Publications

BACKGROUND: Currently, the predictors of readmission after colectomy specifically for ulcerative colitis (UC) are poorly investigated. We sought to determine the rates and predictors of 30-day readmissions after colectomy for UC.

METHODS: Patients undergoing total proctocolectomy and end ileostomy, abdominal colectomy with end ileostomy, proctocolectomy with ileoanal pouch anastomosis (IPAA) formation and diverting ileostomy, one stage IPAA, or abdominal colectomy with ileorectal anastomosis at a tertiary care center between January 2002 and January 2012 for UC were included. Patients were identified using ICD-9 code 556.x. Each record was manually reviewed. The electronic record system was reviewed for demographic information, medical histories, UC history, medications, and data regarding the admission and discharge. Charts were reviewed for readmissions within 30 days of surgery. Univariate and multivariate analyses were performed using Stata v.13.

RESULTS: Two hundred nine patients with UC underwent a colectomy. Forty-three percent had a proctocolectomy with IPAA and diverting ileostomy and 32% had abdominal colectomy with end ileostomy. Seventy-six percent of surgeries were due to failure of medical therapy and 68% of patients were electively admitted for surgery. Thirty-two percent (n = 67/209) of the cohort was unexpectedly readmitted within 30 days. In multivariate model, proctocolectomy with IPAA and diverting ileostomy (odds ratio [OR] = 2.11; 95% CI, 1.06-4.19; P = 0.033) was the only significant predictor of readmission. Hospital length of stay >7 days (OR = 1.82; 95% CI, 0.98-3.41; P = 0.060), presence of limited UC (OR = 2.10; 95% CI, 0.93-4.74; P = 0.074), and steroid before admission (OR = 1.69; 95% CI, 0.90-3.2; P = 0.100) trended toward significance.

CONCLUSIONS: Surgery for UC is associated with a high rate of readmission. Further prospective studies are necessary to determine the means to reduce these readmissions.

BACKGROUND: Dyslipidemia, typically recognized as high serum triglyceride, high low-density lipoprotein cholesterol (LDL-C) or low high-density lipoprotein cholesterol (HDL-C) levels, are associated with nonalcoholic fatty liver disease (NAFLD). However, low LDL-C levels could result from defects in lipoprotein metabolism or impaired liver synthetic function, and may serve as ab initio markers for unrecognized liver diseases. Whether such relationships exist in the general population has not been investigated. We hypothesized that despite common conception that low LDL-C is desirable, it might be associated with elevated liver enzymes due to metabolic liver diseases.

METHODS AND FINDINGS: We examined the associations between alanine aminotransferase (ALT), aspartate aminotransferase (AST) and major components of serum lipid profiles in a nationally representative sample of 23,073 individuals, who had no chronic viral hepatitis and were not taking lipid-lowering medications, from the National Health and Nutrition Examination Survey (NHANES) from 1999 to 2010. ALT and AST exhibited non-linear U-shaped associations with LDL-C and HDL-C, but not with triglyceride. After adjusting for potential confounders, individuals with LDL-C less than 40 and 41-70 mg/dL were associated with 4.2 (95% CI 1.5-11.7, p = 0.007) and 1.6 (95% CI 1.1-2.5, p = 0.03) times higher odds of abnormal liver enzymes respectively, when compared with those with LDL-C values 71-100 mg/dL (reference group). Surprisingly, those with HDL-C levels above 100 mg/dL was associated with 3.2 (95% CI 2.1-5.0, p<0.001) times higher odds of abnormal liver enzymes, compared with HDL-C values of 61-80 mg/dL.

CONCLUSIONS: Both low LDL-C and high HDL-C, often viewed as desirable, were associated with significantly higher odds of elevated transaminases in the general U.S. adult population. Our findings underscore an underestimated biological link between lipoprotein metabolism and liver diseases, and raise a potential need for liver evaluation among over 10 million people with particularly low LDL-C or high HDL-C in the United States.

