De novo design of potent and selective mimics of IL-2 and IL-15.

We describe a de novo computational approach for designing proteins that recapitulate the binding sites of natural cytokines, but are otherwise unrelated in topology or amino acid sequence. We use this strategy to design mimics of the central immune cytokine interleukin-2 (IL-2) that bind to the IL-2 receptor ßγc heterodimer (IL-2Rßγc) but have no binding site for IL-2Rα (also called CD25) or IL-15Rα (also known as CD215). The designs are hyper-stable, bind human and mouse IL-2Rßγc with higher affinity than the natural cytokines, and elicit downstream cell signalling independently of IL-2Rα and IL-15Rα. Crystal structures of the optimized design neoleukin-2/15 (Neo-2/15), both alone and in complex with IL-2Rßγc, are very similar to the designed model. Neo-2/15 has superior therapeutic activity to IL-2 in mouse models of melanoma and colon cancer, with reduced toxicity and undetectable immunogenicity. Our strategy for building hyper-stable de novo mimetics could be applied generally to signalling proteins, enabling the creation of superior therapeutic candidates.

In Vivo Metabolic Regulation of Alternative Oxidase under Nutrient Deficiency-Interaction with Arbuscular Mycorrhizal Fungi and Rhizobium Bacteria.

The interaction of the alternative oxidase (AOX) pathway with nutrient metabolism is important for understanding how respiration modulates ATP synthesis and carbon economy in plants under nutrient deficiency. Although AOX activity reduces the energy yield of respiration, this enzymatic activity is upregulated under stress conditions to maintain the functioning of primary metabolism. The in vivo metabolic regulation of AOX activity by phosphorus (P) and nitrogen (N) and during plant symbioses with Arbuscular mycorrhizal fungi (AMF) and Rhizobium bacteria is still not fully understood. We highlight several findings and open questions concerning the in vivo regulation of AOX activity and its impact on plant metabolism during P deficiency and symbiosis with AMF. We also highlight the need for the identification of which metabolic regulatory factors of AOX activity are related to N availability and nitrogen-fixing legume-rhizobia symbiosis in order to improve our understanding of N assimilation and biological nitrogen fixation.

Rbfox proteins regulate microRNA biogenesis by sequence-specific binding to their precursors and target downstream Dicer.

Rbfox proteins regulate tissue-specific splicing by targeting a conserved GCAUG sequence within pre-mRNAs. We report here that sequence-specific binding of the conserved Rbfox RRM to miRNA precursors containing the same sequence motif in their terminal loops, including miR-20b and miR-107, suppresses their nuclear processing. The structure of the complex between precursor miR-20b and Rbfox RRM shows the molecular basis for recognition, and reveals changes in the stem-loop upon protein binding. In mammalian cells, Rbfox2 downregulates mature miR-20b and miR-107 levels and increases the expression of their downstream targets PTEN and Dicer, respectively, suggesting that Rbfox2 indirectly regulates many more cellular miRNAs. Thus, some of the widespread cellular functions of Rbfox2 protein are attributable to regulation of miRNA biogenesis, and might include the mis-regulation of miR-20b and miR-107 in cancer and neurodegeneration.

Towards the development of standardized methods for comparison, ranking and evaluation of structure alignments.

MOTIVATION: Pairwise alignment of protein structures is a fundamental task in structural bioinformatics. There are numerous computer programs in the public domain that produce alignments for a given pair of protein structures, but the results obtained by the various programs generally differ substantially. Hence, in the application of such programs the question arises which of the alignment programs are the most trustworthy in the sense of overall performance, and which programs provide the best result for a given pair of proteins. The major problem in comparing, evaluating and judging alignment results is that there is no clear notion of the optimality of an alignment. As a consequence, the numeric criteria and scores reported by the individual structure alignment programs are largely incomparable. RESULTS: Here we report on the development and application of a new approach for the evaluation of structure alignment results. The method uses the translation vector and rotation matrix to generate the superposition of two structures but discards the alignment reported by the individual programs. The optimal alignment is then generated in standardized form based on a suitably implemented dynamic programming algorithm where the length of the alignment is the single most informative parameter. We demonstrate that some of the most popular programs in protein structure research differ considerably in their overall performance. In particular, each of the programs investigated here produced in at least in one case the best and the worst alignment compared with all others. Hence, at the current state of development of structure comparison techniques, it is advisable to use several programs in parallel and to choose the optimal alignment in the way reported here. AVAILABILITY AND IMPLEMENTATION: The computer software that implement the method described here is freely available at http://melolab.org/stovca.

