The rockefeller university

In most chemokine receptors, one or multiple tyrosine residues have been identified within the receptor N-terminal domain that are, at least partially, modified by posttranslational tyrosine sulfation. For example, tyrosine sulfation has been demonstrated for Tyr-3, -10, -14, and -15 of CCR5, for Tyr-3, -14, and -15 of CCR8, and for Tyr-7, -12, and -21 of CXCR4. While there is evidence for several chemokine receptors that tyrosine sulfation is required for optimal interaction with the chemokine ligands, the precise role of tyrosine sulfation for chemokine receptor function remains unclear. Furthermore, the function of the chemokine receptor N-terminal domain in chemokine binding and receptor activation is also not well understood. Sulfotyrosine peptides corresponding to the chemokine receptor N-termini are valuable tools to address these important questions both in structural and functional studies. However, due to the lability of the sulfotyrosine modification, these peptides are difficult to obtain using standard peptide chemistry methods. In this chapter, we provide methods to prepare sulfotyrosine peptides by enzymatic in vitro sulfation of peptides using purified recombinant tyrosylprotein sulfotransferase (TPST) enzymes. In addition, we also discuss alternative approaches for the generation of sulfotyrosine peptides and methods for sulfopeptide analysis.

The PRDM [PRDI-BFI (positive regulatory domain l-binding factor 1) and RIZ1 (retinoblastoma protein-interacting zinc finger gene 1) homologous domain containing] protein family is involved in a spectrum of biological processes including cell fate determination and development. These proteins regulate transcription through intrinsic chromatin-modifying activity or by complexing with histone-modifying or other nuclear proteins. Studies have indicated crucial roles for PRDM16 in the determination and function of brown and beige fat as well as in hematopoiesis and cardiac development, highlighting the importance of PRDM16 in developmental processes in different tissues. More recently, PRDM16 mutations were also identified in humans. The substantial progress in understanding the mechanism underlying the action of PRDM16 in adipose biology may have relevance to other PROM family members, and this new knowledge has the potential to be exploited for therapeutic benefit.

Streptococcus pyogenes is a human commensal and a bacterial pathogen responsible for a wide variety of human diseases differing in symptoms, severity, and tissue tropism. The completed genome sequences of >37 strains of S. pyogenes, representing diverse disease-causing serotypes, have been published. The greatest genetic variation among these strains is attributed to numerous integrated prophage and prophage-like elements, encoding several virulence factors. A comparison of isogenic strains, differing in prophage content, would reveal the effects of these elements on streptococcal pathogenesis. However, curing strains of prophage is often difficult and sometimes unattainable. We have applied a novel counter-selection approach to identify rare S. pyogenes mutants spontaneously cured of select prophage. To accomplish this, we first inserted a two-gene cassette containing a gene for kanamycin resistance (Kan(R)) and the rpsL wild-type gene, responsible for dominant streptomycin sensitivity (Sm-S), into a targeted prophage on the chromosome of a streptomycin resistant (Sm-R) mutant of S. pyogenes strain SF370. We then applied antibiotic counter-selection for the re-establishment of the Kan(S)/Sm-R phenotype to select for isolates cured of targeted prophage. This methodology allowed for the precise selection of spontaneous phage loss and restoration of the natural phage attB attachment sites for all four prophage-like elements in this S. pyogenes chromosome. Overall, 15 mutants were constructed that encompassed every permutation of phage knockout as well as a mutant strain, named CEM1 Delta phi, completely cured of all bacteriophage elements (a similar to 10% loss of the genome); the only reported S. pyogenes strain free of prophage-like elements. We compared CEM1 Delta phi to the WT strain by analyzing differences in secreted DNase activity, as well as lytic and lysogenic potential. These mutant strains should allow for the direct examination of bacteriophage relationships within S. pyogenes and further elucidate how the presence of prophage may affect overall streptococcal survival, pathogenicity, and evolution.

Background: A complete genome sequence and the advent of genome editing open up non-traditional model organisms to mechanistic genetic studies. The mosquito Aedes aegypti is an important vector of infectious diseases such as dengue, chikungunya, and yellow fever and has a large and complex genome, which has slowed annotation efforts. We used comprehensive transcriptomic analysis of adult gene expression to improve the genome annotation and to provide a detailed tissue-specific catalogue of neural gene expression at different adult behavioral states. Results: We carried out deep RNA sequencing across all major peripheral male and female sensory tissues, the brain and (female) ovary. Furthermore, we examined gene expression across three important phases of the female reproductive cycle, a remarkable example of behavioral switching in which a female mosquito alternates between obtaining blood-meals from humans and laying eggs. Using genome-guided alignments and de novo transcriptome assembly, our re-annotation includes 572 new putative protein-coding genes and updates to 13.5 and 50.3 % of existing transcripts within coding sequences and untranslated regions, respectively. Using this updated annotation, we detail gene expression in each tissue, identifying large numbers of transcripts regulated by blood-feeding and sexually dimorphic transcripts that may provide clues to the biology of male-and female-specific behaviors, such as mating and blood-feeding, which are areas of intensive study for those interested in vector control. Conclusions: This neurotranscriptome forms a strong foundation for the study of genes in the mosquito nervous system and investigation of sensory-driven behaviors and their regulation. Furthermore, understanding the molecular genetic basis of mosquito chemosensory behavior has important implications for vector control.

