Thyroid axis participates in high-temperature-induced male sex reversal through its activation by the stress response.

Environmental changes alter the sex fate in about 15% of vertebrate orders, mainly in ectotherms such as fish and reptiles. However, the effects of temperature changes on the endocrine and molecular processes controlling gonadal sex determination are not fully understood. Here, we provide evidence that thyroid hormones (THs) act as co-players in heat-induced masculinization through interactions with the stress axis to promote testicular development. We first demonstrated that the thyroid axis (through thyroid-related genes and T3 levels) is highly active in males during the gonadal development in medaka (Oryzias latipes). Similarly, T3 treatments promoted female-to-male sex reversal in XX embryos. Subsequently, embryonic exposure to temperature-induced stress up-regulated the genes related to the thyroid and stress axes with a final increase in T3 levels. In this context, we show that blocking the stress axis response by the loss of function of the corticotropin-releasing hormone receptors suppresses thyroid-stimulating hormone expression, therefore, heat-induced activation of the thyroid axis. Thus, our data showed that early activation of the stress axis and, in consequence, the TH axis, too, leaves us with that both being important endocrine players in inducing female-to-male reversal, which can help predict possible upcoming physiological impacts of global warming on fish populations.

MYC integrates FSH signalling networks in cumulus cells during bovine oocyte maturation.

Follicle-stimulating hormone (FSH) contributes to the acquisition of oocyte competence by modulating signalling pathways in cumulus cells (CCs), albeit much less is known about transcription factors (TFs) that orchestrate the downstream transcriptional changes. This work allowed to prospect TFs involved in FSH-mediated signalling during oocyte in vitro maturation (IVM). Bovine cumulus-oocyte complexes underwent IVM with FSH (FSH+) or without FSH (control/CTL) for 22 h, and CCs were subjected to gene expression profiling. Five software identified reference genes for RT-qPCR (ATP1A1, UBB, and YWHAZ). The transcript levels of FSH-responsive genes HAS2 and PTGS2 (COX2) validated the experimental design. Among candidate TFs, MYC was down-regulated (0.35-fold; P < 0.0001), and THAP11 (RONIN) was up-regulated (1.47-fold; P = 0.016) under FSH+ conditions. In silico analyses predicted binding motifs at MYC and THAP11 genes for previously known FSH-responsive TFs. Signalling pathways (EGFR, ERK, GSK3, PKA, and P38) may execute post-translational regulation due to potential phosphorylation sites in MYC and THAP11 proteins. Prediction of protein-protein interaction networks showed MYC as a core component of FSH signalling, albeit THAP11 acts independently. Hence, MYC integrates FSH signalling networks and may assist in exploring genome-wide transcriptional changes associated with the acquisition of oocyte competence.

Surface-enhanced Raman scattering sensing platform for detecting amyloid-ß peptide interaction with an aggregation inhibitor.

Soluble, small amyloid-ß oligomers (AßO) are recognized as significant contributors to the pathology of Alzheimer's disease (AD). Although drugs for treating AD symptoms have been approved, no therapy targeting amyloid-ß (Aß) capable of modifying the course of the disease is available. In an effort to develop a label-free method for screening new anti-AD therapeutic agents, we show the use of a surface-enhanced Raman scattering (SERS) active substrate for detecting the interactions between Aß peptides and spin-labeled fluorine (SLF), a peptide aggregation inhibitor. Changes in the peak positions and intensity ratios of two spectral peaks near 1600cm-1 and 2900cm-1 can be used to monitor the molecular interactions between SLF and Aß. This study demonstrates the potential of SERS spectroscopy for rapidly screening and identifying new anti-Aß therapeutic agents.

Edema Induced by a Crotalus durissus terrificus Venom Serine Protease (Cdtsp 2) Involves the PAR Pathway and PKC and PLC Activation.