Role of backbone strain in de novo design of complex α/ß protein structures.

We previously elucidated principles for designing ideal proteins with completely consistent local and non-local interactions which have enabled the design of a wide range of new αß-proteins with four or fewer ß-strands. The principles relate local backbone structures to supersecondary-structure packing arrangements of α-helices and ß-strands. Here, we test the generality of the principles by employing them to design larger proteins with five- and six- stranded ß-sheets flanked by α-helices. The initial designs were monomeric in solution with high thermal stability, and the nuclear magnetic resonance (NMR) structure of one was close to the design model, but for two others the order of strands in the ß-sheet was swapped. Investigation into the origins of this strand swapping suggested that the global structures of the design models were more strained than the NMR structures. We incorporated explicit consideration of global backbone strain into the design methodology, and succeeded in designing proteins with the intended unswapped strand arrangements. These results illustrate the value of experimental structure determination in guiding improvement of de novo design, and the importance of consistency between local, supersecondary, and global tertiary interactions in determining protein topology. The augmented set of principles should inform the design of larger functional proteins.

De novo design of ACE2 protein decoys to neutralize SARS-CoV-2.

There is an urgent need for the ability to rapidly develop effective countermeasures for emerging biological threats, such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes the ongoing coronavirus disease 2019 (COVID-19) pandemic. We have developed a generalized computational design strategy to rapidly engineer de novo proteins that precisely recapitulate the protein surface targeted by biological agents, like viruses, to gain entry into cells. The designed proteins act as decoys that block cellular entry and aim to be resilient to viral mutational escape. Using our novel platform, in less than ten weeks, we engineered, validated, and optimized de novo protein decoys of human angiotensin-converting enzyme 2 (hACE2), the membrane-associated protein that SARS-CoV-2 exploits to infect cells. Our optimized designs are hyperstable de novo proteins (∼18-37 kDa), have high affinity for the SARS-CoV-2 receptor binding domain (RBD) and can potently inhibit the virus infection and replication in vitro. Future refinements to our strategy can enable the rapid development of other therapeutic de novo protein decoys, not limited to neutralizing viruses, but to combat any agent that explicitly interacts with cell surface proteins to cause disease.

De novo design of potent and resilient hACE2 decoys to neutralize SARS-CoV-2.

We developed a de novo protein design strategy to swiftly engineer decoys for neutralizing pathogens that exploit extracellular host proteins to infect the cell. Our pipeline allowed the design, validation, and optimization of de novo human angiotensin-converting enzyme 2 (hACE2) decoys to neutralize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The best monovalent decoy, CTC-445.2, bound with low nanomolar affinity and high specificity to the receptor-binding domain (RBD) of the spike protein. Cryo-electron microscopy (cryo-EM) showed that the design is accurate and can simultaneously bind to all three RBDs of a single spike protein. Because the decoy replicates the spike protein target interface in hACE2, it is intrinsically resilient to viral mutational escape. A bivalent decoy, CTC-445.2d, showed ~10-fold improvement in binding. CTC-445.2d potently neutralized SARS-CoV-2 infection of cells in vitro, and a single intranasal prophylactic dose of decoy protected Syrian hamsters from a subsequent lethal SARS-CoV-2 challenge.

Restrictive water condition modifies the root exudates composition during peanut-PGPR interaction and conditions early events, reversing the negative effects on plant growth.

