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1.  Halicephalobus gingivalis: A Rare Cause of Fatal Meningoencephalomyelitis in Humans 
The genus Halicephalobus consists of eight species of free-living nematodes. Only one species (H. gingivalis) has been reported to infect vertebrates. Human infection is extremely rare, and only four cases have been reported in the literature. These nematodes seem to exhibit neurotropism, but their life cycle, mode of infection, and risk factors are poorly understood. Neurohelminthiases are not commonly recognized in the United States and when they do occur, pose great diagnostic challenges because of lack of appropriate non-invasive screening and/or confirmatory tests. We report a challenging case of meningoencephalomyelitis caused by a Halicephalobus sp., in which the patient had a rapidly deteriorating clinical course. The case did not raise any clinical suspicion of neurohelminthiases, although increased eosinophils were present in the cerebrospinal fluid. This case presents an opportunity to highlight the importance of considering parasitic infection in meningoencephalitis or meningoencephalomyelitis presenting atypically.
PMCID: PMC3752803  PMID: 23509120
2.  Assessment and Management of Professionalism Issues in Pathology Residency Training 
Academic pathology  2015;2(3):2374289515592887.
Professionalism issues are common in residency training and can be very difficult to recognize and manage. Almost one-third of the milestones for pathology recently instituted by the Accreditation Council for Graduate Medical Education encompass aspects of professionalism. Program directors are often unsure of how and when to remediate residents for unprofessional behavior. We used a case-based educational approach in a workshop setting to assist program directors in the management of unprofessional behavior in residents. Eight case scenarios highlighting various aspects of unprofessional behavior by pathology residents were developed and presented in an open workshop forum at the annual pathology program director’s meeting. Prior to the workshop, 2 surveys were conducted: (1) to collect data on program directors’ experience with identifying, assessing, and managing unprofessional behavior in their residents and (2) to get feedback from workshop registrants on how they would manage each of the 8 case scenarios. A wide range of unprofessional behaviors have been observed by pathology program directors. Although there is occasionally general agreement on how to manage specific behaviors, there remains wide variation in how to manage many of the presented unprofessional behaviors. Remediation for unprofessional behavior in pathology residents remains a difficult and challenging process. Additional education and research in this area are warranted.
PMCID: PMC5479457
competency; graduate medical education; milestones; pathology training; professionalism; residents; resident remediation; resident training
3.  Loss of N-Myc interactor promotes epithelial-mesenchymal-transition by activation of TGF-β/SMAD signaling 
Oncogene  2013;33(20):2620-2628.
Epithelial-Mesenchymal-Transition (EMT) is one of the critical cellular programs that facilitate the progression of breast cancer to an invasive disease. We have observed that the expression of N-myc interactor (NMI) decreases significantly during progression of breast cancer, specifically in invasive and metastatic stages. Recapitulation of this loss in breast cell lines with epithelial morphology [MCF10A (non-tumorigenic) and T47D (tumorigenic)] by silencing NMI expression causes mesenchymal-like morphological changes in 3-D growth, accompanied by up-regulation of SLUG and ZEB2 and increased invasive properties. Conversely, we found that restoring NMI expression attenuated mesenchymal attributes of metastatic breast cancer cells accompanied by distinctly circumscribed 3-D growth with basement membrane deposition and decreased invasion. Further investigations into the downstream signaling modulated by NMI revealed that NMI expression negatively regulates SMAD signaling, which is a key regulator of cellular plasticity. We demonstrate that NMI blocks TGF-β/SMAD signaling via up-regulation of SMAD7, a negative feedback regulator of the pathway. We also provide evidence that NMI activates STAT signaling which negatively modulates TGF-β/SMAD signaling. Taken together, our findings suggest that loss of NMI during breast cancer progression could be one of the driving factors that enhance invasive ability of breast cancer by aberrant activation of TGF-β/SMAD signaling.
PMCID: PMC4267223  PMID: 23770854
N-Myc interactor; EMT; SMAD; Breast cancer
4.  microRNA-29 negatively regulates EMT regulator N-myc interactor in breast cancer 
Molecular Cancer  2014;13:200.
