Proteomic Profiling in Patients With Peripartum Cardiomyopathy: A Biomarker Study of the ESC EORP PPCM Registry.

BACKGROUND: Peripartum cardiomyopathy (PPCM) remains an important cause of maternal morbidity and mortality globally. The pathophysiology remains incompletely understood, and the diagnosis is often missed or delayed. OBJECTIVES: This study explored the serum proteome profile of patients with newly diagnosed PPCM, as compared with matched healthy postpartum mothers, to unravel novel protein biomarkers that would further an understanding of the pathogenesis of PPCM and improve diagnostic precision. METHODS: Study investigators performed untargeted serum proteome profiling using data-independent acquisition-based label-free quantitative liquid chromatography-tandem mass spectrometry on 84 patients with PPCM, as compared with 29 postpartum healthy controls (HCs). Significant changes in protein intensities were determined with nonpaired Student's t-tests and were further classified by using the Boruta algorithm. The proteins' diagnostic performance was evaluated by area under the curve (AUC) and validated using the 10-fold cross-validation. RESULTS: Patients with PPCM presented with a mean left ventricular ejection fraction of 33.5% ± 9.3% vs 57.0% ± 8.8% in HCs (P < 0.001). Study investigators identified 15 differentially up-regulated and 14 down-regulated proteins in patients with PPCM compared with HCs. Seven of these proteins were recognized as significant by the Boruta algorithm. The combination of adiponectin, quiescin sulfhydryl oxidase 1, inter-α-trypsin inhibitor heavy chain, and N-terminal pro-B-type natriuretic peptide had the best diagnostic precision (AUC: 0.90; 95% CI: 0.84-0.96) to distinguish patients with PPCM from HCs. CONCLUSIONS: Salient biologic themes related to immune response proteins, inflammation, fibrosis, angiogenesis, apoptosis, and coagulation were predominant in patients with PPCM compared with HCs. These newly identified proteins warrant further evaluation to establish their role in the pathogenesis of PPCM and potential use as diagnostic markers.

Bundle of care to drive improvements in palliative and end-of-life care (PEOLC) in an acute tertiary hospital.

OBJECTIVES: There is little evidence to suggest the best model of palliative and end-of-life care (PEOLC) in an acute care hospital. We introduced a bundle of care to drive improvements in PEOLC; this bundle included three full-time nursing positions providing a palliative care clinical consult service with physician backup, as well as educating staff, using the NSW Resuscitation Plan and the Last-Days-of-Life Toolkit. METHODS: Two audits were performed at John Hunter Hospital, a tertiary hospital in Newcastle, Australia, each sampling from all deaths in a 12-month period, one prior to and one after the bundle of care was introduced. Sampling was stratified into deaths that occurred within 4-48 hours of admission and after 48 hours. Key outcomes/data points were recorded and compared across the two time periods. RESULTS: Statistically significant improvements noted included: lower mortality on the wards after 48 hours of admission, better recognition of the dying patient, increased referral to palliative care nurses and physicians, reduction in the number of medical emergency team calls and increase in the use of comfort care and resuscitation plans. Currently, 73% of patients have their end-of-life wishes observed as per their advance care directive. CONCLUSION: A bundle of care involving dedicated nurses with physician backup providing a consult service and education is an effective method for driving improvements in PEOLC.

Delirium Screening and Pharmacotherapy in the ICU: The Patients Are Not the Only Ones Confused.

