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Background & Aims: Anaemia is frequently observed in patients with cirrhosis and was identified as a predictor of adverse outcomes, such as increased mortality and occurrence of acute-on-chronic liver failure. To date, the possible effects of iron supplementation on these adverse outcomes are not well described. We therefore aimed to assess the role of iron supplementation in patients with cirrhosis and its capability to improve prognosis., Methods: Laboratory diagnostics were performed in consecutive outpatients with cirrhosis admitted between July 2018 and December 2019 to the University Hospital Essen. Associations with transplant-free survival were assessed in regression models., Results: A total of 317 outpatients with cirrhosis were included, of whom 61 received a liver transplant (n = 19) or died (n = 42). In multivariate Cox regression analysis, male sex (hazard ratio [HR] = 3.33, 95% CI [1.59, 6.99], p  = 0.001), model for end-stage liver disease score (HR = 1.19, 95% CI [1.11, 1.27], p <0.001) and the increase of haemoglobin levels within 6 months (ΔHb6) (HR = 0.72, 95% CI [0.63, 0.83], p <0.001) were associated with transplant-free survival. Regarding the prediction of haemoglobin increase, intake of rifaximin (beta = 0.50, SD beta = 0.19, p  = 0.007) and iron supplementation (beta = 0.79, SD beta = 0.26, p  = 0.003) were significant predictors in multivariate analysis., Conclusions: An increase of haemoglobin levels is associated with improvement of transplant-free survival in patients with cirrhosis. Because the prediction of haemoglobin increase significantly depends on rifaximin and iron supplementation, application of these two medications can have an important impact on the outcome of these patients., Impact and Implications: Anaemia is very common in patients with cirrhosis and is known to be a predictor of negative outcomes, but little is known about the effect of iron substitution in these individuals. In our cohort, increase of haemoglobin levels improved transplant-free survival of patients with cirrhosis. The increase of haemoglobin levels was mainly induced by iron supplementation and was even stronger in the case of concomitant use of iron and rifaximin., Clinical Trial Registration: UME-ID-10042., Competing Interests: JRA: Speaker fees from AbbVie, travel support from Gilead, all unrelated to the submitted work. CML: Speaker and consulting fees from AbbVie, Gilead, MSD, Norgine, Falk, Eisai, Roche, Behring, and travel support from AbbVie and Gilead, all unrelated to the submitted work. Please refer to the accompanying ICMJE disclosure forms for further details., (© 2023 The Author(s).)

Background: The majority of patients with autoimmune hepatitis (AIH) achieve complete remission with established treatment regiments. In patients with intolerance or insufficient response to these drugs, the remaining options are limited and novel treatment approaches necessary. In primary biliary cholangitis (PBC), ursodeoxycholic acid (UDCA) and fibrates have improved prognosis dramatically, but there remains a proportion of patients with refractory disease.In patients with refractory AIH and/or PBC, we used a novel treatment strategy with the anti -B cell activating factor, belimumab. The first three patients had concomitant Sjögren's disease. The connecting element between all three diseases is B cell activation, including elevated levels of the B cell activating factor (BAFF). Furthermore, belimumab has been shown to be beneficial in Sjögren's disease., Aims and Methods: To retrospectively investigate treatment response in six patients with AIH or PBC with or without concomitant Sjögren's disease treated with the anti -BAFF therapy belimumab at the University Hospital in Bern, Switzerland., Results: In all three patients with AIH, belimumab improved disease control and helped by-pass or reduce problematic side effects from corticosteroids and calcineurin inhibitors. In PBC patients (n = 3), there was no clear improvement of liver function tests, despite reduction or normalization of IgM. All patients with concomitant Sjögren's disease (n = 3) had an improvement of sicca symptoms and two out of three patients experienced an initially marked reduction in fatigue, which lessened over time., Conclusions: Belimumab may be a promising treatment option for patients with AIH and further investigations are needed. In PBC however, response was not convincing. The effects on sicca symptoms and fatigue were encouraging., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Author(s).)

