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(Cell Reports 42, 113311; November 28, 2023)

Short polypeptides encoded by small open reading frames (smORFs) are ubiquitously found in eukaryotic genomes and are important regulators of physiology, development, and mitochondrial processes. Here, we focus on a subset of 298 smORFs that are evolutionarily conserved between Drosophila melanogaster and humans. Many of these smORFs are conserved broadly in the bilaterian lineage, and ∼182 are conserved in plants. We observe remarkably heterogeneous spatial and temporal expression patterns of smORF transcripts-indicating wide-spread tissue-specific and stage-specific mitochondrial architectures. In addition, an analysis of annotated functional domains reveals a predicted enrichment of smORF polypeptides localizing to mitochondria. We conduct an embryonic ribosome profiling experiment and find support for translation of 137 of these smORFs during embryogenesis. We further embark on functional characterization using CRISPR knockout/activation, RNAi knockdown, and cDNA overexpression, revealing diverse phenotypes. This study underscores the importance of identifying smORF function in disease and phenotypic diversity.

Transcription factors (TFs) play a key role in development and in cellular responses to the environment by activating or repressing the transcription of target genes in precise spatial and temporal patterns. In order to develop a catalog of target genes of Drosophila melanogaster TFs, the modERN consortium systematically knocked down the expression of TFs using RNAi in whole embryos followed by RNA-seq. We generated data for 45 TFs which have 18 different DNA-binding domains and are expressed in 15 of the 16 organ systems. The range of inactivation of the targeted TFs by RNAi ranged from log2fold change -3.52 to +0.49. The TFs also showed remarkable heterogeneity in the numbers of candidate target genes identified, with some generating thousands of candidates and others only tens. We present detailed analysis from five experiments, including those for three TFs that have been the focus of previous functional studies (ERR, sens, and zfh2) and two previously uncharacterized TFs (sens-2 and CG32006), as well as short vignettes for selected additional experiments to illustrate the utility of this resource. The RNA-seq datasets are available through the ENCODE DCC (http://encodeproject.org) and the Sequence Read Archive (SRA). TF and target gene expression patterns can be found here: https://insitu.fruitfly.org. These studies provide data that facilitate scientific inquiries into the functions of individual TFs in key developmental, metabolic, defensive, and homeostatic regulatory pathways, as well as provide a broader perspective on how individual TFs work together in local networks during embryogenesis.

Novel approaches are required to address the urgent need to develop lipid-based carriers of paclitaxel (PTX) and other hydrophobic drugs for cancer chemotherapy. Carriers based on cationic liposomes (CLs) with fluid (i.e., chain-melted) membranes (e.g., EndoTAG-1) have shown promise in preclinical and late-stage clinical studies. Recent work found that the addition of a cone-shaped poly(ethylene glycol)-lipid (PEG-lipid) to PTX-loaded CLs (CLsPTX) promotes a transition to sterically stabilized, higher-curvature (smaller) nanoparticles consisting of a mixture of PEGylated CLsPTX and PTX-containing fluid lipid nanodiscs (nanodiscsPTX). These CLsPTX and nanodiscsPTX show significantly improved uptake and cytotoxicity in cultured human cancer cells at PEG coverage in the brush regime (10 mol % PEG-lipid). Here, we studied the PTX loading, in vivo circulation half-life, and biodistribution of systemically administered CLsPTX and nanodiscsPTX and assessed their ability to induce apoptosis in triple-negative breast-cancer-bearing immunocompetent mice. We focused on fluid rather than solid lipid nanodiscs because of the significantly higher solubility of PTX in fluid membranes. At 5 and 10 mol % of a PEG-lipid (PEG5K-lipid, molecular weight of PEG 5000 g/mol), the mixture of PEGylated CLsPTX and nanodiscsPTX was able to incorporate up to 2.5 mol % PTX without crystallization for at least 20 h. Remarkably, compared to preparations containing 2 and 5 mol % PEG5K-lipid (with the PEG chains in the mushroom regime), the particles at 10 mol % (with PEG chains in the brush regime) showed significantly higher blood half-life, tumor penetration, and proapoptotic activity. Our study suggests that increasing the PEG coverage of CL-based drug nanoformulations can improve their pharmacokinetics and therapeutic efficacy.

