MCT4 and CD147 colocalize with MMP14 in invadopodia and support matrix degradation and invasion by breast cancer cells.

Expression levels of the lactate-H+ cotransporter MCT4 (also known as SLC16A3) and its chaperone CD147 (also known as basigin) are upregulated in breast cancers, correlating with decreased patient survival. Here, we test the hypothesis that MCT4 and CD147 favor breast cancer invasion through interdependent effects on extracellular matrix (ECM) degradation. MCT4 and CD147 expression and membrane localization were found to be strongly reciprocally interdependent in MDA-MB-231 breast cancer cells. Overexpression of MCT4 and/or CD147 increased, and their knockdown decreased, migration, invasion and the degradation of fluorescently labeled gelatin. Overexpression of both proteins led to increases in gelatin degradation and appearance of the matrix metalloproteinase (MMP)-generated collagen-I cleavage product reC1M, and these increases were greater than those observed upon overexpression of each protein alone, suggesting a concerted role in ECM degradation. MCT4 and CD147 colocalized with invadopodia markers at the plasma membrane. They also colocalized with MMP14 and the lysosomal marker LAMP1, as well as partially with the autophagosome marker LC3, in F-actin-decorated intracellular vesicles. We conclude that MCT4 and CD147 reciprocally regulate each other and interdependently support migration and invasiveness of MDA-MB-231 breast cancer cells. Mechanistically, this involves MCT4-CD147-dependent stimulation of ECM degradation and specifically of MMP-mediated collagen-I degradation. We suggest that the MCT4-CD147 complex is co-delivered to invadopodia with MMP14.

Dynamic localization of the Na+-HCO3- co-transporter NBCn1 to the plasma membrane, centrosomes, spindle and primary cilia.

Finely tuned regulation of transport protein localization is vital for epithelial function. The Na+-HCO3- co-transporter NBCn1 (also known as SLC4A7) is a key contributor to epithelial pH homeostasis, yet the regulation of its subcellular localization is not understood. Here, we show that a predicted N-terminal ß-sheet and short C-terminal α-helical motif are essential for NBCn1 plasma membrane localization in epithelial cells. This localization was abolished by cell-cell contact disruption, and co-immunoprecipitation (co-IP) and proximity ligation (PLA) revealed NBCn1 interaction with E-cadherin and DLG1, linking it to adherens junctions and the Scribble complex. NBCn1 also interacted with RhoA and localized to lamellipodia and filopodia in migrating cells. Finally, analysis of native and GFP-tagged NBCn1 localization, subcellular fractionation, co-IP with Arl13B and CEP164, and PLA of NBCn1 and tubulin in mitotic spindles led to the surprising conclusion that NBCn1 additionally localizes to centrosomes and primary cilia in non-dividing, polarized epithelial cells, and to the spindle, centrosomes and midbodies during mitosis. We propose that NBCn1 traffics between lateral junctions, the leading edge and cell division machinery in Rab11 endosomes, adding new insight to the role of NBCn1 in cell cycle progression.

The Effect of Patient-Controlled Oral Analgesia for Acute Abdominal Pain after Discharge.