Objective:To determine the prevalence of HIV-associated neurocognitive disorders (HAND) in HIV-infected participants who initiated combination antiretroviral therapy (cART) during primary infection.Design:Cross-sectional observational study.Methods:HIV-infected men without neuropsychiatric confounds who had initiated cART during primary infection were administered a neuropsychological battery as well as questionnaires evaluating depression and quality of life. Eligibility was determined by a medical examination with history and review of records.Results:Twenty-six primarily non-Hispanic white (73%), male (100%) participants were enrolled and underwent neurocognitive assessment. Mean age was 43 (28-71) years, with a median of 17 years of education (13-24). Median current and nadir CD4(+) T-cell counts were 828 (506-1411) and 359 (150-621) cells/l. All participants had plasma HIV-1 RNA less than 50copies/ml. Median duration of cART prior to enrolment was 5.7 years (2.2-9.9). Median global deficit score was 0.17 (0.00-0.60). Only one (4%) participant was impaired.Conclusion:Rates of HAND in this cohort of HIV-infected men without comorbid conditions who initiated early cART are low. Our findings suggest a possible neuroprotective benefit of early cART and an important contribution of comorbidities to observed HAND prevalence.

Plasmodium salivary sporozoites are the infectious form of the malaria parasite and are dormant inside salivary glands of Anopheles mosquitoes. During dormancy, protein translation is inhibited by the kinase UIS1 that phosphorylates serine 59 in the eukaryotic initiation factor 2 alpha (eIF2 alpha). De-phosphorylation of eIF2 alpha-P is required for the transformation of sporozoites into the liver stage. In mammalian cells, the de-phosphorylation of eIF2 alpha-P is mediated by the protein phosphatase 1 (PP1). Using a series of genetically knockout parasites we showed that in malaria sporozoites, contrary to mammalian cells, the eIF2 alpha-P phosphatase is a member of the PP2C/PPM phosphatase family termed UIS2. We found that eIF2 alpha was highly phosphorylated in uis2 conditional knockout sporozoites. These mutant sporozoites maintained the crescent shape after delivery into mammalian host and lost their infectivity. Both uis1 and uis2 were highly transcribed in the salivary gland sporozoites but uis2 expression was inhibited by the Pumilio protein Puf2. The repression of uis2 expression was alleviated when sporozoites developed into liver stage. While most eukaryotic phosphatases interact transiently with their substrates, UIS2 stably bound to phosphorylated eIF2 alpha, raising the possibility that high-throughput searches may identify chemicals that disrupt this interaction and prevent malaria infection.

Environmental stressors induce coping strategies in the majority of individuals. The stress response, involving the activation of the hypothalamic-pituitary-adrenocortical axis and the consequent release of corticosteroid hormones, is indeed aimed at promoting metabolic, functional, and behavioral adaptations. However, behavioral stress is also associated with fast and long-lasting neurochemical, structural, and behavioral changes, leading to long-term remodeling of glutamate transmission, and increased susceptibility to neuropsychiatric disorders. Of note, early-life events, both in utero and during the early postnatal life, trigger reprogramming of the stress response, which is often associated with loss of stress resilience and ensuing neurobehavioral (mal) adaptations. Indeed, adverse experiences in early life are known to induce long-term stress-related neuropsychiatric disorders in vulnerable individuals. Here, we discuss recent findings about stress remodeling of excitatory neurotransmission and brain morphology in animal models of behavioral stress. These changes are likely driven by epigenetic factors that lie at the core of the stress-response reprogramming in individuals with a history of perinatal stress. We propose that reprogramming mechanisms may underlie the reorganization of excitatory neurotransmission in the short- and long-term response to stressful stimuli.

Ant colonies provide well-protected and resource-rich environments for a plethora of symbionts. Historically, most studies of ants and their symbionts have had a narrow taxonomic scope, often focusing on a single ant or symbiont species. Here we discuss the prospects of studying these assemblies in a community ecology context using the framework of ecological network analysis. We introduce three basic network metrics that we consider particularly relevant for improving our knowledge of ant-symbiont communities: interaction specificity, network modularity, and phylogenetic signal. We then discuss army ant symbionts as examples of large and primarily parasitic communities, and symbiotic sternorrhynchans as examples of generally smaller and primarily mutualistic communities in the context of these network analyses. We argue that this approach will provide new and complementary insights into the evolutionary and ecological dynamics between ants and their many associates, and will facilitate comparisons across different ant-symbiont assemblages as well as across different types of ecological networks.

Stem cell technology can allow us to produce human neuronal cell types outside the body, but what exactly are stem cells, and what challenges are associated with their use? Stem cells are a kind of cell that has the capacity to self-renew to produce additional stem cells by mitosis, and also to differentiate into other-more mature-cell types. Stem cells are usually categorized as multipotent (able to give rise to multiple cells within a lineage), pluripotent (able to give rise to all cell types in an adult) and totipotent (able to give rise to all embryonic and adult lineages). Multipotent adult stem cells are found throughout the body, and they include neural stem cells. The challenge in utilizing adult stem cells for disease research is obtaining cells that are genetically matched to people with disease phenotypes, and being able to differentiate them into the appropriate cell types of interest. As adult neural stem cells reside in the brain, their isolation would require considerably invasive and dangerous procedures. In contrast, pluripotent stem cells are easy to obtain, due to the paradigm-shifting work on direct reprogramming of human skin fibroblasts into induced pluripotent stem cells. This work has enabled us to produce neurons that are genetically matched to individual patients. While we are able to isolate pluripotent stem cells from patients in a minimally invasive manner, we do not yet fully understand how to direct these cells to many of the medically important neuroendocrine fates. Progress in this direction continues to be made, on multiple fronts, and it involves using small molecules and proteins to mimic developmentally important signals, as well as building on advances in "reprogramming" to directly convert one cell type into another by forced expression of sets of transcription factors. An additional challenge involves providing these cells with the appropriate environment to induce their normal behavior outside the body. Despite these challenges, the promise of producing human neuroendocrine cell types in vitro gives opportunities for unique insights and is therefore worthwhile.