Transduction Units Research Articles

Development of Sustainable Electronic Device for Non-Enzymatic Glucose Sensing through Extended-Gate-Based Organic Field Effect Transistors

Diabetes is one the most common problem and can lead to kidney failure and other deadly medical conditions if not monitored properly. The pre-existing techniques for monitoring diabetes utilize enzymes, which makes the technique costly, sophisticated, and temperature-dependent storage restricts its broad utilities and to circumvent this drawback of pre-existing techniques, label-free detection of glucose is highly desired. Thus, in this study, CuO/MXenes were synthesized by adopting an ex-situ synthesis approach. Initially, CuO nanoparticles were synthesized using Annona squamosa seed extract as per the previous report. Then, MXenes were synthesized and mixed with CuO nanoparticles in equal ratios, then the mixed solution was kept for stirring at 600 rpm for 8 hours in an inert atmosphere at 80oC. Further, the synthesized CuO/MXenes nano-composites were initially characterized for its optical, structural, and morphological characteristics and then screen printed on indium tin oxide (ITO) glass substrate for utilizing it as an extended-gate electrode. Further, the organic field effect transistor (OFET) was then fabricated by utilizing three different conjugated polymers, namely RR-P3HT, NR-P3HT, and DPP-TTT as a semiconductor material in the bottom gate top contact device architecture and utilized as a transducer for the label-free sensing of glucose via CuO/MXenes nanocomposite. The sensing electrode was connected to a transduction unit (OFET) by extending the gate and all the experiments were performed by applying gate voltage via the reference electrode (schematically shown in Figure 1) under optimized pH condition of 50 mM phosphate buffer saline (PBS; 0.9% NaCl; pH 7) solution containing 5 mM [Fe(CN)6]3−/4−. The main objective of this study is to understand the mobility effect on sensing characteristics, so three different OFETs were fabricated from low to high mobility (order 10-1 to 10-3). Moreover, before sensing glucose, the operation of the OFET was validated before connecting to the extended gate and after connecting to the extended gate (with a blank PBS solution), which demonstrated typical p-type OFET characteristics associated with various polymers. Further, during sensing studies in the presence of glucose solution, there was a clear shift in the threshold voltage (Vth). These extended gate OFETs demonstrated a good response time, but the higher mobility OFET demonstrated rapid determination of glucose in just 20 seconds. There was a linear shift in the threshold voltage (Vth) as a function of the increasing glucose concentration varying from 2 to 100 mM. Details about sensitivity and selectivity will be discussed in detail during the presentation; apart from the mentioned studies, we have also compared the performance of our fabricated transduction unit with the commercially available electrochemical transduction unit for sensing glucose. However, from the results of this study, it is evident that the mobility of OFETs plays a major role in sensing. Figure 1

YeeE-like bacterial SoxT proteins mediate sulfur import for oxidation and signal transduction

Many sulfur-oxidizing prokaryotes oxidize sulfur compounds through a combination of initial extracytoplasmic and downstream cytoplasmic reactions. Facultative sulfur oxidizers adjust transcription to sulfur availability. While sulfur-oxidizing enzymes and transcriptional repressors have been extensively studied, sulfur import into the cytoplasm and how regulators sense external sulfur are poorly understood. Addressing this gap, we show that SoxT1A and SoxT1B, which resemble YeeE/YedE-family thiosulfate transporters and are encoded alongside sulfur oxidation and transcriptional regulation genes, fulfill these roles in the Alphaproteobacterium Hyphomicrobium denitrificans. SoxT1A mutants are sulfur oxidation-negative despite high transcription levels of sulfur oxidation genes, showing that SoxT1A delivers sulfur to the cytoplasm for its further oxidation. SoxT1B serves as a signal transduction unit for the transcriptional repressor SoxR, as SoxT1B mutants are sulfur oxidation-negative due to low transcription unless SoxR is also absent. Thus, SoxT1A and SoxT1B play essential but distinct roles in oxidative sulfur metabolism and its regulation.

Collagen II enrichment through scAAV6-RNAi-mediated inhibition of matrix-metalloproteinases 3 and 13 in degenerative nucleus-pulposus cells degenerative disc disease and biological treatment strategies.

