Quantitative chemical exchange saturation transfer imaging of nuclear overhauser effects in acute ischemic stroke.

PURPOSE: In chemical exchange saturation transfer imaging, saturation effects between - 2 to - 5 ppm (nuclear Overhauser effects, NOEs) have been shown to exhibit contrast in preclinical stroke models. Our previous work on NOEs in human stroke used an analysis model that combined NOEs and semisolid MT; however their combination might feasibly have reduced sensitivity to changes in NOEs. The aim of this study was to explore the information a 4-pool Bloch-McConnell model provides about the NOE contribution in ischemic stroke, contrasting that with an intentionally approximate 3-pool model. METHODS: MRI data from 12 patients presenting with ischemic stroke were retrospectively analyzed, as well as from six animals induced with an ischemic lesion. Two Bloch-McConnell models (4 pools, and a 3-pool approximation) were compared for their ability to distinguish pathological tissue in acute stroke. The association of NOEs with pH was also explored, using pH phantoms that mimic the intracellular environment of naïve mouse brain. RESULTS: The 4-pool measure of NOEs exhibited a different association with tissue outcome compared to 3-pool approximation in the ischemic core and in tissue that underwent delayed infarction. In the ischemic core, the 4-pool measure was elevated in patient white matter ( 1.20±0.20 ) and in animals ( 1.27±0.20 ). In the naïve brain pH phantoms, significant positive correlation between the NOE and pH was observed. CONCLUSION: Associations of NOEs with tissue pathology were found using the 4-pool metric that were not observed using the 3-pool approximation. The 4-pool model more adequately captured in vivo changes in NOEs and revealed trends depending on tissue pathology in stroke.

Integrin ß1 Establishes Liver Microstructure and Modulates Transforming Growth Factor ß during Liver Development and Regeneration.

A unique and complex microstructure underlies the diverse functions of the liver. Breakdown of this organization, as occurs in fibrosis and cirrhosis, impairs liver function and leads to disease. The role of integrin ß1 was examined both in establishing liver microstructure and recreating it after injury. Embryonic deletion of integrin ß1 in the liver disrupts the normal development of hepatocyte polarity, specification of cell-cell junctions, and canalicular formation. This in turn leads to the expression of transforming growth factor ß (TGF-ß) and widespread fibrosis. Targeted deletion of integrin ß1 in adult hepatocytes prevents recreation of normal hepatocyte architecture after liver injury, with resultant fibrosis. In vitro, integrin ß1 is essential for canalicular formation and is needed to prevent stellate cell activation by modulating TGF-ß. Taken together, these findings identify integrin ß1 as a key determinant of liver architecture with a critical role as a regulator of TGF-ß secretion. These results suggest that disrupting the hepatocyte-extracellular matrix interaction is sufficient to drive fibrosis.

Study of common quantification methods of amide proton transfer magnetic resonance imaging for ischemic stroke detection.

PURPOSE: To assess the correlation and differences between common amide proton transfer (APT) quantification methods in the diagnosis of ischemic stroke. METHODS: Five APT quantification methods, including asymmetry analysis and its variants as well as two Lorentzian model-based methods, were applied to data acquired from six rats that underwent middle cerebral artery occlusion scanned at 9.4T. Diffusion and perfusion-weighted images, and water relaxation time maps were also acquired to study the relationship of these conventional imaging modalities with the different APT quantification methods. RESULTS: The APT ischemic area estimates had varying sizes (Jaccard index: 0.544 ≤ J ≤ 0.971) and had varying correlations in their distributions (Pearson correlation coefficient: 0.104 ≤ r ≤ 0.995), revealing discrepancies in the quantified ischemic areas. The Lorentzian methods produced the highest contrast-to-noise ratios (CNRs; 1.427 ≤ CNR ≤ 2.002), but generated APT ischemic areas that were comparable in size to the cerebral blood flow (CBF) deficit areas; asymmetry analysis and its variants produced APT ischemic areas that were smaller than the CBF deficit areas but larger than the apparent diffusion coefficient deficit areas, though having lower CNRs (0.561 ≤ CNR ≤ 1.083). CONCLUSION: There is a need to further investigate the accuracy and correlation of each quantification method with the pathophysiology using a larger scale multi-imaging modality and multi-time-point clinical study. Future studies should include the magnetization transfer ratio asymmetry results alongside the findings of the study to facilitate the comparison of results between different centers and also the published literature.