Snake venom serine proteases (SVSPs) represent an essential group of enzymatic toxins involved in several pathophysiological effects on blood homeostasis. Some findings suggest the involvement of this class of enzymatic toxins in inflammation. In this paper, we purified and isolated a new gyroxin isoform from the Crotalus durissus terrificus (Cdt) venom, designated as Cdtsp 2, which showed significant proinflammatory effects in a murine model. In addition, we performed several studies to elucidate the main pathway underlying the edematogenic effect induced by Cdtsp 2. Enzymatic assays and structural analysis (primary structure analysis and three-dimensional modeling) were closely performed with pharmacological assays. The determination of edematogenic activity was performed using Cdtsp 2 isolated from snake venom, and was applied to mice treated with protein kinase C (PKC) inhibitor, phospholipase C (PLC) inhibitor, dexamethasone (Dexa), antagonists for protease-activated receptors (PARs), or saline (negative control). Additionally, we measured the levels of cyclooxygenase 2 (COX-2), malondialdehyde (MDA), and prostaglandin E2 (PGE2). Cdtsp 2 is characterized by an approximate molecular mass of 27 kDa, an isoelectric point (pI) of 4.5, and significant fibrinolytic activity, as well as the ability to hydrolyze Nα-benzoyl-l-arginine 4-nitroanilide (BAPNA). Its primary and three-dimensional structures revealed Cdtsp 2 as a typical snake venom serine protease that induces significant edema via the metabolism of arachidonic acid (AA), involving PARs, PKC, PLC, and COX-2 receptors, as well as inducing a significant increase in MDA levels. Our results showed that Cdtsp 2 is a serine protease with significant enzymatic activity, and it may be involved in the degradation of PAR1 and PAR2, which activate PLC and PKC to mobilize AA, while increasing oxidative stress. In this article, we provide a new perspective for the role of SVSPs beyond their effects on blood homeostasis.

How pH modulates the dimer-decamer interconversion of 2-Cys peroxiredoxins from the Prx1 subfamily.

2-Cys peroxiredoxins belonging to the Prx1 subfamily are Cys-based peroxidases that control the intracellular levels of H2O2 and seem to assume a chaperone function under oxidative stress conditions. The regulation of their peroxidase activity as well as the observed functional switch from peroxidase to chaperone involves changes in their quaternary structure. Multiple factors can modulate the oligomeric transitions of 2-Cys peroxiredoxins such as redox state, post-translational modifications, and pH. However, the molecular basis for the pH influence on the oligomeric state of these enzymes is still elusive. Herein, we solved the crystal structure of a typical 2-Cys peroxiredoxin from Leishmania in the dimeric (pH 8.5) and decameric (pH 4.4) forms, showing that conformational changes in the catalytic loop are associated with the pH-induced decamerization. Mutagenesis and biophysical studies revealed that a highly conserved histidine (His(113)) functions as a pH sensor that, at acidic conditions, becomes protonated and forms an electrostatic pair with Asp(76) from the catalytic loop, triggering the decamerization. In these 2-Cys peroxiredoxins, decamer formation is important for the catalytic efficiency and has been associated with an enhanced sensitivity to oxidative inactivation by overoxidation of the peroxidatic cysteine. In eukaryotic cells, exposure to high levels of H2O2 can trigger intracellular pH variations, suggesting that pH changes might act cooperatively with H2O2 and other oligomerization-modulator factors to regulate the structure and function of typical 2-Cys peroxiredoxins in response to oxidative stress.

Incidence of apoptosis after retinoids and insulin-like growth factor-I (IGF-I) supplementation during goat in vitro embryo production.

The addition of growth factors and vitamins enhances goat embryonic development in vitro. However, few attempts have been reported trying to identify supplementation regimens for oocyte maturation or embryo culture with additive properties. The present report was aimed to evaluate if retinoids [0.3 µM retinyl acetate (RAc) and 0.5 µM 9-cis-retinoic acid (RA)] supplementation during goat oocyte maturation and retinoids and/or 50 ng mL-1 IGF-I during embryo culture synergically enhanced embryonic development while diminishing the incidence of apoptosis. All combinations of RAc and RA treatment produced blastocysts with similar efficiencies, while IGF-I enhanced embryos yields irrespectively of retinoid addition. Moreover, retinoids and IGF-I supplementation showed similar caspase activity or DNA fragmentation indexes in blastocysts. In conclusion, supplementation with retinoids and IGF-I during goat embryo culture enhances blastocysts development without synergic reduction of apoptosis.

