RNA-Based Sensor Systems for Affordable Diagnostics in the Age of Pandemics.

In the era of the COVID-19 pandemic, the significance of point-of-care (POC) diagnostic tools has become increasingly vital, driven by the need for quick and precise virus identification. RNA-based sensors, particularly toehold sensors, have emerged as promising candidates for POC detection systems due to their selectivity and sensitivity. Toehold sensors operate by employing an RNA switch that changes the conformation when it binds to a target RNA molecule, resulting in a detectable signal. This review focuses on the development and deployment of RNA-based sensors for POC viral RNA detection with a particular emphasis on toehold sensors. The benefits and limits of toehold sensors are explored, and obstacles and future directions for improving their performance within POC detection systems are presented. The use of RNA-based sensors as a technology for rapid and sensitive detection of viral RNA holds great potential for effectively managing (dealing/coping) with present and future pandemics in resource-constrained settings.

High Tumor Mutation Burden Is Associated with Poor Clinical Outcome in EGFR-Mutated Lung Adenocarcinomas Treated with Targeted Therapy.

This study aimed to determine the association between TMB and treatment outcomes in patients with epidermal growth factor receptor (EGFR)-mutated lung cancer that were treated with tyrosine kinase inhibitors (TKIs). The TMB was assessed using a 409-gene targeted next-generation sequencing panel. We compared the response rate (RR), progression-free survival (PFS), overall survival (OS), and frequency of secondary T790M mutations among the different TMB groups. The median TMB of the study population (n = 88) was 3.36/megabases. We divided 52 (59%) and 36 (41%) patients into the low and high TMB groups, respectively. A high TMB level was significantly associated with liver metastasis and more advanced stage (all p < 0.05). RR was significantly lower in the high TMB group than that of the low TMB group (50.0% vs. 80.7%, all p = 0.0384). In multivariate analysis, high TMB was independently associated with a shorter PFS (hazard ratio [HR] = 1.80, p = 0.0427) and shorter OS (HR = 2.05, p = 0.0397) than that of the low TMB group. Further, high TMB was independently associated with decreased T790M mutation development. These results suggest that high TMB may be a predictive biomarker for adverse treatment outcomes and represent a patients' subgroup warranting tailored therapeutic approaches.

Design of RNA-Based Translational Repressors.

The toehold switch is an RNA-based riboregulator that activates translation in response to a cognate trigger RNA and provides high ON/OFF ratios, excellent orthogonality, and logic capabilities. Riboregulators that provide the inverse function - turning off translation in response to a trigger RNA - are also versatile tools for sensing and efficiently implementing logic gates such as NAND or NOR. Toehold and three-way junction (3WJ) repressors are two de novo designed translational repressors devised to provide NOT functions with an easily programmable and intuitive structural design. Toehold and 3WJ repressors repress translation upon binding to cognate trigger RNAs by forming strong hairpin and three-way junction structures, respectively. These two translational repressors can be incorporated into multi-input NAND and NOR gates. This chapter provides methods for designing these translational repressors and protocols for in vivo characterization in E. coli.

Design of Ribocomputing Devices for Complex Cellular Logic.

The ability to control cell function is a critical goal for synthetic biology and motivates the development of ever-improving methods for precise regulation of gene expression. RNA-based systems represent powerful tools for this purpose since they can take full advantage of the predictable and programmable base pairing properties of RNA to control gene expression. This chapter is focused on the computational design of RNA-only biological circuits that can execute complex Boolean logic expressions in living cells. These ribocomputing devices use toehold switches as building blocks for circuit construction, integrating sensing, computation, and signal generation functions within a gate RNA transcript that regulates expression of a gene of interest. The gate RNA in turn assesses the assembly state of networks of interacting input RNAs to execute AND, OR, and NOT operations with high dynamic range in E. coli. Harnessing in silico tools for device design facilitates scaling of the circuits to complex logic expressions, including four-input AND, six-input OR, and disjunctive normal form expressions with up to 12 inputs. This molecular architecture provides an intuitive and modular strategy for devising logic systems that can be readily engineered using RNA sequence design software and applied in vivo and in vitro. In this chapter, we describe the process for designing ribocomputing devices from the generation of orthogonal toehold switch libraries through to their use as building blocks for AND, OR, and NOT circuitry.

