Gonadotropin-releasing hormone analog structural determinants of selectivity for inhibition of cell growth: support for the concept of ligand-induced selective signaling

GnRH and its receptor are expressed in human reproductive tract cancers, and direct antiproliferative effects of GnRH analogs have been demonstrated in cancer cell lines. The intracellular signaling responsible for this effect differs from that mediating pituitary gonadotropin secretion. The GnRH structure-activity relationship is different for the two effects. Here we report a structure-activity relationship study of GnRH agonist antiproliferative action in model cell systems of rat and human GnRH receptors stably expressed in HEK293 cells. GnRH II was more potent than GnRH I in inhibiting cell growth in the cell lines. In contrast, GnRH I was more potent than GnRH II in stimulating inositol phosphate production, the signaling pathway in gonadotropes. The different residues in GnRH II (His(5), Trp(7), Tyr(8)) were introduced singly or in pairs into GnRH I. Tyr(5) replacement by His(5) produced the highest increase in the antiproliferative potency of GnRH I. Tyr(8) substitution of Arg(8) produced the most selective analog, with very poor inositol phosphate generation but high antiproliferative potency. In nude mice bearing tumors of the HEK293 cell line, GnRH II and an antagonist administration was ineffective in inhibiting tumor growth, but D-amino acid stabilized analogs (D-Lys(6) and D-Arg(6)) ablated tumor growth. Docking of GnRH I and GnRH II to the human GnRH receptor molecular model revealed that Arg(8) of GnRH I makes contact with Asp(302), whereas Tyr(8) of GnRH II appears to make different contacts, suggesting these residues stabilize different receptor conformations mediating differential intracellular signaling and effects on gonadotropin and cell growth. These findings provide the basis for the development of selective GnRH analog cancer therapeutics that directly target tumor cells or inhibit pituitary gonadotropins or do both.     

The glucagon-like peptide-1 receptor binding site for the N-terminus of GLP-1 requires polarity at Asp198 rather than negative charge

The mutation of Asp198 to Asn in the receptor for glucagon-like peptide-1(7–36)amide (GLP-1) had no effect upon GLP-1 affinity whereas substitution with Ala greatly reduced affinity, demonstrating the importance of polarity rather than negative charge at Asp198. However, the Asp198-Ala mutation had less effect upon the affinity of Exendin-4, a peptide agonist that has been shown previously not to require its N-terminus for high affinity. Moreover, the affinity of a truncated GLP-1 analogue lacking the first eight residues was not affected by the Asp198-Ala mutation, demonstrating that Asp198 is required for maintaining the binding site of the N-terminal region of GLP-1.     

Moving through three‐dimensional phase diagrams of monoclonal antibodies

Protein phase behavior characterization is a multivariate problem due to the high amount of influencing parameters and the diversity of the proteins. Single influences on the protein are not understood and fundamental knowledge remains to be obtained. For this purpose, a systematic screening method was developed to characterize the influence of fluid phase conditions on the phase behavior of proteins in three‐dimensional phase diagrams. This approach was applied to three monoclonal antibodies to investigate influences of pH, protein and salt concentrations, with five different salts being tested. Although differences exist between the antibodies, this extensive study confirmed the general applicability of the Hofmeister series over the broad parameter range analyzed. The influence of the different salts on the aggregation (crystallization and precipitation) probability was described qualitatively using this Hofmeister series, with a differentiation between crystallization and precipitation being impossible, however. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1103–1113, 2014     

Central Nervous System Paracoccidioidomycosis in an AIDS Patient: Case Report

Up to now, over 200 patients with paracoccidioidomycosis (PCM) associated to HIV infection have already been reported; however, the central nervous system involvement in this coinfection was rarely reported. This paper presents a 35-year-old Brazilian male AIDS patient who developed pulmonary PCM successfully treated with itraconazole. At the antiretroviral therapy starting, he had 32 CD4⁺ T cells baseline count and high viral load levels. After 9 months, he presented severe fungal meningoencephalitis diagnosed by sublenticular enhanced nodular lesion at computerized tomography and magnetic resonance brain imaging and a positive Paracoccidiodes brasiliensis smear and culture from cerebrospinal fluid. At the time, a sixfold increase in CD4⁺ T cell count and undetectable viral load level were evidenced. The patient received amphotericin B during 1 year presenting slow but progressive clinical improvement, and he is currently asymptomatic and without neurological disabilities. To our knowledge, this is the second case report of a patient with neuroparacoccidioidomycosis associated to HIV infection.     

Arctic root‐associated fungal community composition reflects environmental filtering

There is growing evidence that root‐associated fungi have important roles in Arctic ecosystems. Here, we assess the diversity of fungal communities associated with roots of the ectomycorrhizal perennial herb Bistorta vivipara on the Arctic archipelago of Svalbard and investigate whether spatial separation and bioclimatic variation are important structuring factors of fungal community composition. We sampled 160 plants of B. vivipara from 32 localities across Svalbard. DNA was extracted from entire root systems, and 454 pyrosequencing of ITS1 amplicons was used to profile the fungal communities. The fungal communities were predominantly composed of Basidiomycota (55% of reads) and Ascomycota (35%), with the orders Thelephorales (24%), Agaricales (13.8%), Pezizales (12.6%) and Sebacinales (11.3%) accounting for most of the reads. Plants from the same site or region had more similar fungal communities to one another than plants from other sites or regions, and sites clustered together along a weak latitudinal gradient. Furthermore, a decrease in per‐plant OTU richness with increasing latitude was observed. However, no statistically significant spatial autocorrelation between sites was detected, suggesting that environmental filtering, not dispersal limitation, causes the observed patterns. Our analyses suggest that while latitudinal patterns in community composition and richness might reflect bioclimatic influences at global spatial scales, at the smaller spatial scale of the Svalbard archipelago, these changes more likely reflect varied bedrock composition and associated edaphic factors. The need for further studies focusing on identifying those specific bioclimatic and edaphic factors structuring root‐associated fungal community composition at both global and local scales is emphasized.     

