The intestinal MUC2 mucin C-terminus is stabilized by an extra disulfide bond in comparison to von Willebrand factor and other gel-forming mucins.

The MUC2 mucin polymer is the main building unit of the intestinal mucus layers separating intestinal microbiota from the host epithelium. The MUC2 mucin is a large glycoprotein with a C-terminal domain similar to the MUC5AC and MUC5B mucins and the von Willebrand factor (VWF). A structural model of the C-terminal part of MUC2, MUC2-C, was generated by combining Cryo-electron microscopy, AlphaFold prediction, information of its glycosylation, and small angle X-ray scattering information. The globular VWD4 assembly in the N-terminal of MUC2-C is followed by 3.5 linear VWC domains that form an extended flexible structure before the C-terminal cystine-knot. All gel-forming mucins and VWF form tail-tail disulfide-bonded dimers in their C-terminal cystine-knot domain, but interestingly the MUC2 mucin has an extra stabilizing disulfide bond on the N-terminal side of the VWD4 domain, likely essential for a stable intestinal mucus barrier.

Identification of C. elegans ASNA-1 domains and tissue requirements that differentially influence platinum sensitivity and growth control.

ASNA1 plays an essential role in cisplatin chemotherapy response, type 2 diabetes, and heart disease. It is also an important biomarker in the treatment response of many diseases. Biochemically, ASNA1 has two mutually exclusive redox-modulated roles: a tail-anchored protein (TAP) targeting function in the reduced state and a holdase/chaperone function in the oxidized state. Assigning biochemical roles of mammalian ASNA1 to biomedical functions is crucial for successful therapy development. Our previous work showed the relevance of the C. elegans ASNA-1 homolog in modeling cisplatin response and insulin secretion. Here we analyzed two-point mutants in highly conserved residues in C. elegans ASNA-1 and determined their importance in separating the cisplatin response function from its roles in insulin secretion. asna-1(ΔHis164) and asna-1(A63V) point mutants, which both preferentially exist in the oxidized state, displayed cisplatin sensitivity phenotype as well as TAP insertion defect but not an insulin secretion defect. Further, using targeted depletion we analyzed the tissue requirements of asna-1 for C. elegans growth and development. Somatic depletion of ASNA-1 as well as simultaneous depletion of ASNA-1 in neurons and intestines resulted in an L1 arrest. We concluded that, targeting single residues in ASNA-1 affecting Switch I/Switch II domain function, in comparison to complete knockdown counteracted cisplatin resistance without jeopardizing other important biological functions. Taken together, our study shows that effects on health caused by ASNA1 mutations can have different biochemical bases.

A Clinical Trial Protocol to Compare the Effect of Dry Needling and Acupoint Dry Needling on Wrist Flexor Spasticity after Stroke.

Background: Stroke is the leading cause of disability in adults worldwide, with spasticity after stroke being one of the more common complications. Dry needling (DN) has been demonstrated to decrease spasticity in stroke patients, although its effects on improving function remain unclear. The purpose of this study protocol is to compare the short-term effects of DN versus acupoint DN on wrist flexor spasticity and upper extremity function in patients with stroke. Methods: A double-blind, randomized clinical trial will be conducted to include patients with stroke and upper extremity spasticity and functional disability. Twenty-four patients with stroke will be randomly assigned to either the DN or acupoint DN (DNap) group. These groups will receive three DN sessions during a one-week period of the wrist flexor muscles or at the LI4 & TE5 acupoints, respectively. The primary outcome measure is the Modified Modified Ashworth Scale (MMAS) of spasticity. The secondary outcomes are the Fugl-Meyer Assessment of motor function (FMA) and the goniometry to assess the active and passive range of motion of the wrist. Data will be collected at baseline, immediately after the end of three sessions DN, and at a one-week follow-up visit. Discussion: The results of this comparative study will help to determine the more effective method for reducing spasticity and improving motor function in patients with stroke.

The effect of dry needling of myofascial trigger points on muscle stiffness and motoneuron excitability in healthy subjects.

