UC San Diego

The tympanic membrane (TM) forms an impenetrable barrier to medical therapies for middle ear (ME) diseases like otitis media. By screening a phage-displayed peptide library, we have previously discovered rare peptides that mediate the active transport of cargo across the intact membrane of animals and humans. Since the M13 filamentous bacteriophage on which the peptides are expressed are large (nearly 1 µm in length), this offers the possibility of noninvasively delivering drugs, large drug packages, or gene therapy to the ME. To evaluate this possibility, EDC chemistry was employed to covalently attach amoxicillin, or neomycin molecules to phage bearing a trans-TM peptide, as a model for large drug packages. Eight hours after application of antibiotic-phage to the TM of infected rats, ME bacterial titers were substantially reduced compared to untreated animals. As a control, antibiotic was linked to wild-type phage, not bearing any peptide, and application to the TM did not affect ME bacteria. The results support the ability of rare peptides to actively deliver pharmacologically relevant amounts of drugs through the intact TM and into the ME. Moreover, since bacteriophage engineered to express peptides are viral vectors, the trans-TM peptides could also transport other viral vectors into the ME.

Obstructive sleep apnea (OSA) is characterized by intermittent hypoxia/hypercapnia (IHC), affects predominantly obese individuals, and increases atherosclerosis risk. Since we and others have implicated gut microbiota and metabolites in atherogenesis, we dissected their contributions to OSA-induced atherosclerosis. Atherosclerotic lesions were compared between conventionally-reared specific pathogen free (SPF) and germ-free (GF) Apoe-/- mice following a high fat high cholesterol diet (HFHC), with and without IHC conditions. The fecal microbiota and metabolome were profiled using 16S rRNA gene amplicon sequencing and untargeted tandem mass spectrometry (LC-MS/MS) respectively. Phenotypic data showed that HFHC significantly increased atherosclerosis as compared to regular chow (RC) in both aorta and pulmonary artery (PA) of SPF mice. IHC exacerbated lesions in addition to HFHC. Differential abundance analysis of gut microbiota identified an enrichment of Akkermansiaceae and a depletion of Muribaculaceae (formerly S24-7) family members in the HFHC-IHC group. LC-MS/MS showed a dysregulation of bile acid profiles with taurocholic acid, taurodeoxycholic acid, and 12-ketodeoxycholic acid enriched in the HFHC-IHC group, long-chain N-acyl amides, and phosphatidylcholines. Interestingly, GF Apoe-/- mice markedly reduced atherosclerotic formation relative to SPF Apoe-/- mice in the aorta under HFHC/IHC conditions. In contrast, microbial colonization did not show a significant impact on the atherosclerotic progression in PA. In summary, this research demonstrated that (1) IHC acts cooperatively with HFHC to induce atherosclerosis; (2) gut microbiota modulate atherogenesis, induced by HFHC/IHC, in the aorta not in PA; (3) different analytical methods suggest that a specific imbalance between Akkermansiaceae and Muribaculaceae bacterial families mediate OSA-induced atherosclerosis; and (4) derived bile acids, such as deoxycholic acid and lithocholic acid, regulate atherosclerosis in OSA. The knowledge obtained provides novel insights into the potential therapeutic approaches to prevent and treat OSA-induced atherosclerosis.

Non-coding RNAs (ncRNAs) are finely tuned cellular regulators important for human cell growth and cancer progression. DUBR (Dppa2 upstream binding RNA, also known as linc00883) is a nuclear ncRNA first discovered in mice for its role in regulating myoblast differentiation through interactions with chromatin and DNA methyltransferases. High expression levels of this ncRNA are predictive of poor patient outcome in colon adenocarcinoma, suggesting that DUBR may be involved in controlling cancer growth. To elucidate its function, we used RAP-MS and RNA immunoprecipitation techniques which revealed its interaction with epigenetic maintenance proteins in the human colon cancer cell line HCT116. Further, ATAC-seq and RNA-seq were used to address its function in regulating the epigenome and transcriptome of HCT116 cells. Here we report that DUBR is a regulator of human colon cancer cell line HCT116 survival. Additionally, we find that the ncRNA DUBR regulates AP-1 transcription factor binding site accessibility at enhancers of genes involved in differentiation and morphogenesis through interactions with epigenetic proteins such as NuRD complex members HDAC1 and CHD4.

Monocytes infiltrating tumors acquire various states that distinctly impact cancer treatment. Here, we show that resistance of tumors to radiotherapy (RT) is controlled by the accumulation of monocyte-derived dendritic cells (moDCs). These moDCs are characterized by the expression of CD301b and have a superior capacity to generate regulatory T cells (Tregs). Accordingly, moDC depletion limits Treg generation and improves the therapeutic outcome of RT. Mechanistically, we demonstrate that granulocyte-macrophage colony-stimulating factor (GM-CSF) derived from radioresistant tumor cells following RT is necessary for the accumulation of moDCs. Our results unravel the immunosuppressive function of moDCs and identify GM-CSF as an immunotherapeutic target during RT.

The burden of cirrhosis and chronic liver disease is growing, yet there is a projected worsening deficit in hepatology providers. As such, cirrhosis and liver disease have been important inclusions within the core curricula of Internal Medicine. Formal assessments of provider preparedness resulting from the curriculum are lacking though. Prior studies have demonstrated that exposure to cirrhosis in undergraduate medical education is insufficient, as are learner comfort and self-reported knowledge levels. These findings are further corroborated by a multicenter survey of incoming Internal Medicine interns showing that subjective comfort with and objective knowledge of various liver disease topics are lacking compared to other common Internal Medicine topics. This paper also demonstrates how similar surveys may be used to identify additional topics that may require adjustments for curricular improvement.

RATIONALE: Stable isotope analysis of growth layer groups (GLGs) in mammal dentin is an increasingly prevalent and noninvasive approach to study animal foraging ecology. However, empirical evidence to support assumed proper methodologies for sampling GLGs is lacking. Here, we examine the effects of intratooth and intertooth variations with respect to targeted GLGs, as well as the effects of common pretreatments (e.g., formic acid and graphite) to enhance GLG visibility, on stable isotope values (δ13C and δ15N) from dentin. METHODS: We measured the δ13C and δ15N values of killer whale (Orcinus orca) dentin. We used dentin from 37 teeth to compare stable carbon (δ13C) and nitrogen (δ15N) isotope values from multiple locations within a GLG (intratooth variation), from corresponding GLGs among teeth of an individual (intertooth variation), and from treated and untreated teeth. RESULTS: We observed no significant differences in the δ13C or δ15N values when sampling a single GLG from multiple locations (intratooth variation) or when comparing the same GLG across duplicate teeth of individuals (intertooth variation). One tooth in a triplicate set showed a significantly different but likely biologically inconsequential δ13C value. Lastly, formic acid and graphite highlighting to accentuate GLGs did not significantly influence measured stable isotope values. CONCLUSIONS: We validate several previous assumptions in this field of study. First, dentin samples for stable isotope analysis can be sampled from different locations across a GLG. Second, researchers can compare stable isotope values from the same GLGs of different teeth collected from the same individual in most cases, as the δ13C and δ15N values did not vary with the sampled tooth. Third, a common protocol of formic acid and graphite treatment to enhance GLG visibility does not bias the δ13C and δ15N values from dentin. We also describe factors to consider and cautions associated with these conclusions.

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