Abstract: We have previously reported that murine peritoneal macrophages exposed to ultraviolet B (UV-B; 100 mJ/cm2) undergo apoptosis, as indicated by alterations in cell morphology, caspase-3 activation, poly (ADP-ribose) polymerase (PARP) cleavage, DNA fragmentation, sustained activation of p38/c-Jun N-terminal kinase (JNK) mitogen-activated protein kinases (MAPKs) and inactivation of p42/44 MAPKs. It is now reported that macrophages undergoing UV-B-induced apoptosis show enhanced expression of protein kinase Cδ (PKCδ) in a time-dependent manner. Pretreatment of macrophages with PKCδ-specific inhibitor rottlerin prior to the UV-B irradiation inhibits activation of caspase-3, PARP cleavage, DNA fragmentation and release of intracellular Ca2+. Inhibition of PKCδ also blocks the sustained activation of p38 and JNK MAPKs as well as inactivation of p42/44 MAPKs. PKCα and PKCβ1 expression also increases during UV-B-induced apoptosis in macrophages. Inhibition of these two isoforms with Go6976 slightly suppresses caspase-3 activation, PARP cleavage, DNA fragmentation and release of intracellular Ca2+, but has no effect on the sustained activation of p38/JNK MAPKs or inactivation of p42/44 MAPKs. It is, therefore, suggested that activation of PKCδ might play an important role in the UV-B-induced apoptosis and that specific activated isoforms of PKC may have distinct functions in cell death. [Copyright &y& Elsevier]

Abstract: This study demonstrates the upregulation of alkaline phosphatase and the mechanisms involved in experimental colitis. All models of ileal and colonic inflammation examined, which were characterized by significant oxidative stress and neutrophil infiltration, resulted in an increase in alkaline phosphatase activity which was attributable to both epithelial cells and cells of the lamina propria, mainly leukocytes. The increase in alkaline phosphatase sensitivity to the inhibitors levamisole and homoarginine, together with changes in the apparent molecular size and in the sialization of the enzyme, indicated a change in the isoform expressed. An increase in tissue non-specific alkaline phosphatase expression was observed by Western blotting. Treatment with the bone/kidney alkaline phosphatase inhibitor levamisole or a monoclonal antibody resulted in significant protection from colonic inflammation. Taken together, these results indicate that the kidney isoform is a marker of intestinal inflammation and that it might even constitute a target for pharmacological intervention. [Copyright &y& Elsevier]

The human organotypic skin explant culture (hOSEC) model is a promising alternative in vitro model for screening contact allergens. In this model, the chemical-induced migration of Langerhans cells (LCs) out of the epidermis, evaluated after a 24-h exposure period, is used as a measure of sensitizer potential. As skin irritants can also induce LC migration it is essential that concentrations of test chemicals are used that are not even weakly irritant. Using the hOSEC irritation model chemicals are classified as weak irritants if they are toxic after a 48-h exposure period. Toxicity is determined by methyl green-pyronine (MGP) staining of hOSEC. We studied three frequently used non-sensitizing skin irritants and six potent or frequent human sensitizers in a dose–response. A complete discrimination between non-sensitizers and contact sensitizers was obtained for the chemicals tested when the concentrations used were lower than the weak irritant concentrations. Frequency of positive allergen reactions in patch test of human populations correlated with the difference between weak irritant concentrations and the lowest concentration inducing significant LC migration. Sensitizer potency correlated with chemical irritancy as determined by keratinocyte death. For the compounds tested, the hOSEC model predicted allergenicity in humans better than the guinea pig maximization test and the mouse local lymph node assay. [Copyright &y& Elsevier]

Lysophosphatidic acid (LPA) is a lipid mediator with multiple biological activities and is detected in various biological fluids, including human seminal plasma. Due to its cell proliferation stimulatory and anti-apoptotic activities, LPA has been implicated in the progression of some cancers such as ovarian cancer and prostate cancer. Here, we show that prostatic acid phosphatase, which is a non-specific phosphatase and which has been implicated in the progression of prostate cancer, inactivates LPA in human seminal plasma. Human seminal plasma contains both an LPA-synthetic enzyme, lysoPLD, which converts lysophospholipids to LPA and is responsible for LPA production in serum, and its major substrate, lysophosphatidylcholine. In serum, LPA accumulated during incubation at 37 °C. However, in seminal plasma, LPA did not accumulate. This discrepancy is explained by the presence of a strong LPA-degrading activity. Incubation of LPA with seminal plasma resulted in the disappearance of LPA and an accompanying accumulation of monoglyceride showing that LPA is degraded by phosphatase activity present in the seminal plasma. When seminal plasma was incubated in the presence of a phosphatase inhibitor, sodium orthovanadate, LPA accumulated, indicating that LPA is produced and degraded in the fluid. Biochemical characterization of the LPA-phosphatase activity identified two phosphatase activities in human seminal plasma. By Western blotting analysis in combination with several column chromatographies, the major activity was revealed to be identical to prostatic acid phosphatase. The present study demonstrates active LPA metabolism in seminal plasma and indicates the possible role of LPA signaling in male sexual organs including prostate cancer. [Copyright &y& Elsevier]