N-Myc Interactor is an inducible protein whose expression is compromised in advanced stage breast cancer. Downregulation of NMI, a gatekeeper of epithelial phenotype, in breast tumors promotes mesenchymal, invasive and metastatic phenotype of the cancer cells. Thus the mechanisms that regulate expression of NMI are of potential interest for understanding the etiology of breast tumor progression and metastasis.
Web based prediction algorithms were used to identify miRNAs that potentially target the NMI transcript. Luciferase reporter assays and western blot analysis were used to confirm the ability of miR-29 to target NMI. Quantitive-RT-PCRs were used to examine levels of miR29 and NMI from cell line and patient specimen derived RNA. The functional impact of miR-29 on EMT phenotype was evaluated using transwell migration as well as monitoring 3D matrigel growth morphology. Anti-miRs were used to examine effects of reducing miR-29 levels from cells. Western blots were used to examine changes in GSK3β phosphorylation status. The impact on molecular attributes of EMT was evaluated using immunocytochemistry, qRT-PCRs as well as Western blot analyses.
Invasive, mesenchymal-like breast cancer cell lines showed increased levels of miR-29. Introduction of miR-29 into breast cancer cells (with robust level of NMI) resulted in decreased NMI expression and increased invasion, whereas treatment of cells with high miR-29 and low NMI levels with miR-29 antagonists increased NMI expression and decreased invasion. Assessment of 2D and 3D growth morphologies revealed an EMT promoting effect of miR-29. Analysis of mRNA of NMI and miR-29 from patient derived breast cancer tumors showed a strong, inverse relationship between the expression of NMI and the miR-29. Our studies also revealed that in the absence of NMI, miR-29 expression is upregulated due to unrestricted Wnt/β-catenin signaling resulting from inactivation of GSK3β.
Aberrant miR-29 expression may account for reduced NMI expression in breast tumors and mesenchymal phenotype of cancer cells that promotes invasive growth. Reduction in NMI levels has a feed-forward impact on miR-29 levels.
PMCID: PMC4169820  PMID: 25174825
N-Myc interactor; EMT; Breast cancer; miR-29
5.  Towards a Functional Understanding of PGO Waves 
Ponto-Geniculo-Occipital (PGO) waves are biphasic field potentials identified in a range of mammalian species that are ubiquitous with sleep, but can also be identified in waking perception and eye movement. Their role in REM sleep and visual perception more broadly may constitute a promising avenue for further research, however what was once an active field of study has recently fallen into stasis. With the reality that invasive recordings performed on animals cannot be replicated in humans; while animals themselves cannot convey experience to the extent required to elucidate how PGO waves factor into awareness and behavior, innovative solutions are required if significant research outcomes are to ever be realized. Advances in non-invasive imaging technologies and sophistication in imaging methods now offer substantial scope to renew the study of the electrophysiological substrates of waking and dreaming perception. Among these, Magnetoencephalogram (MEG) stands out through its capacity to measure deep brain activations with high temporal resolution. With the current trend in sleep and dream research to produce translational findings of psychopathological and medical significance, in addition to the clear links that PGO wave generation sites share, pharmacologically, with receptors involved in expression of mental illness; there is a strong case to support scientific research into PGO waves and develop a functional understanding of their broader role in human perception.
PMCID: PMC5334507
magnetoencephalogram; rapid eye movements; pontine-geniculate-occipital waves; 5-HT; vision
6.  Bacterial‐induced calcium oscillations are common to nitrogen‐fixing associations of nodulating legumes and non‐legumes 
The New Phytologist  2015;207(3):551-558.
Plants that form root‐nodule symbioses are within a monophyletic ‘nitrogen‐fixing’ clade and associated signalling processes are shared with the arbuscular mycorrhizal symbiosis. Central to symbiotic signalling are nuclear‐associated oscillations in calcium ions (Ca2+), occurring in the root hairs of several legume species in response to the rhizobial Nod factor signal.In this study we expanded the species analysed for activation of Ca2+ oscillations, including non‐leguminous species within the nitrogen‐fixing clade.We showed that Ca2+ oscillations are a common feature of legumes in their association with rhizobia, while Cercis, a non‐nodulating legume, does not show Ca2+ oscillations in response to Nod factors from Sinorhizobium fredii NGR234. Parasponia andersonii, a non‐legume that can associate with rhizobia, showed Nod factor‐induced calcium oscillations to S. fredii NGR234 Nod factors, but its non‐nodulating sister species, Trema tomentosa, did not. Also within the nitrogen‐fixing clade are actinorhizal species that associate with Frankia bacteria and we showed that Alnus glutinosa induces Ca2+ oscillations in root hairs in response to exudates from Frankia alni, but not to S. fredii NGR234 Nod factors.We conclude that the ability to mount Ca2+ oscillations in response to symbiotic bacteria is a common feature of nodulating species within the nitrogen‐fixing clade.