Complex natural product production methods and options.

Natural products have had a major impact upon quality of life, with antibiotics as a classic example of having a transformative impact upon human health. In this contribution, we will highlight both historic and emerging methods of natural product bio-manufacturing. Traditional methods of natural product production relied upon native cellular host systems. In this context, pragmatic and effective methodologies were established to enable widespread access to natural products. In reviewing such strategies, we will also highlight the development of heterologous natural product biosynthesis, which relies instead on a surrogate host system theoretically capable of advanced production potential. In comparing native and heterologous systems, we will comment on the base organisms used for natural product biosynthesis and how the properties of such cellular hosts dictate scaled engineering practices to facilitate compound distribution. In concluding the article, we will examine novel efforts in production practices that entirely eliminate the constraints of cellular production hosts. That is, cell free production efforts will be introduced and reviewed for the purpose of complex natural product biosynthesis. Included in this final analysis will be research efforts made on our part to test the cell free biosynthesis of the complex polyketide antibiotic natural product erythromycin.

Improving E. coli Bactofection by Expression of Bacteriophage ΦX174 Gene E.

Bactofection, a bacterial-mediated form of genetic transfer, is highlighted as an alternative mechanism for gene therapy. A key advantage of this system for immune-reactivity purposes stems from the nature of the bacterial host capable of initiating an immune response by attracting recognition and cellular uptake by antigen-presenting cells (APCs). The approach is also a suitable technique to deliver larger genetic constructs more efficiently as it can transfer plasmids of varying sizes into target mammalian cells. Given these advantages, bacterial vectors are being studied as potential carriers for the delivery of plasmid DNA into target cells to enable expression of heterologous proteins. The bacteria used for bactofection are generally nonpathogenic; however, concerns arise due to the use of a biological agent. To overcome such concerns, enhanced bacterial degradation has been engineered as an attenuation and safety feature for bactofection vectors. In particular, the ΦX174 lysis E (LyE) gene can be repurposed to both minimize bacterial survival within mammalian hosts while also improving overall gene delivery. More specifically, an engineered bacterial vector carrying the LyE gene showed improved gene delivery and safety profiles when tested with murine RAW264.7 macrophage APCs. This chapter outlines steps taken to engineer E. coli for LyE expression as a safer and more effective genetic antigen delivery bactofection vehicle in the context of vaccine utility.

Liposomal Encapsulation of Polysaccharides (LEPS) as an Effective Vaccine Strategy to Protect Aged Hosts Against S. pneumoniae Infection.

Despite the availability of licensed vaccines, pneumococcal disease caused by the bacteria Streptococcus pneumoniae (pneumococcus), remains a serious infectious disease threat globally. Disease manifestations include pneumonia, bacteremia, and meningitis, resulting in over a million deaths annually. Pneumococcal disease disproportionally impacts older adults aged ≥65 years. Interventions are complicated through a combination of complex disease progression and 100 different bacterial capsular polysaccharide serotypes. This has made it challenging to develop a broad vaccine against S. pneumoniae, with current options utilizing capsular polysaccharides as the primary antigenic content. However, current vaccines are substantially less effective in protecting the elderly. We previously developed a Liposomal Encapsulation of Polysaccharides (LEPS) vaccine platform, designed around limitations of current pneumococcal vaccines, that allowed the noncovalent coupling of polysaccharide and protein antigen content and protected young hosts against pneumococcal infection in murine models. In this study, we modified the formulation to make it more economical and tested the novel LEPS vaccine in aged hosts. We found that in young mice (2-3 months), LEPS elicited comparable responses to the pneumococcal conjugate vaccine Prevnar-13. Further, LEPS immunization of old mice (18-22 months) induced comparable antibody levels and improved antibody function compared to Prevnar-13. Importantly, LEPS protected old mice against both invasive and lung localized pneumococcal infections. In summary, LEPS is an alternative and effective vaccine strategy that protects aged hosts against different manifestations of pneumococcal disease.

Grafting Activated Graphene Oxide Nanosheets onto Ultrafiltration Membranes Using Polydopamine to Enhance Antifouling Properties.

