Mass Spectrometry Multiplexed Detection of SARS-CoV-2.

Testing of large populations for virus infection is now a reality worldwide due to the coronavirus (SARS-CoV-2) pandemic. The demand for SARS-CoV-2 testing using alternatives other than PCR led to the development of mass spectrometry (MS)-based assays. However, MS for SARS-CoV-2 large-scale testing have some downsides, including complex sample preparation and slow data analysis. Here, we describe a high-throughput targeted proteomics method to detect SARS-CoV-2 directly from nasopharyngeal and oropharyngeal swabs. This strategy employs fully automated sample preparation mediated by magnetic particles, followed by detection of SARS-CoV-2 nucleoprotein peptides by turbulent flow chromatography coupled with tandem mass spectrometry.

Establishing a mass spectrometry-based system for rapid detection of SARS-CoV-2 in large clinical sample cohorts.

The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is pressing public health systems around the world, and large population testing is a key step to control this pandemic disease. Here, we develop a high-throughput targeted proteomics assay to detect SARS-CoV-2 nucleoprotein peptides directly from nasopharyngeal and oropharyngeal swabs. A modified magnetic particle-based proteomics approach implemented on a robotic liquid handler enables fully automated preparation of 96 samples within 4 hours. A TFC-MS system allows multiplexed analysis of 4 samples within 10 min, enabling the processing of more than 500 samples per day. We validate this method qualitatively (Tier 3) and quantitatively (Tier 1) using 985 specimens previously analyzed by real-time RT-PCR, and detect up to 84% of the positive cases with up to 97% specificity. The presented strategy has high sample stability and should be considered as an option for SARS-CoV-2 testing in large populations.

The Primary Duct of Bothrops jararaca Glandular Apparatus Secretes Toxins.

Despite numerous studies concerning morphology and venom production and secretion in the main venom gland (and some data on the accessory gland) of the venom glandular apparatus of Viperidae snakes, the primary duct has been overlooked. We characterized the primary duct of the Bothrops jararaca snake by morphological analysis, immunohistochemistry and proteomics. The duct has a pseudostratified epithelium with secretory columnar cells with vesicles of various electrondensities, as well as mitochondria-rich, dark, basal, and horizontal cells. Morphological analysis, at different periods after venom extraction, showed that the primary duct has a long cycle of synthesis and secretion, as do the main venom and accessory glands; however, the duct has a mixed mode venom storage, both in the lumen and in secretory vesicles. Mouse anti-B. jararaca venom serum strongly stained the primary duct's epithelium. Subsequent proteomic analysis revealed the synthesis of venom toxins-mainly C-type lectin/C-type lectin-like proteins. We propose that the primary duct's toxin synthesis products complement the final venom bolus. Finally, we hypothesize that the primary duct and the accessory gland (components of the venom glandular apparatus) are part of the evolutionary path from a salivary gland towards the main venom gland.

Bothrops jararaca accessory venom gland is an ancillary source of toxins to the snake.

In Viperidae snakes, it has been attributed to the main venom gland, a component of the venom gland apparatus, the function of synthesizing all venom toxins and storing them inside a basal-central lumen. However, the role of the accessory gland is still unknown. Here, we analyzed the proteome and the transcriptome of the accessory gland during venom production and secretion cycle. We showed that the accessory gland expresses and synthesizes toxins that are similar to those produced by the main venom gland such as C-type lectin/C-type lectin-like proteins, metalloproteinase, phospholipase A2, cysteine rich secretory protein, nerve growth factor, vascular endothelial growth factor, serine proteinase, and l-amino acid oxidase. Our data have shown that toxin synthesis in the accessory gland is asynchronous when compared to the same process in the venom gland. Moreover, this gland also expresses inhibitors of venom phospholipases A2 and metalloproteinases. Transcriptome analysis showed that the transcripts that correspond to toxins in the accessory gland have a good correlation to the main venom gland transcripts. Therefore, it is proposed that the accessory gland is an ancillary source of toxins to the snake, and provides inhibitors that could control venom toxicity (and integrity) during storage. SIGNIFICANCE: In this study, we propose that the accessory venom gland acts as an important ancillary source of toxins to the snake, in lieu of a depleted main venom gland, and provides inhibiting agents that control venom toxicity (and integrity) during its storage.