PMCID: PMC4736677  PMID: 26010117
actinorhizal; calcium oscillations; Frankia; legumes; nitrogen‐fixing clade; nodulation; Parasponia; symbiotic signalling
7.  Molecular markers for tolerance of European ash (Fraxinus excelsior) to dieback disease identified using Associative Transcriptomics 
Scientific Reports  2016;6:19335.
Tree disease epidemics are a global problem, impacting food security, biodiversity and national economies. The potential for conservation and breeding in trees is hampered by complex genomes and long lifecycles, with most species lacking genomic resources. The European Ash tree Fraxinus excelsior is being devastated by the fungal pathogen Hymenoscyphus fraxineus, which causes ash dieback disease. Taking this system as an example and utilizing Associative Transcriptomics for the first time in a plant pathology study, we discovered gene sequence and gene expression variants across a genetic diversity panel scored for disease symptoms and identified markers strongly associated with canopy damage in infected trees. Using these markers we predicted phenotypes in a test panel of unrelated trees, successfully identifying individuals with a low level of susceptibility to the disease. Co-expression analysis suggested that pre-priming of defence responses may underlie reduced susceptibility to ash dieback.
PMCID: PMC4725942  PMID: 26757823
8.  Virtual reality and consciousness inference in dreaming 
Frontiers in Psychology  2014;5:1133.
This article explores the notion that the brain is genetically endowed with an innate virtual reality generator that – through experience-dependent plasticity – becomes a generative or predictive model of the world. This model, which is most clearly revealed in rapid eye movement (REM) sleep dreaming, may provide the theater for conscious experience. Functional neuroimaging evidence for brain activations that are time-locked to rapid eye movements (REMs) endorses the view that waking consciousness emerges from REM sleep – and dreaming lays the foundations for waking perception. In this view, the brain is equipped with a virtual model of the world that generates predictions of its sensations. This model is continually updated and entrained by sensory prediction errors in wakefulness to ensure veridical perception, but not in dreaming. In contrast, dreaming plays an essential role in maintaining and enhancing the capacity to model the world by minimizing model complexity and thereby maximizing both statistical and thermodynamic efficiency. This perspective suggests that consciousness corresponds to the embodied process of inference, realized through the generation of virtual realities (in both sleep and wakefulness). In short, our premise or hypothesis is that the waking brain engages with the world to predict the causes of sensations, while in sleep the brain’s generative model is actively refined so that it generates more efficient predictions during waking. We review the evidence in support of this hypothesis – evidence that grounds consciousness in biophysical computations whose neuronal and neurochemical infrastructure has been disclosed by sleep research.
PMCID: PMC4191565  PMID: 25346710
sleep; consciousness; virtual reality; prediction; free energy; rapid eye movements; pontine-geniculate-occipital waves; neuromodulation
9.  Renal Pharmacology of Netilmicin 
Netilmicin (Sch 20569), a new semisynthetic aminoglycoside, was studied for its effects on kidney function and mechanisms by which it is handled by the kidneys. Measurements of glomerular filtration rate (GFR) and urinalysis in chronic rat studies indicated that the nephrotoxicity of netilmicin was remarkably less than that of gentamicin. Gentamicin caused a dose-related reduction in GFR in association with glucosuria and elevated fractional excretion of K+. By contrast, high doses of netilmicin produced only slight reduction in GFR with increased fractional excretion of K+ but without glucosuria. In separate experiments, rats were shown to excrete 71 to 90% of netilmicin or gentamicin in 24 h after daily intramuscular administration of doses of 20 or 40 mg/kg for 4 days. In acute experiments on anesthetized dogs, GFR and renal plasma flow were unaffected at serum levels of 11.0 ± 0.6 μg/ml maintained by constant infusion of netilmicin for 5 h. The renal clearance of netilmicin was significantly correlated with GFR. The urinary output of netilmicin was 80.0 ± 4.2% of the infusion rate and was independent of urine flow over the range of 0.04 to 0.33 ml/kg per min. Preferential accumulation of netilmicin occurred in the renal cortex; the cortex–serum and medulla–serum ratios were 9.9 ± 1.2 and 4.2 ± 0.6, respectively. In addition, the extraction ratio of netilmicin, which was lower than that of inulin, suggested that netilmicin reabsorption occurs in the proximal tubule and results in cortical accumulation. It is concluded that netilmicin, like gentamicin, is excreted by the dog kidney by glomerular filtration plus limited reabsorption. However, the new drug is characterized by low intrinsic nephrotoxicity in rats.
