A Breath-Based In Vitro Diagnostics for Lower Respiratory Tract Infection.

Lower Respiratory Tract Infections (LRTIs) represent the leading cause of death due to infectious diseases. Current diagnostic modalities primarily depend on clinical symptoms and lack specificity, especially in light of common colonization without overt infection. To address this, we developed a noninvasive diagnostic approach that employs BreathBiomics™, an advanced human breath sampling system, to detect protease activities induced by bacterial infection in the lower respiratory tract. Specifically, we engineered a high-sensitivity and high-specificity molecular sensor for human neutrophil elastase (HNE). The sensor undergoes cleavage in the presence of HNE, an event that is subsequently detected via Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS). Application of this methodology to clinical samples, breath specimens collected from intubated patients with LRTIs, demonstrated the detection of the cleaved sensor by MALDI-TOF MS. Our findings indicate that this novel approach offers a noninvasive and specific diagnostic strategy for people with LRTIs.

Human exhaled air diagnostic markers for respiratory tract infections in subjects receiving mechanical ventilation.

Diagnosing respiratory tract infections (RTIs) in critical care settings is essential for appropriate antibiotic treatment and lowering mortality. The current diagnostic method, which primarily relies on clinical symptoms, lacks sensitivity and specificity, resulting in incorrect or delayed diagnoses, putting patients at a heightened risk. In this study we developed a noninvasive diagnosis method based on collecting non-volatile compounds in human exhaled air. We hypothesized that non-volatile compound profiles could be effectively used for bacterial RTI diagnosis. Exhaled air samples were collected from subjects receiving mechanical ventilation diagnosed with or without bacterial RTI in intensive care units at the Johns Hopkins Hospital. Truncated proteoforms, a class of non-volatile compounds, were characterized by top-down proteomics, and significant features associated with RTI were identified using feature selection algorithms. The results showed that three truncated proteoforms, collagen type VI alpha three chain protein, matrix metalloproteinase-9, and putative homeodomain transcription factor II were independently associated with RTI with thep-values of 2.0 × 10-5, 1.1 × 10-4, and 1.7 × 10-3, respectively, using multiple logistic regression. Furthermore, a score system named 'TrunScore' was constructed by combining the three truncated proteoforms, and the diagnostic accuracy was significantly improved compared to that of individual truncated proteoforms, with an area under the receiver operator characteristic curve of 96.9%. This study supports the ability of this noninvasive breath analysis method to provide an accurate diagnosis for RTIs in subjects receiving mechanical ventilation. The results of this study open the doors to be able to potentially diagnose a broad range of diseases using this non-volatile breath analysis technique.

MALDI-TOF Mass Spectrometric Detection of SARS-CoV-2 Using Cellulose Sulfate Ester Enrichment and Hot Acid Treatment.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the ongoing coronavirus disease 2019 (COVID-19) pandemic. Here we report a novel strategy for the rapid detection of SARS-CoV-2 based on an enrichment approach exploiting the affinity between the virus and cellulose sulfate ester functional groups, hot acid hydrolysis, and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). Virus samples were enriched using cellulose sulfate ester microcolumns. Virus peptides were prepared using the hot acid aspartate-selective hydrolysis and characterized by MALDI-TOF MS. Collected spectra were processed with a peptide fingerprint algorithm, and searching parameters were optimized for the detection of SARS-CoV-2. These peptides provide high sequence coverage for nucleocapsid (N protein) and allow confident identification of SARS-CoV-2. Peptide markers contributing to the detection were rigorously identified using bottom-up proteomics. The approach demonstrated in this study holds the potential for developing a rapid assay for COVID-19 diagnosis and detecting virus variants from a variety of sources, such as sewage and nasal swabs.

Non-volatile organic compounds in exhaled breath particles correspond to active tuberculosis.