BACKGROUND: Myofascial trigger points (MTrPs) are hypersensitive nodules in a taut band (TB) of skeletal muscle. Dry needling (DN) is an invasive technique recommended for the treatment of MTrPs. However, to our knowledge, no studies have investigated the influence of the DN technique on modification of muscle stiffness and neurophysiological properties of MTrPs. OBJECTIVE: The objective was to examine the effect of DN on muscle stiffness and motoneuron excitability of a latent medial MTrP (nodule and TB) of the soleus muscle in non-injured subjects. METHODS: A double-blinded randomised controlled trial of 46 subjects with latent medial MTrPs of the soleus was conducted, in which all received one session of DN. The intervention group (n = 23) were subjected to DN into the MTrP (the nodule), while the control group (n = 23) were subjected to DN into the TB. Assessment was carried out at baseline (pre-test), after the intervention (post-test) and 1 week after the intervention (follow-up). Biomechanical variables (muscle resistive force at 10°/s and 180°/s, muscle extensibility and strength), as measured with an isokinetic dynamometer, and neurophysiological variables (H-reflex), were recorded. RESULTS: There were no statistically significant differences in biomechanical or neurophysiological assessments between groups. Considering the intra-group analysis, subjects in the intervention group exhibited increased maximal isometric voluntary force to ankle plantarflexion (MIVFp) at both post-intervention and follow-up assessment (p < 0.0125; 0.2 < d < 0.5), while no changes were found in the control group. CONCLUSION: One session of DN targeting latent MTrPs did not change muscle stiffness, muscle extensibility or motoneuron excitability. Further research on subjects with muscle tone disorders should be considered to better address the impact of DN on muscle tone. TRIAL REGISTRATION NUMBER: NCT02575586 (ClinicalTrials.gov).

Effects of Dry Needling on Biomechanical Properties of the Myofascial Trigger Points Measured by Myotonometry: A Randomized Controlled Trial.

OBJECTIVE: The purpose of the present study was to examine the effect of dry needling (DN) on the biomechanical properties of a latent medial myofascial trigger point (MTrP) of the soleus muscle compared with an adjacent point within the taut band (TB) measured by myotonometry. METHODS: Fifty asymptomatic volunteers were randomly assigned to an intervention group (n = 26) or control group (n = 24). One session of DN was performed in every group as follows: 10 needle insertions into the MTrP area (intervention group) or TB area (control group). Myotonometric measurements (frequency, decrement, and stiffness) were performed at baseline (pre-intervention) and after the intervention (post-intervention) in both locations (MTrP and TB areas). RESULTS: The results showed that stiffness outcome significantly decreased with a large effect size after DN in the MTrP when measured in the MTrP location (P = .002; d = 0.928) but not when measured in the TB location. In contrast, no significant changes were observed in any location when the TB was needled (P > .05). CONCLUSIONS: The findings suggest that only DN into the MTrP area was effective in decreasing stiffness outcome, therefore a specific puncture was needed to modify myofascial muscle stiffness.

The IgGFc-binding protein FCGBP is secreted with all GDPH sequences cleaved but maintained by interfragment disulfide bonds.

Mucus forms an important protective barrier that minimizes bacterial contact with the colonic epithelium. Intestinal mucus is organized in a complex network with several specific proteins, including the mucin-2 (MUC2) and the abundant IgGFc-binding protein, FCGBP. FCGBP is expressed in all intestinal goblet cells and is secreted into the mucus. It is comprised of repeated von Willebrand D (vWD) domain assemblies, most of which have a GDPH amino acid sequence that can be autocatalytically cleaved, as previously observed in the mucins MUC2 and mucin-5AC. However, the functions of FCGBP in the mucus are not understood. We show that all vWD domains of FCGBP with a GDPH sequence are cleaved and that these cleavages occur early during biosynthesis in the endoplasmic reticulum. All cleaved fragments, however, remain connected via a disulfide bond within each vWD domain. This cleavage generates a C-terminal-reactive Asp-anhydride that could react with other molecules, such as MUC2, but this was not observed. Quantitative analyses by MS showed that FCGBP was mainly soluble in chaotropic solutions, whereas MUC2 was insoluble, and most of the secreted FCGBP was not covalently bound to MUC2. Although FCGBP has been suggested to bind immunoglobulin G, we were unable to reproduce this binding in vitro using purified proteins. In conclusion, while the function of FCGBP is still unknown, our results suggest that it does not contribute to covalent crosslinking in the mucus, nor incorporate immunoglobulin G into mucus, instead the single disulfide bond linking each fragment could mediate controlled dissociation.

