Isolation and identification of bovine tuberculosis in a Brazilian herd (São Paulo)

Mycobacterium was verified in animals from a Brazilian dairy herd, a total of 42 samples from 30 cows were submitted to culture and the isolated strains were analyzed by two polymerase chain reaction (PCR), the first specific for species belonging to the Mycobacterium complex (MTBC) and the other for differentiating M. tuberculosis from M. bovis. Twenty seven samples (64.3%) from 18 animals (60%) were positive for mycobacteria by culture, including samples from 15 retrofaryngeal lymphnodes (55.5%), 9 prescapular lymphnodes (33.3%), 2 lungs (7.4%), and 1 liver (3.7%). All isolated colonies were confirmed by PCR to contain MTBC organisms, and were identified as M. bovis by the same methodology.     

Conditions for Production of Microbial Cell Flocculant by Aspergillus sojae AJ7002

Cultivation is reported on Aspergillus sojae AJ7002 which synthesized an extracellular bio-flocculant. Growth studies in shaking flasks and fermentors were conducted to obtain higher flocculant production. The highest level of polymer accumulation was attained after 48–72 hr cultivation at 30–34°C. The favorable substrates for polymer formation were casein, yeast extract, polypepton and amino acids, such as glutamic acid and alanine. The addition of saccharides to the medium was found to reduce the pH of the culture broths, and hence inhibit the accumulation of flocculant in the culture broth. The finding that the product was a single substance from the early stage of fermentation suggested that the polymer was not a product of cell autolysis. The components of the polymer which were produced by Asp. sojae did not vary even if the medium composition or culture condition changed. The addition of 2-ketogluconic acid, which is one of the constituents of the polymer increased the flocculating activity of the culture medium.     

Pyrrolidine and piperidine analogues of SC-57461A as potent,orally active inhibitors of leukotriene A(4) hydrolase

The synthesis and biological evaluation of a series of functionalized pyrrolidine- and piperidine-containing analogues of our lead LTA(4) hydrolase inhibitor, SC-57461A, is described. A number of compounds showed excellent potency in our in vitro screens and several demonstrated good oral activity in a mouse ex vivo assay. These efforts led to the identification of SC-56938 (14) as a potent, orally active inhibitor of LTA(4) hydrolase.     

Statistical discovery of site inter-dependencies in sub-molecular hierarchical protein structuring

Background

Much progress has been made in understanding the 3D structure of proteins using methods such as NMR and X-ray crystallography. The resulting 3D structures are extremely informative, but do not always reveal which sites and residues within the structure are of special importance. Recently, there are indications that multiple-residue, sub-domain structural relationships within the larger 3D consensus structure of a protein can be inferred from the analysis of the multiple sequence alignment data of a protein family. These intra-dependent clusters of associated sites are used to indicate hierarchical inter-residue relationships within the 3D structure. To reveal the patterns of associations among individual amino acids or sub-domain components within the structure, we apply a k-modes attribute (aligned site) clustering algorithm to the ubiquitin and transthyretin families in order to discover associations among groups of sites within the multiple sequence alignment. We then observe what these associations imply within the 3D structure of these two protein families.

Results

The k-modes site clustering algorithm we developed maximizes the intra-group interdependencies based on a normalized mutual information measure. The clusters formed correspond to sub-structural components or binding and interface locations. Applying this data-directed method to the ubiquitin and transthyretin protein family multiple sequence alignments as a test bed, we located numerous interesting associations of interdependent sites. These clusters were then arranged into cluster tree diagrams which revealed four structural sub-domains within the single domain structure of ubiquitin and a single large sub-domain within transthyretin associated with the interface among transthyretin monomers. In addition, several clusters of mutually interdependent sites were discovered for each protein family, each of which appear to play an important role in the molecular structure and/or function.

Conclusions

Our results demonstrate that the method we present here using a k- modes site clustering algorithm based on interdependency evaluation among sites obtained from a sequence alignment of homologous proteins can provide significant insights into the complex, hierarchical inter-residue structural relationships within the 3D structure of a protein family.

     

Selective inhibition of membrane fusion events in echinoderm gametes and embryos by halenaquinol sulfate.

Halenaquinol sulfate, a hydroquinone sulfate obtained from the sponge Xestospongia sapra, prevented cell membrane fusion events of echinaderm gametes but did not affect early embryonic development of fertilized eggs up to the gastrula stage. However, halenaquinol sulfate inhibited secretion of hatching enzyme, resulting in the formation of gastrulae that were surrounded by the fertilization envelope. Therefore, the use of halenaquinol sulfate offers a unique opportunity to analyze the role of secretory events in complex populations of cells without affecting other cellular functions.     

