Optimizing polymer lab-on-chip platforms for ultrasonic manipulation: Influence of the substrate

The choice of substrate material in a chip that combines ultrasound with microfluidics for handling biological and synthetic microparticles can have a profound effect on the performance of the device. This is due to the high surface-to-volume ratio that exists within such small structures and acquires particular relevance in polymer-based resonators with 3D standing waves. This paper presents three chips developed to perform particle flow-through separation by ultrasound based on a polymeric SU-8 layer containing channelization over three different substrates: Polymethyl methacrylate (PMMA); Pyrex; and a cracked PMMA composite-like structure. Through direct observations of polystyrene microbeads inside the channel, the three checked chips exhibit their potential as disposable continuous concentration devices with different spatial pressure patterns at frequencies of resonance close to 1 Mhz. Chips with Pyrex and cracked PMMA substrates show restrictions on the number of pressure nodes established in the channel associated with the inhibition of 3D modes in the solid structure. The glass-substrate chip presents some advantages associated with lower energy requirements to collect particles. According to the results, the use of polymer-based chips with rigid substrates can be advantageous for applications that require short treatment times (clinical tests handling human samples) and low-cost fabrication. © 2015 by the authors; licensee MDPI, Basel, Switzerland.The study has been performed in the framework of two Spanish National Research Project BIO2011-30535-C04-01,02,03, “Development of a high throughput for isolation of tumor cells circulating in peripheral blood”.We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI).Peer Reviewe

Avidity engineering of human heavy-chain-only antibodies mitigates neutralization resistance of SARS-CoV-2 variants

Emerging SARS-CoV-2 variants have accrued mutations within the spike protein rendering most therapeutic monoclonal antibodies against COVID-19 ineffective. Hence there is an unmet need for broad-spectrum mAb treatments for COVID-19 that are more resistant to antigenically drifted SARS-CoV-2 variants. Here we describe the design of a biparatopic heavy-chain-only antibody consisting of six antigen binding sites recognizing two distinct epitopes in the spike protein NTD and RBD. The hexavalent antibody showed potent neutralizing activity against SARS-CoV-2 and variants of concern, including the Omicron sub-lineages BA.1, BA.2, BA.4 and BA.5, whereas the parental components had lost Omicron neutralization potency. We demonstrate that the tethered design mitigates the substantial decrease in spike trimer affinity seen for escape mutations for the hexamer components. The hexavalent antibody protected against SARS-CoV-2 infection in a hamster model. This work provides a framework for designing therapeutic antibodies to overcome antibody neutralization escape of emerging SARS-CoV-2 variants

Study of the structure and function of the influenza virus RNA-dependent RNA polymerase

Influenza viruses are segmented, negative-sense RNA viruses responsible for influenza disease. Unlike other negative-sense RNA viruses, influenza virus genome replication occurs in the nucleus of infected cells. The virus encodes a heterotrimeric RNA-dependent RNA polymerase (FluPol) that is responsible for transcribing and replicating the viral genome. In order to transcribe, the FluPol generates capped RNA primers by a process termed âcap-snatchingâ. In it, the polymerase binds host capped RNAs and cleaves them 10-13 nucleotides downstream of the cap structure. The catalytic core of FluPol is found in the PB1 (Polymerase Basic protein 1) subunit, which associates with PB2 (Polymerase Basic protein 2) and PA (Polymerase Acidic protein). PB2 and PA contain a cap binding domain and an endonuclease domain respectively, which enable cap-snatching to take place. Viral transcription and replication occur in the context of a ribonucleoprotein (RNP) complex, where an RNA segment is coated by nucleoprotein and bound by a single copy of FluPol. Regulation of transcription and replication during infection is dependent on multiple viral and host factors, including nucleoprotein, cellular DNA-dependent RNA polymerase II (Pol II) and nuclear import factors. While high-resolution structures of the FluPol heterotrimer and low-resolution structures of RNPs are available, multiple aspects of the mechanisms involved in polymerase activity and its modulation remain poorly understood. In this DPhil project, I have employed a combination of functional and structural approaches towards exploring FluPol activity. Here I present the work carried out towards optimising the methods for FluPol structural analysis, more specifically, the use of cryo-electron microscopy to explore both the structure of the heterotrimer in complex with modulators of polymerase activity as well as RNPs. I also describe the work carried out on characterisation of the requirements for transcription and replication by FluPol. This includes the role of Pol II in transcription regulation as well as those of FluPol, nucleoprotein and host factor ANP32 in controlling viral replication. Furthermore, I also present the characterisation of a panel of 24 camelid single-chain antibodies, known as nanobodies, as tools to explore polymerase activity. Within these, nanobodies which exhibited robust inhibition of all polymerase activity were found, which opens the possibility of employing these to identify sites on the polymerase that would be suitable targets for small molecule inhibitor design.</p