Proteomics research has delivered many novel tumor targets. However, due to key limitations, it does not specifically identify targets that are most accessible for drug delivery, such as cell-surface antigens. A novel tumor antigen discovery platform based on screening a single domain antibody (sdAb) library against tumor cells and subsequently identifying the corresponding antigens of the isolated antibodies is described. An sdAb, AFAI, specific for non-small cell lung carcinoma (A549 cell line) was isolated from a phage library derived from the heavy chain antibody repertoire of a llama. The homopentamerization property of a non-toxic verotoxin B-subunit was exploited to make the ES1 pentabody, a pentameric form of AFAI. Pentamerization improved the binding of the AFAI to A549 cells dramatically and greatly facilitated antigen identification by a Western blotting/mass spectrometry approach. The antigen of ES1, which is present only in the hydrophobic, not in the hydrophilic, fraction of A549 cellular proteins, was identified as carcinoembryonic antigen-related cell adhesion molecule 6 (CEA6). CEA6 was observed to be acidic and highly glycosylated, and to exist in multiple glycoforms. The results show that the platform described here should find wide application in antigen discovery, and demonstrated that the pentabodies are very useful immunological reagents for proteomics. [Copyright &y& Elsevier]

Co-crystallization of membrane proteins with antibody fragments may emerge as a general tool to facilitate crystal growth and improve crystal quality. The bound antibody fragment enlarges the hydrophilic part of the mostly hydrophobic membrane protein, thereby increasing the interaction area for possible protein–protein contacts in the crystal. Additionally, it may restrain flexible parts or lock the membrane protein in a defined conformational state. For successful co-crystallization trials, the antibody fragments must be stable in detergents during the extended period of crystal growth and must be easily produced in amounts necessary for crystallography. Therefore, we constructed a library of antibody Fab fragments from a framework subset of the HuCAL GOLD® library (Morphosys, Munich, Germany). By combining the most stable and well expressed frameworks, VH3 and Vκ3, with the further stabilizing constant domains, a Fab library with the desired properties was obtained in a standard phage display format. As a proof of principle, we selected binders with phage display against the detergent-solubilized citrate transporter CitS of Klebsiella pneumoniae. We describe efficient methods for the immobilization of the membrane protein during selection, for ELISA screening, and for BIAcore evaluation. We demonstrate that the selected Fab fragments form stable complexes with native CitS and recognize conformational epitopes with affinities in the low nanomolar range. [Copyright &y& Elsevier]

It is widely assumed that the functional activity of signal sequences has been conserved throughout evolution, at least between Gram-negative bacteria and eukaryotes. The ovalbumin family of serine protease inhibitors (serpins) provides a unique tool to test this assumption, since individual members can be secreted (ovalbumin), cytosolic (leukocyte elastase inhibitor, LEI), or targeted to both compartments (plasminogen activator inhibitor 2, PAI-2). The facultative secretion of PAI-2 is mediated by a signal sequence proposed to be inefficient by design. We show here that the same internal domain that promotes an inefficient translocation of murine PAI-2 in mammalian cells is a weak signal sequence in Escherichia coli. In contrast, the ovalbumin signal sequence is much more efficient, whereas the corresponding sequence elements from LEI, maspin and PI-10 are entirely devoid of signal sequence activity in E. coli. Mutations that improve the activity of the PAI-2 signal sequence and that convert the N-terminal regions of maspin and PI-10 into efficient signal sequences have been characterized. Taken together, these results indicate that several structural features contribute to the weak activity of the PAI-2 signal sequence and provide new insights into the plasticity of the “hydrophobic core” of signal sequences. High-level expression of two chimeric proteins containing the PAI-2 signal sequence is toxic, and the reduced viability is accompanied by a rapid decrease in the membrane proton motive force, in ATP levels and in translation. In unc− cells, which lack the F0F1 ATP-synthase, the chimeric proteins retain their toxicity and their expression only affected the proton motive force. Thus, the properties of these toxic signal sequences offer a new tool to dissect the interactions of signal sequences with the protein export machinery. [Copyright &y& Elsevier]

Aggrecanases are considered to play a key role in the destruction of articular cartilage during the progression of arthritis. Here we report that the N-terminal inhibitory domain of tissue inhibitor of metalloproteinases 3 (N-TIMP-3), but not TIMP-1 or TIMP-2, inhibits glycosaminoglycan release from bovine nasal and porcine articular cartilage explants stimulated with interleukin-1α or retinoic acid in a dose-dependent manner. This inhibition is due to the blocking of aggrecanase activity induced by the catabolic factors. Little apoptosis of primary porcine chondrocytes is observed at an effective concentration of N-TIMP-3. These results suggest that TIMP-3 may be a candidate agent for use against cartilage degradation. [Copyright &y& Elsevier]

