LPS-aggregating proteins GBP1 and GBP2 are each sufficient to enhance caspase-4 activation both in cellulo and in vitro.

The gamma-interferon (IFNγ)-inducible guanylate-binding proteins (GBPs) promote host defense against gram-negative cytosolic bacteria in part through the induction of an inflammatory cell death pathway called pyroptosis. To activate pyroptosis, GBPs facilitate sensing of the gram-negative bacterial outer membrane component lipopolysaccharide (LPS) by the noncanonical caspase-4 inflammasome. There are seven human GBP paralogs, and it is unclear how each GBP contributes to LPS sensing and pyroptosis induction. GBP1 forms a multimeric microcapsule on the surface of cytosolic bacteria through direct interactions with LPS. The GBP1 microcapsule recruits caspase-4 to bacteria, a process deemed essential for caspase-4 activation. In contrast to GBP1, closely related paralog GBP2 is unable to bind bacteria on its own but requires GBP1 for direct bacterial binding. Unexpectedly, we find that GBP2 overexpression can restore gram-negative-induced pyroptosis in GBP1KO cells, without GBP2 binding to the bacterial surface. A mutant of GBP1 that lacks the triple arginine motif required for microcapsule formation also rescues pyroptosis in GBP1KO cells, showing that binding to bacteria is dispensable for GBPs to promote pyroptosis. Instead, we find that GBP2, like GBP1, directly binds and aggregates "free" LPS through protein polymerization. We demonstrate that supplementation of either recombinant polymerized GBP1 or GBP2 to an in vitro reaction is sufficient to enhance LPS-induced caspase-4 activation. This provides a revised mechanistic framework for noncanonical inflammasome activation where GBP1 or GBP2 assembles cytosol-contaminating LPS into a protein-LPS interface for caspase-4 activation as part of a coordinated host response to gram-negative bacterial infections.

The pH Dependence of Niclosamide Solubility, Dissolution, and Morphology: Motivation for Potentially Universal Mucin-Penetrating Nasal and Throat Sprays for COVID19, its Variants and other Viral Infections.

MOTIVATION: With the coronavirus pandemic still raging, prophylactic-nasal and early-treatment throat-sprays could help prevent infection and reduce viral load. Niclosamide has the potential to treat a broad-range of viral infections if local bioavailability is optimized as mucin-penetrating solutions that can reach the underlying epithelial cells. EXPERIMENTAL: pH-dependence of supernatant concentrations and dissolution rates of niclosamide were measured in buffered solutions by UV/Vis-spectroscopy for niclosamide from different suppliers (AK Sci and Sigma), as precipitated material, and as cosolvates. Data was compared to predictions from Henderson-Hasselbalch and precipitation-pH models. Optical-microscopy was used to observe the morphologies of original, converted and precipitated niclosamide. RESULTS: Niclosamide from the two suppliers had different polymorphs resulting in different dissolution behavior. Supernatant concentrations of the "AKSci-polymorph" increased with increasing pH, from 2.53µM at pH 3.66 to 300µM at pH 9.2, reaching 703µM at pH 9.63. However, the "Sigma-polymorph" equilibrated to much lower final supernatant concentrations, reflective of more stable polymorphs at each pH. Similarly, when precipitated from supersaturated solution, or as cosolvates, niclosamide also equilibrated to lower final supernatant concentrations. Polymorph equilibration though was avoided by using a solvent-exchange technique to make the solutions. CONCLUSIONS: Given niclosamide's activity as a host cell modulator, optimized niclosamide solutions could represent universal prophylactic nasal and early treatment throat sprays against COVID19, its more contagious variants, and other respiratory viral infections. They are the simplest and potentially most effective formulations from both an efficacy standpoint as well as manufacturing and distribution, (no cold chain). They now just need testing.

A distinct lineage of giant viruses brings a rhodopsin photosystem to unicellular marine predators.

Giant viruses are remarkable for their large genomes, often rivaling those of small bacteria, and for having genes thought exclusive to cellular life. Most isolated to date infect nonmarine protists, leaving their strategies and prevalence in marine environments largely unknown. Using eukaryotic single-cell metagenomics in the Pacific, we discovered a Mimiviridae lineage of giant viruses, which infects choanoflagellates, widespread protistan predators related to metazoans. The ChoanoVirus genomes are the largest yet from pelagic ecosystems, with 442 of 862 predicted proteins lacking known homologs. They are enriched in enzymes for modifying organic compounds, including degradation of chitin, an abundant polysaccharide in oceans, and they encode 3 divergent type-1 rhodopsins (VirR) with distinct evolutionary histories from those that capture sunlight in cellular organisms. One (VirRDTS) is similar to the only other putative rhodopsin from a virus (PgV) with a known host (a marine alga). Unlike the algal virus, ChoanoViruses encode the entire pigment biosynthesis pathway and cleavage enzyme for producing the required chromophore, retinal. We demonstrate that the rhodopsin shared by ChoanoViruses and PgV binds retinal and pumps protons. Moreover, our 1.65-Å resolved VirRDTS crystal structure and mutational analyses exposed differences from previously characterized type-1 rhodopsins, all of which come from cellular organisms. Multiple VirR types are present in metagenomes from across surface oceans, where they are correlated with and nearly as abundant as a canonical marker gene from Mimiviridae Our findings indicate that light-dependent energy transfer systems are likely common components of giant viruses of photosynthetic and phagotrophic unicellular marine eukaryotes.

