CCAAT/enhancer-binding protein beta (C/EBPß) knockdown reduces inflammation, ER stress, and apoptosis, and promotes autophagy in oxLDL-treated RAW264.7 macrophage cells.

Atherosclerosis is associated with deregulated cholesterol metabolism and formation of macrophage foam cells. CCAAT/enhancer-binding protein beta (C/EBPß) is a transcription factor, and its inhibition has recently been shown to prevent atherosclerosis development and foam cell formation. However, whether C/EBPß regulates inflammation, endoplasmic reticulum (ER) stress, and apoptosis, in macrophage foam cells and its underlying molecular mechanism remains unknown. Here, we investigated the effect of C/EBPß knockdown on proteins and genes implicated in inflammation, ER stress, apoptosis, and autophagy in macrophage foam cells. RAW264.7 macrophage cells were transfected with control and C/EBPß-siRNA and then treated with nLDL and oxLDL. Key proteins and genes involved in inflammation, ER stress, apoptosis, and autophagy were analyzed by western blot and qPCR. We found that short interfering RNA (siRNA)-mediated knockdown of C/EBPß attenuated atherogenic lipid-mediated induction of proteins and genes implicated in inflammation (P-NFkB-p65, NFkB-p65, and TNFα), ER stress (ATF4 and ATF6), and apoptosis (CHOP, caspase 1, 3, and 12). Interestingly, C/EBPß knockdown upregulated the expression of autophagy proteins (LC3A/B-II, ATG5) and genes (LC3B, ATG5) but decreased the mammalian target of rapamycin (mTOR) protein phosphorylation and mTORC1 gene expression in oxLDL-loaded RAW264.7 macrophage cells. More importantly, treatment with rapamycin (inhibitor of mTOR) increased expression of proteins implicated in autophagy and cholesterol efflux in oxLDL-loaded RAW 264.7 macrophage cells. The present results suggest that C/EBPß inactivation regulates macrophage foam cell formation in atherogenesis by reducing inflammation, ER stress, and apoptosis and by promoting autophagy and inactivating mTOR.

VD3 and LXR agonist (T0901317) combination demonstrated greater potency in inhibiting cholesterol accumulation and inducing apoptosis via ABCA1-CHOP-BCL-2 cascade in MCF-7 breast cancer cells.

Obesity is associated with hypercholesterolemia and is a global epidemic. Epidemiological and animal studies revealed cholesterol is an essential regulator of estrogen receptor positive (ER+) breast cancer progression while inhibition of cholesterol accumulation was found to prevent breast tumor growth. Individually, vitamin D and LXR agonist T0901317 showed anticancer properties. The present study investigated the effects of vitamin D3 (VD3, calcitriol), LXR agonist (T0901317) and a combination of VD3 + T0901317 on cholesterol metabolism and cancer progression in ER+ breast cancer (MCF-7) cells. VD3 or T0901317 alone reduced cholesterol accumulation significantly in MCF-7 cells concomitant with an induction of ABCA1 protein and gene expression compared to the control treatment. Most importantly, VD3 + T0901317 combination showed higher effects in reducing cholesterol levels and increasing ABCA1 protein and gene expression compared to individual treatments. Importantly, VD3 + T0901317 combination showed higher effects in increasing apoptosis as measured by annexin apoptosis assay, cell viability and was associated with induction of CHOP protein and gene expression. Additionally, the VD3 + T0901317 exerted higher effects in reducing antiapoptotic BCL-2 while increased pro-apoptotic BAX gene expression compared to the individual treatments. The present results suggest that VD3 and T0901317 combination may have an important therapeutic application to prevent obesity and hyperlipidemia mediated ER+ breast cancer progression.

Predictors of resolution and persistence of renal laboratory abnormalities in pediatric HIV infection.

