Rapid detection of West Nile and Dengue viruses from mosquito saliva by loop-mediated isothermal amplification and displaced probes.

Arthropod-borne viruses are major causes of human and animal disease, especially in endemic low- and middle-income countries. Mosquito-borne pathogen surveillance is essential for risk assessment and vector control responses. Sentinel chicken serosurveillance (antibody testing) and mosquito pool screening (by RT-qPCR or virus isolation) are currently used to monitor arbovirus transmission, however substantial time lags of seroconversion and/or laborious mosquito identification and RNA extraction steps sacrifice their early warning value. As a consequence, timely vector control responses are compromised. Here, we report on development of a rapid arbovirus detection system whereby adding sucrose to reagents of loop-mediated isothermal amplification with displaced probes (DP-LAMP) elicits infectious mosquitoes to feed directly upon the reagent mix and expectorate viruses into the reagents during feeding. We demonstrate that RNA from pathogenic arboviruses (West Nile and Dengue viruses) transmitted in the infectious mosquito saliva was detectable rapidly (within 45 minutes) without RNA extraction. Sucrose stabilized viral RNA at field temperatures for at least 48 hours, important for transition of this system to practical use. After thermal treatment, the DP-LAMP could be reliably visualized by a simple optical image sensor to distinguish between positive and negative samples based on fluorescence intensity. Field application of this technology could fundamentally change conventional arbovirus surveillance methods by eliminating laborious RNA extraction steps, permitting arbovirus monitoring from additional sites, and substantially reducing time needed to detect circulating pathogens.

Multiplex Surveillance Kit Using Sweetened Solid Support to Detect Arboviruses Isothermally in Mosquito Saliva from the Field.

Recently reported "displaceable probe" loop amplification (DP-LAMP) architecture has shown to amplify viral RNA from SARS-CoV-2 with little sample processing. The architecture allows signals indicating the presence of target nucleic acids to be spatially separated, and independent in sequence, from the complicated concatemer that LAMP processes create as part of their amplification process. This makes DP-LAMP an attractive molecular strategy to integrate with trap and sampling innovations to detect RNA from arboviruses carried by mosquitoes in the field. These innovations include (a) development of organically produced carbon dioxide with ethylene carbonate as a bait deployable in mosquito trap, avoiding the need for dry ice, propane tanks, or inorganic carbonates and (b) a process that induces mosquitoes to lay virus-infected saliva on a quaternary ammonium-functionalized paper (Q-paper) matrix, where (c) the matrix (i) inactivates the deposited viruses, (ii) releases their RNA, and (iii) captures viral RNA in a form that keeps it stable for days at ambient temperatures. We report this integration here, with a surprisingly simple workflow. DP-LAMP with a reverse transcriptase was found to amplify arboviral RNA directly from Q-paper, without requiring a separate elution step. This capture-amplification-detection architecture can be multiplexed, with the entire system integrated into a device that can support a campaign of surveillance, in the wild outdoors, that reports the prevalence of arboviruses from field-captured mosquitoes.

IAP and HDAC inhibitors interact synergistically in myeloma cells through noncanonical NF-κB- and caspase-8-dependent mechanisms.

Interactions between the inhibitor of apoptosis protein antagonist LCL161 and the histone deacetylase inhibitor panobinostat (LBH589) were examined in human multiple myeloma (MM) cells. LCL161 and panobinostat interacted synergistically to induce apoptosis in diverse MM cell lines, including those resistant to bortezomib (PS-R). Similar interactions were observed with other histone deacetylase inhibitors (MS-275) or inhibitors of apoptosis protein antagonists (birinapant). These events were associated with downregulation of the noncanonical (but not the canonical) NF-κB pathway and activation of the extrinsic, caspase-8-related apoptotic cascade. Coexposure of MM cells to LCL161/LBH589 induced TRAF3 upregulation and led to TRAF2 and NIK downregulation, diminished expression of BCL-XL, and induction of γH2A.X. Ectopic expression of TRAF2, NIK, or BCL-XL, or short hairpin RNA TRAF3 knock-down, significantly reduced LCL161/LBH589 lethality, as did ectopic expression of dominant-negative FADD. Stromal/microenvironmental factors failed to diminish LCL161/LBH589-induced cell death. The LCL161/LBH589 regimen significantly increased cell killing in primary CD138+ cells (N = 31) and was particularly effective in diminishing the primitive progenitor cell-enriched CD138-/19+/20+/27+ population (N = 23) but was nontoxic to normal CD34+ cells. Finally, combined LCL161/LBH589 treatment significantly increased survival compared with single-agent treatment in an immunocompetent 5TGM1 murine MM model. Together, these findings argue that LCL161 interacts synergistically with LBH589 in MM cells through a process involving inactivation of the noncanonical NF-κB pathway and activation of the extrinsic apoptotic pathway, upregulation of TRAF3, and downregulation of TRAF2/BCL-XL. Notably, this regimen overcomes various forms of resistance, is active against primary MM cells, and displays significant in vivo activity. This strategy warrants further consideration in MM.

Ultra-rapid detection of SARS-CoV-2 in public workspace environments.

Managing the pandemic caused by SARS-CoV-2 requires new capabilities in testing, including the possibility of identifying, in minutes, infected individuals as they enter spaces where they must congregate in a functioning society, including workspaces, schools, points of entry, and commercial business establishments. Here, the only useful tests (a) require no sample transport, (b) require minimal sample manipulation, (c) can be performed by unlicensed individuals, (d) return results on the spot in much less than one hour, and (e) cost no more than a few dollars. The sensitivity need not be as high as normally required by the FDA for screening asymptomatic carriers (as few as 10 virions per sample), as these viral loads are almost certainly not high enough for an individual to present a risk for forward infection. This allows tests specifically useful for this pandemic to trade-off unneeded sensitivity for necessary speed, simplicity, and frugality. In some studies, it was shown that viral load that creates forward-infection risk may exceed 105 virions per milliliter, easily within the sensitivity of an RNA amplification architecture, but unattainable by antibody-based architectures that simply target viral antigens. Here, we describe such a test based on a displaceable probe loop amplification architecture.

