Role of pancreatic cancer-derived exosomes in salivary biomarker development.

Recent studies have demonstrated that discriminatory salivary biomarkers can be readily detected upon the development of systemic diseases such as pancreatic cancer, breast cancer, lung cancer, and ovarian cancer. However, the utility of salivary biomarkers for the detection of systemic diseases has been undermined due to the absence of the biological and mechanistic rationale as to why distal diseases from the oral cavity would lead to the development of discriminatory biomarkers in saliva. Here, we examine the hypothesis that pancreatic tumor-derived exosomes are mechanistically involved in the development of pancreatic cancer-discriminatory salivary transcriptomic biomarkers. We first developed a pancreatic cancer mouse model that yielded discriminatory salivary biomarkers by implanting the mouse pancreatic cancer cell line Panc02 into the pancreas of the syngeneic host C57BL/6. The role of pancreatic cancer-derived exosomes in the development of discriminatory salivary biomarkers was then tested by engineering a Panc02 cell line that is suppressed for exosome biogenesis, implanting into the C56BL/6 mouse, and examining whether the discriminatory salivary biomarker profile was ablated or disrupted. Suppression of exosome biogenesis results in the ablation of discriminatory salivary biomarker development. This study supports that tumor-derived exosomes provide a mechanism in the development of discriminatory biomarkers in saliva and distal systemic diseases.

Direct Detection of 4-Dimensions of SARS-CoV-2: Infection (vRNA), Infectivity (Antigen), Binding Antibody, and Functional Neutralizing Antibody in Saliva.

We developed a 4-parameter clinical assay using Electric Field Induced Release and Measurement (EFIRM) technology to simultaneously assess SARS-CoV-2 RNA (vRNA), nucleocapsid antigen, host binding (BAb) and neutralizing antibody (NAb) levels from a drop of saliva with performance that equals or surpasses current EUA-approved tests. The vRNA and antigen assays achieved lower limit of detection (LOD) of 100 copies/reaction and 3.5 TCID50/mL, respectively. The vRNA assay differentiated between acutely infected (n=10) and infection-naïve patients (n=33) with an AUC of 0.9818, sensitivity of 90%, and specificity of 100%. The antigen assay similarly differentiated these patient populations with an AUC of 1.000. The BAb assay detected BAbs with an LOD of 39 pg/mL and distinguished acutely infected (n=35), vaccinated with prior infection (n=13), and vaccinated infection-naïve patients (n=13) from control (n=81) with AUC of 0.9481, 1.000, and 0.9962, respectively. The NAb assay detected NAbs with an LOD of 31.6 Unit/mL and differentiated between COVID-19 recovered or vaccinated patients (n=31) and pre-pandemic controls (n=60) with an AUC 0.923, sensitivity of 87.10%, and specificity of 86.67%. Our multiparameter assay represents a significant technological advancement to simultaneously address SARS-CoV-2 infection and immunity, and it lays the foundation for tackling potential future pandemics.

Neutrophil elastase acts as a biased agonist for proteinase-activated receptor-2 (PAR2).

Human neutrophil proteinases (elastase, proteinase-3, and cathepsin-G) are released at sites of acute inflammation. We hypothesized that these inflammation-associated proteinases can affect cell signaling by targeting proteinase-activated receptor-2 (PAR(2)). The PAR family of G protein-coupled receptors is triggered by a unique mechanism involving the proteolytic unmasking of an N-terminal self-activating tethered ligand (TL). Proteinases can either activate PAR signaling by unmasking the TL sequence or disarm the receptor for subsequent enzyme activation by cleaving downstream from the TL sequence. We found that none of neutrophil elastase, cathepsin-G, and proteinase-3 can activate G(q)-coupled PAR(2) calcium signaling; but all of these proteinases can disarm PAR(2), releasing the N-terminal TL sequence, thereby preventing G(q)-coupled PAR(2) signaling by trypsin. Interestingly, elastase (but neither cathepsin-G nor proteinase-3) causes a TL-independent PAR(2)-mediated activation of MAPK that, unlike the canonical trypsin activation, does not involve either receptor internalization or recruitment of ß-arrestin. Cleavage of synthetic peptides derived from the extracellular N terminus of PAR(2), downstream of the TL sequence, demonstrated distinct proteolytic sites for all three neutrophil-derived enzymes. We conclude that in inflammation, neutrophil proteinases can modulate PAR(2) signaling by preventing/disarming the G(q)/calcium signal pathway and, via elastase, can selectively activate the p44/42 MAPK pathway. Our data illustrate a new mode of PAR regulation that involves biased PAR(2) signaling by neutrophil elastase and a disarming/silencing effect of cathepsin-G and proteinase-3.

