Glycine‐modified growth hormone secretagogues identified in seized doping material

A number of unknown pharmaceutical preparations seized by Danish customs authorities were submitted for liquid chromatography–high resolution mass spectrometry (LC–HRMS) analysis. Comparison with reference standards unequivocally identified the content of the powders as analogs of the growth hormone secretagogues GHRP‐2 (Pralmorelin), GHRP‐6, Ipamorelin, and modified growth hormone releasing factor (modified GRF 1–29), which can be used as performance‐enhancing substances in sports. In all cases, the detected modification involved the addition of an extra glycine amino acid at the N‐terminus, and analytical methods targeting growth hormone secretagogues should hence be updated accordingly.     

Action research methodology in clinical pharmacy: how to involve and change

Introduction The focus in clinical pharmacy practice is and has for the last 30–35 years been on changing the role of pharmacy staff into service orientation and patient counselling. One way of doing this is by involving staff in change process and as a researcher to take part in the change process by establishing partnerships with staff. On the background of the authors’ widespread action research (AR)-based experiences, recommendations and comments for how to conduct an AR-study is described, and one of their AR-based studies illustrate the methodology and the research methods used. Methodology AR is defined as an approach to research which is based on a problem-solving relationship between researchers and clients, which aims at both solving a problem and at collaboratively generating new knowledge. Research questions relevant in AR-studies are: what was the working process in this change oriented study? What learning and/or changes took place? What challenges/pitfalls had to be overcome? What were the influence/consequences for the involved parts? When to use If you want to implement new services and want to involve staff and others in the process, an AR methodology is very suitable. The basic advantages of doing AR-based studies are grounded in their participatory and democratic basis and their starting point in problems experienced in practice. Limitations Some of the limitations in AR-studies are that neither of the participants in a project steering group are the only ones to decide. Furthermore, the collective process makes the decision-making procedures relatively complex.

     

Availability of Paracetamol Sold Over the Counter in Europe: A Descriptive Cross‐Sectional International Survey of Pack Size Restriction

Due to the risk of hepatotoxicity when excessive amounts of paracetamol are consumed, Poisons Information Centers (PICs) frequently receive paracetamol‐related enquiries. This study examined how widely pack size restrictions of paracetamol sold over the counter have been implemented in Europe and also availability of paracetamol through non‐pharmacy outlets and their possible associations with frequency of poisoning enquiries. A cross‐sectional European multi‐centre questionnaire study was performed using a questionnaire to identify the extent and nature of paracetamol pack size restrictions, non‐pharmacy outlet sales and the frequency of paracetamol‐related enquiries to PICs. In total, 21 European countries participated. All PICs provided telephone hotline services. In 14 (67%) countries, pack size restrictions had been implemented in pharmacies (range: 8–30 g). No significant difference (median difference 0.7%, p‐value = 0.36) was found when comparing median frequencies of paracetamol‐related enquiries in countries with pack size restriction to countries without restrictions. A significantly lower median frequency of paracetamol‐related enquiries was found in countries without non‐pharmacy outlet sales compared to those with such sales (median difference 2.2%, p = 0.02). Pack size restrictions on pharmacy sales of paracetamol have been implemented in two‐thirds of examined countries. There was no difference in the proportion of paracetamol‐related enquiries to PICs among countries with and without pack size restrictions. However, a lower rate of paracetamol‐related enquiries was noted in countries where paracetamol was not available in non‐pharmacy outlets.     

Advanced Electrocardiogram Analysis in the Amitriptyline‐poisoned Pig Treated with Activated Charcoal Haemoperfusion

Coated activated charcoal haemoperfusion (CAC‐HP) does not reduce the plasma concentration in amitriptyline (AT)‐poisoned pigs. The aim of this non‐blinded, randomized, controlled animal trial was to determine if CAC‐HP reduces the pathological ECG changes caused by AT poisoning. Fourteen female Danish Landrace pigs (mean weight 27.7 kg, range 20–35 kg (CAC‐HP) and 24.4 kg, range 18–30 kg (control group, CG), n = 7 in each group) were included. After randomization, the pigs were anaesthetized and intravenously poisoned with AT. The intervention group underwent 4 hr of CAC‐HP plus standard care (oral activated charcoal). Intervention was compared to standard care alone. From each pig, a 12‐lead ECG and haemodynamic variables were obtained at baseline, at full AT loading dose, before and during CAC‐HP. Baseline ECG variables (RR, PR, QRS, QTc, QTp, QTe, TpTe and TpTe/QT) for lead II, v2 and v5 were not significantly different (F = 0.035–0.297, p‐values 0.421–0.919). Differences within groups over time and between groups were tested by anova repeated measures. For all variables, the time‐plus‐group level of significance revealed a p‐value > 0.05. Severe cardiovascular arrhythmias occurred in both groups with 3 in the CAC‐HP group versus 1 incident with premature death in the CG. The attenuating effect of CAC‐HP to orally instilled activated charcoal alone on AT‐induced ECG alterations did not differ significantly. We conclude that the use of modern CAC‐HP as an adjunctive treatment modality in AT‐poisoned pigs is inadequate.     

