Structure-Activity Relationships of Natural and Semisynthetic Plecomacrolides Suggest Distinct Pathways for HIV-1 Immune Evasion and Vacuolar ATPase-Dependent Lysosomal Acidification.

The human immunodeficiency virus (HIV)-encoded accessory protein Nef enhances pathogenicity by reducing major histocompatibility complex I (MHC-I) cell surface expression, protecting HIV-infected cells from immune recognition. Nef-dependent downmodulation of MHC-I can be reversed by subnanomolar concentrations of concanamycin A (1), a well-known inhibitor of vacuolar ATPase, at concentrations below those that interfere with lysosomal acidification or degradation. We conducted a structure-activity relationship study that assessed 76 compounds for Nef inhibition, 24 and 72 h viability, and lysosomal neutralization in Nef-expressing primary T cells. This analysis demonstrated that the most potent compounds were natural concanamycins and their derivatives. Comparison against a set of new, semisynthetic concanamycins revealed that substituents at C-8 and acylation of C-9 significantly affected Nef potency, target cell viability, and lysosomal neutralization. These findings provide important progress toward understanding the mechanism of action of these compounds and the identification of an advanced lead anti-HIV Nef inhibitory compound.

The Effects of Water Volume and Bacterial Concentration on the Water Filtration Assay Used in Zebrafish Health Surveillance.

The number of zebrafish in biomedical research has increased exponentially over the past decades, leading to pressure on the laboratory animal community to develop and refine techniques to monitor zebrafish health so that suitable stocks can be maintained for research. The water filtration assay is a promising technique in which water from a zebrafish system is filtered, and the filter analyzed by PCR. In the present report, we studied how the volume of water tested and the concentration of bacterial pathogens affected test results. To do so, we used stock solutions of 3 zebrafish pathogens: Edwardsiella ictaluri, Aeromonas hydrophila, and Mycobacterium marinum. We used these stocks to create solutions with known concentrations of each pathogen, ranging between 10² and 107 Colony Forming Units (CFU) per ml. One, 2, and 3 L of each solution was filtered using positive pressure, and the filters were submitted to a commercial lab for PCR testing. Results were fit with a logistic regression model, and the probability of obtaining a positive result were calculated. Test sensitivity varied by organism, but in general, test results were positively correlated with the volume of the water filtered and with the concentration of bacteria in solution. We conclude that a positive result can be expected for E. ictaluri at 105 CFU per mL, A. hydrophila at 106 CFU per ml, and M. marinum at 106 CFU per mL, when 3 L of solution are filtered.

Determination of the pKa Values of trans-Resveratrol, a Triphenolic Stilbene, by Singular Value Decomposition. Comparison with Theory.

Several independent determinations of the pKa values of trans-resveratrol in water have led to conflicting results. Singular value decomposition analysis of UV absorption spectra of trans-resveratrol (t-Resv) in N2-outgased aqueous solutions buffered to pH values in the 7.0-13.6 range yielded the UV spectra of the three anionic forms and the corresponding pKa values: pKa1 = 9.16, pKa2 = 9.77, and pKa3 = 10.55 in very good agreement with calculated theoretical values. The analysis of the absorption spectra guided the assignment of the fluorescence spectrum of each anionic form. With the resolved spectra on hand, we applied the Förster equation to estimate pKa* values of 2.5 and 0, respectively, for the p- and m-OH substituents of t-Resv in S1. Theory supports a proposed mechanism for the reaction of t-Resv anions with O2.

Evaluation of Data Analysis Platforms and Compatibility with MALDI-TOF Imaging Mass Spectrometry Data Sets.