Intervertebral disc (IVD) degeneration damaging the extracellular matrix (ECM) of IVDs is the main cause of spine-associated disorders. Degenerative disc disease (DDD) is a multifaceted disorder, where environmental factors, inflammatory cytokines and catabolic enzymes act together. DDD starts typically due to imbalance between ECM biosynthesis and degradation within IVDs, especially through unbalanced degradation of aggrecan and collagen II in nucleus pulposus (NP). Current treatment approaches are primarily based on conservative or surgical therapies, which are insufficient for biological regeneration. The disintegrins and metalloproteinases with thrombospondin motifs (ADAMTSs) and matrix metalloproteinases (MMPs) are the key proteolytic enzymes for degradation of aggrecan and collagens. Previously, high expression levels of ADAMTS4, ADAMTS5, MMP3 and MMP13, which are accompanied with low levels of aggrecan and collagen II, were demonstrated in degenerative human NP cells. Moreover, self-complementary adeno-associated virus type 6 (scAAV6) mediated inhibitions of ADAMTS4 and ADAMTS5 by RNA-interference (RNAi) could specifically enhance aggrecan level. Thus, MMPs are apparently the main degrading enzymes of collagen II in NP. Furthermore, scAAV6-mediated inhibitions of MMP3 and MMP13 have not yet been investigated. Therefore, we attempted to enhance the level of collagen II in degenerative NP cells by scAAV6-RNAi-mediated inhibitions of MMP3 and MMP13. MRI was used to determine preoperative grading of IVD degeneration in patients. After isolation and culturing of NP cells, cells were transduced with scAAV6-shRNAs targeting MMP3 or MMP13; and analysed by fluorescence microscopy, FACS, MTT assay, RT-qPCR, ELISA and western blotting. scAAV6-shRNRs have no impact on cell viability and proliferation, despite high transduction efficiencies (98.6%) and transduction units (1383 TU/Cell). Combined knockdown of MMP3 (92.8%) and MMP13 (90.9%) resulted in highest enhancement of collagen II (143.2%), whereby treatment effects were significant over 56days (p < 0.001). Conclusively, scAAV6-RNAi-mediated inhibitions of MMP3 and MMP13 help to progress less immunogenic and enduring biological treatments in DDD.

Interference-free multimodal biosensing of adrenaline over other neurotransmitters: Role of 2-iminomethylenephenylboronic acid as the signal transduction unit of fluorescence and impedance

Aberrant levels of the neurotransmitters adrenaline (AD), noradrenaline (NA), and dopamine (DA) are linked to various diseases. Therefore, interference-free biosensing of neurotransmitters has become an important area of research. However, structural similarity (presence of diol/amine functionalities), small size, and neutral charge of the catecholamines add complexity to designing recognition systems and make the detection non-specific. Herein, we report a recognition unit 2-iminomethylenephenylboronic acid (2-IMPBA), which appears to be a remarkable transduction unit for the tailor-made development of biosensors for interference-free detection of the neurotransmitter AD via multiple techniques. Integrating a signaling unit 4-methylbenzothiazole-2-amine to 2- formylphenylboronic acid (2-FPBA), yielded a novel fluorosensor, which showed 38- fold fluorescence enhancement selectively with AD without any competition from other bioanalytes, including NA and DA. The attachment of 2-FPBA on the homemade plastic chip electrode surface afforded an impedimetric sensor that detects AD in the picomolar range without any challenge from its competitors. Both optical and electrochemical sensors have been tested for AD measurement in human plasma samples. We are not aware of any report describing the selective discrimination of AD from NA or other biomolecules commonly found in humanbiofluids.

Distance-Regulated Photoelectrochemical Sensor "Signal-On" and "Signal-Off" Transitions for the Multiplexed Detection of Viruses Exposed in the Aquatic Environment.

Photochemical (PEC) sensors were severely limited for multiplex detection applications due to the cross interference between multiplex signals at the single recognition interface. In this work, a distance-regulated PEC sensor was developed for multiplex detection by using an i-Motif sequence with conformational transformation activity as the signal transduction unit. Through dynamic regulation of the spatial distance between the end site of the functional sequence and the electrode material, the photogenerated electrons on the surface of the sensor were directionally transferred. Thus, a PEC sensor with "signal-on" and "signal-off" dual signal output modes was developed for simultaneous detection of multitarget molecules. Combining isothermal nucleic acid amplification, the PEC sensor constructed in this work was successfully applied to the detection of two virus (Norovirus and Rotavirus) nucleic acid sequences. Under the optimal condition, this bioassay protocol exhibits a linear range of 0.01-100 nM for both viruses with detection limits of 0.72 and 0.53 pM, respectively. In this study, a stimulus-mediated distance regulation strategy successfully addressed the transduction of multiplex detection signals at the single recognition interface of the PEC sensor. It is expected that the technical barriers to multiplex detection of PEC sensors will be overcome and the application of PEC sensing technology will be expanded in the field of environmental analysis.