Human breath contains trace amounts of non-volatile organic compounds (NOCs) which might provide non-invasive methods for evaluating individual health. In previous work, we demonstrated that lipids detected in exhaled breath aerosol (EBA) could be used as markers of active tuberculosis (TB). Here, we advanced our analytical platform for characterizing small metabolites and lipids in EBA samples collected from participants enrolled in clinical trials designed to identify molecular signatures of active TB. EBA samples from 26 participants with active TB and 73 healthy participants were processed using a dual-phase extraction method, and metabolites and lipids were identified via mass spectrometry database matching. In total, 13 metabolite and 9 lipid markers were identified with statistically different optimized relative standard deviation values between individuals diagnosed with active TB and the healthy controls. Importantly, EBA lipid profiles can be used to separate the two sample types, indicating the diagnostic potential of the identified molecules. A feature ranking algorithm reduced this number to 10 molecules, with the membrane glycerophospholipid, phosphatidylinositol 24:4, emerging as the top driver of segregation between the two groups. These results support the use of this approach to identify consistent NOC signatures from EBA samples in active TB cases. This suggests the potential to apply this method to other human diseases which alter respiratory NOC release.

Transcriptomics Provides a Genetic Signature of Vineyard Site and Offers Insight into Vintage-Independent Inoculated Fermentation Outcomes.

Ribosomal DNA amplicon sequencing of grape musts has demonstrated that microorganisms occur nonrandomly and are associated with the vineyard of origin, suggesting a role for the vineyard, grape, and wine microbiome in shaping wine fermentation outcomes. Here, ribosomal DNA amplicon sequencing from grape musts and RNA sequencing of eukaryotic transcripts from primary fermentations inoculated with the wine yeast Saccharomyces cerevisiae RC212 were used to profile fermentations from 15 vineyards in California and Oregon across two vintages. These data demonstrate that the relative abundance of fungal organisms detected by ribosomal DNA amplicon sequencing correlated with neither transcript abundance from those same organisms within the RNA sequencing data nor gene expression of the inoculated RC212 yeast strain. These data suggest that the majority of the fungi detected in must by ribosomal DNA amplicon sequencing were not active during the primary stage of these inoculated fermentations and were not a major factor in determining RC212 gene expression. However, unique genetic signatures were detected within the ribosomal DNA amplicon and eukaryotic transcriptomic sequencing that were predictive of vineyard site and region. These signatures included S. cerevisiae gene expression patterns linked to nitrogen, sulfur, and thiamine metabolism. These genetic signatures of site offer insight into specific environmental factors to consider with respect to fermentation outcomes and vineyard site and regional wine characteristics.IMPORTANCE The wine industry generates billions of dollars of revenue annually, and economic productivity is in part associated with regional distinctiveness of wine sensory attributes. Microorganisms associated with grapes and wineries are influenced by region of origin, and given that some microorganisms play a role in fermentation, it is thought that microbes may contribute to the regional distinctiveness of wine. In this work, as in previous studies, it is demonstrated that specific bacteria and fungi are associated with individual wine regions and vineyard sites. However, this work further shows that their presence is not associated with detectable fungal gene expression during the primary fermentation or the expression of specific genes by the inoculate Saccharomyces cerevisiae strain RC212. The detected RC212 gene expression signatures associated with region and vineyard site also allowed the identification of flavor-associated metabolic processes and environmental factors that could impact primary fermentation outcomes. These data offer novel insights into the complexities and subtleties of vineyard-specific inoculated wine fermentation and starting points for future investigations into factors that contribute to regional wine distinctiveness.

Saccharomyces cerevisiae Gene Expression during Fermentation of Pinot Noir Wines at an Industrially Relevant Scale.

Saccharomyces cerevisiae metabolism produces ethanol and other compounds during the fermentation of grape must into wine. Thousands of genes change expression over the course of a wine fermentation, allowing S. cerevisiae to adapt to and dominate the fermentation environment. Investigations into these gene expression patterns previously revealed genes that underlie cellular adaptation to the grape must and wine environments, involving metabolic specialization and ethanol tolerance. However, the majority of studies detailing gene expression patterns have occurred in controlled environments that may not recapitulate the biological and chemical complexity of fermentations performed at production scale. Here, an analysis of the S. cerevisiae RC212 gene expression program is presented, drawing from 40 pilot-scale fermentations (150 liters) using Pinot noir grapes from 10 California vineyards across two vintages. A core gene expression program was observed across all fermentations irrespective of vintage, similar to that of laboratory fermentations, in addition to novel gene expression patterns likely related to the presence of non-Saccharomyces microorganisms and oxygen availability during fermentation. These gene expression patterns, both common and diverse, provide insight into Saccharomyces cerevisiae biology critical to fermentation outcomes under industry-relevant conditions.IMPORTANCE This study characterized Saccharomyces cerevisiae RC212 gene expression during Pinot noir fermentation at pilot scale (150 liters) using industry-relevant conditions. The reported gene expression patterns of RC212 are generally similar to those observed under laboratory fermentation conditions but also contain gene expression signatures related to yeast-environment interactions found in a production setting (e.g., the presence of non-Saccharomyces microorganisms). Key genes and pathways highlighted by this work remain undercharacterized, indicating the need for further research to understand the roles of these genes and their impact on industrial wine fermentation outcomes.