BACKGROUND: Among human immunodeficiency virus (HIV)-infected youth, the role of renal disease (RD) and its management has become increasingly important as these children/adolescents mature into young adults. The identification of predictors of abnormal renal laboratory events (RLE) may be helpful in the management of their HIV infection and its associated renal complications. METHODS: Data collected from HIV-infected youth followed for ≥ 48 months were analyzed to identify predictors of resolution versus persistence of RLE and determine the utility of RLE to predict the onset of RD. Analysis included descriptive and inferential methods using a multivariable extended Cox proportional hazards model. RESULTS: Of the 1,874 at-risk children enrolled in the study, 428 (23 %) developed RLE, which persisted in 229 of these (54 %). CD4 percentages of <25 % [hazard ratio (HR) 0.63, p < 0.002) and an HIV viral load of >100,000 copies/ml (HR 0.31, p < 0.01) were associated with reduced rates of resolution, while in most cases exposure to highly active antiretroviral therapy (HAART)/nephrotoxic HAART prior to or subsequent to RLE were not. Persistence of RLE was 88 % sensitive for identifying new RD. Negative predictive values for RD were >95 % for both the at-risk cohort and those with RLE. CONCLUSIONS: Advanced HIV disease predicted persistence of RLE in HIV-infected youth. Persistent RLE were useful for identifying RD.

Solvent effects on the wood delignification with sustainable solvents.

Solutions of a pure organic solvent acidified with 1% sulfuric acid, and some of their aqueous mixtures were used for lignin extraction in the Pinus radiata sawdust delignification. Organic acid solvents including acetic, lactic and citric acids as well as non-acidic compounds such as γ-valerolactone, 2-methyltetrahydrofuran, glycerol and ethylene glycol were studied. Crude lignin extractions yields (%) ranging between ca. 5-50% were obtained, from which ethylene glycol (33%), γ-valerolactone (48%) and propylene carbonate (52%) showed the greatest effectiveness. The effect of added water on the lignin extraction was investigated in mixtures of an organic solvent with a variable water content (75%w/w, 50% w/w, 25%w/w and 10% w/w) where it was observed that the yield of extraction decreased with the increased water content. Moreover, the purity of extracted lignins were analyzed by spectroscopic methods (UV and IR). Kamlet-Taft solvent polarity parameters, were determined with the solvatochromic probes 4-nitroaniline, N,N-diethyl-4-nitroaniline, Nile Red and 6-propionyl-2-N,N-dimethylaminonaphthalene (PRODAN), and then correlated to lignin extraction yields to explain the influence of the solute-solvent interactions on biomass delignification. A reasonable correlation was found between the medium polarizability-dipolarity π* and the effectiveness of the solvent mixture on the extraction of lignin wood.

Semisynthetic Glycoconjugate Vaccine Candidates against Escherichia coli O25B Induce Functional IgG Antibodies in Mice.

Extraintestinal pathogenic Escherichia coli (ExPEC) is a major health concern due to emerging antibiotic resistance. Along with O1A, O2, and O6A, E. coli O25B is a major serotype within the ExPEC group, which expresses a unique O-antigen. Clinical studies with a glycoconjugate vaccine of the above-mentioned O-types revealed O25B as the least immunogenic component, inducing relatively weak IgG titers. To evaluate the immunological properties of semisynthetic glycoconjugate vaccine candidates against E. coli O25B, we here report the chemical synthesis of an initial set of five O25B glycan antigens differing in length, from one to three repeat units, and frameshifts of the repeat unit. The oligosaccharide antigens were conjugated to the carrier protein CRM197. The resulting semisynthetic glycoconjugates induced functional IgG antibodies in mice with opsonophagocytic activity against E. coli O25B. Three of the oligosaccharide-CRM197 conjugates elicited functional IgGs in the same order of magnitude as a conventional CRM197 glycoconjugate prepared with native O25B O-antigen and therefore represent promising vaccine candidates for further investigation. Binding studies with two monoclonal antibodies (mAbs) revealed nanomolar anti-O25B IgG responses with nanomolar K D values and with varying binding epitopes. The immunogenicity and mAb binding data now allow for the rational design of additional synthetic antigens for future preclinical studies, with expected further improvements in the functional antibody responses. Moreover, acetylation of a rhamnose residue was shown to be likely dispensable for immunogenicity, as a deacylated antigen was able to elicit strong functional IgG responses. Our findings strongly support the feasibility of a semisynthetic glycoconjugate vaccine against E. coli O25B.

A C-RING-like domain participates in protein self-assembly and mineral nucleation.