PMCID: PMC352082  PMID: 879737
10.  Multiple roles of the transcription factor AtMYBR1/AtMYB44 in ABA signaling, stress responses, and leaf senescence 
BMC Plant Biology  2013;13:192.
The transcription factor AtMYBR1 (MYB44) is a member of the MYB family of transcription factors and is expressed throughout the plant life cycle and especially in senescing and wounded leaves. It has previously been shown to be involved in responses to abiotic stress and is regulated by phosphorylation.
When MYBR1 was over-expressed under the control of the constitutive 35S promoter in Arabidopsis thaliana (OxMYBR1), leaf senescence was delayed. In contrast loss-of-function mybr1 plants showed more rapid chlorophyll loss and senescence. The MYBR1 promoter strongly drove β-GLUCURONIDASE reporter gene expression in tissues immediately after wounding and many wounding/pathogenesis genes were downregulated in OxMYBR1. OxMYBR1 plants were more susceptible to injury under water stress than wildtype, which was correlated with suppression of many ABA inducible stress genes in OxMYBR1. Conversely, mybr1 plants were more tolerant of water stress and exhibited reduced rates of water loss from leaves. MYBR1 physically interacted with ABA receptor PYR1-LIKE8 (PYL8) suggesting a direct involvement of MYBR1 in early ABA signaling. MYBR1 appears to exhibit partially redundant functions with AtMYBR2 (MYB77) and double mybr1 X mybr2 mutants exhibited stronger senescence and stress related phenotypes than single mybr1 and mybr2 mutants.
MYBR1 is a negative regulator of ABA, stress, wounding responses and blocks senescence. It appears to have a homeostatic function to maintain growth processes in the event of physical damage or stress.
PMCID: PMC4219380  PMID: 24286353
ABA; Drought stress; Transcription factor; PYL8; Senescence
11.  MicroRNAs and their putative targets in Brassica napus seed maturation 
BMC Genomics  2013;14:140.
MicroRNAs (miRNAs) are 20–21 nucleotide RNA molecules that suppress the transcription of target genes and may also inhibit translation. Despite the thousands of miRNAs identified and validated in numerous plant species, only small numbers have been identified from the oilseed crop plant Brassica napus (canola) – especially in seeds.
Using next-generation sequencing technologies, we performed a comprehensive analysis of miRNAs during seed maturation at 9 time points from 10 days after flowering (DAF) to 50 DAF using whole seeds and included separate analyses of radicle, hypocotyl, cotyledon, embryo, endosperm and seed coat tissues at 4 selected time points. We identified more than 500 conserved miRNA or variant unique sequences with >300 sequence reads and also found 10 novel miRNAs. Only 27 of the conserved miRNA sequences had been previously identified in B. napus (miRBase Release 18). More than 180 MIRNA loci were identified/annotated using the B. rapa genome as a surrogate for the B.napus A genome. Numerous miRNAs were expressed in a stage- or tissue-specific manner suggesting that they have specific functions related to the fine tuning of transcript abundance during seed development. miRNA targets in B. napus were predicted and their expression patterns profiled using microarray analyses. Global correlation analysis of the expression patterns of miRNAs and their targets revealed complex miRNA-target gene regulatory networks during seed development. The miR156 family was the most abundant and the majority of the family members were primarily expressed in the embryo.