Background/Aims: Very little is known about the HFE gene in the rat. The aim of the present study was to determine: (1) the structure of the rat HFE gene; and (2) the tissue expression of the HFE mRNA in the rat, with special emphasis on the liver.Methods: Cloning of the rat HFE gene was performed using library screening and PCR. Exon-intron borders were assigned by DNA sequencing. Parenchymal and non-parenchymal liver cells were isolated by fractionation of normal rat liver. HFE mRNA levels were determined by Northern blot (tissues) and real-time PCR (isolated liver cells).Results: The rat HFE gene contained six exons and five introns. The HFE gene is expressed in multiple tissues in the rat, including bone marrow, with the highest expression in the liver. We observed HFE transcripts in several categories of isolated rat liver cells. Unexpectedly, expression also occurred in rat hepatocytes.Conclusions: The exon-intron pattern of the HFE gene is strongly conserved between rat and mouse. The pattern of tissue expression of the HFE gene is rather similar in humans and rodents. The finding of HFE gene expression in rat hepatocytes raises interesting questions regarding its role in the hepatocyte iron metabolism. [Copyright &y& Elsevier]

This report establishes the conditions for monitoring the intrinsic Trp phosphorescence of proteins encapsulated in silica hydrogels and demonstrates the usefulness of the delayed emission for examining potential perturbations of protein structure-dynamics by the silica matrix. Phosphorescence measurements were conducted both in low temperature (140 K) glasses and at ambient temperature on the proteins apo- and Cd-azurin, alkaline phosphatase and liver alcohol dehydrogenase together with the complexes of liver alcohol dehydrogenase with coenzyme analogs ADPR and H2NADH. While spectral shifts and broadening indicate that alterations of the Trp microenvironment are more marked on superficial regions of the macromolecule the decay kinetics of deeply buried chromophores show that the internal flexibility of the polypeptide in two out of three cases is significantly affected by silica entrapment. Both the intrinsic lifetime and the bimolecular acrylamide quenching constant confirm that, relative to the aqueous solution, in hydrogels the globular fold is more rigid with azurin, looser with alcohol dehydrogenase and substantially unaltered with alkaline phosphatase. It was also noted that large amplitude structural fluctuations, as those involved in coenzyme binding to alcohol dehydrogenase or thermally activated in alkaline phosphatase, were not restricted by gelation. Common features of the three silica entrapped proteins are pronounced conformational heterogeneity and immobilization of rotational motions of the macromolecule in the long time scale of seconds. [Copyright &y& Elsevier]

Dickkopf1 (Dkk1) is a secreted antagonist of the Wnt/β-catenin signaling pathway that acts by direct binding to and inhibiting the Wnt co-receptor LRP6. The related Dkk2, however, can function either as LRP6 agonist or antagonist, depending on the cellular context, suggesting that its activity is modulated by unknown co-factors. We have recently identified the transmembrane proteins Kremen1 and -2 as additional Dkk receptors, which bind to both Dkk1 and Dkk2 with high affinity. Here we show that Kremen2 (Krm2) regulates Dkk2 activity during Wnt signaling. In human 293 fibroblasts transfected dkk2 activates LRP6 signaling. However, co-transfection of krm2 blocks the ability of Dkk2 to activate LRP6 and enhances inhibition of Wnt/Frizzled signaling. Krm2 also co-operates with Dkk4 to inhibit Wnt signaling, but not with Dkk3, which has no effect on Wnt signaling. Likewise, in Xenopus embryos, Dkk2 and Krm2 co-operate in Wnt inhibition leading to anteriorized embryos. Finally, we show that interaction with Krm2 is mediated by the second cysteine-rich domain of Dkks. These results suggest that Krm2 can function as a switch that turns Dkk2 from an activator into an inhibitor of Wnt/lRP6 signaling. [Copyright &y& Elsevier]