TABLE OF CONTENTS ABSTRACT 955 TABLE OF CONTENTS 956 I. INTRODUCTION 957 II. THE TWO COMPETING THEORIES OF THE CORPORATION'S ROLE IN SOCIETY 960 III. THE ODD TIMING OF THE [...], Stakeholder enthusiasm grips public companies and asset managers. Sustainability reports abound, reflecting an appetite for detailed data on company efforts to reduce carbon emissions and water usage and to protect and diversify workforces--while investors still seek returns to finance college tuition and fund retirement. But commentators and those who control public companies fail to engage on fundamental questions: Which stakeholders count? To what degree will companies sacrifice shareholder return to benefit those stakeholders? What happens when the interests of differing sets of non-shareholder stakeholders differ? Is all the commotion really necessary, given the many laws and regulations protecting such stakeholders? Whose priorities will, as a practical matter, govern the application of stakeholder theory, and will those in control of the priorities use this corporate theory to further their personal purposes? Exposition of these questions suggests serious changes in disclosures that director nominees and asset management firms must make, as well as the need for a hard look at whether stakeholder capitalism can be practiced with normative consistency.

Exosomes are cell-secreted vesicles less than ≈150 nm in size that contain gene-encoding and gene-silencing RNA and cytosolic proteins with roles in intercellular communication. Interest in the use of exosomes as targeted drug delivery vehicles has grown since it was shown that they can bind specific cells and deliver intact genetic material to the cytosol of target cells. We isolated extracellular vesicles (EVs), consisting of a mixture of exosomes and microvesicles, from prostate (PC3) and melanoma (M21) cancer cell lines using serial ultracentrifugation. Interrogation via western blot analysis confirmed enrichment of CD63, a widely recognized EV surface protein, in the EV pellet from both cell lines. Nanoparticle tracking analysis (NTA) of EV pellets revealed that the two cell lines produced distinct vesicle size profiles in the ≈30 nm to ≈400 nm range. NTA further showed that the fraction of exosomes to all EVs was constant, suggesting cellular mechanisms that control the fraction of secreted vesicles that are exosomes. Transmission electron microscopy (TEM) images of the unmodified PC3 EVs showed vesicles with cup-like (i.e., nanocapsule) and previously unreported prolate morphologies. The observed non-spherical morphologies for dehydrated exosomal vesicles (size ≈30-100 nm) are most likely related to the dense packing of proteins in exosome membranes. Solubility phase diagram data showed that EVs enhanced the solubility of paclitaxel (PTX) in aqueous solution compared to a water-only control. Combined with their inherent targeting and cytosol delivery properties, these findings highlight the potential advantages of using exosomes as chemotherapeutic drug carriers in vivo.

Lipid carriers of hydrophobic paclitaxel (PTX) are used in clinical trials for cancer chemotherapy. Improving their loading capacity requires enhanced PTX solubilization. We compared the time-dependence of PTX membrane solubility as a function of PTX content in cationic liposomes (CLs) with lipid tails containing one (oleoyl; DOPC/DOTAP) or two (linoleoyl; DLinPC/newly synthesized DLinTAP) cis double bonds by using microscopy to generate kinetic phase diagrams. The DLin lipids displayed significantly increased PTX membrane solubility over DO lipids. Remarkably, 8 mol% PTX in DLinTAP/DLinPC CLs remained soluble for approximately as long as 3 mol% PTX (the solubility limit, which has been the focus of most previous studies and clinical trials) in DOTAP/DOPC CLs. The increase in solubility is likely caused by enhanced molecular affinity between lipid tails and PTX, rather than by the transition in membrane structure from bilayers to inverse cylindrical micelles observed with small-angle X-ray scattering. Importantly, the efficacy of PTX-loaded CLs against prostate cancer cells (their IC50 of PTX cytotoxicity) was unaffected by changing the lipid tails, and toxicity of the CL carrier was negligible. Moreover, efficacy was approximately doubled against melanoma cells for PTX-loaded DLinTAP/DLinPC over DOTAP/DOPC CLs. Our findings demonstrate the potential of chemical modifications of the lipid tails to increase the PTX membrane loading while maintaining (and in some cases even increasing) the efficacy of CLs. The increased PTX solubility will aid the development of liposomal PTX carriers that require significantly less lipid to deliver a given amount of PTX, reducing side effects and costs.

