[Foot reflexology for caregivers in palliative care]. / La réflexologie plantaire au service des soignants de soins palliatifs.

Foot reflexology is the use of massage and acupressure techniques on the feet, which represent each organ of the human body. The reflexologist knows precisely the anatomy of the body and the reflex points on the feet in order to relieve and treat the person's problems. A team from the University Hospital of Clermont-Ferrand shares a very positive experience, unfortunately interrupted by the Covid-19 health crisis.

[Advocacy for food and wine in palliative care]. / Plaidoyer pour la nourriture et le vin en soins palliatifs.

In the palliative care unit, the care and treatment provided are aimed at optimizing the quality of life and not the quantity of life. Food is adjusted to the patient's condition and is primarily oriented towards pleasure food.

[Connectivity and care, innovative tools for the benefit of patients]. / Connectivité et soins, des outils innovants au bénéfice des patients.

The first department within Clermont-Ferrand university hospital to have undergone the introduction of electronic patient records, the pulmonology department again served as the pilot for studying connectivity solutions. Time savings, increased use of the electronic patient records and improved quality of care are the outcomes highlighted by a survey of the nurses in this unit.

Cyclodipeptide synthases are a family of tRNA-dependent peptide bond-forming enzymes.

Cyclodipeptides and their derivatives belong to the diketopiperazine (DKP) family, which is comprised of a broad array of natural products that exhibit useful biological properties. In the few known DKP biosynthetic pathways, nonribosomal peptide synthetases (NRPSs) are involved in the synthesis of cyclodipeptides that constitute the DKP scaffold, except in the albonoursin (1) pathway. Albonoursin, or cyclo(alpha,beta-dehydroPhe-alpha,beta-dehydroLeu), is an antibacterial DKP produced by Streptomyces noursei. In this pathway, the formation of the cyclo(Phe-Leu) (2) intermediate is catalyzed by AlbC, a small protein unrelated to NRPSs. We demonstrated that AlbC uses aminoacyl-tRNAs as substrates to catalyze the formation of the DKP peptide bonds. Moreover, several other bacterial proteins, presenting moderate similarity to AlbC, also use aminoacyl-tRNAs to synthesize various cyclodipeptides. Therefore, AlbC and these related proteins belong to a newly defined family of enzymes that we have named cyclodipeptide synthases (CDPSs).

Crystallization and halide phasing of the C-terminal domain of human KIN17.

Here, the crystallization and initial phasing of the C-terminal domain of human KIN17, a 45 kDa protein mainly expressed in response to ionizing radiation and overexpressed in certain tumour cell lines, are reported. Crystals diffracting to 1.4 A resolution were obtained from 10% ethylene glycol, 27% PEG 6000, 500 mM LiCl and 100 mM sodium acetate pH 6.3 in space group P2(1)2(1)2(1), with unit-cell parameters a = 45.75, b = 46.31, c = 60.80 A and one molecule in the asymmetric unit. Since this domain has a basic pI, heavy-atom derivatives were obtained by soaking the crystals with negatively charged ions such as tungstate and iodine. The replacement of LiCl by KI in the cryosolution allowed the determination of phases from iodide ions to give an interpretable electron-density map.

Induction of body weight loss through RNAi-knockdown of APOBEC1 gene expression in transgenic rabbits.

In the search of new strategies to fight against obesity, we targeted a gene pathway involved in energy uptake. We have thus investigated the APOB mRNA editing protein (APOBEC1) gene pathway that is involved in fat absorption in the intestine. The APOB gene encodes two proteins, APOB100 and APOB48, via the editing of a single nucleotide in the APOB mRNA by the APOBEC1 enzyme. The APOB48 protein is mandatory for the synthesis of chylomicrons by intestinal cells to transport dietary lipids and cholesterol. We produced transgenic rabbits expressing permanently and ubiquitously a small hairpin RNA targeting the rabbit APOBEC1 mRNA. These rabbits exhibited a moderately but significantly reduced level of APOBEC1 gene expression in the intestine, a reduced level of editing of the APOB mRNA, a reduced level of synthesis of chylomicrons after a food challenge, a reduced total mass of body lipids and finally presented a sustained lean phenotype without any obvious physiological disorder. Interestingly, no compensatory mechanism opposed to the phenotype. These lean transgenic rabbits were crossed with transgenic rabbits expressing in the intestine the human APOBEC1 gene. Double transgenic animals did not present any lean phenotype, thus proving that the intestinal expression of the human APOBEC1 transgene was able to counterbalance the reduction of the rabbit APOBEC1 gene expression. Thus, a moderate reduction of the APOBEC1 dependent editing induces a lean phenotype at least in the rabbit species. This suggests that the APOBEC1 gene might be a novel target for obesity treatment.

Enteropeptidase: a gene associated with a starvation human phenotype and a novel target for obesity treatment.

