Saliva-based detection of COVID-19 infection in a real-world setting using reagent-free Raman spectroscopy and machine learning.

SIGNIFICANCE: The primary method of COVID-19 detection is reverse transcription polymerase chain reaction (RT-PCR) testing. PCR test sensitivity may decrease as more variants of concern arise and reagents may become less specific to the virus. AIM: We aimed to develop a reagent-free way to detect COVID-19 in a real-world setting with minimal constraints on sample acquisition. The machine learning (ML) models involved could be frequently updated to include spectral information about variants without needing to develop new reagents. APPROACH: We present a workflow for collecting, preparing, and imaging dried saliva supernatant droplets using a non-invasive, label-free technique-Raman spectroscopy-to detect changes in the molecular profile of saliva associated with COVID-19 infection. RESULTS: We used an innovative multiple instance learning-based ML approach and droplet segmentation to analyze droplets. Amongst all confounding factors, we discriminated between COVID-positive and COVID-negative individuals yielding receiver operating coefficient curves with an area under curve (AUC) of 0.8 in both males (79% sensitivity and 75% specificity) and females (84% sensitivity and 64% specificity). Taking the sex of the saliva donor into account increased the AUC by 5%. CONCLUSION: These findings may pave the way for new rapid Raman spectroscopic screening tools for COVID-19 and other infectious diseases.

Increased EEG alpha peak frequency in adolescents with idiopathic scoliosis during balance control in normal upright standing.

Adolescent idiopathic scoliosis (AIS) is a multifactorial disorder characterized by a tridimensional deformation of the spine. AIS pathophysiology is still unclear and its aetiology is unknown. Results from several studies revealed balance control alterations in adolescents with AIS suggesting cortical sensorimotor processing impairments. Studies assessing cortical activity involved in balance control revealed an increase in alpha peak frequency (APF), which is a neurophysiological marker of thalamo-cortical transmission, related to a more challenging balance task. The objective of this study is to assess APF of adolescents with AIS during balance control in upright standing posture using electroencephalography (EEG). EEG was recorded in 16 girls with AIS and 15 control girls in normal standing posture on a force platform. The participants stood upright for 2 min with eyes open and 2 min with eyes closed. Fast Fourier transformations of EEG data were calculated to obtain APF. Balance performances were assessed through the area of an ellipse covering the center of pressure (COP) displacement and the root mean square value of the COP velocity. Compared to the control group, APF was higher in the AIS group at central, frontal, parietal and occipital regions. Further, COP analyses did not reveal any difference between AIS and control groups. A higher APF may indicate the need for increased cortical processing to maintain balance control in normal upright standing in adolescents with AIS compared to healthy controls. We suggest that this may be a compensatory strategy to overcome balance control challenges.

Regulation of protein L-isoaspartyl methyltransferase by cell-matrix interactions: involvement of integrin alphavbeta3, PI 3-kinase, and the proteasome.

The enzyme L-isoaspartyl methyltransferase (PIMT) is known to repair damaged proteins that have accumulated abnormal aspartyl residues during cell aging. However, little is known about the mechanisms involved in the regulation of PIMT expression. Here we report that PIMT expression in bovine aortic endothelial cells is regulated by cell detachment and readhesion to a substratum. During cell detachment, the PIMT level was rapidly and strongly increased and correlated with a stimulation of protein synthesis. Aside from endothelial cells, PIMT levels were also regulated by cell adhesion in various cancer cell lines. The upregulation of PIMT expression could be prevented by an anti-alphavbeta3 antibody (LM609) or by a cyclic RGD peptide (XJ735) specific to integrin alphavbeta3, indicating that this integrin was likely involved in PIMT regulation. Moreover, we found that PIMT expression returned to the basal level when cells were replated on a substratum after detachment, though downregulation of PIMT expression could be partly prevented by the PI3K inhibitors LY294002 and wortmannin, as well as by the proteasome inhibitors MG-132, lactacystin, and beta-lactone. These findings support the assumption that the PIMT level was downregulated by proteasomal degradation, involving the PI3K pathway, during cell attachment. This study reports new insights on the molecular mechanisms responsible for the regulation of PIMT expression in cells. The regulation of PIMT level upon cell-substratum contact suggests a potential role for PIMT in biological processes such as wound healing, cell migration, and tumor metastasis dissemination.

Expression and activity of l-isoaspartyl methyltransferase decrease in stage progression of human astrocytic tumors.

