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1.  The CLO3403/CLO3404 Two-Component System of Clostridium botulinum E1 Beluga Is Important for Cold Shock Response and Growth at Low Temperatures 
In order to survive a temperature downshift, bacteria have to sense the changing environment and adjust their metabolism and structure. Two-component signal transduction systems (TCSs) play a central role in sensing and responding to many different environmental stimuli. Although the nonproteolytic (group II) Clostridium botulinum represents a major hazard in chilled foods, the cold adaption mechanisms of group II C. botulinum organisms are not known. Here, we show that the CLO3403/CLO3404 TCS of C. botulinum E1 Beluga is involved in the cold shock response and growth at 12°C. Cold shock induced the expression of the genes encoding the histidine kinase (clo3403) and the response regulator (clo3404) by more than 100-fold after 5 h relative to their expression in a nonshocked culture at the corresponding time point. The involvement of CLO3403/CLO3404 in growth at low temperature was demonstrated by impaired growth of the insertional clo3403 and clo3404 knockout mutants at 12°C compared to the growth of the wild-type culture. Additionally, the inactivation of clo3403 had a negative effect on motility. The growth efficiency at 12°C of the TCS mutants and the motility of the kinase mutants were restored by introducing a plasmid harboring the operon of the CLO3403/CLO3404 TCS. The results suggest that the CLO3403/CLO3404 TCS is important for the cold tolerance of C. botulinum E1 Beluga.
doi:10.1128/AEM.03204-13
PMCID: PMC3911019  PMID: 24185852
2.  Alternative Sigma Factor SigK Has a Role in Stress Tolerance of Group I Clostridium botulinum Strain ATCC 3502 
Applied and Environmental Microbiology  2013;79(12):3867-3869.
The role of the alternative sigma factor SigK in cold and osmotic stress tolerance of Clostridium botulinum ATCC 3502 was demonstrated by induction of sigK after temperature downshift and exposure to hyperosmotic conditions and by impaired growth of the sigK mutants under the respective conditions.
doi:10.1128/AEM.04036-12
PMCID: PMC3675920  PMID: 23563953
3.  Mathematical Model for Radial Expansion and Conflation of Intratumoral Infectious Centers Predicts Curative Oncolytic Virotherapy Parameters 
PLoS ONE  2013;8(9):e73759.
Simple, inductive mathematical models of oncolytic virotherapy are needed to guide protocol design and improve treatment outcomes. Analysis of plasmacytomas regressing after a single intravenous dose of oncolytic vesicular stomatitis virus in myeloma animal models revealed that intratumoral virus spread was spatially constrained, occurring almost exclusively through radial expansion of randomly distributed infectious centers. From these experimental observations we developed a simple model to calculate the probability of survival for any cell within a treated tumor. The model predicted that small changes to the density of initially infected cells or to the average maximum radius of infected centers would have a major impact on treatment outcome, and this was confirmed experimentally. The new model provides a useful and flexible tool for virotherapy protocol optimization.
doi:10.1371/journal.pone.0073759
PMCID: PMC3770695  PMID: 24040057
4.  Whole-body scanning PCR; a highly sensitive method to study the biodistribution of mRNAs, noncoding RNAs and therapeutic oligonucleotides 
Nucleic Acids Research  2013;41(15):e145.
Efficient tissue-specific delivery is a crucial factor in the successful development of therapeutic oligonucleotides. Screening for novel delivery methods with unique tissue-homing properties requires a rapid, sensitive, flexible and unbiased technique able to visualize the in vivo biodistribution of these oligonucleotides. Here, we present whole body scanning PCR, a platform that relies on the local extraction of tissues from a mouse whole body section followed by the conversion of target-specific qPCR signals into an image. This platform was designed to be compatible with a novel RT-qPCR assay for the detection of siRNAs and with an assay suitable for the detection of heavily chemically modified oligonucleotides, which we termed Chemical-Ligation qPCR (CL-qPCR). In addition to this, the platform can also be used to investigate the global expression of endogenous mRNAs and non-coding RNAs. Incorporation of other detection systems, such as aptamers, could even further expand the use of this technology.
doi:10.1093/nar/gkt515
PMCID: PMC3753639  PMID: 23766292
5.  Involvement of Clostridium botulinum ATCC 3502 Sigma Factor K in Early-Stage Sporulation 
Applied and Environmental Microbiology  2012;78(13):4590-4596.