Comparison of vitrification and conventional freezing for cryopreservation of caprine embryos.

The experiment aimed to compare conventional freezing and different vitrification protocols for cryopreservation of caprine embryos at morphological, ultrastructural, and functional levels. Caprine embryos produced in vivo were allocated randomly to three groups: (1) conventional freezing with ethylene glycol (EG); (2) dimethyl sulfoxide + EG (DMSO/EG) vitrification; and (3) dimethylformamide + EG (DMF/EG) vitrification. All groups were scored for cell viability (propidium iodide staining and ultrastructural levels) and re-expansion rate after thawing or warming. Embryos subjected to DMSO/EG vitrification showed higher cell viability (73.33%), compared with DMF/EG vitrification and conventional freezing group embryos (40.00 and 66.66%, respectively). The ultrastructural study revealed that vitrified embryos had greater preservation of cellular structure than embryos from conventional freezing with EG. DMSO/EG vitrification resulted in higher rates of re-expansion in vitro (47.36%) than DMF/EG vitrification (31.58%), and conventional freezing (25.00%). In conclusion, caprine embryos produced in vivo are better cryopreserved after vitrification than conventional freezing, therefore we conclude that DMSO/EG vitrification is the most effective protocol for cryopreservation.

Use of retinoids during oocyte maturation diminishes apoptosis in caprine embryos.

Exposure of caprine oocytes and embryos to retinoids enhances embryonic development, but the mechanisms governing this phenomenon have not been characterised. The aim of the present study was to evaluate if the incidence of apoptosis is affected by the addition of retinyl acetate (RAc) and 9-cis-retinoic acid (RA) during in vitro maturation (IVM) of caprine oocytes. Embryonic development was recorded on days 3 and 8 post-fertilisation, and apoptosis was measured by caspase activity and DNA fragmentation (TUNEL assay). Control zygotes had lower capacity to cleave and reach the blastocyst stage (24.45 ± 2.32 and 5.32 ± 0.81, respectively) than those of RAc- (29.96 ± 1.62 and 7.94 ± 0.93, respectively) and RA-treated groups (30.12 ± 1.51 and 7.36 ± 1.02, respectively). Oocytes and blastocysts positive for TUNEL assay were more frequent, respectively, in the controls (8.20 ± 0.78, 8.70 ± 1.05) than in RAc (5.60 ± 0.52, 4.80 ± 0.51) and RA (6.40 ± 0.69, 5.40 ± 0.69). Caspase activity did not differ between control oocytes (7.20 ± 0.91), RAc (6.60 ± 0.68) and RA (7.30 ± 0.67), but it was reduced in RAc- (5.05 ± 0.62) and RA-treated blastocysts (5.75 ± 0.22) compared to controls (8.35 ± 0.71). These results indicate that the addition of retinoids during IVM increases the developmental potential of goat embryos with a concomitant reduction in apoptosis rates.

The First Anti-Snakebite and Hepatoprotective Characterization of a Trypsin Kunitz-like Inhibitor (EcTI) from the Plant Enterolobium contortisiliquum; A Case of Two Soul Mates Meeting.

Snake venom serine protease (SVSP) interferes with the regulation and control of important biological reactions in homeostasis and can be classified as an activator of the fibrinolytic system and platelet aggregation. Our group has recently isolated a new serine protease from Crotalus durissus terrificus total venom (Cdtsp-2). This protein exhibits edematogenic capacity and myotoxic activity. A Kunitz-like EcTI inhibitor protein with a molecular mass of 20 kDa was isolated from Enterolobium contortisiliquum and showed high trypsin inhibition. Thus, the objective of this work is to verify the possible inhibition of the pharmacological activities of Cdtsp-2 by the Kutinz-type inhibitor EcTI. To isolate Cdtsp-2 from total C. d. terrificus venom, we used three-step chromatographic HPLC. Using the mice paw edema model, we observed an edematogenic effect, myotoxicity and hepatotoxicity caused by Cdtsp-2. In vitro and in vivo experiments showed that the alterations in hemostasis caused by Cdtsp-2 are crucial for the development of marked hepatotoxicity and that EcTI significantly inhibits the enzymatic and pharmacological activities of Cdtsp-2. Kunitz-like inhibitor may be a viable alternative for the development of ancillary treatments against the biological activities of venoms.