Comprehensive single-PCR 16S and 18S rRNA community analysis validated with mock communities, and estimation of sequencing bias against 18S.

Universal primers for SSU rRNA genes allow profiling of natural communities by simultaneously amplifying templates from Bacteria, Archaea, and Eukaryota in a single PCR reaction. Despite the potential to show relative abundance for all rRNA genes, universal primers are rarely used, due to various concerns including amplicon length variation and its effect on bioinformatic pipelines. We thus developed 16S and 18S rRNA mock communities and a bioinformatic pipeline to validate this approach. Using these mocks, we show that universal primers (515Y/926R) outperformed eukaryote-specific V4 primers in observed versus expected abundance correlations (slope = 0.88 vs. 0.67-0.79), and mock community members with single mismatches to the primer were strongly underestimated (threefold to eightfold). Using field samples, both primers yielded similar 18S beta-diversity patterns (Mantel test, p < 0.001) but differences in relative proportions of many rarer taxa. To test for length biases, we mixed mock communities (16S + 18S) before PCR and found a twofold underestimation of 18S sequences due to sequencing bias. Correcting for the twofold underestimation, we estimate that, in Southern California field samples (1.2-80 µm), there were averages of 35% 18S, 28% chloroplast 16S, and 37% prokaryote 16S rRNA genes. These data demonstrate the potential for universal primers to generate comprehensive microbiome profiles.

Manipulating Single Microdroplets of NaCl Solutions: Solvent Dissolution, Microcrystallization, and Crystal Morphology.

A new "three-micropipette manipulation technique" for forming, dehydrating, crystallizing, and resolvating nanograms of salt material has been developed to study supersaturated single microdroplets and microcrystals. This is the first report of studies that have measured in situ both supersaturation (as homogeneous nucleation) and saturation (as microcrystal redissolution) for single microdroplets of NaCl solution using the micropipette technique. This work reports a measure of the critical supersaturation concentration for homogeneous nucleation of NaCl (10.3 ± 0.3 M) at a supersaturation fraction of S = 1.9, the saturation concentration of NaCl in aqueous solution as measured with nanograms of material (5.5 ± 0.1 M), the diffusion coefficient for water in octanol, D = (1.96 ± 0.10) × 10-6 cm2/s, and the effect of the solvent's activity on dissolution kinetics. It is further shown that the same Epstein-Plesset (EP) model, which was originally developed for diffusion-controlled dissolution and uptake of gas, and successfully applied to liquid-in-liquid dissolution, can now also be applied to describe the diffusion-controlled uptake of water from a water-saturated environment using the extended activity-based model of Bitterfield et al. This aspect of the EP model has not previously been tested using single microdroplets. Finally, it is also reported how the water dissolution rate, rate of NaCl concentration change, resulting crystal structure, and the time frame of initial crystal growth are affected by changing the bathing medium from octanol to decane. A much slower loss of water-solvent and concomitant slower up-concentration of the NaCl solute resulted in a lower tendency to nucleate and slower crystal growth because much less excess material was available at the onset of nucleation in the decane system as compared to the octanol system. Thus, the crystal structure is reported to be dendritic for NaCl solution microdroplets dissolving rapidly and nucleating violently in octanol, while they are formed as single cubic crystals in a gentler way for solution-dissolution in decane. These new techniques and analyses can now also be used for any other system where all relevant parameters are known. An example of this is control of drug/hydrogel/emulsion particle size change due to solvent uptake.

Vertical and Seasonal Patterns Control Bacterioplankton Communities at Two Horizontally Coherent Coastal Upwelling Sites off Galicia (NW Spain).