Synthesis and Structural/Functional Characterization of Selective M14 Metallocarboxypeptidase Inhibitors Based on Phosphinic Pseudopeptide Scaffold: Implications on the Design of Specific Optical Probes.

Metallocarboxypeptidases (MCPs) of the M14 family are Zn2+-dependent exoproteases present in almost every tissue or fluid in mammals. These enzymes perform a large variety of physiological functions and are involved in several pathologies, such as pancreatic diseases, inflammation, fibrinolysis, and cancer. Here, we describe the synthesis and functional/structural characterization of a series of reversible tight-binding phosphinic pseudopeptide inhibitors that show high specificity and potency toward these proteases. Characterization of their inhibitory potential against a large variety of MCPs, combined with high-resolution crystal structures of three selected candidates in complex with human carboxypeptidase A (CPA)1, allowed to decipher the structural determinants governing selectivity for type-A of the M14A MCP family. Further, the phosphinic pseudopeptide framework was exploited to generate an optical probe selectively targeting human CPAs. The phosphinic pseudopeptides presented here constitute the first example of chemical probes useful to selectively report on type-A MCPs activity in complex media.

Discovery of Mechanism-Based Inactivators for Human Pancreatic Carboxypeptidase A from a Focused Synthetic Library.

Metallocarboxypeptidases (MCPs) are involved in many biological processes such as fibrinolysis or inflammation, development, Alzheimer's disease, and various types of cancer. We describe the synthesis and kinetic characterization of a focused library of 22 thiirane- and oxirane-based potential mechanism-based inhibitors, which led to discovery of an inhibitor for the human pro-carboxypeptidase A1. Our structural analyses show that the thiirane-based small-molecule inhibitor penetrates the barrier of the pro-domain to bind within the active site. This binding leads to a chemical reaction that covalently modifies the catalytic Glu270. These results highlight the importance of combined structural, biophysical, and biochemical evaluation of inhibitors in design strategies for the development of spectroscopically nonsilent probes as effective beacons for in vitro, in cellulo, and/or in vivo localization in clinical and industrial applications.

Cavity filling mutations at the thyroxine-binding site dramatically increase transthyretin stability and prevent its aggregation.

More than a hundred different Transthyretin (TTR) mutations are associated with fatal systemic amyloidoses. They destabilize the protein tetrameric structure and promote the extracellular deposition of TTR as pathological amyloid fibrils. So far, only mutations R104H and T119M have been shown to stabilize significantly TTR, acting as disease suppressors. We describe a novel A108V non-pathogenic mutation found in a Portuguese subject. This variant is more stable than wild type TTR both in vitro and in human plasma, a feature that prevents its aggregation. The crystal structure of A108V reveals that this stabilization comes from novel intra and inter subunit contacts involving the thyroxine (T4) binding site. Exploiting this observation, we engineered a A108I mutation that fills the T4 binding cavity, as evidenced in the crystal structure. This synthetic protein becomes one of the most stable TTR variants described so far, with potential application in gene and protein replacement therapies.