Apical and basolateral pools of proteinase-activated receptor-2 direct distinct signaling events in the intestinal epithelium.

Studies suggest that there are two distinct pools of proteinase-activated receptor-2 (PAR2) present in intestinal epithelial cells: an apical pool accessible from the lumen, and a basolateral pool accessible from the interstitial space and blood. Although introduction of PAR2 agonists such as 2-furoyl-LIGRL-O-NH2 (2fAP) to the intestinal lumen can activate PAR2, the presence of accessible apical PAR2 has not been definitively shown. Furthermore, some studies have suggested that basolateral PAR2 responses in the intestinal epithelium are mediated indirectly by neuropeptides released from enteric nerve fibers, rather than by intestinal PAR2 itself. Here we identified accessible pools of both apical and basolateral PAR2 in cultured Caco2-BBe monolayers and in mouse ileum. Activation of basolateral PAR2 transiently increased short-circuit current by activating electrogenic Cl⁻ secretion, promoted dephosphorylation of the actin filament-severing protein, cofilin, and activated the transcription factor, AP-1, whereas apical PAR2 did not. In contrast, both pools of PAR2 activated extracellular signal-regulated kinase 1/2 (ERK1/2) via temporally and mechanistically distinct pathways. Apical PAR2 promoted a rapid, biphasic PLCß/Ca²(+)/PKC-dependent ERK1/2 activation, resulting in nuclear localization, whereas basolateral PAR2 promoted delayed ERK1/2 activation which was predominantly restricted to the cytosol, involving both PLCß/Ca²(+) and ß-arrestin-dependent pathways. These results suggest that the outcome of PAR2 activation is dependent on the specific receptor pool that is activated, allowing for fine-tuning of the physiological responses to different agonists.

A conserved protein interaction network involving the yeast MAP kinases Fus3 and Kss1.

The Saccharomyces cerevisiae mitogen-activated protein kinases (MAPKs) Fus3 and Kss1 bind to multiple regulators and substrates. We show that mutations in a conserved docking site in these MAPKs (the CD/7m region) disrupt binding to an important subset of their binding partners, including the Ste7 MAPK kinase, the Ste5 adaptor/scaffold protein, and the Dig1 and Dig2 transcriptional repressors. Supporting the possibility that Ste5 and Ste7 bind to the same region of the MAPKs, they partially competed for Fus3 binding. In vivo, some of the MAPK mutants displayed reduced Ste7-dependent phosphorylation, and all of them exhibited multiple defects in mating and pheromone response. The Kss1 mutants were also defective in Kss1-imposed repression of Ste12. We conclude that MAPKs contain a structurally and functionally conserved docking site that mediates an overall positively acting network of interactions with cognate docking sites on their regulators and substrates. Key features of this interaction network appear to have been conserved from yeast to humans.

Decision-to-delivery intervals and total duration of surgery for Caesarean sections in a tertiary general hospital.