Analysis of seasonal patterns of marine bacterial community structure along horizontal and vertical spatial scales can help to predict long-term responses to climate change. Several recent studies have shown predictable seasonal reoccurrence of bacterial assemblages. However, only a few have assessed temporal variability over both horizontal and vertical spatial scales. Here, we simultaneously studied the bacterial community structure at two different locations and depths in shelf waters of a coastal upwelling system during an annual cycle. The most noticeable biogeographic patterns observed were seasonality, horizontal homogeneity, and spatial synchrony in bacterial diversity and community structure related with regional upwelling-downwelling dynamics. Water column mixing eventually disrupted bacterial community structure vertical heterogeneity. Our results are consistent with previous temporal studies of marine bacterioplankton in other temperate regions and also suggest a marked influence of regional factors on the bacterial communities inhabiting this coastal upwelling system. Bacterial-mediated carbon fluxes in this productive region appear to be mainly controlled by community structure dynamics in surface waters, and local environmental factors at the base of the euphotic zone.

Genome and epigenome of a novel marine Thaumarchaeota strain suggest viral infection, phosphorothioation DNA modification and multiple restriction systems.

Marine Thaumarchaeota are abundant ammonia-oxidizers but have few representative laboratory-cultured strains. We report the cultivation of Candidatus Nitrosomarinus catalina SPOT01, a novel strain that is less warm-temperature tolerant than other cultivated Thaumarchaeota. Using metagenomic recruitment, strain SPOT01 comprises a major portion of Thaumarchaeota (4-54%) in temperate Pacific waters. Its complete 1.36 Mbp genome possesses several distinguishing features: putative phosphorothioation (PT) DNA modification genes; a region containing probable viral genes; and putative urea utilization genes. The PT modification genes and an adjacent putative restriction enzyme (RE) operon likely form a restriction modification (RM) system for defence from foreign DNA. PacBio sequencing showed >98% methylation at two motifs, and inferred PT guanine modification of 19% of possible TGCA sites. Metagenomic recruitment also reveals the putative virus region and PT modification and RE genes are present in 18-26%, 9-14% and <1.5% of natural populations at 150 m with ≥85% identity to strain SPOT01. The presence of multiple probable RM systems in a highly streamlined genome suggests a surprising importance for defence from foreign DNA for dilute populations that infrequently encounter viruses or other cells. This new strain provides new insights into the ecology, including viral interactions, of this important group of marine microbes.

Every base matters: assessing small subunit rRNA primers for marine microbiomes with mock communities, time series and global field samples.

Microbial community analysis via high-throughput sequencing of amplified 16S rRNA genes is an essential microbiology tool. We found the popular primer pair 515F (515F-C) and 806R greatly underestimated (e.g. SAR11) or overestimated (e.g. Gammaproteobacteria) common marine taxa. We evaluated marine samples and mock communities (containing 11 or 27 marine 16S clones), showing alternative primers 515F-Y (5'-GTGYCAGCMGCCGCGGTAA) and 926R (5'-CCGYCAATTYMTTTRAGTTT) yield more accurate estimates of mock community abundances, produce longer amplicons that can differentiate taxa unresolvable with 515F-C/806R, and amplify eukaryotic 18S rRNA. Mock communities amplified with 515F-Y/926R yielded closer observed community composition versus expected (r(2) = 0.95) compared with 515F-Y/806R (r(2) ∼ 0.5). Unexpectedly, biases with 515F-Y/806R against SAR11 in field samples (∼4-10-fold) were stronger than in mock communities (∼2-fold). Correcting a mismatch to Thaumarchaea in the 515F-C increased their apparent abundance in field samples, but not as much as using 926R rather than 806R. With plankton samples rich in eukaryotic DNA (> 1 µm size fraction), 18S sequences averaged ∼17% of all sequences. A single mismatch can strongly bias amplification, but even perfectly matched primers can exhibit preferential amplification. We show that beyond in silico predictions, testing with mock communities and field samples is important in primer selection.

Micropipette Technique Study of Natural and Synthetic Lung Surfactants at the Air-Water Interface: Presence of a SP-B Analog Peptide Promotes Membrane Aggregation, Formation of Tightly Stacked Lamellae, and Growth of Myelin Figures.

