Mycobacterial Ser/Thr protein kinases (STPKs) play a critical role in signal transduction pathways that ultimately determine mycobacterial growth and metabolic adaptation. Identification of key physiological substrates of these protein kinases is, therefore, crucial to better understand how Ser/Thr phosphorylation contributes to mycobacterial environmental adaptation, including response to stress, cell division, and host-pathogen interactions. Various substrate detection methods have been employed with limited success, with direct targets of STPKs remaining elusive. Recently developed mass spectrometry (MS)-based phosphoproteomic approaches have expanded the list of potential STPK substrate identifications, yet further investigation is required to define the most functionally significant phosphosites and their physiological importance. Prior to the application of MS workflows, for instance, GarA was the only known and validated physiological substrate for protein kinase G (PknG) from pathogenic mycobacteria. A subsequent list of at least 28 candidate PknG substrates has since been reported with the use of MS-based analyses. Herein, we integrate and critically review MS-generated datasets available on novel STPK substrates and report new functional and subcellular localization enrichment analyses on novel candidate protein kinase A (PknA), protein kinase B (PknB) and PknG substrates to deduce the possible physiological roles of these kinases. In addition, we assess substrate specificity patterns across different mycobacterial STPKs by analyzing reported sets of phosphopeptides, in order to determine whether novel motifs or consensus regions exist for mycobacterial Ser/Thr phosphorylation sites. This review focuses on MS-based techniques employed for STPK substrate identification in mycobacteria, while highlighting the advantages and challenges of the various applications.

The use of protein biomarkers as surrogates for clinical endpoints requires extensive multilevel validation including development of robust and sensitive assays for precise measurement of protein concentration. Multiple reaction monitoring (MRM) is a well-established mass-spectrometric method that can be used for reproducible protein-concentration measurements in biological specimens collected via microsampling. The dried blood spot (DBS) microsampling technique can be performed non-invasively without the expertise of a phlebotomist, and can enhance analyte stability which facilitate the application of this technique in retrospective studies while providing lower storage and shipping costs, because cold-chain logistics can be eliminated. Thus, precise, sensitive, and multiplexed methods for measuring protein concentrations in DBSs can be used for de novo biomarker discovery and for biomarker quantification or verification experiments. To achieve this goal, MRM assays were developed for multiplexed concentration measurement of proteins in DBSs.

The lower limit of quantification (LLOQ) was found to have a median total coefficient of variation (CV) of 18% for 245 proteins, whereas the median LLOQ was 5 fmol of peptide injected on column, and the median inter-day CV over 4 days for measuring endogenous protein concentration was 8%. The majority (88%) of the assays displayed parallelism, whereas the peptide standards remained stable throughout the assay workflow and after exposure to multiple freeze-thaw cycles. For 190 proteins, the measured protein concentrations remained stable in DBS stored at ambient laboratory temperature for up to 2 months. Finally, the developed assays were used to measure the concentration ranges for 200 proteins in twenty same sex, same race and age matched individuals.

Mass spectrometry (MS) and proteomics offer comprehensive characterization and identification of microorganisms and discovery of protein biomarkers that are applicable for diagnostics of infectious diseases. The use of biomarkers for diagnostics is widely applied in the clinic and the use of peptide biomarkers is increasingly being investigated for applications in the clinical laboratory. Respiratory-tract infections are a predominant cause for medical treatment, although, clinical assessments and standard clinical laboratory protocols are time-consuming and often inadequate for reliable diagnoses. Novel methods, preferably applied directly to clinical samples, excluding cultivation steps, are needed to improve diagnostics of infectious diseases, provide adequate treatment and reduce the use of antibiotics and associated development of antibiotic resistance. This study applied nano-liquid chromatography (LC) coupled with tandem MS, with a bioinformatics pipeline and an in-house database of curated high-quality reference genome sequences to identify species-unique peptides as potential biomarkers for four bacterial pathogens commonly found in respiratory tract infections (RTIs): Staphylococcus aureus; Moraxella catarrhalis; Haemophilus influenzae and Streptococcus pneumoniae. The species-unique peptides were initially identified in pure cultures of bacterial reference strains, reflecting the genomic variation in the four species and, furthermore, in clinical respiratory tract samples, without prior cultivation, elucidating proteins expressed in clinical conditions of infection. For each of the four bacterial pathogens, the peptide biomarker candidates most predominantly found in clinical samples, are presented. Data are available via ProteomeXchange with identifier PXD014522. As proof-of-principle, the most promising species-unique peptides were applied in targeted tandem MS-analyses of clinical samples and their relevance for identifications of the pathogens, i.e. proteotyping, was validated, thus demonstrating their potential as peptide biomarker candidates for diagnostics of infectious diseases.