Does the magnetization transfer effect bias chemical exchange saturation transfer effects? Quantifying chemical exchange saturation transfer in the presence of magnetization transfer.

PURPOSE: Chemical exchange saturation transfer (CEST) is an MRI technique sensitive to the presence of low-concentration solute protons exchanging with water. However, magnetization transfer (MT) effects also arise when large semisolid molecules interact with water, which biases CEST parameter estimates if quantitative models do not account for macromolecular effects. This study establishes under what conditions this bias is significant and demonstrates how using an appropriate model provides more accurate quantitative CEST measurements. METHODS: CEST and MT data were acquired in phantoms containing bovine serum albumin and agarose. Several quantitative CEST and MT models were used with the phantom data to demonstrate how underfitting can influence estimates of the CEST effect. CEST and MT data were acquired in healthy volunteers, and a two-pool model was fit in vivo and in vitro, whereas removing increasing amounts of CEST data to show biases in the CEST analysis also corrupts MT parameter estimates. RESULTS: When all significant CEST/MT effects were included, the derived parameter estimates for each CEST/MT pool significantly correlated (P < .05) with bovine serum albumin/agarose concentration; minimal or negative correlations were found with underfitted data. Additionally, a bootstrap analysis demonstrated that significant biases occur in MT parameter estimates (P < .001) when unmodeled CEST data are included in the analysis. CONCLUSIONS: These results indicate that current practices of simultaneously fitting both CEST and MT effects in model-based analyses can lead to significant bias in all parameter estimates unless a sufficiently detailed model is utilized. Therefore, care must be taken when quantifying CEST and MT effects in vivo by properly modeling data to minimize these biases.

SOCS2 Balances Metabolic and Restorative Requirements during Liver Regeneration.

After significant injury, the liver must maintain homeostasis during the regenerative process. We hypothesized the existence of mechanisms to limit hepatocyte proliferation after injury to maintain metabolic and synthetic function. A screen for candidates revealed suppressor of cytokine signaling 2 (SOCS2), an inhibitor of growth hormone (GH) signaling, was strongly induced after partial hepatectomy. Using genetic deletion and administration of various factors we investigated the role of SOCS2 during liver regeneration. SOCS2 preserves liver function by restraining the first round of hepatocyte proliferation after partial hepatectomy by preventing increases in growth hormone receptor (GHR) via ubiquitination, suppressing GH pathway activity. At later times, SOCS2 enhances hepatocyte proliferation by modulating a decrease in serum insulin-like growth factor 1 (IGF-1) that allows GH release from the pituitary. SOCS2, therefore, plays a dual role in modulating the rate of hepatocyte proliferation. In particular, this is the first demonstration of an endogenous mechanism to limit hepatocyte proliferation after injury.

Dicer-dependent production of microRNA221 in hepatocytes inhibits p27 and is required for liver regeneration in mice.