The present study is a microscopic interfacial characterization of a series of lung surfactant materials performed with the micropipette technique. The advantages of this technique include the measurement of equilibrium and dynamic surface tensions while acquiring structural and dynamic information at microscopic air-water interfaces in real time and upon compression. Here, we characterized a series of animal-derived and synthetic lung surfactant formulations, including native surfactant obtained from porcine lungs (NS); the commercial Curosurf, Infasurf, and Survanta; and a synthetic Super Mini-B (SMB)-containing formulation. It was observed that the presence of the natural hydrophobic proteins and, more strikingly, the peptide SMB, promoted vesicle condensation as thick membrane stacks beneath the interface. Only in the presence of SMB, these stacks underwent spontaneous structural transformations, consisting of the nucleation and growth of microtubes and in some cases their subsequent coiling into helices. The dimensions of these tubes (2-15 µm diameter) and their linear (2-3 µm/s) and volumetric growth rates (20-30 µm3/s) were quantified, and no specific effects were found on them for increasing SMB concentrations from 0.1 to 4%. Nevertheless, a direct correlation between the number of tubes and SMB contents was found, suggesting that SMB molecules are the promoters of tube nucleation in these membranes. A detailed analysis of the tube formation process was performed following previous models for the growth of myelin figures, proposing a combined mechanism between dehydration-rehydration of the lipid bilayers and induction of mechanical defects by SMB that would act as nucleation sites for the tubes. The formation of tubes was also observed in Infasurf, and in NS only after subsequent expansion and compression but neither in the other clinical surfactants nor in protein-free preparations. Finally, the connection between this data and the observations from the lung surfactant literature concerning the widely reported "near-zero surface tension" for lung surfactant films and intact alveolar surfaces is also discussed.

An Activity-Based Dissolution Model for Solute-Containing Microdroplets.

When a solute is present in an aqueous droplet, the water activity in the droplet and the rate of droplet dissolution are both decreased (as compared to a pure water droplet). One of the main parameters that controls this effect is the dynamically changing solute concentration, and therefore water activity and chemical potential, at the droplet interface. This work addresses the importance of understanding how water activity changes during solution droplet dissolution. A model for dissolution rate is presented that accounts for the kinetic effects of changing water activity at the droplet interface during the dissolution of an aqueous salt solution microdroplet into a second immiscible liquid phase. The important underlying question in this model is whether the dissolving component can be considered in local equilibrium on both sides of the droplet interface and whether this assumption is sufficient to account for the kinetics of dissolution. The dissolution model is based on the Epstein-Plesset equation, which has previously been applied to pure gas (bubble) and liquid (droplet) dissolution into liquid phases, but not to salt solutions. The model is tested by using the micropipet technique to form and observe the dehydration of single NaCl solution microdroplets in octanol or butyl acetate. The model successfully predicts the droplet diameter as a function of time in both organic solvents. The NaCl concentration in water is measured well into the supersaturated area >5.4 M, and the supersaturation limit at which NaCl nucleation happens is reported to be 10.24 ± 0.31 M.

Two phase I dose-escalation/pharmacokinetics studies of low temperature liposomal doxorubicin (LTLD) and mild local hyperthermia in heavily pretreated patients with local regionally recurrent breast cancer.

PURPOSE: Unresectable chest wall recurrences of breast cancer (CWR) in heavily pretreated patients are especially difficult to treat. We hypothesised that thermally enhanced drug delivery using low temperature liposomal doxorubicin (LTLD), given with mild local hyperthermia (MLHT), will be safe and effective in this population. PATIENTS AND METHODS: This paper combines the results of two similarly designed phase I trials. Eligible CWR patients had progressed on the chest wall after prior hormone therapy, chemotherapy, and radiotherapy. Patients were to get six cycles of LTLD every 21-35 days, followed immediately by chest wall MLHT for 1 hour at 40-42 °C. In the first trial 18 subjects received LTLD at 20, 30, or 40 mg/m2; in the second trial, 11 subjects received LTLD at 40 or 50 mg/m2. RESULTS: The median age of all 29 patients enrolled was 57 years. Thirteen patients (45%) had distant metastases on enrolment. Patients had received a median dose of 256 mg/m2 of prior anthracyclines and a median dose of 61 Gy of prior radiation. The median number of study treatments that subjects completed was four. The maximum tolerated dose was 50 mg/m2, with seven subjects (24%) developing reversible grade 3-4 neutropenia and four (14%) reversible grade 3-4 leucopenia. The rate of overall local response was 48% (14/29, 95% CI: 30-66%), with. five patients (17%) achieving complete local responses and nine patients (31%) having partial local responses. CONCLUSION: LTLD at 50 mg/m2 and MLHT is safe. This combined therapy produces objective responses in heavily pretreated CWR patients. Future work should test thermally enhanced LTLD delivery in a less advanced patient population.

Niclosamide: A career builder PART I: (Almost) Everything You Wanted To Know About Niclosamide But Were Too Afraid To Ask PART II: Nanomedicines? A Carrier-Free Nanomedicine for Cancer and a Simple Buffered Solution to Prevent COVID19 and Other Respiratory Infections: They Just Need Testing Invited Manuscript: J Controlled Release VSI in Honor of Prof. Park.

