δßγ-ENaC function. Molecular docking identified two putative binding sites in the extracellular domain of ENaC for both CFTR blockers. Together, our results indicate that caution is needed when using these two CFTR inhibitors to dissect the role of CFTR, and potentially ENaC, in physiological processes.

Series resistance errors in whole cell voltage clamp measured directly with dual patch-clamp recordings: not as bad as you think.

Whole cell patch clamp has provided much insight into the function of voltage-gated ion channels in central neurons. However, voltage errors caused by the resistance of the recording electrode [series resistance (Rs)] limit its application to relatively small ionic currents. Ohm's law is often applied to estimate and correct the membrane potential for these voltage errors. We tested this assumption in brainstem motoneurons of adult frogs with dual patch-clamp recordings, one performing whole cell voltage clamp of K+ currents and the other directly recording the membrane potential. We hypothesized that Ohm's law-based correction would approximate the measured voltage error. We found that voltage errors averaged <5 mV for currents considered to be large for patch clamp (∼7-13 nA) and <10 mV for massive currents thought to be experimentally intractable (25-30 nA), each error falling within commonly accepted inclusion limits. In most cases Ohm's law-based correction overpredicted these measured voltage errors by roughly 2.5-fold. Consequently, the use of Ohm's law to correct for voltage errors led to erroneous current-voltage (I-V) relationships, showing the greatest distortion for inactivating currents. Finally, recordings with low electrode Rs compensated moderately by the amplifier circuitry appeared to have smaller voltage errors than those with larger Rs and high compensation despite the same "effective Rs" and current magnitude. Therefore, when Rs is low, large currents may be studied with better-than-expected voltage control. These results suggest that patch-clamp may be used to study ionic currents often interpreted to be off limits because of size.NEW & NOTEWORTHY Voltage errors occur in whole cell voltage clamp. We make, to our knowledge, the first direct measurements of these errors and find that voltage errors are far smaller than standard calculations would predict. Since voltage errors were often minimal during the measurement of large ion channel currents, this technique may be applied to large neurons of adults to gain insight into ion channel function across the life span and progression of disease.

Incorporating a polygenic risk score-triaged coronary calcium score into cardiovascular disease examinations to identify subclinical coronary artery disease (ESCALATE): Protocol for a prospective, nonrandomized implementation trial.

BACKGROUND: Identifying and targeting established modifiable risk factors has been a successful strategy for reducing the burden of coronary artery disease (CAD) at the population-level. However, up to 1-in-4 patients who present with ST elevation myocardial infarction do so in the absence of such risk factors. Polygenic risk scores (PRS) have demonstrated an ability to improve risk prediction models independent of traditional risk factors and self-reported family history, but a pathway for implementation has yet to be clearly identified. The aim of this study is to examine the utility of a CAD PRS to identify individuals with subclinical CAD via a novel clinical pathway, triaging low or intermediate absolute risk individuals for noninvasive coronary imaging, and examining the impact on shared treatment decisions and participant experience. TRIAL DESIGN: The ESCALATE study is a 12-month, prospective, multicenter implementation study incorporating PRS into otherwise standard primary care CVD risk assessments, to identify patients at increased lifetime CAD risk for noninvasive coronary imaging. One-thousand eligible participants aged 45 to 65 years old will enter the study, which applies PRS to those considered low or moderate 5-year absolute CVD risk and triages those with CAD PRS ≥80% for a coronary calcium scan. The primary outcome will be the identification of subclinical CAD, defined as a coronary artery calcium score (CACS) >0 Agatston units (AU). Multiple secondary outcomes will be assessed, including baseline CACS ≥100 AU or ≥75th age-/sex-matched percentile, the use and intensity of lipid- and blood pressure-lowering therapeutics, cholesterol and blood pressure levels, and health-related quality of life (HRQOL). CONCLUSION: This novel trial will generate evidence on the ability of a PRS-triaged CACS to identify subclinical CAD, as well as subsequent differences in traditional risk factor medical management, pharmacotherapy utilization, and participant experience. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry, ACTRN12622000436774. Trial was prospectively registered on March 18, 2022. https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=383134.

Clinical trial protocol for PanDox: a phase I study of targeted chemotherapy delivery to non-resectable primary pancreatic tumours using thermosensitive liposomal doxorubicin (ThermoDox®) and focused ultrasound.

