The synthesis of solid supports carrying base labile linkers to generate 3'-phosphate oligonucleotides.

Oligonucleotides carrying 3'-terminal phosphates and conjugates are important tools in molecular biology and diagnostic purposes. We described the preparation of solid supports carrying the base labile linker 4-((2-hydroxyethyl)sulfonyl)benzamide for the solid-phase synthesis of 3'-phosphorylated oligonucleotides. These supports are fully compatible with the phosphoramidite chemistry yielding the desired 3'-phosphate oligonucleotides in excellent yields. The use of mild deprotection conditions allows the generation of partially protected DNA fragments.

Modeling Dynamics of Human Gut Microbiota Derived from Gluten Metabolism: Obtention, Maintenance and Characterization of Complex Microbial Communities.

Western diets are rich in gluten-containing products, which are frequently poorly digested. The human large intestine harbors microorganisms able to metabolize undigested gluten fragments that have escaped digestion by human enzymatic activities. The aim of this work was obtaining and culturing complex human gut microbial communities derived from gluten metabolism to model the dynamics of healthy human large intestine microbiota associated with different gluten forms. For this purpose, stool samples from six healthy volunteers were inoculated in media containing predigested gluten or predigested gluten plus non-digested gluten. Passages were carried out every 24 h for 15 days in the same medium and community composition along time was studied via V3-V4 16S rDNA sequencing. Diverse microbial communities were successfully obtained. Moreover, communities were shown to be maintained in culture with stable composition for 14 days. Under non-digested gluten presence, communities were enriched in members of Bacillota, such as Lachnospiraceae, Clostridiaceae, Streptococcaceae, Peptoniphilaceae, Selenomonadaceae or Erysipelotrichaceae, and members of Actinomycetota, such as Bifidobacteriaceae and Eggerthellaceae. Contrarily, communities exposed to digested gluten were enriched in Pseudomonadota. Hence, this study shows a method for culture and stable maintenance of gut communities derived from gluten metabolism. This method enables the analysis of microbial metabolism of gluten in the gut from a community perspective.

Sustainable Protocol for the Synthesis of 2',3'-Dideoxynucleoside and 2',3'-Didehydro-2',3'-dideoxynucleoside Derivatives.

An improved protocol for the transformation of ribonucleosides into 2',3'-dideoxynucleoside and 2',3'-didehydro-2',3'-dideoxynucleoside derivatives, including the anti-HIV drugs stavudine (d4T), zalcitabine (ddC) and didanosine (ddI), was established. The process involves radical deoxygenation of xanthate using environmentally friendly and low-cost reagents. Bromoethane or 3-bromopropanenitrile was the alkylating agent of choice to prepare the ribonucleoside 2',3'-bisxanthates. In the subsequent radical deoxygenation reaction, tris(trimethylsilyl)silane and 1,1'-azobis(cyclohexanecarbonitrile) were used to replace hazardous Bu3SnH and AIBN, respectively. In addition, TBAF was substituted for camphorsulfonic acid in the deprotection step of the 5'-O-silyl ether group, and an enzyme (adenosine deaminase) was used to transform 2',3'-dideoxyadenosine into 2',3'-dideoxyinosine (ddI) in excellent yield.

Oligonucleotides Containing 1-Aminomethyl or 1-Mercaptomethyl-2-deoxy-d-ribofuranoses: Synthesis, Purification, Characterization, and Conjugation with Fluorophores and Lipids.

Oligonucleotide conjugates are widely used as therapeutic drugs, gene analysis, and diagnostic tools. A critical step in the biologically relevant oligonucleotide conjugates is the design and synthesis of functional molecules that connect oligonucleotide with ligands. Here, we report the synthesis and application for oligonucleotide functionalization of novel tethers based on aminomethyl and mercaptomethyl sugar derivatives. Starting from a common cyano sugar precursor, three novel phosphoramidites have been prepared in the two α- and ß-anomeric forms. The mercaptomethyl sugar was protected with the S-acetyl group, while two different protecting groups have been developed for the aminomethyl sugar. These two protecting groups are orthogonal, as they can be removed independently using photolysis or ammonolysis. This combination allowed the introduction of two different ligands in a single oligonucleotide.