A key survival mechanism of Clostridium botulinum, the notorious neurotoxic food pathogen, is the ability to form heat-resistant spores. While the genetic mechanisms of sporulation are well understood in the model organism Bacillus subtilis, nothing is known about these mechanisms in C. botulinum. Using the ClosTron gene-knockout tool, sigK, encoding late-stage (stage IV) sporulation sigma factor K in B. subtilis, was disrupted in C. botulinum ATCC 3502 to produce two different mutants with distinct insertion sites and orientations. Both mutants were unable to form spores, and their elongated cell morphology suggested that the sporulation pathway was blocked at an early stage. In contrast, sigK-complemented mutants sporulated successfully. Quantitative real-time PCR analysis of sigK in the parent strain revealed expression at the late log growth phase in the parent strain. Analysis of spo0A, encoding the sporulation master switch, in the sigK mutant and the parent showed significantly reduced relative levels of spo0A expression in the sigK mutant compared to the parent strain. Similarly, sigF showed significantly lower relative transcription levels in the sigK mutant than the parent strain, suggesting that the sporulation pathway was blocked in the sigK mutant at an early stage. We conclude that σK is essential for early-stage sporulation in C. botulinum ATCC 3502, rather than being involved in late-stage sporulation, as reported for the sporulation model organism B. subtilis. Understanding the sporulation mechanism of C. botulinum provides keys to control the public health risks that the spores of this dangerous pathogen cause through foods.
doi:10.1128/AEM.00304-12
PMCID: PMC3370484  PMID: 22544236
6.  IMMEDIATE EFFECTS OF LOCALIZED VIBRATION ON HAMSTRING AND QUADRICEP MUSCLE PERFORMANCE 
Purpose/Background:
A reduction in the maximal force output of muscles following pre-performance stretching has been reported. Several studies have suggested that localized vibration may enhance or replace stretching for gaining flexibility. It is important to know if localized vibration may also compromise muscle output. The purpose of this investigation was to determine the immediate effects of localized hamstring vibration on hamstrings (HAM) and quadriceps (QUAD) performance.
Methods:
Thirty asymptomatic participants, 19 female and 11 male, mean age 25.4 years (±SD 2.7) received either five minutes of localized vibration to the right hamstrings at 30 Hz and 6 mm amplitude, or sham. One week later, each participant received the alternate treatment. Following treatments, right (R) and left (L) isometric HAM and QUAD strength was measured twice by handheld dynamometer and maximal horizontal hop distance of each lower extremity was measured by single leg hop test (SLH). Treatment outcomes were compared using paired t-tests. Treatment order effect was measured by independent T-test. Pre-study intrarater reliability for dynamometry was established using ICC(3,2).
Results:
Mean (±SD) values for strength following vibration were 58.7 kg (15.7), 60.4 kg (14.0), 45.5 kg (14.2), 45.8 kg (13.2) for R QUAD, L QUAD, R HAM, L HAM respectively. SLH mean values were R SLH 153.8 cm (35 cm) and L SLH 155.4 cm (36 cm). There were no significant differences in means between vibration and sham treatment for any outcomes on either leg (p-values ranged .412-.971); p<.001 for all comparisons. Order had no significant effect (p-values .370–1.0). Intrarater ICCs were .888, .762, .884, .960 for R HAM, L HAM, R QUAD, L QUAD.
Conclusions:
Unilateral application of localized vibration to the hamstrings at a duration previously reported to increase flexibility did not diminish the isometric performance of the hamstrings or quadriceps of either leg.
Level of Evidence:
1b
PMCID: PMC3414070  PMID: 22893858
hamstring; muscle performance; vibration
7.  PreImplantation Factor (PIF) promoting role in embryo implantation: increases endometrial Integrin-α2β3, amphiregulin and epiregulin while reducing betacellulin expression via MAPK in decidua 
Background
Viable embryos secrete preimplantation factor (PIF), a peptide that has autocrine effects where levels correlate with cultured embryos development. sPIF (PIF synthetic analog) promotes implantation by regulating decidual-cells immunity, adhesion, apoptosis and enhances trophoblastic cell invasion. Herein sPIF priming effects on non-decidualized endometrium and decidualized-stroma are investigated, assessing elements critical for effective embryo-maternal cross-talk, prior to and at implantation.
Methods
We tested sPIF effect on human non-pregnant endometrial epithelial and non-decidualized stroma α2β3 integrin expression (IHC and flow cytometry), comparing with scrambled PIF (PIFscr-control). We examined sPIF effect on decidualized non-pregnant human endometrial stromal cells (HESC) determining pro-inflammatory mediators expression and secretion (ELISA) and growth factors (GFs) expression (Affymetrix global gene array). We tested sPIF effect on HESC Phospho-kinases (BioPlex) and isolated kinases activity (FastKinase).