In a previous communication, we reported the isolation of a novel cDNA clone (pA6) from a library constructed from squid axonal mRNAs. The partial cDNA clone contained a unique open reading frame that encoded 84 amino acids and was complementary to a moderately abundant mRNA approximately 550–600 nucleotides in length [Chun et al., J. Neurosci. Res. 49 (1997) 144–153]. In this report, we identify the pA6 gene product, and characterize its expression in the squid and rodent brain. Results of immunoblot analyses conducted in squid, using a polyclonal antibody raised against a synthetic peptide corresponding to the C-terminus of the putative protein, established the presence of two pA6 immunoreactive proteins of approximately 14 kDa and 26 kDa in size. In contrast, mouse brain contained only a single 26-kDa immunoreactive species. In both the squid and mouse brain, the expression of pA6 appears highly selective, being detected in certain neurons but not in non-neuronal cells, as judged by both in situ hybridization and immunocytochemistry. Findings derived from light microscopic, double-label immunohistofluorescence studies indicate that pA6 protein co-localizes with prohibitin, a mitochondrial marker protein. Consistent with these results, electron microscopy localized pA6 immunoreactivity to several membrane compartments to include the outer membrane of mitochondria, as well as to the smooth endoplasmic reticulum and tubulovesicles in dendrites, axons, and axon terminals of neurons in the rat brain.Taken together, these findings indicate that pA6 is a novel, membrane-associated protein, which is expressed in the distal structural/functional domains of neurons in both the invertebrate and vertebrate nervous systems. [Copyright &y& Elsevier]

The peroxisome proliferator (PP) ciprofibrate stimulates gastrin-producing cells (G-cells) in the rat stomach by an unknown mechanism, inducing hypergastrinemia and secondary enterochromaffin-like (ECL) cell hyperplasia. Ciprofibrate is a specific ligand for the nuclear peroxisome proliferator-activated receptor α (PPARα). To see whether the effects of ciprofibrate could be imitated, rats were given another PPARα ligand WY-14643 or the PPARγ ligand troglitazone by gastric intubations daily for 28 and 56 days. Troglitazone failed to raise gastrin levels. WY-14643 increased gastrin mRNA abundance, G-cell density and induced hypergastrinemia, but to a lesser extent than ciprofibrate. ECL cell parameters increased in proportion with the relative hypergastrinemia. Ciprofibrate and WY-14643 altered the levels of acyl CoA-oxidase mRNA and PPARα mRNA in antrum, but had no effect in corpus. The PPARα receptor was found in at least some G-cells by immunostaining. This study supports the hypothesis that PPARα specific ligands could stimulate the G-cells by acting locally from the stomach lumen through antral PPARα. [Copyright &y& Elsevier]

The soluble N-ethylmaleimide-sensitive factor-attached protein receptor (SNARE) proteins syntaxin 1 and synaptosomal-associated protein-25 have been implicated in axonal outgrowth. Neuronal Sec1 (nSec1), also called murine unc18a (Munc18a), is a syntaxin 1-binding protein involved in the regulation of SNARE complex formation in synaptic vesicle membrane fusion. Here we analysed whether nSec1/Munc18a is involved in neurite formation. nSec1/Munc18a expressed under the control of an inducible promoter in differentiated PC12 cells as well as in hippocampal neurons appears first in the cell body, and at later times after induction along neurites and in growth cones. It is localised to distinct tubular and punctated structures. In addition, exogenous nSec1/Munc18a inhibited regulated secretion in PC12 cells. Overexpression in PC12 cells of nSec1/Munc18a or its homologue Munc18b, reduced the total length of neurites. This effect was enhanced with nSec1-T574A, a mutant that lacks a cyclin-dependent kinase 5 phosphorylation site and displays an increased binding to syntaxin 1. In contrast, in hippocampal neurons the total length of all primary neurites and branches was increased upon transfection of nSec1/Munc18a. Detailed morphometric analysis revealed that this was a consequence of an increased number of axonal side branches, while the average lengths in primary neurites and of side branches were not affected.From these results we suggest that nSec1/Munc18a is involved in the regulation of SNARE complex-dependent membrane fusion events implicated in the ramification of axonal processes in neurons. [Copyright &y& Elsevier]

Expression of the normal cellular form of prion protein is both necessary and rate-limiting in the spread of prion disease, yet its cellular expression in vivo is poorly understood. To optimise immunohistochemical labelling of this protein in mouse brain, we have developed novel antibodies that recognise cellular prion protein in glutaraldehyde-fixed tissue. Expression was found to be predominantly neuronal, and to differ between different classes of neurone. Thus, neurones immunoreactive for GABA expressed very high levels of normal prion protein; most projection neurones expressed much lower levels, particularly on their axons in the major fibre tracts, and some neurones (e.g. those positive for dopamine) displayed no detectable prion protein. In marked contrast, all neurones, even those that were immunonegative, expressed high levels of message for prion protein, shown by non-radioactive in situ hybridisation. Glia expressed very low levels of message, and undetectable levels of prion protein.We conclude that the steady-state level of prion protein, which differs so markedly between different neuronal types, is primarily controlled post-transcriptionally, possibly by differences in protein trafficking or degradation. These marked differences in the way different neurones produce and/or degrade their normal cellular prion protein may influence the selective spread and neurotoxic targeting of prion diseases within the CNS. [Copyright &y& Elsevier]