The gut microbiome produces vitamins, nutrients, and neurotransmitters, and helps to modulate the host immune system-and also plays a major role in the metabolism of many exogenous compounds, including drugs and chemical toxicants. However, the extent to which specific microbial species or communities modulate hazard upon exposure to chemicals remains largely opaque. Focusing on the effects of collateral dietary exposure to the widely used herbicide atrazine, we applied integrated omics and phenotypic screening to assess the role of the gut microbiome in modulating host resilience in Drosophila melanogaster. Transcriptional and metabolic responses to these compounds are sex-specific and depend strongly on the presence of the commensal microbiome. Sequencing the genomes of all abundant microbes in the fly gut revealed an enzymatic pathway responsible for atrazine detoxification unique to Acetobacter tropicalis. We find that Acetobacter tropicalis alone, in gnotobiotic animals, is sufficient to rescue increased atrazine toxicity to wild-type, conventionally reared levels. This work points toward the derivation of biotic strategies to improve host resilience to environmental chemical exposures, and illustrates the power of integrative omics to identify pathways responsible for adverse health outcomes.

To improve uptake of influenza vaccine in pregnancy, it is important to understand the factors that predict prenatal vaccination. The aim of this study was to test the capability of the theory of planned behavior, augmented with information constructs, to predict and explain influenza vaccination uptake in a sample of 600 pregnant individuals in Canada. A baseline survey at the start of influenza season assessed beliefs, norms, perceived control, and information-seeking behavior related to influenza vaccination in pregnancy, as well as respondent demographics. A follow-up survey at the conclusion of influenza season assessed self-reported influenza vaccine uptake as well as infant vaccination intentions. Multivariable analysis indicated that attitudes toward influenza vaccination in pregnancy, subjective norms, information seeking, and past vaccination behavior predicted intentions to be vaccinated, and intentions predicted vaccine uptake. Neither perceived control nor demographics were significant predictors of intentions or vaccine uptake. These findings suggest that presumptive offering of vaccination in pregnancy by health care providers, as well as patient and public health educational interventions, may be effective in communicating norms and strengthening positive attitudes and intentions concerning influenza vaccination in pregnancy, resulting in higher vaccine coverage.

Lipid-based carriers of the hydrophobic drug paclitaxel (PTX) are used in clinical trials as next-generation agents for cancer chemotherapy. Improving the loading capacity of these carriers requires enhanced PTX solubilization. We compared the solubility of PTX in cationic liposomes (CLs) with lipid tails containing one (oleoyl; C18:1 Δ 9 ; DOTAP/DOPC) or two (linoleoyl; C18:2 Δ 9 ; DLinTAP/DLinPC) cis double bonds with newly synthesized cationic DLinTAP (2,3-dilinoleoyloxypropyltrimethylammonium methylsufate). We used differential-interference-contrast microscopy to directly observe PTX crystal formation and generate kinetic phase diagrams representing the time-dependence of PTX solubility as a function of PTX content in the membrane. Replacing tails bearing one cis double bond (DO lipids) with those bearing two (DLin lipids) significantly increased PTX membrane solubility in CLs. Remarkably, 8 mol% PTX in DLinTAP/DLinPC CLs remained soluble for approximately as long as 3 mol% PTX (the membrane solubility limit which has been the focus of most previous fundamental studies and clinical trials) in DOTAP/DOPC CLs. The large increase in solubility is likely caused by enhanced molecular affinity between lipid tails and PTX upon replacement of oleoyl by linoleoyl tails, rather than by the transition in membrane structure from lipid bilayers to inverse cylindrical micelles observed in small-angle X-ray scattering. Importantly, the efficacy of PTX-loaded CLs against human prostate cancer (PC3) cells from measurements of the IC50 of PTX cytotoxicity was unaffected by changing the lipid tails, and toxicity of the CL carrier alone was negligible. Moreover, efficacy was approximately doubled against human melanoma (M21) cells for PTX-loaded DLinTAP/DLinPC over DOTAP/DOPC CLs. The findings demonstrate the potential of chemical modifications of the lipid tails to increase the PTX membrane loading well over the typically used 3 mol% while maintaining (and in some cases even increasing) the efficacy of CLs. The increased PTX solubility will aid the development of liposomal PTX carriers that require significantly less lipid to deliver a given amount of PTX, reducing side effects and costs.