Strategic orientations and cooperation of external agents in the innovation process of rural enterprises / As orientações estratégicas e a cooperação dos agentes externos no processo de inovação das empresas rurais

ABSTRACT: This article provides empirical evidence for the relationship between the market orientation, entrepreneurial orientation, and collaboration of external actors in the processes of incremental and radical innovations in rural enterprises. The research tested two sets of assumptions: first, the relation between contributions of strategic orientation and innovation processes; and second, collaborations of external stakeholders in the implementation of innovation processes. Data were collected from 208 rural enterprises and analyzed with the use of techniques of partial least squares structural equation modeling. Results showed that the market orientation contributes to the development of incremental innovation, and that the entrepreneurial orientation contributes to the implementation of the incremental and radical innovations. Specific agents (buyers, suppliers of goods and services, consultants, and others producers), collaborate positively in implementing incremental innovation; however, generic agents do not. The generic agents (universities and specialized public organizations) are the ones that most intervene in radical innovations. The use of consistent theory in the areas of strategy, marketing and management in identifying that some of their causal relationships are confirmed for rural businesses unlike others, due to the structure of the market and the products produced by them.

RESUMO: Este artigo fornece evidências empíricas sobre as relações entre a orientação para o mercado, a orientação empreendedora e a colaboração dos agentes externos nos processos de inovações incrementais e radicais em empresas rurais. A pesquisa testou três conjuntos de hipóteses: no primeiro, relacionaram-se as contribuições das orientações estratégicas e os processos de inovação; no segundo, verificaram-se as colaborações dos agentes externos na implementação dos processos de inovação e, no terceiro, avaliaram-se os efeitos de moderação do ambiente e da intensidade competitiva no relacionamento entre as orientações estratégicas e os processos de inovações. Os dados foram coletados em 208 empresas rurais e analisados com o emprego de técnicas da modelagem de equações estruturais pelo método de mínimos quadrados parciais. Os resultados mostram que a orientação para o mercado contribui para o desenvolvimento da inovação incremental, e que a orientação empreendedora contribui na implementação das inovações incrementais e radicais. Os agentes específicos (compradores, fornecedores de bens e serviços, consultores e outros agricultores), colaboram positivamente na execução da inovação incremental, mas os agentes genéricos, não. Os agentes genéricos (universidades e organizações públicas) são as que intervêm mais na inovação radical. Destaca-se a utilização de teoria consistente na área de estratégia, marketing e gestão na identificação de que algumas das suas relações causais são confirmadas para as empresas rurais, e outras não, em decorrência da estrutura, do mercado e dos produtos produzidos por elas.

Repositioning tolcapone as a potent inhibitor of transthyretin amyloidogenesis and associated cellular toxicity.

Transthyretin (TTR) is a plasma homotetrameric protein implicated in fatal systemic amyloidoses. TTR tetramer dissociation precedes pathological TTR aggregation. Native state stabilizers are promising drugs to treat TTR amyloidoses. Here we repurpose tolcapone, an FDA-approved molecule for Parkinson's disease, as a potent TTR aggregation inhibitor. Tolcapone binds specifically to TTR in human plasma, stabilizes the native tetramer in vivo in mice and humans and inhibits TTR cytotoxicity. Crystal structures of tolcapone bound to wild-type TTR and to the V122I cardiomyopathy-associated variant show that it docks better into the TTR T4 pocket than tafamidis, so far the only drug on the market to treat TTR amyloidoses. These data indicate that tolcapone, already in clinical trials for familial amyloid polyneuropathy, is a strong candidate for therapeutic intervention in these diseases, including those affecting the central nervous system, for which no small-molecule therapy exists.

Mechanistic basis of Nek7 activation through Nek9 binding and induced dimerization.

Mitotic spindle assembly requires the regulated activities of protein kinases such as Nek7 and Nek9. Nek7 is autoinhibited by the protrusion of Tyr97 into the active site and activated by the Nek9 non-catalytic C-terminal domain (CTD). CTD binding apparently releases autoinhibition because mutation of Tyr97 to phenylalanine increases Nek7 activity independently of Nek9. Here we find that self-association of the Nek9-CTD is needed for Nek7 activation. We map the minimal Nek7 binding region of Nek9 to residues 810-828. A crystal structure of Nek7(Y97F) bound to Nek9(810-828) reveals a binding site on the C-lobe of the Nek7 kinase domain. Nek7(Y97F) crystallizes as a back-to-back dimer between kinase domain N-lobes, in which the specific contacts within the interface are coupled to the conformation of residue 97. Hence, we propose that the Nek9-CTD activates Nek7 through promoting back-to-back dimerization that releases the autoinhibitory tyrosine residue, a mechanism conserved in unrelated kinase families.