Background: Delirium is difficult to measure in the Intensive Care Unit (ICU). It is possible that by considering the rate of screening, incidence, and rate of treatment with antipsychotic medications (APMs) for suspected delirium, a clearer picture can emerge. Methods: A retrospective, observational study was conducted at two ICUs in Australia, between April and June of 2020. All adult ICU patients were screened; those who spoke English and did not have previous neurocognitive pathology or intracranial pathology were included in the analysis. Data were collected from the hospitals' electronic medical records. The primary outcome was incidence of delirium based on the use of the Confusion Assessment Method for ICU (CAM-ICU). Secondary outcomes included measures of screening for delirium, treatment of suspected delirium with APMs, and identifying clinical factors associated with both delirium and the use of APMs. Results: From 736 patients that were screened, 665 were included in the analysis. The incidence of delirium was 11.3% (75/665); on average, the Richmond Agitation and Sedation Scale (RASS) was performed every 2.9 h and CAM-ICU every 40 h. RASS was not performed in 8.4% (56/665) of patients and CAM-ICU was not performed in 40.6% (270/665) of patients. A total of 17% (113/665) of patients were prescribed an APM, with quetiapine being the most used. ICU length of stay (LOS), APACHE-III score, and the use of alpha-2 agonists were associated with the presence of delirium, while ICU LOS, the use of alpha-2 agonists, and the presence of delirium were associated with patients receiving APMs. Conclusions: The incidence of delirium was lower than previously reported, at 11.3%. The rate of screening for delirium was low, while the use of APMs for delirium was higher than the incidence of delirium. It is possible that the true incidence is higher than what was measured. Critical prospective assessment is required to optimize APM indications in the ICU.

Lactic acid from vaginal microbiota enhances cervicovaginal epithelial barrier integrity by promoting tight junction protein expression.

BACKGROUND: Women with a cervicovaginal microbiota dominated by Lactobacillus spp. are at reduced risk of acquiring sexually transmitted infections including HIV, but the biological mechanisms involved remain poorly defined. Here, we performed metaproteomics on vaginal swab samples from young South African women (n = 113) and transcriptomics analysis of cervicovaginal epithelial cell cultures to examine the ability of lactic acid, a metabolite produced by cervicovaginal lactobacilli, to modulate genital epithelial barrier function. RESULTS: Compared to women with Lactobacillus-depleted microbiota, women dominated by vaginal lactobacilli exhibit higher abundance of bacterial lactate dehydrogenase, a key enzyme responsible for lactic acid production, which is independently associated with an increased abundance of epithelial barrier proteins. Physiological concentrations of lactic acid enhance epithelial cell culture barrier integrity and increase intercellular junctional molecule expression. CONCLUSIONS: These findings reveal a novel ability of vaginal lactic acid to enhance genital epithelial barrier integrity that may help prevent invasion by sexually transmitted pathogens. Video abstract.

The epidemiology of overtransfusion of red cells in trauma resuscitation patients in the context of a mature massive transfusion protocol.

PURPOSE: Packed red blood cell (PRBC) transfusion remains an integral part of trauma resuscitation and an independent predictor of unfavourable outcomes. It is often administered urgently based on clinical judgement. These facts put trauma patients at high risk of potentially dangerous overtransfusion. We hypothesised that trauma patients are frequently overtransfused and overtransfusion is associated with worse outcomes. METHODS: Trauma patients who received PRBCs within 24 h of admission were identified from the trauma registry during the period January 1 2011-December 31 2018. Overtransfusion was defined as haemoglobin concentration of greater than or equal to 110 g/L at 24 h post ED arrival (± 12 h). Demographics, injury severity, injury pattern, shock severity, blood gas values and outcomes were compared between overtransfused and non-overtransfused patients. RESULTS: From the 211 patients (mean age 45 years, 71% male, ISS 27, mortality 12%) who met inclusion criteria 27% (56/211) were overtransfused. Patients with a higher pre-hospital systolic blood pressure (112 vs 99 mmHg p < 0.01) and a higher initial haemoglobin concentration (132 vs 124 p = 0.02) were more likely to be overtransfused. Overtransfused patients received smaller volumes of packed red blood cells (5 vs 7 units p = 0.049), fresh frozen plasma (4 vs 6 units p < 0.01) and cryoprecipitate (6 vs 9 units p = 0.01) than non-overtransfused patients. CONCLUSION: More than a quarter of patients in our cohort were potentially given more blood products than required without obvious clinical consequences. There were no clinically relevant associations with overtransfusion.

Effect of polyethylene glycol 20 000 on protein extraction efficiency of formalin-fixed paraffin-embedded tissues in South Africa.