Simulated fine-needle aspiration diagnosis of follicular thyroid nodules by hyperspectral Raman microscopy and chemometric analysis.

SIGNIFICANCE: Follicular thyroid carcinoma carries a substantially poor prognosis due to its unique biological behavior and less favorable outcomes. In particular, fine-needle aspiration (FNA) biopsies, which play a key role in screening thyroid nodules, cannot differentiate benign from malignant follicular neoplasm. AIM: We report on the use of hyperspectral Raman microscopy in combination with chemometric analysis for identifying and classifying single cells obtained from clinical samples of human follicular thyroid neoplasms. APPROACH: We used a method intended to simulate the FNA procedure to obtain single cells from thyroid nodules. A total of 392 hyperspectral Raman images of single cells from follicular thyroid neoplasms were collected. RESULTS: Malignant cells were identified based on their intrinsic Raman spectral signatures with an overall diagnostic accuracy of up to 83.7%. CONCLUSIONS: Our findings indicate that hyperspectral Raman microscopy can potentially be developed into an ancillary test for analyzing single cells from thyroid FNA biopsies to better stratify "indeterminate" nodules and other cytologically challenging cases.

Transcervical Versus Laparotomy Embryo Recovery: What Strategy Is Best for Embryo Bank Formation in the Canindé Goat Conservation Program?

By allowing for the creation of embryo banks, reproductive biotechnologies play an essential role in the preservation of endangered goat breeds' genetic diversity. This study focused on comparing both available embryo collection methods [laparotomy (LAP) vs. nonsurgical embryo recovery (NSER)] in Canindé goats to create an embryo bank for later use in a breed conservation program. Twelve females were superovulated and subjected to either the LAP or NSER technique for embryo recovery. The recovery rate was similar (p > 0.05) between NSER (86.8% ± 5.6%) and LAP (92.8% ± 4.0%). Moreover, there were no differences (p > 0.05) in the number of structures recovered, the viable embryos, and the freezable embryos per goat, respectively, for NSER (11.7 ± 1.3, 11.2 ± 1.5, and 10.2 ± 1.1) and LAP (10.3 ± 1.0, 8.7 ± 0.7, and 8.0 ± 0.8). Overall, 132 structures were collected out of 151 ovulations (∼12.6 ± 1.2 corpora lutea per goat). Finally, the procedure duration time was also similar (p > 0.05) for NSER versus LAP, respectively: 32.3 ± 3.3 versus 30.8 ± 3.9 minutes. In conclusion, the NSER method results proved to be similar to the LAP technique in small-sized Canindé goats. It was noticeable, however, that the NSER technique is simpler and provides the possibility for successive procedures with few health risks and sequels for females. This study may hopefully boost in vivo embryo production programs in the Canindé breed, facilitating the formation of embryo banks and so assuring the availability of genetic diversity before any decline becomes irreversible.

Upconversion ultraviolet random lasing in Nd3+ doped fluoroindate glass powder.

An upconversion random laser (RL) operating in the ultraviolet is reported for Nd3+ doped fluoroindate glass powder pumped at 575 nm. The RL is obtained by the resonant excitation of the Nd3+ state 2G7/2 followed by energy transfer among two excited ions such that one ion in the pair decays to a lower energy state and the other is promoted to state 4D7/2 from where it decays emitting light at 381 nm. The RL threshold of 30 kW/cm2 was determined by monitoring the photoluminescence intensity as a function of the pump laser intensity. The RL pulses have time duration of 29 ns that is 50 times smaller than the decay time of the upconversion signal when the sample is pumped with intensities below the RL laser threshold.

Developing an international industrial advanced pharmacy practice experience in Brazil.