The Human Digestive Tract Is Capable of Degrading Gluten from Birth.

The human gastrointestinal system has the capacity to metabolize dietary gluten. The capacity to degrade gliadin-derived peptide is present in humans from birth and increases during the first stages of life (up to 6-12 months of age). Fecal samples from 151 new-born and adult non-celiac disease (NCD) volunteers were collected, and glutenase and glianidase activities were evaluated. The capacity of total fecal proteins to metabolize 33-mer, 19-mer, and 13-mer gliadin peptides was also evaluated by high-performance liquid chromatography (HPLC). Feces from new-borns (meconium) showed glutenase and gliadinase activities, and peptidase activity against all three gliadin peptides. Maximal gluten degradative activity was observed in fecal samples from the youngest volunteers (0-12 months old). After the age of nine months, the gluten digestive capacity of gastrointestinal tract decreases and, from ±8 years old, individuals lose the ability to completely degrade toxic peptides. The gastrointestinal proteases involved in gluten digestion: elastase 2A, elastase 3B, and carboxipeptidase A1 are present from earlier stages of life. The human digestive tract contains the proteins capable of metabolizing gluten from birth, even before starting gluten intake. Humans are born with the ability to digest gluten and to completely degrade the potentially toxic gliadin-derived peptides (33-, 19-, and 13-mer).

Does an early mobilization and immediate home-based self-therapy exercise program displace proximal humeral fractures in conservative treatment? Observational study.

BACKGROUND: Nonoperative management of proximal humeral fractures (PHFs) is the most common treatment, but its functional outcome may improve with early mobilization. In frail osteoporotic patients, quick recovery of prefracture independency is mandatory. This study assessed fracture displacement in PHFs managed with conservative treatment after early mobilization and a home-based self-exercise program. METHODS: We retrospectively analyzed the radiologic displacement of fracture fragments of PHFs treated conservatively with early mobilization and a home-based self-exercise program. RESULTS: Included were 99 patients with 26 one-part, 32 two-part, 32 three-part, and 9 four-part PHFs managed conservatively, followed by early mobilization and a home-based self-exercise program. In the x-ray examinations, the head displaced from varus into valgus 55° ± 23° to 42° ± 22°, in the normal range of anatomic values. The medial hinge displaced from medial to the diaphysis (+1 ± 6 mm) to lateral to the head (-0.6 ± 6 mm). The greater tuberosity displaced cranially from -1 ± 7 mm to 2 ± 5 mm. The Constant score at the 1-year follow-up was 79.69 ± 16.3. DISCUSSION AND CONCLUSIONS: The home-based self-exercise program for conservative treatment of PHFs displaces the head-diaphysis angle and the medial hinge toward anatomic reduction, but there is a risk of greater tuberosity cranial displacement. Functional results are fairly good, allowing frail patients to keep on with their independency and life style. Because a large number of patients might need further physiotherapy, the quality of the home-based self-exercises should be supervised.

Factors influencing recurrence and progression of Dupuytren's disease treated by Collagenase Clostridium histolitycum.