BACKGROUND: The dense stroma of pancreatic ductal adenocarcinomas is a major barrier to drug delivery. To increase the local drug diffusion gradient, high doses of chemotherapeutic agent doxorubicin can be released from thermally-sensitive liposomes (ThermoDox®) using ultrasound-mediated hyperthermia at the tumour target. PanDox is designed as a Phase 1 single centre study to investigate enhancing drug delivery to adult patients with non-operable pancreatic ductal adenocarcinomas. The study compares a single cycle of either conventional doxorubicin alone or ThermoDox® with focused ultrasound-induced hyperthermia for targeted drug release. METHODS: Adults with non-resectable pancreatic ductal adenocarcinoma are allocated to receive a single cycle of either doxorubicin alone (Arm A) or ThermoDox® with focused ultrasound-induced hyperthermia (Arm B), based on patient- and tumour-specific safety conditions. Participants in Arm B will undergo a general anaesthetic and pre-heating of the tumour by extra-corporal focused ultrasound (FUS). Rather than employing invasive thermometry, ultrasound parameters are derived from a patient-specific treatment planning model to reach the 41 °C target temperature for drug release. ThermoDox® is then concurrently infused with further ultrasound exposure. Tumour biopsies at the targeted site from all patients are analysed post-treatment using high performance liquid chromatography to quantify doxorubicin delivered to the tumour. The primary endpoint is defined as a statistically significant enhancement in concentration of total intra-tumoural doxorubicin, comparing samples from patients receiving liposomal drug with FUS to free drug alone. Participants are followed for 21 days post-treatment to assess secondary endpoints, including radiological assessment to measure changes in tumour activity by Positron Emission Tomography Response Criteria in Solid Tumours (PERCIST) criteria, adverse events and patient-reported symptoms. DISCUSSION: This early phase study builds on previous work targeting tumours in the liver to investigate whether enhancement of chemotherapy delivery using ultrasound-mediated hyperthermia can be translated to the stroma-dense environment of pancreatic ductal adenocarcinoma. If successful, it could herald a new approach towards managing these difficult-to-treat tumours. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT04852367 . Registered 21st April 2022. EudraCT number: 2019-003950-10 (Registered 2019) Iras Project ID: 272253 (Registered 2019) Ethics Number: 20/EE/0284.

The SLC26A9 inhibitor S9-A13 provides no evidence for a role of SLC26A9 in airway chloride secretion but suggests a contribution to regulation of ASL pH and gastric proton secretion.

The solute carrier 26 family member A9 (SLC26A9) is an epithelial anion transporter that is assumed to contribute to airway chloride secretion and surface hydration. Whether SLC26A9 or CFTR is responsible for airway Cl- transport under basal conditions is still unclear, due to the lack of a specific inhibitor for SLC26A9. In the present study, we report a novel potent and specific inhibitor for SLC26A9, identified by screening of a drug-like molecule library and subsequent chemical modifications. The most potent compound S9-A13 inhibited SLC26A9 with an IC50 of 90.9 ± 13.4 nM. S9-A13 did not inhibit other members of the SLC26 family and had no effects on Cl- channels such as CFTR, TMEM16A, or VRAC. S9-A13 inhibited SLC26A9 Cl- currents in cells that lack expression of CFTR. It also inhibited proton secretion by HGT-1 human gastric cells. In contrast, S9-A13 had minimal effects on ion transport in human airway epithelia and mouse trachea, despite clear expression of SLC26A9 in the apical membrane of ciliated cells. In both tissues, basal and stimulated Cl- secretion was due to CFTR, while acidification of airway surface liquid by S9-A13 suggests a role of SLC26A9 for airway bicarbonate secretion.

A Cell-Based Optimised Approach for Rapid and Efficient Gene Editing of Human Pluripotent Stem Cells.

Introducing or correcting disease-causing mutations through genome editing in human pluripotent stem cells (hPSCs) followed by tissue-specific differentiation provide sustainable models of multiorgan diseases, such as cystic fibrosis (CF). However, low editing efficiency resulting in extended cell culture periods and the use of specialised equipment for fluorescence activated cell sorting (FACS) make hPSC genome editing still challenging. We aimed to investigate whether a combination of cell cycle synchronisation, single-stranded oligodeoxyribonucleotides, transient selection, manual clonal isolation, and rapid screening can improve the generation of correctly modified hPSCs. Here, we introduced the most common CF mutation, ΔF508, into the CFTR gene, using TALENs into hPSCs, and corrected the W1282X mutation using CRISPR-Cas9, in human-induced PSCs. This relatively simple method achieved up to 10% efficiency without the need for FACS, generating heterozygous and homozygous gene edited hPSCs within 3-6 weeks in order to understand genetic determinants of disease and precision medicine.

High-Throughput Measure of Mitochondrial Superoxide Levels as a Marker of Coronary Artery Disease to Accelerate Drug Translation in Patient-Derived Endothelial Cells Using Opera Phenix® Technology.

