Validation of ion mobility spectrometry - mass spectrometry as a screening tool to identify type II kinase inhibitors of FGFR1 kinase.

RATIONALE: The protein kinase FGFR1 regulates cellular processes in human development. As over-activity of FGFR1 is implicated with cancer, effective inhibitors are in demand. Type I inhibitors, which bind to the active form of FGFR1, are less effective than type II inhibitors, which bind to the inactive form. Screening to distinguish between type I and type II inhibitors is required. METHODS: X-ray crystallography was used to indicate whether a range of potential inhibitors bind to the active or inactive FGFR1 kinase conformation. The binding affinity of each ligand to FGFR1 was measured using biochemical methods. Electrospray ionisation - ion mobility spectrometry - mass spectrometry (ESI-IMS-MS) in conjunction with collision-induced protein unfolding generated a conformational profile of each FGFR1-ligand complex. The results indicate that the protein's conformational profile depends on whether the inhibitor is type I or type II. RESULTS: X-ray crystallography confirmed which of the kinase inhibitors bind to the active or inactive form of FGFR1 kinase. Collision-induced unfolding combined with ESI-IMS-MS showed distinct differences in the FGFR1 folding landscape for type I and type II inhibitors. Biochemical studies indicated a similar range of FGFR1 affinities for both types of inhibitors, thus providing confidence that the conformational variations detected using ESI-IMS-MS can be interpretated unequivocally and that this is an effective screening method. CONCLUSIONS: A robust ESI-IMS-MS method has been implemented to distinguish between the binding mode of type I and type II inhibitors by monitoring the conformational unfolding profile of FGFR1. This rapid method requires low sample concentrations and could be used as a high-throughput screening technique for the characterisation of novel kinase inhibitors.

Hypopharyngeal Perforation Mimicking Esophageal Atresia: A Case Report of an Extremely Low Birth Weight Infant Emphasizing the Need for Preoperative Endoscopy.

We report a case of an extremely low birth weight premature infant born at 27 weeks of gestation, transferred to our tertiary pediatric referral center for surgical repair of an esophageal atresia. Endoscopic evaluation before the start of surgery revealed a hypopharyngeal perforation, resulting in the false impression of esophageal atresia. If no tracheoesophageal fistula is found during tracheoscopy, esophagoscopy should be done before surgical intervention as the inability to pass a nasogastric tube into the stomach is not sufficiently reliable for a correct diagnosis of esophageal atresia.

[Hand burn injuries in children]. / Thermische Handverletzungen im Kindesalter.

A consistently high proportion of thermal injuries in children are to the hand, and scalds and contact burns are the main causes. While most thermal Injuries to the hand in children can be treated conservatively, deep burns can result in scary contractures and syndactylies that cause functional impairments to the hand. Therefore, thermal injuries to the hand in children should be treated in a specialised centre, thus ensuring a differentiated approach with respect to the localisation and extent of the thermal injury.Besides acute therapy, regular follow-up consultations - including splint and compression treatments -, physiotherapy, ergotherapy and, if necessary, corrective surgical measures are of immense importance. Only adherence to this treatment regime can guarantee optimal functional and aesthetic results and minimise daily restrictions for the young patients. The purpose of this article is to illustrate/outline the essential aspects of this treatment of thermal injuries to the infantile hand.

Functional Brush Poly(2-ethyl-2-oxazine)s: Synthesis by CROP and RAFT, Thermoresponsiveness and Grafting onto Iron Oxide Nanoparticles.

Brush polymers are highly functional polymeric materials combining the properties of different polymer classes and have found numerous applications, for example, in nanomedicine. Here, the synthesis of functional phosphonate-ester-bearing brush polymers based on poly(2-oxazine)s is reported through a combination of cationic ring-opening polymerization (CROP) of 2-ethyl-2-oxazine and reversible addition-fragmentation chain transfer (RAFT) polymerization. In this way, a small library of well-defined (D ≤ 1.17) poly(oligo(2-ethyl-2-oxazine) methacrylate) P(OEtOzMA)n brushes with tunable lower critical solution temperature (LCST) behavior and negligible cell toxicity is prepared. Upon deprotection, the phosphonic acid end-group of the P(OEtOzMA)n brush enables the successful grafting-onto iron oxide nanoparticles (IONPs). Colloidal stability of the particle suspension in combination with suitable magnetic resonance imaging (MRI) relaxivities demonstrates the potential of these particles for future applications as negative MRI contrast agents.

