Development and testing of a low cost simulation manikin for extracorporeal cardiopulmonary resuscitation (ECPR) using 3-dimensional printing.

BACKGROUND: There has been an explosive growth of ECPR within new and established ECMO programs worldwide with the concomitant need for simulation trainers. However, current commercially available ECMO simulation models are expensive and lack many standard cardiorespiratory resuscitative (CPR) features. OBJECTIVE: To use 3-dimensional (3D) printing to develop a training manikin for comprehensive ECPR simulation. METHODS: A standard commercially available CPR manikin with airway model was used as the base model for modification. An inexpensive 3D printer was used to print a modular plastic pelvis. A medical silicone gel incorporated silicone femoral vasculature component was manufactured with connection to a gravity fed vascular system. RESULTS: The resulting modified manikin included the modular in-house designed ECMO cannulation and vascular structures wedded to the commercially available airway and CPR components. In simulation exercise involving first responders, paramedics, and emergency and critical care physicians, the model was reported as realistic with ultrasound views, cannulation, and resuscitative components functional. The entire cost for development of the ECMO component was estimated at $2000 Australian dollars AUD, including the printer purchase and supplies. Future reuse of components is estimated to cost less than $5 AUD per simulation run. CONCLUSIONS: A novel in-house modified manikin for ECPR was developed that was cost-efficient and realistic to use from first response through to establishment of ECMO circulation.

NPC1 regulates ER contacts with endocytic organelles to mediate cholesterol egress.

Transport of dietary cholesterol from endocytic organelles to the endoplasmic reticulum (ER) is essential for cholesterol homoeostasis, but the mechanism and regulation of this transport remains poorly defined. Membrane contact sites (MCS), microdomains of close membrane apposition, are gaining attention as important platforms for non-vesicular, inter-organellar communication. Here we investigate the impact of ER-endocytic organelle MCS on cholesterol transport. We report a role for Niemann-Pick type C protein 1 (NPC1) in tethering ER-endocytic organelle MCS where it interacts with the ER-localised sterol transport protein Gramd1b to regulate cholesterol egress. We show that artificially tethering MCS rescues the cholesterol accumulation that characterises NPC1-deficient cells, consistent with direct lysosome to ER cholesterol transport across MCS. Finally, we identify an expanded population of lysosome-mitochondria MCS in cells depleted of NPC1 or Gramd1b that is dependent on the late endosomal sterol-binding protein STARD3, likely underlying the mitochondrial cholesterol accumulation in NPC1-deficient cells.

Distinct roles for Tsg101 and Hrs in multivesicular body formation and inward vesiculation.

In mammalian cells, epidermal growth factor (EGF) stimulation promotes multivesicular body (MVB) formation and inward vesiculation within MVB. Annexin 1 is required for EGF-stimulated inward vesiculation but not MVB formation, demonstrating that MVB formation (the number of MVBs/unit cytoplasm) and inward vesiculation (the number of internal vesicles/MVB) are regulated by different mechanisms. Here, we show that EGF-stimulated MVB formation requires the tumor susceptibility gene, Tsg101, a component of the ESCRT (endosomal sorting complex required for transport) machinery. Depletion of Tsg101 potently inhibits EGF degradation and MVB formation and causes the vacuolar domains of the early endosome to tubulate. Although Tsg101 depletion inhibits MVB formation and alters the morphology of the early endosome in unstimulated cells, these effects are much greater after EGF stimulation. In contrast, depletion of hepatocyte growth factor receptor substrate (Hrs) only modestly inhibits EGF degradation, does not induce tubulation of the early endosome, and causes the generation of enlarged MVBs that retain the ability to fuse with the lysosome. Together, these results indicate that Tsg101 is required for the formation of stable vacuolar domains within the early endosome that develop into MVBs and Hrs is required for the accumulation of internal vesicles within MVBs and that both these processes are up-regulated by EGF stimulation.

Hydroxyapatite promotes superior keratocyte adhesion and proliferation in comparison with current keratoprosthesis skirt materials.