BACKGROUND: Optimal protocols for efficient and reproducible protein extraction from formalin-fixed paraffin-embedded (FFPE) tissues are not yet standardised and new techniques are continually developed and improved. The effect of polyethylene glycol (PEG) 20 000 on protein extraction efficiency has not been evaluated using human FFPE colorectal cancer tissues and there is no consensus on the protein extraction solution required for efficient, reproducible extraction. OBJECTIVE: The impact of PEG 20 000 on protein extraction efficiency, reproducibility and protein selection bias was evaluated using FFPE colonic tissue via liquid chromatography tandem mass spectrometry analysis. METHODS: This study was conducted from August 2017 to July 2019 using human FFPE colorectal carcinoma tissues from the Anatomical Pathology department at Tygerberg Hospital in South Africa. Samples were analysed via label-free liquid chromatography tandem mass spectrometry to determine the impact of using PEG 20 000 in the protein extraction solution. Data were assessed regarding peptide and protein identifications, method efficiency, reproducibility, protein characteristics and organisation relating to gene ontology categories. RESULTS: Polyethylene glycol 20 000 exclusion increased peptides and proteins identifications and the method was more reproducible compared to the samples processed with PEG 20 000. However, no differences were observed with regard to protein selection bias. We found that higher protein concentrations (> 10 µg) compromised the function of PEG. CONCLUSION: This study indicates that protocols generating high protein yields from human FFPE tissues would benefit from the exclusion of PEG 20 000 in the protein extraction solution.

Evaluation of Protein Purification Techniques and Effects of Storage Duration on LC-MS/MS Analysis of Archived FFPE Human CRC Tissues.

To elucidate cancer pathogenesis and its mechanisms at the molecular level, the collecting and characterization of large individual patient tissue cohorts are required. Since most pathology institutes routinely preserve biopsy tissues by standardized methods of formalin fixation and paraffin embedment, these archived FFPE tissues are important collections of pathology material that include patient metadata, such as medical history and treatments. FFPE blocks can be stored under ambient conditions for decades, while retaining cellular morphology, due to modifications induced by formalin. However, the effect of long-term storage, at resource-limited institutions in developing countries, on extractable protein quantity/quality has not yet been investigated. In addition, the optimal sample preparation techniques required for accurate and reproducible results from label-free LC-MS/MS analysis across block ages remains unclear. This study investigated protein extraction efficiency of 1, 5, and 10-year old human colorectal carcinoma resection tissue and assessed three different gel-free protein purification methods for label-free LC-MS/MS analysis. A sample size of n = 17 patients per experimental group (with experiment power = 0.7 and α = 0.05, resulting in 70% confidence level) was selected. Data were evaluated in terms of protein concentration extracted, peptide/protein identifications, method reproducibility and efficiency, sample proteome integrity (due to storage time), as well as protein/peptide distribution according to biological processes, cellular components, and physicochemical properties. Data are available via ProteomeXchange with identifier PXD017198. The results indicate that the amount of protein extracted is significantly dependent on block age (p < 0.0001), with older blocks yielding less protein than newer blocks. Detergent removal plates were the most efficient and overall reproducible protein purification method with regard to number of peptide and protein identifications, followed by the MagReSyn® SP3/HILIC method (with on-bead enzymatic digestion), and lastly the acetone precipitation and formic acid resolubilization method. Overall, the results indicate that long-term storage of FFPE tissues (as measured by methionine oxidation) does not considerably interfere with retrospective proteomic analysis (p > 0.1). Block age mainly affects initial protein extraction yields and does not extensively impact on subsequent label-free LC-MS/MS analysis results.

Proteomic Identification and Meta-Analysis in Salvia hispanica RNA-Seq de novo Assemblies.

