Human DDX3X Unwinds Japanese Encephalitis and Zika Viral 5′ Terminal Regions

Flavivirus genus includes many deadly viruses such as the Japanese encephalitis virus (JEV) and Zika virus (ZIKV). The 5′ terminal regions (TR) of flaviviruses interact with human proteins and such interactions are critical for viral replication. One of the human proteins identified to interact with the 5′ TR of JEV is the DEAD-box helicase, DDX3X. In this study, we in vitro transcribed the 5′ TR of JEV and demonstrated its direct interaction with recombinant DDX3X (K_d of 1.66 ± 0.21 µM) using microscale thermophoresis (MST). Due to the proposed structural similarities of 5′ and 3′ TRs of flaviviruses, we investigated if the ZIKV 5′ TR could also interact with human DDX3X. Our MST studies suggested that DDX3X recognizes ZIKV 5′ TR with a K_d of 7.05 ± 0.75 µM. Next, we performed helicase assays that suggested that the binding of DDX3X leads to the unwinding of JEV and ZIKV 5′ TRs. Overall, our data indicate, for the first time, that DDX3X can directly bind and unwind in vitro transcribed flaviviral TRs. In summary, our work indicates that DDX3X could be further explored as a therapeutic target to inhibit Flaviviral replication     

Fluctuating NMDA Receptor Subunit Levels in Perirhinal Cortex Relate to Their Dynamic Roles in Object Memory Destabilization and Reconsolidation

Reminder cues can destabilize consolidated memories, rendering them modifiable before they return to a stable state through the process of reconsolidation. Older and stronger memories resist this process and require the presentation of reminders along with salient novel information in order to destabilize. Previously, we demonstrated in rats that novelty-induced object memory destabilization requires acetylcholine (ACh) activity at M₁ muscarinic receptors. Other research predominantly has focused on glutamate, which modulates fear memory destabilization and reconsolidation through GluN2B- and GluN2A-containing NMDARs, respectively. In the current study, we demonstrate the same dissociable roles of GluN2B- and N2A-containing NMDARs in perirhinal cortex (PRh) for object memory destabilization and reconsolidation when boundary conditions are absent. However, neither GluN2 receptor subtype was required for novelty-induced destabilization of remote, resistant memories. Furthermore, GluN2B and GluN2A subunit proteins were upregulated selectively in PRh 24 h after learning, but returned to baseline by 48 h, suggesting that NMDARs, unlike muscarinic receptors, have only a temporary role in object memory destabilization. Indeed, activation of M₁ receptors in PRh at the time of reactivation effectively destabilized remote memories despite inhibition of GluN2B-containing NMDARs. These findings suggest that cholinergic activity at M₁ receptors overrides boundary conditions to destabilize resistant memories when other established mechanisms are insufficient.     

An Engineered Biliverdin-Compatible Cyanobacteriochrome Enables a Unique Ultrafast Reversible Photoswitching Pathway

Cyanobacteriochromes (CBCRs) are promising optogenetic tools for their diverse absorption properties with a single compact cofactor-binding domain. We previously uncovered the ultrafast reversible photoswitching dynamics of a red/green photoreceptor AnPixJg2, which binds phycocyanobilin (PCB) that is unavailable in mammalian cells. Biliverdin (BV) is a mammalian cofactor with a similar structure to PCB but exhibits redder absorption. To improve the AnPixJg2 feasibility in mammalian applications, AnPixJg2_BV4 with only four mutations has been engineered to incorporate BV. Herein, we implemented femtosecond transient absorption (fs-TA) and ground state femtosecond stimulated Raman spectroscopy (GS-FSRS) to uncover transient electronic dynamics on molecular time scales and key structural motions responsible for the photoconversion of AnPixJg2_BV4 with PCB (Bpcb) and BV (Bbv) cofactors in comparison with the parent AnPixJg2 (Apcb). Bpcb adopts the same photoconversion scheme as Apcb, while BV4 mutations create a less bulky environment around the cofactor D ring that promotes a faster twist. The engineered Bbv employs a reversible clockwise/counterclockwise photoswitching that requires a two-step twist on ~5 and 35 picosecond (ps) time scales. The primary forward P_fr → P_o transition displays equal amplitude weights between the two processes before reaching a conical intersection. In contrast, the primary reverse P_o → P_fr transition shows a 2:1 weight ratio of the ~35 ps over 5 ps component, implying notable changes to the D-ring-twisting pathway. Moreover, we performed pre-resonance GS-FSRS and quantum calculations to identify the Bbv vibrational marker bands at ~659,797, and 1225 cm⁻¹. These modes reveal a stronger H-bonding network around the BV cofactor A ring with BV4 mutations, corroborating the D-ring-dominant reversible photoswitching pathway in the excited state. Implementation of BV4 mutations in other PCB-binding GAF domains like AnPixJg4, AM1_1870g3, and NpF2164g5 could promote similar efficient reversible photoswitching for more directional bioimaging and optogenetic applications, and inspire other bioengineering advances.     