Peptidoglycan-binding protein TsaP functions in surface assembly of type IV pili

Type IV pili (T4P) are ubiquitous and versatile bacterial cell surface structures involved in adhesion to host cells, biofilm formation, motility, and DNA uptake. In Gram-negative bacteria, T4P pass the outer membrane (OM) through the large, oligomeric, ring-shaped secretin complex. In the β-proteobacterium Neisseria gonorrhoeae, the native PilQ secretin ring embedded in OM sheets is surrounded by an additional peripheral structure, consisting of a peripheral ring and seven extending spikes. To unravel proteins important for formation of this additional structure, we identified proteins that are present with PilQ in the OM. One such protein, which we name T4P secretin-associated protein (TsaP), was identified as a phylogenetically widely conserved component of the secretin complex that co-occurs with genes for T4P in Gram-negative bacteria. TsaP contains an N-terminal carbohydrate-binding lysin motif (LysM) domain and a C-terminal domain of unknown function. In N. gonorrhoeae, lack of TsaP results in the formation of membrane protrusions containing multiple T4P, concomitant with reduced formation of surface-exposed T4P. Lack of TsaP did not affect the oligomeric state of PilQ, but resulted in loss of the peripheral structure around the PilQ secretin. TsaP binds peptidoglycan and associates strongly with the OM in a PilQ-dependent manner. In the δ-proteobacterium Myxococcus xanthus, TsaP is also important for surface assembly of T4P, and it accumulates and localizes in a PilQ-dependent manner to the cell poles. Our results show that TsaP is a novel protein associated with T4P function and suggest that TsaP functions to anchor the secretin complex to the peptidoglycan.Type IV pili systems (T4PSs) are involved in the assembly of long, thin fibers, which are found on the surfaces of many bacteria and archaea (1). Type IV pili (T4P) function in host cell adhesion, twitching motility, virulence, DNA uptake, and biofilm formation and are evolutionary related to type II secretion systems (T2SSs), bacterial transformation systems, and the archaellum (2–4). T4PSs can be divided into T4aPSs and T4bPSs that are distinguished based on pilin size and assembly systems (5, 6). T4aPSs form the most abundant class, and the T4P formed by these systems can undergo cycles of extension, adhesion, and retraction, which is a feature that distinguishes them from the other bacterial surface structures (7, 8). T4aP retract at rates up to 1 μm/s and can generate forces up to 150 pN (9, 10). Generally, T4bPSs are not associated with retraction. Here, we focus on T4aPSs and refer to these as T4PSs unless specifically indicated. T4PSs have been studied extensively in many bacteria but are especially well characterized in Neisseria and Pseudomonas spp. and in Myxococcus xanthus. Different nomenclature is used for different T4PSs (Table S1). Here, the Neisseria gonorrhoeae nomenclature is used.T4P are composed of major (e.g., PilE) and minor (in N. gonorrhoeae; e.g., PilV, PilX, ComP) pilins that are synthesized as preproteins with a type III signal peptide. After cleavage of the signal peptide by the prepilin peptidase PilD (11, 12), the T4P are assembled by a multiprotein complex (13). In Gram-negative bacteria, the proteins of T4PSs can be divided into three subcomplexes: the inner membrane (IM) motor complex, the alignment complex, and the outer membrane (OM) pore complex (6). The IM motor complex drives both the assembly and the retraction of T4P. Pilin subunits are extruded from the IM by the platform protein PilG (14) and the hexameric ATPase PilF (15). Disassembly of T4P with retraction occurs when PilF is replaced by the hexameric ATPase PilT (7, 16). PilU, a PilT paralog, is involved in retraction to a lesser extent (17). The alignment complex consisting of PilM, PilN, PilO, and PilP is proposed to connect the IM motor complex and the OM pore complex, and it is also thought to be involved in the stability and/or gating of the OM complex (18–20). In the OM, PilQ forms a homooligomeric ring that serves as a conduit for T4P (21–23).PilQ is a member of the secretin protein family. Proteins belonging to this family are present in many Gram-negative bacteria and are components of T4PSs, T2SSs, type III secretion systems (T3SSs), and extrusion systems of filamentous phages (24). Secretins are multidomain proteins with a signal sequence and a conserved C-terminal OM-spanning domain. Most secretins contain multiple