C9ORF72-associated Motor Neuron Disease patients feature abnormal expression of 5 dipeptide repeat (DPR) polymers. Here we used quantitative proteomics in a mouse neuronal-like cell line (Neuro2a) to demonstrate that the Arg residues in the most toxic DPRS, PR and GR, leads to a promiscuous binding to the proteome compared with a relative sparse binding of the more inert AP and GA. Notable targets included ribosomal proteins, translation initiation factors and translation elongation factors. PR and GR comprising more than 10 repeats appeared to robustly stall on ribosomes during translation suggesting Arg-rich peptide domains can electrostatically jam the ribosome exit tunnel during synthesis. Poly-GR also recruited arginine methylases, induced hypomethylation of endogenous proteins, and induced a profound destabilization of the actin cytoskeleton. Our findings point to arginine in GR and PR polymers as multivalent toxins to translation as well as arginine methylation that may explain the dysfunction of biological processes including ribosome biogenesis, mRNA splicing and cytoskeleton assembly.

The redox-based modifications of cysteine residues in proteins regulate their function in many biological processes. The gas molecule H₂S has been shown to persulfidate redox sensitive cysteine residues resulting in an H₂S-modified proteome known as the sulfhydrome. Tandem Mass Tags (TMT) multiplexing strategies for large-scale proteomic analyses have become increasingly prevalent in detecting cysteine modifications. Here we developed a TMT-based proteomics approach for selectively trapping and tagging cysteine persulfides in the cellular proteomes. We revealed the natural protein sulfhydrome of two human cell lines, and identified insulin as a novel substrate in pancreatic beta cells. Moreover, we showed that under oxidative stress conditions, increased H₂S can target enzymes involved in energy metabolism by switching specific cysteine modifications to persulfides. Specifically, we discovered a Redox Thiol Switch, from protein S-glutathioinylation to S-persulfidation (RTS^GS). We propose that the RTS^GS from S-glutathioinylation to S-persulfidation is a potential mechanism to fine tune cellular energy metabolism in response to different levels of oxidative stress.

When you run 32-bit SAS on Windows and disconnect from the Oracle database server, you might see the error: "ERROR: ORACLE disconnect error: ORA-03135: connection lost contact."

If the following conditions are met in PROC QLIM: the SELECT option and DISCRETE option are specified in the same MODEL statement or ENDOGENOUS statement the same dependent variable with S

The developing nervous system is remarkably sensitive to environmental signals, including disruptive toxins, such as polybrominated diphenyl ethers (PBDEs). PBDEs are an environmentally pervasive class of brominated flame retardants whose neurodevelopmental toxicity mechanisms remain largely unclear. Using dissociated cortical neurons from embryonic Rattus norvegicus, we found here that chronic exposure to 6-OH–BDE-47, one of the most prevalent hydroxylated PBDE metabolites, suppresses both spontaneous and evoked neuronal electrical activity. On the basis of our previous work on mitogen-activated protein kinase (MAPK)/extracellular signal-related kinase (ERK) (MEK) biology and our observation that 6-OH–BDE-47 is structurally similar to kinase inhibitors, we hypothesized that certain hydroxylated PBDEs mediate neurotoxicity, at least in part, by impairing the MEK–ERK axis of MAPK signal transduction. We tested this hypothesis on three experimental platforms: 1) in silico, where modeling ligand–protein docking suggested that 6-OH–BDE-47 is a promiscuous ATP-competitive kinase inhibitor; 2) in vitro in dissociated neurons, where 6-OH–BDE-47 and another specific hydroxylated BDE metabolite similarly impaired phosphorylation of MEK/ERK1/2 and activity-induced transcription of a neuronal immediate early gene; and 3) in vivo in Drosophila melanogaster, where developmental exposures to 6-OH–BDE-47 and a MAPK inhibitor resulted in offspring displaying similarly increased frequency of mushroom-body β–lobe midline crossing, a metric of axonal guidance. Taken together, our results support that certain ortho-hydroxylated PBDE metabolites are promiscuous kinase inhibitors and can cause disruptions of critical neurodevelopmental processes, including neuronal electrical activity, pre-synaptic functions, MEK–ERK signaling, and axonal guidance.

