In-hospital and 90-day outcomes after total pancreatectomy with islet autotransplantation for pediatric chronic and acute recurrent pancreatitis.

Total pancreatectomy with islet autotransplantation (TPIAT) is used to treat debilitating chronic pancreatitis (CP) and acute recurrent pancreatitis (ARP) that has failed medical and endoscopic therapy. We performed a retrospective review of TPIAT patients at a free-standing children's hospital to evaluate perioperative outcomes. Twenty patients (median age 13, 65% female) underwent TPIAT (2015 through 2017). Of the 20 patients, 95% had CP and 1 patient (5%) had ARP alone. Seventy-five percent of the patients had a pancreatitis-associated genetic mutation; 40% had pancreas divisum. The median surgical time was 757 (IQR 657 to 835) minutes. Median islet equivalents per kg of body weight (IEQ/kg) were 6404 (IQR 5018 to 7554). At 90 days postoperatively vs preoperatively, significantly fewer patients were receiving parenteral nutrition (0% vs 25%, P = .006) and opioids (45% vs 75%, P = .01). Short Form 36-Item Health Survey (SF-36) physical health module scores and total scores improved (34.0 preoperatively vs 54.6 at 90 days, P = .008, and 47.1 vs 65.3, P = .007, respectively); SF-10 physical health scores also improved (13.4 vs 33.1, P = .02). Insulin requirement decreased from 0.5 unit/kg/day to 0.4 unit/kg/day between discharge and 90 days (P = .02). TPIAT is an effective option when debilitating disease persists despite maximal medical and endoscopic therapy. Opioid, parenteral nutrition, and exogenous insulin use can successfully be weaned within 90 days after TPIAT, with gains in health-related quality of life.

Differential eNOS-signalling by platelet subpopulations regulates adhesion and aggregation.

AIMS: In addition to maintaining haemostasis, circulating blood platelets are the cellular culprits that form occlusive thrombi in arteries and veins. Compared to blood leucocytes, which exist as functionally distinct subtypes, platelets are considered to be relatively simple cell fragments that form vascular system plugs without a differentially regulated cellular response. Hence, investigation into platelet subpopulations with distinct functional roles in haemostasis/thrombosis has been limited. In our present study, we investigated whether functionally distinct platelet subpopulations exist based on their ability to generate and respond to nitric oxide (NO), an endogenous platelet inhibitor. METHODS AND RESULTS: Utilizing highly sensitive and selective flow cytometry protocols, we demonstrate that human platelet subpopulations exist based on the presence and absence of endothelial nitric oxide synthase (eNOS). Platelets lacking eNOS (approximately 20% of total platelets) fail to produce NO and have a down-regulated soluble guanylate cyclase-protein kinase G (sGC-PKG)-signalling pathway. In flow chamber and aggregation experiments eNOS-negative platelets primarily initiate adhesion to collagen, more readily activate integrin αIIbß3 and secrete matrix metalloproteinase-2, and form larger aggregates than their eNOS-positive counterparts. Conversely, platelets having an intact eNOS-sGC-PKG-signalling pathway (approximately 80% of total platelets) form the bulk of an aggregate via increased thromboxane synthesis and ultimately limit its size via NO generation. CONCLUSION: These findings reveal previously unrecognized characteristics and complexity of platelets and their regulation of adhesion/aggregation. The identification of platelet subpopulations also has potentially important consequences to human health and disease as impaired platelet NO-signalling has been identified in patients with coronary artery disease.

Axial super-resolution evanescent wave tomography.

Optical tomographic reconstruction of a three-dimensional (3D) nanoscale specimen is hindered by the axial diffraction limit, which is 2-3 times worse than the focal plane resolution. We propose and experimentally demonstrate an axial super-resolution evanescent wave tomography method that enables the use of regular evanescent wave microscopes like the total internal reflection fluorescence microscope beyond surface imaging and achieve a tomographic reconstruction with axial super-resolution. Our proposed method based on Fourier reconstruction achieves axial super-resolution by extracting information from multiple sets of 3D fluorescence images when the sample is illuminated by an evanescent wave. We propose a procedure to extract super-resolution features from the incremental penetration of an evanescent wave and support our theory by one-dimensional (along the optical axis) and 3D simulations. We validate our claims by experimentally demonstrating tomographic reconstruction of microtubules in HeLa cells with an axial resolution of ∼130 nm. Our method does not require any additional optical components or sample preparation. The proposed method can be combined with focal plane super-resolution techniques like stochastic optical reconstruction microscopy and can also be adapted for THz and microwave near-field tomography.

Bile Salts Modulate the Mucin-Activated Type VI Secretion System of Pandemic Vibrio cholerae.

