Engineering Nucleotide Specificity of Succinyl-CoA Synthetase in Blastocystis: The Emerging Role of Gatekeeper Residues.

PublishedJournal ArticleThis is the final version of the article. Available from American Chemical Society via the DOI in this record.Charged, solvent-exposed residues at the entrance to the substrate binding site (gatekeeper residues) produce electrostatic dipole interactions with approaching substrates, and control their access by a novel mechanism called "electrostatic gatekeeper effect". This proof-of-concept study demonstrates that the nucleotide specificity can be engineered by altering the electrostatic properties of the gatekeeper residues outside the binding site. Using Blastocystis succinyl-CoA synthetase (SCS, EC 6.2.1.5), we demonstrated that the gatekeeper mutant (ED) resulted in ATP-specific SCS to show high GTP specificity. Moreover, nucleotide binding site mutant (LF) had no effect on GTP specificity and remained ATP-specific. However, via combination of the gatekeeper mutant with the nucleotide binding site mutant (ED+LF), a complete reversal of nucleotide specificity was obtained with GTP, but no detectable activity was obtained with ATP. This striking result of the combined mutant (ED+LF) was due to two changes; negatively charged gatekeeper residues (ED) favored GTP access, and nucleotide binding site residues (LF) altered ATP binding, which was consistent with the hypothesis of the "electrostatic gatekeeper effect". These results were further supported by molecular modeling and simulation studies. Hence, it is imperative to extend the strategy of the gatekeeper effect in a different range of crucial enzymes (synthetases, kinases, and transferases) to engineer substrate specificity for various industrial applications and substrate-based drug design.Work is supported by the National Institute of Malaria Research, Indian Council of Medical Research, New Delhi and Dept. of Biotechnology, New Delhi. K.C.P. is a recipient of the Prof. Ramalingaswami Fellowship (Department of Biotechnology, Government of India (BT/HRD/35/02/2006), K.V. is a recipient of UGC Senior Research Fellowship, M.v.d.G. is grateful for support from the University of Exeter and the Wellcome Trust (078566/A/05/Z)

Generation of Functional CLL-Specific Cord Blood CTL Using CD40-Ligated CLL APC

PMCID: PMC3526610This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Human Platelet-Rich Plasma- and Extracellular Matrix-Derived Peptides Promote Impaired Cutaneous Wound Healing In Vivo

Previous work in our laboratory has described several pro-angiogenic short peptides derived from endothelial extracellular matrices degraded by bacterial collagenase. Here we tested whether these peptides could stimulate wound healing in vivo. Our experiments demonstrated that a peptide created as combination of fragments of tenascin X and fibrillin 1 (comb1) applied into cranial dermal wounds created in mice treated with cyclophosphamide to impair wound healing, can improve the rate of wound closure. Furthermore, we identify and characterize a novel peptide (UN3) created and modified from two naturally-occurring peptides, which are present in human platelet-rich plasma. In vitro testing of UN3 demonstrates that it causes a 50% increase in endothelial proliferation, 250% increase in angiogenic response and a tripling of epithelial cell migration in response to injury. Results of in vivo experiments where comb1 and UN3 peptides were added together to cranial wounds in cyclophosphamide-treated mice leads to improvement of wound vascularization as shown by an increase of the number of blood vessels present in the wound beds. Application of the peptides markedly promotes cellular responses to injury and essentially restores wound healing dynamics to those of normal, acute wounds in the absence of cyclophosphamide impairment. Our current work is aimed at understanding the mechanisms underlying the stimulatory effects of these peptides as well as identification of the cellular receptors mediating these effects.National Institutes of Health (U.S.) (Grant EY15125)National Institutes of Health (U.S.) (Grant EY19533)Wound Care Partners, LL