Lethal infections by strains of the highly-pathogenic avian influenza virus (HPAIV) H5N1 pose serious threats to both the poultry industry and public health worldwide. A lack of confirmed HPAIV epitopes recognized by cytotoxic T lymphocytes (CTLs) has hindered the utilization of CD8+ T-cell–mediated immunity and has precluded the development of effectively diversified epitope-based vaccination approaches. In particular, an HPAIV H5N1 CTL-recognized epitope based on the peptide MHC-I–β2m (pMHC-I) complex has not yet been designed. Here, screening a collection of selected peptides of several HPAIV strains against a specific pathogen-free pMHC-I (pBF2*1501), we identified a highly-conserved HPAIV H5N1 CTL epitope, named HPAIV–PA123–130. We determined the structure of the BF2*1501–PA123–130 complex at 2.1 Å resolution to elucidate the molecular mechanisms of a preferential presentation of the highly-conserved PA123–130 epitope in the chicken B15 lineage. Conformational characteristics of the PA123–130 epitope with a protruding Tyr-7 residue indicated that this epitope has great potential to be recognized by specific TCRs. Moreover, significantly increased numbers of CD8+ T cells specific for the HPAIV–PA123–130 epitope in peptide-immunized chickens indicated that a repertoire of CD8+ T cells can specifically respond to this epitope. We anticipate that the identification and structural characterization of the PA123–130 epitope reported here could enable further studies of CTL immunity against HPAIV H5N1. Such studies may aid in the development of vaccine development strategies using well-conserved internal viral antigens in chickens.

Luigi Fontana
Apr 1, 2007; 56:1010-1013
Metabolism

Many chemicals and cellular processes cause oxidative stress that can damage lipids, proteins, or DNA (1). To quickly sense and respond to this ubiquitous threat, organisms have evolved enzymes that neutralize harmful oxidants such as reactive oxygen species and electrophilic compounds (including xenobiotics and their breakdown products) in cells.These antioxidant enzymes include GSH S-transferase (GST),2 NADPH:quinone oxidoreductase 1, thioredoxin, hemeoxygenase-1, and others (2, 3). Many of these proteins are commonly expressed in cells exposed to oxidative stress.The antioxidant response element (ARE) is a major regulatory component of this cellular stress response. The ARE is a conserved, 11-nucleotide-long DNA motif present in the 5'-flanking regions of many genes encoding antioxidant proteins. The laboratory of Cecil Pickett (Fig. 1) at the Merck Frosst Centre for Therapeutic Research in Quebec discovered ARE, a finding reported in the early 1990s in two JBC papers recognized as Classics here (4, 5).jbc;295/12/3929/F1F1F1Figure 1.Cecil Pickett (pictured) and colleagues first described the ARE motif, present in the 5' regions of many genes whose expression is up-regulated by oxidative stress and xenobiotics. Photo courtesy of Cecil Pickett.ARE's discovery was spurred in large part by Pickett's career choice. After completing a PhD in biology and a 2-year postdoc at UCLA in the mid-1970s, he began to work in the pharmaceutical industry.Recruited to Merck in 1978 by its then head of research and development (and later CEO), Roy Vagelos, “I became interested in how drug-metabolizing enzymes were induced by various xenobiotics,” Pickett says.According to Pickett, Vagelos encouraged researchers at the company...

