Chemical Transformations of 2D Kaolinic Clay Mineral Surfaces from Sulfuric Acid Exposure.

A combined experimental and computational approach is used to investigate the chemical transformations of kaolinite and metakaolin surfaces when exposed to sulfuric acid. These clay minerals are hydrated ternary metal oxides and are shown to be susceptible to degradation by loss of Al as the water-soluble salt Al2(SO4)3, due to interactions between H2SO4 and aluminum cations. This degradation process results in a silica-rich interfacial layer on the surfaces of the aluminosilicates, most prominently observed in metakaolin exposed to pH environments of less than 4. Our observations are supported by XPS, ATR-FTIR, and XRD experiments. Concurrently, DFT methodologies are used to probe the interactions between the clay mineral surfaces and H2SO4 as well as other sulfur-containing adsorbates. An analysis performed using a DFT + thermodynamics model shows that the surface transformation processes that lead to the loss of Al and SO4 from metakaolin are favorable at pH below 4; however, such transformations are not favorable for kaolinite, a result that agrees with our experimental efforts. The data obtained from both experimental techniques and computational studies support that the dehydrated surface of metakaolin interacts more strongly with sulfuric acid and provide atomistic insight into the acid-induced transformations of these mineral surfaces.

Extending the viability of human precision-cut intestinal slice model for drug metabolism studies.

Human Precision-cut intestinal slices (hPCIS) are used to study intestinal physiology, pathophysiology, drug efficacy, toxicology, kinetics, and metabolism. However, the use of this ex vivo model is restricted to approximately a 24 h timeframe because of declining viability of the hPCIS during traditional culture. We hypothesized that we could extend the hPCIS viability by using organoid medium. Therefore, we cultured hPCIS for up to 72 h in organoid media [expansion medium (Emed) and differentiation medium (Dmed)]. After incubation, we assessed culture-induced changes on viability markers, specific cell type markers and we assessed the metabolic activity of enterocytes by measuring midazolam metabolite formation. We show that the adenosine triphosphate (ATP)/protein ratio of Emed-cultured hPCIS and morphology of both Emed- and Dmed-cultured hPCIS was improved compared to WME-cultured hPCIS. Emed-cultured hPCIS showed an increased expression of proliferation and stem cell markers, whereas Dmed-cultured hPCIS showed an increased expression of proliferation and enterocyte markers, along with increased midazolam metabolism. Using the Emed, the viability of hPCIS could be extended for up to 72 h, and proliferating stem cells remained preserved. Using Dmed, hPCS also remained viable for up to 72 h, and specifically rescued the metabolizing enterocytes during culture. In conclusion, by using two different organoid culture media, we could extend the hPCIS viability for up to 72 h of incubation and specifically steer stem cells or enterocytes towards their original function, metabolism, and proliferation, potentially allowing pharmacokinetic and toxicology studies beyond the 24 h timeframe.

Fracture Diodes: Directional Asymmetry of Fracture Toughness.

Toughness describes the ability of a material to resist fracture or crack propagation. It is demonstrated here that fracture toughness of a material can be asymmetric, i.e., the resistance of a medium to a crack propagating from right to left can be significantly different from that to a crack propagating from left to right. Such asymmetry is unknown in natural materials, but we show that it can be built into artificial materials through the proper control of microstructure. This paves the way for control of crack paths and direction, where fracture-when unavoidable-can be guided through predesigned paths to minimize loss of critical components.

Comparison of sporadic and familial behavioral variant frontotemporal dementia (FTD) in a North American cohort.

INTRODUCTION: Behavioral variant frontotemporal dementia (bvFTD) may present sporadically or due to an autosomal dominant mutation. Characterization of both forms will improve understanding of the generalizability of assessments and treatments. METHODS: A total of 135 sporadic (s-bvFTD; mean age 63.3 years; 34% female) and 99 familial (f-bvFTD; mean age 59.9; 48% female) bvFTD participants were identified. f-bvFTD cases included 43 with known or presumed chromosome 9 open reading frame 72 (C9orf72) gene expansions, 28 with known or presumed microtubule-associated protein tau (MAPT) mutations, 14 with known progranulin (GRN) mutations, and 14 with a strong family history of FTD but no identified mutation. RESULTS: Participants with f-bvFTD were younger and had earlier age at onset. s-bvFTD had higher total Neuropsychiatric Inventory Questionnaire (NPI-Q) scores due to more frequent endorsement of depression and irritability. DISCUSSION: f-bvFTD and s-bvFTD cases are clinically similar, suggesting the generalizability of novel biomarkers, therapies, and clinical tools developed in either form to the other.