BACKGROUND: During hospitalization, patients who were admitted with acute abdominal pain must be prepared to care for themselves at home after discharge to continue established treatment, promote recovery, and avoid readmission. AIMS: Our aim was to investigate the quality of pain management after discharge, when patient-controlled oral analgesia was compared with standard care for patients admitted to hospital with acute abdominal pain. The primary outcome measures were pain intensity and patient perception of care. The secondary outcome measures were pain interference with activity, affective experiences, side effects, and use of analgesics. DESIGN: A questionnaire study measuring the effect of an intervention on patient-controlled oral analgesics. SETTINGS: An emergency department and a surgical department in Denmark. PARTICIPANTS: Patients admitted to hospital with acute abdominal pain. METHODS: A pre- and postintervention study was conducted in an emergency department and a surgical department with three subunits. Data were collected using a Danish modified Revised American Pain Society Patient Outcome Questionnaire with five subscales (scale 0-10) completed in weeks 1 and 4 after discharge. RESULTS: In total, 117 patients were included. The median scores at week 1 and week 4 in the control and intervention groups were, respectively, 2/1 and 1/0 on the pain subscale (p = .11/.16), 3/0 and 3/0 on the activity subscale (p = .19/.80), 1/0 and 0/0 on the emotional subscale (p = .02/.72), 1/0 and 1/0 on the side effect subscale (p = .95/.99), and 8/5 and 7/7 on the patient perception subscale (p = .35/.49). There was no significant difference in the use of analgesics at week 1. CONCLUSIONS: Patient-controlled oral analgesia during the hospital stay did not improve the quality of pain management after discharge.

Patient-controlled oral analgesia at acute abdominal pain: A before-and-after intervention study of pain management during hospital stay.

AIM: To investigate the patient experience of pain management, when patient-controlled oral analgesia was compared with standard care for patients admitted to hospital with acute abdominal pain. The primary outcome measures were pain intensity and patient perception of care. BACKGROUND: Pain management of patients admitted to hospital with acute abdominal pain can be insufficient. Patient involvement in health care has been seen to have benefits for patients. METHODS: A before-and-after intervention study was conducted in an emergency department observation unit and a surgical department. Data were collected from a questionnaire (APS-POQ-R-D) with the six subscales: pain severity, perception of care, interference with activity, interference with emotions, side effects and patient-related barriers. RESULTS: A total of 156 patients were included. During admission the median score (0-10 scale) for the pain intensity and patient perception of care subscale was 4 (p = 0.96) and 8 (p = 0.92), respectively, in both the control and intervention group. On the activity subscale, the median scores were 6 and 5 (p = 0.17); on the emotion subscale, the scores were 5 and 4 (p = 0.31); and on the side effect subscale, the scores were 3 and 4 (p = 0.18) in the control and intervention group, respectively. Overall, the score was 5-8 at one item about being allowed to participate in decisions about pain treatment as much as wanted. CONCLUSION: Patient-controlled oral analgesia did not improve patient experience of pain management for patients admitted to hospital with acute abdominal pain.

A diverse range of bacterial and eukaryotic chitinases hydrolyzes the LacNAc (Galß1-4GlcNAc) and LacdiNAc (GalNAcß1-4GlcNAc) motifs found on vertebrate and insect cells.

There is emerging evidence that chitinases have additional functions beyond degrading environmental chitin, such as involvement in innate and acquired immune responses, tissue remodeling, fibrosis, and serving as virulence factors of bacterial pathogens. We have recently shown that both the human chitotriosidase and a chitinase from Salmonella enterica serovar Typhimurium hydrolyze LacNAc from Galß1-4GlcNAcß-tetramethylrhodamine (LacNAc-TMR (Galß1-4GlcNAcß(CH2)8CONH(CH2)2NHCO-TMR)), a fluorescently labeled model substrate for glycans found in mammals. In this study we have examined the binding affinities of the Salmonella chitinase by carbohydrate microarray screening and found that it binds to a range of compounds, including five that contain LacNAc structures. We have further examined the hydrolytic specificity of this enzyme and chitinases from Sodalis glossinidius and Polysphondylium pallidum, which are phylogenetically related to the Salmonella chitinase, as well as unrelated chitinases from Listeria monocytogenes using the fluorescently labeled substrate analogs LacdiNAc-TMR (GalNAcß1-4GlcNAcß-TMR), LacNAc-TMR, and LacNAcß1-6LacNAcß-TMR. We found that all chitinases examined hydrolyzed LacdiNAc from the TMR aglycone to various degrees, whereas they were less active toward LacNAc-TMR conjugates. LacdiNAc is found in the mammalian glycome and is a common motif in invertebrate glycans. This substrate specificity was evident for chitinases of different phylogenetic origins. Three of the chitinases also hydrolyzed the ß1-6 bond in LacNAcß1-6LacNAcß-TMR, an activity that is of potential importance in relation to mammalian glycans. The enzymatic affinities for these mammalian-like structures suggest additional functional roles of chitinases beyond chitin hydrolysis.