6 November 2019

Although some digital platforms now have an impact on more people’s lives than does any one state authority, the international community has been slow to hold to account these platforms’ activities by reference to human rights law. This paper examines how human rights frameworks should guide digital technology.

As we inch closer and closer to Opening Day, we'll continue to take a position-by-position look at the Orioles roster options and remaining questions. Now that pitchers and catchers have officially reported to Spring Training, now feels like a good time to take a look behind the plate, where the Orioles have a ton of options but no clear answer.

In the US the licence, or marketing authorisation, for alteplase is limited to 0-3 hours after onset of stroke, but some other countries - including the UK and Australia - have extended the licence to 4.5 hours. In an analysis article on thebmj.com Brian Alper, vice president of evidence based medicine research and development at Dynamed, and...

It’s been called “the universal panacea” - exercise has a positive effect on almost all health measures, and governments are actively campaigning for us to do more. But at the opposite end of the scale, the realisation that some people may be addicted to exercise is gaining traction. In this podcast we're joined by Heather Hausenblas - professor...

There comes a tipping point in all campaigns when the evidence is overwhelming and the only way to proceed is with action. According to David Williams, it’s time to tackle the disproportionate effects of race on patients in the UK. David Williams, from Harvard University, developed the Everyday Discrimination Scale that, in 1997, launched a new...

D Dabelea
Dec 1, 2000; 49:2208-2211
Articles

Reds starting pitcher Anthony DeSclafani hated how his 2018 season ended and didn't need any motivation to make improvements over the winter.

THE EDITOR, Madam: The devastating news of Jodian Fearon’s death has resulted in public outrage and concern. The controversy and seemingly negligent actions of the hospitals involved have left many Jamaicans anxious about our health system and...

Despite a stiff neck, which he says came on a recent airplane flight, D-backs ace Zack Greinke said Saturday that he feels much better physically than he did last year when he reported to Spring Training. Here are five takeaways from Greinke's Saturday session with reporters.

We aimed to assess the value of ¹¹C-choline PET in patients with primary hyperparathyroidism and negative or discordant results on ^99mTc-sestamibi imaging and neck ultrasound. Methods: Eighty-seven such patients were assessed and subsequently underwent parathyroidectomy. PET/CT image data were analyzed semiquantitatively using SUV_max and SUV ratios (target to contralateral thyroid gland and carotid artery). A positive PET/CT result was defined as focal uptake significantly higher than regular thyroid tissue. Ectopic foci were also considered positive. Inconclusive PET/CT cases were defined as a lesion with uptake equal to normal thyroid tissue. If no prominent or ectopic uptake was detectable, the PET/CT result was considered negative. Results: When dichotomizing the ¹¹C-choline PET/CT imaging results by defining lesions with both positive and inconclusive uptake as positive, we found 84 of 92 lesions (91.3%) to have true-positive uptake whereas 8 lesions (8.7%) had false-positive uptake. One lesion showed false-negative uptake; the sensitivity was 98.8%. The corresponding positive predictive value for lesions was 91.3%. The mean SUV_max was 6.15 ± 4.92 in 72 lesions with positive uptake (70 patients) and 2.96 ± 2.32 in 20 lesions with inconclusive uptake (18 patients). Conclusion: These results in a large group of patients indicate that ¹¹C-choline PET/CT is a promising tool for parathyroid adenoma localization when ultrasound and ^99mTc-sestamibi imaging yield negative or discordant results.

Research Event

This awards ceremony, honoring the 2011 recipients of the E Pluribus Unum Prizes — a national awards program for exceptional immigrant integration initiatives — featured panel discussions with the awardees and federal officials and remarks by White House Director of Intergovernmental Affairs Cecilia Muñoz and Assistant Secretary of Education Brenda Dann-Messier.

This awards ceremony, honoring the 2011 recipients of the E Pluribus Unum Prizes -- a national awards program for exceptional immigrant integration initiatives -- featured panel discussions with the awardees and federal officials and remarks by White House Director of Intergovernmental Affairs Cecilia Muñoz and Assistant Secretary of Education Brenda Dann-Messier.