SYVN1 attenuates ferroptosis and alleviates spinal cord ischemia–reperfusion injury in rats by regulating the HMGB1/NRF2/HO-1 axis

BackgroundThe ferroptosis of neurons is an important pathological mechanism of spinal cord ischemia reperfusion injury (SCIRI). Previous studies showed that synoviolin 1 (SYVN1) is a good prognostic marker of neurodegenerative diseases, but its mechanism is still unclear. This study aims to explore the role of SYVN1 in the ferroptosis of neurons and to clarify its internal mechanism. MethodsRat primary spinal cord neurons were treated with oxygen-glucose deprivation (OGD) for 1, 4 or 8 h, and then cell viability, ROS and MDA levels, glutathione peroxidase (GSH-Px) activity, and the expression of ferroptosis-related proteins GPX4, FTH1 and PTGS2 were detected. OGD/R-induced neurons were transfected with pcDNA-SYVN1 or si-HMGB1, and then cell functions were detected. Transmission electron microscope (TEM) was used to detect cell ferroptosis. The interplay between SYVN1 and high mobility group box 1 (HMGB1) was confirmed with Co-immunoprecipitation (Co-IP) assay. The stability of HMGB1 was measured by ubiquitination assay. Also, cells were treated with pcDNA-SYVN1 or together with ubiquitination inhibitor MG132, as well as treated with pcDNA-SYVN1 and pcDNA-HMGB1 or together with NRF2 activator dimethyl fumarate (DMF), and then Western blotting was used to detect the expression of HMGB1, nuclear NRF2 and HO-1 proteins. In addition, SD rats were occluded left common carotid artery and aortic arch to establish a SCIRI rat model. And rats were injected intrathecal with adenovirus-mediated SYVN1 overexpression vector (Ad-SYVN1, 2 μL, virus titer 5 × 1013 transduction unit [TU]/mL) to overexpress SYVN1. The motion function of rats was quantified using the Basso Rat Scale (BMS) for Locomotion. The ferroptosis and the number of neurons in the spinal cord tissue of rats were detected. ResultsSYVN1 overexpression inhibited ferroptosis of SCIRI rats and OGD/R-treated primary spinal cord neurons, and down-regulated the expression of HMGB1. In terms of mechanism, the binding of SYVN1 and HMGB1 promoted the ubiquitination and degradation of HMGB1, and negatively regulated the expression of HMGB1. Moreover, under OGD/R conditions, MG132 treatment or HMGB1 overexpression eliminated the inhibitory effect of SYVN1 overexpression on the ferroptosis of neurons and the activation of the NRF2/HO-1 pathway, and DMF treatment abolished the inhibition of HMGB1 overexpression on the NRF2/HO-1 pathway. Finally, in vivo experiments showed that SYVN1 overexpression could alleviate the spinal cord ischemia–reperfusion injury in rats by down-regulating HMGB1 and promoting the activation of the NRF2/HO-1 pathway. ConclusionSYVN1 regulates ferroptosis through the HMGB1/NRF2/HO-1 axis to prevent spinal cord ischemia–reperfusion injury.

Synergetic enrichment of aggrecan in nucleus pulposus cells by scAAV6-shRNA-mediated knockdown of aggrecanase-1 and aggrecanase-2.