Imaging mass spectrometry (IMS) has proven to be a useful tool when investigating the spatial distributions of metabolites and proteins in a biological system. One of the biggest advantages of IMS is the ability to maintain the 3D chemical composition of a sample and analyze it in a label-free manner. However, acquiring the spatial information leads to an increase in data size. Due to the increased availability of commercial mass spectrometers capable of IMS, there has been an exciting development of different statistical tools that can help decipher the spatial relevance of an analyte in a biological sample. To address this need, software packages like SCiLS and the open source R package Cardinal have been designed to perform unbiased spectral grouping based on the similarity of spectra in an IMS data set. In this note, we evaluate SCiLS and Cardinal compatibility with MALDI-TOF IMS data sets of the Gram-negative pathogen Pseudomonas aeruginosa PA14. Both software were able to perform unsupervised segmentation with similar performance. There were a few notable differences which are discussed related to the identification of statistically significant features which required optimization of preprocessing steps, region of interest, and manual analysis.

Addition of insoluble fiber to isolation media allows for increased metabolite diversity of lab-cultivable microbes derived from zebrafish gut samples.

There is a gap in measured microbial diversity when comparing genomic sequencing techniques versus cultivation from environmental samples in a laboratory setting. Standardized methods in artificial environments may not recapitulate the environmental conditions that native microbes require for optimal growth. For example, the intestinal tract houses microbes at various pH values as well as minimal oxygen and light environments. These microbes are also exposed to an atypical source of carbon: dietary fiber compacted in fecal matter. To investigate how the addition of insoluble fiber to isolation media could affect the cultivation of microbes from zebrafish intestines, an isolate library was built and analyzed using the bioinformatics pipeline IDBac. While all isolation media encouraged the growth of species from several phyla, the extent of growth was greater with the addition of fiber allowing for easier isolation. Furthermore, fiber addition altered the metabolism of the cultivated gut-derived microbes and induced the production of unique metabolites that were not produced when microbes were otherwise grown on standard isolation media. Addition of this inexpensive carbon source to the media supported the cultivation of a diverse community whose secondary metabolite production may more closely replicate their metabolite production in vivo.

Biofilm Inhibitor Taurolithocholic Acid Alters Colony Morphology, Specialized Metabolism, and Virulence of Pseudomonas aeruginosa.

Biofilm inhibition by exogenous molecules has been an attractive strategy for the development of novel therapeutics. We investigated the biofilm inhibitor taurolithocholic acid (TLCA) and its effects on the specialized metabolism, virulence, and biofilm formation of the clinically relevant bacterium Pseudomonas aeruginosa strain PA14. Our study shows that TLCA alters the specialized metabolism, thereby affecting P. aeruginosa colony biofilm physiology. We observed an upregulation of metabolites correlated to virulence such as the siderophore pyochelin. A wax moth virulence assay confirmed that treatment with TLCA increases the virulence of P. aeruginosa. On the basis of our results, we believe that future endeavors to identify biofilm inhibitors must consider how a putative lead alters the specialized metabolism of a bacterial community to prevent pathogens from entering a highly virulent state.

Calling all hosts: Bacterial communication in situ.

Bacteria are cosmopolitan organisms that in recent years have demonstrated many roles in maintaining host equilibrium. In this review, we discuss three roles bacteria can occupy in a host: pathogenic, symbiotic, and transient, with a specific focus on how bacterial small molecules contribute to homeostasis or dysbiosis. First, we will dissect how small molecules produced by pathogenic bacteria can be used as a source for communication during colonization and as protection against host immune responses. The ability to achieve a higher level of organization through small molecule communication gives pathogenic bacteria an opportunity for increased virulence and fitness. Conversely, in symbiotic relationships with hosts, small molecules are used in the initial acquisition, colonization, and maintenance of this beneficial population. Chemical signals can come from both the host and symbiont, and it is often observed that these interKingdom symbioses result in coevolution of both species involved. Furthermore, the transition from transient to commensal or opportunistic likely relies on molecular mechanisms. The small molecules utilized and produced by transient bacteria are desirable for both the immune and nutritional benefits they provide to the host. Finally, the advantages and disadvantages of modern analytical techniques that are available to researchers in order to study small molecules in situ is an important aspect of this review. It is our opinion that small molecules produced by bacteria are central to many biological processes and a larger focus on uncovering the function and identity of these small molecules is required to gain a deeper understanding of host-microbe associations.