Effect of the first wave of COVID-19 on Poison Control Centre activities in 21 European countries: an EAPCCT initiative.

BACKGROUND: Public health emergencies often affect Poison Control Centre (PCC) operations. We examined possible effects of the coronavirus disease 2019 (COVID-19) pandemic on call volume, call characteristics, and workload in European PCCs. METHOD: All 65 individual European PCCs were requested to supply data on the number of calls and call characteristics (caller, age groups, reason and specific exposures) from March to June in 2018, 2019, and 2020 (Part 1). Number of calls with specific characteristics was normalised to all calls. Calls (N) and call characteristics (%) were compared between 2020 and 2018/2019 (average), within PCCs/countries and grouped. Correlation between call volume and COVID-19 cases per PCC/country was examined. All PCCs received a survey on workload (Part 2). Parts 1 and 2 were independent. RESULTS: For Part 1, 36 PCCs (21 countries) supplied 26 datasheets. PCCs in the UK and in France merged data and supplied one datasheet each with national data. Summed data showed an increase of 4.5% in call volume from 228.794 in 2018/2019 (average) to 239.170 in 2020 (p < 0.001). Within PCCs/countries, calls significantly increased for 54% of PCCs/countries (N = 14/26) and decreased for 19% (N = 5/26), three of which (N = 3/5) only serve medical professionals. Correlation between call volume and COVID-19 cases was (non-significant) positive (Rho >0.7) in 5/26 PCCs/countries (19%), and negative in 6/26 (23%). Call characteristics (median proportion of grouped data in 2018/2019 vs. 2020) changed: fewer medical professionals called (40 vs. 34%, p < 0.001), calls on intentional exposures decreased (20 vs. 17%, p < 0.012), as did calls on patients between 13 and 17 years (5 vs. 4%, p < 0.05). Calls on specific exposures increased; disinfectants from 1.9 to 5.2%, and cleaning products from 4.4 to 5.7% (p < 0.001). For Part 2, 38 PCCs (24 countries) filled the survey on workload (number/length of shifts and time on PCC duties), which increased in 23/38 PCCs (61%), while 10/38 (26%) worked with fewer employees. CONCLUSIONS: Obtaining aggregated European PCC data proved challenging but showed an increase in overall call volume and workload during the first COVID-19 wave. Call characteristics changed including fewer calls from professionals and more calls on specific exposures. Within single PCCs/countries a variety of effects was observed.

Gender and gut microbiota composition determine hepatic bile acid, metabolic and inflammatory response to a single fast-food meal in healthy adults.

BACKGROUND & AIMS: Regular consumption of fast-food (FF) as a form of typical Western style diet is associated with obesity and the metabolic syndrome, including its hepatic manifestation nonalcoholic fatty liver disease. Currently, it remains unclear how intermittent excess FF consumption may influence liver metabolism. The study aimed to characterize the effects of a single FF binge on hepatic steatosis, inflammation, bile acid (BA), glucose and lipid metabolism. METHODS: Twenty-five healthy individuals received a FF meal and were asked to continue eating either for a two-hour period or until fully saturated. Serum levels of transaminases, fasting BA, lipid profile, glucose and cytokine levels as well as transient elastography and controlled attenuation parameter (CAP; to assess hepatic steatosis) were analyzed before (day 0) and the day after FF binge (day 1). Feces was collected prior and after the FF challenge for microbiota analysis. RESULTS: The FF meal induced a modest increase in CAP, which was accompanied by a robust increase of fasting serum BA levels. Surprisingly, levels of cholesterol and bilirubin were significantly lower after the FF meal. Differentiating individuals with a relevant delta BA (>1 µmol/l) increase vs. individuals without (delta BA ≤1 µmol/l), identified several gut microbiota, as well as gender to be associated with the BA increase and the observed alterations in liver function, metabolism and inflammation. CONCLUSION: A single binge FF meal leads to a robust increase in serum BA levels and alterations in parameters of liver injury and metabolism, indicating a novel metabolic aspect of the gut-liver axis.