Bacterial chitinases and chitin-binding proteins as virulence factors.

Bacterial chitinases (EC 3.2.1.14) and chitin-binding proteins (CBPs) play a fundamental role in the degradation of the ubiquitous biopolymer chitin, and the degradation products serve as an important nutrient source for marine- and soil-dwelling bacteria. However, it has recently become clear that representatives of both Gram-positive and Gram-negative bacterial pathogens encode chitinases and CBPs that support infection of non-chitinous mammalian hosts. This review addresses this biological role of bacterial chitinases and CBPs in terms of substrate specificities, regulation, secretion and involvement in cellular and animal infection.

Characterization of a novel Salmonella Typhimurium chitinase which hydrolyzes chitin, chitooligosaccharides and an N-acetyllactosamine conjugate.

Salmonella contain genes annotated as chitinases; however, their chitinolytic activities have never been verified. We now demonstrate such an activity for a chitinase assigned to glycoside hydrolase family 18 encoded by the SL0018 (chiA) gene in Salmonella enterica Typhimurium SL1344. A C-terminal truncated form of chiA lacking a putative chitin-binding domain was amplified by PCR, cloned and expressed in Escherichia coli BL21 (DE3) with an N-terminal (His)(6) tag. The purified enzyme hydrolyzes 4-nitrophenyl N,N'-diacetyl-ß-D-chitobioside, 4-nitrophenyl ß-D-N,N',N″-triacetylchitotriose and carboxymethyl chitin Remazol Brilliant Violet but does not act on 4-nitrophenyl N-acetyl-ß-D-glucosaminide, peptidoglycan or 4-nitrophenyl ß-D-cellobioside. Enzyme activity was also characterized by directly monitoring product formation using (1)H-nuclear magnetic resonance which showed that chitin is a substrate with the release of N,N'-diacetylchitobiose. Hydrolysis occurs with the retention of configuration and the enzyme acts on only the ß-anomers of chitooligosaccharide substrates. The enzyme also released N-acetyllactosamine disaccharide from Galß1 → 4GlcNAcß-O-(CH(2))(8)CONH(CH(2))(2)NHCO-tetramethylrhodamine, a model substrate for LacNAc terminating glycoproteins and glycolipids.

Psychometric evaluation of the Danish version of a modified Revised American Pain Society Patient Outcome Questionnaire (APS-POQ-R-D) for patients hospitalized with acute abdominal pain.

