PLPHP deficiency: clinical, genetic, biochemical, and mechanistic insights.

Biallelic pathogenic variants in PLPBP (formerly called PROSC) have recently been shown to cause a novel form of vitamin B6-dependent epilepsy, the pathophysiological basis of which is poorly understood. When left untreated, the disease can progress to status epilepticus and death in infancy. Here we present 12 previously undescribed patients and six novel pathogenic variants in PLPBP. Suspected clinical diagnoses prior to identification of PLPBP variants included mitochondrial encephalopathy (two patients), folinic acid-responsive epilepsy (one patient) and a movement disorder compatible with AADC deficiency (one patient). The encoded protein, PLPHP is believed to be crucial for B6 homeostasis. We modelled the pathogenicity of the variants and developed a clinical severity scoring system. The most severe phenotypes were associated with variants leading to loss of function of PLPBP or significantly affecting protein stability/PLP-binding. To explore the pathophysiology of this disease further, we developed the first zebrafish model of PLPHP deficiency using CRISPR/Cas9. Our model recapitulates the disease, with plpbp-/- larvae showing behavioural, biochemical, and electrophysiological signs of seizure activity by 10 days post-fertilization and early death by 16 days post-fertilization. Treatment with pyridoxine significantly improved the epileptic phenotype and extended lifespan in plpbp-/- animals. Larvae had disruptions in amino acid metabolism as well as GABA and catecholamine biosynthesis, indicating impairment of PLP-dependent enzymatic activities. Using mass spectrometry, we observed significant B6 vitamer level changes in plpbp-/- zebrafish, patient fibroblasts and PLPHP-deficient HEK293 cells. Additional studies in human cells and yeast provide the first empirical evidence that PLPHP is localized in mitochondria and may play a role in mitochondrial metabolism. These models provide new insights into disease mechanisms and can serve as a platform for drug discovery.

Elevated viscosities in a simulated moving bed for γ-aminobutyric acid recovery.

Process streams of agro-food industries are often large and viscous. In order to fractionate such a stream the viscosity can be reduced by either a high temperature or dilution, the former is not an option in case of temperature sensitive components. Such streams are diluted prior to chromatographic fractionation, resulting in even larger volumes and high energy costs for sub-sequential water removal. The influence of feed viscosity on the performance of simulated moving bed chromatography has been investigated in a case study of the recovery of a γ-aminobutyric acid rich fraction from tomato serum. This work addresses the chromatographic system design, evaluates results from a pilot scale operation, and uses these to calculate the productivity and water use at elevated feed concentration. At the two higher feed viscosities (2.5 and 4 mPa·s) water use is lower and productivity higher, compared to the lowest feed viscosity (1 mPa·s). The behavior of the sugars for different feed viscosities can be described well by the model using the ratio of feed to eluent as dilution factor. The behavior of γ-aminobutyric acid is highly concentration dependent and the recovery could not be accurately predicted.

IgG is elevated in obese white adipose tissue but does not induce glucose intolerance via Fcγ-receptor or complement.

BACKGROUND/OBJECTIVES: In obesity, B cells accumulate in white adipose tissue (WAT) and produce IgG, which may contribute to the development of glucose intolerance. IgG signals by binding to Fcγ receptors (FcγR) and by activating the complement system. The aim of our study was to investigate whether activation of FcγR and/or complement C3 mediates the development of high-fat diet-induced glucose intolerance. METHODS: We studied mice lacking all four FcγRs (FcγRI/II/III/IV-/-), only the inhibitory FcγRIIb (FcγRIIb-/-), only the central component of the complement system C3 (C3-/-), and mice lacking both FcγRs and C3 (FcγRI/II/III/IV/C3-/-). All mouse models and wild-type controls were fed a high-fat diet (HFD) for 15 weeks to induce obesity. Glucose metabolism was assessed and adipose tissue was characterized for inflammation and adipocyte functionality. RESULTS: In obese WAT of wild-type mice, B cells (+142%, P<0.01) and IgG (+128% P<0.01) were increased compared to lean WAT. Macrophages of FcγRI/II/III/IV-/-mice released lower levels of cytokines compared to wild-type mice upon IgG stimulation. Only C3-/- mice showed reduced HFD-induced weight gain as compared to controls (-18%, P<0.01). Surprisingly, FcγRI/II/III/IV-/- mice had deteriorated glucose tolerance (AUC +125%, P<0.001) despite reduced leukocyte number (-30%, P<0.05) in gonadal WAT (gWAT), whereas glucose tolerance and leukocytes within gWAT in the other models were unaffected compared to controls. Although IgG in gWAT was increased (+44 to +174%, P<0.05) in all mouse models lacking FcγRIIb, only FcγRI/II/III/IV/C3-/- mice exhibited appreciable alterations in immune cells in gWAT, for example, increased macrophages (+36%, P<0.001). CONCLUSIONS: Lack of FcγRs reduces the activity of macrophages upon IgG stimulation, but neither FcγR nor C3 deficiency protects against HFD-induced glucose intolerance or reduces adipose tissue inflammation. This indicates that if obesity-induced IgG contributes to the development of glucose intolerance, this is not mediated by FcγR or complement activation.