Value of hospital antimicrobial stewardship programs [ASPs]:a systematic review

Abstract Background Hospital antimicrobial stewardship programs (ASPs) aim to promote judicious use of antimicrobials to combat antimicrobial resistance. For ASPs to be developed, adopted, and implemented, an economic value assessment is essential. Few studies demonstrate the cost-effectiveness of ASPs. This systematic review aimed to evaluate the economic and clinical impact of ASPs. Methods An update to the Dik et al. systematic review (2000–2014) was conducted on EMBASE and Medline using PRISMA guidelines. The updated search was limited to primary research studies in English (30 September 2014–31 December 2017) that evaluated patient and/or economic outcomes after implementation of hospital ASPs including length of stay (LOS), antimicrobial use, and total (including operational and implementation) costs. Results One hundred forty-six studies meeting inclusion criteria were included. The majority of these studies were conducted within the last 5 years in North America (49%), Europe (25%), and Asia (14%), with few studies conducted in Africa (3%), South America (3%), and Australia (3%). Most studies were conducted in hospitals with 500–1000 beds and evaluated LOS and change in antibiotic expenditure, the majority of which showed a decrease in LOS (85%) and antibiotic expenditure (92%). The mean cost-savings varied by hospital size and region after implementation of ASPs. Average cost savings in US studies were $732 per patient (range:$2.50 to $2640), with similar trends exhibited in European studies. The key driver of cost savings was from reduction in LOS. Savings were higher among hospitals with comprehensive ASPs which included therapy review and antibiotic restrictions. Conclusions Our data indicates that hospital ASPs have significant value with beneficial clinical and economic impacts. More robust published data is required in terms of implementation, LOS, and overall costs so that decision-makers can make a stronger case for investing in ASPs, considering competing priorities. Such data on ASPs in lower- and middle-income countries is limited and requires urgent attention

HS1, a Lyn Kinase Substrate, Is Abnormally Expressed in B-Chronic Lymphocytic Leukemia and Correlates with Response to Fludarabine-Based Regimen

In B-Chronic Lymphocytic Leukemia (B-CLL) kinase Lyn is overexpressed, active, abnormally distributed, and part of a cytosolic complex involving hematopoietic lineage cell-specific protein 1 (HS1). These aberrant properties of Lyn could partially explain leukemic cells’ defective apoptosis, directly or through its substrates, for example, HS1 that has been associated to apoptosis in different cell types. To verify the hypothesis of HS1 involvement in Lyn-mediated leukemic cell survival, we investigated HS1 protein in 71 untreated B-CLL patients and 26 healthy controls. We found HS1 overexpressed in leukemic as compared to normal B lymphocytes (1.38±0.54 vs 0.86±0.29, p<0.01), and when HS1 levels were correlated to clinical parameters we found a higher expression of HS1 in poor-prognosis patients. Moreover, HS1 levels significantly decreased in ex vivo leukemic cells of patients responding to a fludarabine-containing regimen. We also observed that HS1 is partially localized in the nucleus of neoplastic B cells. All these data add new information on HS1 study, hypothesizing a pivotal role of HS1 in Lyn-mediated modulation of leukemic cells’ survival and focusing, one more time, the attention on the BCR-Lyn axis as a putative target for new therapeutic strategies in this disorder

Genetic Structure, Linkage Disequilibrium and Signature of Selection in Sorghum: Lessons from Physically Anchored DArT Markers

Population structure, extent of linkage disequilibrium (LD) as well as signatures of selection were investigated in sorghum using a core sample representative of worldwide diversity. A total of 177 accessions were genotyped with 1122 informative physically anchored DArT markers. The properties of DArTs to describe sorghum genetic structure were compared to those of SSRs and of previously published RFLP markers. Model-based (STRUCTURE software) and Neighbor-Joining diversity analyses led to the identification of 6 groups and confirmed previous evolutionary hypotheses. Results were globally consistent between the different marker systems. However, DArTs appeared more robust in terms of data resolution and bayesian group assignment. Whole genome linkage disequilibrium as measured by mean r2 decreased from 0.18 (between 0 to 10 kb) to 0.03 (between 100 kb to 1 Mb), stabilizing at 0.03 after 1 Mb. Effects on LD estimations of sample size and genetic structure were tested using i. random sampling, ii. the Maximum Length SubTree algorithm (MLST), and iii. structure groups. Optimizing population composition by the MLST reduced the biases in small samples and seemed to be an efficient way of selecting samples to make the best use of LD as a genome mapping approach in structured populations. These results also suggested that more than 100,000 markers may be required to perform genome-wide association studies in collections covering worldwide sorghum diversity. Analysis of DArT markers differentiation between the identified genetic groups pointed out outlier loci potentially linked to genes controlling traits of interest, including disease resistance genes for which evidence of selection had already been reported. In addition, evidence of selection near a homologous locus of FAR1 concurred with sorghum phenotypic diversity for sensitivity to photoperiod

Assessment of potential effects of the electromagnetic fields of mobile phones on hearing