Alkaline phosphatases are non-specific phosphomonoesterases that are distributed widely in species ranging from bacteria to man. This study has concentrated on the tissue-nonspecific alkaline phosphatase from arctic shrimps (shrimp alkaline phosphatase, SAP). Originating from a cold-active species, SAP is thermolabile and is used widely in vitro, e.g. to dephosphorylate DNA or dNTPs, since it can be inactivated by a short rise in temperature.Since alkaline phosphatases are zinc-containing enzymes, a multiwavelength anomalous dispersion (MAD) experiment was performed on the zinc K edge, which led to the determination of the structure to a resolution of 1.9 A˚. Anomalous data clearly showed the presence of a zinc triad in the active site, whereas alkaline phosphatases usually contain two zinc and one magnesium ion per monomer.SAP shares the core, an extended β-sheet flanked by α-helices, and a metal triad with the currently known alkaline phosphatase structures (Escherichia coli structures and a human placental structure). Although SAP lacks some features specific for the mammalian enzyme, their backbones are very similar and may therefore be typical for other higher organisms. Furthermore, SAP possesses a striking feature that the other structures lack: surface potential representations show that the enzyme's net charge of −80 is distributed such that the surface is predominantly negatively charged, except for the positively charged active site. The negatively charged substrate must therefore be directed strongly towards the active site. It is generally accepted that optimization of the electrostatics is one of the characteristics related to cold-adaptation. SAP demonstrates this principle very clearly. [Copyright &y& Elsevier]

This study identifies a complement component C3-like protein in the solitary tunicate, Styela plicata. Three different polyclonal antibodies raised against C3 molecules from two species (humans and the tunicate, Halocynthia roretzi) were used to identify the C3-like protein in S. plicata hemolymph. The C3 cross-reactive protein is a 170 kDa heterodimer composed of polypeptides (116 and 80 kDa) that have molecular weights comparable to those of C3α and C3β from other species. Amino acid sequencing and amino acid composition analysis confirmed that the C3-like protein from S. plicata is closely related to C3 from H. roretzi. [Copyright &y& Elsevier]

The hyperthermophilic bacterium Thermotoga maritima encodes a gene sharing sequence similarities with several known genes for alkaline phosphatase (AP). The putative gene was isolated and the corresponding protein expressed in Escherichia coli, with and without a predicted signal sequence. The recombinant protein showed phosphatase activity toward the substrate p-nitrophenyl-phosphate with a kcat of 16 s−1 and a Km of 175 μM at a pH optimum of 8.0 when assayed at 25°C. T. maritima phosphatase activity increased at high temperatures, reaching a maximum kcat of 100 s−1, with a Km of 93 μM at 65°C. Activity was stable at 65°C for >24 h and at 90°C for 5 h. Phosphatase activity was dependent on divalent metal ions, specifically Co(II) and Mg(II). Circular dichroism spectra showed that the enzyme gains secondary structure on addition of these metals. Zinc, the most common divalent metal ion required for activity in known APs, was shown to inhibit the T. maritima phosphatase enzyme at concentrations above 0.3 moles Zn: 1 mole monomer. All activity was abolished in the presence of 0.1 mM EDTA. The T. maritima AP primary sequence is 28% identical when compared with E. coli AP. Based on a structural model, the active sites are superimposable except for two residues near the E. coli AP Mg binding site, D153 and K328 ( E. coli numbering) corresponding to histidine and tryptophan in T. maritima AP, respectively. Sucrose-density gradient sedimentation experiments showed that the protein exists in several quaternary forms predominated by an octamer. [ABSTRACT FROM AUTHOR]

The first problem in gene expression profiling to be solved is choosing the appropriate gene array, detection procedure, image analysis and data generation depending on the organism of interest, equipment and budget. The next one is how to deduce biologically meaningful data. We assessed gene expression data from chemiluminescent detection and empirically found criteria for the reliable identification of biologically meaningful expression ratios. Current statistical assessments are often applied unreflectedly concerning problems occurring in practice. So interesting results are considered to be irrelevant. This requires a laborious data check. We suggest automation. Our empirically found criteria were transformed into and validated by a knowledge-based system. This system is adaptable to all other methods of expression profiling. We compared the experience-based and new knowledge-based assessment of the expression data from our chemiluminescent and additionally radioactive detection of several experiments with published data to evaluate our entire procedure. With our adaptation of chemiluminescence detection to commercially available Escherichia coli gene arrays we present a useful alternative to common procedures in gene expression monitoring. Moreover, with our consideration of plasmid-harbouring E. coli strains we provide the opportunity to monitor gene expression during processes requiring any plasmids (e.g. recombinant protein expression). [Copyright &y& Elsevier]