Influence of variations in arterial PCO2 on surgical conditions during laparoscopic retroperitoneal surgery.

BACKGROUND: Although deep neuromuscular block (post-tetanic-count 1-2 twitches) improves surgical conditions during laparoscopic retroperitoneal surgery compared with standard block (train-of-four 1-2 twitches), the quality of surgical conditions varies widely, often related to diaphragmatic contractions. Hypocapnia may improve surgical conditions. Therefore we studied the effect of changes in arterial carbon dioxide concentrations on surgical conditions in patients undergoing laparoscopic surgery under general anaesthesia and deep neuromuscular block. METHODS: Forty patients undergoing elective laparoscopic surgery for prostatectomy or nephrectomy received propofol/remifentanil anaesthesia and deep neuromuscular block with rocuronium. Patients were randomized to surgery under hypocapnic or hypercapnic conditions. During surgery, the surgical conditions were evaluated using the 5-point Leiden-Surgical Rating Scale (L-SRS) ranging from 1 (extremely poor conditions) to 5 (optimal conditions) by the surgeon, who was blinded to group. RESULTS: Mean (sd) arterial carbon dioxide concentrations were 4.5 (0.6) [range: 3.8-5.6] kPa under hypocapnic and 6.9 (0.6) [6.1-8.1] kPa under hypercapnic conditions. The L-SRS did not differ between groups: 4.84 (0.4) [4-5] in hypocapnia and 4.77 (0.4) [3.9-5] in hypercapnia. Ninety-nine percent of ratings were good or excellent irrespective of treatment. CONCLUSIONS: Deep neuromuscular block provides good to optimal surgical conditions in laparoscopic retroperitoneal urological surgery, independent of the level of arterial [Formula: see text]. CLINICAL TRIAL REGISTRATION: NCT01968447.

HDL functionality in South Asians as compared to white Caucasians.

BACKGROUND AND AIMS: South Asians have an exceptionally high risk of developing cardiovascular disease compared to white Caucasians. A contributing factor might be dysfunction of high density lipoprotein (HDL). We aimed to compare HDL function in different age groups of both ethnicities. METHODS AND RESULTS: HDL functionality with respect to cholesterol efflux, anti-oxidation and anti-inflammation was determined using fasting, apoB-depleted, plasma samples from South Asian and white Caucasian neonates (n = 14 each), adolescent healthy men (n = 12 each, 18-25 y), and adult overweight men (n = 12 each, 40-50 y). Adolescents were subjected to a 5-day high fat high calorie diet (HCD) and adults to an 8-day very low calorie diet (LCD). Additionally, HDL composition was measured in adolescents and adults using (1)H-NMR spectroscopy. Anti-oxidative capacity was lower in South Asian adults before LCD (19.4 ± 2.1 vs. 25.8 ± 1.2%, p = 0.045, 95%-CI = [0.1; 12.7]) and after LCD (16.4 ± 2.4 vs. 27.6 ± 2.7%, p = 0.001, 95%-CI = [4.9; 17.5]). Anti-inflammatory capacity was reduced in South Asian neonates (23.8 ± 1.2 vs. 34.9 ± 1.3%, p = 0.000001, 95%-CI = [-14.6; -7.5]), and was negatively affected by an 8-day LCD only in South Asian adults (-12.2 ± 4.3%, p = 0.005, 95%-CI = [-5.9; -1.2]). Cholesterol efflux capacity was increased in response to HCD in adolescents (South Asians: +6.3 ± 2.9%, p = 0.073, 95%-CI = [-0.02; 0.46], Caucasians: +11.8 ± 3.4%, p = 0.002, 95%-CI = [0.17;0.65]) and decreased after LCD in adults (South Asians: -10.3 ± 2.4%, p < 0.001, 95%-CI = [-0.57; -0.20], Caucasians: -13.7 ± 1.9%, p < 0.00001, 95%-CI = [-0.67; -0.33]). Although subclass analyses of HDL showed no differences between ethnicities, cholesterol efflux correlated best with cholesterol and phospholipid within small HDL compared to other HDL subclasses and constituents. CONCLUSION: Impaired HDL functionality in South Asians may be a contributing factor to their high CVD risk. CLINICAL TRIAL REGISTRATION: NTR 2473 (URL: http://www.trialregister.nl/).