BACKGROUND: Obesity research focuses essentially on gene targets associated with the obese phenotype. None of these targets have yet provided a viable drug therapy. Focusing instead on genes that are involved in energy absorption and that are associated with a "human starvation phenotype", we have identified enteropeptidase (EP), a gene associated with congenital enteropeptidase deficiency, as a novel target for obesity treatment. The advantages of this target are that the gene is expressed exclusively in the brush border of the intestine; it is peripheral and not redundant. METHODOLOGY/PRINCIPAL FINDINGS: Potent and selective EP inhibitors were designed around a boroarginine or borolysine motif. Oral administration of these compounds to mice restricted the bioavailability of dietary energy, and in a long-term treatment it significantly diminished the rate of increase in body weight, despite ad libitum food intake. No adverse reactions of the type seen with lipase inhibitors, such as diarrhea or steatorrhea, were observed. This validates EP as a novel, druggable target for obesity treatment. CONCLUSIONS: In vivo testing of novel boroarginine or borolysine-based EP inhibitors validates a novel approach to the treatment of obesity.

'Energy expenditure genes' or 'energy absorption genes': a new target for the treatment of obesity and Type II diabetes.

Several hundred genes associated or linked to obesity have been described in the scientific literature. Whereas many of these genes are potential targets for the treatment of obesity and associated conditions, none of them have permitted the developement of an efficient drug therapy. As proposed by the 'thrifty genotype' theory, obesity genes may have conferred an evolutionary advantage in times of food shortage through efficient energy exploitation, while 'lean' or 'energy expenditure' genes may have become very rare during the same periods. It is therefore a challenge to identify 'energy expenditure genes' or 'energy absorption genes,' whose mutations or single nucleotide polymorphisms do result in reduced energy intake. We submit that such 'energy absorption' or 'energy expenditure' genes (crucial genes) are potential new targets for the treatment of obesity. These genes can be identified in rare genetic diseases that produce a lean, failure-to-thrive, energy malabsorption or starvation phenotype.

Effect of SsrA (tmRNA) tagging system on translational regulation in Streptomyces.

ssrA genes encoding tmRNA with transfer and messenger RNA functions are ubiquitous in bacteria. In a process called trans-translation, tmRNA enters a stalled ribosome and allows release of the original mRNA, then tmRNA becomes the template for translation of a short tag that signals for proteolytic degradation. We provide here the first evidences that the tmRNA tagging system (ssrA and cohort smpB) is active in Streptomyces. Transcription of the genes was shown and construction of a genetic probe allowed detection of a tmRNA-tagged peptide. Obtention of ssrA and smpB mutants of Streptomyces lividans showed that the ssrA system is dispensable in Streptomyces. Morphologies of the mutants colonies were similar to the wild type, thus tmRNA-mediated tagging does not seem to have, under conditions used, a significant effect in the Streptomyces differentiation.

The carboxyl-terminal nucleoplasmic region of MAN1 exhibits a DNA binding winged helix domain.

MAN1 is an integral protein of the inner nuclear membrane that interacts with nuclear lamins and emerin, thus playing a role in nuclear organization. It also binds to chromatin-associated proteins and transcriptional regulators, including the R-Smads, Smad1, Smad2, and Smad3. Mutations in the human gene encoding MAN1 cause sclerosing bone dysplasias, which sometimes have associated skin abnormalities. At the molecular level, these mutations lead to loss of the MAN1-R-Smads interaction, thus perturbing transforming growth factor beta superfamily signaling pathway. As a first step to understanding the physical basis of MAN1 interaction with R-Smads, we here report the structural characterization of the carboxyl-terminal nucleoplasmic region of MAN1, which is responsible for Smad binding. This region exhibits an amino-terminal globular domain adopting a winged helix fold, as found in several Smad-associated sequence-specific DNA binding factors. Consistently, it binds to DNA through the positively charged recognition helix H3 of its winged helix motif. However, it does not show the predicted carboxyl-terminal U2AF homology domain in solution, suggesting that the folding and stability of such a domain in MAN1 depend upon binding to an unidentified partner. Modeling the complex between DNA and the winged helix domain shows that the regions involved in DNA binding are essentially distinct from those reported to be involved in Smad binding. This suggests that MAN1 binds simultaneously to R-Smads and their targeted DNA sequences.

A variable residue in the pore of Kv1 channels is critical for the high affinity of blockers from sea anemones and scorpions.