Dicer processes microRNAs (miRs) into active forms in a wide variety of tissues, including the liver. To determine the role of Dicer in liver regeneration, we performed a series of in vivo and in vitro studies in a murine 2/3 hepatectomy model. Dicer was downregulated after 2/3 hepatectomy, and loss of Dicer inhibited liver regeneration associated with decreased cyclin A2 and miR-221, as well as increased levels of the cell cycle inhibitor p27. In vitro, miR-221 inhibited p27 production in primary hepatocytes and increased hepatocyte proliferation. Specific reconstitution of miR-221 in hepatocyte-specific Dicer-null mice inhibited p27 and restored liver regeneration. In wild type mice, targeted inhibition of miR-221 using a cholesterol-conjugated miR-221 inhibited hepatocyte proliferation after 2/3 hepatectomy. These results identify Dicer production of miR-221 as an essential component of a miRNA-dependent mechanism for suppression of p27 that controls the rate of hepatocyte proliferation after partial hepatectomy.NEW & NOTEWORTHY Our findings demonstrate a direct role for microRNAs in controlling the rate of liver regeneration after injury. By deleting Dicer, an enzyme responsible for processing microRNAs into mature forms, we determined miR-221 is a critical microRNA in the physiological process of restoration of liver mass after injury. miR-221 suppresses p27, releasing its inhibitory effects on hepatocyte proliferation. Pharmaceuticals based on miR-221 may be useful to modulate hepatocyte proliferation in the setting of liver injury.

Absolute Protein Quantification by Mass Spectrometry: Not as Simple as Advertised.

Stable isotopically labeled (SIL) tryptic peptides, cleavable SIL peptides, and a full-length SIL protein were compared for internal calibration (i.e., as internal calibrators) and external calibration (i.e., as internal standards) when quantifying three forms of unlabeled, human thyroglobulin (Tg) by bottom-up protein analysis. All SIL materials and human proteins were standardized by amino acid analysis to ensure traceability of measurements and allow confident assignment of accuracy. The three forms of human Tg quantified were (1) the primary reference material BCR457-a native protein purified from human thyroids, (2) a commercially available form also purified from human thyroids, and (3) a full-length recombinant form expressed and purified from a human embryonic kidney 293 cell-line. Collectively, the results unequivocally demonstrate the lack of commutability of tryptic and cleavable SIL peptides as internal calibrators across various bottom-up assays (i.e., denaturing/digestion conditions). Further, the results demonstrate the potential during external calibration for surrogate protein calibrators (i.e., recombinant proteins) to produce inaccurate concentration assignments of native protein analytes by bottom-up analysis due to variance in digestion efficiency, which is not alleviated by altering denaturation/digestion stringency and indicates why protein calibrators may not be commutable in bottom-up protein assays. These results have implications regarding the veracity of "absolute" protein concentration assignments by bottom-up assays using peptide calibrators, as well as protein calibrators, given that absolute accuracy was not universally observed. Nevertheless, these results support the use of recombinant SIL proteins as internal standards over SIL peptides due to their ability to better mimic the digestion of human-derived proteins and mitigate bias due to digestion-based matrix effects that were observed during external calibration.

Determination of an optimally sensitive and specific chemical exchange saturation transfer MRI quantification metric in relevant biological phantoms.

The purpose of this study was to develop realistic phantom models of the intracellular environment of metastatic breast tumour and naïve brain, and using these models determine an analysis metric for quantification of CEST MRI data that is sensitive to only labile proton exchange rate and concentration. The ability of the optimal metric to quantify pH differences in the phantoms was also evaluated. Novel phantom models were produced, by adding perchloric acid extracts of either metastatic mouse breast carcinoma cells or healthy mouse brain to bovine serum albumin. The phantom model was validated using 1 H NMR spectroscopy, then utilized to determine the sensitivity of CEST MRI to changes in pH, labile proton concentration, T1 time and T2 time; six different CEST MRI analysis metrics (MTRasym , APT*, MTRRex , AREX and CESTR* with and without T1 /T2 compensation) were compared. The new phantom models were highly representative of the in vivo intracellular environment of both tumour and brain tissue. Of the analysis methods compared, CESTR* with T1 and T2 time compensation was optimally specific to changes in the CEST effect (i.e. minimal contamination from T1 or T2 variation). In phantoms with identical protein concentrations, pH differences between phantoms could be quantified with a mean accuracy of 0.6 pH units. We propose that CESTR* with T1 and T2 time compensation is the optimal analysis method for these phantoms. Analysis of CEST MRI data with T1 /T2 time compensated CESTR* is reproducible between phantoms, and its application in vivo may resolve the intracellular alkalosis associated with breast cancer brain metastases without the need for exogenous contrast agents.