Study of the structure and function of the influenza virus RNA-dependent RNA polymerase

Influenza viruses are segmented, negative-sense RNA viruses responsible for influenza disease. Unlike other negative-sense RNA viruses, influenza virus genome replication occurs in the nucleus of infected cells. The virus encodes a heterotrimeric RNA-dependent RNA polymerase (FluPol) that is responsible for transcribing and replicating the viral genome. In order to transcribe, the FluPol generates capped RNA primers by a process termed “cap-snatching”. In it, the polymerase binds host capped RNAs and cleaves them 10-13 nucleotides downstream of the cap structure. The catalytic core of FluPol is found in the PB1 (Polymerase Basic protein 1) subunit, which associates with PB2 (Polymerase Basic protein 2) and PA (Polymerase Acidic protein). PB2 and PA contain a cap binding domain and an endonuclease domain respectively, which enable cap-snatching to take place. Viral transcription and replication occur in the context of a ribonucleoprotein (RNP) complex, where an RNA segment is coated by nucleoprotein and bound by a single copy of FluPol. Regulation of transcription and replication during infection is dependent on multiple viral and host factors, including nucleoprotein, cellular DNA-dependent RNA polymerase II (Pol II) and nuclear import factors. While high-resolution structures of the FluPol heterotrimer and low-resolution structures of RNPs are available, multiple aspects of the mechanisms involved in polymerase activity and its modulation remain poorly understood. In this DPhil project, I have employed a combination of functional and structural approaches towards exploring FluPol activity. Here I present the work carried out towards optimising the methods for FluPol structural analysis, more specifically, the use of cryo-electron microscopy to explore both the structure of the heterotrimer in complex with modulators of polymerase activity as well as RNPs. I also describe the work carried out on characterisation of the requirements for transcription and replication by FluPol. This includes the role of Pol II in transcription regulation as well as those of FluPol, nucleoprotein and host factor ANP32 in controlling viral replication. Furthermore, I also present the characterisation of a panel of 24 camelid single-chain antibodies, known as nanobodies, as tools to explore polymerase activity. Within these, nanobodies which exhibited robust inhibition of all polymerase activity were found, which opens the possibility of employing these to identify sites on the polymerase that would be suitable targets for small molecule inhibitor design.</p

Flavivirus maturation leads to the formation of an occupied lipid pocket in the surface glycoproteins

Here, the authors provide cryo-EM structures of mature and immature Spondweni virus, defining the furin recognition site at high resolution, and identifying a lipid that binds E upon capsid maturation and is also present in Zika and Dengue virions

Avidity engineering of human heavy-chain-only antibodies mitigates neutralization resistance of SARS-CoV-2 variants

Emerging SARS-CoV-2 variants have accrued mutations within the spike protein rendering most therapeutic monoclonal antibodies against COVID-19 ineffective. Hence there is an unmet need for broad-spectrum mAb treatments for COVID-19 that are more resistant to antigenically drifted SARS-CoV-2 variants. Here we describe the design of a biparatopic heavy-chain-only antibody consisting of six antigen binding sites recognizing two distinct epitopes in the spike protein NTD and RBD. The hexavalent antibody showed potent neutralizing activity against SARS-CoV-2 and variants of concern, including the Omicron sub-lineages BA.1, BA.2, BA.4 and BA.5, whereas the parental components had lost Omicron neutralization potency. We demonstrate that the tethered design mitigates the substantial decrease in spike trimer affinity seen for escape mutations for the hexamer components. The hexavalent antibody protected against SARS-CoV-2 infection in a hamster model. This work provides a framework for designing therapeutic antibodies to overcome antibody neutralization escape of emerging SARS-CoV-2 variants

CT or Invasive Coronary Angiography in Stable Chest Pain.