Monocytes and their pathophysiological role in Crohn's disease.

Our immune system shows a stringent dichotomy, on the one hand displaying tolerance towards commensal bacteria, but on the other hand vigorously combating pathogens. Under normal conditions the balance between flora tolerance and active immunity is maintained via a plethora of dynamic feedback mechanisms. If, however, the balancing act goes faulty, an inappropriate immune reaction towards an otherwise harmless intestinal flora causes disease, Crohn's disease for example. Recent developments in the immunology and genetics of mucosal diseases suggest that monocytes and their derivative cells play an important role in the pathophysiology of Crohn's disease. In our review, we summarize the recent studies to discuss the dual function of monocytes - on the one hand the impaired monocyte function initiating Crohn's disease, and on the other hand the overactivation of monocytes and adaptive immunity maintaining the disease. With a view to developing new therapies, both aspects of monocyte functions need to be taken into account.

Pex22p of Pichia pastoris, essential for peroxisomal matrix protein import, anchors the ubiquitin-conjugating enzyme, Pex4p, on the peroxisomal membrane.

We isolated a Pichia pastoris mutant that was unable to grow on the peroxisome-requiring media, methanol and oleate. Cloning the gene by complementation revealed that the encoded protein, Pex22p, is a new peroxin. A Deltapex22 strain does not grow on methanol or oleate and is unable to import peroxisomal matrix proteins. However, this strain targets peroxisomal membrane proteins to membranes, most likely peroxisomal remnants, detectable by fluorescence and electron microscopy. Pex22p, composed of 187 amino acids, is an integral peroxisomal membrane protein with its NH2 terminus in the matrix and its COOH terminus in the cytosol. It contains a 25-amino acid peroxisome membrane-targeting signal at its NH2 terminus. Pex22p interacts with the ubiquitin-conjugating enzyme Pex4p, a peripheral peroxisomal membrane protein, in vivo, and in a yeast two-hybrid experiment. Pex22p is required for the peroxisomal localization of Pex4p and in strains lacking Pex22p, the Pex4p is cytosolic and unstable. Therefore, Pex22p anchors Pex4p at the peroxisomal membrane. Strains that do not express Pex4p or Pex22p have similar phenotypes and lack Pex5p, suggesting that Pex4p and Pex22p act at the same step in peroxisome biogenesis. The Saccharomyces cerevisiae hypothetical protein, Yaf5p, is the functional homologue of P. pastoris Pex22p.

Quantitative comparison of the neutralizing capacity, immunogenicity and cross-reactivity of anti-TNF-α biologicals and an Infliximab-biosimilar.

INTRODUCTION: TNF-α-neutralizing antibodies, such as infliximab (IFX) and adalimumab (ADA), are effective in the treatment of inflammatory bowel diseases (IBD), but they are expensive and become ineffective when patients develop anti-IFX or anti-ADA antibodies (ATI and ATA, respectively). Second-generation anti-TNF-α antibodies, such as Golimumab, Etanercept, Certolizumab-pegol and IFX biosimilars, may solve these issues. AIM: To determine the neutralizing capacity of first- and second generation anti-TNF-α antibodies and to determine whether ATI show cross-reactivity with the IFX biosimilar CT-P13 (Inflectra). METHODS: TNF-α neutralization was measured using a quantitative TNF-α sensor assay consisting of HeLa 8D8 cells that express the Green Fluorescence Protein (GFP) under control of a NF-кB response element. All available anti-TNF-α drugs and the IFX biosimilar CT-P13 (Inflectra) were tested for their TNF-α-neutralizing capacity. In addition, patient sera with ATI were tested for their potential to block the activity of IFX, IFX (F)ab2-fragment, biosimilar CT-P13 (Inflectra) and ADA. RESULTS: TNF-α strongly induced GFP expression in Hela 8D8 cells. Higher concentrations of first-generation anti-TNF-α drugs were required to neutralize TNF-α compared to the second-generation anti-TNF-α drugs. Serum of IBD patients with proven ATI blocked TNF-α-neutralizing properties of IFX biosimilar CT-P13 (Inflectra), whereas such sera did not block the effect of ADA. CONCLUSION: The second-generation anti-TNF-α drugs show increased TNF-α-neutralizing potential compared to first-generation variants. ATI show cross-reactivity toward IFX biosimilar CT-P13 (Inflectra), consequently patients with ATI are unlikely to benefit from treatment with this IFX biosimilar.