The causative agent of cholera, Vibrio cholerae, regulates its diverse virulence factors to thrive in the human small intestine and environmental reservoirs. Among this pathogen's arsenal of virulence factors is the tightly regulated type VI secretion system (T6SS). This system acts as an inverted bacteriophage to inject toxins into competing bacteria and eukaryotic phagocytes. V. cholerae strains responsible for the current 7th pandemic activate their T6SS within the host. We established that T6SS-mediated competition occurs upon T6SS activation in the infant mouse, and that this system is functional under anaerobic conditions. When investigating the intestinal host factors mucins (a glycoprotein component of mucus) and bile for potential regulatory roles in controlling the T6SS, we discovered that once mucins activate the T6SS, bile acids can further modulate T6SS activity. Microbiota modify bile acids to inhibit T6SS-mediated killing of commensal bacteria. This interplay is a novel interaction between commensal bacteria, host factors, and the V. cholerae T6SS, showing an active host role in infection.

Epithelial gaps in a rodent model of inflammatory bowel disease: a quantitative validation study.

OBJECTIVES: Confocal laser endomicroscopy (CLE) is a non-invasive imaging modality of the gastrointestinal tract. Epithelial gaps in the small intestine of patients and rodents have been demonstrated using CLE. The goal of this study was to quantitatively validate the findings of epithelial gap density observed with CLE against confocal microscopy (CM) and light microscopy. METHODS: Two strains of mice (control 129 Sv/Ev and interleukin 10 knockout (IL-10(-/-))) underwent CLE of the terminal ileum. Adjacent ileal tissues were examined using CM and light microscopy. The total number of gaps and cells in the villi were manually counted from the three-dimensional reconstruction of cross-sectional CLE and CM images. The histology specimens were reviewed for epithelial gap and cell counts by a pathologist blinded to the study groups. The inter- and intra-observer variability for cell and gap counts were determined. RESULTS: For CLE, the gap densities (mean±s.d.) in the ileum for control and IL-10(-/-) mice were: 9.5±1.3 gaps per 1,000 cells and 20.6±2.1 gaps per 1,000 cells counted (P<0.001), respectively. For CM, the ileal gap densities were 7.3±1.3 gaps per 1,000 cells and 22.8±6.2 gaps per 1,000 cells (P=0.03), respectively. For light microscopy, the ileal gap densities were 29.2±5.9 gaps per 1,000 cells and 51.5±6.4 gaps per 1,000 cells for the two strains. CONCLUSION: CLE can be used to quantitatively assess epithelial cells and gaps with accuracy comparable to CM and light microscopy. In a mouse model of inflammatory bowel disease, the epithelial gap density in the terminal ileum is significantly increased when examined using all three modalities.

In vivo identification of human small ubiquitin-like modifier polymerization sites by high accuracy mass spectrometry and an in vitro to in vivo strategy.

The length and precise linkage of polyubiquitin chains is important for their biological activity. Although other ubiquitin-like proteins have the potential to form polymeric chains their identification in vivo is challenging and their functional role is unclear. Vertebrates express three small ubiquitin-like modifiers, SUMO-1, SUMO-2, and SUMO-3. Mature SUMO-2 and SUMO-3 are nearly identical and contain an internal consensus site for sumoylation that is missing in SUMO-1. Combining state-of-the-art mass spectrometry with an "in vitro to in vivo" strategy for post-translational modifications, we provide direct evidence that SUMO-1, SUMO-2, and SUMO-3 form mixed chains in cells via the internal consensus sites for sumoylation in SUMO-2 and SUMO-3. In vitro, the chain length of SUMO polymers could be influenced by changing the relative amounts of SUMO-1 and SUMO-2. The developed methodology is generic and can be adapted for the identification of other sumoylation sites in complex samples.

The Human Intermediate Filament Database: comprehensive information on a gene family involved in many human diseases.

We describe a revised and expanded database on human intermediate filament proteins, a major component of the eukaryotic cytoskeleton. The family of 70 intermediate filament genes (including those encoding keratins, desmins, and lamins) is now known to be associated with a wide range of diverse diseases, at least 72 distinct human pathologies, including skin blistering, muscular dystrophy, cardiomyopathy, premature aging syndromes, neurodegenerative disorders, and cataract. To date, the database catalogs 1,274 manually-curated pathogenic sequence variants and 170 allelic variants in intermediate filament genes from over 459 peer-reviewed research articles. Unrelated cases were collected from all of the six sequence homology groups and the sequence variations were described at cDNA and protein levels with links to the related diseases and reference articles. The mutations and polymorphisms are presented in parallel with data on protein structure, gene, and chromosomal location and basic information on associated diseases. Detailed statistics relating to the variants records in the database are displayed by homology group, mutation type, affected domain, associated diseases, and nucleic and amino acid substitutions. Multiple sequence alignment algorithms can be run from queries to determine DNA or protein sequence conservation. Literature sources can be interrogated within the database and external links are provided to public databases. The database is freely and publicly accessible online at www.interfil.org (last accessed 13 September 2007). Users can query the database by various keywords and the search results can be downloaded. It is anticipated that the Human Intermediate Filament Database (HIFD) will provide a useful resource to study human genome variations for basic scientists, clinicians, and students alike.

Distinct and overlapping sets of SUMO-1 and SUMO-2 target proteins revealed by quantitative proteomics.