A novel system for the comprehensive collection of nonvolatile molecules from human exhaled breath.

Characterization of nonvolatile molecules in exhaled breath particles can be used for respiratory disease monitoring and diagnosis. Conventional methods for the collection of nonvolatile molecules in breath heavily rely on the physical properties of exhaled breath particles. Strategies taking advantage of their chemical properties have not yet been explored. In the present study, we developed a column system in which the surface chemistry between organic nonvolatile molecules and octadecyl carbon chain was exploited for the comprehensive collection of metabolites, lipids, and proteins. We demonstrated that the collection system had the capture efficiency of 99% and the capacity to capture representative nonvolatile molecules. The collection system was further evaluated using human subjects and proteins collected from human exhaled breath were characterized and identified using gel electrophoresis and bottom-up proteomics. The identified 303 proteins from mass spectrometry were further searched against reported bronchoalveolar lavage fluid proteomes and it was shown that 60 proteins have the tissue origin of lower respiratory airways. In summary, we demonstrate that our collection system can collect nonvolatile molecules from human exhaled breath in an efficient and comprehensive manner and has the potential to be used for the study of respiratory diseases.

Automated bioacoustics: methods in ecology and conservation and their potential for animal welfare monitoring.

Vocalizations carry emotional, physiological and individual information. This suggests that they may serve as potentially useful indicators for inferring animal welfare. At the same time, automated methods for analysing and classifying sound have developed rapidly, particularly in the fields of ecology, conservation and sound scene classification. These methods are already used to automatically classify animal vocalizations, for example, in identifying animal species and estimating numbers of individuals. Despite this potential, they have not yet found widespread application in animal welfare monitoring. In this review, we first discuss current trends in sound analysis for ecology, conservation and sound classification. Following this, we detail the vocalizations produced by three of the most important farm livestock species: chickens ( Gallus gallus domesticus), pigs ( Sus scrofa domesticus) and cattle ( Bos taurus). Finally, we describe how these methods can be applied to monitor animal welfare with new potential for developing automated methods for large-scale farming.

TrxR1, Gsr, and oxidative stress determine hepatocellular carcinoma malignancy.

Thioredoxin reductase-1 (TrxR1)-, glutathione reductase (Gsr)-, and Nrf2 transcription factor-driven antioxidant systems form an integrated network that combats potentially carcinogenic oxidative damage yet also protects cancer cells from oxidative death. Here we show that although unchallenged wild-type (WT), TrxR1-null, or Gsr-null mouse livers exhibited similarly low DNA damage indices, these were 100-fold higher in unchallenged TrxR1/Gsr-double-null livers. Notwithstanding, spontaneous cancer rates remained surprisingly low in TrxR1/Gsr-null livers. All genotypes, including TrxR1/Gsr-null, were susceptible to N-diethylnitrosamine (DEN)-induced liver cancer, indicating that loss of these antioxidant systems did not prevent cancer cell survival. Interestingly, however, following DEN treatment, TrxR1-null livers developed threefold fewer tumors compared with WT livers. Disruption of TrxR1 in a marked subset of DEN-initiated cancer cells had no effect on their subsequent contributions to tumors, suggesting that TrxR1-disruption does not affect cancer progression under normal care, but does decrease the frequency of DEN-induced cancer initiation. Consistent with this idea, TrxR1-null livers showed altered basal and DEN-exposed metabolomic profiles compared with WT livers. To examine how oxidative stress influenced cancer progression, we compared DEN-induced cancer malignancy under chronically low oxidative stress (TrxR1-null, standard care) vs. elevated oxidative stress (TrxR1/Gsr-null livers, standard care or phenobarbital-exposed TrxR1-null livers). In both cases, elevated oxidative stress was correlated with significantly increased malignancy. Finally, although TrxR1-null and TrxR1/Gsr-null livers showed strong Nrf2 activity in noncancerous hepatocytes, there was no correlation between malignancy and Nrf2 expression within tumors across genotypes. We conclude that TrxR1, Gsr, Nrf2, and oxidative stress are major determinants of liver cancer but in a complex, context-dependent manner.