Specific activin receptor-like kinase 3 inhibitors enhance liver regeneration.

Pharmacologic agents to enhance liver regeneration after injury would have wide therapeutic application. Based on previous work suggesting inhibition of bone morphogenetic protein (BMP) signaling stimulates liver regeneration, we tested known and novel BMP inhibitors for their ability to accelerate regeneration in a partial hepatectomy (PH) model. Compounds were produced based on the 3,6-disubstituted pyrazolo[1,5-a] pyrimidine core of the BMP antagonist dorsomorphin and evaluated for their ability to inhibit BMP signaling and enhance liver regeneration. Antagonists of the BMP receptor activin receptor-like kinase 3 (ALK3), including LDN-193189 (LDN; 4-[6-[4-(1-piperazinyl)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]-quinoline), DMH2 (4-(2-(4-(3-(quinolin-4-yl)pyrazolo[1,5-a]pyrimidin-6-yl)phenoxy)ethyl)morpholine; VU0364849), and the novel compound VU0465350 (7-(4-isopropoxyphenyl)-3-(1H-pyrazol-4-yl)imidazo[1,2-a]pyridine; VU5350), blocked SMAD phosphorylation in vitro and in vivo, and enhanced liver regeneration after PH. In contrast, an antagonist of the BMP receptor ALK2, VU0469381 (5-(6-(4-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinolone; 1LWY), did not affect liver regeneration. LDN did not affect liver synthetic or metabolic function. Mechanistically, LDN increased serum interleukin-6 levels and signal transducer and activator of transcription 3 phosphorylation in the liver, and modulated other factors known to be important for liver regeneration, including suppressor of cytokine signaling 3 and p53. These findings suggest that inhibition of ALK3 may be part of a therapeutic strategy for treating human liver disease.

Adult pancreatic acinar cells give rise to ducts but not endocrine cells in response to growth factor signaling.

Studies in both humans and rodents have found that insulin(+) cells appear within or near ducts of the adult pancreas, particularly following damage or disease, suggesting that these insulin(+) cells arise de novo from ductal epithelium. We have found that insulin(+) cells are continuous with duct cells in the epithelium that makes up the hyperplastic ducts of both chronic pancreatitis and pancreatic cancer in humans. Therefore, we tested the hypothesis that both hyperplastic ductal cells and their associated insulin(+) cells arise from the same cell of origin. Using a mouse model that develops insulin(+) cell-containing hyperplastic ducts in response to the growth factor TGFalpha, we performed genetic lineage tracing experiments to determine which cells gave rise to both hyperplastic ductal cells and duct-associated insulin(+) cells. We found that hyperplastic ductal cells arose largely from acinar cells that changed their cell fate, or transdifferentiated, into ductal cells. However, insulin(+) cells adjacent to acinar-derived ductal cells arose from pre-existing insulin(+) cells, suggesting that islet endocrine cells can intercalate into hyperplastic ducts as they develop. We conclude that apparent pancreatic plasticity can result both from the ability of acinar cells to change fate and of endocrine cells to reorganize in association with duct structures.

Artificial intelligence based software facilitates spirometry quality control in asthma and COPD clinical trials.