Water deficit is one of the most serious environmental factors that affect the productivity of crops in the world. Arachis hypogaea is a legume with a high nutritional value and 70% is cultivated in semi-arid regions. This research aimed to study the effect of water deficit on peanut root exudates composition, analyzing the importance of exudates on peanut-PGPR interaction under restrictive water condition. Peanut seedlings were subjected to six treatments: 0 and 15 mM PEG, in combination with non-inoculated, Bradyrhizobium sp. and Bradyrhizobium-Azospirillum brasilense inoculated treatments. We analyzed the 7-day peanut root exudate in response to a water restrictive condition and the presence of bacterial inocula. Molecular analysis was performed by HPLC, UPLC and GC. Bacteria motility, chemotaxis, bacterial adhesion to peanut roots and peanut growth parameters were analyzed. Restrictive water condition modified the pattern of molecules exuded by roots, increasing the exudation of Naringenin, oleic FA, citric and lactic acid, and stimulation the release of terpenes of known antioxidant and antimicrobial activity. The presence of microorganisms modified the composition of root exudates. Water deficit affected the first events of peanut-PGPR interaction and the root exudates favored bacterial mobility, the chemotaxis and attachment of bacteria to peanut roots. Changes in the profile of molecules exuded by roots allowed A. hypogaea-Bradyrhizobium and A.hypogaea-Bradyrhizobium-Azospirillum interaction thus reversing the negative effects of restrictive water condition on peanut growth. These findings have a future potential application to improve plant-PGPR interactions under water deficit by formulating inoculants containing key molecules exuded during stress.

Overexpression of the arginine decarboxylase gene promotes the symbiotic interaction Medicago truncatula-Sinorhizobium meliloti and induces the accumulation of proline and spermine in nodules under salt stress conditions.

Legumes have the capacity to fix nitrogen in symbiosis with soil bacteria known as rhizobia by the formation of root nodules. However, nitrogen fixation is highly sensitive to soil salinity with a concomitant reduction of the plant yield and soil fertilization. Polycationic aliphatic amines known as polyamines (PAs) have been shown to be involved in the response to a variety of stresses in plants including soil salinity. Therefore, the generation of transgenic plants overexpressing genes involved in PA biosynthesis have been proposed as a promising tool to improve salt stress tolerance in plants. In this work we tested whether the modulation of PAs in transgenic Medicago truncatula plants was advantageous for the symbiotic interaction with Sinorhizobium meliloti under salt stress conditions, when compared to wild type plants. Consequently, we characterized the symbiotic response to salt stress of the homozygous M. truncatula plant line L-108, constitutively expressing the oat adc gene, coding for the PA biosynthetic enzyme arginine decarboxylase, involved in PAs biosynthesis. In a nodulation kinetic assay, nodule number incremented in L-108 plants under salt stress. In addition, these plants at vegetative stage showed higher nitrogenase and nodule biomass and, under salt stress, accumulated proline (Pro) and spermine (Spm) in nodules, while in wt plants, the accumulation of glutamic acid (Glu), γ-amino butyric acid (GABA) and 1-aminocyclopropane carboxylic acid (ACC) (the ethylene (ET) precursor) were the metabolites involved in the salt stress response. Therefore, overexpression of oat adc gene favours the symbiotic interaction between plants of M. truncatula L-108 and S. meliloti under salt stress and the accumulation of Pro and Spm, seems to be the molecules involved in salt stress tolerance.

[Total duodenal section. A reliable indicator of severe physical abuse]. / Sección duodenal total. Indicador confiable de maltrato fisico severo.

BACKGROUND: Rupture of the hollow viscera due to battered child syndrome is an unusual clinical finding and it is even less likely when we encounter total duodenum section. The literature on child abuse does not include visceral lesions as part of the spectrum. The aim of this study was to analize if complete duodenal section is a reliable indicator of severe physical abuse. Data were statistically analyzed using chi-square tests, likelihood ratios and the Cochran-Mantel-Haentzel test. CLINICAL CASES: Four children were admitted with a battered child syndrome diagnosis. RESULTS: All participants were surgically treated and the finding was total section of the duodenum. The postoperative course was normal. Statistical tests were p<0.0001 and the likelihood ratio 18.7 CONCLUSION: Duodenal rupture is a statistically reliable indicator of a severe form of physical abuse in children.

Gemcitabine plus docetaxel as first-line chemotherapy in patients with advanced non-small cell lung cancer: a lung cancer Galician group phase II study.