AP7 is a nacre-associated protein of the mollusk shell that forms supramolecular assemblies that nucleate single-crystal aragonite in vitro. AP7 possesses two major sequence regions: a random coil 30-amino acid N-terminal domain (AP7N) and a partially disordered 36-amino acid C-terminal domain (AP7C) that exhibits imperfect sequence homology to the C subclass of the intracellular RING domain family. We report here new findings that implicate the C-RING domain in AP7-mediated supramolecular assembly and single-crystal mineral formation. AP7 protein spontaneously self-assembles over a pH range of 4-9 and is monomeric at pH >9.5. AP7N and AP7C both oligomerize over the pH range of 4-9, with the AP7C sequence closely resembling AP7 in terms of particle morphology and size. In vitro mineralization experiments demonstrate that both AP7N and AP7C form supramolecular assemblies that nucleate single-crystal calcium carbonates. Comparison of previously published nuclear magnetic resonance-based structures of AP7C and AP7N reveals the significant presence of complementary anionic-cationic electrostatic molecular surfaces on AP7C that are not found on AP7N, and this may explain the noted discrepancies between the two domains in terms of self-assembly and single-crystal nucleation. We conclude that the C-RING-like sequence is an important site for AP7 self-association and mineral nucleation, and this represents the first known instance of a RING-like sequence performing these functions within an extracellular protein.

Formation of framework nacre polypeptide supramolecular assemblies that nucleate polymorphs.

The formation of aragonite in the mollusk shell nacre layer is linked to the assembly of framework protein complexes that interact with ß-chitin polysaccharide. What is not yet understood is how framework nacre proteins control crystal growth. Recently, a 30 AA intrinsically disordered nacre protein sequence (n16N) derived from the n16 framework nacre protein was found to form aragonite, vaterite, or ACC deposits when adsorbed onto ß-chitin. Our present study now establishes that n16N assembles to form amorphous nonmineralized supramolecular complexes that nucleate calcium carbonate polymorphs in vitro. These complexes contain unfolded or disordered (54% random coil, 46% ß structures) n16N polypeptide chains that self-assemble in response to alkaline pH shift. The pH-dependent assembly process involves two stages, and it is likely that side chain salt-bridging interactions are a major driving force in n16N self-association. Intriguingly, Ca(II) ions are not required for n16N assembly but do shift the assembly process to higher pH values, and it is likely that Ca(II) plays some role in stabilizing the monomeric form of n16N. Using preassembled fibril-spheroid n16N assemblies on Si wafers or polystyrene supports, we were able to preferentially nucleate vaterite at higher incidence compared to control scenarios, and it is clear that the n16N assemblies are in contact with the nucleating crystals. We conclude that the framework nacre protein sequence n16N assembles to form supramolecular complexes whose surfaces act as nucleation sites for crystal growth. This may represent a general mineralization mechanism employed by framework nacre proteins in general.

Intrinsically disordered mollusk shell prismatic protein that modulates calcium carbonate crystal growth.

The formation of calcite prism architecture in the prismatic layer of the mollusk shell involves the participation of a number of different proteins. One protein family, Asprich, has been identified as a participant in amorphous calcium carbonate stabilization and calcite architecture in the prismatic layer of the mollusk, Atrina rigida . However, the functional role(s) of this protein family are not fully understood due to the fact that insufficient quantities of these proteins are available for experimentation. To overcome this problem, we employed stepwise solid-phase synthesis to recreate one of the 10 members of the Asprich family, the 61 AA single chain protein, Asprich "3". We find that the Asprich "3" protein inhibits the formation of rhombohedral calcite crystals and induces the formation of round calcium carbonate deposits in vitro that contain calcite and amorphous calcium carbonate (ACC). This mineralization behavior does not occur under control conditions, and the formation of ACC and calcite is similar to that reported for the recombinant form of the Asprich "g" protein. Circular dichroism studies reveal that Asprich "3" is an intrinsically disordered protein, predominantly random coil (66%), with 20-30% ß-strand content, a small percentage of ß-turn, and little if any α-helical content. This protein is not extrinsically stabilized by Ca(II) ions but can be stabilized by 2,2,2-trifluoroethanol to form a structure consisting of turn-like and random coil characteristics. This finding suggests that Asprich "3" may require other extrinsic interactions (i.e., with mineral or ionic clusters or other macromolecules) to achieve folding. In conclusion, Asprich "3" possesses in vitro functional and structural qualities that are similar to other reported for other Asprich protein sequences.