Degenerative disk disease (DDD) that aggravates structural deterioration of intervertebral disks (IVDs) can be accompanied by painful inflammation and immunopathological progressions. Current surgical or pharmacological therapies cannot repair the structure and function of IVDs. Nucleus pulposus (NP) cells are crucial for the preservation or restoration of IVDs by balancing the anabolic and catabolic factors affecting the extracellular matrix. Imbalanced anabolic and catabolic factors cause increased degradation of aggrecan. Aggrecanases A Disintegrin And Metalloproteinase with ThromboSpondin motifs (ADAMTS)4 and ADAMTS5 are the main degrading enzymes of aggrecan. Previously, we characterized adeno-associated virus (AAV6) as the most suitable serotype with marked NP cellular tropism and demonstrated that ADAMTS4 could be silenced by self-complementary adeno-associated virus grade 6 small helix ribonucleic acid (scAAV6-shRNA) in NP cells of degeneration grade III, which resulted in enrichment of aggrecan. Nonetheless, neither scAAV6-shRNA-mediated inhibition of ADAMTS5 nor joint inhibitions of ADAMTS4 and ADAMTS5 have been investigated, although both enzymes are regulated by analogous proinflammatory cytokines and have the same cleavage sites in aggrecan. Therefore, we attempted scAAV6-shRNA-mediated inhibitions of both enzymes in NP cells of degeneration grade IV to increase efficacies in treatments of DDD. The degeneration grade of IVDs in patients was determined by magnetic resonance imaging (MRI) before surgical operations. After isolation and culturing of NP cells, cells were transduced with scAAV6-shRNAs targeting ADAMTS4 or ADAMTS5. Transduced cells were analyzed by reverse transcription quantitative polymerase chain reaction (RT-qPCR), fluorescence microscopy, flow cytometry-assisted cell sorting (FACS), MTT assay (3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide assay), immunoblotting, and enzyme-linked immunosorbent assay (ELISA). Joint transduction of NP cells exhibited high transduction efficacies (98.1%), high transduction units (TU) (1381 TU/Cell), and no effect on cell viability or proliferation. Above all joint treatments resulted in effective knockdown of ADAMTS4 (92.8%) and ADAMTS5 (93.4%) along with additive enrichment of aggrecan (113.9%). Treatment effects were significant for more than 56 days after transduction (P < 0.001). In conclusion, scAAV6-shRNA-mediated combined molecular therapy could be very valuable for more effective, durable, and less immunogenic treatment approaches in DDD.

Packaging cells for lentiviral vectors generated using the cumate and coumermycin gene induction systems and nanowell single-cell cloning

Lentiviral vectors (LVs) are important for cell therapy because of their capacity to stably modify the genome after integration. This study describes a novel and relatively simple approach to generate packaging cells and producer clones for self-inactivating (SIN) LVs pseudotyped with the vesicular stomatitis virus glycoprotein (VSV-G). A novel gene regulation system, based on the combination of the cumate and coumermycin induction systems, was developed to ensure tight control for the expression of cytotoxic packaging elements. To accelerate clone isolation and ensure monoclonality, the packaging genes were transfected simultaneously into human embryonic kidney cells (293SF-3F6) previously engineered with the induction system, and clones were isolated after limiting dilution into nanowell arrays using a robotic cell picking instrument with scanning capability. The method's effectiveness to isolate colonies derived from single cells was demonstrated using mixed populations of cells labeled with two different fluorescent markers. Because the recipient cell line grew in suspension culture, and all the procedures were performed without serum, the resulting clones were readily adaptable to serum-free suspension culture. The best producer clone produced LVs expressing GFP at a titer of 2.3×108 transduction units (TU)/mL in the culture medium under batch mode without concentration.

Piezoelectric energy harvesting from extremely low-frequency vibrations via gravity induced self-excited resonance

In this paper, we originally present a gravity induced self-excited vibration generator based on a cantilever piezoelectric resonator. The configuration is composed of a sliding rod, a sleeve, a transduction unit. To estimate output voltage and investigate dynamic responses of the generator, an equivalent model based on mechanical structure was established and numerically solved. The effects of the cantilever length, tip mass and linear coupling factor on the open circuit voltage were discussed. In addition, we also conducted series of experiments to validate the theoretical responses. In a 10s cycle (namely 0.1Hz excitation frequency) scenario, the transduction unit responds with structural resonant frequency of 8.621Hz. The results of the experiments show that the open circuit voltage of the generator increases with increment of the cantilever beam length and the tip mass, which agrees well with simulation estimations. We also conducted impedance matching experiments to examine power response in the load spectrum, of which the results indicate that the prototype peak power reaches maximum 1.897 mW. We also discussed the change of gravity potential energy of the prototype and compared it with electric energy. This work provides a novel approach for piezoelectric energy harvesting from extremely low frequency excitations.

