The rockefeller university

Background Urine is a potential source of biomarkers for diseases of the kidneys and urinary tract. RNA, including microRNA, is present in the urine enclosed in detached cells or in extracellular vesicles (EVs) or bound and protected by extracellular proteins. Detection of cell-and disease-specific microRNA in urine may aid early diagnosis of organ-specific pathology. In this study, we applied barcoded deep sequencing to profile microRNAs in urine of healthy volunteers, and characterized the effects of sex, urine fraction (cells vs. EVs) and repeated voids by the same individuals. Results Compared to urine-cell-derived small RNA libraries, urine-EV-derived libraries were relatively enriched with miRNA, and accordingly had lesser content of other small RNA such as rRNA, tRNA and sn/snoRNA. Unsupervised clustering of specimens in relation to miRNA expression levels showed prominent bundling by specimen type (urine cells or EVs) and by sex, as well as a tendency of repeated (first and second void) samples to neighbor closely. Likewise, miRNA profile correlations between void repeats, as well as fraction counterparts (cells and EVs from the same specimen) were distinctly higher than correlations between miRNA profiles overall. Differential miRNA expression by sex was similar in cells and EVs. Conclusions miRNA profiling of both urine EVs and sediment cells can convey biologically important differences between individuals. However, to be useful as urine biomarkers, careful consideration is needed for biofluid fractionation and sex-specific analysis, while the time of voiding appears to be less important.

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.

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.

Type 1 interferon (IFN) is a key mediator of organismal responses to pathogens, eliciting prototypical "interferon signature genes'' that encode antiviral and inflammatory mediators. For a global view of IFN signatures and regulatory pathways, we performed gene expression and chromatin analyses of the IFN-induced response across a range of immunocyte lineages. These distinguished ISGs by cell-type specificity, kinetics, and sensitivity to tonic IFN and revealed underlying changes in chromatin configuration. We combined 1,398 human and mouse datasets to computationally infer ISG modules and their regulators, validated by genetic analysis in both species. Some ISGs are controlled by Stat1/2 and Irf9 and the ISRE DNA motif, but others appeared dependent on non-canonical factors. This regulatory framework helped to interpret JAK1 blockade pharmacology, different clusters being affected under tonic or IFN-stimulated conditions, and the IFN signatures previously associated with human diseases, revealing unrecognized subtleties in disease footprints, as affected by human ancestry.

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: Infectious diseases, such as hepatitis C and community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA), are emerging health issues. Objectives: The CA-MRSA Project (CAMP1) extended its learning collaborative to the barbershop/hair salon settings to increase awareness and prevention of CA-MRSA and hepatitis C infections. Methods: Education sessions on CA-MRSA and hepatitis C were conducted with 43 estheticians at nine barbershop/hair salons in New York City. All completed pre-post intervention knowledge tests. Low-cost primary care referral cards were also distributed in the CA-MRSA education project. Results: Knowledge about CA-MRSA risks (p < .0003) and infection prevention measures (p < .0001), as well as hepatitis C knowledge and prevention (both p < .0001) increased. Nine shops received referral cards (n = 500) and 4% of the cards (n = 19) were distributed to clients. No self-referrals were reported. Conclusions: CAMP I successfully recruited and trained a cadre of estheticians on CA-MRSA and hepatitis C prevention increasing their health knowledge deepening our engagement with the community.

Brain channelopathies represent a growing class of brain disorders that usually result in paroxysmal disorders, although their role in other neurological phenotypes, including the recently described NALCN-related infantile encephalopathy, is increasingly recognized. In three Saudi Arabian families and one Egyptian family all affected by a remarkably similar phenotype (infantile encephalopathy and largely normal brain MRI) to that of NALCN-related infantile encephalopathy, we identified a locus on 2q34 in which whole-exome sequencing revealed three, including two apparently loss-of-function, recessive mutations in UNC80. UNC80 encodes a large protein that is necessary for the stability and function of NALCN and for bridging NALCN to UNC79 to form a functional complex. Our results expand the clinical relevance of the UNC79-UNC80-NALCN channel complex.

The brain is the central organ for adaptation to experiences, including stressors, which are capable of changing brain architecture as well as altering systemic function through neuroendocrine, autonomic, immune, and metabolic systems. Because the brain is the master regulator of these systems, as well as of behavior, alterations in brain function by chronic stress can have direct and indirect effects on cumulative allostatic overload, which refers to the cost of adaptation. There is much new knowledge on the neural control of systemic physiology and the feedback actions of physiologic mediators on brain regions regulating higher cognitive function, emotional regulation, and self-regulation. The healthy brain has a considerable capacity for resilience, based upon its ability to respond to interventions designed to open "windows of plasticity" and redirect its function toward better health. As a result, plasticity-facilitating treatments should be given within the framework of a positive behavioral intervention; negative experiences during this window may even make matters worse. Indeed, there are no magic bullets and drugs cannot substitute for targeted interventions that help an individual become resilient, of which mindfulness-based stress reduction and meditation are emerging as useful tools.

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.

Background: Atopic dermatitis (AD) and psoriasis pathogeneses involve skin barrier impairment and immune dysregulation; however, the contribution of B-cell imbalances to these diseases has not yet been determined. Objective: We sought to quantify B-cell populations and antibody-secreting cells in the blood of patients with AD, patients with psoriasis, and control subjects. Methods: We studied 34 adults with moderate-to-severe AD (mean SCORAD score, 65), 24 patients with psoriasis (mean Psoriasis Area and Severity Index score, 16), and 27 healthy subjects using an 11-color flow cytometric antibody panel. IgD/CD27 and CD24/CD38 core gating systems were used to determine frequencies of plasmablasts and naive, memory, transitional, and activated B cells. Results: We measured increased CD19(+)CD20(+) B-cell counts in the skin and blood of patients with AD (P < .01). Significantly higher frequencies of chronically activated CD27(+) memory and nonswitched memory B cells were observed in patients with AD (P < .05), with lower values of double-negative populations (4% for patients with AD vs 7% for patients with psoriasis [P = .001] and 6% for control subjects [P = .02]). CD23 expression was highest in patients with AD and correlated with IgE levels (P < .01) and disease severity (r = 0.6, P = .0002). Plasmablast frequencies and IgE expression were highest in all memory subsets of patients with AD (P < .01). Finally, CD19(+)CD24(++)CD38(++) transitional and CD19(+)CD24(-)CD38(-) new memory B-cell counts were higher in patients with AD versus those in patients with psoriasis (2.8% vs 1.4% [P = .001] and 9.2% vs 5.7% [P = .02], respectively). Conclusions: AD is accompanied by systemic expansion of transitional and chronically activated CD27(+) memory, plasmablast, and IgE-expressing memory subsets. These data create a critical basis for the future understanding of this debilitating skin disease.