INTRODUCTION: In this commentary, we advocate for implementing international industry-focused advanced pharmacy practice experiences (APPEs) that engage pharmacy students and schools with the pharmaceutical industry to develop products for the global market to broaden pharmacy student experiences in non-patient care electives. COMMENTARY: Our five-year experience suggests the following structural components are essential to the success of the APPE: (1) identification of suitable international industry partners through non-traditional methods, such as the local, United States (US) chamber of commerce; (2) commitment by the company and educational institutions to develop key personnel to work with international students in the host country; (3) development of a global regulatory affairs course and workshops that target the product development stage; (4) development of student experiences based on projects directly linked to a new product(s); (5) use of technology platforms to support weekly video conferencing and translation; (6) engagement of students in community service; (7) cooperative evaluation of students and the program. The aggregate of student projects led to the development of a line of dietary supplement products introduced to the US market. IMPLICATIONS: The implementation of this APPE benefits pharmacy students but also the academic and industry hosts. Pharmacy students obtained global manufacturing experience, an appreciation for a different culture, and supported commercial product development. The educational institutions developed joint courses and workshops. Students were embedded into various departments, carried out Food & Drug Administration regulations research, prepared comparative regulatory process maps, and provided company employees with an understanding of American consumers.

Diagnosing Hirschsprung disease by detecting intestinal ganglion cells using label-free hyperspectral microscopy.

Hirschsprung disease (HD) is a congenital disorder in the distal colon that is characterized by the absence of nerve ganglion cells in the diseased tissue. The primary treatment for HD is surgical intervention with resection of the aganglionic bowel. The accurate identification of the aganglionic segment depends on the histologic evaluation of multiple biopsies to determine the absence of ganglion cells in the tissue, which can be a time-consuming procedure. We investigate the feasibility of using a combination of label-free optical modalities, second harmonic generation (SHG); two-photon excitation autofluorescence (2PAF); and Raman spectroscopy (RS), to accurately locate and identify ganglion cells in murine intestinal tissue without the use of exogenous labels or dyes. We show that the image contrast provided by SHG and 2PAF signals allows for the visualization of the overall tissue morphology and localization of regions that may contain ganglion cells, while RS provides detailed multiplexed molecular information that can be used to accurately identify specific ganglion cells. Support vector machine, principal component analysis and linear discriminant analysis classification models were applied to the hyperspectral Raman data and showed that ganglion cells can be identified with a classification accuracy higher than 95%. Our findings suggest that a near real-time intraoperative histology method can be developed using these three optical modalities together that can aid pathologists and surgeons in rapid, accurate identification of ganglion cells to guide surgical decisions with minimal human intervention.

Follicle-stimulating hormone mediates the consumption of serum-derived glycogen by bovine cumulus-oocyte complexes during in vitro maturation.

BACKGROUND AND AIM: Oocyte in vitro maturation (IVM) is an appealing approach for several assisted reproductive technologies and dissecting oocyte maturation. Nonetheless, IVM leads to lower developmental competence and usually relies on undefined, serum-containing media. Therefore, biochemical profiling aimed to explore fluctuations in IVM media content during the acquisition of oocyte developmental competence. MATERIALS AND METHODS: Bovine cumulus-oocyte complexes (COCs) underwent IVM in TCM199 medium with Earle's salts, supplemented with 2.0 mM L-glutamine, 10% fetal bovine serum, antibiotics, and 0.05 IU/mL porcine follicle-stimulating hormone (FSH+) or vehicle control (CTL) medium for 22 h. RESULTS: FSH withdrawal (CTL) diminished several processes associated with the acquisition of oocyte developmental competence, such as reduced cumulus cell expansion, diminished estradiol synthesis (FSH+: 116.0±0.0 pg/mL vs. CTL: 97.6±18.0 pg/mL), and lower oocyte nuclear maturation rate (FSH+: 96.47% vs. CTL: 88.76%). Fresh media formulations (i.e., TCM199 with FSH or vehicle) were indistinguishable under biochemical profiling threshold conditions. Biochemical profiling showed similar total protein and lipid concentrations between groups. Further, total sugar concentrations diminished from fresh media to their post-IVM counterparts, albeit in an FSH-independent manner. Glycogen concentrations remained unaltered after IVM within CTL media, albeit were substantially lower after IVM under FSH+ conditions. CONCLUSION: FSH mediates the consumption of serum-derived glycogen by bovine COCs during IVM and implies that serum-free media should contain increased glucose concentrations to facilitate the acquisition of oocyte developmental competence.