Improved Lentiviral In Vivo Gene Therapy for Chronic Granulomatous Disease

Background: Chronic granulomatous disease (CGD) is a congenital immunodeficiency characterized by severe life-threatening infections. CGD patients lack reactive oxygen species (ROS) in phagocytic leukocytes and often develop widespread tissue granulomas. The current treatment for CGD is lifelong antibiotic prophylaxis or hematopoietic stem cell transplantation (HSCT). However, most patients cannot find matched donors, and HSCT is known to have increased risk in CGD patients. Gene therapy has become a promising treatment for CGD. At present, "ex vivo" HSC gene therapy is the widely applied strategy. "In vivo" gene therapy is based on direct delivery of gene therapy vectors into patients, which could overcome the difficulties in HSCT. Methods: We have developed an in vivo CGD gene therapy strategy by intravenous (iv) injection of lentiviral vectors (LVs) carrying an universal EF1a promoter or a myeloid-specific miR223 promoter driving the expression of CYBB (cytochrome B-245 beta chain, or p91-phox) in X-CGD mice (X-CGD; B6.129S-Cybbtm1Din/J). Each mouse received 1x109 transduction units (TU) of LV-CYBB via iv injection after non-myeloablative conditioning. A second injection of 1x109 TU of LV-CYBB after re-conditioning was given 12 weeks later. Results: We developed a chemotherapy conditioning regimen to improve gene delivery efficiency by using busulfan, cyclophosphamide and dexamethasone. The X-CGD mice received two iv injections of LVs, and the expression and function of the LV-CYBB gene were analyzed by intracellular gp91-phox staining and DHR123 assay 4 weeks after each injection. Based on flow cytometry, we detected gp91-phox positive cells in the blood as the following, EF1a: 1.14-5.18% and 5.27-14.36%, and miR223: 1.65-5.17% and 4.59-17.89% after the first and the second injections, respectively, as compared with the WT mice: 79.9-87.6%. Functional analyses of ROS showed the following, EF1a: 1.93-5.2% and 2.39-18.51%, miR223: 2.65-6.67% and 3.75-19.8%, after the first and the second injections respectively, as compared with WT: 72.6-85.31% (Figure A). The LV vector copy number (VCN) in the peripheral blood mononuclear cells was detected by qPCR as the following, EF1a: 1.11-2.32% and 1.35-9.32%, and miR223: 1.01-2.39% and 2.71-8.51% after the first and the second injections, respectively. These results indicated that iv LV injection effectively delivered and expressed the LV-CYBB gene in vivo, and repeated injection of the LVs could increase the gene transfer efficiency in the CGD mice. Importantly, after the second injection, we found no antibody response to the LV particle-associated protein p24. However, after 3-12 months, markedly reduced VCN was found in the blood of the treated mice, suggesting exhaustion of the transgenes in the somatic cells. To improve the gene delivery into HSCs, we treated the mice with AMD3100 to mobilize HSCs into peripheral blood. The mice were treated with chemo-conditioning and AMD3100 prior to LV ivinjection. After four weeks, the gp91-phox positive HSCs of total Sca1+ HSCs in blood with or without (+/-) AMD3100 treatment were examined by flow cytometry, and the results showed the following, EF1a: (-) 9.52-15.43%, (+) 16.1-29.4%, and miR223: (-) 4.49-10.35% and (+) 7.06-16.84%. To confirm stem cell gene transfer, we transplanted the bone marrow cells from the LV-CYBB-treated CGD mice into untreated CGD mice. The VCN in blood was examined before (donor mice) and 4 weeks after (recipient mice) the HSCT, and the results showed the following, EF1a: before 4.49% (in the donor) and after 1.05-2.57% (in the recipients), and miR223: before 4.91% (in the donor) and after 1.04-2.69% (in the recipients) (Figure B). This transplantation study confirmed that the AMD3100 treatment effectively increased iv LV gene delivery into HSCs. Conclusion: We have developed an improved LV iv gene delivery strategy by applying chemo-conditioning and AMD3100 mobilization prior to the injection of the LVs into CGD mice. This in vivo LV gene therapy strategy could potentially be translated into future CGD gene therapy applications. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

An In Vivo CRISPR Screening Platform to Evaluate the Therapeutic Impact of DNA Damage Response Gene Mutations