AIM OF THE STUDY: The purpose of this study was to determine the recurrence rate, possible adverse reactions and factors influencing recurrence and progression of Dupuytren's disease (DD) treated with Collagenase from Clostridium histolyticum (CCH). METHOD: This was a prospective study of 71 patients with DD treated with CCH from 2011 to February 2013, with a minimum follow-up period of four years. Clinical, functional, patient satisfaction, drug safety and factors influencing recurrence and disease progression were evaluated. RESULTS: In all patients, the rupture of the cord was achieved after the injection, reducing joint contracture. In five patients (7%) we verified the existence of disease recurrence during the follow-up. In 11 patients (15.5%) there was a disease progression. Three patients have been surgically operated on, without added surgery difficulty; the rate of recurrence and progression was higher in grades III and IV of Tubiana, in proximal interphalangeal (PIP) punctures, and was earlier in patients younger than 60 years. DISCUSSION: No serious local complications or general complications were observed with this method. The recurrence of DD, following criteria of Felici, is mainly observed in young patients with greater severity of the disease and at the PIP level. Progression is influenced by the same factors. Patients operated on after recurrence have no added difficulty in the surgical technique, as it has also been published in other studies. CONCLUSIONS: Patients with the lowest rates of recurrence and progression were those with a single cord in the metacarpophalangeal (MCP), a grade II of Tubiana, and were older than 60 years.

Study of conjunctival flora in anophthalmic patients: influence on the comfort of the socket.

PURPOSE: To investigate the relationship between conjunctival flora and comfort of the socket in anophthalmic patients. METHODS: A cross-sectional clinical study including 60 patients with unilateral anophthalmia who wear a prosthetic eye. From each patient three microbiological samples were taken from the lower conjunctival sac (healthy eye, pre-prosthesis, and retro-prosthesis space of socket). The 180 samples obtained were cultured. Samples from a randomized subgroup of 29 patients were measured by spectrophotometry at 540 nm after 48 h of growth, to determine their microbial density (MD). The grade of comfort of the socket (GCS) of each patient was established by a questionnaire. Epidemiological and clinical data of the anophthalmic socket and artificial eye care of each patient were also collected. RESULTS: MD decreased in healthy eyes (0.213 ± 0.201, P = 0.004) compared with the pre-prosthesis (0.402 ± 0.323) and retro-prosthesis (0.438 ± 0.268) samples. Pre-prosthesis MD correlated with retro-prosthesis MD (R = 0.401, P = 0.031) and healthy eye MD (R = 0.482, P = 0.008), and it was also related to poor GCS (P = 0.017). Aerobic Gram-negative bacteria in retro-prosthesis samples of patients with poor GCS was higher than in patients with good or fair GCS (P = 0.008). In the same samples, coagulase-negative staphylococci proportion (excluding S. epidermidis) increased in patients with good GCS (P = 0.030). CONCLUSIONS: Socket microflora is related to GCS. Increased pathogenic flora, especially Gram-negative bacteria, and high MD are related to discomfort, while coagulase-negative staphylococci (other than S. epidermidis) are associated with comfort.

Differences in gluten metabolism among healthy volunteers, coeliac disease patients and first-degree relatives.

Coeliac disease (CD) is an immune-mediated enteropathy resulting from exposure to gluten in genetically predisposed individuals. Gluten proteins are partially digested by human proteases generating immunogenic peptides that cause inflammation in patients carrying HLA-DQ2 and DQ8 genes. Although intestinal dysbiosis has been associated with patients with CD, bacterial metabolism of gluten has not been studied in depth thus far. The aim of this study was to analyse the metabolic activity of intestinal bacteria associated with gluten intake in healthy individuals, CD patients and first-degree relatives of CD patients. Faecal samples belonging to twenty-two untreated CD patients, twenty treated CD patients, sixteen healthy volunteers on normal diet, eleven healthy volunteers on gluten-free diet (GFD), seventy-one relatives of CD patients on normal diet and sixty-nine relatives on GFD were tested for several proteolytic activities, cultivable bacteria involved in gluten metabolism, SCFA and the amount of gluten in faeces. We detected faecal peptidasic activity against the gluten-derived peptide 33-mer. CD patients showed differences in faecal glutenasic activity (FGA), faecal tryptic activity (FTA), SCFA and faecal gluten content with respect to healthy volunteers. Alterations in specific bacterial groups metabolising gluten such as Clostridium or Lactobacillus were reported in CD patients. Relatives showed similar parameters to CD patients (SCFA) and healthy volunteers (FTA and FGA). Our data support the fact that commensal microbial activity is an important factor in the metabolism of gluten proteins and that this activity is altered in CD patients.