The small ubiquitin-like modifier (SUMO) family in vertebrates includes three different family members that are conjugated as post-translational modifications to target proteins. SUMO-2 and -3 are nearly identical but differ substantially from SUMO-1. We used quantitative proteomics to investigate the target protein preferences of SUMO-1 and SUMO-2. HeLa cells were established that stably express His6-SUMO-1 or His6-SUMO-2. These cell lines and control HeLa cells were labeled with stable arginine isotopes, and His6-SUMOs were enriched from lysates using immobilized metal affinity chromatography. 53 SUMO-conjugated proteins were identified, including 44 novel SUMO targets. 25 proteins were preferentially conjugated to SUMO-1, 19 were preferentially conjugated to SUMO-2, and nine proteins were conjugated to both SUMO-1 and SUMO-2. SART1 was confirmed by immunoblotting to have both SUMO-1- and SUMO-2-linked forms at similar levels. SUMO-1 and SUMO-2 are thus shown to have distinct and overlapping sets of target proteins, indicating that SUMO-1 and SUMO-2 may have both redundant and non-redundant cellular functions. Interestingly, 14 of the 25 SUMO-1-conjugated proteins contain zinc fingers. Although both SUMO family members play roles in many cellular processes, our data show that sumoylation is strongly associated with transcription because nearly one-third of the identified target proteins are putative transcriptional regulators.

Characterization of hCINAP, a novel coilin-interacting protein encoded by a transcript from the transcription factor TAFIID32 locus.

Coilin is a marker protein for the Cajal body, a subnuclear domain acting as a site for assembly and maturation of nuclear RNA-protein complexes. Using a yeast two-hybrid screen to identify coilin-interacting proteins, we have identified hCINAP (human coilin interacting nuclear ATPase protein), a nuclear factor of 172 amino acids with a P-loop nucleotide binding motif and ATPase activity. The hCINAP protein sequence is highly conserved across its full-length from human to plants and yeast and is ubiquitously expressed in all human tissues and cell lines tested. The yeast orthologue of CINAP is a single copy, essential gene. Tagged hCINAP is present in complexes containing coilin in mammalian cells and recombinant, Escherichia coli expressed hCINAP binds directly to coilin in vitro. The 214 carboxyl-terminal residues of coilin appear essential for the interaction with hCINAP. Both immunofluorescence and fluorescent protein tagging show that hCINAP is specifically nuclear and distributed in a widespread, diffuse nucleoplasmic pattern, excluding nucleoli, with some concentration also in Cajal bodies. Overexpression of hCINAP in HeLa cells results in a decrease in the average number of Cajal bodies per nucleus, consistent with it affecting either the stability of Cajal bodies and/or their rate of assembly. The hCINAP mRNA is an alternatively spliced transcript from the TAF9 locus, which encodes the basal transcription factor subunit TAFIID32. However, hCINAP and TAFIID32 mRNAs are translated from different ATG codons and use distinct reading frames, resulting in them having no identity in their respective protein sequences.

A proteomic study of SUMO-2 target proteins.

The SUMO family in vertebrates includes at least three distinct proteins (SUMO-1, -2, and -3) that are added as post-translational modifications to target proteins. A considerable number of SUMO-1 target proteins have been identified, but little is known about SUMO-2. A stable HeLa cell line expressing His6-tagged SUMO-2 was established and used to label and purify novel endogenous SUMO-2 target proteins. Tagged forms of SUMO-2 were functional and localized predominantly in the nucleus. His6-tagged SUMO-2 conjugates were affinity-purified from nuclear fractions and identified by mass spectrometry. Eight novel potential SUMO-2 target proteins were identified by at least two peptides. Three of these proteins, SART1, heterogeneous nuclear ribonucleoprotein (RNP) M, and the U5 small nuclear RNP 200-kDa helicase, play a role in RNA metabolism. SART1 and heterogeneous nuclear RNP M were both shown to be genuine SUMO targets, confirming the validity of the approach.

Cajal body proteins SMN and Coilin show differential dynamic behaviour in vivo.

Analysis of stable cell lines expressing fluorescently tagged survival of motor neurons protein (SMN) and coilin shows striking differences in their dynamic behaviour, both in the nucleus and during mitosis. Cajal bodies labelled with either FP-SMN or FP-coilin show similar behaviour and frequency of movements. However, fluorescence recovery after photobleaching (FRAP) studies show that SMN returns approximately 50-fold more slowly to Cajal bodies than does coilin. Time-lapse studies on cells progressing from prophase through to G1 show further differences between SMN and coilin, both in their localisation in telophase and in the timing of their re-entry into daughter nuclei. The data reveal similarities between Cajal bodies and nucleoli in their behaviour during mitosis. This in vivo study indicates that SMN and coilin interact differentially with Cajal bodies and reveals parallels in the pathway for reassembly of nucleoli and Cajal bodies following mitosis.

Cajal bodies and coilin--moving towards function.

Many nuclear factors are concentrated within nonmembrane-bound subnuclear bodies. The Cajal body is an example of a conserved nuclear compartment that has been linked to molecular disease. Recent studies have shown Cajal bodies to be surprisingly mobile and offer clues about their function in the cell.