The Effects of Insulin-Like Growth Factor I and BTP-2 on Acute Lung Injury

Acute lung injury (ALI) afflicts approximately 200,000 patients annually and has a 40% mortality rate. The COVID-19 pandemic has massively increased the rate of ALI incidence. The pathogenesis of ALI involves tissue damage from invading microbes and, in severe cases, the overexpression of inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). This study aimed to develop a therapy to normalize the excess production of inflammatory cytokines and promote tissue repair in the lipopolysaccharide (LPS)-induced ALI. Based on our previous studies, we tested the insulin-like growth factor I (IGF-I) and BTP-2 therapies. IGF-I was selected, because we and others have shown that elevated inflammatory cytokines suppress the expression of growth hormone receptors in the liver, leading to a decrease in the circulating IGF-I. IGF-I is a growth factor that increases vascular protection, enhances tissue repair, and decreases pro-inflammatory cytokines. It is also required to produce anti-inflammatory 1,25-dihydroxyvitamin D. BTP-2, an inhibitor of cytosolic calcium, was used to suppress the LPS-induced increase in cytosolic calcium, which otherwise leads to an increase in proinflammatory cytokines. We showed that LPS increased the expression of the primary inflammatory mediators such as toll like receptor-4 (TLR-4), IL-1β, interleukin-17 (IL-17), TNF-α, and interferon-γ (IFN-γ), which were normalized by the IGF-I + BTP-2 dual therapy in the lungs, along with improved vascular gene expression markers. The histologic lung injury score was markedly elevated by LPS and reduced to normal by the combination therapy. In conclusion, the LPS-induced increases in inflammatory cytokines, vascular injuries, and lung injuries were all improved by IGF-I + BTP-2 combination therapy.     

Microstructure Evolution and Tensile Properties of a Selectively Laser Melted CoNi-Base Superalloy

Metallurgical and Materials Transactions A - A high $$\gamma ^{\prime }$$ volume fraction CoNi-base superalloy with roughly equal amounts of cobalt and nickel was successfully processed through...     

Growth of sculptured polymer submicronwire assemblies by vapor deposition

We have grown helical nanowire assemblies of parylene C, thereby demonstrating that polymeric sculptured thin films (STFs) can be fabricated by a combination of physical and chemical vapor deposition processes. The deposition method is explained in detail and electron micrographs of 200-400 nm size sculptured thin film of parylene are given. The shapes of the submicron and nanowire assemblies can be engineered so that the polymeric STF acts as a template for preferential attachment of biomolecules.     

Hydrogen and Oxygen Bubble Attachment to a Bitumen Drop

Air bubble – bitumen attachment is a critical step in the flotation of bitumen from mined oil sand. In this study, single bubble – bitumen drop attachment was observed directly using a novel experimental technique. Induction time is determined and used as an indication of bubble‐bitumen attachment potency for both hydrogen and oxygen bubbles. The attachment tests were conducted in deaerated municipal water (City of Edmonton tap water) at temperatures ranging from 22–50°C. Induction times measured for hydrogen bubble attachment were shorter than those for oxygen bubbles. Coalescence tests were also conducted in the absence of bitumen, and showed that hydrogen bubbles coalesced more rapidly than oxygen bubbles in both deaerated municipal water and clear (solids‐free) process water.     

Implications of variable fluid resistance caused by start-up flow in microfluidic networks

Electrical circuit analogies are often used to design microfluidic systems because they simplify device design, providing simple relationships between fluid flow rate, driving forces, and channel dimensions. However, such approximations often significantly overestimate flow rates in situations where start-up energy losses from establishing kinetic head are similar in magnitude to the energy required to overcome viscous shear stresses, as is often the case within complex microfluidic networks. These reduced flows can be more accurately predicted within an electrical analogy framework that accounts for the nonlinear flow resistance generated on the transient regime of start-up flow. In this work, standard flow resistance expressions are modified to account for such effects, and the onset of nonlinear resistance is predicted by a dimensionless parameter, $\xi = Re\frac{D}{L},$ which is dependent on the Reynolds number and the channel length. As a demonstration, variable fluid resistance is shown to dramatically affect the flow performance of common microfluidic units such as T-junctions and serpentine channels, and the change in performance is accurately predicted. Experimental and theoretical analysis of T-junctions further shows that variable flow resistance causes the ratio of flows through the junction to converge toward unity with respect to an increasing total flow rate. In addition, serpentine channels are shown to exaggerate these start-up effects, owing to compounded energetic demand associated with changing a flow’s direction. As a result, serpentine channels cause the ratio of flow rates exiting a T-junction to diverge from unity with respect to an increasing flow rate.     

An optimality criteria based method for discrete design optimization taking into account buildability constraints

This paper presents a heuristic design optimization method specifically developed for practicing structural engineers. Practical design optimization problems are often governed by buildability constraints. The majority of optimization methods that have recently been proposed for design optimization under buildability constraints are based on evolutionary computing. While these methods are generally easy to implement, they require a large number of function evaluations (finite element analyses), and they involve algorithmic parameters that require careful tuning. As a consequence, both the computation time and the engineering time are high. The discrete design optimization algorithm presented in this paper is based on the optimality criteria method for continuous optimization. It is faster than an evolutionary algorithm and it is free of tuning parameters. The algorithm is successfully applied to two classical benchmark problems (the design of a ten-bar truss and an eight-story frame) and to a practical truss design optimization problem.     

Kinetic Study of the Steam Reforming of Isobutane Using a Pt–CeO2–Gd2O3 Catalyst

Steam reforming of isobutane on a 0.5% Pt–Ce_0.8Gd_0.2O_1.9 catalyst was carried out from 300 to 700 °C under integral conditions with a gas hourly space velocity (GHSV) of 12,000 h⁻¹. The major products were H₂, CO₂, CO and CH₄. The other products produced were ethane, ethylene, propane and propylene with a total molar composition of less than 1.5%. A complete conversion of isobutane was achieved at 700 °C, Kinetic data was obtained by changing the partial pressure of the reactants and the temperature under differential conditions with a GHSV of 55,400 h⁻¹. This was done after observing stable isobutane steam reforming for 160 h and under conditions where the mass transfer limitations were insignificant. An empirical Langmuir–Hinshelwood type model that best fit the kinetic data available was developed.