My contribution to honoring Professor Kinam Park celebrates and resonates with his scholarly career in drug delivery, his commitment to encouraging the next generation(s), and his efforts to keep us focused on clinically effective formulations. To do this I take as my example, niclosamide, a small molecule protonophore that, uniquely, can "target" all cell membranes, both plasma and organelle. As such, it acts upstream of many cell pathways and so has the potential to affect many of the essential events that a cell, and particularly a diseased cell or other entities like a virus, use to stay alive and prosper. Literature shows that it has so far been discovered to positively influence (at least): cancer, bacterial and viral infection, metabolic diseases such as Type II diabetes, NASH and NAFLD, artery constriction, endometriosis, neuropathic pain, rheumatoid arthritis, sclerodermatous graft-versus-host disease, systemic sclerosis, Parkinson's, and COPD. With such a fundamental action and broad-spectrum activity, I believe that studying niclosamide in all its manifestations, discovering if and to what extent it can contribute positively to disease control (and also where it can't), formulating it as effective therapeutics, and testing them in preclinical and clinical trials is a career builder for our next generation(s). The article is divided into two parts: Part I introduces niclosamide and other proton shunts mainly in cancer and viral infections and reviews an exponentially growing literature with some concepts and physicochemical properties that lead to its proton shunt mechanism. Part II focuses on repurposing by reformulation of niclosamide. I give two examples of "carrier-free formulations", - one for cancer (as a prodrug therapeutic of niclosamide stearate for i.v. and other administration routes, exemplified by our recent work on Osteosarcoma in mice and canine patients), and the other as a niclosamide solution formulation (that could provide the basis for a preventative nasal spray and early treatment option for COVID19 and other respiratory virus infections). My goal is to excite and enthuse, encourage, and motivate all involved in the drug development and testing process in academia, institutes, and industry, to learn more about this interesting molecule and others like it. To enable such endeavors, I give many proposed ideas throughout the document, that have been stimulated and inspired by gaps in the literature, urgent needs in disease, and new studies arising from our own work. The hope is that, by reading through this document and studying the suggested topics and references, the drug delivery and development community will continue our lineage and benefit from our legacy to achieve niclosamide's potential as an effective contributor to the treatment and control of many diseases and conditions.

Gradients of bacteria in the oceanic water column reveal finely-resolved vertical distributions.

Bacterial communities directly influence ecological processes in the ocean, and depth has a major influence due to the changeover in primary energy sources between the sunlit photic zone and dark ocean. Here, we examine the abundance and diversity of bacteria in Monterey Bay depth profiles collected from the surface to just above the sediments (e.g., 2000 m). Bacterial abundance in these Pacific Ocean samples decreased by >1 order of magnitude, from 1.22 ±0.69 ×106 cells ml-1 in the variable photic zone to 1.44 ± 0.25 ×105 and 6.71 ± 1.23 ×104 cells ml-1 in the mesopelagic and bathypelagic, respectively. V1-V2 16S rRNA gene profiling showed diversity increased sharply between the photic and mesopelagic zones. Weighted Gene Correlation Network Analysis clustered co-occurring bacterial amplicon sequence variants (ASVs) into seven subnetwork modules, of which five strongly correlated with depth-related factors. Within surface-associated modules there was a clear distinction between a 'copiotrophic' module, correlating with chlorophyll and dominated by e.g., Flavobacteriales and Rhodobacteraceae, and an 'oligotrophic' module dominated by diverse Oceanospirillales (such as uncultured JL-ETNP-Y6, SAR86) and Pelagibacterales. Phylogenetic reconstructions of Pelagibacterales and SAR324 using full-length 16S rRNA gene data revealed several additional subclades, expanding known microdiversity within these abundant lineages, including new Pelagibacterales subclades Ia.B, Id, and IIc, which comprised 4-10% of amplicons depending on the subclade and depth zone. SAR324 and Oceanospirillales dominated in the mesopelagic, with SAR324 clade II exhibiting its highest relative abundances (17±4%) in the lower mesopelagic (300-750 m). The two newly-identified SAR324 clades showed highest relative abundances in the photic zone (clade III), while clade IV was extremely low in relative abundance, but present across dark ocean depths. Hierarchical clustering placed microbial communities from 900 m samples with those from the bathypelagic, where Marinimicrobia was distinctively relatively abundant. The patterns resolved herein, through high resolution and statistical replication, establish baselines for marine bacterial abundance and taxonomic distributions across the Monterey Bay water column, against which future change can be assessed.

Mass transfer in the dissolution of a multicomponent liquid droplet in an immiscible liquid environment.