The Cologne pouch procedure for continent anal urinary diversion in children with bladder exstrophy-epispadias complex.

INTRODUCTION: In children who remain incontinent after reconstruction of bladder exstrophy-epispadias complex (BEEC), continent anal urinary diversion (CAD) is one option to achieve continence. Known problems after CAD are an increased stool frequency and ureterointestinal stenosis. We devised a new surgical technique of CAD that we named the "Cologne pouch procedure" (CPP) that renders the possibility of separate evacuation of urine and feces. Furthermore, we connect the bladder plate to the rectosigmoid pouch instead of performing a ureterosigmoidostomy to reduce the rate of ureterointestinal stenosis. In this study, we want to introduce the CCP and critically evaluate our results. STUDY DESIGN: In CPP a detubularized sigmoid-bladder pouch is created, which is naturally connected to the rectum. A retrospective study was performed including all patients with BEEC and CPP treated in our hospital between January 1, 2007, and December 31, 2016. Epidemiological and surgical key data, complications, and the need for alkaline supplementation were assessed. At follow-up examinations, we evaluated continence, ability of independent urine and feces evacuation, need for bicarbonate supplementation, status of the upper urinary tract, and complications such as urinary tract infections or urolithiasis. RESULTS: In total, 29 patients with BEEC and CPP were included. The mean age at surgery was 4.2 ± 3.3 years (range 0.1-12.7 years). Overall, 14 short-term complications occurred in nine patients. Postoperatively, all patients were continent for urine and feces during daytime and only one child occasionally lost small portions of urine at night. An independent evacuation of urine and feces was accomplished in 22 patients (81.5%). Continued bicarbonate supplementation was necessary in 15 patients (55.6%). During the follow-up period six patients (22.2%) had a single urinary tract infection and four patients (14.8%) calculi of the urinary tract. No urinary tract abnormalities-especially no vesicoureteral reflux (VUR) or stenosis-were detected during follow-up ultrasound examination. In two children, a preoperatively known hydronephrosis decreased after CPP. CONCLUSION: CPP is a novel technique that yields excellent results concerning continence. In contrast to other forms of rectosigmoid urinary diversion, functional separation of defecation and urination can be achieved in most patients.

Hydrolyzable Poly[Poly(Ethylene Glycol) Methyl Ether Acrylate]-Colistin Prodrugs through Copper-Mediated Photoinduced Living Radical Polymerization.

Through the recently developed copper-mediated photoinduced living radical polymerization (CP-LRP), a novel and well-defined polymeric prodrug of the antimicrobial lipopeptide colistin has been developed. A colistin initiator (Boc5-col-Br2) was synthesized through the modification of colistin on both of its threonine residues using a cleavable initiator linker, 2-(2-bromo-2-methylpropanoyloxy) acetic acid (BMPAA), and used for the polymerization of acrylates via CP-LRP. Polymerization proceeds from both sites of the colistin initiator, and through the polymerization of poly(ethylene glycol) methyl ether acrylate (PEGA480), three water-soluble polymer-colistin conjugates (col-PPEGA, having degrees of polymerization of 5, 10, and 20) were achieved with high yield (conversion of ≥93%) and narrow dispersities (D < 1.3) in 2-4 h. Little or no effect on the structure and activity of the colistin was observed during the synthesis, and most of the active colistin can be recovered from the conjugates in vitro within 2 days. Furthermore, in vitro biological analyses including disk diffusion, broth microdilution, and time-kill studies suggested that all of the conjugates have the ability to inhibit the growth of multidrug-resistant (MDR) Gram-negative bacteria, of which col-PPEGA DP5 and DP10 showed similar or better antibacterial performance compared to the clinically relevant colistin prodrug CMS, indicating their potential as an alternative antimicrobial therapy. Moreover, considering the control over the polymerization, the CP-LRP technique has the potential to provide an alternative platform for the development of polymer bioconjugates.

Immobilization of Cysteine-Tagged Proteins on Electrode Surfaces by Thiol-Ene Click Chemistry.