BACKGROUND: Numerous phase II and III clinical trials have demonstrated a higher activity of combined gemcitabine plus docetaxel schedules against non-small cell lung cancer (NSCLC) than that of both agents in monotherapy. METHODS: This phase II study evaluated a 3-week based schedule of docetaxel 85 mg/m(2) (1-h i.v. infusion, d8) combined with gemcitabine 1,000 mg/m(2) (30-min i.v. infusion; d1,8) as first-line chemotherapy for patients with advanced NSCLC. RESULTS: Forty-one patients with non-resectable, stage IIIB/IV, and bidimensionally measurable disease were enrolled. A total of 182 chemotherapy cycles (median 6, range 1-6) was administered to 40 patients during the study; one patient did not receive chemotherapy due to a protocol deviation. Two patients were not evaluable for treatment efficacy. The overall response rate found was 44% (95% CI, 29-59%): three patients (7%) had a complete response and 15 patients (37%) had a partial response (median duration of response = 4.0 months). With a median follow-up of 8.7 months, the median time to disease progression was 4.4 months and the median overall survival was 7.3 months. The combined gemcitabine plus docetaxel chemotherapy was well tolerated except for pulmonary toxicity. The main grade 3-4 hematological toxicity was neutropenia (28% of patients, 9% of cycles). Two cases of febrile neutropenia were reported. The main grade 3-4 non-hematological toxicity was pulmonary toxicity (23% of patients, 6% of cycles). CONCLUSION: Gemcitabine 1,000 mg/m(2) on days 1 and 8 in combination with docetaxel 85 mg/m(2) on day 8 given in 3-week cycles is an active and well-tolerated first-line chemotherapeutic regimen for advanced NSCLC.

Influence of seat tilt motion on discomfort perception during a simulated driving activity.

BACKGROUND: Discomfort perceived in activities where there is a prolonged sitting posture are normally compensated in a natural way by means of macro-repositioning movements in the seat. Nevertheless, evidence shows that such movements are not able to palliate discomfort due to lumbar pain. OBJECTIVE: This study involves research performed to demonstrate whether induced postural changes are able to mitigate this type of discomfort during a simulated driving activity. METHODS: Twenty-four subjects with lumbar pain (LBP) and without lumbar pain (WLBP) underwent 90 min of simulated driving activities while periodic variations of seat tilt (Tt) were implemented. RESULTS: Discomfort perception due to lumbar pain significantly decreased in the case of Tt compared with the case of WTt (without seat tilt), and significant differences were found (p = 0.02). However, treatments with Tt indicated that no substantial differences exist between LBP and WLBP subjects when considering discomfort perception due to lumbar pain and the erector spinae activity. CONCLUSIONS: This study revealed that periodic variations on seat tilt can help to reduce discomfort perception due to lumbar pain during driving activities, regardless of the health condition of the subject.

Polyamines contribute to salinity tolerance in the symbiosis Medicago truncatula-Sinorhizobium meliloti by preventing oxidative damage.

Polyamines (PAs) such as spermidine (Spd) and spermine (Spm) are small ubiquitous polycationic compounds that contribute to plant adaptation to salt stress. The positive effect of PAs has been associated to a cross-talk with other anti-stress hormones such as brassinosteroids (BRs). In this work we have studied the effects of exogenous Spd and Spm pre-treatments in the response to salt stress of the symbiotic interaction between Medicago truncatula and Sinorhizobium meliloti by analyzing parameters related to nitrogen fixation, oxidative damage and cross-talk with BRs in the response to salinity. Exogenous PAs treatments incremented the foliar and nodular Spd and Spm content which correlated with an increment of the nodule biomass and nitrogenase activity. Exogenous Spm treatment partially prevented proline accumulation which suggests that this polyamine could replace the role of this amino acid in the salt stress response. Additionally, Spd and Spm pre-treatments reduced the levels of H2O2 and lipid peroxidation under salt stress. PAs induced the expression of genes involved in BRs biosynthesis which support a cross-talk between PAs and BRs in the salt stress response of M. truncatula-S. meliloti symbiosis. In conclusion, exogenous PAs improved the response to salinity of the M. truncatula-S. meliloti symbiosis by reducing the oxidative damage induced under salt stress conditions. In addition, in this work we provide evidences of the cross-talk between PAs and BRs in the adaptive responses to salinity.