Graphene oxide (GO) nanosheets are negatively charged and exhibit excellent antifouling properties. However, their hydrophilicity makes it challenging for their grafting onto membrane surfaces to improve antifouling properties for long-term underwater operation. Herein, we demonstrate a versatile approach to covalently graft GO onto ultrafiltration membrane surfaces in aqueous solutions at ≈22 °C. The membrane surface is first primed using dopamine and then reacted with activated GO (aGO) containing amine-reactive esters. The aGO grafting improves the membrane surface hydrophilicity without decreasing water permeance. When the membranes are challenged with 1.0 g/L sodium alginate in a constant-flux crossflow system, the aGO grafting increases the critical flux by 20% and reduces the fouling rate by 63% compared with the pristine membrane. The modified membranes demonstrate stability for 48 h operation and interval cleanings using NaOH solutions.

Heterologous biosynthesis as a platform for producing new generation natural products.

Natural products have demonstrated value across numerous application areas, with antibiotics a notable historical example. Native cellular hosts provide an initial option in efforts to harness natural product production. However, various complexities associated with native hosts, including fastidious growth traits and limited molecular biology tools, have prompted an alternative approach termed heterologous biosynthesis that relies upon a surrogate biological system to reconstitute the biosynthetic sequence stemming from transplanted genetic blueprint. In turn, heterologous biosynthesis offers the benefit of enzymatically driven complex natural product formation combined with the prospect of improved compound access via scalable cellular production. In this review, we conduct a literature meta-analysis of heterologous natural product biosynthesis over the period of 2011-2020 with the goal of identifying trends in heterologous natural product host selection, target natural products, and compound-host selection tendencies, with associated commentary on the research directions of heterologous biosynthesis based upon this analysis.

Extended Polysaccharide Analysis within the Liposomal Encapsulation of Polysaccharides System.

The Liposomal Encapsulation of Polysaccharides (LEPS) dual antigen vaccine carrier system was assessed across two distinct polysaccharides for encapsulation efficiency, subsequent liposomal surface adornment with protein, adjuvant addition, and size and charge metrics. The polysaccharides derive from two different serotypes of Streptococcus pneumoniae and have traditionally served as the active ingredients of vaccines against pneumococcal disease. The LEPS system was designed to mimic glycoconjugate vaccines that covalently couple polysaccharides to protein carriers; however, the LEPS system uses a noncovalent co-localization mechanism through protein liposomal surface attachment. In an effort to more thoroughly characterize the LEPS system across individual vaccine components and thus support broader future utility, polysaccharides from S. pneumoniae serotypes 3 and 4 were systematically compared within the LEPS framework both pre- and post-surface protein attachment. For both polysaccharides, ≥85% encapsulation efficiency was achieved prior to protein surface attachment. Upon protein attachment with either a model protein (GFP) or a pneumococcal disease antigen (PncO), polysaccharide encapsulation was maintained at ≥61% encapsulation efficiency. Final LEPS carriers were also evaluated with and without alum as an included adjuvant, with encapsulation efficiency maintained at ≥30%, while protein surface attachment efficiency was maintained at ≥~50%. Finally, similar trends and distributions were observed across the different polysaccharides when assessed for liposomal zeta potential and size.

Crystal structure of proteolyzed VapBC and DNA-bound VapBC from Salmonella enterica Typhimurium LT2 and VapC as a putative Ca2+ -dependent ribonuclease.

Bacterial toxin-antitoxin (TA) system has gained attention for its essential roles in cellular maintenance and survival under harsh environmental conditions such as nutrient deficiency and antibiotic treatment. There are at least 14 TA systems in Salmonella enterica serovar Typhimurium LT2, a pathogenic bacterium, and none of the structures of these TA systems have been determined. We determined the crystal structure of the VapBC TA complex from S. Typhimurium LT2 in proteolyzed and DNA-bound forms at 2.0 Å and 2.8 Å resolution, respectively. The VapC toxin possesses a pilT N-terminal domain (PIN-domain) that shows ribonuclease activity, and the VapB antitoxin has an AbrB-type DNA binding domain. In addition, the structure revealed details of interaction mode between VapBC and the cognate promoter DNA, including the inhibition of VapC by VapB and linear conformation of bound DNA in the VapBC complex. The complexation of VapBC with the linear DNA is not consistent with known structures of VapBC homologs in complex with bent DNA. We also identified VapC from S. Typhimurium LT2 as a putative Ca2+ -dependent ribonuclease, which differs from previous data showing that VapC homologs have Mg2+ or Mn2+ -dependent ribonuclease activities. The present studies could provide structural understanding of the physiology of VapBC systems and foundation for the development of new antibiotic drugs against Salmonella infection.