While proteomics has demonstrated its value for model organisms and for organisms with mature genome sequence annotations, proteomics has been of less value in nonmodel organisms that are unaccompanied by genome sequence annotations. This project sought to determine the value of RNA-Seq experiments as a basis for establishing a set of protein sequences to represent a nonmodel organism, in this case, the pseudocereal chia. Assembling four publicly available chia RNA-Seq datasets produced transcript sequence sets with a high BUSCO completeness, though the number of transcript sequences and Trinity "genes" varied considerably among them. After six-frame translation, ProteinOrtho detected substantial numbers of orthologs among other species within the taxonomic order Lamiales. These protein sequence databases demonstrated a good identification efficiency for three different LC-MS/MS proteomics experiments, though a seed proteome showed considerable variability in the identification of peptides based on seed protein sequence inclusion. If a proteomics experiment emphasizes a particular tissue, an RNA-Seq experiment incorporating that same tissue is more likely to support a database search identification of that proteome.

Microbial function and genital inflammation in young South African women at high risk of HIV infection.

BACKGROUND: Female genital tract (FGT) inflammation is an important risk factor for HIV acquisition. The FGT microbiome is closely associated with inflammatory profile; however, the relative importance of microbial activities has not been established. Since proteins are key elements representing actual microbial functions, this study utilized metaproteomics to evaluate the relationship between FGT microbial function and inflammation in 113 young and adolescent South African women at high risk of HIV infection. Women were grouped as having low, medium, or high FGT inflammation by K-means clustering according to pro-inflammatory cytokine concentrations. RESULTS: A total of 3186 microbial and human proteins were identified in lateral vaginal wall swabs using liquid chromatography-tandem mass spectrometry, while 94 microbial taxa were included in the taxonomic analysis. Both metaproteomics and 16S rRNA gene sequencing analyses showed increased non-optimal bacteria and decreased lactobacilli in women with FGT inflammatory profiles. However, differences in the predicted relative abundance of most bacteria were observed between 16S rRNA gene sequencing and metaproteomics analyses. Bacterial protein functional annotations (gene ontology) predicted inflammatory cytokine profiles more accurately than bacterial relative abundance determined by 16S rRNA gene sequence analysis, as well as functional predictions based on 16S rRNA gene sequence data (p < 0.0001). The majority of microbial biological processes were underrepresented in women with high inflammation compared to those with low inflammation, including a Lactobacillus-associated signature of reduced cell wall organization and peptidoglycan biosynthesis. This signature remained associated with high FGT inflammation in a subset of 74 women 9 weeks later, was upheld after adjusting for Lactobacillus relative abundance, and was associated with in vitro inflammatory cytokine responses to Lactobacillus isolates from the same women. Reduced cell wall organization and peptidoglycan biosynthesis were also associated with high FGT inflammation in an independent sample of ten women. CONCLUSIONS: Both the presence of specific microbial taxa in the FGT and their properties and activities are critical determinants of FGT inflammation. Our findings support those of previous studies suggesting that peptidoglycan is directly immunosuppressive, and identify a possible avenue for biotherapeutic development to reduce inflammation in the FGT. To facilitate further investigations of microbial activities, we have developed the FGT-DB application that is available at http://fgtdb.org/ . Video Abstract.

The molecular identity of fleas (Siphonaptera) carrying Rickettsia felis, Bartonella clarridgeiae and Bartonella rochalimae from dogs and cats in Northern Laos.

Cat fleas (Ctenocephalides felis) are the most commonly recognised ectoparasites of domestic pets globally and are frequently implicated in the transmission of a variety of zoonotic vector-borne pathogens. The aim of the present study was to investigate the morphological and molecular identity of fleas parasitising cats and dogs in Northern Laos and screen them for a range of bacterial pathogens. Fleas (n = 120) were collected from dogs and cats and morphologically identified as Ctenocephalides felis (115/120), Ctenocephalides orientis (4/120) and Pulex irritans (1/120). Molecular barcoding using the cytochrome c oxidase subunit I gene (cox1) was used to confirmed species identity of 21 selected fleas. The cat flea (C. felis) was the most dominant flea identified. Rickettsia and Bartonella spp. DNA was detected in 21/21 and 7/21 samples, respectively, via a multiplex real-time PCR targeting gltA and ssrA. Sequencing of the seven Bartonella-positive samples and ten Rickettsia-positive samples revealed Bartonella clarridgeiae, Bartonella rochalimae, Rickettsia felis and Rickettsia sp. genotype RF2125 DNA. Anaplasma platys DNA was detected in a single C. felis after 20 of the 21 DNA samples were screened using a commercial PCR panel for vector-borne pathogens. The detection of a range of bacterial pathogens in fleas from owned cats and dogs in Northern Laos provides further evidence to the importance of these ectoparasites as vectors of zoonotic diseases in the region.