Placebo, meaning, and health

Placebos are boon and bane to medical theory and clinical practice. On the one hand, randomized controlled trials employ concealed allocations of placebo to control for effects not due to specific pharmacological mechanisms. As a result, nearly all of evidence-based medicine derives from principles and practices based on placebo. On the other hand, medical researchers and physicians have tended to ignore, minimize, or deride placebos and placebo effects, perhaps due to values emphasizing scientific understanding of mechanistic pathways. We argue that intention, expectation, culture, and meaning are central to placebo-effect phenomena and are substantive determinants of health. We introduce three dualities that are integral to placebo/meaning phenomena: body-mind, subconscious-conscious, and passive-active. These placebo-related dualities should be acknowledged, explored with research, and incorporated in theory. While we view consideration of placebo and meaning effects as essential to any adequate understanding of human health, we feel that lessons from this area of inquiry may already provide practical tools for astute clinicians. Toward this end, we list eight specific clinical actions: speak positively about treatments, provide encouragement, develop trust, provide reassurance, support relationships, respect uniqueness, explore values, and create ceremony. These clinical actions can empower patients to seek greater health and may provide a healthful sense of being cared for.     

ITS1 versus ITS2 as DNA metabarcodes for fungi

The nuclear ribosomal Internal Transcribed Spacer ITS region is widely used as a DNA metabarcoding marker to characterize the diversity and composition of fungal communities. In amplicon pyrosequencing studies of fungal diversity, one of the spacers ITS1 or ITS2 of the ITS region is normally used. In this methodological study we evaluate the usability of ITS1 vs. ITS2 as a DNA metabarcoding marker for fungi. We analyse three data sets: two comprising ITS1 and ITS2 sequences of known taxonomic affiliations and a third comprising ITS1 and ITS2 environmental amplicon pyrosequencing data. Clustering analyses of sequences with known taxonomy using the bioinformatics pipeline ClustEx revealed that a 97% similarity cut‐off represent a reasonable threshold for estimating the number of known species in the data sets for both ITS1 and ITS2. However, no single threshold value worked well for all fungi at the same time within the curated UNITE database, and we found that the Operational Taxonomic Unit (OTU) concept is not easily translated into the level of species because many species are distributed over several clusters. Clustering analyses of the 134 692 ITS1 and ITS2 pyrosequences using a 97% similarity cut‐off revealed a high similarity between the two data sets when it comes to taxonomic coverage. Although some groups are under‐ or unrepresented in the two data sets due to, e.g. primer mismatches, our results indicate that ITS1 and ITS2 to a large extent yield similar results when used as DNA metabarcodes for fungi.     

Primary succession of Bistorta vivipara (L.) Delabre (Polygonaceae) root‐associated fungi mirrors plant succession in two glacial chronosequences

Glacier chronosequences are important sites for primary succession studies and have yielded well‐defined primary succession models for plants that identify environmental resistance as an important determinant of the successional trajectory. Whether plant‐associated fungal communities follow those same successional trajectories and also respond to environmental resistance is an open question. In this study, 454 amplicon pyrosequencing was used to compare the root‐associated fungal communities of the ectomycorrhizal (ECM) herb Bistorta vivipara along two primary succession gradients with different environmental resistance (alpine versus arctic) and different successional trajectories in the vascular plant communities (directional replacement versus directional non‐replacement). At both sites, the root‐associated fungal communities were dominated by ECM basidiomycetes and community composition shifted with increasing time since deglaciation. However, the fungal community's successional trajectory mirrored the pattern observed in the surrounding plant community at both sites: the alpine site displayed a directional‐replacement successional trajectory, and the arctic site displayed a directional‐non‐replacement successional trajectory. This suggests that, like in plant communities, environmental resistance is key in determining succession patterns in root‐associated fungi. The need for further replicated study, including in other host species, is emphasized.     

Diffusional Properties of Methanogenic Granular Sludge: 1H NMR Characterization

The diffusive properties of anaerobic methanogenic and sulfidogenic aggregates present in wastewater treatment bioreactors were studied using diffusion analysis by relaxation time-separated pulsed-field gradient nuclear magnetic resonance (NMR) spectroscopy and NMR imaging. NMR spectroscopy measurements were performed at 22°C with 10 ml of granular sludge at a magnetic field strength of 0.5 T (20 MHz resonance frequency for protons). Self-diffusion coefficients of H₂O in the investigated series of mesophilic aggregates were found to be 51 to 78% lower than the self-diffusion coefficient of free water. Interestingly, self-diffusion coefficients of H₂O were independent of the aggregate size for the size fractions investigated. Diffusional transport occurred faster in aggregates growing under nutrient-rich conditions (e.g., the bottom of a reactor) or at high (55°C) temperatures than in aggregates cultivated in nutrient-poor conditions or at low (10°C) temperatures. Exposure of aggregates to 2.5% glutaraldehyde or heat (70 or 90°C for 30 min) modified the diffusional transport up to 20%. In contrast, deactivation of aggregates by HgCl₂ did not affect the H₂O self-diffusion coefficient in aggregates. Analysis of NMR images of a single aggregate shows that methanogenic aggregates possess a spin-spin relaxation time and self-diffusion coefficient distribution, which are due to both physical (porosity) and chemical (metal sulfide precipitates) factors.