Modulating amyloid-ß aggregation: The effects of peptoid side chain placement and chirality.

Alzheimer's disease (AD) is characterized by the buildup of insoluble aggregated amyloid-ß protein (Aß) into plaques that accumulate between the neural cells in the brain. AD is the sixth leading cause of death in the United States and is the only cause of death among the top ten that cannot currently be treated or cured (Alzheimer's Association, 2011; Selkoe, 1996). Researchers have focused on developing small molecules and peptides to prevent Aß aggregation; however, while some compounds appear promising in vitro, the research has not resulted in a viable therapeutic treatment. We previously reported a peptoid-based mimic (JPT1) of the peptide KLVFF (residues 16-20 of Aß) that modulates Aß40 aggregation, specifically reducing the total number of fibrillar, ß-sheet structured aggregates formed. In this study, we investigate two new variants of JPT1 that probe the importance of aromatic side chain placement (JPT1s) and side chain chirality (JPT1a). Both JPT1s and JPT1a modulate Aß40 aggregation by reducing total ß-sheet aggregates. However, JPT1a also has a pronounced effect on the morphology of fibrillar Aß40 aggregates. These results suggest that Aß40 aggregation may follow a different pathway in the presence of peptoids with different secondary structures. A better understanding of the interactions between peptoids and Aß will allow for improved design of AD treatments.

Controlled electrochemical etching of nanoporous Si anodes and its discharge behavior in alkaline Si-air batteries.

We report the fabrication of nanoporous silicon (nPSi) electrodes via electrochemical etching to form a porous Si layer with controllable thickness and pore size. Varying the etching time and ethanolic HF concentration results in different surface morphologies, with various degrees of electrolyte access depending on the pore characteristics. Optimizing the etching condition leads to well-developed nPSi electrodes, which have thick porous layers and smaller pore diameter and exhibit improved discharge behavior as anodes in alkaline Si-air cells in contrast to flat Si anode. Although electrochemical etching is effective in improving the interfacial characteristics of Si in terms of high surface area, we observed that mild anodization occurs and produces an oxide overlayer. We then show that this oxide layer in nPSi anodes can be effectively removed to produce an nPSi anode with good discharge behavior in an actual alkaline Si-air cell. In the future, the combination of high surface area nPSi anodes with nonaqueous electrolytes (e.g., room-temperature ionic liquid electrolyte) to minimize the strong passivation behavior and self-discharge in Si could lead to Si-air cells with a stable voltage profile and high anode utilization.

Quasi-photonic crystal effect of TiCl3/electrolyte matrix in unipolar dye-absorber devices.

Effectiveness of TiCl3 pre- and post-treatments on dye-sensitized solar cells (DSCs) and interfacial charge-transfer properties were investigated. It was confirmed that a yield of current collection was strongly dependent on the position of the TiCl3/electrolyte matrix in the DSC configuration. The interfacial charge-transfer properties were studied using thermionic emission-diffusion process and electrochemical impedance spectroscopy analysis. The TiCl3/electrolyte matrix was considered to be a three-dimensional quasi-photonic crystal with a photonic band gap, which reinforces electric field and facilitates current collection from the TiCl3/electrolyte matrix to the FTO by accelerating electron motion, whereas the potential barrier blocks current collection from the TiO2 bulk region to the FTO and decreases current.

Functionalized graphene-based cathode for highly reversible lithium-sulfur batteries.

In this article, we highlight the salient issues in the development of lithium-sulfur battery (LSB) cathodes, present different points of view in solving them, and argue, why in the future, functionalized graphene or graphene oxide might be the ultimate solution towards LSB commercialization. As shown by previous studies and also in our recent work, functionalized graphene and graphene oxide enhance the reversibility of the charge-discharge process by trapping polysulfides in the oxygen functional groups on the graphene surface, thus minimizing polysulfide dissolution. This will be helpful for the rational design of new cathode structures based on graphene for LSBs with minimal capacity fading, low extra cost, and without the unnecessary weight increase caused by metal/metal oxide additives.