Background and aims This paper forms part of a study evaluating the effect of patient-controlled oral analgesia for patients admitted to hospital with acute abdominal pain. Pain is a subjective experience, and a multifaceted evaluation tool concerning patient-reported outcome measures is needed to monitor, evaluate, and guide health care professionals in the quality of pain management. The Revised American Pain Society Patient Outcome Questionnaire (APS-POQ-R) is a validated multifaceted evaluation tool for measuring patient-reported pain experiences to evaluate different pain management interventions. The aim of this study was to evaluate the psychometric properties of a modified Danish version of the Revised American Pain Society Patient Outcome Questionnaire (APS-POQ-R-D) used during and after hospitalization for patients with acute abdominal pain. Methods The APS-POQ-R was translated into Danish and two slightly different questionnaires were formed. Questionnaire one had 39 items and the six subscales pain severity (pain), perception of care (satisfaction), pain interference with function (activity) and emotions (emotion), side effects of treatment (safety), and patient-related barriers to pain management. The questionnaire focused on time during hospital stay and was to be completed at discharge. Questionnaire two included 25 items and the five subscales pain, satisfaction, activity, emotion, and safety and focused on time at home and was to be completed daily 1 week after discharge. The questionnaires were tested on 156 patients with acute abdominal pain. Internal consistency reliability and construct validity was examined. Results In both questionnaires, the results of correlations and tests for internal consistency reliability showed a Cronbach's alpha of >0.7 for the pain, activity, and emotion subscales, but the value was ≥0.69 for the satisfaction subscale. In questionnaire one, Cronbach's alpha was ≤0.64 for the safety subscale, but this was 0.73 when the item "itching" was deleted. In questionnaire two, Cronbach's alpha was ≤0.51 for the safety subscale. For the patient-barrier subscale in questionnaire one, Cronbach's alpha was ≤0.62 for any combination of the items in the subscale. The results of the construct validity and factor analysis showed a five-factor structure in questionnaire one and a three-factor structure in questionnaire two. In questionnaire one, items from the pain, activity, emotion, and safety subscales, except for the items "least pain" and "itching," loaded on factor one. In questionnaire two, all items from the pain, activity, and emotion subscales loaded on factor one. Conclusions The modified APS-POQ-R-D demonstrated adequate psychometric properties for the five subscales pain severity (pain), perception of care (satisfaction), pain interference with function (activity) and emotions (emotion), side effects of treatment (safety), but not for the patient-barrier subscale for patients hospitalized with acute abdominal pain. Consequently, the APS-POQ-R-D may be used without the patient-barrier subscale. Implications The clinical implications of this study may help clinicians with investigating how acute patients manage pain during and after hospital admission.

Human chitotriosidase CHIT1 cross reacts with mammalian-like substrates.

Humans do not synthesize chitin, yet they produce a number of active and inactive chitinases. One of the active enzymes is chitotriosidase whose serum levels are elevated in a number of diseases such as Gaucher's disease and upon fungal infection. Since the biological role of chitotriosidase in disease pathogenesis is not understood we screened a panel of mammalian GlcNAc-containing glycoconjugates as alternate substrates. LacNAc and LacdiNAc-terminating substrates are hydrolyzed, the latter with a turnover comparable to that of pNP-chitotriose. Glycolipids or glycoproteins with LacNAc and LacdiNAc represent potential chitinase substrates and the subsequent alteration of glycosylation pattern could be a factor in disease pathogenesis.

The importance of dietary composition for efficacy of iron absorption measured in a whole diet that includes rye bread fortified with ferrous fumerate: a radioisotope study in young women.

Fe absorption is affected by many dietary factors. The objective of the present study was to measure the effects of high v. low content of vitamin C, meat and phytic acid in whole diets with Fe-fortified bread on the efficacy of Fe absorption. Thirty-two healthy women with low Fe stores were randomised to three groups, each of which was given two of six test diets containing either low/high amounts of vitamin C, meat or phytic acid, respectively, in a cross-over design. Each diet was served throughout a 5 d period. Fe-fortified rye bread, extrinsically labelled with (59)Fe, was given with all main meals. Fe absorption was determined from whole-body counter measurements of (59)Fe retention. The fractional non-haem Fe absorption (corrected to a 40 % standard absorption by measurements from the reference dose) was 1.9 % v. 3.4 % (P=0.04) for the low/high vitamin C diets, 3.0 % v. 3.5 % (P=0.58) on the low/high meat diets and 4.9 % v. 3.8 % (P=0.24) on the low/high phytic acid diet, respectively. The total Fe absorbed (geometric mean with standard error) varied from 0.43 (se 0.11) mg from the diet with lowest bioavailability to 1.09 (se 0.18) mg from the diet with highest bioavailability (P<0.001). The present whole-diet study indicates that diet composition is a strong predictor of Fe absorption. In the diet with a low content of enhancers and a high content of inhibitors, vitamin C improved non-haem Fe absorption. The total Fe absorption varied 2.5-fold after small alterations of the content of enhancers and inhibitors in the diet.