Jeffrey S. Prince

Design and analysis of multiple choice feeding preference data

Traditional analyses of feeding experiments that test consumer preference for an array of foods suffer from several defects. We have modified the experimental design to incorporate into a multivariate analysis the variance due to autogenic change in control replicates. Our design allows the multiple foods to be physically paired with their control counterparts. This physical proximity of the multiple food choices in control/experimental pairs ensures that the variance attributable to external environmental factors jointly affects all combinations within each replicate. Our variance term, therefore, is not a contrived estimate as is the case for the random pairing strategy proposed by previous studies. The statistical analysis then proceeds using standard multivariate statistical tests. We conducted a multiple choice feeding experiment using our experimental design and utilized a Monte Carlo analysis to compare our results with those obtained from an experimental design that employed the random pairing strategy. Our experimental design allowed detection of moderate differences among feeding means when the random design did not.

Novel “Superspreader” Bacteriophages Promote Horizontal Gene Transfer by Transformation

ABSTRACT Bacteriophages infect an estimated 1023 to 1025 bacterial cells each second, many of which carry physiologically relevant plasmids (e.g., those encoding antibiotic resistance). However, even though phage-plasmid interactions occur on a massive scale and have potentially significant evolutionary, ecological, and biomedical implications, plasmid fate upon phage infection and lysis has not been investigated to date. Here we show that a subset of the natural lytic phage population, which we dub “superspreaders,” releases substantial amounts of intact, transformable plasmid DNA upon lysis, thereby promoting horizontal gene transfer by transformation. Two novel Escherichia coli phage superspreaders, SUSP1 and SUSP2, liberated four evolutionarily distinct plasmids with equal efficiency, including two close relatives of prominent antibiotic resistance vectors in natural environments. SUSP2 also mediated the extensive lateral transfer of antibiotic resistance in unbiased communities of soil bacteria from Maryland and Wyoming. Furthermore, the addition of SUSP2 to cocultures of kanamycin-resistant E. coli and kanamycin-sensitive Bacillus sp. bacteria resulted in roughly 1,000-fold more kanamycin-resistant Bacillus sp. bacteria than arose in phage-free controls. Unlike many other lytic phages, neither SUSP1 nor SUSP2 encodes homologs to known hydrolytic endonucleases, suggesting a simple potential mechanism underlying the superspreading phenotype. Consistent with this model, the deletion of endonuclease IV and the nucleoid-disrupting protein ndd from coliphage T4, a phage known to extensively degrade chromosomal DNA, significantly increased its ability to promote plasmid transformation. Taken together, our results suggest that phage superspreaders may play key roles in microbial evolution and ecology but should be avoided in phage therapy and other medical applications. IMPORTANCE Bacteriophages (phages), viruses that infect bacteria, are the planet’s most numerous biological entities and kill vast numbers of bacteria in natural environments. Many of these bacteria carry plasmids, extrachromosomal DNA elements that frequently encode antibiotic resistance. However, it is largely unknown whether plasmids are destroyed during phage infection or released intact upon phage lysis, whereupon their encoded resistance could be acquired and manifested by other bacteria (transformation). Because phages are being developed to combat antibiotic-resistant bacteria and because transformation is a principal form of horizontal gene transfer, this question has important implications for biomedicine and microbial evolution alike. Here we report the isolation and characterization of two novel Escherichia coli phages, dubbed “superspreaders,” that promote extensive plasmid transformation and efficiently disperse antibiotic resistance genes. Our work suggests that phage superspreaders are not suitable for use in medicine but may help drive bacterial evolution in natural environments. Bacteriophages (phages), viruses that infect bacteria, are the planet’s most numerous biological entities and kill vast numbers of bacteria in natural environments. Many of these bacteria carry plasmids, extrachromosomal DNA elements that frequently encode antibiotic resistance. However, it is largely unknown whether plasmids are destroyed during phage infection or released intact upon phage lysis, whereupon their encoded resistance could be acquired and manifested by other bacteria (transformation). Because phages are being developed to combat antibiotic-resistant bacteria and because transformation is a principal form of horizontal gene transfer, this question has important implications for biomedicine and microbial evolution alike. Here we report the isolation and characterization of two novel Escherichia coli phages, dubbed “superspreaders,” that promote extensive plasmid transformation and efficiently disperse antibiotic resistance genes. Our work suggests that phage superspreaders are not suitable for use in medicine but may help drive bacterial evolution in natural environments.