Effects of different immunosuppressive regimens on regulatory T-cells in noninflamed colon of liver transplant recipients.

BACKGROUND: Regulatory T-cells (Treg) are natural suppressors of autoimmunity. Previous studies indicate that immunosuppressive drugs, especially calcineurin-inhibitors, may interfere with Treg homeostasis. Inflammatory bowel disease (IBD) can relapse or develop de novo after liver transplantation. IBD is associated with a relative deficiency of Treg. The aim of this study was to determine the effect of long-term immunosuppression on the presence of Treg in the noninflamed colonic mucosa of liver transplant recipients. METHODS: Colonic biopsies of normal mucosa of 36 liver transplant recipients on different types of immunosuppression and 11 controls were studied. Treg marker Foxp3 and Treg products transforming growth factor-beta (TGF-beta) and interleukin-10 (IL-10) were studied by quantitative polymerase chain reaction (Q-PCR) and immunohistochemistry. TGF-beta-induced Smad-protein 3 and 7 were studied by Q-PCR. RESULTS: No significant differences between controls and patients were observed in IL-10, TGF-beta, and Smad expression. Mucosal Foxp3 mRNA levels and Foxp3+CD3+ cells were significantly reduced in transplant recipients using prednisone/azathioprine/tacrolimus compared with controls but no direct relationship between Foxp3 expression and 1 specific drug was detected. CONCLUSIONS: These results challenge the hypothesis that calcineurin-induced reduction of Treg or TGF-beta expression predisposes nontransplanted tissue to inflammation, but indicate that combined immunosuppression hampers Treg development in the intestine.

Two AAA family peroxins, PpPex1p and PpPex6p, interact with each other in an ATP-dependent manner and are associated with different subcellular membranous structures distinct from peroxisomes.

Two peroxins of the AAA family, PpPex1p and PpPex6p, are required for peroxisome biogenesis in the yeast Pichia pastoris. Cells from the corresponding deletion strains (Pp delta pex1 and Pp delta pex6) contain only small vesicular remnants of peroxisomes, the bulk of peroxisomal matrix proteins is mislocalized to the cytosol, and these cells cannot grow in peroxisome-requiring media (J. A. Heyman, E. Monosov, and S. Subramani, J. Cell Biol. 127:1259-1273, 1994; A. P. Spong and S. Subramani, J. Cell Biol. 123:535-548, 1993). We demonstrate that PpPex1p and PpPex6p interact in an ATP-dependent manner. Genetically, the interaction was observed in a suppressor screen with a strain harboring a temperature-sensitive allele of PpPEX1 and in the yeast two-hybrid system. Biochemially, these proteins were coimmunoprecipitated with antibodies raised against either of the proteins, but only in the presence of ATP. The protein complex formed under these conditions was 320 to 400 kDa in size, consistent with the formation of a heterodimeric PpPex1p-PpPex6p complex. Subcellular fractionation revealed PpPex1p and PpPex6p to be predominantly associated with membranous subcellular structures distinct from peroxisomes. Based on their behavior in subcellular fractionation experiments including flotation gradients and on the fact that these structures are also present in a Pp delta pex3 strain in which no morphologically detectable peroxisomal remnants have been observed, we propose that these structures are small vesicles. The identification of vesicle-associated peroxins is novel and implies a role for these vesicles in peroxisome biogenesis. We discuss the possible role of the ATP-dependent interaction between PpPex1p and PpPex6p in regulating peroxisome biogenesis events.

Pex19p interacts with Pex3p and Pex10p and is essential for peroxisome biogenesis in Pichia pastoris.