The young patient and the medial unicompartmental knee replacement.

PURPOSE: Younger patients with unicompartmental degenerative knee joint diseases present a therapeutic dilemma. The purpose of this study is to evaluate the long-term results of the Oxford phase III unicompartmental knee replacement through a minimally invasive approach in the young patient. METHODS: We have studied a total of 51 patients (59 interventions) who underwent the Oxford Phase III Unicompartmental Knee Replacement. A clinical, radiographical and functional evaluation was performed twelve years after intervention. RESULTS: According to the American Knee Society Score using Insall's criteria overall results of unicompartmental knee arthroplasty showed an excellent or good outcome for 53 knees (96.36%), fair for 1 (1.81%) and poor for 1 (1.81%) in the postoperative long term. The survival rate of the implants was close to 95% at follow-up. CONCLUSIONS: Twelve years follow-up results of UKA through a minimally invasive exposure in young patients demonstrate predictably good outcomes.

Single nucleotide polymorphism (SNP) analysis used for the phylogeny of the Mycobacterium tuberculosis complex based on a pyrosequencing assay.

BACKGROUND: Different polymorphisms have been described as markers to classify the lineages of the Mycobacterium tuberculosis complex. The analysis of nine single nucleotide polymorphisms (SNPs) was used to describe seven SNPs cluster groups (SCGs). We attempted to classify those strains that could not been categorized into lineages by the genotyping methods used in the routine testing. RESULTS: The M. tuberculosis complex isolates collected in 2010 in our region were analysed. A new method based on multiplex-PCRs and pyrosequencing to analyse these SNPs was designed. For the pyrosequencing assay nine SNPs that defined the seven SCGs were selected from the literature: 1977, 74092, 105139, 232574, 311613, 913274, 2460626, 3352929 and gyrA95. In addition, SNPs in katG(463), mgtC(182), Ag85C(103) and RD(Rio) deletion were detected. CONCLUSIONS: This work has permitted to achieve a better classification of Aragonian strains into SCGs and in some cases, to assign strains to its certain lineage. Besides, the description of a new pattern shared by two isolates "SCG-6c" reinforces the interest of SNPs to follow the evolution of M. tuberculosis complex.

Whole-cell biotransformation with recombinant cytochrome P450 for the selective oxidation of Grundmann's ketone.

25-Hydroxy-Grundmann's ketone is a key building block in the chemical synthesis of vitamin D3 and its derivatives through convergent routes. Generally, the chemical synthesis of this compound involves tedious procedures and results in a mixture of several products. Recently, the selective hydroxylation of Grundmann's ketone at position C25 by cytochrome P450 (CYP) 154E1 from Thermobifida fusca YX was described. In this study a recombinant whole-cell biocatalyst was developed and applied for hydroxylation of Grundmann's ketone. Biotransformation was performed by Escherichia coli cells expressing CYP154E1 along with two redox partner systems, Pdx/PdR and YkuN/FdR. The system comprising CYP154E1/Pdx/PdR showed the highest production of 25-hydroxy-Grundmann's ketone and resulted in 1.1mM (300mgL(-1)) product concentration.

Radial tunnel syndrome: results of surgical decompression by a postero-lateral approach.

PURPOSE: Our purpose is to describe the results obtained in surgical treatment of a series of patients with symptoms of radial tunnel syndrome. METHODS: We performed a prospective study on 42 patients (43 limbs) operated for radial tunnel syndrome between 1996 and 2010, using a posterior-external approach. RESULTS: Using the Roles and Maudsley criteria, 21 patients had excellent results (48.8%), 16 good (37.2%) and six fair results (13.9%). Most patients were satisfied with the surgery, reporting symptom relief and improved functionality. CONCLUSIONS: Radial tunnel syndrome consists of intermittent compression of the posterior interosseous nerve in the forearm, with pain and functional disability of the forearm, without motor or sensory electromyogram alterations. Because it is often confused with enthesitis of the epicondyle muscle insertions (an entity often occurring simultaneously), differential diagnosis is necessary with treatment-resistant epicondylitis. The most effective treatment is surgical, releasing all possible nerve compression sites.