Rapid uptake of synaptically released glutamate via the high affinity glutamate transporter 1 (GLT1; EAAT2) is important for limiting transmitter signalling and prevents a harmful receptor overstimulation. So far, in the adult brain GLT1 protein has only been detected in astrocytes. Here, we describe the cDNA cloning of a variant of GLT1 from rat brain which is generated by alternative splicing at the 3′-end of the GLT1 cDNA. Reverse transcription-polymerase chain reaction revealed that the GLT1 variant message was present not only in brain, but also in peripheral organs. Northern blot analysis showed that in brain the mRNA of GLT1 (∼11 kb) is predominant while in the retina the mRNA of GLT1 variant (∼12.5 kb) prevails. In situ hybridization using cRNA and oligonucleotide probes, and immunocytochemistry using an antibody against a synthetic GLT1v peptide were applied in order to identify the cell types expressing GLT1 variant in the adult rat nervous system. GLT1 variant is preferentially expressed in neurons of the CNS and PNS, but is also detected in glial cells (oligodendrocytes, ependymal cells, epithelial cells of the plexus choroideus, satellite cells, and Schwann cells). In contrast to GLT1, GLT1 variant was only occasionally detected in astrocytes. Immunolabelling revealed a preferentially cytoplasmic (frequently granular) staining of neurons and glial cells, suggesting a localization of GLT1 variant protein in vesicle membranes.The studies provide evidence that the cellular expression of the GLT1 variant in the CNS is almost complementary to that of GLT1 and that the GLT1 variant does not seem to be restricted to the CNS. [Copyright &y& Elsevier]

Objectives: Causative variants in genes responsible for Alström syndrome (ALMS) and Bardet-Biedl syndrome (BBS) cause damage to primary cilia associated with correct functioning of cell signaling pathways in many tissues. Despite differences in genetic background, both syndromes affect multiple organs and numerous clinical manifestations are common including obesity, retinal degeneration, insulin resistance, type 2 diabetes and many others. The aim of the study was to evaluate bone metabolism abnormalities and their relation to metabolic disorders based on bone turnover markers and presence of mandibular atrophy in patients with ALMS and BBS syndromes., Material and Methods: In 18 patients (11 with ALMS and 7 with BBS aged 5-29) and in 42 age-matched ( p  < 0.05) healthy subjects, the following markers of bone turnover were assessed: serum osteocalcin (OC), osteoprotegerin (OPG), s-RANKL and urinary deoxypyridinoline - DPD. In addition, a severity of alveolar atrophy using dental panoramic radiograms was evaluated., Results: Lower serum OC ( p  = 0.0004) and urinary DPD levels ( p  = 0.0056) were observed in the study group compared to controls. In ALMS and BBS patients, serum OC and urinary DPD values negatively correlated with the HOMA-IR index, while a positive correlation between the OC and 25-OHD levels and a negative correlation between s-RANKL and fasting glucose concentrations were found. A significant difference in the incidence of low-grade mandibular atrophy between patients with ALMS and BBS and controls ( p  < 0.0001) was observed., Conclusions: The identification of bone metabolism disorders in patients with ALMS and BBS syndromes indicates the necessity to provide them with appropriate diagnosis and treatment of these abnormalities., Competing Interests: The authors declare that they have no conflict of interest., (© 2022 The Authors. Published by Elsevier Inc.)