Effect of castration monotherapy on the levels of adrenal androgens in cancerous prostatic tissues

The mechanism accounting for the development of castration-resistant prostate cancer (CRPC) remains unclear. Studies in CRPC tissues suggest that, after androgen deprivation therapy (ADT), the adrenal androgens may be an important source of testosterone (T) and 5-alpha dihydrotestosterone (DHT) in CRPC tissues. To clarify the role of adrenal androgens in the prostatic tissues (prostatic tissue adrenal androgens) during ADT, we developed a high sensitive and specific quantification method for the levels of androgens in prostatic tissue using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Human prostatic tissues were purified using mixed-mode reversed-phase, strong anion exchange Oasis cartridges (Oasis MAX). Analysis of steroids was performed using LC-MS/MS after picolinic acid derivatization. The validation tests showed that our method of quantitative analysis was precise and sensitive enough for the quantification of dehydroepiandrosterone (DHEA), androstenedione, androstenediol, T, and DHT in the prostatic tissue. The levels of adrenal androgens in prostate cancer tissues after ADT were similar to those in untreated PCa. Especially, DHEA was the most existing androgen precursor in PCa tissues after ADT. The levels of DHEA were high in PCa tissues, irrespective of ADT. We assumed that DHEA played a significant role in the synthesis of T and DHT in PCa tissues after ADT.     

Imidazo[1,5-a]quinoxalines as irreversible BTK inhibitors for the treatment of rheumatoid arthritis

Imidazo[1,5-a]quinoxalines were synthesized that function as irreversible Bruton's tyrosine kinase (BTK) inhibitors. The syntheses and SAR of this series of compounds are presented as well as the X-ray crystal structure of the lead compound 36 in complex with a gate-keeper variant of ITK enzyme. The lead compound showed good in vivo efficacy in preclinical RA models.     

Assessing the Pulsatility of Luteinizing Hormone in Female Vervet Monkeys (Chlorocebus aethiops sabaeus)

Specific alterations in the pulsatility of luteinizing hormone (LH) are linked to obesity-related subfertility in ovulatory women. Vervet monkeys (Chlorocebus aethiops sabaeus) are an Old World nonhuman primate that develops obesity and has a menstrual cycle similar to humans. We evaluated follicular-phase LH pulses in 12 adult normal-weight female vervets. Serum was collected every 10 min for 4 h by using a tether device in conscious, freely moving monkeys on menstrual cycle days 2 through 5. Serum estradiol was collected daily during the follicular phase to identify the luteal–follicular transition. For comparison, we used data from 12 ovulatory normal-weight women who had undergone frequent blood sampling of early-follicular LH. LH pulse frequency was similar, with 2.8 ± 0.7 LH pulses during 4 h in vervets compared with 2.3 ± 0.7 LH pulses during 4 h in women. The LH pulse mass (percentage change in the pulse peak over the preceding nadir) was 123.2% ± 27.4% in vervets and 60.9% ± 14.9% in humans. The first day of low serum estradiol after the follicular-phase peak was denoted as the day of the luteal–follicular transition. Luteectomy was performed on luteal days 7 through 9, and corpora lutea were confirmed by histology. We demonstrate that follicular LH patterns in vervets are similar to those in humans and that the luteal phase is easily identified by monitoring daily serum estradiol. These findings demonstrate that vervet monkeys are a suitable animal model for evaluating LH pulse dynamics longitudinally in studies of diet-induced obesity.Abbreviations: CL, corpus luteum; LH, luteinizing hormoneNonhuman primates have been used in biomedical research for decades and have enabled advancements in many areas, including HIV–AIDS, Alzheimer disease, diabetes, asthma, and endometriosis.²³ Neuroendocrine research in menstruating nonhuman primates, such as rhesus and cynomolgus macaques, have provided valuable information regarding the hypothalamic– pituitary–ovarian axis, including modulating factors of pulsatile gonadotropin-releasing hormone secretion and the negative and positive feedback mechanisms of sex steroids.^20,25,33Normal reproductive physiology in women involves highly coordinated communication between the hypothalamus, pituitary gland, and the end organ of female reproduction, the ovary. These processes are governed by the magnitude and frequency of secretory outbursts (pulses) of gonadotropin-releasing hormone from the hypothalamus. The activity of gonadotropin-releasing hormone results in a pulsatile mode of secretion of follicle-stimulating hormone and luteinizing hormone (LH) from the anterior pituitary. In females, follicle-stimulating hormone drives ovarian follicle growth during the follicular phase of the menstrual cycle. The midcycle LH surge results in ovulation and the subsequent formation of a corpus luteum (CL). Secretion of estradiol, produced by the developing follicles, progressively increases over the course of the follicular (proliferative) phase of the menstrual cycle and peaks prior to ovulation. Progesterone, secreted by the CL, is the dominant sex steroid during the luteal (secretory) phase.¹² Both estradiol and progesterone exert tightly regulated negative feedback on the hypothalamus and pituitary and affect gonadotropin release. Alterations in this intricate system can result in anovulation or infertility.Obesity is a growing worldwide hazard that has many adverse health outcomes, including subfertility. Endocrine alterations associated with obesity include relative hypogonadotropic hypogonadism^29,34 and selective impairment of LH pulse amplitude.¹⁴ Progesterone metabolite excretion in morbidly obese women is reduced by 70% compared with that in normal-weight women,²⁹ and pulsatile LH amplitude is suppressed by half in frequent blood-sampling studies.¹⁴ However, despite the recent advances in understanding the endocrine pathophysiology of obesity-related subfertility,¹⁵ its molecular mechanisms are poorly understood.Animal models for obesity-related subfertility are needed for mechanistic studies but are currently unavailable. The hormonal control of the menstrual cycle has been extensively studied in rhesus and cynomolgus macaques and is similar to that of humans.^12,22,26 These nonhuman primates have also been shown to develop obesity and resultant metabolic disturbances.¹ However, demand for rhesus and cynomolgus macaques is high, and the NIH has espoused the need to identify other species of nonhuman primate that are suitable for research.⁶Vervet monkeys (Chlorocebus aethiops sabaeus) are a small, Old World nonhuman primate with an ovarian cycle similar to that in humans; therefore vervets may be an appropriate alternative species in which to do neuroendocrine research. LH pulsatility in this species has not been assessed comprehensively. Our objective in the current study was to characterize the follicular LH pulse pattern in vervet monkeys, to establish the feasibility of using this model in future studies to assess the effect of body mass on pituitary function.     