Bothrops jararaca accessory venom gland is an ancillary source of toxins to the snake

In Viperidae snakes, it has been attributed to the main venom gland, a component of the venom gland apparatus, the function of synthesizing all venom toxins and storing them inside a basal-central lumen. However, the role of the accessory gland is still unknown. Here, we analyzed the proteome and the transcriptome of the accessory gland during venom production and secretion cycle. We showed that the accessory gland expresses and synthesizes toxins that are similar to those produced by the main venom gland such as C-type lectin/C-type lectin-like proteins, metalloproteinase, phospholipase A(2), cysteine rich secretory protein, nerve growth factor, vascular endothelial growth factor, serine proteinase, and L-amino acid oxidase. Our data have shown that toxin synthesis in the accessory gland is asynchronous when compared to the same process in the venom gland. Moreover, this gland also expresses inhibitors of venom phospholipases A(2) and metalloproteinases. Transcriptome analysis showed that the transcripts that correspond to toxins in the accessory gland have a good correlation to the main venom gland transcripts. Therefore, it is proposed that the accessory gland is an ancillary source of toxins to the snake, and provides inhibitors that could control venom toxicity (and integrity) during storage. Significance: In this study, we propose that the accessory venom gland acts as an important ancillary source of toxins to the snake, in lieu of a depleted main venom gland, and provides inhibiting agents that control venom toxicity (and integrity) during its storage.

The primary duct of Bothrops jararaca glandular apparatus secretes toxins

Despite numerous studies concerning morphology and venom production and secretion in the main venom gland (and some data on the accessory gland) of the venom glandular apparatus of Viperidae snakes, the primary duct has been overlooked. We characterized the primary duct of the Bothrops jararaca snake by morphological analysis, immunohistochemistry and proteomics. The duct has a pseudostratified epithelium with secretory columnar cells with vesicles of various electrondensities, as well as mitochondria-rich, dark, basal, and horizontal cells. Morphological analysis, at different periods after venom extraction, showed that the primary duct has a long cycle of synthesis and secretion, as do the main venom and accessory glands; however, the duct has a mixed mode venom storage, both in the lumen and in secretory vesicles. Mouse anti-B. jararaca venom serum strongly stained the primary duct's epithelium. Subsequent proteomic analysis revealed the synthesis of venom toxins-mainly C-type lectin/C-type lectin-like proteins. We propose that the primary duct's toxin synthesis products complement the final venom bolus. Finally, we hypothesize that the primary duct and the accessory gland (components of the venom glandular apparatus) are part of the evolutionary path from a salivary gland towards the main venom gland.

Bothrops jararaca accessory venom gland is an ancillary source of toxins to the snake

In Viperidae snakes, it has been attributed to the main venom gland, a component of the venom gland apparatus, the function of synthesizing all venom toxins and storing them inside a basal-central lumen. However, the role of the accessory gland is still unknown. Here, we analyzed the proteome and the transcriptome of the accessory gland during venom production and secretion cycle. We showed that the accessory gland expresses and synthesizes toxins that are similar to those produced by the main venom gland such as C-type lectin/C-type lectin-like proteins, metalloproteinase, phospholipase A(2), cysteine rich secretory protein, nerve growth factor, vascular endothelial growth factor, serine proteinase, and L-amino acid oxidase. Our data have shown that toxin synthesis in the accessory gland is asynchronous when compared to the same process in the venom gland. Moreover, this gland also expresses inhibitors of venom phospholipases A(2) and metalloproteinases. Transcriptome analysis showed that the transcripts that correspond to toxins in the accessory gland have a good correlation to the main venom gland transcripts. Therefore, it is proposed that the accessory gland is an ancillary source of toxins to the snake, and provides inhibitors that could control venom toxicity (and integrity) during storage. Significance: In this study, we propose that the accessory venom gland acts as an important ancillary source of toxins to the snake, in lieu of a depleted main venom gland, and provides inhibiting agents that control venom toxicity (and integrity) during its storage.