Rationale: Acquiring high-quality spirometry data in clinical trials is important, particularly when using forced expiratory volume in 1 s or forced vital capacity as primary end-points. In addition to quantitative criteria, the American Thoracic Society (ATS)/European Respiratory Society (ERS) standards include subjective evaluation which introduces inter-rater variability and potential mistakes. We explored the value of artificial intelligence (AI)-based software (ArtiQ.QC) to assess spirometry quality and compared it to traditional over-reading control. Methods: A random sample of 2000 sessions (8258 curves) was selected from Chiesi COPD and asthma trials (n=1000 per disease). Acceptability using the 2005 ATS/ERS standards was determined by over-reader review and by ArtiQ.QC. Additionally, three respiratory physicians jointly reviewed a subset of curves (n=150). Results: The majority of curves (n=7267, 88%) were of good quality. The AI agreed with over-readers in 91% of cases, with 97% sensitivity and 93% positive predictive value. Performance was significantly better in the asthma group. In the revised subset, n=50 curves were repeated to assess intra-rater reliability (κ=0.83, 0.86 and 0.80 for each of the three reviewers). All reviewers agreed on 63% of 100 unique tests (κ=0.5). When reviewers set the consensus (gold standard), individual agreement with it was 88%, 94% and 70%. The agreement between AI and "gold-standard" was 73%; over-reader agreement was 46%. Conclusion: AI-based software can be used to measure spirometry data quality with comparable accuracy as experts. The assessment is a subjective exercise, with intra- and inter-rater variability even when the criteria are defined very precisely and objectively. By providing consistent results and immediate feedback to the sites, AI may benefit clinical trial conduct and variability reduction.

Hepatic stellate cells maintain liver homeostasis through paracrine neurotrophin-3 signaling that induces hepatocyte proliferation.

Organ size is maintained by the controlled proliferation of distinct cell populations. In the mouse liver, hepatocytes in the midlobular zone that are positive for cyclin D1 (CCND1) repopulate the parenchyma at a constant rate to preserve liver mass. Here, we investigated how hepatocyte proliferation is supported by hepatic stellate cells (HSCs), pericytes that are in close proximity to hepatocytes. We used T cells to ablate nearly all HSCs in the murine liver, enabling the unbiased characterization of HSC functions. In the normal liver, complete loss of HSCs persisted for up to 10 weeks and caused a gradual reduction in liver mass and in the number of CCND1+ hepatocytes. We identified neurotrophin-3 (Ntf-3) as an HSC-produced factor that induced the proliferation of midlobular hepatocytes through the activation of tropomyosin receptor kinase B (TrkB). Treating HSC-depleted mice with Ntf-3 restored CCND1+ hepatocytes in the midlobular region and increased liver mass. These findings establish that HSCs form the mitogenic niche for midlobular hepatocytes and identify Ntf-3 as a hepatocyte growth factor.

Investigation of relayed nuclear Overhauser enhancement effect at -1.6 ppm in an ischemic stroke model.

Background: When an ischemic stroke happens, it triggers a complex signalling cascade that may eventually lead to neuronal cell death if no reperfusion. Recently, the relayed nuclear Overhauser enhancement effect at -1.6 ppm [NOE(-1.6 ppm)] has been postulated may allow for a more in-depth analysis of the ischemic injury. This study assessed the potential utility of NOE(-1.6 ppm) in an ischemic stroke model. Methods: Diffusion-weighted imaging, perfusion-weighted imaging, and chemical exchange saturation transfer (CEST) magnetic resonance imaging (MRI) data were acquired from five rats that underwent scans at 9.4 T after middle cerebral artery occlusion. Results: The apparent diffusion coefficient (ADC), cerebral blood flow (CBF), and apparent exchange-dependent relaxations (AREX) at 3.5 ppm and NOE(-1.6 ppm) were quantified. AREX(3.5 ppm) and NOE(-1.6 ppm) were found to be hypointense and exhibited different signal patterns within the ischemic tissue. The NOE(-1.6 ppm) deficit areas were equal to or larger than the ADC deficit areas, but smaller than the AREX(3.5 ppm) deficit areas. This suggested that NOE(-1.6 ppm) might further delineate the acidotic tissue estimated using AREX(3.5 ppm). Since NOE(-1.6 ppm) is closely related to membrane phospholipids, NOE(-1.6 ppm) potentially highlighted at-risk tissue affected by lipid peroxidation and membrane damage. Altogether, the ADC/NOE(-1.6 ppm)/AREX(3.5 ppm)/CBF mismatches revealed four zones of increasing sizes within the ischemic tissue, potentially reflecting different pathophysiological information. Conclusions: Using CEST coupled with ADC and CBF, the ischemic tissue may thus potentially be separated into four zones to better understand the pathophysiology after stroke and improve ischemic tissue fate definition. Further verification of the potential utility of NOE(-1.6 ppm) may therefore lead to a more precise diagnosis.