Apolipoprotein B (apoB) is the principal protein component of triacylglyceride (TAG)-rich lipoproteins, including chylomicrons and very low density lipoprotein, which is the precursor to LDL (the "bad cholesterol"). TAG-rich lipoprotein assembly is initiated by the N-terminal βα1 superdomain of apoB, which co-translationally binds and remodels the luminal leaflet of the rough endoplasmic reticulum. The βα1 superdomain contains four domains and is predicted to interact directly with lipids. Using drop tensiometry, we examined the interfacial properties of the α-helical and C-sheet domains and several subdomains to establish a detailed structure-function relationship at the lipid/water interface. The adsorption, stress response, exchangeability, and pressure (Π)-area relationship were studied at both triolein/water and triolein/1-palmitoyl, 2-oleoylphosphatidylcholine/water interfaces that mimic physiological environments. The α-helical domain spontaneously adsorbed to a triolein/water interface and formed a viscoelastic surface. It was anchored to the surface by helix 6, and the other helices were ejected and/or remodeled on the surface as a function of surface pressure. The C-sheet instead formed an elastic film on a triolein/water interface and was irreversibly anchored to the lipid surface, which is consistent with the behavior of amphipathic β-strands. When both domains were adsorbed together on the surface, the C-sheet shielded a portion of the α-helical domain from the surface, which retained its globular structure. Overall, the unique secondary and tertiary structures of the N-terminal domains of apoB support the intrinsic capability of co-translational lipid recruitment. The evidence presented here allows the construction of a detailed model of the initiation of TAG-rich lipoprotein assembly.

Phosphohydrolysis of extracellular ATP and ADP is an essential step in purinergic signaling that regulates key pathophysiological processes, such as those linked to inflammation. Classically, this reaction has been known to occur in the pericellular milieu catalyzed by membrane bound cellular ecto-nucleotidases, which can be released in the form of both soluble ecto-enzymes as well as being associated with exosomes. Circulating ecto-nucleoside triphosphate diphosphohydrolase 1 (NTPDase 1/CD39) and adenylate kinase 1 (AK1) activities have been shown to be present in plasma. However, other ecto-nucleotidases have not been characterized in depth. An in vitro ADPase assay was developed to probe the ecto-enzymes responsible for the ecto-nucleotidase activity in human platelet-free plasma, in combination with various specific biochemical inhibitors. Identities of ecto-nucleotidases were further characterized by chromatography, immunoblotting, and flow cytometry of circulating exosomes. We noted that microparticle-bound E-NTPDases and soluble AK1 constitute the highest levels of ecto-nucleotidase activity in human plasma. All four cell membrane expressed E-NTPDases are also found in circulating microparticles in human plasma, inclusive of: CD39, NTPDase 2 (CD39L1), NTPDase 3 (CD39L3), and NTPDase 8. CD39 family members and other ecto-nucleotidases are found on distinct microparticle populations. A significant proportion of the microparticle-associated ecto-nucleotidase activity is sensitive to POM6, inferring the presence of NTPDases, either -2 or/and -3. We have refined ADPase assays of human plasma from healthy volunteers and have found that CD39, NTPDases 2, 3, and 8 to be associated with circulating microparticles, whereas soluble AK1 is present in human plasma. These ecto-enzymes constitute the bulk circulating ADPase activity, suggesting a broader implication of CD39 family and other ecto-enzymes in the regulation of extracellular nucleotide metabolism.

Nonalcoholic fatty liver disease (NAFLD), an escalating health problem worldwide, covers a spectrum of pathologies characterized by fatty accumulation in hepatocytes in early stages, with potential progression to liver inflammation, fibrosis, and failure. A close, yet poorly understood link exists between NAFLD and dyslipidemia, a constellation of abnormalities in plasma lipoproteins including triglyceride-rich very low density lipoproteins. Apolipoproteins are a group of primarily liver-derived proteins found in serum lipoproteins; they not only play an extracellular role in lipid transport between vital organs through circulation, but also play an important intracellular role in hepatic lipoprotein assembly and secretion. The liver functions as the central hub for lipoprotein metabolism, as it dictates lipoprotein production and to a significant extent modulates lipoprotein clearance. Lipoprotein metabolism is an integral component of hepatocellular lipid homeostasis and is implicated in the pathogenesis, potential diagnosis, and treatment of NAFLD.