Thiol-ene click chemistry can be exploited for the immobilization of cysteine-tagged dehydrogenases in an active form onto carbon electrodes (glassy carbon and carbon felt). The electrode surfaces have been first modified with vinylphenyl groups by electrochemical reduction of the corresponding diazonium salts generated in situ from 4-vinylaniline. The grafting process has been optimized in order to not hinder the electrochemical regeneration of NAD(+)/NADH cofactor and soluble mediators such as ferrocenedimethanol and [Cp*Rh(bpy)Cl](+). Having demonstrated the feasibility of thiol-ene click chemistry for attaching ferrocene moieties onto those carbon surfaces, the same approach was then applied to the immobilization of d-sorbitol dehydrogenases with cysteine tag. These proteins can be effectively immobilized (as pointed out by XPS), and the cysteine tag (either 1 or 2 cysteine moieties at the N terminus of the polypeptide chain) was proven to maintain the enzymatic activity of the dehydrogenase upon grafting. The bioelectrode was applied to electroenzymatic enantioselective reduction of d-fructose to d-sorbitol, as a case study.

An experimental study in six fresh human cadavers using a novel approach to avoid abdominal wall incisions in total colectomy: totally transanal laparoendoscopic single-site pull-through colectomy with J-pouch creation.

BACKGROUND: The use of transanal laparoscopic access to completely avoid abdominal wall incisions represents the most current evolution in minimally invasive surgery. The combination of single-site surgery and natural orifice transluminal endoscopic surgery (NOTES™) can be used for totally transanal laparoendoscopic pull-through colectomy with J-pouch creation (TLPC-J). The aim of the present study was to provide evidence for the feasibility of TLPC-J in adult human cadavers. METHODS: TLPC-J was performed in six fresh adult human cadavers. The procedure involved endorectal submucosal dissection from 1 cm above the dentate line to a point above the peritoneal reflection, where the rectal muscle was divided circumferentially. The edge of the mucosal cuff was closed distally in order to prevent fecal contamination and the endorectal tube was placed back into the abdomen. A Triport+™ or QuadPort+™ system was introduced transanally, and it served as a multiport device (MD). Resection of the entire colon, mobilization of the distal ileal segment, and extracorporeal suture of the ileal J-loop were performed via the transanal approach. The J-pouch was created using Endo GIA™. After removal of the MD, the J-pouch was sutured to the rectal wall. RESULTS: TLPC-J was performed in all cadavers, with a mean operation duration of 236 ± 22 min. Conversion to either transabdominal laparoscopy or laparotomy was not required in any of the cadavers. No bowel perforation or damage to other organs was observed. The use of a curved endoscope greatly facilitated visualization during transanal laparoscopic dissection for partial and total colectomy, making the procedure feasible. All specimens were retrieved through the anus, eliminating the need for additional transabdominal incisions. CONCLUSIONS: TLPC-J was technically feasible in adult human cadavers, and abdominal wall incisions were not required. However, clinical studies are needed to determine its feasibility in living adults.

Experiences with LESS-appendectomy in Children.

PURPOSE: To evaluate the outcome of laparoendoscopic single-site (LESS-A) through one transumbilical port vs. 3-port laparoscopic (3TA) appendectomy in children. METHODS: We reviewed the records of 309 children (65 LESS-A, 244 3TA) operated on between 2008 and 2012. One hundered forty-nine patients had acute catarrhalis (CA), 133 phlegmonous (PLA), and 27 perforated appendicitis (PA). We compared the duration of operation (DO) the incidence of abdominal abscesses (AA) and wound infections (WI), as well as the degree of appendiceal inflammation (DI) among surgeons with and without board certification. RESULTS: For all DI, LESS-A resulted in a shorter DO than 3TA (CA 57.9 ± 22.8 vs. 68.5 ± 23.2, P = 0.014; PLA 51.5±16.5 vs. 68.4±33.0, P = 0.006; PA 66.0 ± 29.0 vs. 97.3 ± 41.8, P = 0.039). LESS-A was not used for less complicated cases when compared to 3TA (CA 50.8% vs. 47.5%; PLA 33.8% vs. 45.5%; PA 15.4% vs. 7.0%; CA vs. PLA, P = 0.292; CA vs. PA, P = 0.142; PLA vs. PA, P = 0.031). Surgeons without board certification were assigned to a similar percentage to perform both techniques for any DI (CA 30.3% vs. 37.1%, P = 0.541; PLA 31.8% vs. 40.5%, P= 0.484; PA 40% vs. 35.3%, P = 1.0). We found no significant differences concerning AA (1.5% vs. 1.2%, P = 1.0) and WI (3.1% vs. 1.6%, P = 0.61). CONCLUSIONS: LESS-A can be done by surgeons with and without board certification for all DI, with shorter DO and similar complication rates as compared to 3TA.