<h3>Purpose/Objective(s)</h3> Alterations in DNA damage response (DDR) genes are prevalent in human cancers. How these genetic changes modify responses to DNA-directed therapeutics (DDTs) is poorly understood. Here we describe an <i>in vivo</i> CRISPR screening platform to systemically identify DDR gene mutations that confer sensitivity or resistance to different classes of DDTs in a transgenic breast cancer model. <h3>Materials/Methods</h3> Murine breast tumors were induced by mammary intraductal injection of <i>Rosa26^{LSL-Myc;LSL-Cas9};Trp53^flox/flox</i> 6-10 week old female mice with 5 × 10⁵ transduction units of lentivirus expressing Cre recombinase and a library of small guide RNAs targeting 310 DDR genes ("DDR-CRISPR" library). Mammary tumors were analyzed for sgRNA representation by amplicon-based next generation sequencing. A pool of 39 DDR-CRISPR mammary tumor lines was orthotopically injected into a cohort of female <i>NOD/RAG1^−/-</i> mice. When tumors reached 8-10mm in maximal dimension, cohorts of six tumors each were treated with the following DDTs: doxorubicin/hydroxydaunorubicin, Paclitaxel, Capecitabine, Olaparib, AZD6738, 2Gyx4, and 8Gyx1. Tumors were harvested when they reached 15mm in maximal dimension. Statistically significant changes in sgRNA abundance in drug treated tumors versus mock treated controls were identified using two-tailed t-tests. <i>FANCA-</i>knockout clones of MDA-MB-231 cells were generated by dual Cas9-sgRNA targeting. Sensitivity to Olaparib and AZD6738 were assessed by colony forming assay and <i>in vivo</i> by tumor growth analyses in female <i>NOD/RAG1^−/−</i> mice. <h3>Results</h3> Mock-treated mammary tumor pools contained approximately 500 sgRNAs targeting 230 distinct DDR genes. Each type of DDT resulted in distinct yet reproducible fluctuations in sgRNA representation, likely reflecting the variable biological effects of DDR gene mutations on therapeutic sensitivity. Olaparib and AZD6738 treatments resulted in depletion of sgRNAs targeting genes in the canonical homologous recombination (HR) repair pathway (e.g., <i>Brca2, Mre11, Nbn</i>, and <i>Rad54b</i>). However, non-HR genes such as <i>FANCA</i> were also significantly depleted by PARP and ATR inhibitor treatments. Using an isogenic MDA-MB-231 cell line model, we show that <i>FANCA</i> deficiency does not impair HR but results in significant accumulation of single-stranded gaps upon PARP and ATR inhibitor treatment. Single and dual treatment with PARP and/or ATR inhibitors resulted in substantial effects on tumor growth delay and mouse survival. <h3>Conclusion</h3> Our findings illustrate that genetic mixtures of DDR-deficient breast cancers generated through an <i>in vivo</i> CRISPR screen can be used to efficiently quantify the impact of DDR gene mutations on therapeutic sensitivity. Using this platform, we discovered <i>FANCA</i> deficiency as a determinant of sensitivity to PARP and ATR inhibitors due to an accumulation of single-stranded DNA gaps, despite being proficient in HR repair.

Sensitive Autocatalytic Hybridization Circuit for Reliable In Situ Intracellular Polynucleotide Kinase Imaging.

Exploring the characteristic functions of polynucleotide kinase (PNK) could substantially promote the elucidation of PNK-related mechanistic pathways. Yet, the sensitive and reliable detection of intracellular PNK still presents a challenging goal. Herein, we propose a simple autocatalytic hybridization circuit (AHC) for in situ intracellular imaging of PNK with high reliability. The AHC amplifier consists of two mutually activated hybridization chain reaction (HCR) modules for magnified signal transduction. The PNK is transduced into initiator I by phosphorylation and cleavage of mediator Hp. Initiator I activates the initial HCR-1 module, leading to the formation of long dsDNA nanowires that carry numerous initiator T. Then, T-initiated feedback HCR-2 module generates branched products that contain plentiful initiator I, thus realizing an autocatalytic HCR amplification reaction. Simultaneously, the HCR-2 module is also assembled as a versatile signal transduction unit for generating the amplified readout. Based on the mutually sustained accumulation of two initiators for the reciprocal activation of two reaction modules, continuous signal amplification and assembly of high-molecular-weight copolymers endow the AHC system with high sensitivity and robustness for the PNK assay. Moreover, the PNK-sensing AHC system achieves reliable imaging of intracellular PNK, thus showing great potential to decipher the correlation between PNK and related diseases.