The N-terminal sequence of apolipoprotein B (apoB) is critical in triacylglycerol-rich lipoprotein assembly. The first 17% of apoB (B17) is thought to consist of three domains: B5.9, a beta-barrel, B6.4-13, a series of 17 alpha-helices, and B13-17, a putative beta-sheet. B5.9 does not bind to lipid, while B6.4-13 and B13-17 contain hydrophobic interfaces that can interact with lipids. To understand how B6.4-13 and B13-17 might interact with triacylglycerol during lipoprotein assembly, the interfacial properties of both peptides were studied at the triolein/water interface. Both B6.4-13 and B13-17 are surface active. Once bound, the peptides can be neither exchanged nor pushed off the interface. Some residues of the peptides can be ejected from the interface upon compression but readsorb on expansion. B13-17 binds to the interface more strongly. The maximum pressure the peptide can withstand without being partially ejected (Pi(max)) is 19.2 mN/m for B13-17 compared to 16.7 mN/m for B6.4-13. B13-17 is purely elastic at the interface, while B6.4-13 forms a viscous-elastic film. When they are spread at an air/water interface, the limiting area and the collapse pressures are 16.6 A(2)/amino acid and 31 mN/m for B6.4-13 and 17.8 A(2)/amino acid and 35 mN/m for B13-17, respectively. The alpha-helical B6.4-13 contains some hydrophobic helices that stay bound and prevent the peptide from leaving the surface. The beta-sheets of B13-17 bind irreversibly to the surface. We suggest that during lipoprotein assembly, the N-terminal apoB starts recruiting lipid as early as B6.4, but additional sequences are essential for formation of a lipid pocket that can stabilize lipoprotein emulsion particles for secretion.

We have shown that expression of apolipoprotein (apo) C-III promotes VLDL secretion from transfected McA-RH7777 cells under lipid-rich conditions. To determine structural elements within apoC-III that confer to this function, we contrasted wild-type apoC-III with a mutant Ala23Thr originally identified in hypotriglyceridemia subjects. Although synthesis of [(3)H]glycerol-labeled TAG was comparable between cells expressing wild-type apoC-III (C3wt cells) or Ala23Thr mutant (C3AT cells), secretion of [(3)H]TAG from C3AT cells was markedly decreased. The lowered [(3)H]TAG secretion was associated with an inability of C3AT cells to assemble VLDL(1). Moreover, [(3)H]TAG within the microsomal lumen in C3AT cells was 60% higher than that in C3wt cells, yet the activity of microsomal triglyceride-transfer protein in C3AT cells was not elevated. The accumulated [(3)H]TAG in C3AT microsomal lumen was mainly associated with lumenal IDL/LDL-like lipoproteins. Phenotypically, this [(3)H]TAG fractionation profiling resembled what was observed in cells treated with brefeldin A, which at low dose specifically blocked the second-step VLDL(1) maturation. Furthermore, lumenal [(35)S]Ala23Thr protein accumulated in IDL/LDL fractions and was absent in VLDL fractions in C3AT cells. These results suggest that the presence of Ala23Thr protein in lumenal IDL/LDL particles might prevent effective fusion between lipid droplets and VLDL precursors. Thus, the current study reveals an important structural element residing within the N-terminal region of apoC-III that governs the second step VLDL(1) maturation.