Can a simplified algorithm prevent incomplete laparoscopic pyloromyotomy?

PURPOSE: The purpose of this study is to analyze an algorithm intended to prevent incomplete pyloromyotomy in 3-port laparoscopic (3TP) and laparoendoscopic single-site (LESS-P) procedures in a teaching hospital. METHODS: We defined the pyloroduodenal and pyloroantral junctions as anatomical margins prior pyloromyotomy by palpating and coagulating the serosa with the hook cautery instrument. Incomplete pyloromyotomies, mucosa perforations, serosa lacerations, and wound infections were recorded for pediatric surgical trainees (PST) and board-certified pediatric surgeons (BC). RESULTS: We reviewed the medical files of 233 infants, who underwent LESS-P (n=21), 3TP (n=71), and open pyloromyotomy (OP, n=141). No incomplete pyloromyotomies occurred. In contrast to OP, mucosa perforations did not occur in the laparoscopic procedures during the study period (6.38% vs. 0%, P=.013). OP had insignificantly more serosal lacerations (3.5% vs. 1.4%, P=.407). There was no difference in the rate of wound infections between OP and laparoscopic procedures (2.8% vs. 4.3%, P=.715). In the latter, all wound infections were associated with the use of skin adhesive. CONCLUSIONS: This algorithm helps avoiding incomplete laparoscopic pyloromyotomy during the learning curve and in a teaching setting. It is not risky to assist 3TP and LESS-P to PST as this led to a decreased rate of mucosa perforations without experiencing incomplete pyloromyotomies.

Surgical Repair of a Superior Sternal Cleft in an Infant.

Sternal cleft is a rare congenital malformation with little more than 100 cases published worldwide. Incomplete sternal clefting in a female newborn is the most frequent form seen. First-line treatment is the surgical defect closure in the neonatal period. Presurgical examination has to focus on common associated malformations, in particular cardiac defects. The surgical repair of sternal cleft itself shows satisfying functional and cosmetic results with low complication rates. We present the case of a 4-month-old male infant with a superior sternal cleft.

Structural insights into FGFR kinase isoform selectivity: diverse binding modes of AZD4547 and ponatinib in complex with FGFR1 and FGFR4.

The fibroblast growth factor receptor (FGFR) family of receptor tyrosine kinases has been implicated in a wide variety of cancers. Despite a high level of sequence homology in the ATP-binding site, the majority of reported inhibitors are selective for the FGFR1-3 isoforms and display much reduced potency toward FGFR4, an exception being the Bcr-Abl inhibitor ponatinib. Here we present the crystal structure of the FGFR4 kinase domain and show that both FGFR1 and FGFR4 kinase domains in complex with ponatinib adopt a DFG-out activation loop conformation. Comparison with the structure of FGFR1 in complex with the candidate drug AZD4547, combined with kinetic characterization of the binding of ponatinib and AZD4547 to FGFR1 and FGFR4, sheds light on the observed differences in selectivity profiles and provides a rationale for developing FGFR4-selective inhibitors.

FGFR1 Kinase Inhibitors: Close Regioisomers Adopt Divergent Binding Modes and Display Distinct Biophysical Signatures.

The binding of a ligand to its target protein is often accompanied by conformational changes of both the protein and the ligand. This is of particular interest, since structural rearrangements of the macromolecular target and the ligand influence the free energy change upon complex formation. In this study, we use X-ray crystallography, isothermal titration calorimetry, and surface-plasmon resonance biosensor analysis to investigate the binding of pyrazolylaminopyrimidine inhibitors to FGFR1 tyrosine kinase, an important anticancer target. Our results highlight that structurally close analogs of this inhibitor series interact with FGFR1 with different binding modes, which are a consequence of conformational changes in both the protein and the ligand as well as the bound water network. Together with the collected kinetic and thermodynamic data, we use the protein-ligand crystal structure information to rationalize the observed inhibitory potencies on a molecular level.