Synthesis of vitamin D3 analogues with A-ring modifications to directly measure vitamin D levels in biological samples.

C-3-substituted 25-hydroxyvitamin D3 analogues were synthesized as tools to directly measure levels of vitamin D in biological samples. The strategy involves vinyloxycarbonylation of the 3ß-hydroxy group and formation of a carbamate bond with a hydroxyl or amino group at the end of the alkyl chain. Biotinylated conjugates of synthesized derivatives were generated to be linked with vitamin D binding protein (DBP). The spacer group present in the alkyl chain is important in the binding of antibodies to the analogue-DBP complex. When compared to 25-hydroxyvitamin D3-DBP, the binding of some antibodies to the analogue-DBP complex of the 25-hydroxyvitamin D3 derivative 10 that posses an 8-aminoctyl alkyl chain is significantly reduced, but this analogue displaced [26,27-(3)H]-25-hydroxyvitamin D3 from DBP. In contrast, the 8-hydroxyoctyl alkyl chain analogue 9 showed less displacement.

Long-term results of the retrocapital metatarsal percutaneous osteotomy for hallux valgus.

PURPOSE: The current trend in hallux valgus surgery is directed toward percutaneous procedures. However, no evidence that any of these methods of treatment are superior to the others has been described, excepting studies in the long term. The aim of this study was to analyse a series of patients who had undergone a percutaneous distal retrocapital osteotomy of the first metatarsal, and had been followed up for ten years. METHODS: We carried out a clinical and radiological evaluation of 115 feet ten years after surgery. RESULTS: The AOFAS scale results in the tenth postoperative year remained significantly favourable compared to their corresponding values in the preoperative period, yielding an improvement of 42.2 points overall on average. In relation to radiological findings, the mean hallux angle was maintained below 20 °, with a mean intermetatarsal angle of 8.1 °. CONCLUSION: Percutaneous retrocapital metatarsal osteotomy for treatment of mild to moderate hallux valgus is effective in the long term, with the advantages of a minimally invasive procedure.

Oxford phase 3 unicondylar knee arthroplasty through a minimally invasive approach: long-term results.

PURPOSE: Surgical treatment options for medial compartment osteoarthritis of the knee include high tibial osteotomy, total knee arthroplasty or unicompartmental knee arthroplasty (UKA), depending on the patient's age, level of physical activity and the degree of deformity. METHODS: In this study, we evaluated the long-term results of patients who underwent the Oxford cemented meniscal-bearing unicondylar knee prosthesis through a minimally invasive approach including a clinical, functional and radiographic assessment. RESULTS: Favourable clinical and radiological outcomes were registered overall at ten years after surgery. Overall results of UKA according to the American Knee Society (AKS) using Insall's criteria showed an excellent or good outcome for 492 knees (96.28 %), fair for 11 (2.15 %) and poor for eight (1.57 %) in the post-operative long term. CONCLUSIONS: We believe that with appropriate surgical technique, patient selection, prosthetic design and specific training, surgeons should achieve good outcomes with the added advantages of a minimally invasive approach. High volume for this technique is important in our opinion.

Synthesis of 2'-O-Methyl/2'-O-MOE-L-Nucleoside Derivatives and Their Applications: Preparation of G-Quadruplexes, Their Characterization, and Stability Studies.