AIM: Published clinical series suggest the osteoodontokeratoprosthesis (OOKP) may have a lower extrusion rate than current synthetic keratoprostheses. The OOKP is anchored in the eye wall by autologous tooth. The authors' aim was to compare adhesion, proliferation, and morphology for telomerase transformed keratocytes seeded on calcium hydroxyapatite (the principal mineral constituent of tooth) and materials used in the anchoring elements of commercially available synthetic keratoprostheses. METHODS: Test materials were hydroxyapatite, polytetrafluoroethylene (PTFE), polyhydroxyethyl methacrylate (HEMA), and glass (control). Cell adhesion and viability were quantified at 4 hours, 24 hours, and 1 week using a calcein-AM/EthD-1 viability/cytotoxicity assay. Focal contact expression and cytoskeletal organisation were studied at 24 hours by confocal microscopy with immunoflourescent labelling. Further studies of cell morphology were performed using light and scanning electron microscopy. RESULTS: Live cell counts were significantly greater on hydroxyapatite surfaces at each time point (p<0.04). Dead cell counts were significantly higher for PTFE at 7 days (p<0.002). ss(1) integrin expression was highest on hydroxyapatite. Adhesion structures were well expressed in flat, spread out keratocytes on both HA and glass. Keratocytes tended to be thinner and spindle shaped on PTFE. The relatively few keratocytes visible on HEMA test surfaces were rounded and poorly adherent. CONCLUSIONS: Keratocyte adhesion, spreading, and viability on hydroxyapatite test surfaces is superior to that seen on PTFE and HEMA. Improving the initial cell adhesion environment in the skirt element of keratoprostheses may enhance tissue integration and reduce device failure rates.

Do pre-operative abdominal exercises prevent post-operative donor site complications for women undergoing DIEP flap breast reconstruction? A two-centre, prospective randomised controlled trial.

The deep inferior epigastric perforator (DIEP) flap is the gold standard for breast reconstruction using abdominal tissue. Unlike the transverse rectus abdominis myocutaneous (TRAM) flap, no rectus abdominis muscle is removed with the flap, but intra-muscular scarring can still cause post-operative complications. Strong abdominal muscles have been advocated as a prerequisite for surgery, but without any evidence as to the potential benefits. This study aimed to investigate the effect of pre-operative abdominal exercises on inpatient pain levels, length of hospital stay, post-operative abdominal muscle strength and function following a DIEP flap.Ninety-three women undergoing delayed breast reconstruction with a DIEP flap between October 1999 and November 2000 were randomly allocated to either a control or exercise group. The exercise group performed pre-operative exercises using the Abdotrim abdominal exerciser. Pre-operatively, outcome measures included trunk muscle strength measured on an isokinetic dynamometer, SF-36, rectus muscle thickness measured using ultrasound, and submaximal fitness. Post-operative pain and length of hospital stay were recorded. Subjects were reassessed using the same outcome measures 1 year post-operatively. There was a statistically significant increase in static (isometric) muscle strength and thickness pre-operatively for the exercise group. One year following surgery, there was a significant decrease in dynamic (concentric and eccentric) flexion strength for both groups, although the clinical significance of this is questionable as the majority of women had returned to pre-operative fitness and the surgery had no impact on functional activities. The static flexion strength of the control group was reduced at 1 year, whereas it was maintained in the exercise group, although this was not statistically significant. One third of women in the control group complained of functional problems or abdominal pain post-operatively compared to one fifth of the exercise group. Overall, the DIEP flap had no major impact on abdominal muscle strength for either group, demonstrating its superiority over the TRAM flap. There was no statistically significant benefit to the exercise group of the pre-operative exercises 1 year following surgery. However, there was a subjective benefit, albeit statistically nonsignificant, in terms of reduced functional problems post-operatively and improved well-being prior to surgery.

Human VPS34 is required for internal vesicle formation within multivesicular endosomes.

After internalization from the plasma membrane, activated EGF receptors (EGFRs) are delivered to multivesicular bodies (MVBs). Within MVBs, EGFRs are removed from the perimeter membrane to internal vesicles, thereby being sorted from transferrin receptors, which recycle back to the plasma membrane. The phosphatidylinositol (PI) 3'-kinase inhibitor, wortmannin, inhibits internal vesicle formation within MVBs and causes EGFRs to remain in clusters on the perimeter membrane. Microinjection of isotype-specific inhibitory antibodies demonstrates that the PI 3'-kinase required for internal vesicle formation is hVPS34. In the presence of wortmannin, EGFRs continue to be delivered to lysosomes, showing that their removal from the recycling pathway and their delivery to lysosomes does not depend on inward vesiculation. We showed previously that tyrosine kinase-negative EGFRs fail to accumulate on internal vesicles of MVBs but are recycled rather than delivered to lysosomes. Therefore, we conclude that selection of EGFRs for inclusion on internal vesicles requires tyrosine kinase but not PI 3'-kinase activity, whereas vesicle formation requires PI 3'-kinase activity. Finally, in wortmannin-treated cells there is increased EGF-stimulated tyrosine phosphorylation when EGFRs are retained on the perimeter membrane of MVBs. Therefore, we suggest that inward vesiculation is involved directly with attenuating signal transduction.