ApolipoproteinB (ApoB) is a lipid binding protein that is a nonexchangeable component of chylomicrons, VLDL, and LDL. In the liver and intestinal cells ApoB recruits lipid to form nascent triacylglycerol rich particles cotranslationally in the endoplasmic reticulum membrane which are then processed and secreted to form plasma lipoproteins. The N-terminal domain, which comprises the first 22% of apoB, recruits lipid in a controlled manner. The first 6% (residues 1-291) of the N-terminus does not bind lipid. The first lipid binding domain, including residues 292-782 (B6-17), forms a lipid binding pocket which is predicted to consist of 17 alpha-helices and 6 beta-strands. A structural model based on the X-ray structure of the homologues protein lipovitellin suggests that the N-terminal 6-8 helices and the beta-sheet interact with lipid while the C-terminal helices form a structural unit stabilizing the beta-sheet. Using isothermal drop tensiometry we showed that ApoB6.4-17 is surface active and binds to a triolein/water interface and exerts 16-19 mN/m of pressure (Pi) on that surface. The protein initially adsorbs slowly from aqueous solution to the surface but following compression and re-expansion it reaches equilibrium much faster. When Pi exceeds 16.9 mN/m part of the protein is ejected from the surface, but when compressed to high Pi the protein is never completely ejected indicating that part of the peptide is irreversibly anchored to the interface. The surface dilation modulus (epsilon) varies between 25-38 mN/m, and is predominantly elastic with a small viscous component. When compressed at an air/water interface ApoB6.4-17 has a limiting area of approximately 11 A2 per amino acid at lift off and only approximately 7 A2 per amino acid at the collapse Pi (28 mN/m). These values are about half the anticipated values if all the residues are at the surface. This suggests that ApoB6.4-17 retains some globular structure at an interface and does not completely denature at the surface, as many other globular proteins do. We suggest that while bound to the surface ApoB6.4-17 exhibits properties of both alpha and beta structure giving it unique and versatile characteristics at a hydrophobic interface.

Dematin is an actin-binding protein originally identified in the junctional complex of the erythrocyte plasma membrane, and is present in many nonerythroid cells. Dematin headpiece knockout mice display a spherical red cell phenotype and develop a compensated anemia. Dematin has two domains: a 315-residue, proline-rich "core" domain and a 68-residue carboxyl-terminal villin-type "headpiece" domain. Expression of full-length dematin in E. coli as a GST recombinant protein results in truncation within a proline, glutamic acid, serine, threonine rich region (PEST). Therefore, we designed a mutant construct that replaces the PEST sequence. The modified dematin has high actin binding activity as determined by actin sedimentation assays. Negative stain electron microscopy demonstrates that the modified dematin also exhibits actin bundling activity like that of native dematin. Circular dichroism (CD) and NMR spectral analysis, however, show little secondary structure in the modified dematin. The lack of secondary structure is also observed in native dematin purified from human red blood cells. (15)N-HSQC NMR spectra of modified dematin indicate that the headpiece domain is fully folded whereas the core region is primarily unfolded. Our finding suggests that the core is natively unfolded and may serve as a scaffold to organize the components of the junctional complex.

The synthesis of apolipoprotein B (apoB) dictates the formation of chylomicrons and very low-density lipoproteins, two major lipoprotein precursors in the human plasma. Despite its biological significance, the mechanism of the assembly of these apoB-containing lipoproteins remains elusive. An essential obstacle is the lack of systems that allow fine dissection of key components during assembly, including nascent apoB peptide, lipids in defined forms, chaperones, and microsomal triglyceride transfer protein (MTP). In this study, we used a prokaryotic cell-free expression system to reconstitute early events in the assembly of apoB-containing lipoprotein that involve the N-terminal domains of apoB. Our study shows that N-terminal domains larger than 20.5% of apoB (B20.5) have an intrinsic ability to remodel vesicular phospholipid bilayers into discrete protein-lipid complexes. The presence of appropriate lipid substrates during apoB translation plays a pivotal role for successful lipid recruitment, and similar lipid recruitment fails to occur if the lipids are added posttranslationally. Cotranslational presence of MTP can dramatically promote the folding of B6.4-20.5 and B6.4-22. Furthermore, apoB translated in the presence of MTP retains its phospholipid recruitment capability posttranslationally. Our data suggest that during the synthesis of apoB, the N-terminal domain has a short window for intrinsic phospholipid recruitment, the time frame of which is predetermined by the environment where apoB synthesis occurs. The presence of MTP prolongs this window of time by acting as a chaperone. The absence of either proper lipid substrate or MTP may result in the improper folding of apoB and, consequently, its degradation.