Redox signaling modulates Rho activity and tissue contractility in the Caenorhabditis elegans spermatheca.

Actomyosin-based contractility in smooth muscle and nonmuscle cells is regulated by signaling through the small GTPase Rho and by calcium-activated pathways. We use the myoepithelial cells of the Caenorhabditis elegans spermatheca to study the mechanisms of coordinated myosin activation in vivo. Here, we show that redox signaling modulates RHO-1/Rho activity in this contractile tissue. Exogenously added as well as endogenously generated hydrogen peroxide decreases spermathecal contractility by inhibition of RHO-1, which depends on a conserved cysteine in its nucleotide binding site (C20). Further, we identify an endogenous gradient of H2O2 across the spermathecal tissue, which depends on the activity of cytosolic superoxide dismutase, SOD-1. Collectively, we show that SOD-1-mediated H2O2 production regulates the redox environment and fine tunes Rho activity across the spermatheca through oxidation of RHO-1 C20.

Challenges and Opportunities for Biological Mass Spectrometry Core Facilities in the Developing World.

The developing world is seeing rapid growth in the availability of biological mass spectrometry (MS), particularly through core facilities. As proteomics and metabolomics becomes locally feasible for investigators in these nations, application areas associated with high burden in these nations, such as infectious disease, will see greatly increased research output. This article evaluates the rapid growth of MS in South Africa (currently approaching 20 laboratories) as a model for establishing MS core facilities in other nations of the developing world. Facilities should emphasize new services rather than new instruments. The reduction of the delays associated with reagent and other supply acquisition would benefit both facilities and the users who make use of their services. Instrument maintenance and repair, often mediated by an in-country business for an international vendor, is also likely to operate on a slower schedule than in the wealthiest nations. A key challenge to facilities in the developing world is educating potential facility users in how best to design experiments for proteomics and metabolomics, what reagents are most likely to introduce problematic artifacts, and how to interpret results from the facility. Here, we summarize the experience of 6 different institutions to raise the level of biological MS available to researchers in South Africa.

TB-IRIS: Proteomic analysis of in vitro PBMC responses to Mycobacterium tuberculosis and response modulation by dexamethasone.

Paradoxical tuberculosis-associated immune reconstitution inflammatory syndrome (TB-IRIS) occurs in 8-54% of South African patients undergoing treatment for tuberculosis/human immunodeficiency virus co-infection. Improved TB-IRIS molecular pathogenesis understanding would enhance risk stratification, diagnosis, prognostication, and treatment. We assessed how TB-IRIS status and dexamethasone influence leukocyte proteomic responses to Mycobacterium tuberculosis (Mtb). Patient blood was obtained three weeks post-anti-retroviral therapy initiation. Isolated mononuclear cells were stimulated ex vivo with heat-killed Mtb in the presence/absence of dexamethasone. Mass spectrometry-based proteomic comparison of TB-IRIS and non-IRIS patient-derived cells facilitated generation of hypotheses regarding pathogenesis. Few represented TB-IRIS-group immune-related pathways achieved significant activation, with relative under-utilisation of "inter-cellular interaction" and "Fcγ receptor-mediated phagocytosis" (but a tendency towards apoptosis-related) pathways. Dexamethasone facilitated significant activation of innate-related pathways. Differentially-expressed non-IRIS-group proteins suggest focused and co-ordinated immunological pathways, regardless of dexamethasone status. Findings suggest a relative deficit in TB-IRIS-group responses to and clearance of Mtb antigens, ameliorated by dexamethasone.