Large numbers of miRNAs with diverse expression patterns, multiple-targeting and co-targeting of many miRNAs, and complex relationships between expression of miRNAs and targets were identified in this study. Several key miRNA-target expression patterns were identified and new roles of miRNAs in regulating seed development are suggested. miR156, miR159, miR172, miR167, miR158 and miR166 are the major contributors to the network controlling seed development and maturation through their pivotal roles in plant development. miR156 may regulate the developmental transition to germination.
PMCID: PMC3602245  PMID: 23448243
Seed development; Embryo; Next generation sequencing
12.  Loss of Tumor Suppressor Merlin in Advanced Breast Cancer Is due to Post-translational Regulation* 
The Journal of Biological Chemistry  2011;286(46):40376-40385.
Background: The role of Merlin in breast cancer is unknown.
Results: Merlin protein is degraded in advanced breast cancer due to osteopontin-initiated signaling.
Conclusion: Merlin is regulated at the post-translational level in breast tumors.
Significance: We have defined a functional role for Merlin in limiting breast tumor growth and elucidated the utility of Merlin as an important biomarker in breast cancer.
Unlike malignancies of the nervous system, there have been no mutations identified in Merlin in breast cancer. As such, the role of the tumor suppressor, Merlin, has not been investigated in breast cancer. We assessed Merlin expression in breast cancer tissues by immunohistochemistry and by real-time PCR. The expression of Merlin protein (assessed immunohistochemically) was significantly decreased in breast cancer tissues (although the transcript levels were comparable) simultaneous with increased expression of the tumor-promoting protein, osteopontin (OPN). We further demonstrate that the loss of Merlin in breast cancer is brought about, in part, due to OPN-initiated Akt-mediated phosphorylation of Merlin leading to its proteasomal degradation. Restoring expression of Merlin resulted in reduced malignant attributes of breast cancer, characterized by reduced invasion, migration, motility, and impeded tumor (xenograft) growth in immunocompromised mice. The possibility of developing a model using the relationship between OPN and Merlin was tested with a logistic regression model applied to immunohistochemistry data. This identified consistent loss of immunohistochemical expression of Merlin in breast tumor tissues. Thus, we demonstrate for the first time a role for Merlin in impeding breast malignancy, identify a novel mechanism for the loss of Merlin protein in breast cancer, and have developed a discriminatory model using Merlin and OPN expression in breast tumor tissues.
PMCID: PMC3220570  PMID: 21965655
Akt PKB; Breast Cancer; Protein Degradation; Tumor Promoter; Tumor Suppressor Gene; Merlin; NF2; Osteopontin; Post-translational Regulation; Ubiquitination
13.  Nonlinear Time Series Analysis of Nodulation Factor Induced Calcium Oscillations: Evidence for Deterministic Chaos? 
PLoS ONE  2009;4(8):e6637.
Legume plants form beneficial symbiotic interactions with nitrogen fixing bacteria (called rhizobia), with the rhizobia being accommodated in unique structures on the roots of the host plant. The legume/rhizobial symbiosis is responsible for a significant proportion of the global biologically available nitrogen. The initiation of this symbiosis is governed by a characteristic calcium oscillation within the plant root hair cells and this signal is activated by the rhizobia. Recent analyses on calcium time series data have suggested that stochastic effects have a large role to play in defining the nature of the oscillations. The use of multiple nonlinear time series techniques, however, suggests an alternative interpretation, namely deterministic chaos. We provide an extensive, nonlinear time series analysis on the nature of this calcium oscillation response. We build up evidence through a series of techniques that test for determinism, quantify linear and nonlinear components, and measure the local divergence of the system. Chaos is common in nature and it seems plausible that properties of chaotic dynamics might be exploited by biological systems to control processes within the cell. Systems possessing chaotic control mechanisms are more robust in the sense that the enhanced flexibility allows more rapid response to environmental changes with less energetic costs. The desired behaviour could be most efficiently targeted in this manner, supporting some intriguing speculations about nonlinear mechanisms in biological signaling.
PMCID: PMC2722092  PMID: 19675679
16.  A Common Genomic Framework for a Diverse Assembly of Plasmids in the Symbiotic Nitrogen Fixing Bacteria 
PLoS ONE  2008;3(7):e2567.