Flight simulation using a Brain-Computer Interface: A pilot, pilot study.

As Brain-Computer Interface (BCI) systems advance for uses such as robotic arm control it is postulated that the control paradigms could apply to other scenarios, such as control of video games, wheelchair movement or even flight. The purpose of this pilot study was to determine whether our BCI system, which involves decoding the signals of two 96-microelectrode arrays implanted into the motor cortex of a subject, could also be used to control an aircraft in a flight simulator environment. The study involved six sessions in which various parameters were modified in order to achieve the best flight control, including plane type, view, control paradigm, gains, and limits. Successful flight was determined qualitatively by evaluating the subject's ability to perform requested maneuvers, maintain flight paths, and avoid control losses such as dives, spins and crashes. By the end of the study, it was found that the subject could successfully control an aircraft. The subject could use both the jet and propeller plane with different views, adopting an intuitive control paradigm. From the subject's perspective, this was one of the most exciting and entertaining experiments she had performed in two years of research. In conclusion, this study provides a proof-of-concept that traditional motor cortex signals combined with a decoding paradigm can be used to control systems besides a robotic arm for which the decoder was developed. Aside from possible functional benefits, it also shows the potential for a new recreational activity for individuals with disabilities who are able to master BCI control.

Comparison between an independent midwifery program and a district hospital in rural Tanzania: observations regarding the treatment of female patients.

Tanzania faces a significant shortage of physicians. In light of this, nurse-midwives have been critical in reducing maternal mortality in Tanzania in recent years. Despite the importance of both entities in providing health care to women in Tanzania, there have been few studies addressing the cultural competency of each entity. We shadowed and assisted both an independent nurse-midwife as well as physicians and nurse-midwives at a large district hospital in rural Tanzania. In this article we describe our observations regarding the treatment of female patients within the culture of an independent midwifery practice and at a large district hospital.

Recapitulating flesh with silicon and steel: advancements in upper extremity robotic prosthetics.

With the loss of function of an upper extremity because of stroke or spinal cord injury or a physical loss from amputation, an individual's life is forever changed, and activities that were once routine become a magnitude more difficult. Much research and effort have been put into developing advanced robotic prostheses to restore upper extremity function. For patients with upper extremity amputations, previously crude prostheses have evolved to become exceptionally functional. Because the upper extremities can perform a wide variety of activities, several types of upper extremity prostheses are available ranging from passive cosmetic limbs to externally powered robotic limbs. In addition, new developments in brain-machine interface are poised to revolutionize how patients can control these advanced prostheses using their thoughts alone. For patients with spinal cord injury or stroke, functional electrical stimulation promises to provide the most sophisticated prosthetic limbs possible by reanimating paralyzed arms of these patients. Advances in technology and robotics continue to help patients recover vital function. This article examines the latest neurorestorative technologies for patients who have either undergone amputation or lost the use of their upper extremities secondary to stroke or spinal cord injury.

The relationship between physical activity and brain responses to pain in fibromyalgia.