Improved human-relevant preclinical models of coronary artery disease (CAD) are needed to improve translational research and drug discovery. Mitochondrial dysfunction and associated oxidative stress contribute to endothelial dysfunction and are a significant factor in the development and progression of CAD. Endothelial colony-forming cells (ECFCs) can be derived from peripheral blood mononuclear cells (PBMCs) and offer a unique potentially personalised means for investigating new potential therapies targeting important components of vascular function. We describe the application of the high-throughput and confocal Opera Phenix® High-Content Screening System to examine mitochondrial superoxide (mROS) levels, mitochondrial membrane potential, and mitochondrial area in both established cell lines and patient-derived ECFCs simultaneously. Unlike traditional plate readers, the Opera Phenix® is an imaging system that integrates automated confocal microscopy, precise fluorescent detection, and multi-parameter algorithms to visualize and precisely quantify targeted biological processes at a cellular level. In this study, we measured mROS production in human umbilical vein endothelial cells (HUVECs) and patient-derived ECFCs using the mROS production probe, MitoSOXTM Red. HUVECs exposed to oxidized low-density lipoprotein (oxLDL) increased mROS levels by 47.7% (p < 0.0001). A pooled group of patient-derived ECFCs from participants with CAD (n = 14) exhibited 30.9% higher mROS levels compared to patients with no CAD when stimulated with oxLDL (n = 14; p < 0.05). When tested against a small group of candidate compounds, this signal was attenuated by PKT-100 (36.22% reduction, p = 0.03), a novel P2X7 receptor antagonist. This suggests the P2X7 receptor as a valid target against excess mROS levels. As such, these findings highlight the potential of the MitoSOX-Opera Phenix technique to be used for drug discovery efforts in CAD.

Sonosensitive Cavitation Nuclei-A Customisable Platform Technology for Enhanced Therapeutic Delivery.

Ultrasound-mediated cavitation shows great promise for improving targeted drug delivery across a range of clinical applications. Cavitation nuclei-sound-sensitive constructs that enhance cavitation activity at lower pressures-have become a powerful adjuvant to ultrasound-based treatments, and more recently emerged as a drug delivery vehicle in their own right. The unique combination of physical, biological, and chemical effects that occur around these structures, as well as their varied compositions and morphologies, make cavitation nuclei an attractive platform for creating delivery systems tuned to particular therapeutics. In this review, we describe the structure and function of cavitation nuclei, approaches to their functionalization and customization, various clinical applications, progress toward real-world translation, and future directions for the field.

An Unexpectedly Complex Mitoribosome in Andalucia godoyi, a Protist with the Most Bacteria-like Mitochondrial Genome.

The mitoribosome, as known from studies in model organisms, deviates considerably from its ancestor, the bacterial ribosome. Deviations include substantial reduction of the mitochondrial ribosomal RNA (mt-rRNA) structure and acquisition of numerous mitochondrion-specific (M) mitoribosomal proteins (mtRPs). A broadly accepted view assumes that M-mtRPs compensate for structural destabilization of mt-rRNA resulting from its evolutionary remodeling. Since most experimental information on mitoribosome makeup comes from eukaryotes having derived mitochondrial genomes and mt-rRNAs, we tested this assumption by investigating the mitochondrial translation machinery of jakobids, a lineage of unicellular protists with the most bacteria-like mitochondrial genomes. We report here proteomics analyses of the Andalucia godoyi small mitoribosomal subunit and in silico transcriptomic and comparative genome analyses of four additional jakobids. Jakobids have mt-rRNA structures that minimally differ from their bacterial counterparts. Yet, with at least 31 small subunit and 44 large subunit mtRPs, the mitoriboproteome of Andalucia is essentially as complex as that in animals or fungi. Furthermore, the relatively high conservation of jakobid sequences has helped to clarify the identity of several mtRPs, previously considered to be lineage-specific, as divergent homologs of conserved M-mtRPs, notably mS22 and mL61. The coexistence of bacteria-like mt-rRNAs and a complex mitoriboproteome refutes the view that M-mtRPs were ancestrally recruited to stabilize deviations of mt-rRNA structural elements. We postulate instead that the numerous M-mtRPs acquired in the last eukaryotic common ancestor allowed mt-rRNAs to pursue a broad range of evolutionary trajectories across lineages: from dramatic reduction to acquisition of novel elements to structural conservatism.

The role of nitrogen-responsive regulators in controlling inorganic polyphosphate synthesis in Escherichia coli.