This work centres on the genomic comparisons of two closely-related nitrogen-fixing symbiotic bacteria, Rhizobium leguminosarum biovar viciae 3841 and Rhizobium etli CFN42. These strains maintain a stable genomic core that is also common to other rhizobia species plus a very variable and significant accessory component. The chromosomes are highly syntenic, whereas plasmids are related by fewer syntenic blocks and have mosaic structures. The pairs of plasmids p42f-pRL12, p42e-pRL11 and p42b-pRL9 as well large parts of p42c with pRL10 are shown to be similar, whereas the symbiotic plasmids (p42d and pRL10) are structurally unrelated and seem to follow distinct evolutionary paths. Even though purifying selection is acting on the whole genome, the accessory component is evolving more rapidly. This component is constituted largely for proteins for transport of diverse metabolites and elements of external origin. The present analysis allows us to conclude that a heterogeneous and quickly diversifying group of plasmids co-exists in a common genomic framework.
PMCID: PMC2434198  PMID: 18596979
17.  From synapse to gene product: Prolonged expression of c-fos induced by a single microinjection of carbachol in the pontomesencephalic tegmentum 
It is not known how the brain modifies its regulatory systems in response to the application of a drug, especially over the long term of weeks and months. We have developed a model system approach to this question by manipulating cholinergic cell groups of the laterodorsal and pedunculopontine tegmental (LDT/PPT) nuclei in the pontomesencephalic tegmentum (PMT), which are known to be actively involved in the timing and quantity of rapid eye movement (REM) sleep. In a freely moving feline model, a single microinjection of the cholinergic agonist carbachol conjugated to a latex nanosphere delivery system into the caudolateral PMT elicits a long-term enhancement of one distinguishing phasic event of REM sleep, ponto-geniculo-occipital (PGO) waves, lasting 5 days but without any significant change in REM sleep or other behavioral state. Here, we test the hypothesis that cholinergic activation within the caudolateral PMT alters the postsynaptic excitability of the PGO network, stimulating the prolonged expression of c-fos that underlies this long-term PGO enhancement (LTPE) effect. Using quantitative Fos immunohistochemistry, we found that the number of Fos-immunoreactive (Fos-IR) neurons surrounding the caudolateral PMT injection site decreased sharply by postcarbachol day 03, while the number of Fos-IR neurons in the more rostral LDT/PPT increased >30-fold and remained at a high level following the course of LTPE. These results demonstrate a sustained c-fos expression in response to pharmacological stimulation of the brain and suggest that carbachol's acute effects induce LTPE via cholinergic receptors, with subsequent transsynaptic activation of the LDT/PPT maintaining the LTPE effect.
PMCID: PMC1570022  PMID: 15893601
Immediate early gene; c-fos; Carbachol nanospheres; Brainstem; Ponto-geniculo-occipital waves; Sleep
18.  Protein domains and architectural innovation in plant-associated Proteobacteria 
BMC Genomics  2005;6:17.
Evolution of new complex biological behaviour tends to arise by novel combinations of existing building blocks. The functional and evolutionary building blocks of the proteome are protein domains, the function of a protein being dependent on its constituent domains. We clustered completely-sequenced proteomes of prokaryotes on the basis of their protein domain content, as defined by Pfam (release 16.0). This revealed that, although there was a correlation between phylogeny and domain content, other factors also have an influence. This observation motivated an investigation of the relationship between an organism's lifestyle and the complement of domains and domain architectures found within its proteome.
We took a census of all protein domains and domain combinations (architectures) encoded in the completely-sequenced proteobacterial genomes. Nine protein domain families were identified that are found in phylogenetically disparate plant-associated bacteria but are absent from non-plant-associated bacteria. Most of these are known to play a role in the plant-associated lifestyle, but they also included domain of unknown function DUF1427, which is found in plant symbionts and pathogens of the alpha-, beta- and gamma-Proteobacteria, but not known in any other organism. Further, several domains were identified as being restricted to phytobacteria and Eukaryotes. One example is the RolB/RolC glucosidase family, which is found only in Agrobacterium species and in plants. We identified the 0.5% of Pfam protein domain families that were most significantly over-represented in the plant-associated Proteobacteria with respect to the background frequencies in the whole set of available proteobacterial proteomes. These included guanylate cyclase, domains implicated in aromatic catabolism, cellulase and several domains of unknown function.