Molecular characterization and expression analysis of anti-Müllerian hormone in common carp (Cyprinus carpio) adult testes.

Anti-Müllerian hormone (Amh) is a member of the transforming growth factor-ß (Tgf-ß) superfamily required in the regression of Müllerian ducts during gonadal sex differentiation of higher vertebrates. Teleost fish lack Müllerian ducts, but identified Amh orthologs have been shown to exert crucial functions during sex determination and differentiation of several species of teleosts. However, the function of Amh during gametogenesis in adult fish remains poorly investigated. Therefore, to expand present knowledge on the role of Amh in teleosts, the present study aimed to isolate and clone full-length amh cDNA in the common carp, Cyprinus carpio, and examine its expression levels throughout the male reproductive cycle and in response to different hormone treatments of testicular explants. Molecular cloning and characterization showed that the common carp Amh precursor amino acid sequence shared common features to other fish Amh precursors, including a conserved C-terminus (Tgf-ß domain) and a double proteolytic cleavage site (R-X-X-R-X-X-R) upstream to the Tgf-ß domain. Expression analysis showed amh dimorphic expression in the adult gonads with higher expression in the testes than ovaries. In testes, amh mRNA was detected in Sertoli cells contacting different types of germ cells, although the expression was greatest in Sertoli cells associated with type A undifferentiated spermatogonia. Expression analysis during the reproductive cycle showed that amh transcripts were down-regulated during the developing phase, which is characterized by an increased proliferation of type A undifferentiated spermatogonia and Sertoli cells and appearance of spermatocytes (meiosis) in the testes. Furthermore, ex vivo experiments showed that a 7 day exposure to Fsh or estrogens was required to decrease amh mRNA levels in common carp testicular explants. In summary, this study provided information on the molecular characterization and transcript abundance of amh in common carp adult testes. Altogether, these data will be useful for further investigations on sex determination and differentiation in this species, and also to improved strategies for improved carp aquaculture, such as inhibiting precocious maturation of males.

A duplicated copy of id2b is an unusual sex-determining candidate gene on the Y chromosome of arapaima (Arapaima gigas).

Arapaima gigas is one of the largest freshwater fish species of high ecological and economic importance. Overfishing and habitat destruction are severe threats to the remaining wild populations. By incorporating a chromosomal Hi-C contact map, we improved the arapaima genome assembly to chromosome-level, revealing an unexpected high degree of chromosome rearrangements during evolution of the bonytongues (Osteoglossiformes). Combining this new assembly with pool-sequencing of male and female genomes, we identified id2bbY, a duplicated copy of the inhibitor of DNA binding 2b (id2b) gene on the Y chromosome as candidate male sex-determining gene. A PCR-test for id2bbY was developed, demonstrating that this gene is a reliable male-specific marker for genotyping. Expression analyses showed that this gene is expressed in juvenile male gonads. Its paralog, id2ba, exhibits a male-biased expression in immature gonads. Transcriptome analyses and protein structure predictions confirm id2bbY as a prime candidate for the master sex-determiner. Acting through the TGFß signaling pathway, id2bbY from arapaima would provide the first evidence for a link of this family of transcriptional regulators to sex determination. Our study broadens our current understanding about the evolution of sex determination genetic networks and provide a tool for improving arapaima aquaculture for commercial and conservation purposes.

RADSex: A computational workflow to study sex determination using restriction site-associated DNA sequencing data.