Background: In the diagnosis of obstructive coronary artery disease (CAD), computed tomography (CT) is an accurate, noninvasive alternative to invasive coronary angiography (ICA). However, the comparative effectiveness of CT and ICA in the management of CAD to reduce the frequency of major adverse cardiovascular events is uncertain. Methods: We conducted a pragmatic, randomized trial comparing CT with ICA as initial diagnostic imaging strategies for guiding the treatment of patients with stable chest pain who had an intermediate pretest probability of obstructive CAD and were referred for ICA at one of 26 European centers. The primary outcome was major adverse cardiovascular events (cardiovascular death, nonfatal myocardial infarction, or nonfatal stroke) over 3.5 years. Key secondary outcomes were procedure-related complications and angina pectoris. Results: Among 3561 patients (56.2% of whom were women), follow-up was complete for 3523 (98.9%). Major adverse cardiovascular events occurred in 38 of 1808 patients (2.1%) in the CT group and in 52 of 1753 (3.0%) in the ICA group (hazard ratio, 0.70; 95% confidence interval [CI], 0.46 to 1.07; P = 0.10). Major procedure-related complications occurred in 9 patients (0.5%) in the CT group and in 33 (1.9%) in the ICA group (hazard ratio, 0.26; 95% CI, 0.13 to 0.55). Angina during the final 4 weeks of follow-up was reported in 8.8% of the patients in the CT group and in 7.5% of those in the ICA group (odds ratio, 1.17; 95% CI, 0.92 to 1.48). Conclusions: Among patients referred for ICA because of stable chest pain and intermediate pretest probability of CAD, the risk of major adverse cardiovascular events was similar in the CT group and the ICA group. The frequency of major procedure-related complications was lower with an initial CT strategy. (Funded by the European Union Seventh Framework Program and others; DISCHARGE ClinicalTrials.gov number, NCT02400229.)

Evolution over Time of Ventilatory Management and Outcome of Patients with Neurologic Disease∗

OBJECTIVES: To describe the changes in ventilator management over time in patients with neurologic disease at ICU admission and to estimate factors associated with 28-day hospital mortality. DESIGN: Secondary analysis of three prospective, observational, multicenter studies. SETTING: Cohort studies conducted in 2004, 2010, and 2016. PATIENTS: Adult patients who received mechanical ventilation for more than 12 hours. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Among the 20,929 patients enrolled, we included 4,152 (20%) mechanically ventilated patients due to different neurologic diseases. Hemorrhagic stroke and brain trauma were the most common pathologies associated with the need for mechanical ventilation. Although volume-cycled ventilation remained the preferred ventilation mode, there was a significant (p &lt; 0.001) increment in the use of pressure support ventilation. The proportion of patients receiving a protective lung ventilation strategy was increased over time: 47% in 2004, 63% in 2010, and 65% in 2016 (p &lt; 0.001), as well as the duration of protective ventilation strategies: 406 days per 1,000 mechanical ventilation days in 2004, 523 days per 1,000 mechanical ventilation days in 2010, and 585 days per 1,000 mechanical ventilation days in 2016 (p &lt; 0.001). There were no differences in the length of stay in the ICU, mortality in the ICU, and mortality in hospital from 2004 to 2016. Independent risk factors for 28-day mortality were age greater than 75 years, Simplified Acute Physiology Score II greater than 50, the occurrence of organ dysfunction within first 48 hours after brain injury, and specific neurologic diseases such as hemorrhagic stroke, ischemic stroke, and brain trauma. CONCLUSIONS: More lung-protective ventilatory strategies have been implemented over years in neurologic patients with no effect on pulmonary complications or on survival. We found several prognostic factors on mortality such as advanced age, the severity of the disease, organ dysfunctions, and the etiology of neurologic disease