Animal toxins are associated with well defined selectivity profiles; however the molecular basis for this property is not understood. To address this issue we refined our previous three-dimensional models of the complex between the sea anemone toxin BgK and the S5-S6 region of Kv1.1 (Gilquin, B., Racape, J., Wrisch, A., Visan, V., Lecoq, A., Grissmer, S., Ménez, A., and Gasparini, S. (2002) J. Biol. Chem. 277, 37406-37413) using a docking procedure that scores and ranks the structures by comparing experimental and back-calculated values of coupling free energies DeltaDeltaGint obtained from double-mutant cycles. These models further highlight the interaction between residue 379 of Kv1.1 and the conserved dyad tyrosine residue of BgK. Because the nature of the residue at position 379 varies from one channel subtype to another, we explored how these natural mutations influence the sensitivity of Kv1 channel subtypes to BgK using binding and electrophysiology experiments. We demonstrated that mutations at this single position indeed suffice to abolish or enhance the sensitivity of Kv1 channels for BgK and other sea anemone and scorpion toxins. Altogether, our data suggest that the residue at position 379 of Kv1 channels controls the affinity of a number of blocking toxins.

Dual expression system suitable for high-throughput fluorescence-based screening and production of soluble proteins.

Many studies that aim to characterize the proteome structurally or functionally require the production of pure protein in a high-throughput format. We have developed a fast and flexible integrated system for cloning, protein expression in Escherichia coli, solubility screening and purification that can be completely automated in a 96-well microplate format. We used recombination cloning in custom-designed vectors including (i) a (His)(6) tag-encoding sequence, (ii) a variable solubilizing partner gene, (iii) the DNA sequence corresponding to the TEV protease cleavage site, (iv) the gene (or DNA fragment) of interest, (v) a suppressible amber stop codon, and (vi) an S.tag peptide-encoding sequence. First, conditions of bacterial culture in microplates (250 microL) were optimized to obtain expression and solubility patterns identical to those obtained in a 1-L flask (100-mL culture). Such conditions enabled the screening of various parameters in addition to the fusion partners (E. coli strains, temperature, inducer...). Second, expression of fusion proteins in amber suppressor strains allowed quantification of soluble and insoluble proteins by fluorescence through the detection of the S.tag. This technique is faster and more sensitive than other commonly used methods (dot blots, Western blots, SDS-PAGE). The presence of the amber suppressor tRNA was shown to affect neither the expression pattern nor the solubility of the target proteins. Third, production of the most interesting soluble fusion proteins, as detected by our screening method, could be performed in nonsuppressor strains. After cleavage with the TEV protease, the target proteins were obtained in a native form with a unique additional N-terminal glycine.

The stratagem utilized by the plasminogen activator from the snake Trimeresurus stejnegeri to escape serpins.

The plasminogen activator isolated from the venom of the snake Trimeresurus stejnegeri (TSV-PA) triggers plasmin production, along with tissue-type plasminogen activators (t-PA) and urokinase (u-PA). The half-life of TSV-PA in plasma is remarkable. We unveil in this paper two of the molecular mechanisms allowing TSV-PA to escape inhibition by plasma serpins. The first involves a phenylalanine at position 193 (chymotrypsinogen numbering system). Phe(193) distinguishes TSV-PA from nearly all trypsin-like proteinases, having glycine at this position. A mutant of TSV-PA (F193G), in which Phe(193) had been replaced by a glycine, was inactivated by plasminogen activator inhibitor 1 (PAI-1) and alpha(2)-antiplasmin 100-fold more rapidly than the wild-type enzyme. The second mechanism originates from the 37-loop of TSV-PA. Swapping the 37-loop of TSV-PA for either that of t-PA or that of u-PA also increased dramatically the rate of inactivation by PAI-1. Loop swapping and F193G mutations were additive, resulting in a rate of inactivation by PAI-1 that was 4 orders of magnitude higher than for the wild-type enzyme. The potential role of Phe(193) and of the 37-loop in the immunity of TSV-PA toward alpha(1)-antitrypsin and antithrombin is also discussed.

BgK, a disulfide-containing sea anemone toxin blocking K+ channels, can be produced in Escherichia coli cytoplasm as a functional tagged protein.

BgK, a sea anemone peptide consisting of 37 amino acid residues and 3 disulfide bonds, blocks voltage-gated potassium (Kv1) channels. Here, we report a method for producing tagged BgK in Escherichia coli, as a soluble cytoplasmic protein. First, using peptidic synthesis, we show that addition of a 15 residue peptide (S.Tag) at the BgK C-terminus does not affect its biological activity. Then, a synthetic DNA sequence encoding BgK was constructed and cloned to produce a BgK-S.Tag hybrid in the cytoplasm of E. coli. The presence of S.Tag did not only facilitate detection, quantification, and purification of the recombinant protein, but also increased the production yield by more than two orders of magnitude. Moreover, use of an E. coli OrigamiB(DE3)pLacI strain also increased production; up to 5.8-7.5mg of BgK-S.Tag or mutated BgK(F6A)-S.Tag was produced per liter of culture and could be functionally characterized in crude extracts. Using a two-step purification procedure (affinity chromatography and RP-HPLC), we obtained 1.8-2.8mg of purified recombinant protein per liter of culture. The recombinant peptides displayed functional properties similar to those of native BgK or BgK(F6A).