The Epstein-Plesset equation has recently been shown to predict accurately the dissolution of a pure liquid microdroplet into a second immiscible solvent, such as oil into water. Here, we present a series of new experiments and a modification to this equation to model the dissolution of a two-component oil-mixture microdroplet into a second immiscible solvent in which the two materials of the droplet have different solubilities. The model is based on a reduced surface area approximation and the assumption of ideal homogeneous mixing [mass flux d(m(i))/dt = A(frac(i))D(i)(c(i) - c(s)){(1/R) + (1/(πD(i)t)(1/2)}] where A(frac(i)) is the area fraction of component i, c(i) and c(s) are the initial and saturation concentrations of the droplet material in the surrounding medium, R is the radius of the droplet, t is time, and D(i) is the coefficient of diffusion of component i in the surrounding medium. This new model has been tested by the use of a two-chamber micropipet-based method, which measured the dissolution of single individual microdroplets of mutually miscible liquid mixtures (ethyl acetate/butyl acetate and butyl acetate/amyl acetate) in water. We additionally measured the diffusion coefficient of the pure materials-ethyl acetate, butyl acetate, and amyl acetate-in water at 22 °C. Diffusion coefficients for the pure acetates in water were 8.65 × 10(-6), 7.61 × 10(-6), and 9.14 × 10(-6) cm(2)/s, respectively. This model accurately predicts the dissolution of microdroplets for the ethyl acetate/butyl acetate and butyl acetate/amyl acetate systems given the solubility and diffusion coefficients of each of the individual components in water as well as the initial droplet radius. The average mean squared error was 8.96%. The dissolution of a spherical ideally mixed multicomponent droplet closely follows the modified Epstein-Plesset model presented here.

A method to convert MRI images of temperature change into images of absolute temperature in solid tumours.

PURPOSE: During hyperthermia (HT), the therapeutic response of tumours varies substantially within the target temperature range (39-43 °C). Current thermometry methods are either invasive or measure only temperature change, which limits the ability to study tissue responses to HT. This study combines manganese-containing low temperature sensitive liposomes (Mn-LTSL) with proton resonance frequency shift (PRFS) thermometry to measure absolute temperature in tumours with high spatial and temporal resolution using MRI. METHODS: Liposomes were loaded with 300 mM MnSO(4). The phase transition temperature (T(m)) of Mn-LTSL samples was measured by differential scanning calorimetry (DSC). The release of manganese from Mn-LTSL in saline was characterised with inductively coupled plasma atomic emission spectroscopy. A 2T GE small animal scanner was used to acquire dynamic T1-weighted images and temperature change images of Mn-LTSL in saline phantoms and fibrosarcoma-bearing Fisher-344 rats receiving hyperthermia after Mn-LTSL injection. RESULTS: The T(m) of Mn-LTSL in rat blood was 42.9 ± 0.2 °C (DSC). For Mn-LTSL samples (0.06 mM-0.5 mM Mn(2+) in saline) heated monotonically from 30 °C to 50 °C, a peak in the rate of MRI signal enhancement occurred at 43.1° ± 0.3 °C. The same peak in signal enhancement rate was observed during heating of fibrosarcoma tumours (N = 3) after injection of Mn-LTSL, and the peak was used to convert temperature change images into absolute temperature. Accuracies of calibrated temperature measurements were in the range 0.9-1.8 °C. CONCLUSION: The release of Mn(2+) from Mn-LTSL affects the rate of MR signal enhancement which enables conversion of MRI-based temperature change images to absolute temperature.

Extraction of niclosamide from commercial approved tablets into aqueous buffered solution creates potentially approvable oral and nasal sprays against COVID-19 and other respiratory infections.