A system of miniaturized stirred bioreactors for parallel continuous cultivation of yeast with online measurement of dissolved oxygen and off-gas.

Chemostat cultivation is a powerful tool for physiological studies of microorganisms. We report the construction and application of a set of eight parallel small-scale bioreactors with a working volume of 10 mL for continuous cultivation. Hungate tubes were used as culture vessels connected to multichannel-peristaltic pumps for feeding fresh media and removal of culture broth and off-gas. Water saturated air is sucked into the bioreactors by applying negative pressure, and small stirrer bars inside the culture vessels allow sufficient mixing and oxygen transfer. Optical sensors are used for non-invasive online measurement of dissolved oxygen, which proved to be a powerful indicator of the physiological state of the cultures, particularly of steady-state conditions. Analysis of culture exhaust-gas by means of mass spectrometry enables balancing of carbon. The capacity of the developed small-scale bioreactor system was validated using the fission yeast Schizosaccharomyces pombe, focusing on the metabolic shift from respiratory to respiro-fermentative metabolism, as well as studies on consumption of different substrates such as glucose, fructose, and gluconate. In all cases, an almost completely closed carbon balance was obtained proving the reliability of the experimental setup.

Evaluation of the use of lower body perfusion at 28°C in aortic arch surgery.

OBJECTIVES: Although hypothermic circulatory arrest (HCA) and selective cerebral perfusion (SCP) are widely used for cerebral protection during aortic arch surgery, these strategies offer no protection for mesenteric ischaemia during prolonged circulatory arrest. This study explored mesenteric haemodynamics, metabolism, oxidative stress and inflammatory response levels during isolated SCP and combined cerebral and lower body perfusion (CLBP) in pigs. METHODS: Fourteen pigs (35-45 kg) were cooled on CPB to 28°C. After 10 min of HCA, they were randomized to 60 min of isolated SCP (n = 7) and CLBP (n = 7) at low-flow pump rates: 10 ml/kg/min (SCP) and 20 ml/kg/min (LBP). Microspheres were injected at baseline, 5 and 60 min of SCP/CLBP and 5 and 60 min off CPB, to calculate mesenteric regional blood flow (RBF). Lactate levels and Oxy-DNA expression [fluorescence activated cell sorting (FACS)] in the portal venous blood were determined at the same time points. Semi-quantitative assessment of inflammatory cytokines was performed using real-time polymerase chain reaction (PCR) and immunhistochemical analyses. RESULTS: At baseline mesenteric, RBF was 61 ± 31 ml/min/100 g in the jejunum and 78 ± 43 ml/min/100 g in the colon. Whereas SCP provided a residual mesenteric RBF of 5%, CLBP offered 47% of the baseline jejunal (34 ± 10 ml/min/100 g) and 68% of the colonic RBF (52 ± 34 ml/min/100 g; P = 0.001). Lactate levels were significantly higher in then SCP group (15 ± 2 vs. 11 ± 3 mmol/l; P = 0.01). Oxy-DNA increased, reaching 137% of baseline (SCP) and 129% (CLBP) at 60 min SCP/CLBP, but recovered promptly during reperfusion. Real-time PCR revealed a massive increase in early cytokine expression vs. baseline, showing significant higher interleukin (IL) -6 (29 vs.2; P = 0.027) and COX-relative expression (7 vs. 3, P = 0.016) in the SCP group. Immunhistochemical analysis confirmed a higher immunological activity in the SCP group, showing more intensive signal for tumour necrosis factor-α, IL-6 and p38 when compared with the CLBP group. CONCLUSIONS: Low-flow CLBP provides a diminished but considerable mesenteric RBF, leading to lower lactate and oxidative stress levels and a diminished local inflammatory response reaction than isolated SCP.

Comparison of real-time elastography with grey-scale ultrasonography for detection of organ-confined prostate cancer and extra capsular extension: a prospective analysis using whole mount sections after radical prostatectomy.