A retrospective comparison of abdominal muscle strength following breast reconstruction with a free TRAM or DIEP flap.

Abdominal weakness is a known potential complication of breast reconstruction with a pedicled or free TRAM flap. It has been presumed that the DIEP flap, which involves no muscle resection, does not compromise abdominal muscle strength but little objective research exists to substantiate this. The aims of this retrospective study were to compare abdominal muscle strength following free TRAM flap and DIEP flap, to compare both groups with a control group and to establish the effect of both procedures on functional activities. Fifty women (23 with a DIEP flap, 27 with a free TRAM flap) plus 32 non-operated controls underwent assessment of their abdominal and back extensor muscle strength on a KIN COM isokinetic dynamometer. Two questionnaires were used to establish the impact on function. The TRAM flap group had significant weakness of the abdominal and back extensor muscles compared with the DIEP flap group and the control group. The trend was for the DIEP flap group to have weaker abdominal muscles than the control group. There was a higher level of abdominal pain and a greater number of reported functional difficulties in the TRAM flap group than in the DIEP flap group. This study demonstrates that whilst the DIEP flap can reduce the strength deficit caused by the free TRAM flap, abdominal weakness can still result from the DIEP flap. A randomised controlled trial is currently underway to investigate the effect of preoperative abdominal exercises in preventing/minimising postoperative abdominal muscle weakness in this group.

Sorting mechanisms regulating membrane protein traffic in the apical transcytotic pathway of polarized MDCK cells.

The transcytotic pathway followed by the polymeric IgA receptor (pIgR) carrying its bound ligand (dIgA) from the basolateral to the apical surface of polarized MDCK cells has been mapped using morphological tracers. At 20 degreesC dIgA-pIgR internalize to interconnected groups of vacuoles and tubules that comprise the endosomal compartment and in which they codistribute with internalized transferrin receptors (TR) and epidermal growth factor receptors (EGFR). Upon transfer to 37 degreesC the endosome vacuoles develop long tubules that give rise to a distinctive population of 100-nm-diam cup-shaped vesicles containing pIgR. At the same time, the endosome gives rise to multivesicular endosomes (MVB) enriched in EGFR and to 60-nm-diam basolateral vesicles. The cup-shaped vesicles carry the dIgA/pIgR complexes to the apical surface where they exocytose. Using video microscopy and correlative electron microscopy to study cells grown thin and flat we show that endosome vacuoles tubulate in response to dIgA/pIgR but that the tubules contain TR as well as pIgR. However, we show that TR are removed from these dIgA-induced tubules via clathrin-coated buds and, as a result, the cup-shaped vesicles to which the tubules give rise become enriched in dIgA/pIgR. Taken together with the published information available on pIgR trafficking signals, our observations suggest that the steady-state concentrations of TR and unoccupied pIgR on the basolateral surface of polarized MDCK cells are maintained by a signal-dependent, clathrin-based sorting mechanism that operates along the length of the transcytotic pathway. We propose that the differential sorting of occupied receptors within the MDCK endosome is achieved by this clathrin-based mechanism continuously retrieving receptors like TR from the pathways that deliver pIgR to the apical surface and EGFR to the lysosome.

In polarized MDCK cells basolateral vesicles arise from clathrin-gamma-adaptin-coated domains on endosomal tubules.

Human transferrin receptors (TR) and receptors for polymeric immunoglobulins (pIgR) expressed in polarized MDCK cells maintain steady-state, asymmetric distributions on the separate basolateral and apical surfaces even though they are trafficking continuously into and across these cells. The intracellular mechanisms required to maintain these asymmetric distributions have not been located. Here we show that TR and pIgR internalize from both surfaces to a common interconnected endosome compartment that includes tubules with buds coated with clathrin lattices. These buds generate vesicles that carry TR to the basolateral border. The lattices contain gamma-adaptin and are dispersed by treatment with brefeldin A (BFA). Since BFA treatment abrogates the vectorial trafficking of TR in polarized MDCK cells, we propose that the clathrin-coated domains of the endosome tubules contain the polarized sorting mechanism responsible for their preferential basolateral distribution.

The kinetics of mannose 6-phosphate receptor trafficking in the endocytic pathway in HEp-2 cells: the receptor enters and rapidly leaves multivesicular endosomes without accumulating in a prelysosomal compartment.