24-Epibrassinolide ameliorates salt stress effects in the symbiosis Medicago truncatula-Sinorhizobium meliloti and regulates the nodulation in cross-talk with polyamines.

Brassinosteroids (BRs) are steroid plant hormones that have been shown to be involved in the response to salt stress in cross-talk with other plant growth regulators such as polyamines (PAs). In addition, BRs are involved in the regulation of the nodulation in the rhizobium-legume symbiosis through the alteration of the PAs content in leaves. In this work, we have studied the effect of exogenous 24-epibrassinolide (EBL) in the response to salinity of nitrogen fixation in the symbiosis Medicago truncatula-Sinorhizobium meliloti. Foliar spraying of EBL restored the growth of plants subjected to salt stress and provoked an increment of the nitrogenase activity. In general, PAs levels in leaves and nodules decreased by the salt and EBL treatments, however, the co-treatment with NaCl and EBL augmented the foliar spermine (Spm) concentration. This increment of the Spm levels was followed by a reduction of the membrane oxidative damage and a diminution of the proline accumulation. The effect of BRs on the symbiotic interaction was evaluated by the addition of 0.01, 0.1 and 0.5 µM EBL to the growing solution, which provoked a reduction of the nodule number and an increment of the PAs levels in shoot. In conclusion, foliar treatment with EBL had a protective effect against salt stress in the M. truncatula-S. meliloti symbiosis mediated by an increment of the Spm levels. Treatment of roots with EBL incremented PAs levels in shoot and reduced the nodule number which suggests a cross-talk between PAs and BRs in the nodule suppression and the protection against salt stress.

[Esophageal atresia: a second-level hospital experience]. / Atresia esofágica: experiencia de un hospital de segundo nivel de atención.

INTRODUCTION: In recent years, surgical correction of esophageal atresia with distal tracheoesophageal fistula has become increasingly successful. However, there is still a group of high-risk patients with specific factors in whom the mortality remains appreciable. These associated factors include weight, gestational age, associated malformations and respiratory distress. MATERIAL AND METHODS: This report analyzes the mortality in 80 newborn infants with variants of esophageal atresia with or without tracheoesophageal fistula who were treated from 1999 to 2003. Data were collected restrospectively from hospital and office records. RESULTS: We observed 42 male patients, 69 patients were C variety, all had more than 12 h of postnatal life, 34 were preterm newborn, 41 were classified A or B according to Waterston, and 41 died. A logistic regression analysis and chi2 of the influence of each risk factor on mortality was performed. Relevant statistical significance was found in the studied variables. CONCLUSIONS: Morbidity and mortality of esophageal atresia was higher due to identified risk factors.

Docetaxel plus epirubicin is a highly active, well-tolerated, first-line chemotherapy for metastatic breast cancer: results of a large, multicentre phase II study.

PURPOSE: In this multicentre phase II study, the efficacy and safety profile of the combination of docetaxel and epirubicin as first-line chemotherapy for metastatic breast cancer (MBC) were evaluated. METHODS: Epirubicin (75 mg/m(2)) and docetaxel (75 mg/m(2)) were given intravenously once every 3 weeks for six cycles to 133 patients with MBC. RESULTS: The overall clinical response rate was 67% (complete and partial responses were 23% and 44%, respectively). The median time to progression was 10.8 months (95% CI 9.7-12.6) and the median overall survival was 19.5 months. Granulocyte colony-stimulating factor support was administered to 32% of patients and in 22% of cycles. Grade 3/4 neutropenia occurred in 35% of patients and febrile neutropenia in 19%. The most frequent grade 3/4 non-haematological toxicities (as percent of patients) were asthenia (6%), vomiting (5%) and nausea (5%). No patients developed congestive heart failure. CONCLUSIONS: The combination of docetaxel and epirubicin was highly active as first-line treatment for MBC and showed a manageable toxicity profile.