Motivation: The low solubility, weak acid drug, niclosamide is a host cell modulator with broad-spectrum anti-viral cell-activity against many viruses, including stopping the SARS-CoV-2 virus from infecting cells in cell culture. As a result, a simple universal nasal spray preventative was proposed and investigated in earlier work regarding the dissolution of niclosamide into simple buffers. However, starting with pharmaceutical grade, niclosamide represents a new 505(b)(2) application. The motivation for this second paper in the series was therefore to explore if and to what extent niclosamide could be extracted from commercially available and regulatory-approved niclosamide oral tablets that could serve as a preventative nasal spray and an early treatment oral/throat spray, with possibly more expeditious testing and regulatory approval. Experimental: Measurements of supernatant niclosamide concentrations were made by calibrated UV-Vis for the dissolution of niclosamide from commercially available Yomesan crushed into a powder for dissolution into Tris Buffer (TB) solutions. Parameters tested were as follows: time (0-2 days), concentration (300 µM to -1 mM), pH (7.41 to 9.35), and anhydrous/hydrated state. Optical microscopy was used to view the morphologies of the initial crushed powder, and the dissolving and equilibrating undissolved excess particles to detect morphologic changes that might occur. Results: Concentration dependence: Niclosamide was readily extracted from powdered Yomesan at pH 9.34 TB at starting Yomesan niclosamide equivalents concentrations of 300 µM, 600 µM, and 1 mM. Peak dissolved niclosamide supernatant concentrations of 264 µM, 216 µM, and 172 µM were achieved in 1 h, 1 h, and 3 h respectively. These peaks though were followed by a reduction in supernatant concentration to an average of 112.3 µM ± 28.4 µM after overnight stir on day 2. pH dependence: For nominal pHs of 7.41, 8.35, 8.85, and 9.35, peak niclosamide concentrations were 4 µM, 22.4 µM, 96.2 µM, and 215.8 µM, respectively. Similarly, the day 2 values all reduced to 3 µM, 12.9 µM, 35.1 µM, and 112.3 µM. A heat-treatment to 200 °C dehydrated the niclosamide and showed a high 3 h concentration (262 µM) and the least day-2 reduction (to 229 µM). This indicated that the presence, or formation during exposure to buffer, of lower solubility polymorphs was responsible for the reductions in total solubilities. These morphologic changes were confirmed by optical microscopy that showed initially featureless particulate-aggregates of niclosamide could grow multiple needle-shaped crystals and form needle masses, especially in the presence of Tris-buffered sodium chloride, where new red needles were rapidly made. Scale up: A scaled-up 1 L solution of niclosamide was made achieving 165 µM supernatant niclosamide in 3 h by dissolution of just one fifth (100 mg niclosamide) of a Yomesan tablet. Conclusion: These comprehensive results provide a guide as to how to utilize commercially available and approved tablets of niclosamide to generate aqueous niclosamide solutions from a simple dissolution protocol. As shown here, just one 4-tablet pack of Yomesan could readily make 165 L of a 20 µM niclosamide solution giving 16,500 10 mL bottles. One million bottles, from just 60 packs of Yomesan, would provide 100 million single spray doses for distribution to mitigate a host of respiratory infections as a universal preventative-nasal and early treatment oral/throat sprays throughout the world. Graphical Abstract: pH dependence of niclosamide extraction from crushed Yomesan tablet material into Tris buffer (yellow-green in vial) and Tris-buffered saline solution (orange-red in vial). Initial anhydrous dissolution concentration is reduced by overnight stirring to likely monohydrate niclosamide; and is even lower if in TBSS forming new niclosamide sodium needle crystals grown from the original particles. Supplementary Information: The online version contains supplementary material available at 10.1186/s41120-023-00072-x.

Low-Density Lipoprotein Pathway Is a Ubiquitous Metabolic Vulnerability in High Grade Glioma Amenable for Nanotherapeutic Delivery.

Metabolic reprogramming, through increased uptake of cholesterol in the form of low-density lipoproteins (LDL), is one way by which cancer cells, including high grade gliomas (HGG), maintain their rapid growth. In this study, we determined LDL receptor (LDLR) expression in HGGs using immunohistochemistry on tissue microarrays from intra- and inter tumour regions of 36 adult and 133 paediatric patients to confirm LDLR as a therapeutic target. Additionally, we analysed expression levels in three representative cell line models to confirm their future utility to test LDLR-targeted nanoparticle uptake, retention, and cytotoxicity. Our data show widespread LDLR expression in adult and paediatric cohorts, but with significant intra-tumour variation observed between the core and either rim or invasive regions of adult HGG. Expression was independent of paediatric tumour grade or identified clinicopathological factors. LDLR-expressing tumour cells localized preferentially within perivascular niches, also with significant adult intra-tumour variation. We demonstrated variable levels of LDLR expression in all cell lines, confirming their suitability as models to test LDLR-targeted nanotherapy delivery. Overall, our study reveals the LDLR pathway as a ubiquitous metabolic vulnerability in high grade gliomas across all ages, amenable to future consideration of LDL-mediated nanoparticle/drug delivery to potentially circumvent tumour heterogeneity.

Phase separation behavior of fusidic acid and rifampicin in PLGA microspheres.