SEXUAL REPRODUCTIO IN CODIUM FRAGILE SSP. TOMENTOSOIDES (CHLOROPHYCEAE) FROM THE NORTHEAST COAST OF NORTH AMERICA1

Male and female gametes were formed within the same gametangiurn on reproductive plants of Codium fragile (Sur.) Hariot ssp. tomentosoides (Van Goor) Silva growing off Appledore Island in the Gulf of Maine, USA. The female gametes were approximately twice the diameter of male gametes and outnumbered male gametes in the same gametangium by approximately six times. Fusion appears to require gametes from different gametangia if not different plants.

Exploiting ROS and metabolic differences to kill cisplatin resistant lung cancer

Cisplatin resistance remains a major problem in the treatment of lung cancer. We have discovered that cisplatin resistant (CR) lung cancer cells, regardless of the signaling pathway status, share the common parameter which is an increase in reactive oxygen species (ROS) and undergo metabolic reprogramming. CR cells were no longer addicted to the glycolytic pathway, but rather relied on oxidative metabolism. They took up twice as much glutamine and were highly sensitive to glutamine deprivation. Glutamine is hydrolyzed to glutamate for glutathione synthesis, an essential factor to abrogate high ROS via xCT antiporter. Thus, blocking glutamate flux using riluzole (an amyotropic lateral sclerosis approved drug) can selectively kill CR cells in vitro and in vivo. However, we discovered here that glutathione suppression is not the primary pathway in eradicating the CR cells. Riluzole can lead to further decrease in NAD+ (nicotinamide adenine dinucleotide) and lactate dehydrogenase-A (LDHA) expressions which in turn further heightened oxidative stress in CR cells. LDHA knocked-down cells became hypersensitive to riluzole treatments and possessed increased levels of ROS. Addition of NAD+ re-stabilized LDHA and reversed riluzole induced cell death. Thus far, no drugs are available which could overcome cisplatin resistance or kill cisplatin resistant cells. CR cells possess high levels of ROS and undergo metabolic reprogramming. These metabolic adaptations can be exploited and targeted by riluzole. Riluzole may serve as a dual-targeting agent by suppression LDHA and blocking xCT antiporter. Repurposing of riluzole should be considered for future treatment of cisplatin resistant lung cancer patients.

Direct connection between myelinosomes, endoplasmic reticulum and nuclear envelope in mouse hepatocytes grown with the amphiphilic drug, quinacrine

Mouse hepatocytes grown in 4 microM quinacrine had numerous myelinosomes which were directly connected to expanded cisternae of the rough endoplasmic reticulum (RER). The cisternae of the RER either subtended the electron transparent space of the myelinosome, expanded to form the outer membrane of the myelinosome or penetrated into it. Material of low electron density was frequently seen within the area where the cisternae penetrated into the electron transparent space of the myelinosome. Myelinosomes were also associated with the nuclear envelope in a pattern similar to that of the RER. Quinacrine appears to bind with the phospholipids of the membranes of the endoplasmic reticulum and nuclear envelope and this drug-lipid complex is then moved into myelinosomes effectively removing the drug from the cell.

Seed Coat Morphology in Gentianopsis (Gentianaceae)

Abstract Seed coat morphology is examined in 16 taxa of Gentianopsis, Pterygocalyx, and Gentianella, including representatives from 14 taxa of Gentianopsis, to resolve inconsistencies in previous reports and make new observations using a low vacuum mode of SEM. Four characters are proposed to describe variation in seed coat morphology of this group: (1) the outer periclinal walls of testa; (2) sculpting of inner periclinal walls; (3) seed shape; and (4) seed length. The distinctive papillate seeds of many species of Gentianopsis are the result of inflated outer periclinal walls of testa cells. In some species, the outer periclinal walls of the testa are collapsed inwards, revealing previously unreported sculpting on the inner periclinal walls. Seeds are irregularly angular-ovate or fusiform in Gentianopsis and discoid in Pterygocalyx. Seed length varies from 0.22 to 1.0 mm. These data provide another line of evidence for phylogenetic relationships of Gentianopsis and Pterygocalyx to other members of Swertiinae, and illuminate the affinities of fossil seeds from New England that have been assigned to Gentianopsis.