UNLABELLED: The relationship between physical activity and central nervous system mechanisms of pain in fibromyalgia (FM) is unknown. This study determined whether physical activity was predictive of brain responses to experimental pain in FM using functional magnetic resonance imaging (fMRI). Thirty-four participants (n = 16 FM; n = 18 Control) completed self-report and accelerometer measures of physical activity and underwent fMRI of painful heat stimuli. In FM patients, positive relationships (P < .005) between physical activity and brain responses to pain were observed in the dorsolateral prefrontal cortex, posterior cingulate cortex, and the posterior insula, regions implicated in pain regulation. Negative relationships (P < .005) were found for the primary sensory and superior parietal cortices, regions implicated in the sensory aspects of pain. Greater physical activity was significantly (P < .05) associated with decreased pain ratings to repeated heat stimuli for FM patients. A similar nonsignificant trend was observed in controls. In addition, brain responses to pain were significantly (P < .005) different between FM patients categorized as low active and those categorized as high active. In controls, positive relationships (P < .005) were observed in the lateral prefrontal, anterior cingulate, and superior temporal cortices and the posterior insula. Our results suggest an association between measures of physical activity and central nervous system processing of pain. PERSPECTIVE: Our data suggest that brain responses to pain represent a dynamic process where perception and modulation co-occur and that physical activity plays a role in balancing these processes. Physically active FM patients appear to maintain their ability to modulate pain while those who are less active do not.

Are women with fibromyalgia less physically active than healthy women?

PURPOSE: The primary purpose was to quantify and compare physical activity in fibromyalgia (FM) patients to age-matched healthy controls using both objective and self-report measures. Secondary purposes were to compare self-reported and objective measurement of physical activity and to evaluate the relationship between physical activity and pain and mood. METHOD: Patients with FM (n=39) and healthy controls (n=40) completed the International Physical Activity Questionnaire and wore an accelerometer at the hip for 7 d. Pain and mood were measured using the McGill Pain Questionnaire, Pain Catastrophizing Scale, Beck Depression Inventory, State-Trait Anxiety Inventory, Profile of Mood States, and Fibromyalgia Impact Questionnaire. RESULTS: FM patients had significantly lower physical activity than controls measured by both the International Physical Activity Questionnaire and accelerometer (P<0.05). Both groups self-reported significantly greater moderate and vigorous physical activities than were measured by the accelerometer (P < 0.05). Self-reported and objective measures of time spent in different intensities of activity showed significant correlations in healthy controls (r=0.41-0.51, ρ=0.41, P<0.05). No significant correlations between measures were found in FM patients (P>0.05). Finally, physical activity levels were negatively related (r=-0.37, P<0.05) to depressed mood for FM patients and positively related (r=-0.41, P<0.05) to self-reported vigor for healthy controls. CONCLUSIONS: This controlled study objectively demonstrates that FM patients are less physically active than healthy controls, thus extending on two earlier investigations that did not show differences in total physical activity levels using wrist-mounted actigraphy methods. Physical activity levels were not predictive of pain in FM but were significantly related to depressed mood. FM patients may also have a greater variability in their manner of self-report than healthy controls. Therefore, physical activity measurement in FM patients should not be limited solely to self-report measures.

Biopharmaceutical production: Applications of surface plasmon resonance biosensors.

Surface plasmon resonance (SPR) permits the quantitative analysis of therapeutic antibody concentrations and impurities including bacteria, Protein A, Protein G and small molecule ligands leached from chromatography media. The use of surface plasmon resonance has gained popularity within the biopharmaceutical industry due to the automated, label free, real time interaction that may be exploited when using this method. The application areas to assess protein interactions and develop analytical methods for biopharmaceutical downstream process development, quality control, and in-process monitoring are reviewed.

Imaging pain of fibromyalgia.

Brain imaging studies have provided objective evidence of abnormal central regulation of pain in fibromyalgia (FM). Resting brain blood flow studies have reported mixed findings for several brain regions, whereas decreased thalamic blood flow has been noted by several investigators. Studies examining the function of the nociceptive system in FM have reported augmented brain responses to both painful and non-painful stimuli that may be influenced by psychologic dispositions such as depressed mood and catastrophizing. Treatment approaches are beginning to demonstrate the potential for brain imaging to improve our understanding of pain-alleviating mechanisms. Data from other chronic conditions suggest that idiopathic pain may be maintained by similar central abnormalities as in FM, whereas chronic pain conditions with a known nociceptive source may not be. Future neuroimaging research in FM is clearly warranted and should continue to improve our understanding of factors involved in pain maintenance and symptom exacerbation.