Effect of cultivar and growing location on the trypsin inhibitors, tannins, and lectins of common beans (Phaseolus vulgaris L.) grown in the semiarid highlands of Mexico.

Trypsin inhibitors (TI), tannins, and lectins appear to have a role in preventing chronic diseases in humans. The genetic variability of these traits in common bean needs to be ascertained in order to increase levels through breeding. The variability of TI, tannin, and lectins was determined in five bean cultivars grown at five locations in Mexico. TI and tannins contents in colored beans that belong to the Jalisco race were higher (11.1-11.9 trypsin units inhibited (TUI)/mg and 29.0-38.1 mg catechin equivalent (CE)/g, respectively) than cultivars of the Durango race (7.9-8.3 TUI/mg and 16.8-19.9 CE/mg, respectively). Bayo Victoria, a Durango race cultivar, had three times more lectins than levels reported for soybean. Cultivar influenced TI and tannins contents (p < 0.001), whereas site affected lectins (p < 0.001). An increase in levels of TI and tannins could be enhanced through breeding.

Occurrence of polyamines in root nodules of Phaseolus vulgaris in symbiosis with Rhizobium tropici in response to salt stress.

Polyamines (PAs) are low molecular weight aliphatic compounds that have been shown to be an important part of plant responses to salt stress. For that reason in this work we have investigated the involvement of PAs in the response to salt stress in root nodules of Phaseolus vulgaris in symbiosis with Rhizobium tropici. The level and variety of PAs was higher in nodules, compared to leaves and roots, and in addition to the common PAs (putrescine, spermidine and spermine) we found homospermidine (Homspd) as the most abundant polyamine in nodules. UPLC-mass spectrometry analysis revealed the presence of 4-aminobutylcadaverine (4-ABcad), only described in nodules of Vigna angularis before. Indeed, the analysis of different nodular fractions revealed higher level of 4-ABcad, as well as Homspd, in bacteroids which indicate the production of these PAs by the bacteria in symbiosis. The genes involved in PAs biosynthesis in nodules displayed an induction under salt stress conditions which was not consistent with the decline of free PAs levels, probably due to the nitrogen limitations provoked by the nitrogenase activity depletion and/or the conversion of free PAs to theirs soluble conjugated forms, that seems to be one of the mechanisms involved in the regulation of PAs levels. On the contrary, cadaverine (Cad) and 4-ABcad concentrations augmented by the salinity, which might be due to their involvement in the response of bacteroids to hyper-osmotic conditions. In conclusion, the results shown in this work suggest the alteration of the bacteroidal metabolism towards the production of uncommon PAs such as 4-ABcad in the response to salt stress in legume root nodules.

Identification of novel transcripts with differential dorso-ventral expression in Xenopus gastrula using serial analysis of gene expression.

BACKGROUND: Recent evidence from global studies of gene expression indicates that transcriptomes are more complex than expected. Xenopus has been typically used as a model organism to study early embryonic development, particularly dorso-ventral patterning. In order to identify novel transcripts involved in dorso-ventral patterning, we compared dorsal and ventral transcriptomes of Xenopus tropicalis at the gastrula stage using serial analysis of gene expression (SAGE). RESULTS: Of the experimental tags, 54.5% were confidently mapped to transcripts and 125 showed a significant difference in their frequency of occurrence between dorsal and ventral libraries. We selected 20 differentially expressed tags and assigned them to specific transcripts using bioinformatics and reverse SAGE. Five mapped to transcripts with known dorso-ventral expression and the frequency of appearance for these tags in each library is in agreement with the expression described by other methods. The other 15 tags mapped to transcripts with no previously described asymmetric expression along the dorso-ventral axis. The differential expression of ten of these novel transcripts was validated by in situ hybridization and/or RT-PCR. We can estimate that this SAGE experiment provides a list of at least 86 novel transcripts with differential expression along the dorso-ventral axis. Interestingly, the expression of some novel transcripts was independent of beta-catenin. CONCLUSIONS: Our SAGE analysis provides a list of novel transcripts with differential expression in the dorso-ventral axis and a large number of orphan tags that can be used to identify novel transcripts and to improve the current annotation of the X. tropicalis genome.