Synthesis and Evaluation of Biphenyl Compounds as Kinesin Spindle Protein Inhibitors

Kinesin spindle protein (KSP), an ATP‐dependent motor protein, plays an essential role in bipolar spindle formation during the mitotic phase (M phase) of the normal cell cycle. KSP has emerged as a novel target for antimitotic anticancer drug development. In this work, we synthesized a range of new biphenyl compounds and investigated their properties in vitro as potential antimitotic agents targeting KSP expression. Antiproliferation (MTT (=3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐2H‐tetrazolium bromide)) assays, combined with fluorescence‐assisted cell sorting (FACS) and Western blot studies analyzing cell‐cycle arrest confirmed the mechanism and potency of these biphenyl compounds in a range of human cancer cell lines. Structural variants revealed that functionalization of biphenyl compounds with bulky aliphatic or aromatic groups led to a loss of activity. However, replacement of the urea group with a thiourea led to an increase in antiproliferative activity in selected cell lines. Further studies using confocal fluorescence microscopy confirmed that the most potent biphenyl derivative identified thus far, compound 7 , exerts its pharmacologic effect specifically in the M phase and induces monoaster formation. These studies confirm that chemical scope remains for improving the potency and treatment efficacy of antimitotic KSP inhibition in this class of biphenyl compounds.     

Cultivable bacteria isolated from apple trees cultivated under different crop systems: Diversity and antagonistic activity against Colletotrichum gloeosporioides

This study evaluated the diversity of cultivable plant growth-promoting (PGP) bacteria associated with apple trees cultivated under different crop management systems and their antagonistic ability against Colletotrichum gloeosporioides. Samples of roots and rhizospheric soil from apple trees cultivated in organic and conventional orchards in southern Brazil were collected, together with soil samples from an area never used for agriculture (native field). Bacteria were identified at the genus level by PCR-RFLP and partial sequencing of the 16S rRNA, and were evaluated for some PGP abilities. The most abundant bacterial genera identified were Enterobacter (27.7%), Pseudomonas (18.7%), Burkholderia (13.7%), and Rahnella (12.3%). Sixty-nine isolates presented some antagonist activity against C. gloeosporioides. In a greenhouse experiment, five days after exposure to C. gloeosporioides, an average of 30% of the leaf area of plants inoculated with isolate 89 (identified as Burkholderia sp.) were infected, whereas 60 to 73% of the leaf area of untreated plants was affected by fungal attack. Our results allowed us to infer how anthropogenic activity is affecting the bacterial communities in soil associated with apple tree crop systems, and to obtain an isolate that was able to delay the emergence of an important disease for this culture.     

Evolutionary divergence of β–expansin structure and function in grasses parallels emergence of distinctive primary cell wall traits

Expansins are wall‐loosening proteins that promote the extension of primary cell walls without the hydrolysis of major structural components. Previously, proteins from the EXPA (α–expansin) family were found to loosen eudicot cell walls but to be less effective on grass cell walls, whereas the reverse pattern was found for EXPB (β–expansin) proteins obtained from grass pollen. To understand the evolutionary and structural bases for the selectivity of EXPB action, we assessed the extension (creep) response of cell walls from diverse monocot families to EXPA and EXPB treatments. Cell walls from Cyperaceae and Juncaceae (families closely related to grasses) displayed a typical grass response (‘β–response’). Walls from more distant monocots, including some species that share with grasses high levels of arabinoxylan, responded preferentially to α–expansins (‘α–response’), behaving in this regard like eudicots. An expansin with selective activity for grass cell walls was detected in Cyperaceae pollen, coinciding with the expression of genes from the divergent EXPB–I branch that includes grass pollen β–expansins. The evolutionary origin of this branch was located within Poales on the basis of phylogenetic analyses and its association with the ‘sigma’ whole‐genome duplication. Accelerated evolution in this branch has remodeled the protein surface in contact with the substrate, potentially for binding highly substituted arabinoxylan. We propose that the evolution of the divergent EXPB–I group made a fundamental change in the target and mechanism of wall loosening in the grass lineage possible, involving a new structural role for xylans and the expansins that target them.     