The purpose of this study was to characterize the phase separation behavior of fusidic acid (FA) and rifampicin (RIF) in poly(d,l-lactic acid-co-glycolic acid) (PLGA) using a model microsphere formulation. To accomplish this, microspheres containing 20% FA with 0%, 5%, 10%, 20%, and 30% RIF and 20% RIF with 30%, 20% 10%, 5%, and 0% FA were prepared by solvent evaporation. Drug-polymer and drug-drug compatibility and miscibility were characterized using laser confocal microscopy, Raman spectroscopy, XRPD, DSC, and real-time video recordings of single-microsphere formation. The encapsulation of FA and RIF alone, or in combination, results in a liquid-liquid phase separation of solvent-and-drug-rich microdomains that are excluded from the polymer bulk during microsphere hardening, resulting in amorphous spherical drug-rich domains within the polymer bulk and on the microsphere surface. FA and RIF phase separate from PLGA at relative droplet volumes of 0.311 ± 0.014 and 0.194 ± 0.000, respectively, predictive of the incompatibility of each drug and PLGA. When coloaded, FA and RIF phase separate in a single event at the relative droplet volume 0.251 ± 0.002, intermediate between each of the monoloaded formulations and dependent on the relative contribution of FA or RIF. The release of FA and RIF from phase-separated microspheres was characterized exclusively by a burst release and was dependent on the phase exclusion of surface drug-rich domains. Phase separation results in coalescence of drug-rich microdroplets and polymer phase exclusion, and it is dependent on the compatibility between FA and RIF and PLGA. FA and RIF are mutually miscible in all proportions as an amorphous glass, and they phase separate from the polymer as such. These drug-rich domains were excluded to the surface of the microspheres, and subsequent release of both drugs from the microspheres was rapid and reflected this surface location.

Genomes from Uncultivated Pelagiphages Reveal Multiple Phylogenetic Clades Exhibiting Extensive Auxiliary Metabolic Genes and Cross-Family Multigene Transfers.

For the abundant marine Alphaproteobacterium Pelagibacter (SAR11), and other bacteria, phages are powerful forces of mortality. However, little is known about the most abundant Pelagiphages in nature, such as the widespread HTVC023P-type, which is currently represented by two cultured phages. Using viral metagenomic data sets and fluorescence-activated cell sorting, we recovered 80 complete, undescribed Podoviridae genomes that form 10 phylogenomically distinct clades (herein, named Clades I to X) related to the HTVC023P-type. These expanded the HTVC023P-type pan-genome by 15-fold and revealed 41 previously unknown auxiliary metabolic genes (AMGs) in this viral lineage. Numerous instances of partner-AMGs (colocated and involved in related functions) were observed, including partners in nucleotide metabolism, DNA hypermodification, and Curli biogenesis. The Type VIII secretion system (T8SS) responsible for Curli biogenesis was identified in nine genomes and expanded the repertoire of T8SS proteins reported thus far in viruses. Additionally, the identified T8SS gene cluster contained an iron-dependent regulator (FecR), as well as a histidine kinase and adenylate cyclase that can be implicated in T8SS function but are not within T8SS operons in bacteria. While T8SS are lacking in known Pelagibacter, they contribute to aggregation and biofilm formation in other bacteria. Phylogenetic reconstructions of partner-AMGs indicate derivation from cellular lineages with a more recent transfer between viral families. For example, homologs of all T8SS genes are present in syntenic regions of distant Myoviridae Pelagiphages, and they appear to have alphaproteobacterial origins with a later transfer between viral families. The results point to an unprecedented multipartner-AMG transfer between marine Myoviridae and Podoviridae. Together with the expansion of known metabolic functions, our studies provide new prospects for understanding the ecology and evolution of marine phages and their hosts. IMPORTANCE One of the most abundant and diverse marine bacterial groups is Pelagibacter. Phages have roles in shaping Pelagibacter ecology; however, several Pelagiphage lineages are represented by only a few genomes. This paucity of data from even the most widespread lineages has imposed limits on the understanding of the diversity of Pelagiphages and their impacts on hosts. Here, we report 80 complete genomes, assembled directly from environmental data, which are from undescribed Pelagiphages and render new insights into the manipulation of host metabolism during infection. Notably, the viruses have functionally related partner genes that appear to be transferred between distant viruses, including a suite that encode a secretion system which both brings a new functional capability to the host and is abundant in phages across the ocean. Together, these functions have important implications for phage evolution and for how Pelagiphage infection influences host biology in manners extending beyond canonical viral lysis and mortality.

High-throughput, single-microbe genomics with strain resolution, applied to a human gut microbiome.

Characterizing complex microbial communities with single-cell resolution has been a long-standing goal of microbiology. We present Microbe-seq, a high-throughput method that yields the genomes of individual microbes from complex microbial communities. We encapsulate individual microbes in droplets with microfluidics and liberate their DNA, which we then amplify, tag with droplet-specific barcodes, and sequence. We explore the human gut microbiome, sequencing more than 20,000 microbial single-amplified genomes (SAGs) from a single human donor and coassembling genomes of almost 100 bacterial species, including several with multiple subspecies strains. We use these genomes to probe microbial interactions, reconstructing the horizontal gene transfer (HGT) network and observing HGT between 92 species pairs; we also identify a significant in vivo host-phage association between crAssphage and one strain of Bacteroides vulgatus. Microbe-seq contributes high-throughput culture-free capabilities to investigate genomic blueprints of complex microbial communities with single-microbe resolution.