A Single Kinase Generates the Majority of the Secreted Phosphoproteome.

The existence of extracellular phosphoproteins has been acknowledged for over a century. However, research in this area has been undeveloped largely because the kinases that phosphorylate secreted proteins have escaped identification. Fam20C is a kinase that phosphorylates S-x-E/pS motifs on proteins in milk and in the extracellular matrix of bones and teeth. Here, we show that Fam20C generates the majority of the extracellular phosphoproteome. Using CRISPR/Cas9 genome editing, mass spectrometry, and biochemistry, we identify more than 100 secreted phosphoproteins as genuine Fam20C substrates. Further, we show that Fam20C exhibits broader substrate specificity than previously appreciated. Functional annotations of Fam20C substrates suggest roles for the kinase beyond biomineralization, including lipid homeostasis, wound healing, and cell migration and adhesion. Our results establish Fam20C as the major secretory pathway protein kinase and serve as a foundation for new areas of investigation into the role of secreted protein phosphorylation in human biology and disease.

Deciphering the immunopeptidome in vivo reveals new tumour antigens.

Immunosurveillance of cancer requires the presentation of peptide antigens on major histocompatibility complex class I (MHC-I) molecules1-5. Current approaches to profiling of MHC-I-associated peptides, collectively known as the immunopeptidome, are limited to in vitro investigation or bulk tumour lysates, which limits our understanding of cancer-specific patterns of antigen presentation in vivo6. To overcome these limitations, we engineered an inducible affinity tag into the mouse MHC-I gene (H2-K1) and targeted this allele to the KrasLSL-G12D/+Trp53fl/fl mouse model (KP/KbStrep)7. This approach enabled us to precisely isolate MHC-I peptides from autochthonous pancreatic ductal adenocarcinoma and from lung adenocarcinoma (LUAD) in vivo. In addition, we profiled the LUAD immunopeptidome from the alveolar type 2 cell of origin up to late-stage disease. Differential peptide presentation in LUAD was not predictable by mRNA expression or translation efficiency and is probably driven by post-translational mechanisms. Vaccination with peptides presented by LUAD in vivo induced CD8+ T cell responses in naive mice and tumour-bearing mice. Many peptides specific to LUAD, including immunogenic peptides, exhibited minimal expression of the cognate mRNA, which prompts the reconsideration of antigen prediction pipelines that triage peptides according to transcript abundance8. Beyond cancer, the KbStrep allele is compatible with other Cre-driver lines to explore antigen presentation in vivo in the pursuit of understanding basic immunology, infectious disease and autoimmunity.

Phosphorylation of spore coat proteins by a family of atypical protein kinases.

The modification of proteins by phosphorylation occurs in all life forms and is catalyzed by a large superfamily of enzymes known as protein kinases. We recently discovered a family of secretory pathway kinases that phosphorylate extracellular proteins. One member, family with sequence similarity 20C (Fam20C), is the physiological Golgi casein kinase. While examining distantly related protein sequences, we observed low levels of identity between the spore coat protein H (CotH), and the Fam20C-related secretory pathway kinases. CotH is a component of the spore in many bacterial and eukaryotic species, and is required for efficient germination of spores in Bacillus subtilis; however, the mechanism by which CotH affects germination is unclear. Here, we show that CotH is a protein kinase. The crystal structure of CotH reveals an atypical protein kinase-like fold with a unique mode of ATP binding. Examination of the genes neighboring cotH in B. subtilis led us to identify two spore coat proteins, CotB and CotG, as CotH substrates. Furthermore, we show that CotH-dependent phosphorylation of CotB and CotG is required for the efficient germination of B. subtilis spores. Collectively, our results define a family of atypical protein kinases and reveal an unexpected role for protein phosphorylation in spore biology.

Volatile Profile and Physico-Chemical Analysis of Acacia Honey for Geographical Origin and Nutritional Value Determination.

Honey composition and color depend greatly on the botanical and geographical origin. Water content, water activity and color of 50 declared acacia samples, collected from three different geographical zones of Romania, together with chromatographic determination of sugar spectrum were analyzed. A number of 79 volatile compounds from the classes of: Alcohols, aldehydes, esters, ketones, sulphur compounds, aliphatic hydrocarbons, nitrogen compounds, carboxylic acids, aromatic acids and ethers were identified by solid-phase micro-extraction and gas-chromatography mass spectrometry. The overall volatile profile and sugar spectrum of the investigated honey samples allow the differentiation of geographical origin for the acacia honey samples subjected to analysis. The statistical models of the chromatic determination, physicochemical parameters and volatile profile was optimal to characterize the honey samples and group them into three geographical origins, even they belong to the same botanical origin.