The primary duct of Bothrops jararaca glandular apparatus secretes toxins

Despite numerous studies concerning morphology and venom production and secretion in the main venom gland (and some data on the accessory gland) of the venom glandular apparatus of Viperidae snakes, the primary duct has been overlooked. We characterized the primary duct of the Bothrops jararaca snake by morphological analysis, immunohistochemistry and proteomics. The duct has a pseudostratified epithelium with secretory columnar cells with vesicles of various electrondensities, as well as mitochondria-rich, dark, basal, and horizontal cells. Morphological analysis, at different periods after venom extraction, showed that the primary duct has a long cycle of synthesis and secretion, as do the main venom and accessory glands; however, the duct has a mixed mode venom storage, both in the lumen and in secretory vesicles. Mouse anti-B. jararaca venom serum strongly stained the primary duct’s epithelium. Subsequent proteomic analysis revealed the synthesis of venom toxins—mainly C-type lectin/C-type lectin-like proteins. We propose that the primary duct’s toxin synthesis products complement the final venom bolus. Finally, we hypothesize that the primary duct and the accessory gland (components of the venom glandular apparatus) are part of the evolutionary path from a salivary gland towards the main venom gland.

Bothrops jararaca venom gland secretory cells in culture: Effects of noradrenaline on toxin production and secretion.

Primary culture of snake venom gland secretory cells could be a good model to study the mechanism(s) of toxin(s) production. These cells can produce and secrete venom to the medium with a hemorrhagic activity comparable to that induced by venom collected from snakes. Production of new venom is triggered by the sympathetic outflow, through the release of noradrenaline, but the importance of this neurotransmitter on toxin synthesis has not been addressed. This work led to the identification and comparison of the toxin panel produced by cultured secretory cells, during a 12-day time-course analysis, as well as to the effects of noradrenaline on the process. The results showed that in our culture model the synthesis of new toxins is asynchronous, mimicking data previously published from proteomic analyses of venom glands harvested from animal experimentation. Furthermore, noradrenaline did regulate the synthesis and/or secretion of venom toxins over the analyzed period. Finally, we demonstrated that snake venom metalloproteinases present in these cultured cells secretome were mostly in their zymogen forms; consequently, processing occurs after secretion to the gland lumen. Overall, the data support the use of venom gland secretory cells as a reliable model to investigate the mechanism(s) of toxin(s) synthesis and secretion.

Bothrops jararaca venom gland secretory cells in culture: effects of noradrenaline on toxin production and secretion

Primary culture of snake venom gland secretory cells could be a good model to study the mechanism(s) of toxin(s) production. These cells can produce and secrete venom to the medium with a hemorrhagic activity comparable to that induced by venom collected from snakes. Production of new venom is triggered by the sympathetic outflow, through the release of noradrenaline, but the importance of this neurotransmitter on toxin synthesis has not been addressed. This work led to the identification and comparison of the toxin panel produced by cultured secretory cells, during a 12-day time-course analysis, as well as to the effects of noradrenaline on the process. The results showed that in our culture model the synthesis of new toxins is asynchronous, mimicking data previously published from proteomic analyses of venom glands harvested from animal experimentation. Furthermore, noradrenaline did regulate the synthesis and/or secretion of venom toxins over the analyzed period. Finally, we demonstrated that snake venom metalloproteinases present in these cultured cells secretome were mostly in their zymogen forms; consequently, processing occurs after secretion to the gland lumen. Overall, the data support the use of venom gland secretory cells as a reliable model to investigate the mechanism(s) of toxin(s) synthesis and secretion.

Insulin-regulated aminopeptidase in adipocyte is Cys-specific and affected by obesity.

Insulin-regulated aminopeptidase (IRAP, EC 3.4.11.3) in adipocytes is well known to traffic between high (HDM) and low (LDM) density microsomal fractions toward the plasma membrane (MF) under stimulation by insulin. However, its catalytic preference for aminoacyl substrates with N-terminal Leu or Cys is controversial. Furthermore, possible changes in its traffic under metabolic challenges are unknown. The present study investigated the catalytic activity attributable to EC 3.4.11.3 in HDM, LDM and MF from isolated adipocytes of healthy (C), food deprived (FD) and monosodium glutamate (MSG) obese rats on aminoacyl substrates with N-terminal Cys or Leu, in absence or presence of insulin. Efficacy and reproducibility of subcellular adipocyte fractionation procedure were demonstrated. Comparison among HDM vs LDM vs MF intragroup revealed that hydrolytic activity trafficking from LDM to MF under influence of insulin in C, MSG and FD is only on N-terminal Cys. In MSG the same pattern of anterograde traffic and aminoacyl preference occurred independently of insulin stimulation. The pathophysiological significance of IRAP in adipocytes seems to be linked to comprehensive energy metabolism related roles of endogenous substrates with N-terminal cysteine pair such as vasopressin and oxytocin.