We have previously shown that in HEp-2 cells, multivesicular bodies (MVBs) processing internalized epidermal growth factor-epidermal growth factor receptor complexes mature and fuse directly with lysosomes in which the complexes are degraded. The MVBs do not fuse with a prelysosomal compartment enriched in mannose 6-phosphate receptor (M6PR) as has been described in other cell types. Here we show that the cation-independent M6PR does not become enriched in the endocytic pathway en route to the lysosome, but if a pulse of M6PR or an M6PR ligand, cathepsin D, is followed, a significant fraction of these proteins are routed from the trans-Golgi to MVBs. Accumulation of M6PR does not occur because when the ligand dissociates, the receptor rapidly leaves the MVB. At steady state, most M6PR are distributed within the trans-Golgi and trans-Golgi network and in vacuolar structures distributed in the peripheral cytoplasm. We suggest that these M6PR-rich vacuoles are on the return route from MVBs to the trans-Golgi network and that a separate stable M6PR-rich compartment equivalent to the late endosome/prelysosome stage does not exist on the endosome-lysosome pathway in these cells.

The MAP kinase kinase kinase MLK2 co-localizes with activated JNK along microtubules and associates with kinesin superfamily motor KIF3.

The MLK (mixed lineage) ser/thr kinases are most closely related to the MAP kinase kinase kinase family. In addition to a kinase domain, MLK1, MLK2 and MLK3 each contain an SH3 domain, a leucine zipper domain and a potential Rac/Cdc42 GTPase-binding (CRIB) motif. The C-terminal regions of the proteins are essentially unrelated. Using yeast two-hybrid analysis and in vitro dot-blots, we show that MLK2 and MLK3 interact with the activated (GTP-bound) forms of Rac and Cdc42, with a slight preference for Rac. Transfection of MLK2 into COS cells leads to strong and constitutive activation of the JNK (c-Jun N-terminal kinase) MAP kinase cascade, but also to activation of ERK (extracellular signal-regulated kinase) and p38. When expressed in fibroblasts, MLK2 co-localizes with active, dually phosphorylated JNK1/2 to punctate structures along microtubules. In an attempt to identify proteins that affect the activity and localization of MLK2, we have screened a yeast two-hybrid cDNA library. MLK2 and MLK3 interact with members of the KIF3 family of kinesin superfamily motor proteins and with KAP3A, the putative targeting component of KIF3 motor complexes, suggesting a potential link between stress activation and motor protein function.

Multivesicular endosomes containing internalized EGF-EGF receptor complexes mature and then fuse directly with lysosomes.

We have followed the transfer of EGF-EGF receptor (EGFR) complexes from endosomal vacuoles that contain transferrin receptors (TfR) to lysosome vacuoles identified by their content of HRP loaded as a 15-min pulse 4 h previously. We show that the HRP-loaded lysosomes are lysosomal-associated membrane protein-1 (LAMP-1) positive, mannose-6-phosphate receptor (M6PR) negative. and contain active acid hydrolase. EGF-EGFR complexes are delivered to these lysosomes intact and are then rapidly degraded. Preactivating the HRP contained within the preloaded lysosomes inhibits the delivery of EGFR and degradation of EGF, and results in the accumulation of EGFR-containing multivesicular bodies (MVB). With time these accumulating MVB undergo a series of maturation changes that include the loss of TfR, the continued recruitment of EGFR, and the accumulation of internal vesicles, but they remain LAMP-1 and M6PR negative. The mature MVB are often seen to make direct contact with lysosomes containing preactivated HRP, but their perimeter membranes remain intact. Together our observations suggest that the transfer of EGF-EGFR complexes from the TfR-containing endosome compartment to the lysosomes that degrade them employs a single vacuolar intermediate, the maturing MVB, and can be achieved by a single heterotypic fusion step.

Newly synthesized transferrin receptors can be detected in the endosome before they appear on the cell surface.

It is well established that a proportion of newly synthesized lysosomal enzymes and class II major histocompatibility complex antigens are delivered directly to the endocytic pathway from the Golgi complex. Here we show that a significant proportion of newly synthesized transferrin receptors can be detected in endosomes before reaching the cell surface. These newly synthesized transferrin receptors are delivered to the endosome more efficiently than either constitutively secreted soluble proteins or glycophosphatidylinositol-anchored plasma membrane proteins suggesting that their transfer to the endosome is signal-dependent. Identification of a signal-dependent transfer step for proteins like the transferrin receptor operating on the exocytic pathway has important implications for membrane biogenesis, especially in the establishment of cell surface polarity.

Transport into and out of the Golgi complex studied by transfecting cells with cDNAs encoding horseradish peroxidase.