Guidelines for the Cytopathologic Diagnosis of Epithelioid and Mixed-Type Malignant Mesothelioma: a secondary publication.

OBJECTIVE: To provide practical guidelines for the cytopathologic diagnosis of malignant mesothelioma. DATA SOURCES: Cytopathologists with an interest in the field involved in the International Mesothelioma Interest Group (IMIG) and the International Academy of Cytology (IAC) contributed to this update. Reference material includes peer-reviewed publications and textbooks. RATIONALE: This article is the result of discussions during and after the IMIG 2012 conference in Boston, followed by thorough discussions during the 2013 IAC meeting in Paris. Additional contributions have been obtained from cytopathologists and scientists who could not attend these meetings, with final discussions and input during the IMIG 2014 conference in Cape Town.

Cell response to PEGylated poly(dopamine) coated liposomes considering shear stress.

BACKGROUND: Liposomes have gained immerse attention in the field of drug delivery as carriers of therapeutic molecules. Their modification with a polymer either to make them stealth (e.g. using PEG) and/or more stable (e.g. using poly(dopamine) (PDA)) is a crucial aspect to improve their performance e.g. the blood circulation time. Despite their potential, there are only a few commercialized liposome-based formulations for intravenous drug delivery. Hence, there is still considerable need to address the challenges involved in the design and characterization of liposomal therapeutics. In the latter case, it is of paramount importance to consider the dynamic in vivo environment, e.g. the interstitial fluidic pressure in tumors, blood flow, or bile flow in the liver. METHODS: The PEGylation of PDA films was characterized by quartz crystal microbalance with dissipation monitoring, and the optimized protocol was used to assemble PEGylated PDA coated liposomes (LPDA_PEG). Dynamic light scattering, a plate reader, a flow cytometer and a cytotoxicity assay were used to characterize the liposomes and quantify cellular association/uptake and cell viability in the presence and absence of shear stress after 30min and 4h. The immortalized skeletal mouse myoblast (C2C12) cell line was chosen as model cancer cells, and a hepatic cell line (HepG2) was selected due to their importance in nanosized drug carrier clearance from the system in the liver. RESULTS: The presence of hydrophilic cargo did not affect the PDA assembly process. In the absence of shear stress, there was no difference in cellular uptake/association of both PDA coated liposomes (LPDA) and LPDA_PEG for hepatocytes while myoblasts preferentially internalized/associated with LPDA. In the presence of shear stress, hepatocytes preferentially internalized/associated with LPDA after 30min, while there was only a significant difference for myoblasts after 4h. The cell viability remained unaffected in all cases. CONCLUSIONS: LPDA_PEG are a promising platform towards drug delivery. The nature of cells and fluidic flow are important factors to be considered in their characterization using cell cultures. GENERAL SIGNIFICANCE: These findings will contribute in the better understanding of polymer coated liposomes with cells. The importance of microfluidics in cell culture based characterization is demonstrated, and this will eventually affect the way advanced drug delivery vehicles are designed and characterized prior to animal experiments.

Biocatalytic polymer coatings: on-demand drug synthesis and localized therapeutic effect under dynamic cell culture conditions.