Results
sPIF up-regulates α2β3 integrin expression in epithelial cells, (P < 0.05) while PIFscr had no effect. In contrast, in stromal cell cultures sPIF had no effect on the same. In HESC, sPIF up-regulates pro-inflammatory cytokines; IL8, IL1β and IL6 expression. The major increase in GRO-α, ICAM-1 and MCP-3 expression is coupled with same ligands secretion (P < 0.05). sPIF modulates in HESC GFs expression: up-regulates amphiregulin and epiregulin- critical for implantation and enhances several fibroblast growth factors (FGF) relevant for decidual function. In contrast, sPIF down-regulates major pro-proliferative ligands, betacellulin and IGF1 expression. sPIF modulatory effect on GFs is exerted by down-regulating pro-proliferative phospho-activated MAPkinases, p-MEK1 and p-ERK (P < 0.01, P < 0.04, respectively). Stress-induced p-38-MAPK (P = 0.04) and c-Jun kinase signaling involved MAPK8IP2 (−2.1 fold) expression decreased which protects against reactive oxygen species. Although pro-inflammatory p-NFkB (P = 0.06) decrease was mild, its promoter TNFRS11 expression markedly (−25-fold) decreased. In contrast, anti-proliferative phosphatases PTPRZ1 and PPP2R2C expression increased.
Conclusions
sPIF post-fertilization primes endometrial-epithelium, while during implantation creates a beneficial pro-inflammatory milieu. PIF acts by balancing decidual pro-implantation properties while controlling excessive pro-proliferative and inflammatory signals expression. Overall, PIF influences critical peri-implantation events in a sequential coordinated fashion which facilitates embryo implantation.
doi:10.1186/1477-7827-10-50
PMCID: PMC3444419  PMID: 22788113
Preimplantation Factor (PIF); Endometrium; Decidua; Gene Expression
8.  Genomic Analysis of Organismal Complexity in the Multicellular Green Alga Volvox carteri 
Science (New York, N.Y.)  2010;329(5988):223-226.
The multicellular green alga Volvox carteri and its morphologically diverse close relatives (the volvocine algae) are well suited for the investigation of the evolution of multicellularity and development. We sequenced the 138–mega–base pair genome of V. carteri and compared its ~14,500 predicted proteins to those of its unicellular relative Chlamydomonas reinhardtii. Despite fundamental differences in organismal complexity and life history, the two species have similar protein-coding potentials and few species-specific protein-coding gene predictions. Volvox is enriched in volvocine-algal–specific proteins, including those associated with an expanded and highly compartmentalized extracellular matrix. Our analysis shows that increases in organismal complexity can be associated with modifications of lineage-specific proteins rather than large-scale invention of protein-coding capacity.
doi:10.1126/science.1188800
PMCID: PMC2993248  PMID: 20616280
9.  The Neighborhood Context of Racial and Ethnic Disparities in Arrest 
Demography  2008;45(1):55-77.
This study assesses the role of social context in explaining racial and ethnic disparities in arrest, with a focus on how distinct neighborhood contexts in which different racial and ethnic groups reside explain variations in criminal outcomes. To do so, I utilize a multilevel, longitudinal research design, combining individual-level data with contextual data from the Project on Human Development in Chicago Neighborhoods (PHDCN). Findings reveal that black youths face multiple layers of disadvantage relative to other racial and ethnic groups, and these layers work to create differences in arrest. At the family level, results show that disadvantages in the form of unstable family structures explain much of the disparities in arrest across race and ethnicity. At the neighborhood level, black youths tend to reside in areas with both significantly higher levels of concentrated poverty than other youths as well as lower levels of collective efficacy than white youths. Variations in neighborhood tolerance of deviance across groups explain little of the arrest disparities, yet tolerance of deviance does influence the frequency with which a crime ultimately ends in an arrest. Even after accounting for relevant demographic, family, and neighborhood-level predictors, substantial residual arrest differences remain between black youths and youths of other racial and ethnic groups.
PMCID: PMC2831379  PMID: 18390291
10.  Mutation of Capsid Protein Phosphorylation Sites Abolishes Cauliflower Mosaic Virus Infectivity 
Journal of Virology  2002;76(22):11748-11752.
The cauliflower mosaic virus (CaMV) capsid protein is derived by bidirectional processing of the precapsid protein (CP56). We expressed several derivatives of CP56 in Escherichia coli and used them as substrates for virus-associated kinase and casein kinase II purified from plant cells. Three serine residues located at the N terminus of the mature viral protein CP44 were identified as phosphorylation targets. A mutation of one of them in the viral context had little or no effect on viral infectivity, but a mutation of all three serines abolished infectivity. The mapping of phosphorylation sites in CP44, but not CP39 or CP37, and immunodetection of the Zn finger motif in CP44 and CP39, but not CP37, support the model that CP39 is produced from CP44 by N-terminal processing and CP37 is produced from CP39 by C-terminal processing. We discuss the possible role of phosphorylation in the processing and assembly of CaMV capsid protein.
doi:10.1128/JVI.76.22.11748-11752.2002
PMCID: PMC136793  PMID: 12388736

Results 1-12 (12)