Point-of-care ultrasound: Closing guideline gaps in screening for valvular heart disease.

BACKGROUND: A linear increase in the number of valvular heart disease is expected due to the aging population, yet most patients with severe valvular heart disease remain undiagnosed. HYPOTHESIS: POCUS can serve as a screening tool for valvular heart disease. METHODS: We reviewed the literature to assess the strengths and limitations of POCUS in screening and diagnosing valvular heart disease. RESULTS: POCUS is an accurate, affordable, accessible, and comprehensive tool. It has a fast learning curve and can prevent unnecessary and more expensive imaging. Challenges include training availability, lack of simplified screening protocols, and reimbursement. Large scale valvular screening data utilizing POCUS is not available. CONCLUSION: POCUS can serve as a screening tool and guide the management of patients with valvular heart disease. More data is needed about its efficacy and cost-effectiveness in the screening of patients with valvular heart disease.

The contribution of cholesterol and epigenetic changes to the pathophysiology of breast cancer.

Breast cancer​ is one of the most commonly diagnosed cancers in women. Accumulating evidence suggests that cholesterol plays an important role in the development of breast cancer. Even though the mechanistic link between these two factors is not well understood, one possibility is that dysregulated cholesterol metabolism may affect lipid raft and membrane fluidity and can promote tumor development. Current studies have shown oxysterol 27-hydroxycholesterol (27-HC) as a critical regulator of cholesterol and breast cancer pathogenesis. This is supported by the significantly higher expression of CYP27A1 (cytochrome P450, family 27, subfamily A, polypeptide 1) in breast cancers. This enzyme is responsible for 27-HC synthesis from cholesterol. It has been shown that 27-HC can not only increase the proliferation of estrogen receptor (ER)-positive breast cancer cells but also stimulate tumor growth and metastasis in several breast cancer models. This phenomenon is surprising since 27-HC and other oxysterols generally reduce intracellular cholesterol levels by activating the liver X receptors (LXRs). Resolving this paradox will elucidate molecular pathways by which cholesterol, ER, and LXR are connected to breast cancer. These findings will also provide the rationale for evaluating pharmaceutical approaches that manipulate cholesterol or 27-HC synthesis in order to mitigate the impact of cholesterol on breast cancer pathophysiology. In addition to cholesterol, epigenetic changes including non-coding RNAs, and microRNAs, DNA methylation, and histone modifications, have all been shown to control tumorigenesis. The purpose of this review is to discuss the link between altered cholesterol metabolism and epigenetic modification during breast cancer progression.

Assessing Emotional Suffering in Palliative Care: Use of a Structured Note Template to Improve Documentation.

CONTEXT: Documentation of the emotional or psychological needs of seriously ill patients receiving specialty palliative care is endorsed by the "Measuring What Matters" project as a quality performance metric and recommended for use by hospice and palliative care programs for program improvement. OBJECTIVES: The aim of this study was to increase the proportion of inpatient palliative care team encounters in which emotional or psychological needs of patients and family members were documented and to qualitatively enrich the nature of this documentation. METHODS: This is a mixed-methods retrospective study of 200 patient charts reviewed before and after implementation of a structured note template (SmartPhrase) for palliative care encounters. Patterns of documentation of emotional needs pre- and post-implementation were assessed quantitatively and qualitatively using thematic analysis. RESULTS: A total of 158 of 200 pre-intervention charts and 185 of 200 post-intervention charts included at least one note from the palliative care team. Documentation of emotional assessment increased after SmartPhrase implementation (63.9% [101 of 158] vs. 74.6% [138 of 185]; P < 0.03). Qualitative analysis revealed a post-intervention reduction in the use of generic phrases ("emotional support provided") and an increase in the breadth and depth of emotion-related documentation. CONCLUSION: A structured note template with a prompt for emotional assessment increases the overall quantity and richness of documentation related to patient and family emotions. However, this documentation remains mostly descriptive. Additional prompting for documentation of recommendations to address identified emotional needs, and the use of screening tools for depression and anxiety, when appropriate, may be necessary for clinically meaningful quality improvements in patient care.