BMP4 is a novel paracrine inhibitor of liver regeneration.

Transforming growth factor (TGF)-ß family members exert strong effects on restoration of liver mass after injury. Bone morphogenetic proteins (BMPs) are members of the TGF-ß family and are found in the liver, suggesting that these proteins may play a role in liver regeneration. We examined BMP signaling in the liver during hepatectomy. We found that BMP4 is constitutively expressed in the peribiliary stroma and endothelial cells of the liver and that expression is decreased after hepatectomy. Mice driven to maintain BMP4 expression in the liver display inhibited hepatocyte proliferation and restoration of liver mass after hepatectomy, suggesting that reduced BMP4 is necessary for normal regeneration. Consistent with this finding, hepatocyte-specific deletion of the BMP receptor activin receptor-like kinase 3 (Alk3) enhances regeneration and reduces phosphorylation of SMAD1/5/8, a transducer of BMP signaling. In contrast to experiments in wild-type mice, maintaining BMP4 levels has no effect on liver regeneration in hepatocyte-specific Alk3 null mice, providing evidence that BMP4 signals through Alk3 to inhibit liver regeneration. Consistent with these findings, the BMP4 antagonist Noggin enhances regeneration. Furthermore, high-dose BMP4 inhibits proliferation of primary hepatocytes and HepG2 cells in culture. These findings elucidate a new, potentially clinically relevant paradigm in which a constitutively expressed paracrine inhibitory factor plays a critical role in liver regeneration.

Using the Decrease in Trauma Admissions During the COVID-19 Pandemic to Evaluate Compliance With Stay-at-Home and Social Distancing Guidelines.

Introduction The coronavirus disease 2019 (COVID-19) virus was declared a pandemic on March 10, 2020 by the World Health Organization (WHO) and has massively burdened healthcare systems with cases exponentially rising throughout the United States and the rest of the world. Since implementing precautions to reduce the spread of this disease, emergency departments have seen a decrease in the number of traumas. By evaluating the differences in the number of trauma admissions in different subgroups of patients, we can assess where to target messaging to increase compliance with these precautions. In this study, we aim to analyze the effect of the COVID-19 pandemic on trauma admissions. Methodology This was a retrospective review of the trauma database at our institution, a level 2 trauma center in Southern California, to assess the impact of COVID-19 on the number of traumas. The inclusion criteria were patients activated as traumas, regardless of age. Patients were excluded from the study if they did not have complete information in the trauma database. Data were stratified by date into two groups: (a) COVID period (January to April 2020) and (b) pre-COVID period (January to April 2019). The primary endpoint of this study was to determine whether there was a significant change in the number of patients presenting as trauma during the COVID-19 pandemic. This difference was analyzed and divided into subgroups based on age and trauma type. Results In our review, an average of 279 patients per month presented as trauma from January to April in 2019, and an average of 222 patients per month presented as trauma from January to April 2020 (p = 0.049). We found a significant decrease in the number of patients presenting with the chief complaint of fall and vehicular accident, but a nonsignificant difference in patients presenting as assaults or pedestrian accidents. There was also a significant decrease in the number of traumas in the 18-39 and 65+ age groups and a nonsignificant decrease in the 40-64 age group. It was also noted that the number of trauma admissions in May 2020 increased to 253 compared to 269 in 2019. This increase was most notable in the 18-39 and 40-64 age groups. Discussion As seen in the data, the most notable year-over-year difference was seen in March and April. In California specifically, a stay-at-home order was set in place in March, which was in conjunction with the WHO's declaration of a pandemic. An interesting finding was the significant decrease in patients with traumas in the age groups of 18-39 and 65+ from 2019 to 2020. There was a smaller, nonsignificant decrease in patients aged 40-64. This would be a good group to target with future messaging to increase compliance with health advisories. There was also a notable increase in the number of traumas in May 2020, signaling an end to the cooperation of the majority of people, also indicating that further measures needed to be enacted in all groups. Conclusions COVID-19 has disrupted social structures worldwide. As the pandemic continued, even the observers of stay-at-home and social distancing measures, the 18-39 age group, became fatigued with the guidelines and ventured out into the warming weather and summer activities. This difference in trauma admission due to COVID-19 between subsequent years can highlight the behavioral changes in our patient population and can be further extrapolated to target additional messaging to help reduce the spread of COVID-19.