We identified 459 unique domain architectures found in phylogenetically diverse plant pathogens and symbionts that were absent from non-pathogenic and non-symbiotic relatives. The vast majority of these were restricted to a single species or several closely related species and so their distributions could be better explained by phylogeny than by lifestyle. However, several architectures were found in two or more very distantly related phytobacteria but absent from non-plant-associated bacteria. Many of the proteins with these unique architectures are predicted to be secreted.
In Pseudomonas syringae pathovar tomato, those genes encoding genes with novel domain architectures tended to have atypical GC contents and were adjacent to insertion sequence elements and phage-like sequences, suggesting acquisition by horizontal transfer.
By identifying domains and architectures unique to plant pathogens and symbionts, we highlighted candidate proteins for involvement in plant-associated bacterial lifestyles. Given that characterisation of novel gene products in vivo and in vitro is time-consuming and expensive, this computational approach may be useful for reducing experimental search space. Furthermore we discuss the biological significance of novel proteins highlighted by this study in the context of plant-associated lifestyles.
PMCID: PMC554113  PMID: 15715905
19.  Chemotherapeutic Evaluation of 5-Episisomicin (Sch 22591), a New Semisynthetic Aminoglycoside 
5-episisomicin (Sch 22591) is a novel semisynthetic aminoglycoside with a spectrum and potency similar to gentamicin in its activity against susceptible bacterial strains, but with increased potency against Pseudomonas, Providencia, and Proteus rettgeri. It is also more active than tobramycin and amikacin against these last-mentioned species.
Against resistant strains, Sch 22591 is significantly more active than gentamicin or tobramycin. Against resistant gram-negative bacteria other than Pseudomonas, Sch 22591 has activity similar to that of amikacin, but Sch 22591 is more potent. Against Pseudomonas strains, it is active against most gentamicin- and tobramycin-resistant strains and is more active than the other three antibiotics. Some Pseudomonas strains are resistant to Sch 22591, but susceptible to amikacin. Against a selection of aminoglycoside-resistant staphylococci, Sch 22591 has very good activity against strains resistant to tobramycin, amikacin, and gentamicin. The superior in vitro potency of Sch 22591 against Pseudomonas has been confirmed in vivo in experimental infections in mice. Absorption in dogs is similar to that of other aminoglycoside antibiotics. The acute toxicity of Sch 22591 in mice is greater than that of gentamicin; its vestibular toxicity potential and nephrotoxicity potential in cats and rats appear to be similar to those of gentamicin.
PMCID: PMC352182  PMID: 626490
20.  Biological Activity of Netilmicin, a Broad-Spectrum Semisynthetic Aminoglycoside Antibiotic 
Netilmicin (Sch 20569) is a new broad-spectrum semisynthetic aminoglycoside derived from sisomicin. Netilmicin was compared to gentamicin, tobramycin, and amikacin in a variety of in vitro test systems as well as in mouse protection tests. Netilmicin was found to be similar in activity to gentamicin against aminoglycoside-susceptible strains in both in vitro and in vivo tests. Netilmicin was also active against many aminoglycoside-resistant strains of gram-negative bacteria, particularly those known to possess adenylating enzymes (ANT 2′) or those with a similar resistance pattern. Netilmicin was found to be markedly less toxic than gentamicin in chronic studies in cats, although gentamicin appeared less toxic in acute toxicity tests in mice. The concentrations of netilmicin and gentamicin in serum were compared in dogs after intramuscular dosing, and the pharmacokinetics including peak concentrations in serum were found to be similar.
PMCID: PMC429844  PMID: 1008541
21.  Comparative Activity of Sisomicin, Gentamicin, Kanamycin, and Tobramycin 
Gentamicin, sisomicin, tobramycin, and kanamycin were compared in parallel tests in vitro and in vivo against a variety of bacterial strains and species. A number of differences were seen in vitro, in particular: (i) the lower activity of kanamycin, (ii) the greater activity of tobramycin against Pseudomonas, (iii) the greater activity of gentamicin and sisomicin against Serratia, and (iv) the generally similar results with tobramycin, gentamicin, and sisomicin against species other than Pseudomonas and Serratia, with the ranking in order of decreasing activity being sisomicin, gentamicin, and tobramycin. Analysis of disc test results suggested that the gentamicin disc is not adequate for testing the susceptibility of all bacteria to sisomicin or tobramycin. In vivo tests did not confirm all specifics of in vitro tests; results of in vivo tests indicated that sisomicin may be the most active. It is suggested that the place of each of the antibiotics in human therapy can best be evaluated by more rigorous in vivo tests and clinical studies rather than extensive in vitro comparisons.