OBJECTIVE: •To evaluate whether transrectal real-time elastography (RTE) improves the detection of intraprostatic prostate cancer (PCa) lesions and extracapsular extension (ECE) compared with conventional grey-scale ultrasonography (GSU). PATIENTS AND METHODS: •In total, 229 patients with biopsy-proven PCa were prospectively screened for cancer-suspicious areas and ECE using GSU and RTE. •The largest tumour focus detected by RTE was defined as the index lesion. •The prostate gland was stratified into six sectors on GSU and RTE, which were compared with histopathological whole mount sections after radical prostatectomy. RESULTS: •Histopathologically, PCa was confirmed in 894 out of 1374 (61.8%) evaluated sectors and ECE was identified in 47 (21%) patients. •Of these 894 sectors, RTE correctly detected 594 (66.4%) and GSU 215 (24.0%) cancer suspicious lesions. •Sensitivity was 51% and specificity 72% using RTE compared to 18% and 90% for GSU. •RTE identified the largest side specific tumour focus in 68% of patients. •ECE was identified with a sensitivity of 38% and specificity of 96% using RTE compared to 15% and 97% using GSU. CONCLUSIONS: •Compared with GSU, RTE provides a statistically significant improvement in detection of PCa lesions and ECE. •RTE enhances GSU, although improvement is still needed to achieve a clinically meaningful sensitivity.

Bicyclic substituted hydroxyphenylmethanone type inhibitors of 17 ß-hydroxysteroid dehydrogenase Type†1 (17 ß-HSD1): the role of the bicyclic moiety.

An attractive target that has still to be explored for the treatment of estrogen-dependent diseases, such as breast cancer and endometriosis, is the enzyme responsible for the last step in the biosynthesis of estradiol (E2): 17ß-hydroxysteroid dehydrogenase type 1 (17ß-HSD1). It catalyzes the reduction of the weakly active estrone (E1) into E2, which is the most potent estrogen in humans. Inhibition of 17ß-HSD1 lowers intracellular E2 concentrations and thus presents a therapeutic target for estrogen-dependent pathologies. Recently, we reported a new class of highly active and selective 17ß-HSD1 inhibitors: bicyclic substituted hydroxyphenylmethanones. Here, further structural variations on the bicyclic moiety are described, especially focusing on the exchange of its hydroxy function. Twenty-nine novel inhibitors were synthesized and evaluated for 17ß-HSD1 inhibition in a cell-free and cellular assay, for selectivity toward 17ßHSD2 and estrogen receptors (ER) alpha and beta, as well as for metabolic stability. The best compound exhibited IC50 values of 12 nM (cell-free assay) and 78 nM (cellular assay), high selectivity for 17ß-HSD1, and reasonable metabolic stability. A molecular docking study provided insight into the protein-ligand interactions of this compound with 17ß-HSD1.

17ß-Hydroxysteroid dehydrogenases (17ß-HSDs) as therapeutic targets: protein structures, functions, and recent progress in inhibitor development.

17ß-Hydroxysteroid dehydrogenases (17ß-HSDs) are oxidoreductases, which play a key role in estrogen and androgen steroid metabolism by catalyzing final steps of the steroid biosynthesis. Up to now, 14 different subtypes have been identified in mammals, which catalyze NAD(P)H or NAD(P)(+) dependent reductions/oxidations at the 17-position of the steroid. Depending on their reductive or oxidative activities, they modulate the intracellular concentration of inactive and active steroids. As the genomic mechanism of steroid action involves binding to a steroid nuclear receptor, 17ß-HSDs act like pre-receptor molecular switches. 17ß-HSDs are thus key enzymes implicated in the different functions of the reproductive tissues in both males and females. The crucial role of estrogens and androgens in the genesis and development of hormone dependent diseases is well recognized. Considering the pivotal role of 17ß-HSDs in steroid hormone modulation and their substrate specificity, these proteins are promising therapeutic targets for diseases like breast cancer, endometriosis, osteoporosis, and prostate cancer. The selective inhibition of the concerned enzymes might provide an effective treatment and a good alternative to the existing endocrine therapies. Herein, we give an overview of functional and structural aspects for the different 17ß-HSDs. We focus on steroidal and non-steroidal inhibitors recently published for each subtype and report on existing animal models for the different 17ß-HSDs and the respective diseases. Article from the Special issue on Targeted Inhibitors.