Supermeres are functional extracellular nanoparticles replete with disease biomarkers and therapeutic targets.

Extracellular vesicles and exomere nanoparticles are under intense investigation as sources of clinically relevant cargo. Here we report the discovery of a distinct extracellular nanoparticle, termed supermere. Supermeres are morphologically distinct from exomeres and display a markedly greater uptake in vivo compared with small extracellular vesicles and exomeres. The protein and RNA composition of supermeres differs from small extracellular vesicles and exomeres. Supermeres are highly enriched with cargo involved in multiple cancers (glycolytic enzymes, TGFBI, miR-1246, MET, GPC1 and AGO2), Alzheimer's disease (APP) and cardiovascular disease (ACE2, ACE and PCSK9). The majority of extracellular RNA is associated with supermeres rather than small extracellular vesicles and exomeres. Cancer-derived supermeres increase lactate secretion, transfer cetuximab resistance and decrease hepatic lipids and glycogen in vivo. This study identifies a distinct functional nanoparticle replete with potential circulating biomarkers and therapeutic targets for a host of human diseases.

Transmembrane and soluble isoforms of heparin-binding epidermal growth factor-like growth factor regulate distinct processes in the pancreas.

BACKGROUND & AIMS: Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is produced as a type-I, single-pass transmembrane protein that can be cleaved to release a diffusible peptide. HB-EGF, often overexpressed in damaged or diseased epithelium, is normally expressed in pancreatic islets, but its function is not understood. METHODS: To understand the function of each isoform of HB-EGF, we made transgenes expressing either a constitutively transmembrane or a constitutively secreted protein. RESULTS: The transmembrane isoform was not an inert precursor protein, but a functional molecule, downregulating the glucose-sensing apparatus of pancreatic islets. Conversely, the secreted form of HB-EGF improved islet function, but had severe fibrotic and neoplastic effects on surrounding tissues. Each isoform had a more severe phenotype than that of full-length HB-EGF, even though the full-length protein was efficiently cleaved, thus producing both isoforms, suggesting that a level of regulation was lost by separating the isoforms. CONCLUSIONS: This work demonstrates that islet function depends on the ratio of cleaved to uncleaved HB-EGF and that the transmembrane intermediate, while deleterious to islet function, is necessary to restrict action of soluble HB-EGF away from surrounding tissue.

Improved mass spectrometric characterization of protein glycosylation reveals unusual glycosylation of maize-derived bovine trypsin.

Although bottom-up proteomics using tryptic digests is widely used to locate post-translational modifications (PTM) in proteins, there are cases where the protein has several potential modification sites within a tryptic fragment and MS(2) strategies fail to pinpoint the location. We report here a method using two proteolytic enzymes, trypsin and pepsin, in combination followed by tandem mass spectrometric analysis to provide fragments that allow one to locate the modification sites. We used this strategy to find a glycosylation site on bovine trypsin expressed in maize (TrypZean). Several glycans are present, and all are attached to a nonconsensus N-glycosylation site on the protein.

Epidermal growth factor receptor regulates pancreatic fibrosis.