PMCID: PMC444335  PMID: 4670437
22.  Chemotherapeutic Evaluation of Clotrimazole [Bay b 5097, 1 (o-Chloro-α-α-Diphenylbenzyl) Imidazole] 1 
Applied Microbiology  1971;22(5):891-898.
Clotrimazole has a broad spectrum of activity against yeast and filamentous fungi in vitro and also in vivo when given orally or parenterally to experimentally infected mice and when administered orally or topically to infected guinea pigs. In vitro a distinct inoculum effect has been observed with a number of strains of Candida and Torulopsis; minimal inhibitory concentrations have tended to increase with increased incubation time. With prolonged incubation times, resistance can be developed to clotrimazole in vitro, but this resistance is readily reversible upon passage in drug-free broth. The degree of in vivo activity of clotrimazole against Candida depends on the severity of infection used. Orally it appears to be more effective when administered by gavage than when given mixed in the diet. Pretreatment with the agent may decrease its efficacy because of drug inactivation. Against dermatophytes, clotrimazole has a degree of activity similar to griseofulvin when given orally, but it is less active than tolnaftate topically in cutaneous infection of Trichophyton mentagrophytes in guinea pigs. In vitro, but not in vivo, some gram-positive and gram-negative bacteria are inhibited by low concentrations of clotrimazole.
PMCID: PMC376440  PMID: 4943591
23.  Ash dieback epidemic in Europe: How can molecular technologies help? 
PLoS Pathogens  2017;13(7):e1006381.
PMCID: PMC5519178  PMID: 28727852
24.  Manganese transport is essential for N2‐fixation by Rhizobium leguminosarum in bacteroids from galegoid but not phaseoloid nodules 
Environmental Microbiology  2017;19(7):2715-2726.
Rhizobium leguminosarum has two high‐affinity Mn2+ transport systems encoded by sitABCD and mntH. In symbiosis, sitABCD and mntH were expressed throughout nodules and also strongly induced in Mn2+‐limited cultures of free‐living cells. Growth of a sitA mntH double mutant was severely reduced under Mn2+ limitation and sitA and mntH single mutants were more sensitive to oxidative stress. The double sitA mntH mutant of R. leguminosarum was unable to fix nitrogen (Fix‐) with legumes belonging to the galegoid clade (Pisum sativum, Vicia faba and Vicia hirsuta). The presence of infection thread‐like structures and sparsely‐packed plant cells in nodules suggest that bacteroid development was blocked, either at a late stage of infection thread progression or during bacteroid‐release. In contrast, a double sitA mntH mutant was Fix+ on common bean (Phaseoli vulgaris), a member of the phaseoloid clade of legumes, indicating a host‐specific symbiotic requirement for Mn2+ transport.
PMCID: PMC5575495  PMID: 28447383
25.  Loss of Merlin induces metabolomic adaptation that engages dependence on Hedgehog signaling 
Scientific Reports  2017;7:40773.
The tumor suppressor protein Merlin is proteasomally degraded in breast cancer. We undertook an untargeted metabolomics approach to discern the global metabolomics profile impacted by Merlin in breast cancer cells. We discerned specific changes in glutathione metabolites that uncovered novel facets of Merlin in impacting the cancer cell metabolome. Concordantly, Merlin loss increased oxidative stress causing aberrant activation of Hedgehog signaling. Abrogation of GLI-mediated transcription activity compromised the aggressive phenotype of Merlin-deficient cells indicating a clear dependence of cells on Hedgehog signaling. In breast tumor tissues, GLI1 expression enhanced tissue identification and discriminatory power of Merlin, cumulatively presenting a powerful substantiation of the relationship between these two proteins. We have uncovered, for the first time, details of the tumor cell metabolomic portrait modulated by Merlin, leading to activation of Hedgehog signaling. Importantly, inhibition of Hedgehog signaling offers an avenue to target the vulnerability of tumor cells with loss of Merlin.
PMCID: PMC5256100  PMID: 28112165

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