We have developed a novel technique with which to investigate the morphological basis of exocytotic traffic. We have used expression of HRP from cDNA in a variety of cells in combination with peroxidase cytochemistry to outline traffic into and out of the Golgi apparatus at the electron microscopic level with very high sensitivity. A secretory form of the peroxidase (ssHRP) is active from the beginning of the secretory pathway and the activity is efficiently cleared from cells. Investigation of the morphological elements involved in the itinerary of soluble ER proteins using ssHRP tagged with the ER retention motif (ssHRPKDEL) shows that it progresses through the Golgi stack no further than the cis-most element. Traffic between the RER and the Golgi stack as outlined by ssHRPKDEL occurs via vesicular carriers as well as by tubular elements. ssHRP has also been used to investigate the trans side of the Golgi complex, where incubation at reduced temperatures outlines the trans-Golgi network with HRP reaction product. Tracing the endosomal compartment with transferrin receptor in double-labeling experiments with ssHRP fails to show any overlap between these two compartments.

Annexin I is phosphorylated in the multivesicular body during the processing of the epidermal growth factor receptor.

We have previously shown that an active epidermal growth factor receptor (EGF-R) kinase is necessary for efficient sorting of the EGF-R to the lysosome, and we have shown that this occurs in the multivesicular body (MVB), where EGF-R are sorted away from recycling receptors by being removed to the internal vesicles of the MVB. The aim of the present study was to identify substrates of the EGF-R kinase associated with MVBs which might play a role in this sorting process. We used a density shift technique to isolate MVBs and show that the major substrates phosphorylated in vitro within MVBs which contain an active EGF-R kinase are the EGF-R itself and annexin I. Annexin I is associated with both plasma membrane and MVBs in a calcium-independent manner but can be phosphorylated in vitro only in MVBs. Phosphorylation of calcium-independent annexin I in isolated MVBs converts it to a form that requires calcium for membrane association. In cells with an active EGF-R kinase the amount of calcium-independent annexin I in MVBs is reduced, suggesting that a phosphorylation-induced conversion of the calcium independent to the calcium-dependent form also occurs in vivo. Our observations, together with the known properties of annexin I in mediating membrane fusion, suggest that inward vesiculation in MVBs is induced by the EGF-R and is mediated by phosphorylated annexin I.

Mechanism of action of cytotoxic antibodies to adipocytes on adipose tissue, liver and food intake in the rat.

The aim of this study was to characterize in greater detail the effects of anti-(rat adipocyte plasma membrane) antisera (A/S 83 and A/S 164) that had previously been shown to cause large reductions in total body fat. Both antisera produced lymphocytic infiltration of adipose tissue and reduction of adipocyte numbers seven days after treatment as well as a reduction in food intake and body weight on the first day of treatment. In addition A/S 164 produced a sedative effect for 2-4 h after treatment and induced gross abnormalities of the liver after seven days. Antibody-mediated complement activation was shown to be a critical requirement for all of these effects since animals treated with cobra venom factor (CVF) to deplete serum complement levels showed none of the described effects. Further evidence in support of a role for complement was the large decrease in serum complement levels 12 h after antiserum treatment. The effects of A/S 164 on liver morphology could be successfully dissociated from those on adipose tissue by adsorption of the antiserum with liver membranes. The adsorbed antiserum, which retained in vitro reactivity with adipose tissue but not with liver, induced a significant reduction in adipose tissue mass in vivo whilst the effect on liver morphology was almost completely abolished. Serum free fatty acid and triglyceride levels increased 6-24 h after treatment but then returned to normal, suggesting a very transient release of adipose tissue triglycerides. These results indicate that complement activation is a common pathway for all of the effects produced.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased growth and protein deposition in rats treated with antibodies to adipocytes.

Young growing rats treated with an antiserum to adipocytes showed marked reductions in several adipose tissue depots but surprisingly demonstrated increased body weight gain. During the first 3 wk after treatment body weight gain increased by 17% with no effect on food intake, whereas during weeks 3-7 body weight gain increased by 40-50% and was accompanied by a 15% increase in food intake. These animals thus exhibited increased food conversion efficiencies (intake/gain) of approximately 15-20% for almost 2 mo. Subsequently, food intake and body weight gain returned to normal (although body weight remained elevated) up to 6 mo. By this time several fat depots were still reduced in size, although total (chemical) fat was identical in treated and control groups. These results suggest that 1) reduction of body fat depots can be achieved using antibodies to adipocytes, 2) compensatory increases in lean body mass can occur, and 3) total fat mass may be regulated.