INTRODUCTION: This study aimed to determine the decision-to-delivery intervals (DDIs), total duration of surgery and factors influencing these for Caesarean sections (CSs). METHODS: A retrospective study was conducted of all CSs performed from August 2013 to June 2014 at a single tertiary general hospital. Data collected included maternal demographics, indications for CS, category of urgency, DDI, total duration of surgery, grade of first surgeon and number of previous CSs. RESULTS: In total, 488 CSs (Category 1: n = 28; Category 2: n = 137; Category 3: n = 184; Category 4: n = 139) were studied. Overall mean duration of surgery was 41.7 minutes. Mean DDI was 23.9 minutes and 64.5 minutes for Category 1 and Category 2 CSs, respectively. For Category 1 CSs, deliveries during office hours had a significantly shorter DDI than deliveries out of office hours (p < 0.05). For Category 2 CSs, deliveries during office hours had a significantly longer DDI (p < 0.05). Total duration of surgery for senior surgeons was significantly shorter than for trainee surgeons (p < 0.05). Women with no previous CSs had a significantly shorter duration of surgery than those who had one or more (p < 0.05). CONCLUSION: The majority of the deliveries were within the recommended DDI corresponding to the degree of urgency of CS. The influence of time of day on DDI might be due to challenges of time taken to transfer patients to operating theatres. Total duration of surgery was influenced by surgical experience, history of previous CS and individual surgical styles and preferences.

A review of Caesarean section techniques and postoperative thromboprophylaxis at a tertiary hospital.

INTRODUCTION: Although Caesarean sections (CSs) are among the most commonly undertaken procedures in the world, there are wide variations in the surgical techniques used. This study aimed to: (a) review the surgical techniques used for CS by obstetricians working in a tertiary hospital in Singapore; (b) compare the techniques with those recommended in evidence-based guidelines; and (c) examine the relationship between the technique used and the level of seniority of the surgeons. METHODS: Data on 490 CSs performed in Singapore General Hospital (SGH) between 1 August 2013 and 30 June 2014 was collected from the Delivery Suite database and reviewed. The surgical techniques studied were closure of the pelvic and parietal peritoneum, closure of the uterine layer, use of surgical drains and use of postoperative thromboprophylaxis. RESULTS: A total of 486 CSs were analysed after four cases were excluded due to missing data. Most fetal head deliveries were manual. The majority of surgeons did not close the peritoneum; most of those who did were senior surgeons. Double-layer uterine closures were done for all cases and drain usage was rare. 2.0% of the patients received grossly inadequate thromboprophylaxis. CONCLUSION: The surgical techniques currently practised in SGH are closely aligned with those of the evidence-based guidelines. Peritoneal closure appears to be associated with the surgeon's early training, with a greater number of senior surgeons being less willing to abandon this step. Greater vigilance in implementing appropriate thromboprophylaxis is recommended.

Breast cancer exosome-like microvesicles and salivary gland cells interplay alters salivary gland cell-derived exosome-like microvesicles in vitro.

Saliva is a useful biofluid for the early detection of disease, but how distal tumors communicate with the oral cavity and create disease-specific salivary biomarkers remains unclear. Using an in vitro breast cancer model, we demonstrated that breast cancer-derived exosome-like microvesicles are capable of interacting with salivary gland cells, altering the composition of their secreted exosome-like microvesicles. We found that the salivary gland cells secreted exosome-like microvesicles encapsulating both protein and mRNA. We also showed that the interaction with breast cancer-derived exosome-like microvesicles communicated and activated the transcriptional machinery of the salivary gland cells. Thus, the interaction altered the composition of the salivary gland cell-derived exosome-like microvesicles on both the transcriptomically and proteomically.

Computer-calculated kanamycin dosage regimen and monitoring.

The applicability of a computerized dosage regimen determination based on a recently developed method for drugs following the minimum inhibitory concentration pattern (bacteriostatic drugs) has been tested for kanamycin injected intramuscularly in 8 patients with varying degrees of renal impairment. The dosage regimens for maintaining a minimum therapeutic kanamycin concentration of 5.0 micron g/ml was determined with the Wang 700 C Computer. After the 1st, 2nd, 3rd, 6th, 9th, and 12th dosing interval blood samples were taken and the acual kanamycin serum concentrations determined. The expected multiple dose serum level curves for each patient were simulated using the Comdyna Dose Generator Analog Computer and the individual actual serum concentrations were monitored. The actually found serum level data were compared to the expected values based on the dosage regimen equations and to the steady state equations. Additionally, the expected peak maxima were calculated. The proposed method resulted in effective and safe kanamycin serum levels.