Impact of pump flow rate during selective cerebral perfusion on cerebral hemodynamics and metabolism.

BACKGROUND: Although hypothermic selective cerebral perfusion (SCP) is widely used for cerebral protection during aortic surgery, little is known about the ideal pump-flow management during this procedure. This study explored cerebral hemodynamics and metabolism at two different flow rates. METHODS: Fourteen pigs (33 to 38 kg) were cooled on cardiopulmonary bypass to 25°C. After 10 minutes of hypothermic circulatory arrest, the animals were randomly assigned to 60 minutes of SCP at two different pump flow rates: 8 mL·kg(-1)·min(-1) (n = 7) and 18 mL·kg(-1)·min(-1) (n = 7). Microspheres were injected at baseline, coolest temperature, and at 5, 15, 25, and 60 minutes of SCP to calculate cerebral blood flow, cerebral vascular resistance, metabolic rate, and intracranial pressure. RESULTS: Cerebral blood flow decreased during cooling to 41% of the baseline value (from 57 ± 10 to 23 ± 4 mL·min(-1)·100 g(-1)). It recovered during the initial 15 minutes of SCP, showing a significantly higher increase (p = 0.017) at high-flow versus low-flow perfusion (139 ± 41 versus 75 ± 22 mL·min(-1)·100 g(-1)). After 60 minutes of SCP the cerebral blood flow almost returned to baseline values in the low-flow group (43 ± 25 mL·min(-1)·100 g(-1)), but showed an unexpected decrease (30 ± 7 mL·min(-1)·100 g(-1)) in the high-flow group. The highest regional cerebral blood flow was seen in the cortex (66 ± 12 mL·min(-1)·100 g(-1)), followed by the cerebellum (63 ± 12 mL·min(-1)·100 g(-1)), the pons (51 ± 17 mL·min(-1)·100 g(-1)), and the hippocampus (36 ± 9 mL·min(-1)·100 g(-1)). Intracranial pressure increased from 11 ± 3 to 13 ± 5 mm Hg during cooling on cardiopulmonary bypass. During low-flow SCP, it stayed stable at baseline values, whereas high-flow perfusion resulted in significantly higher intracranial pressures (17 ± 3 mm Hg; p = 0.001). Changes in cerebral vascular resistance and metabolic rate showed no significant differences between the groups. CONCLUSIONS: High-flow SCP provides no benefit during long-term SCP at 25°C. Higher cerebral blood flow during the initial SCP period leads to cerebral edema, with no profit in metabolic rate.

Multilayer coating of gold nanoparticles with drug-polymer coadsorbates.

The aim of our present study was the development of a drug multilayer-based carrier system for delivery of water-insoluble drugs. As drug, we applied the anticancer drug 5,10,15,20-tetrakis(3-hydroxyphenyl)porphyrin, mTHPP, which is a model photosensitizer for photodynamic therapy. Gold nanoparticles (AuNP) with a diameter of 14.5 ± 0.9 nm were prepared and used as template for the layer-by-layer approach. The drug and the negatively charged polyelectrolyte (PE) poly(styrene sulfonate) sodium salt (PSS) were complexed with a new developed method using freeze-drying. The complexation efficiency was determined to be ∼11-12 monomers PSS per mTHPP molecule by CHNS analysis and UV/vis measurement. Molecular docking simulations revealed π-π interactions and H-bonding to be the responsible mechanisms. A drug multilayer system based on the layer-by-layer (LbL) technique utilized the water-soluble complex as anionic layer material and poly(allylamine hydrochloride) (PAH) as cationic layer. The modified AuNP were characterized by different physicochemical techniques such as UV/vis, ζ-potential, ICP-OES, and TEM. To the best of our knowledge, we could demonstrate for the first time the adsorption of three drug layers to a nanoparticulate system. Furthermore, the adaptation of the LbL-technique resulted in drastically increased drug deposition efficiency (factor of 100). Furthermore, we developed a new and comfortable way to solubilize water-insoluble drugs in water.