Temporal map of the pig polytrauma plasma proteome with fluid resuscitation and intravenous vitamin C treatment.

BACKGROUND: Fluid resuscitation plays a prominent role in stabilizing trauma patients with hemorrhagic shock yet there remains uncertainty with regard to optimal administration time, volume, and fluid composition (e.g., whole blood, component, colloids) leading to complications such as trauma-induced coagulopathies (TIC), acidosis, and poor oxygen transport. Synthetic fluids in combination with antioxidants (e.g., vitamin C) may resolve some of these problems. OBJECTIVES: We applied quantitative mass spectrometry-based proteomics [liquid chromatography-mass spectrometry (LC-MS/MS)] to map the effects of fluid resuscitation and intravenous vitamin C (VitC) in a pig model of polytrauma (hemorrhagic shock, tissue injury, liver reperfusion, hypothermia, and comminuted bone fracture). The goal was to determine the effects of VitC on plasma protein expression, with respect to changes associated with coagulation and trauma-induced coagulopathy (TIC). METHODS: Longitudinal blood samples were drawn from nine male Sinclair pigs at baseline, 2 h post trauma, and 0.25, 2, and 4 h post fluid resuscitation with 500 mL hydroxyethyl starch. Pigs were treated intravenously (N = 3/treatment group) with saline, 50 mg VitC/kg (Lo-VitC), or 200 mg VitC/kg (Hi-VitC) during fluid resuscitation. RESULTS: A total of 436 plasma proteins were quantified of which 136 changed following trauma and resuscitation; 34 were associated with coagulation, complement cascade, and glycolysis. Unexpectedly, Lo-VitC and Hi-VitC treatments stabilized ADAMTS13 levels by ~4-fold (P = .056) relative to saline and enhanced ADAMTS13/von Willebrand factor (VWF) cleavage efficiency based on LC-MS/MS evidence for the semitryptic VWF cleavage product (VWF1275-1286 ). CONCLUSIONS: This study provides the first comprehensive map of trauma-induced changes to the plasma proteome, especially with respect to proteins driving the development of TIC.

Informing efficient pilot development of animal trauma models through quality improvement strategies.

Poor quality data in preclinical trials can result from inconsistent and unstandardized experimental processes. Unpredictable pre-intervention variability generates unreliable data, biases outcomes and results in needless waste of animals and resources. We applied Define-Measure-Analyse-Improve-Control (DMAIC) quality improvement processes to pilot development of a swine model of trauma, haemorrhagic shock and coagulopathy. The goal was to reduce variability through protocol standardization and error reduction. Six male Sinclair swine were sequentially anesthetized, intubated, mechanically ventilated and instrumented, then subjected to multiple-hit injury, followed by fluid resuscitation monitoring and coagulation testing. Experimental tasks were defined and mapped. Performance measures were task performance times, subject stabilization time and number of task execution errors. Process improvement was assessed by reduced times and errors, and subject stability at target physiological values. Previously-overlooked performance errors and deficiencies were identified. 'Mistake-proofing' actions included personnel retraining, revisions of standard operating procedures and use of checklists. The quality improvement pilot trial produced a stable model with reduced protocol deviations. Data quality can be improved and animal waste minimized, if experimental planning incorporates strategies to ensure protocol adherence and reduced operator performance variation and errors. Properly designed pilot trials can be essential components of refinement and reduction strategies in animal-based research.

Interventional vitamin C: A strategy for attenuation of coagulopathy and inflammation in a swine multiple injuries model.

BACKGROUND: Coagulopathy and inflammation induced by hemorrhagic shock and traumatic injury are associated with increased mortality and morbidity. Vitamin C (VitC) is an antioxidant with potential protective effects on the proinflammatory and procoagulant pathways. We hypothesized that high-dose VitC administered as a supplement to fluid resuscitation would attenuate inflammation, coagulation dysfunction, and end-organ tissue damage in a swine model of multiple injuries and hemorrhage. METHODS: Male Sinclair swine (n = 24; mean body weight, 27 kg) were anesthetized, intubated, mechanically ventilated, and instrumented for physiologic monitoring. Following stabilization, swine were subjected to shock/traumatic injury (hypothermia, liver ischemia and reperfusion, comminuted femur fracture, hemorrhagic hypotension), resuscitated with 500 mL of hydroxyethyl starch, and randomized to receive either intravenous normal saline (NS), low-dose VitC (50 mg/kg; LO), or high-dose VitC (200 mg/kg; HI). Hemodynamics, blood chemistry, hematology, and coagulation function (ROTEM) were monitored to 4 hours postresuscitation. Histological and molecular analyses were obtained for liver, kidney, and lung. RESULTS: Compared with VitC animals, NS swine showed significant histological end-organ damage, elevated acute lung injury scores, and increased mRNA expression of tissue proinflammatory mediators (IL-1ß, IL-8, TNFα), plasminogen activation inhibitor-1 and tissue factor. There were no statistically significant differences between treatment groups on mean arterial pressure or univariate measures of coagulation function; however, NS showed impaired multivariate clotting function at 4 hours. CONCLUSION: Although correction of coagulation dysfunction was modest, intravenous high-dose VitC may mitigate the proinflammatory/procoagulant response that contributes to multiple organ failure following acute severe multiple injuries. LEVEL OF EVIDENCE: Prospective randomized controlled blinded trial study, Preclinical (animal-based).