A Multidisciplinary Approach to Incorporate Bedside Nurses into Antimicrobial Stewardship and Infection Prevention.

BACKGROUND: Antimicrobial stewardship programs exist to promote appropriate antimicrobial use. The Joint Commission has reported that although many US hospitals have implemented basic components of antimicrobial stewardship programs, there now exists a need for innovative, multidisciplinary approaches, including involving frontline clinicians such as bedside nurses. METHODS: A retrospective evaluation of bedside nurse-driven antimicrobial stewardship and infection prevention rounds was conducted on a 31-bed telemetry unit of a community regional medical center. Rounds were managed by a nurse coordinator and attended by an infectious diseases pharmacist, an infection preventionist, and a nurse practitioner. Primary outcome measures were antimicrobial and acid suppressant medication and invasive catheter use. RESULTS: In the 12-month intervention period the nurse-driven rounds team reviewed of a total of 472 antimicrobial medication, 480 acid suppressant medication, 321 urinary catheter, and 61 central venous catheter therapies over 867 total patient encounters. Compared with the 12-month preintervention period, significant reductions in unit antimicrobial use (791.2 vs. 697.1 days of therapy per 1,000 patient-days; p = 0.03), acid suppressant medication use (708.1 vs. 372.4 days of therapy per 1,000 patient-days; p = 0.0001), and urinary catheter use (0.3 vs. 0.2 catheter-days per patient-day; p = 0.002) were observed. CONCLUSION: This study demonstrates successful engagement of bedside nurses in antimicrobial stewardship and infection prevention activities and a measurable impact on meaningful outcomes. More studies of strategies to integrate bedside nurses in antimicrobial stewardship are needed.

The life-course origins of mastery among older people.

In this article, we aim to identify the sources of mastery--the understanding that individuals hold about their ability to control the circumstances of their lives. The sample for our inquiry was drawn from the Medicare beneficiary files of people 65 and older living in Washington, DC, and two adjoining Maryland counties. We find that past circumstances, particularly those reflecting status attainment and early exposure to intractable hardships, converge with stressors experienced in late life to influence elders' level of mastery. The impact of past conditions, however, does not necessarily directly affect the current mastery of older people. Instead, the effect of prior experiences on current mastery is mediated by what we refer to as life-course mastery: one's belief that one has directed and managed the trajectories that connect one's past to the present. Our analyses show that life-course mastery largely serves as the mediating channel through which individuals connect their past to their present.

Early pregnancy factor treatment suppresses the inflammatory response and adhesion molecule expression in the spinal cord of SJL/J mice with experimental autoimmune encephalomyelitis and the delayed-type hypersensitivity reaction to trinitrochlorobenzene in normal BALB/c mice.

Early pregnancy factor (EPF) is a secreted protein, present in serum during early pregnancy and essential for maintaining viability of the embryo. It is a homologue of chaperonin 10 (Cpn10) but, unlike Cpn10, it has an extracellular role. EPF has immunosuppressive and growth regulatory properties. Previously we have reported the preparation of recombinant EPF (rEPF) and shown that treatment with rEPF will suppress clinical signs of MBP-EAE in Lewis rats and PLP-EAE in SJL/J mice. In the present study, these findings have been extended to investigate possible mechanisms involved in the action of EPF. Following treatment of mice with rEPF from the day of inoculation, there were fewer infiltrating CD3+ and CD4+ cells in the parenchyma of the spinal cord during the onset of disease and after the initial episode, compared with mice treated with vehicle. Expression of the integrins LFA-1, VLA-4 and Mac-1 and of members of the immunoglobulin superfamily of adhesion molecules ICAM-1 and VCAM-1 was suppressed in the central nervous system (CNS) following rEPF treatment. The expression of PECAM-1 was not affected. To determine if rEPF suppressed T cell activation in the periphery, the delayed-type hypersensitivity (DTH) reaction of normal BALB/c mice to trinitrochlorobenzene (TNCB) following treatment with rEPF was studied. The results showed that treatment with rEPF suppressed the DTH reaction, demonstrating the ability of EPF to downregulate the cell-mediated immune response. These results indicate that suppression of immunological mechanisms by rEPF plays a major role in the reduction of clinical signs of disease in experimental autoimmune encephalomyelitis (EAE).