BACKGROUND: Mobile phones have become indispensable as communication tools; however, to date there is only a limited knowledge about interaction between electromagnetic fields (EMF) emitted by mobile phones and auditory function. The aim of the study was to assess potential changes in hearing function as a consequence of exposure to low-intensity EMF's produced by mobile phones at frequencies of 900 and 1800 MHz. METHODS: The within-subject study was performed on thirty volunteers (age 18–30 years) with normal hearing to assess possible acute effect of EMF. Participants attended two sessions: genuine and sham exposure of EMF. Hearing threshold levels (HTL) on pure tone audiometry (PTA) and transient evoked otoacoustic emissions (TEOAE's) were recorded before and immediately after 10 min of genuine and/or sham exposure of mobile phone EMF. The administration of genuine or sham exposure was double blind and counterbalanced in order. RESULTS: Statistical analysis revealed no significant differences in the mean HTLs of PTA and mean shifts of TEOAE's before and after genuine and/or sham mobile phone EMF 10 min exposure. The data collected showed that average TEOAE levels (averaged across a frequency range) changed less than 2.5 dB between pre- and post-, genuine and sham exposure. The greatest individual change was 10 dB, with a decrease in level from pre- to post- real exposure. CONCLUSION: It could be concluded that a 10-min close exposure of EMFs emitted from a mobile phone had no immediate after-effect on measurements of HTL of PTA and TEOAEs in young human subjects and no measurable hearing deterioration was detected in our study

Genome-Wide Association Study SNPs in the Human Genome Diversity Project Populations: Does Selection Affect Unlinked SNPs with Shared Trait Associations?

Genome-wide association studies (GWAS) have identified more than 2,000 trait-SNP associations, and the number continues to increase. GWAS have focused on traits with potential consequences for human fitness, including many immunological, metabolic, cardiovascular, and behavioral phenotypes. Given the polygenic nature of complex traits, selection may exert its influence on them by altering allele frequencies at many associated loci, a possibility which has yet to be explored empirically. Here we use 38 different measures of allele frequency variation and 8 iHS scores to characterize over 1,300 GWAS SNPs in 53 globally distributed human populations. We apply these same techniques to evaluate SNPs grouped by trait association. We find that groups of SNPs associated with pigmentation, blood pressure, infectious disease, and autoimmune disease traits exhibit unusual allele frequency patterns and elevated iHS scores in certain geographical locations. We also find that GWAS SNPs have generally elevated scores for measures of allele frequency variation and for iHS in Eurasia and East Asia. Overall, we believe that our results provide evidence for selection on several complex traits that has caused changes in allele frequencies and/or elevated iHS scores at a number of associated loci. Since GWAS SNPs collectively exhibit elevated allele frequency measures and iHS scores, selection on complex traits may be quite widespread. Our findings are most consistent with this selection being either positive or negative, although the relative contributions of the two are difficult to discern. Our results also suggest that trait-SNP associations identified in Eurasian samples may not be present in Africa, Oceania, and the Americas, possibly due to differences in linkage disequilibrium patterns. This observation suggests that non-Eurasian and non-East Asian sample populations should be included in future GWAS

In vivo fluorescence imaging of the transport of charged chlorine6 conjugates in a rat orthotopic prostate tumour

Polymeric drug conjugates are used in cancer therapy and, varying their molecular size and charge, will affect their in vivo transport and extravasation in tumours. Partitioning between tumour vasculature and tumour tissue will be of particular significance in the case of photosensitizer conjugates used in photodynamic therapy, where this partitioning can lead to different therapeutic effects. Poly-l-lysine chlorine6 conjugates (derived from polymers of averageMr 5000 and 25 000) were prepared both in a cationic state and by poly-succinylation in an anionic state. A fluorescence scanning laser microscope was used to follow the pharmacokinetics of these conjugates in vivo in an orthotopic rat prostate cancer model obtained with MatLyLu cells. Fluorescence was excited with the 454–528 nm group of lines of an argon laser and a 570 nm long pass filter used to isolate the emission. Results showed that the conjugates initially bound to the walls of the vasculature, before extravasating into the tissue, and eventually increasing in fluorescence. The anionic conjugates produced tissue fluorescence faster than the cationic ones, and surprisingly, the largerMr conjugates produced tissue fluorescence faster than the smaller ones with the same charge. These results are consistent with differences in aggregation state between conjugates. © 1999 Cancer Research Campaig