Biocatalytic surface coatings are prepared herein for localized synthesis of drugs and their on-demand, site-specific delivery to adhering cells. This novel approach is based on the incorporation of an enzyme into multilayered polymer coatings to accomplish enzyme-prodrug therapy (EPT). The build-up of enzyme-containing multilayered coatings is characterized and correlations are drawn between the multilayer film assembly conditions and the enzymatic activity of the resulting coatings. Therapeutic effect elicited by the substrate mediated EPT (SMEPT) strategy is investigated using a prodrug for an anticancer agent, SN-38. The performance of biocatalytic coatings under flow conditions is investigated and it is demonstrated that EPT allows synthesizing the drugs on-demand, at the time desired and in a controllable amount to suit particular applications. Finally, using cells cultured in sequentially connected flow chambers, it is demonstrated that SMEPT affords a site-specific drug delivery, that is, exerts a higher therapeutic effect in cells adhering directly to the biocatalytic coatings than in the cells cultured "downstream". Taken together, these data illustrate biomedical opportunities made possible by engineering tools of EPT into multilayered polymer coatings and present a novel, highly versatile tool for surface mediated drug delivery.

Poly(N-isopropylacrylamide)/poly(dopamine) capsules.

Polymer capsules are an interesting concept considered in nanobiotechnology. Approaches that facilitate their assembly remain sought after. Poly(dopamine) (PDA) has been considered and successfully applied in this context. We recently demonstrated that PDA could be copolymerized with different types of poly(N-isopropylacrylamide) (pNiPAAm) to assemble mixed films on planar substrates. Herein, we transferred this approach onto colloidal substrates and characterized the film thickness depending on the film composition and template particles size. While the membrane of capsules assembled using 5 µm template particles exhibited strong dependency on the film composition, smaller templates led to capsules with similar membrane thickness. We then compared the permeability of different capsules using fluorescently labeled dextran and fluorescein. We found that the permeability of capsules was heavily dependent on the polymer composition and the template particle size. These fundamental findings contribute to the potential of these capsules, assembled in one-step, for biomedical applications.

Evaluation of surgical conditions during laparoscopic surgery in patients with moderate vs deep neuromuscular block.

BACKGROUND: The routine use of neuromuscular blocking agents reduces the occurrence of unacceptable surgical conditions. In some surgeries, such as retroperitoneal laparoscopies, deep neuromuscular block (NMB) may further improve surgical conditions compared with moderate NMB. In this study, the effect of deep NMB on surgical conditions was assessed. METHODS: Twenty-four patients undergoing elective laparoscopic surgery for prostatectomy or nephrectomy were randomized to receive moderate NMB (train-of-four 1-2) using the combination of atracurium/mivacurium, or deep NMB (post-tetanic count 1-2) using high-dose rocuronium. After surgery, NMB was antagonized with neostigmine (moderate NMB), or sugammadex (deep NMB). During all surgeries, one surgeon scored the quality of surgical conditions using a five-point surgical rating scale (SRS) ranging from 1 (extremely poor conditions) to 5 (optimal conditions). Video images were obtained and 12 anaesthetists rated a random selection of images. RESULTS: Mean (standard deviation) SRS was 4.0 (0.4) during moderate and 4.7 (0.4) during deep NMB (P<0.001). Moderate block resulted in 18% of scores at the low end of the scale (Scores 1-3); deep block resulted in 99% of scores at the high end of the scale (Scores 4 and 5). Cardiorespiratory conditions were similar during and after surgery in both groups. Between anaesthetists and surgeon, there was poor agreement between scores of individual images (average κ statistic 0.05). CONCLUSIONS: Application of the five-point SRS showed that deep NMB results in an improved quality of surgical conditions compared with moderate block in retroperitoneal laparoscopies, without compromise to the patients' peri- and postoperative cardiorespiratory conditions. Trial registration The study was registered at clinicaltrials.gov under number NCT01361149.

Assembly of poly(dopamine)/poly(N-isopropylacrylamide) mixed films and their temperature-dependent interaction with proteins, liposomes, and cells.