The study of sex determination and sex chromosome organization in nonmodel species has long been technically challenging, but new sequencing methodologies now enable precise and high-throughput identification of sex-specific genomic sequences. In particular, restriction site-associated DNA sequencing (RAD-Seq) is being extensively applied to explore sex determination systems in many plant and animal species. However, software specifically designed to search for and visualize sex-biased markers using RAD-Seq data is lacking. Here, we present RADSex, a computational analysis workflow designed to study the genetic basis of sex determination using RAD-Seq data. RADSex is simple to use, requires few computational resources, makes no prior assumptions about the type of sex-determination system or structure of the sex locus, and offers convenient visualization through a dedicated R package. To demonstrate the functionality of RADSex, we re-analysed a published data set of Japanese medaka, Oryzias latipes, where we uncovered a previously unknown Y chromosome polymorphism. We then used RADSex to analyse new RAD-Seq data sets from 15 fish species spanning multiple taxonomic orders. We identified the sex determination system and sex-specific markers in six of these species, five of which had no known sex-markers prior to this study. We show that RADSex greatly facilitates the study of sex determination systems in nonmodel species thanks to its speed of analyses, low resource usage, ease of application and visualization options. Furthermore, our analysis of new data sets from 15 species provides new insights on sex determination in fish.

Insights into the specificity of thioredoxin reductase-thioredoxin interactions. A structural and functional investigation of the yeast thioredoxin system.

The enzymatic activity of thioredoxin reductase enzymes is endowed by at least two redox centers: a flavin and a dithiol/disulfide CXXC motif. The interaction between thioredoxin reductase and thioredoxin is generally species-specific, but the molecular aspects related to this phenomenon remain elusive. Here, we investigated the yeast cytosolic thioredoxin system, which is composed of NADPH, thioredoxin reductase (ScTrxR1), and thioredoxin 1 (ScTrx1) or thioredoxin 2 (ScTrx2). We showed that ScTrxR1 was able to efficiently reduce yeast thioredoxins (mitochondrial and cytosolic) but failed to reduce the human and Escherichia coli thioredoxin counterparts. To gain insights into this specificity, the crystallographic structure of oxidized ScTrxR1 was solved at 2.4 A resolution. The protein topology of the redox centers indicated the necessity of a large structural rearrangement for FAD and thioredoxin reduction using NADPH. Therefore, we modeled a large structural rotation between the two ScTrxR1 domains (based on the previously described crystal structure, PDB code 1F6M ). Employing diverse approaches including enzymatic assays, site-directed mutagenesis, amino acid sequence alignment, and structure comparisons, insights were obtained about the features involved in the species-specificity phenomenon, such as complementary electronic parameters between the surfaces of ScTrxR1 and yeast thioredoxin enzymes and loops and residues (such as Ser(72) in ScTrx2). Finally, structural comparisons and amino acid alignments led us to propose a new classification that includes a larger number of enzymes with thioredoxin reductase activity, neglected in the low/high molecular weight classification.

Polyamines are required for the expression of key Hms proteins important for Yersinia pestis biofilm formation.

We previously showed that mutations in the genes encoding the two main biosynthetic enzymes responsible for polyamine production, arginine decarboxylase (SpeA) and ornithine decarboxylase (SpeC) cause a loss of biofilm formation in Yersinia pestis. In Y. pestis the development of a biofilm is dependent on 6 Hms (hemin storage) proteins (HmsH, F, R, S, T and P) grouped into 3 operons; hmsHFRS, hmsT and hmsP. In this article we show that polyamines are necessary to maintain the levels of key Hms proteins. In the absence of polyamines there is an approximately 93%, approximately 43% and approximately 90% reduction in protein levels of HmsR, HmsS and HmsT respectively. Overexpression of hmsR and hmsT from plasmids alone can restore biofilm formation to a SpeA(-)SpeC(-) mutant. Addition of exogenous putrescine also restores normal levels of HmsR, HmsS, HmsT and biofilm production. Analyses using transcriptional reporters and quantitative RT-PCR indicate that the initiation of transcription and mRNA stability are not reduced by polyamine deficiency. Instead, translational reporters indicate that polyamines function at least in part by modulating the translation of HmsR and HmsT. Although construction of a consensus Shine-Dalgarno sequence upstream of hmsT modestly reduced the stimulation of translation by putrescine, additional mechanisms likely contribute to the polyamine-dependent expression of HmsT. Finally, we have shown that polyamines play a role in bubonic plague.