BRAF inhibitor resistance enhances vulnerability to arginine deprivation in melanoma

BRAF inhibitor (BRAFi) has been used for treatment of melanomas harboring V600E mutation. Despite a high initial response rate, resistance to BRAFi is inevitable. Here, we demonstrate that BRAFi-resistant (BR) melanomas are susceptible to arginine deprivation due to inability to initiate re-expression of argininosuccinate synthetase (ASS1, a key enzyme for arginine synthesis) as well as ineffective autophagy. Autophagy and ASS1 re-expression are known to protect melanoma cells from cell death upon arginine deprivation. When melanoma cells become BR cells by long-term in vitro incubation with BRAFi, c-Myc-mediated ASS1 re-expression and the levels of autophagy-associated proteins (AMPK-α1 and Atg5) are attenuated. Furthermore, our study uncovers that downregulation of deubiquitinase USP28 which results in more active c-Myc degradation via ubiquitin-proteasome machinery is the primary mechanism for inability to re-express ASS1 upon arginine deprivation in BR cells. Overexpression of USP28 in BR cells enhances c-Myc expression and hence increases ASS1 transcription upon arginine deprivation, and consequently leads to cell survival. On the other hand, overexpression of Atg5 or AMPK-α1 in BR cells can redirect arginine deprivation-induced apoptosis toward autophagy. The xenograft models also confirm that BR tumors possess lower expression of ASS1 and are hypersensitive to arginine deprivation. These biochemical changes in BRAFi resistance which make them vulnerable to arginine deprivation can be exploited for the future treatment of BR melanoma patients.

Role of the Digestive Gland in Ink Production in Four Species of Sea Hares: An Ultrastructural Comparison

The ultrastructure of the digestive gland of several sea hare species that produce different colored ink (Aplysia californica produces purple ink, A. juliana white ink, A. parvula both white and purple ink, while Dolabrifera dolabrifera produces no ink at all) was compared to determine the digestive gland’s role in the diet-derived ink production process. Rhodoplast digestive cells and their digestive vacuoles, the site of digestion of red algal chloroplast (i.e., rhodoplast) in A. californica, were present and had a similar ultrastructure in all four species. Rhodoplast digestive cell vacuoles either contained a whole rhodoplast or fragments of one or were empty. These results suggest that the inability to produce colored ink in some sea hare species is not due to either an absence of appropriate digestive machinery, that is, rhodoplast digestive cells, or an apparent failure of rhodoplast digestive cells to function. These results also propose that the digestive gland structure described herein occurred early in sea hare evolution, at least in the common ancestor to the genera Aplysia and Dolabrifera. Our data, however, do not support the hypothesis that the loss of purple inking is a synapomorphy of the white-ink-producing subgenus Aplysia.

Combined fluorescence and ultrastructural mapping of living cells

The topographic analysis of fluorescence distribution has been carried out pixel-by-pixel by one dimensional, two-dimensional microspectrofluorometry and three-dimensional confocal fluorescence microscopy. Fluorescence emission spectra of NAD(P)H and benzo(a)pyrene (or metabolites) were recorded at different excitation wavelengths. Cell bioenergetics are monitored in normal and malignant cells as well as cells with genetic defects by coenzyme responses to microinjections of substrates and modifiers from key metabolic pathways in presence and absence of inhibitors and drugs active on mitochondrial structure and function. Cooperative interactions between organelles involved in detoxification mechanisms are observed in cells treated with fluorescent cytotoxic agents. Such interactions can be directly mapped by the fluorescence of cytotoxic agents, their reaction products or vital probes such as NBD ceramide for the Golgi apparatus. To identify the organelles involved parallel electron microscopic studies are carried out in cells first treated with the cytotoxic agent and then incubated with an electron opaque material. A recently developed combined X-ray laser microscope (COXRALM) holds the promise of carrying out combined phase-fluorescence-and X-ray microscopic observations of fluorescence and ultrastructural correlations in live cell probing. As further versatility is gained in such methods it may become possible to obtain a very detailed structure and function mapping of living cells within the context of cytomatrix analysis, metabolic compartmentation and organelle interactions.

Ultrastructural Comparison of Processing of Protein and Pigment in the Ink Gland of Four Species of Sea Hares

The ink glands of four sea hare species (Aplysia californica, A. parvula, A. juliana, and Dolabrifera dolabrifera) were compared to determine where ink protein is synthesized, how it is incorporated into protein storage vesicles, and the degree of variation in the structure of the ink gland. Ink protein was synthesized in RER cells and stored in amber and white vesicles. Lack of competent RER cells in the ink gland of D. dolabrifera was correlated with the absence of ink protein. Ink protein had similar characteristics in all three Aplysia species but, again, it was absent in D. dolabrifera. Its uptake involved pinocytosis by protein vesicle cell membranes. Granulate cells showed little variation in structure among the four species, the opposite was the case for RER cells. The conversion of the red algal pigment, phycoerythrin, to phycoerythrobilin (PEB) occurs in the digestive gland but the change of PEB to aplysioviolin (APV), the form of pigment released by the ink gland, occurs in the ink gland itself by both granulate cells and pigment vesicles. The literature describes five types of vesicles based upon color and contents in the ink gland of these four species. We report only three types of vesicle: colored (purple), protein (white and amber), and transparent (includes clear vesicles).