copies of an N-terminal α/β domain (the N domains). PilQ proteins are integral OM proteins and form large gated channels. Oligomeric secretin complexes with different symmetries have been identified. Structural characterization by EM of purified PilQ from Neisseria meningitidis showed a dodecameric structure with a chamber sealed at both ends (25, 26), whereas the T2SS secretins PulD (27) and GspD (28) of the Klebsiella oxytoca pullanase and Vibrio cholerae toxin secretion systems, respectively, showed dodecameric structures with a chamber open at the periplasmic side and closed at the OM side. The structure of the InvG secretin complex of the T3SS of the Salmonella typhimurium needle complex showed 15-fold symmetry and is open at both ends (29), and the phage pIV secretin showed 14-fold symmetry (30). The structure of the C-terminal OM-spanning domain involved in multimer formation is currently not known. Crystal structures of the periplasmic N domains of GspD of the T2SS of enterotoxigenic Escherichia coli (31), of EscC of the T3SS of S. typhimurium (32), and of N. meningitidis PilQ (25) showed that these domains consist of α-helices packed against three-stranded β-sheets. Secretins of T4P systems also contain B domains, which are not present in other secretins and are located N-terminal to the N domains. The structure of the B2 domain of N. meningitidis PilQ consists of several β-strands (25). Remarkably, when the sequence conservation of the B2 domain was mapped to the structure of the B2 domain of N. meningitidis PilQ, a highly conserved patch was identified that was proposed to form the binding site for a currently unidentified T4PS protein (25).Secretins interact with several other proteins. Pilotin proteins are small lipoproteins that interact with the extreme C terminus of secretins and are responsible for OM targeting and oligomerization of secretins (33–38). Secretins of T4PSs also interact with the alignment complex. For N. meningitidis, Pseudomonas aeruginosa, and M. xanthus PilQ, a direct interaction was demonstrated between the respective PilPs and the N0 domains of the PilQs (25, 39, 40). Recently, ExeA of the T2SS of Aeromonas hydrophila (41) and FimV of the T4PS of P. aeruginosa (42) were also implicated in secretin assembly. They contain, respectively, PF01471 and LysM peptidoglycan (PG)-binding domains that might attach them to the PG. However, neither of these two proteins is ubiquitously conserved in bacteria assembling T4P.We have previously shown that the PilQ secretin of N. gonorrhoeae embedded in OM sheets is surrounded by a peripheral structure, which is formed by an additional peripheral ring as well as spikes (43). The proteins that make up these structures are not known. Here, we identify a widely conserved protein, which we name T4P secretin-associated protein (TsaP), that is important for the formation of the peripheral structure. Phylogenomic analysis of 450 genomes of Proteobacteria showed that the presence of the tsaP gene is strongly linked to the presence of genes for T4aPSs. We characterize the TsaP protein and demonstrate the importance of TsaP for T4aP assembly in the two phylogenetically widely separated model organisms N. gonorrhoeae and M. xanthus.     

A mutation update on the LDS‐associated genes TGFB2/3 and SMAD2/3

The Loeys–Dietz syndrome (LDS) is a connective tissue disorder affecting the cardiovascular, skeletal, and ocular system. Most typically, LDS patients present with aortic aneurysms and arterial tortuosity, hypertelorism, and bifid/broad uvula or cleft palate. Initially, mutations in transforming growth factor‐β (TGF‐β) receptors (TGFBR1 and TGFBR2) were described to cause LDS, hereby leading to impaired TGF‐β signaling. More recently, TGF‐β ligands, TGFB2 and TGFB3, as well as intracellular downstream effectors of the TGF‐β pathway, SMAD2 and SMAD3, were shown to be involved in LDS. This emphasizes the role of disturbed TGF‐β signaling in LDS pathogenesis. Since most literature so far has focused on TGFBR1/2, we provide a comprehensive review on the known and some novel TGFB2/3 and SMAD2/3 mutations. For TGFB2 and SMAD3, the clinical manifestations, both of the patients previously described in the literature and our newly reported patients, are summarized in detail. This clearly indicates that LDS concerns a disorder with a broad phenotypical spectrum that is still emerging as more patients will be identified. All mutations described here are present in the corresponding Leiden Open Variant Database.