The development of pancreatic fibrosis has been shown to be a major component in several diseases of the pancreas including pancreatic cancer, chronic pancreatitis, and type 2 diabetes mellitus, but its actual role in the progression of these disorders is still unknown. This fibrosis is characterized by stromal expansion and the excessive deposition of extracellular matrix (ECM) that replaces pancreatic tissue. This eventually leads to dysregulation of ECM turnover, production of cytokines, restriction of blood flow, and often exocrine and endocrine insufficiencies. Activated pancreatic stellate cells (PSCs) have been identified as key mediators in the progression of pancreatic fibrosis, serving as the predominant source of excess ECM proteins. Previously, we found that overexpression of the growth factor heparin-binding epidermal growth factor-like growth factor (HB-EGF) in pancreatic islets led to intraislet fibrosis. HB-EGF binds to and activates two receptors, epidermal growth factor receptor (EGFR) and ErbB4, as well as heparin moieties and CD9/DRAP27. To understand the mechanism underlying the induction of fibrogenesis by HB-EGF, we utilized a hypomorphic allele of Egfr, the Waved-2 allele, to demonstrate that EGFR signaling regulates fibrogenesis in vivo. Using an in vitro cell migration assay, we show that HB-EGF regulates both chemoattraction and stimulation of proliferation of PSCs via EGFR activation.

Infections in Deep Brain Stimulator Surgery.

INTRODUCTION: Deep brain stimulation has emerged as an effective treatment for movement disorders such as Parkinson's disease, dystonia, and essential tremor with estimates of >100,000 deep brain stimulators (DBSs) implanted worldwide since 1980s. Infections rates vary widely in the literature with rates as high as 25%. Traditional management of infection after deep brain stimulation is systemic antibiotic therapy with wound incision and debridement (I&D) and removal of implanted DBS hardware. The aim of this study is to evaluate the infections occurring after DBS placement and implantable generator (IPG) placement in order to better prevent and manage these infections. MATERIALS/METHODS: We conducted a retrospective review of 203 patients who underwent implantation of a DBS at a single institution. For initial electrode placement, patients underwent either unilateral or bilateral electrode placement with implantation of the IPG at the same surgery and IPG replacements occurred as necessary. For patients with unilateral electrodes, repeat surgery for placement of contralateral electrode was performed when desired. Preoperative preparation with ethyl alcohol occurred in all patients while use of intra-operative vancomycin powder was surgeon dependent. All patients received 24 hours of postoperative antibiotics. Primary endpoint was surgical wound infection or brain abscess located near the surgically implanted DBS leads. Infections were classified as early (<90 days) or late (>90 days). Infectious organisms were recorded based on intra-operative wound cultures. Number of lead implantations, IPG replacements and choice of presurgical, intra-operative, and postsurgical antibiotics were recorded and outcomes compared. RESULTS: Two hundred and three patients underwent 391 electrode insertions and 244 IPG replacements. Fourteen patients developed an infection (10 early versus 4 late); 12 after implantation surgery (3%) and 2 after IPG replacement surgery (0.8%). No intracranial abscesses were found. Most common sites were the chest and connector. Staphylococcus aureus (MSSA) was the most common organism. Intra-operative vancomycin powder did not decrease infection risk. Vancomycin powder use was shown to increase risk of infection after electrode implantation surgery (Relative Risk 5.5080, p = 0.02063). Complete hardware removal occurred in eight patients, one patient had electrode only removal, three patients with I&D and no removal of hardware, and two patients with removal of IPG and extensor cables only. All patients were treated with postoperative intravenous antibiotics and no recurrent infections were found in patients with hardware left in place. DISCUSSION/CONCLUSION: Infections after DBS implantation and IPG replacement occurred in 3% and 0.8% of patients respectively in our study which is lower than reported historically. Early infections were more common. No intracranial infections were found. Intra-operative use of vancomycin was not shown to decrease risk of infection after electrode implantation surgery or IPG replacement. However, in our study it was shown to increase risk of infection after electrode implantation surgery. Treatment includes antibiotic therapy and debridement with or without removal of hardware. DBS hardware can be safely left in place in select patients who may have significant adverse effects if it is removed.