Inorganic polyphosphate (polyP) is synthesized by bacteria under stressful environmental conditions and acts by a variety of mechanisms to promote cell survival. While the kinase that synthesizes polyP (PPK, encoded by the ppk gene) is well known, ppk transcription is not activated by environmental stress and little is understood about how environmental stress signals lead to polyP accumulation. Previous work has shown that the transcriptional regulators DksA, RpoN (σ54) and RpoE (σ24) positively regulate polyP production, but not ppk transcription, in Escherichia coli. In this work, we examine the role of the alternative sigma factor RpoN and nitrogen starvation stress response pathways in controlling polyP synthesis. We show that the RpoN enhancer binding proteins GlnG and GlrR impact polyP production, and uncover a new role for the nitrogen phosphotransferase regulator PtsN (EIIANtr) as a positive regulator of polyP production, acting upstream of DksA, downstream of RpoN and apparently independently of RpoE. However, neither these regulatory proteins nor common nitrogen metabolites appear to act directly on PPK, and the precise mechanism(s) by which polyP production is modulated after stress remain(s) unclear. Unexpectedly, we also found that the genes that impact polyP production vary depending on the composition of the rich media in which the cells were grown before exposure to polyP-inducing stress. These results constitute progress towards deciphering the regulatory networks driving polyP production under stress, and highlight the remarkable complexity of this regulation and its connections to a broad range of stress-sensing pathways.

Effects of human tissue acoustic properties, abdominal wall shape, and respiratory motion on ultrasound-mediated hyperthermia for targeted drug delivery to pancreatic tumors.

BACKGROUND: PanDox is a Phase-1 trial of chemotherapeutic drug delivery to pancreatic tumors using ultrasound-mediated hyperthermia to release doxorubicin from thermally sensitive liposomes. This report describes trial-related hyperthermia simulations featuring: (i) new ultrasonic properties of human pancreatic tissues, (ii) abdomen deflections imposed by a water balloon, and (iii) respiration-driven organ motion. METHODS: Pancreas heating simulations were carried out using three patient body models. Pancreas acoustic properties were varied between values found in the literature and those determined from our human tissue study. Acoustic beam distortion was assessed with and without balloon-induced abdomen deformation. Target heating was assessed for static, normal respiratory, and jet-ventilation-controlled pancreas motion. RESULTS: Human pancreatic tumor attenuation is 63% of the literature values, so that pancreas treatments require commensurately higher input intensity to achieve adequate hyperthermia. Abdominal wall deformation decreased the peak field pressure by as much as 3.5 dB and refracted the focal spot by as much as 4.5 mm. These effects were thermally counteracted by sidelobe power deposition, so the net impact on achieving mild hyperthermia was small. Respiratory motion during moving beam hyperthermia produced localized regions overheated by more than 8.0 °C above the 4.0 °C volumetric goal. The use of jet ventilation reduced this excess to 0.7 °C and yielded temperature field uniformity that was nearly identical to having no respiratory motion. CONCLUSION: Realistic modeling of the ultrasonic propagation environment is critical to achieving adequate mild hyperthermia without the use of real time thermometry for targeted drug delivery in pancreatic cancer patients.

Polyphosphate is a primordial chaperone.

Composed of up to 1,000 phospho-anhydride bond-linked phosphate monomers, inorganic polyphosphate (polyP) is one of the most ancient, conserved, and enigmatic molecules in biology. Here we demonstrate that polyP functions as a hitherto unrecognized chaperone. We show that polyP stabilizes proteins in vivo, diminishes the need for other chaperone systems to survive proteotoxic stress conditions, and protects a wide variety of proteins against stress-induced unfolding and aggregation. In vitro studies reveal that polyP has protein-like chaperone qualities, binds to unfolding proteins with high affinity in an ATP-independent manner, and supports their productive refolding once nonstress conditions are restored. Our results uncover a universally important function for polyP and suggest that these long chains of inorganic phosphate may have served as one of nature's first chaperones, a role that continues to the present day.

Cryo-EM structure of the highly atypical cytoplasmic ribosome of Euglena gracilis.

Ribosomal RNA is the central component of the ribosome, mediating its functional and architectural properties. Here, we report the cryo-EM structure of a highly divergent cytoplasmic ribosome from the single-celled eukaryotic alga Euglena gracilis. The Euglena large ribosomal subunit is distinct in that it contains 14 discrete rRNA fragments that are assembled non-covalently into the canonical ribosome structure. The rRNA is substantially enriched in post-transcriptional modifications that are spread far beyond the catalytic RNA core, contributing to the stabilization of this highly fragmented ribosome species. A unique cluster of five adenosine base methylations is found in an expansion segment adjacent to the protein exit tunnel, such that it is positioned for interaction with the nascent peptide. As well as featuring distinctive rRNA expansion segments, the Euglena ribosome contains four novel ribosomal proteins, localized to the ribosome surface, three of which do not have orthologs in other eukaryotes.