Background & Aims: Increased serum IgG and autoantibodies suggest involvement of B cells in autoimmune hepatitis (AIH). The aim of this study was to assess levels of B cell activating factor of the tumour necrosis family (BAFF), IL-21, and circulating B cell populations in AIH and correlate these to treatment response., Methods: BAFF and IL-21 levels were determined in 66 patients with AIH before treatment and 10 healthy controls. Flow cytometry was performed on circulating B cells of 10 patients with AIH and 12 healthy controls., Results: Based on BAFF and IL-21 levels, untreated patients with AIH were divided into 3 groups: 27 (41%) patients with normal BAFF and IL-21 (normal BAFF), 27 (41%) patients with elevated BAFF but normal IL-21 (high BAFF), and 12 (18%) patients with elevated IL-21 (high IL-21). The high BAFF group presented with higher bilirubin compared with the normal BAFF and high IL-21 groups (159 vs . 26 vs . 89 μmol/L; p  = 0.001; Mann-Whitney U test). After 12 months of treatment, 54% of the high BAFF group reached remission compared with 34% of the normal BAFF group and 0% of the high IL-21 group ( p  = 0.006, Chi-square test). During follow-up, 3 patients (25%) with high IL-21 developed primary sclerosing cholangitis (PSC) variant syndrome. Autoimmune-associated B cells were increased in patients with AIH compared with healthy controls (4.4 vs . 1.4%; p  = 0.003, Mann-Whitney U test). BAFF levels were correlated positively with naïve B cells ( p  = 0.01) and negatively with class-switched B cells ( p  = 0.003) and nonclass-switched B cells ( p  = 0.005, Spearman correlation)., Discussion: Using BAFF and IL-21, we identified different immunological phenotypes of AIH with a different presentation, treatment response, and outcome. Patients with high IL-21 had the poorest treatment response and a risk of developing PSC variant syndrome. BAFF level was related to shifts in circulating B-cell populations., Lay Summary: In patients with untreated autoimmune hepatitis (AIH), circulating B-cell activating factor of the tumour necrosis family (BAFF), IL-21, and B-cell populations were determined. Three subgroups were identified: with (1) normal BAFF and IL-21, (2) elevated BAFF and normal IL-21, and (3) elevated IL-21. Remission after 1-year treatment occurred in 54, 34, and 0% in Groups 1, 2, and 3, respectively. Group 2 had higher bilirubin, indicating more liver dysfunction. In 25% of patients with high IL-21, AIH-PSC variant syndrome developed, but none in the other groups. Autoimmune-associated B cells were elevated and BAFF levels correlated with certain B cells., Competing Interests: MB was supported by unrestricted grants from Zambon Pharma. No other potential conflict of interests. Please refer to the accompanying ICMJE disclosure forms for further details., (© 2022 The Authors.)

The shortest functional domains of growth factor B one M orphogenetic P rotein 2 (BMP-2) that are dynamical implicated in osteogenesis have been investigated and well characterized. In particular, the broad C-terminal region expanding from Val63 to Arg114 as well as its shorter sequence 86-AISMLYLDEN-95 exhibited the highest osteogenic ability for regeneration and reconstruction of bone tissue. In addition, the amino acids Ser88 and Leu90 have been identified as crucial for receptor binding and osteogenic efficacy. Furthermore, the above-mentioned domains in contrary to full length BMP-2 protein signal mainly through the Smad pathway as it is evidenced by phosphorylation decrease of Extracellular-signal-Regulated Kinase (ERK1/2). Taking together, our results are significant for clinical applications regarding the generation of biomaterials and healing of orthopedic fractures., Competing Interests: None., (© 2021 The Author(s).)

Objective: To determine the prevalence of Charcot triad, Reynolds pentad, and Tokyo Guidelines criteria and clinical outcomes among patients with cholangitis across different age groups., Patients and Methods: We conducted a retrospective analysis of 257 consecutive hospitalized adult patients with acute cholangitis due to endoscopic retrograde cholangiopancreatography-confirmed choledocholithiasis between January 1, 2015, and December 31, 2019. Patients were divided into 3 age groups: less than 65 years, 65 to 79 years, and 80 years or older. Symptoms, vital signs, and laboratory data on admission were collected. Outcomes included length of hospitalization, intensive care unit stay, and 3-month mortality. Nominal variables were tested with the Pearson χ 2 test, and continuous variables were tested with the Wilcoxon rank sum test., Results: Charcot triad decreased with older ages. In the group that was age 80 years or older, malaise was the most common symptom; 33.6% (37 of 110) presented with altered sensorium, 9.1% (10 of 110) had no pain, fever, or jaundice, and positive blood culture results were more frequent. Tokyo cholestasis criterion was present in 96.0% (247 of 257), while inflammation (considered essential for diagnosis) was present in 75.9% (195 of 257). Patients 80 years or older had significantly higher mean length of hospital stay ( P <.001) and mean length of intensive care unit stay ( P =.021)., Conclusion: Compared with patients in younger age groups, patients with cholangitis who are 80 years or older are less likely to have Charcot triad, are more likely to have features of Reynolds pentad, or present with unexplained malaise. Within the Tokyo Guidelines, cholestasis should replace inflammation as an essential diagnostic criterion., (© 2021 Mayo Foundation for Medical Education and Research. Published by Elsevier Inc.)