Many biomedical applications benefit from responsive polymer coatings. The properties of poly(dopamine) (PDA) films can be affected by codepositing dopamine (DA) with the temperature-responsive polymer poly(N-isopropylacrylamide) (pNiPAAm). We characterize the film assembly at 24 and 39 °C using DA and aminated or carboxylated pNiPAAm by a quartz crystal microbalance with dissipation monitoring (QCM-D), X-ray photoelectron spectroscopy, UV-vis, ellipsometry, and atomic force microscopy. It was found that pNiPAAm with both types of end groups are incorporated into the films. We then identified a temperature-dependent adsorption behavior of proteins and liposomes to these PDA and pNiPAAm containing coatings by QCM-D and optical microscopy. Finally, a difference in myoblast cell response was found when these cells were allowed to adhere to these coatings. Taken together, these fundamental findings considerably broaden the potential biomedical applications of PDA films due to the added temperature responsiveness.

Pulmonary alveolar microlithiasis: a case report and review of the literature.

A 12-year-old girl of Turkish descent was referred 6 weeks after an influenza A infection because of persistent chest X-ray abnormalities compatible with interstitial lung disease. The clinically suspected diagnosis of pulmonary alveolar microlithiasis (PAM) supported by pathognomonic radiological abnormalities was confirmed by genetic analysis. The clinical presentation of PAM is illustrated by a case and review of the current literature on this subject: you only see what you know.

Comparison of neuromuscular block measured by compressomyography at the upper arm and electromyography at the adductor pollicis muscle in obese and non-obese patients: An observational study.

STUDY OBJECTIVE: Quantitative neuromuscular monitoring is traditionally evaluated at the adductor pollicis muscle. By contrast, the TOF-Cuff compressomyograph evaluates neuromuscular block (NMB) at the upper arm. However, compressomyography has not been fully validated against other monitoring entities. This study evaluates the agreement between NMB measured by compressomyography at the upper arm and electromyography at the adductor pollicis muscle during various levels of neuromuscular block in patients with and without obesity. INTERVENTIONS: NMB was measured at the upper arm by compressomyography (TOF-Cuff) and by electromyography (GE-NMT) at the adductor pollicis. DESIGN: Prospective, multicenter, observational study. SETTING: Secondary and tertiary care hospitals' operating theatres. PATIENTS: 200 non-obese and 50 obese patients. MEASUREMENTS: During onset and offset of deep (post-tetanic-count 1-15 twitches), moderate (Train-of-Four-count 1-3 twitches) and shallow (Train-of-Four-ratio 0.01-1.0) depths of NMB were measured in obese and non-obese patients. The bias and limits of agreement of both devices were calculated using a Bland-Altman analysis for repeated measurements. Data obtained during spontaneous recovery (i.e. without the use of reversal agents) were used in the primary analyses. MAIN RESULTS: Data from enrolled patients yielded 942 paired post-tetanic-counts, 1175 paired train-of-four-counts and 1574 paired train-of-four ratios during spontaneous recovery. In non-obese patients, mean bias (95% CI) between the two devices was 3.405 (2.294 to 4.517) during deep NMB; -0.023 (-0.205 to 0.160) during moderate NMB and 0.312 (0.287 to 0.338) during shallow NMB. In obese patients, bias was -0.170 (-2.872 to 2.531); 0.178 (-0.202 to 0.558); 0.384 (0.299 to 0.469) for deep, moderate and shallow NMB respectively. CONCLUSIONS: There is variable disagreement between the level of NMB measured at the upper arm by compressomyography and at the adductor pollicis muscle measured by electromyography, throughout the various stages of NMB in obese and non-obese patients. Recovery of NMB on compressomyography preceded recovery on electromyography, which may have consequences for reversal and extubation decisions in clinical practice.

Disease course and outcome of acute disseminated encephalomyelitis is more severe in adults than in children.

BACKGROUND: Acute disseminated encephalomyelitis (ADEM) affects children more frequently than adults. Current studies investigating ADEM in different age groups are difficult to compare. OBJECTIVE: To investigate whether the clinical presentation, outcome and disease course of ADEM differ between adults and children. METHODS: Disease characteristics of 25 adults and 92 children suffering from ADEM between 1988 and 2008 were compared. RESULTS: The most common presenting symptoms of ADEM in both groups were pyramidal signs and encephalopathy. Ataxia occurred more frequently in children (p = 0.002). In general, MRI showed ill-defined and large white matter lesions in both groups, whereas periventricular lesions were more prevalent in adults (p = 0.001). In adults, duration of hospitalization was longer (p = 0.002) and intensive care unit (ICU) admission was more frequently required (p = 0.043). Three adults (12%) and one child (1%) died (p = 0.030). Fewer adults had complete motor recovery after their first clinical event (p < 0.001). In 73 patients follow-up time was ≥ 2 years and most of these patients remained monophasic. Although relapses after ADEM can occur, only one adult (5%) and five children (6%) converted to MS. CONCLUSIONS: The clinical presentations in children and adults share similarities, but the disease course and outcome of ADEM is more severe in adults with respect to hospitalization, ICU admission, recovery and mortality.