Structural analysis of the regulation of the DYNLL/LC8 binding to Nek9 by phosphorylation.

The NIMA family protein kinases Nek9/Nercc1, Nek6, and Nek7 constitute a signaling module activated in early mitosis involved in the control of spindle organization. DYNLL/LC8 (dynein light chain 8) was originally described as a component of the dynein complex, but the recent discovery of multiple interaction partners for LC8 has suggested that it has a general role as a dimerization hub that organizes different protein partners. Recent experiments suggested that LC8 binding to Nek9 was regulated by Nek9 autophosphorylation on Ser(944), a residue immediately located N-terminal to the LC8 conserved (K/R)xTQT binding motif, and that this was crucial for the control of signal transduction through the Nek/Nek6/7 module. In the present work, we present two crystal structures of LC8 with a peptide corresponding to the Nek9 binding region with and without a phosphorylation on Ser(944). Structural analysis of LC8 with both Nek9 peptides, together with different biophysical experiments, explains the observed diminished binding affinity of Nek9 to LC8 upon phosphorylation on Ser(944) within the Nek9 sequence, thus shedding light into a novel phosphorylation regulatory mechanism that interferes with LC8 protein · protein complex formation.

Structural characterization of the enzymes composing the arginine deiminase pathway in Mycoplasma penetrans.

The metabolism of arginine towards ATP synthesis has been considered a major source of energy for microorganisms such as Mycoplasma penetrans in anaerobic conditions. Additionally, this pathway has also been implicated in pathogenic and virulence mechanism of certain microorganisms, i.e. protection from acidic stress during infection. In this work we present the crystal structures of the three enzymes composing the gene cluster of the arginine deiminase pathway from M. penetrans: arginine deiminase (ADI), ornithine carbamoyltransferase (OTC) and carbamate kinase (CK). The arginine deiminase (ADI) structure has been refined to 2.3 Å resolution in its apo-form, displaying an "open" conformation of the active site of the enzyme in comparison to previous complex structures with substrate intermediates. The active site pocket of ADI is empty, with some of the catalytic and binding residues far from their active positions, suggesting major conformational changes upon substrate binding. Ornithine carbamoyltransferase (OTC) has been refined in two crystal forms at 2.5 Å and 2.6 Å resolution, respectively, both displaying an identical dodecameric structure with a 23-point symmetry. The dodecameric structure of OTC represents the highest level of organization in this protein family and in M.penetrans it is constituted by a novel interface between the four catalytic homotrimers. Carbamate kinase (CK) has been refined to 2.5 Å resolution and its structure is characterized by the presence of two ion sulfates in the active site, one in the carbamoyl phosphate binding site and the other in the ß-phosphate ADP binding pocket of the enzyme. The CK structure also shows variations in some of the elements that regulate the catalytic activity of the enzyme. The relatively low number of metabolic pathways and the relevance in human pathogenesis of Mycoplasma penetrans places the arginine deiminase pathway enzymes as potential targets to design specific inhibitors against this human parasite.

S-shape relationship between customer satisfaction and willingness to pay premium prices for high quality cured pork products in Spain.

This paper explores 2 different probabilistic models explaining willingness to pay premium prices for high-quality cured products from the swine industry. Seven cured pork products (sausage, fuet, ham, loin, shoulder, salami and pepperoni) were studied in 9 food-stores in Valladolid, Spain. Consumers of the products were interviewed (686 completed surveys). It was found by using mixed effect statistical models that the relationship between willingness to pay a premium price and customer satisfaction had nonlinear behavior, following an S-shape with inverted slope which was the first empirical evidence of this type of behavior in meat products in real market conditions. It was also established that the interaction between satisfaction and current expenditure on the product was significant and indispensable for explaining consumers' willingness to pay premium price for cured pork products.