Protein l-isoaspartyl methyltransferase (PIMT) functions as a repair enzyme that acts upon damaged proteins bearing abnormal aspartyl residues. We previously reported that PIMT expression and activity are reduced by half in human epileptic hippocampus. Here we investigated PIMT regulation in astrocytic tumors, which are the most common human brain tumors. PIMT expression and enzyme activity were significantly decreased in all grades of human astrocytic tumors. More precisely, PIMT levels were significantly lower by 76% in pilocytic astrocytomas (grade I), 46% in astrocytomas (grade II), 69% in anaplastic astrocytomas (grade III), and a marked 80% in glioblastomas (grade IV) as compared to normal brains. RT-PCR analysis showed that levels of type I PIMT mRNA were up-regulated while those of type II PIMT mRNA were down-regulated in glioblastomas. Furthermore, the reduced PIMT levels correlated closely with a decrease in the number of neuron cells in astrocytic tumors as assessed by measuring the neuron-specific enolase level. Many proteins with abnormal aspartyl residues accumulated in brain tumors and some were specific to individual grades of astrocytic tumors. Similar results were obtained, either by measuring the reduction in PIMT activity and expression or by measuring the formation of abnormal proteins, in an orthotopic rat brain tumor model implanted with invasive CNS-1 glioma cells. The novelty of these findings was to provide the first evidence for a marked reduction of PIMT expression and activity during stage progression of astrocytic tumors in humans.

Protein L-isoaspartyl methyltransferase repairs abnormal aspartyl residues accumulated in vivo in type-I collagen and restores cell migration.

Abnormal aspartyl residue formation such as L-isoaspartates occurs frequently during aging in long-lived proteins, resulting in the alteration of their structures and biological functions. In this study, we investigated the alteration of aspartyl residues in extracellular matrix (ECM) proteins, type-I collagen and fibronectin, and in integrin- and ECM-binding motifs during aging, as well as the resulting effects on cell biological functions such as migration and attachment. Using protein L-isoaspartyl methyltransferase (PIMT) to monitor the presence of L-isoaspartyl residues, we showed their accumulation during in vivo aging in type-I collagen from rats. In vitro aging of fibronectin as well as of peptides containing an integrin- or ECM-binding motif such as RGDSR, KDGEA and KDDL also resulted in the formation of L-isoaspartyl residues. While aged fibronectin does not alter cell adhesion and migration, type-I collagen aged 20 months reduced by 65% cell motility, but not adhesion, when compared to 3-month-aged type-I collagen. Finally, by repairing 20-month-old type-I collagen with recombinant PIMT (rPIMT), cell migration was recovered by 72%. These results strongly suggest that L-isoaspartyl residue formation in ECM proteins such as type-I collagen could play an important role in reducing cell migration and that PIMT could be a therapeutic tool to restore normal cell migration in pathological conditions where cell motility is crucial.

High transcytosis of melanotransferrin (P97) across the blood-brain barrier.

The blood-brain barrier (BBB) performs a neuroprotective function by tightly controlling access to the brain; consequently it also impedes access of proteins as well as pharmacological agents to cerebral tissues. We demonstrate here that recombinant human melanotransferrin (P97) is highly accumulated into the mouse brain following intravenous injection and in situ brain perfusion. Moreover, P97 transcytosis across bovine brain capillary endothelial cell (BBCEC) monolayers is at least 14-fold higher than that of holo-transferrin, with no apparent intra-endothelial degradation. This high transcytosis of P97 was not related to changes in the BBCEC monolayer integrity. In addition, the transendothelial transport of P97 was sensitive to temperature and was both concentration- and conformation-dependent, suggesting that the transport of P97 is due to receptor-mediated endocytosis. In spite of the high degree of sequence identity between P97 and transferrin, a different receptor than the one for transferrin is involved in P97 transendothelial transport. A member of the low-density lipoprotein receptor protein family, likely LRP, seems to be involved in P97 transendothelial transport. The brain accumulation, high rate of P97 transcytosis and its very low level in the blood suggest that P97 could be advantageously employed as a new delivery system to target drugs directly to the brain.

Down-regulation of protein L-isoaspartyl methyltransferase in human epileptic hippocampus contributes to generation of damaged tubulin.

Protein L-isoaspartyl methyltransferase (PIMT) repairs the damaged proteins which have accumulated abnormal aspartyl residues during cell aging. Gene targeting has elucidated a physiological role for PIMT by showing that mice lacking PIMT died prematurely from fatal epileptic seizures. Here we investigated the role of PIMT in human mesial temporal lobe epilepsy. Using surgical specimens of hippocampus and neocortex from controls and epileptic patients, we showed that PIMT activity and expression were 50% lower in epileptic hippocampus than in controls but were unchanged in neocortex. Although the protein was down-regulated, PIMT mRNA expression was unchanged in epileptic hippocampus, suggesting post-translational regulation of the PIMT level. Moreover, several proteins with abnormal aspartyl residues accumulate in epileptic hippocampus. Microtubules component beta-tubulin, one of the major PIMT substrates, had an increased amount (two-fold) of L-isoaspartyl residues in the epileptic hippocampus. These results demonstrate that the down-regulation of PIMT in epileptic hippocampus leads to a significant accumulation of damaged tubulin that could contribute to neuron dysfunction in human mesial temporal lobe epilepsy.