Fluoromycobacteriophages for rapid, specific, and sensitive antibiotic susceptibility testing of Mycobacterium tuberculosis

Rapid antibiotic susceptibility testing of Mycobacterium tuberculosis is of paramount importance as multiple- and extensively- drug resistant strains of M. tuberculosis emerge and spread. We describe here a virus-based assay in which fluoromycobacteriophages are used to deliver a GFP or ZsYellow fluorescent marker gene to M. tuberculosis, which can then be monitored by fluorescent detection approaches including fluorescent microscopy and flow cytometry. Pre-clinical evaluations show that addition of either Rifampicin or Streptomycin at the time of phage addition obliterates fluorescence in susceptible cells but not in isogenic resistant bacteria enabling drug sensitivity determination in less than 24 hours. Detection requires no substrate addition, fewer than 100 cells can be identified, and resistant bacteria can be detected within mixed populations. Fluorescence withstands fixation by paraformaldehyde providing enhanced biosafety for testing MDR-TB and XDR-TB infections. © 2009 Piuri et al

Spirituality and end-of-life care in disadvantaged men dying of prostate cancer

Despite the positive influence of spiritual coping on the acceptance of a cancer diagnosis, higher spirituality is associated with receipt of more high intensity care at the end of life. The purpose of our study was to assess the association between spirituality and type of end-of-life care received by disadvantaged men with prostate cancer. We studied low-income, uninsured men in IMPACT, a state-funded public assistance program, who had died since its inception in 2001. Of the 60 men who died, we included the 35 who completed a spirituality questionnaire at program enrollment. We abstracted sociodemographic and clinical information as well as treatment within IMPACT, including zolendroic acid, chemotherapy, hospice use, and palliative radiation therapy. We measured spirituality with the Functional Assessment of Chronic Illness Therapy—Spiritual Well-Being questionnaire (FACIT-Sp) and compared end-of-life care received between subjects with low and high FACIT-Sp scores using chi-squared analyses. A higher proportion of men with high (33%) versus low (13%) spirituality scores enrolled in hospice, although our analysis was not adequately powered to demonstrate statistical significance. Likewise, we saw a trend toward increased receipt of palliative radiation among those with higher spirituality (37% vs. 25%, P = 0.69). The differences in end-of-life care received among those with low and high spirituality varied little by the FACIT-Sp peace and faith subscales. End-of-life care was similar between men with lower and higher spirituality. Men with higher spirituality trended toward greater hospice use, suggesting that they redirected the focus of their care from curative to palliative goals

Determining the neurotransmitter concentration profile at active synapses

Establishing the temporal and concentration profiles of neurotransmitters during synaptic release is an essential step towards understanding the basic properties of inter-neuronal communication in the central nervous system. A variety of ingenious attempts has been made to gain insights into this process, but the general inaccessibility of central synapses, intrinsic limitations of the techniques used, and natural variety of different synaptic environments have hindered a comprehensive description of this fundamental phenomenon. Here, we describe a number of experimental and theoretical findings that has been instrumental for advancing our knowledge of various features of neurotransmitter release, as well as newly developed tools that could overcome some limits of traditional pharmacological approaches and bring new impetus to the description of the complex mechanisms of synaptic transmission

Subcellular peptide localization in single identified neurons by capillary microsampling mass spectrometry

Single cell mass spectrometry (MS) is uniquely positioned for the sequencing and identification of peptides in rare cells. Small peptides can take on different roles in subcellular compartments. Whereas some peptides serve as neurotransmitters in the cytoplasm, they can also function as transcription factors in the nucleus. Thus, there is a need to analyze the subcellular peptide compositions in identified single cells. Here, we apply capillary microsampling MS with ion mobility separation for the sequencing of peptides in single neurons of the mollusk Lymnaea stagnalis, and the analysis of peptide distributions between the cytoplasm and nucleus of identified single neurons that are known to express cardioactive Phe-Met-Arg-Phe amide-like (FMRFamide-like) neuropeptides. Nuclei and cytoplasm of Type 1 and Type 2 F group (Fgp) neurons were analyzed for neuropeptides cleaved from the protein precursors encoded by alternative splicing products of the FMRFamide gene. Relative abundances of nine neuropeptides were determined in the cytoplasm. The nuclei contained six of these peptides at different abundances. Enabled by its relative enrichment in Fgp neurons, a new 28-residue neuropeptide was sequenced by tandem MS