An MPI Leadership Visions discussion with the Foreign Minister of Mexico, Claudia Ruiz-Massieu, for her first public appearance in Washington, DC. 

Jan 1, 2020; 43:S205-S206
Disclosures

OBJECTIVE

The MHC region harbors the strongest loci for latent autoimmune diabetes in adults (LADA); however, the strength of association is likely attenuated compared with that for childhood-onset type 1 diabetes. In this study, we recapitulate independent effects in the MHC class I region in a population with type 1 diabetes and then determine whether such conditioning in LADA yields potential genetic discriminators between the two subtypes within this region.

The ADA and the Wisconsin Dental Association are partnering up with other fluoridated water proponents to address February anti-fluoridation movements in three Wisconsin municipalities: Lake Delton, Berlin and Hayward.

Two of the authors of the American Dental Association's antibiotics guideline will discuss C. diff infections and antibiotic stewardship in dentistry April 21 as part of a free, online conference organized by the Peggy Lillis Foundation.

Wisconsin dentists have answered the call for personal protective equipment by organizing drives and donating their surplus supplies to hospitals and urgent care facilities treating patients with COVID-19.

Overfill of medication or obturation materials in endodontic treatment can cause permanent neurologic injury, and there are steps clinicians can take to help prevent that, according to an article published in the May issue of The Journal of the American Dental Association.

A microbe found in mosquitoes that appears to block malaria could be used to control spread of the disease in humans, according to researchers in Kenya and Britain.

OBJECTIVE

In patients with type 2 diabetes (T2D) and critical limb ischemia (CLI), migration of circulating CD34⁺ cells predicted cardiovascular mortality at 18 months after revascularization. This study aimed to provide long-term validation and mechanistic understanding of the biomarker.

An MPI Leadership Visions discussion with the Foreign Minister of Mexico, Claudia Ruiz-Massieu, for her first public appearance in Washington, DC. 

I have a 23 day cycle and around day 19 up until my period I produce a ovulation like discharge, it’s clear/ white and super stretchy. I don’t get this during the “ovulation” time frame, it tends to be thick and cloudy then. Why? And when am I ovulating?

This indulgent cheesecake is as light as air, with all the ingredients of an afternoon tea rolled into one - coffee, chocolate and caramel biscuits.

The Portuguese have a double gene for flavours. They keep it simple, they rely on extremely good produce. This dish has such a unique flavour. It's ideal with all wines. A good Lisbon paste is the trick!

Ireland rugby captain Brian O'Driscoll has been ruled out of action for up to six months and will miss his country's Six Nations campaign when the annual tournament kicks off in February.

OBJECTIVE

Prevalence and prognostic impact of cardiovascular disease differ between patients with or without diabetes. We aimed to explore differences in the prevalence and prognosis of myocardial ischemia by automated quantification of total perfusion deficit (TPD) among patients with and without diabetes.

David Willens
Apr 1, 2011; 29:60-68
Feature Articles

Before the coronavirus pandemic, bioengineer Jim Collins and his team combined the power of AI with synthetic biology in an effort to combat a different looming crisis: antibiotic-resistant superbugs. Collins explains how they pivoted their efforts to begin developing a series of tools and antiviral compounds to help fight COVID-19 -- and shares their plan to discover seven new classes of antibiotics over the next seven years. (This ambitious plan is a part of The Audacious Project, TED's initiative to inspire and fund global change.)

DEADLINE FOR ORAL PRESENTATIONS: FEB 15, 2020 On June 5-6 2020, Temple University will host the 10th Annual Interdisciplinary Symposium on Decision Neuroscience (ISDN) in Philadelphia, PA. This symposium is unique in that it brings together a range of constituencies involved in the use of neuroscience techniques to understand consumer decision making – world renowned […]

The post Annual Interdisciplinary Symposium on Decision Neuroscience (ISDN), Philadelphia, June 5-6, 2020 appeared first on Decision Science News.

In addition to a delay, Wisconsin had to eliminate certain questions from its Smarter Balanced exam, after opting not to use the adaptive testing feature of the test.