Nucleosides and their analogues constitute an important family of molecules with potential antiviral and antiproliferative activity. The enantiomers of natural nucleosides, l-nucleoside derivatives, which have comparable biological activity but more favorable toxicological properties and greater metabolic stability than d-nucleosides, have emerged as a new class of therapeutic agents. Furthermore, l-nucleosides can be used as a building block to prepare l-oligonucleotides, which have identical physical properties in terms of solubility, hybridization kinetics, and duplex thermal stability as d-oligonucleotides but completely orthogonal in nature. Consequently, they are resistant to nuclease degradation, nontoxic, and immunologically passive, which are desirable properties for biomedical applications. Herein, we describe the synthesis of several 2'-O-methyl/2'-O-MOE-l-nucleoside pyrimidine derivatives and their incorporation into G-rich oligonucleotides. Finally, we evaluated the stability and resistance against nucleases of these new G-quadruplexes, demonstrating the potential of the l-nucleosides described in this work in providing enhanced nuclease resistance with a minimal impact in the nucleic acid structural properties.

Design and divergent synthesis of aza nucleosides from a chiral imino sugar.

Several novel nucleoside analogues as potential inhibitors of glycosidases and purine nucleoside phosphorylase (PNP) have been synthesized via selective coupling of an appropriate nucleobase at different positions of an orthogonally protected imino sugar as a common precursor. This synthetic strategy offers a straightforward protocol for the assembly of imino sugar containing nucleosides, establishing a new repertoire of molecules as potential therapeutics.

Synthesis, evaluation of anti-HIV-1 and anti-HCV activity of novel 2',3'-dideoxy-2',2'-difluoro-4'-azanucleosides.

A series of 2',3'-dideoxy-2',2'-difluoro-4'-azanucleosides of both pyrimidine and purine nucleobases were synthesized in an efficient manner starting from commercially available L-pyroglutamic acid via glycosylation of difluorinated pyrrolidine derivative 15. Several 4'-azanucleosides were prepared as a separable mixture of α- and ß-anomers. The 6-chloropurine analogue was obtained as a mixture of N(7) and N(9) regioisomers and their structures were identified based on NOESY and HMBC spectral data. Among the 4'-azanucleosides tested as HIV-1 inhibitors in primary human lymphocytes, four compounds showed modest activity and the 5-fluorouracil analogue (18d) was found to be the most active compound (EC(50)=36.9µM) in this series. None of the compounds synthesized in this study demonstrated anti-HCV activity.

A gluten metabolism study in healthy individuals shows the presence of faecal glutenasic activity.

PURPOSE: To study the gluten metabolism in healthy individuals and its effect over the intestinal microbial activity. METHODS: The faeces of eleven healthy subjects were analysed under 4 diet regimens: their normal gluten diet, a strict gluten-free diet (GFD), a GFD with a supplemental intake of 9 g gluten/day and a GFD with a supplemental intake of 30 g gluten/day. Gluten content, faecal tryptic activity (FTA), short-chain fatty acids (SCFAs) and faecal glutenasic activity (FGA) were analysed in faecal samples. RESULTS: Faecal gluten contents, FTA, SCFAs and FGA varied significantly with different levels of gluten intake in the diet. When high gluten doses (30 g/day) were administered in the diet, SCFA concentrations (70.5 mmoles/kg faeces) were significantly different from those from the GFD period (33.8 mmoles/kg faeces) of the experiment. However, the FTA showed significant differences between the GFD (34 units) and the normal gluten-containing diet (60 units) and also between the GFD and the GFD + 30 g of gluten/day (67 units). When gluten was present in the diet, gluten was detected in the faeces, showing that at least a portion of the gluten ingested is eliminated in the large intestine, providing a substrate for intestinal microbial proteases. We have also shown the presence of faecal glutenasic activity that increased proportionally with the gluten intake in the diet, showing an enzymatic activity of 993 units in DSG, 2,063 units in DSG + 9 g and 6,090 units in DSG + 30 g. CONCLUSIONS: The activity of the intestinal microbiota is modified by gluten intake in the diet. The incorporation of gluten in the diet increases the activity of a gluten proteolytic activity in the faeces.