We report the cloning and characterization of Pichia pastoris PEX19 by complementation of a peroxisome-deficient mutant strain. Import of peroxisomal targeting signal 1- and 2-containing peroxisomal matrix proteins is defective in pex19 mutants. PEX19 encodes a hydrophilic 299-amino acid protein with sequence similarity to Saccharomyces cerevisiae Pex19p and human and Chinese hamster PxF, all farnesylated proteins, as well as hypothetical proteins from Caenorhabditis elegans and Schizosaccharomyces pombe. The farnesylation consensus is conserved in PpPex19p but dispensable for function and appears unmodified under the conditions tested. Pex19p localizes predominantly to the cytosolic fraction. Biochemical and two-hybrid analyses confirmed that Pex19p interacts with Pex3p, as seen in S. cerevisiae, but unexpectedly also with Pex10p. Two-hybrid analysis demonstrated that the amino-terminal 42 amino acids of Pex19p interact with the carboxyl-terminal 335 amino acids of Pex3p. In addition, the extreme carboxyl terminus of Pex19p (67 amino acids) is required for interaction with the amino-terminal 380 amino acids of Pex10p. Biochemical and immunofluorescence microscopy analyses of pex19Delta cells identified the membrane protein Pex3p in peroxisome remnants that were not previously observed in S. cerevisiae. These small vesicular and tubular (early) remnants are morphologically distinct from other Pppex mutant (late) remnants, suggesting that Pex19p functions at an early stage of peroxisome biogenesis.

Sorting and function of peroxisomal membrane proteins.

Peroxisomes are subcellular organelles and are present in virtually all eukaryotic cells. Characteristic features of these organelles are their inducibility and their functional versatility. Their importance in the intermediary metabolism of cells is exemplified by the discovery of several inborn, fatal peroxisomal errors in man, the so-called peroxisomal disorders. Recent findings in research on peroxisome biogenesis and function have demonstrated that peroxisomal matrix proteins and peroxisomal membrane proteins (PMPs) follow separate pathways to reach their target organelle. This paper addresses the principles of PMP sorting and summarizes the current knowledge of the role of these proteins in organelle biogenesis and function.

Validation and determination of taselisib, a ß-sparing phosphoinositide 3-kinase (PI3K) inhibitor, in human plasma by LC-MS/MS.

A liquid chromatographic-tandem mass spectrometry (LC-MS/MS) method for the determination of taselisib (GDC-0032, RO5537381) concentrations in human plasma has been developed and validated to support bioanalysis of clinical samples. Solid phase extraction (SPE) was used to extract plasma samples (50µL) and the resulting samples were analyzed using reversed phase chromatography and mass spectrometry coupled with an atmospheric pressure chemical ionization interface. The mass analysis of taselisib was performed using multiple reaction monitoring transitions in positive ionization mode. The method was validated over the calibration curve range 0.400-400ng/mL using linear regression and 1/x(2) weighting. The within-run relative standard deviation (%RSD) ranged from 1.3 to 5.6%, while the between-run %RSD varied from 2.0 to 4.5% for LLOQ, low, medium, medium high and high QCs. The accuracy ranged from 94.7 to 100.3% of nominal for within-run and 96.0-99.0% of nominal for between-run for the same QCs. Extraction recovery of taselisib was between 83.8% and 92.9%. Stability of taselisib was established in human plasma for 977days at -20°C and -70°C and established in sample extracts for 96h when stored at 2 - 8°C. Stable-labeled internal standard was used to minimize matrix effects. Mean single dose pharmacokinetic parameters determined using this method for a phase I/II clinical trial were: Cmax=35.2ng/mL, AUC0-inf=1570ngh/mL, and T1/2=39.3h.

Stereoselectivities of microbial epoxide hydrolases.

Epoxide hydrolases from bacterial and fungal sources are highly versatile biocatalysts for the asymmetric hydrolysis of epoxides on a preparative scale. Besides kinetic resolution, which yields the corresponding enantiomerically enriched vicinal diol and the remaining nonconverted epoxide, enantioconvergent processes are also possible, which lead to the formation of a single enantiomeric diol from a racemic oxirane. The data available to date indicate that the enantioselectivities of enzymes from certain microbial sources can be correlated to the substitutional pattern of various types of substrates: red yeasts (e.g. Rhodotorula or Rhodosporidium sp.) give best enantioselectivities with monosubstituted oxiranes; fungal cells (e.g. from Aspergillus and Beauveria sp.) are best suited for styrene oxide-type substrates; bacterial enzymes, on the other hand (in particular from Actinomycetes such as Rhodococcus and Nocardia sp.) are the biocatalysts of choice for more highly substituted 2,2- and 2,3-disubstituted epoxides.