Intra-tumor genetic heterogeneity and alternative driver genetic alterations in breast cancers with heterogeneous HER2 gene amplification

BackgroundHER2 is overexpressed and amplified in approximately 15% of invasive breast cancers, and is the molecular target and predictive marker of response to anti-HER2 agents. In a subset of these cases, heterogeneous distribution of HER2 gene amplification can be found, which creates clinically challenging scenarios. Currently, breast cancers with HER2 amplification/overexpression in just over 10% of cancer cells are considered HER2-positive for clinical purposes; however, it is unclear as to whether the HER2-negative components of such tumors would be driven by distinct genetic alterations. Here we sought to characterize the pathologic and genetic features of the HER2-positive and HER2-negative components of breast cancers with heterogeneous HER2 gene amplification and to define the repertoire of potential driver genetic alterations in the HER2-negative components of these cases.ResultsWe separately analyzed the HER2-negative and HER2-positive components of 12 HER2 heterogeneous breast cancers using gene copy number profiling and massively parallel sequencing, and identified potential driver genetic alterations restricted to the HER2-negative cells in each case. In vitro experiments provided functional evidence to suggest that BRF2 and DSN1 overexpression/amplification, and the HER2 I767M mutation may be alterations that compensate for the lack of HER2 amplification in the HER2-negative components of HER2 heterogeneous breast cancers.ConclusionsOur results indicate that even driver genetic alterations, such as HER2 gene amplification, can be heterogeneously distributed within a cancer, and that the HER2-negative components are likely driven by genetic alterations not present in the HER2-positive components, including BRF2 and DSN1 amplification and HER2 somatic mutations.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-015-0657-6) contains supplementary material, which is available to authorized users.     

Competence and natural transformation in vibrios

Natural transformation is a major mechanism of horizontal gene transfer in bacteria. By incorporating exogenous DNA elements into chromosomes, bacteria are able to acquire new traits that can enhance their fitness in different environments. Within the past decade, numerous studies have revealed that natural transformation is prevalent among members of the Vibrionaceae, including the pathogen Vibrio cholerae. Four environmental factors: (i) nutrient limitation, (ii) availability of extracellular nucleosides, (iii) high cell density and (iv) the presence of chitin, promote genetic competence and natural transformation in Vibrio cholerae by co‐ordinating expression of the regulators CRP, CytR, HapR and TfoX respectively. Studies of other Vibrionaceae members highlight the general importance of natural transformation within this bacterial family.