Threat captures attention, but not automatically: Top-down goals modulate attentional orienting to threat distractors

The rapid orienting of attention to potential threats has been proposed to proceed outside of top-down control. However, paradigms that have been used to investigate this have struggled to separate the rapid orienting of attention (i.e. capture) from the later disengagement of focal attention that may be subject to top-down control. Consequently, it remains unclear whether and to what extent orienting to threat is contingent on top-down goals. The current study manipulated the goal-relevance of threat distractors (spiders), whilst a strict top-down attentional set was encouraged by presenting the saliently colored target and the threat distracter simultaneously for a limited time. The goal-relevance of threatening distractors was manipulated by including a spider amongst the possible target stimuli (Experiment 1: spider/cat targets) or excluding it (Experiment 2: bird/fish targets). Orienting and disengagement were disentangled by cueing attention away from or towards the threat prior to its onset. The results indicated that the threatening spider distractors elicited rapid orienting of attention when spiders were potentially goal-relevant (Experiment 1) but did so much less when they were irrelevant to the task goal (Experiment 2). Delayed disengagement from the threat distractors was even more strongly contingent on the task goal and occurred only when a spider was a possible target. These results highlight the role of top-down goals in attentional orienting to and disengagement from threat. © 2016 The Psychonomic Society, Inc

Differential Requirement for Utrophin in the Induced Pluripotent Stem Cell Correction of Muscle versus Fat in Muscular Dystrophy Mice

Duchenne muscular dystrophy (DMD) is an incurable degenerative muscle disorder. We injected WT mouse induced pluripotent stem cells (iPSCs) into mdx and mdx∶utrophin mutant blastocysts, which are predisposed to develop DMD with an increasing degree of severity (mdx <<< mdx∶utrophin). In mdx chimeras, iPSC-dystrophin was supplied to the muscle sarcolemma to effect corrections at morphological and functional levels. Dystrobrevin was observed in dystrophin-positive and, at a lesser extent, utrophin-positive areas. In the mdx∶utrophin mutant chimeras, although iPSC-dystrophin was also supplied to the muscle sarcolemma, mice still displayed poor skeletal muscle histopathology, and negligible levels of dystrobrevin in dystrophin- and utrophin-negative areas. Not only dystrophin-expressing tissues are affected by iPSCs. Mdx and mdx∶utrophin mice have reduced fat/body weight ratio, but iPSC injection normalized this parameter in both mdx and mdx∶utrophin chimeras, despite the fact that utrophin was compromised in the mdx∶utrophin chimeric fat. The results suggest that the presence of utrophin is required for the iPSC-corrections in skeletal muscle. Furthermore, the results highlight a potential (utrophin-independent) non-cell autonomous role for iPSC-dystrophin in the corrections of non-muscle tissue like fat, which is intimately related to the muscle

Adult-Age Inflammatory Pain Experience Enhances Long-Term Pain Vigilance in Rats

Background: Previous animal studies have illustrated a modulatory effect of neonatal pain experience on subsequent painrelated behaviors. However, the relationship between chronic pain status in adulthood and future pain perception remains unclear. Methodology/Principal Findings: In the current study, we investigated the effects of inflammatory pain experience on subsequent formalin-evoked pain behaviors and fear conditioning induced by noxious stimulation in adult rats. Our results demonstrated an increase of the second but not the first phase of formalin-induced pain behaviors in animals with a history of inflammatory pain that have recovered. Similarly, rats with persistent pain experience displayed facilitated acquisition and prolonged retention of pain-related conditioning. These effects of prior pain experience on subsequent behavior were prevented by repeated morphine administration at an early stage of inflammatory pain. Conclusions/Significance: These results suggest that chronic pain diseases, if not properly and promptly treated, may have a long-lasting impact on processing and perception of environmental threats. This may increase the susceptibility of patients to subsequent pain-related disorders, even when chronic pain develops in adulthood. These data highlight th