Microbial epoxide hydrolases for preparative biotransformations.

Epoxide hydrolases from microbial sources are highly versatile biocatalysts for the asymmetric hydrolysis of epoxides on a preparative scale. Besides kinetic resolution, which furnishes the corresponding vicinal diol and remaining non-hydrolysed epoxide in nonracemic form, enantioconvergent processes are possible: these are highly attractive as they lead to the formation of a single enantiomeric diol from a racemic oxirane. The data accumulated over recent years reveal a common picture of the substrate structure selectivity pattern of microbial epoxide hydrolases and indicate that substrates of various structural types can be selectively hydrolysed with enzymes from certain microbial sources.

The detection of loose bodies in the elbow: the value of MRI and CT arthrography.

Our aim was to determine the clinical value of MRI and CT arthrography in predicting the presence of loose bodies in the elbow. A series of 26 patients with mechanical symptoms in the elbow had plain radiography, MRI and CT arthrography, followed by routine arthroscopy of the elbow. The location and number of loose bodies determined by MRI and CT arthrography were recorded. Pre-operative plain radiography, MRI and CT arthrography were compared with arthroscopy. Both MRI and CT arthrography had excellent sensitivity (92% to 100%) but low to moderate specificity (15% to 77%) in identifying posteriorly-based loose bodies. Neither MRI nor CT arthrography was consistently sensitive (46% to 91%) or specific (13% to 73%) in predicting the presence or absence of loose bodies anteriorly. The overall sensitivity for the detection of loose bodies in either compartment was 88% to 100% and the specificity 20% to 70%. Pre-operative radiography had a similar sensitivity and specificity of 84% and 71%, respectively. Our results suggest that neither CT arthrography nor MRI is reliable or accurate enough to be any more effective than plain radiography alone in patients presenting with mechanical symptoms in the elbow.

The prevalence of wrist ganglia in an asymptomatic population: magnetic resonance evaluation.

Magnetic resonance imaging (MRI) was performed on the wrists of 103 asymptomatic volunteers. The images were evaluated independently by two musculoskeletal radiologists and one orthopaedic surgeon. Wrist ganglia were identified in 53 out of the 103 wrists. The average long and short axes measurements were 8 mm (range 3-22) and 3 mm (range 2-10), respectively. Seventy per cent of the ganglia originated from the palmar capsule in the region of the interval between the radioscaphocapitate ligament and the long radiolunate ligament. Fourteen per cent of the ganglia were dorsal and originated from the dorsal, distal fibres of the scapholunate ligament. Two ganglia had surrounding soft tissue oedema and one had an associated intraosseous component. Unlike previous surgical and pathological series, our study showed that palmar wrist ganglia are more common than dorsal wrist ganglia. The vast majority of these asymptomatic ganglia occur without associated ligamentous disruption, soft tissue oedema or intraosseous communication.

Prospects for the increased application of biocatalysts in organic transformations.

Predicting the future use of biocatalysts for the transformation of organic compounds--both natural substrates and other compounds--needs to take many factors into account. To date, relatively few biotransformations have been developed to the industrial scale, primarily because there has been little economic incentive to replace existing successful processes with biocatalysts, with their inherent problems of instability, lack of selectivity and narrow operational range. However, advances that improve biocatalyst performance, coupled with the increasing emphasis on 'chirotechnology', are driving the development of biocatalysis as a complementary, if not a rival technology to existing chemical approaches.

The N-terminus of amine oxidase of Hansenula polymorpha contains a peroxisomal targeting signal.

Here we describe the identification of the targeting sequence of peroxisomal amine oxidase (AMO) of H. polymorpha. Deletion analysis revealed that essential targeting information is located within the extreme N-terminal 16 amino acids. Moreover, this sequence can direct a reporter protein to the peroxisomal matrix of H. polymorpha. The N-terminal 16 amino acids of AMO contain a sequence with strong homology to the conserved PTS2 sequence. Therefore, AMO is considered to be a PTS2 protein.