A BioBrick compatible strategy for genetic modification of plants

ABSTRACT: BACKGROUND: Plant biotechnology can be leveraged to produce food, fuel, medicine, and materials. Standardized methods advocated by the synthetic biology community can accelerate the plant design cycle, ultimately making plant engineering more widely accessible to bioengineers who can contribute diverse creative input to the design process. RESULTS: This paper presents work done largely by undergraduate students participating in the 2010 International Genetically Engineered Machines (iGEM) competition. Described here is a framework for engineering the model plant Arabidopsis thaliana with standardized, BioBrick compatible vectors and parts available through the Registry of Standard Biological Parts (www.partsregistry.org). This system was used to engineer a proof-of-concept plant that exogenously expresses the taste-inverting protein miraculin. CONCLUSIONS: Our work is intended to encourage future iGEM teams and other synthetic biologists to use plants as a genetic chassis. Our workflow simplifies the use of standardized parts in plant systems, allowing the construction and expression of heterologous genes in plants within the timeframe allotted for typical iGEM projects.     

<Emphasis Type="Italic">Gr</Emphasis> and <Emphasis Type="Italic">hp</Emphasis>-<Emphasis Type="Italic">1</Emphasis> tomato mutants unveil unprecedented interactions between arbuscular mycorrhizal symbiosis and fruit ripening

Main conclusion

Systemic responses to an arbuscular mycorrhizal fungus reveal opposite phenological patterns in two tomato ripening mutants depending whether ethylene or light reception is involved. The availability of tomato ripening mutants has revealed many aspects of the genetics behind fleshy fruit ripening, plant hormones and light signal reception. Since previous analyses revealed that arbuscular mycorrhizal symbiosis influences tomato berry ripening, we wanted to test the hypothesis that an interplay might occur between root symbiosis and fruit ripening. With this aim, we screened seven tomato mutants affected in the ripening process for their responsiveness to the arbuscular mycorrhizal fungus Funneliformis mosseae. Following their phenological responses we selected two mutants for a deeper analysis: Green ripe (Gr), deficient in fruit ethylene perception and high-pigment-1 (hp-1), displaying enhanced light signal perception throughout the plant. We investigated the putative interactions between ripening processes, mycorrhizal establishment and systemic effects using biochemical and gene expression tools. Our experiments showed that both mutants, notwithstanding a normal mycorrhizal phenotype at root level, exhibit altered arbuscule functionality. Furthermore, in contrast to wild type, mycorrhization did not lead to a higher phosphate concentration in berries of both mutants. These results suggest that the mutations considered interfere with arbuscular mycorrhiza inducing systemic changes in plant phenology and fruits metabolism. We hypothesize a cross talk mechanism between AM and ripening processes that involves genes related to ethylene and light signaling.

     

In vitro assessment of the antimicrobial activity of new N-acyl-thiourea derivatives

The qualitative screening of the susceptibility spectra of different microbial strains to the newly synthesized substances complexes was performed by adapted disk diffusion techniques, while the quantitative assay of the minimal inhibitory concentration (M.I.C., microg/cm3) value was based on liquid medium serial microdilutions. The compounds were solubilized in dimethylsulfoxide (DMSO). The in vitro biological screening effects were tested against a microbial inoculum of approximately 1.5 x 10(8) UFC/cm3, corresponding to 0.5 McFarland standard density, obtained from Gram positive (Staphylococcus aureus, Bacillus subtilis), Gram negative bacteria (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa) and fungal strains (Candida albicans). In order to investigate the influence of the subinhibitory concentration of the tested substances on the expression of different virulence features, the strains were incubated overnight in the presence of the newly synthesized thiourea derivatives (vol:vol) and different virulence features were investigated, i.e: adherence capacity to the cellular substrate represented by HeLa cells and to inert substrata quantified by slime test and soluble enzymatic virulence factors (haemolysins and other pore-forming toxins, proteases activity, DN-ase and siderophores production). The cytotoxicity was assessed microscopically, by observing the effect of the tested compounds on the cellular substratum integrity.     