Sonochemical synthesis of magnetic Janus nanoparticles.

The sonochemical synthesis of nanosized surface-dissymmetrical (Janus) particles is described. The Janus particles were composed of silica and polystyrene, with the polystyrene portion loaded with nanosized magnetite particles. It is shown that the Janus particles can be used to form kinetically stable oil-in-water emulsions that can be spontaneously broken on application of an external magnetic field. The one-pot synthetic process used to prepare the Janus particles has several advantages over other conventional methods of producing such particles.

Sonochemical polymerization of miniemulsions in organic liquids/water mixtures.

The sonochemical oil-in-water miniemulsion polymerization of n-butyl methacrylate (BMA) has been studied in mixtures with a range of aliphatic and aromatic hydrocarbon liquids under ambient conditions. Measurements of monomer conversion percentage and molecular weights of the BMA polymers were performed to investigate the effect of the various organic liquids on the kinetics of the polymerization process and on the properties of the resultant polymers. Both the rates of polymerization and the molecular weights of the polymers formed were found to be dependent on the amount and type of the organic liquid present in the emulsion. The experimental results revealed that when the organic liquids were aliphatic, there were no significant changes in the rates of BMA polymerization whereas when the organic liquids were aromatic, the rates of polymerization were greatly reduced. Molecular weight data of the BMA polymers showed that in the presence of an organic liquid, the size of the polymer significantly decreased. The results have been interpreted in terms of the formation of a radical complex between the propagating radical and the organic liquid in the oil mixture, as well as chain transfer reactions that affect the kinetics of the polymerization process.

Increased diagnostic accuracy of atypical glandular cells in cervical liquid-based cytology using cell blocks.

OBJECTIVE: The purpose of this study was to reduce the number of diagnoses of atypical glandular cells (AGC). Residual material from the cervical ThinPrep® samples (Hologic, Marlboruogh, MA, USA) was used for cell blocks (CB) and immunohistochemistry (IHC). METHODS: In 2007 there were 87 patients (0.12% of tests) with AGC on liquid-based cytology (LBC) in the Leiden Cytology and Pathology Laboratory (LCPL) using the Bethesda System 2001 (TBS). CB with IHC was used for 26 of these cases. The vials still containing the brush (Cervex-Brush(®) Combi) were placed in a shaker for 10 minutes to dislodge the material trapped between the bristles. The residual sampling fluid was used to prepare paraffin sections (Shandon Cytoblock(®)) stained with Papanicolaou and immunostaining. RESULTS: Four of five cases with AGC not otherwise specified (NOS) were diagnosed with CB/IHC as benign mimics (endometrium, tubal metaplasia, follicular cervicitis, microglandular hyperplasia) and one of four with AGC-favour neoplasia (FN) (endocervical polyp). In one of five cases with AGC-NOS and in two of seven with AGC-FN, CIN3 was found on subsequent histological biopsy. Of six cases diagnosed as adenocarcinoma in situ (AIS) on LBC with CB/IHC the diagnosis was confirmed in four; one was adenocarcinoma and one glandular atypia. Of eight cases diagnosed as adenocarcinoma on cytology and CB/IHC, the diagnosis was confirmed in three. The other five cases were found to be one each of AIS, squamous cell carcinoma, CIN3, CIN2 with glandular atypia, and cervical endometriosis. CONCLUSIONS: By reducing the number of benign mimics of AGC, we achieved a high proportion (16/26; 61.5%) of neoplastic or preneoplastic lesions (glandular or squamous) on histological outcome potentially avoiding colposcopy. Histological biopsy verification by the gynaecologist is needed for final diagnosis of AGC-FN, AIS and adenocarcinoma.