Group membership and racial bias modulate the temporal estimation of in-group/out-group body movements

Social group categorization has been mainly studied in relation to ownership manipulations involving highly-salient multisensory cues. Here, we propose a novel paradigm that can implicitly activate the embodiment process in the presence of group affiliation information, whilst participants complete a task irrelevant to social categorization. Ethnically White participants watched videos of White- and Black-skinned models writing a proverb. The writing was interrupted 7, 4 or 1 s before completion. Participants were tasked with estimating the residual duration following interruption. A video showing only hand kinematic traces acted as a control condition. Residual duration estimates for out-group and control videos were significantly lower than those for in-group videos only for the longest duration. Moreover, stronger implicit racial bias was negatively correlated to estimates of residual duration for out-group videos. The underestimation bias for the out-group condition might be mediated by implicit embodiment, affective and attentional processes, and finalized to a rapid out-group categorization

Variability of Female Responses to Conspecific vs. Heterospecific Male Mating Calls in Polygynous Deer: An Open Door to Hybridization?

Males of all polygynous deer species (Cervinae) give conspicuous calls during the reproductive season. The extreme interspecific diversity that characterizes these vocalizations suggests that they play a strong role in species discrimination. However, interbreeding between several species of Cervinae indicates permeable interspecific reproductive barriers. This study examines the contribution of vocal behavior to female species discrimination and mating preferences in two closely related polygynous deer species known to hybridize in the wild after introductions. Specifically, we investigate the reaction of estrous female red deer (Cervus elaphus) to playbacks of red deer vs. sika deer (Cervus nippon) male mating calls, with the prediction that females will prefer conspecific calls. While on average female red deer preferred male red deer roars, two out of twenty females spent more time in close proximity to the speaker broadcasting male sika deer moans. We suggest that this absence of strict vocal preference for species-specific mating calls may contribute to the permeability of pre-zygotic reproductive barriers observed between these species. Our results also highlight the importance of examining inter-individual variation when studying the role of female preferences in species discrimination and intraspecific mate selection

Streptococcus iniae M-Like Protein Contributes to Virulence in Fish and Is a Target for Live Attenuated Vaccine Development

Streptococcus iniae is a significant pathogen in finfish aquaculture, though knowledge of virulence determinants is lacking. Through pyrosequencing of the S. iniae genome we have identified two gene homologues to classical surface-anchored streptococcal virulence factors: M-like protein (simA) and C5a peptidase (scpI).S. iniae possesses a Mga-like locus containing simA and a divergently transcribed putative mga-like regulatory gene, mgx. In contrast to the Mga locus of group A Streptococcus (GAS, S. pyogenes), scpI is located distally in the chromosome. Comparative sequence analysis of the Mgx locus revealed only one significant variant, a strain with an insertion frameshift mutation in simA and a deletion mutation in a region downstream of mgx, generating an ORF which may encode a second putative mga-like gene, mgx2. Allelic exchange mutagenesis of simA and scpI was employed to investigate the potential role of these genes in S. iniae virulence. Our hybrid striped bass (HSB) and zebrafish models of infection revealed that M-like protein contributes significantly to S. iniae pathogenesis whereas C5a peptidase-like protein does not. Further, in vitro cell-based analyses indicate that SiMA, like other M family proteins, contributes to cellular adherence and invasion and provides resistance to phagocytic killing. Attenuation in our virulence models was also observed in the S. iniae isolate possessing a natural simA mutation. Vaccination of HSB with the Delta simA mutant provided 100% protection against subsequent challenge with a lethal dose of wild-type (WT) S. iniae after 1,400 degree days, and shows promise as a target for live attenuated vaccine development.Analysis of M-like protein and C5a peptidase through allelic replacement revealed that M-like protein plays a significant role in S. iniae virulence, and the Mga-like locus, which may regulate expression of this gene, has an unusual arrangement. The M-like protein mutant created in this research holds promise as live-attenuated vaccine