Higher Prevalence of Klebsiella pneumoniae Extended-Spectrum ß-Lactamase in Patients on Renal Replacement Therapy.

The prevalence of antibiotic resistance is higher in patients undergoing renal replacement therapy (RRT) than in patients who did not undergo RRT. We investigated the presence of KP (Klebsiella pneumoniae) in patients who underwent RRT. All data were collected retrospectively by accessing patient medical records from 2004 to 2011 for the culture results of all patients who were positive for KP. We grouped the patients by the presence of extended-spectrum ß-lactamase (ESBL) into a KP ESBL(-) group (KP[-]) and a KP ESBL(+) group (KP[+]). In total, 292 patients (23.1%) were in the KP(+) group, and 974 patients (76.9%) were in the KP(-) group. A greater percentage of KP(+) was found in patients who underwent RRT (7.5%) than in patients who did not undergo RRT (3.2%) (OR, 2.479; 95% CI,1.412-4.352). A Cox's hazard proportional model analysis was performed, and for patients with pneumonia, the risk of KP(+) was 0.663 times higher in patients who had lower albumin levels, 2.796 times higher in patients who had an inserted Levin tube, and 4.551 times higher in patients who underwent RRT. In conclusion, RRT can be a risk factor for KP(+) in patients with pneumonia.

Fabrication and photovoltaic properties of heterostructured TiO2 nanowires.

One-dimensional heterostructured TiO2 nanowires were successfully fabricated by an electrospinning technique and modified by hydrolysis. We investigated their structure, morphology, chemical composition, and optical properties by using the X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS), and UV-vis spectroscopy. In the case of the photovoltaic performance, the short-circuit current density and cell efficiency of the DSSCs employing single TiO2 nanowires and heterostructured TiO2 nanowires improve from 6.90 to 11.38 mA/cm2 and from 2.56 to 4.29%, respectively. The results show that the photoconversion efficiency of the heterostructured TiO2 nanowires could be improved by more than approximately 67% compared to that of the single TiO2 nanowires because of the enhanced specific surface area that facilitates dye adsorption.

Field modulation in Na-incorporated Cu(In,Ga)Se2 (CIGS) polycrystalline films influenced by alloy-hardening and pair-annihilation probabilities.

The influence of Na on Cu(In,Ga)Se2 (CIGS) solar cells was investigated. A gradient profile of the Na in the CIGS absorber layer can induce an electric field modulation and significantly strengthen the back surface field effect. This field modulation originates from a grain growth model introduced by a combination of alloy-hardening and pair-annihilation probabilities, wherein the Cu supply and Na diffusion together screen the driving force of the grain boundary motion (GBM) by alloy hardening, which indicates a specific GBM pinning by Cu and Na. The pair annihilation between the ubiquitously evolving GBMs has a coincident probability with the alloy-hardening event.PACS: 88. 40. H-, 81. 10. Aj, 81. 40. Cd.

Vertically aligned carbon nanotube electrodes directly grown on a glassy carbon electrode.

Three-dimensional microelectrodes were fabricated using glassy carbon electrodes combined with vertically aligned carbon nanotubes (VACNTs). VACNTs were grown on various conducting electrode patterns including a carbon electrode fabricated by pyrolysis of a negative photoresist, with plasma-enhanced chemical vapor deposition using a bilayer Fe/Al catalyst. VACNT electrodes grown on the glassy carbon showed excellent electrochemical behavior, whereas VACNT electrodes grown on Pt showed poor electrochemical performance, presumably due to the poor contact between VACNTs and the Pt electrode. Electron microscopy showed that the VACNT layer was strongly bound to the carbon electrode, while that on Pt tended to peel away. The versatility of the all-carbon microelectrodes was also tested by using them for interfacing stem cells. Their superior mechanical properties and the electrical connectivity between the carbon electrode and the VACNTs, along with the simple fabrication process, suggest that glassy carbon may be a good conducting substrate for VACNT electrodes.

Carbon nanotube biosensors with aptamers as molecular recognition elements.

In this chapter, we discuss in detail the fabrication of carbon nanotube biosensors that use a single-walled carbon nanotube field-effect transistor (SWNT-FET) as a transducer, and aptamers as molecular recognition elements. We use a patterned growth technique to grow SWNTs on Si/SiO(2) substrates, and standard microfabrication procedures are then employed to fabricate sensing devices.