Routine Clinical-Pathologic Correlation of Pigmented Skin Tumors Can Influence Patient Management

Background

Several studies have demonstrated the benefit of integrating clinical with pathologic information, to obtain a confident diagnosis for melanocytic tumors. However, all those studies were conducted retrospectively and no data are currently available about the role of a clinical-pathologic correlation approach on a daily basis in clinical practice.

Aim of the Study

In our study, we evaluated the impact of a routine clinical-pathologic correlation approach for difficult skin tumors seen over 3 years in a tertiary referral center.

Results

Interestingly, a re-appraisal was requested for 158 out of 2015 (7.7%) excised lesions because clinical-pathologic correlation was missing. Of note, in 0.6% of them (13 out of 2045) the first histologic diagnosis was revised in the light of clinical information that assisted the Pathologist to re-evaluate the histopathologic findings that might be bland or inconspicuous per se.

Conclusion

In conclusion, our study demonstrated that an integrated approach involving clinicians and pathologists allows improving management of selected patients by shifting from a simply disease-focused management (melanoma versus nevus) to a patient-centered approach.     

Dopamine receptor D4 allele distribution in Amerindians: A reflection of past behavior differences?

The DRD4 variable number of tandem repeats (VNTR) allele distribution of 172 Guarani (Kaiowá and Ñandeva subgroups) and Kaingang Brazilian Amerindians is reported. These results are integrated with those previously obtained for this ethnic group. Allele frequencies for the three populations are within the interval observed for 15 other Native American populations and show intermediate values between those observed in Amazonia and Patagonia. Significant differences in allele distribution between recent past hunter–gatherer and agriculturalist populations are observed, with an increase of the 7R allele among hunter–gatherers (P < 0.001). Analysis of molecular variance (AMOVA) and pairwise F_ST data suggest three distinct sectors for the genetic landscape of Native South America: Andes, Center/Southeast region, and Amazonia. Common traits among hunter–gatherers such as novelty‐seeking temperament, hyperactivity, and impulsivity could have been important and advantageous in new environments during America's prehistoric colonization. Am J Phys Anthropol, 2010. © 2010 Wiley‐Liss, Inc.     

Voice and handgrip strength predict reproductive success in a group of indigenous african females

Evolutionary accounts of human traits are often based on proxies for genetic fitness (e.g., number of sex partners, facial attractiveness). Instead of using proxies, actual differences in reproductive success is a more direct measure of Darwinian fitness. Certain voice acoustics such as fundamental frequency and measures of health such as handgrip strength correlate with proxies of fitness, yet there are few studies showing the relation of these traits to reproduction. Here, we explore whether the fundamental frequency of the voice and handgrip strength account for differences in actual reproduction among a population of natural fertility humans. Our results show that both fundamental frequency and handgrip strength predict several measures of reproductive success among a group of indigenous Namibian females, particularly amongst the elderly, with weight also predicting reproductive outcomes among males. These findings demonstrate that both hormonally regulated and phenotypic quality markers can be used as measures of Darwinian fitness among humans living under conditions that resemble the evolutionary environment of Homo sapiens. We also argue that these findings provide support for the Grandmother Hypothesis.     

A unique arginine cluster in PolDIP2 enhances nucleotide binding and DNA synthesis by PrimPol

Replication forks often stall at damaged DNA. To overcome these obstructions and complete the DNA duplication in a timely fashion, replication can be restarted downstream of the DNA lesion. In mammalian cells, this repriming of replication can be achieved through the activities of primase and polymerase PrimPol. PrimPol is stimulated in DNA synthesis through interaction with PolDIP2, however the exact mechanism of this PolDIP2-dependent stimulation is still unclear. Here, we show that PrimPol uses a flexible loop to interact with the C-terminal ApaG-like domain of PolDIP2, and that this contact is essential for PrimPol''s enhanced processivity. PolDIP2 increases primer-template and dNTP binding affinities of PrimPol, which concomitantly enhances its nucleotide incorporation efficiency. This stimulation is dependent on a unique arginine cluster in PolDIP2. Since the polymerase activity of PrimPol alone is very limited, this mechanism, where the affinity for dNTPs gets increased by PolDIP2 binding, might be critical for the in vivo function of PrimPol in tolerating DNA lesions at physiological nucleotide concentrations.