Advanced Bio‐Inspired Mechanical Sensing Technology: Learning from Nature but Going beyond Nature

AbstractThe high‐performance optimized design of industrial sensor has been a challenging point of breakthrough in high‐end equipment manufacturing technology since its invention. Highly sensitive detectors have emerged during a long evolutionary period and have shown to be essential tools for ensuring species survival and reproduction. As a result, bio‐sensors have served as a great source of inspiration for high‐sensitivity and high‐performance sensor. Mechanism of bio‐sensors in mechanical detection of three common arthropods, as well as the most recent research results in bio‐mimetic devices, have been discussed in this study. The structure of bio‐detectors and bio‐sensors has been creatively split into environmental information capture device and transduction unit from the standpoint of biological sensing mechanisms and engineering technology. And development of bio‐sensors will rely on design and exploitation of mechanical structures and functional materials with various couplings. Exploration of bionic mechanical sensors is in its early stages and further research is required. High‐performance research on biological detectors and multi‐level bionic design on industrial sensors have been enhanced by revealing mechanism of environmental information capture in environment capture device from basic science and synthesizing new multi‐functional materials of transduction unit with a combination of biological materials and production technology.

Correction of a chronic pulmonary disease through lentiviral vector-mediated protein expression

We developed a novel lentiviral vector, pseudotyped with the F and HN proteins from Sendai virus (rSIV.F/HN), that produces long-lasting, high-efficiency transduction of the respiratory epithelium. Here we addressed whether this platform technology can secrete sufficient levels of a therapeutic protein into the lungs to ameliorate a fatal pulmonary disease as an example of its translational capability. Pulmonary alveolar proteinosis (PAP) results from alveolar granulocyte-macrophage colony-stimulating factor (GM-CSF) insufficiency, resulting in abnormal surfactant homeostasis and consequent ventilatory problems. Lungs of GM-CSF knockout mice were transduced with a single dose of rSIV.F/HN-expressing murine GM-CSF (mGM-CSF; 1e5–92e7 transduction units [TU]/mouse); mGM-CSF expression was dose related and persisted for at least 11 months. PAP disease biomarkers were rapidly and persistently corrected, but we noted a narrow toxicity/efficacy window. rSIV.F/HN may be a useful platform technology to deliver therapeutic proteins for lung diseases requiring long-lasting and stable expression of secreted proteins.

Graphene for Nanobiosensors and Nanobiochips.

Graphene family nanomaterials have interesting electronic structures which determine their electrical, optical, and mechanical properties. Especially, their unique chemical properties enable interactions with biological substances and chemical reagents, and the interactions have further an influence on the observable properties of the graphene family nanomaterials. Such aspects render graphene family nanomaterials versatile for various types of biosensing as a target recognition unit and a recognition-to-signal transduction unit. In this chapter, we look over the recent progress on the graphene-based biosensors, which is categorized in terms of (1) the role of graphene family nanomaterials (target recognition, signal transduction), (2) the sensing mechanisms and modes (electrochemical, electrical, fluorescent, Raman scattering), and (3) the formats of sensing devices (paper, lab-on-a-chip, wearable devices).

The TBC1D31/praja2 complex controls primary ciliogenesis through PKA‐directed OFD1 ubiquitylation

The primary cilium is a microtubule‐based sensory organelle that dynamically links signalling pathways to cell differentiation, growth, and development. Genetic defects of primary cilia are responsible for genetic disorders known as ciliopathies. Orofacial digital type I syndrome (OFDI) is an X‐linked congenital ciliopathy caused by mutations in the OFD1 gene and characterized by malformations of the face, oral cavity, digits and, in the majority of cases, polycystic kidney disease. OFD1 plays a key role in cilium biogenesis. However, the impact of signalling pathways and the role of the ubiquitin‐proteasome system (UPS) in the control of OFD1 stability remain unknown. Here, we identify a novel complex assembled at centrosomes by TBC1D31, including the E3 ubiquitin ligase praja2, protein kinase A (PKA), and OFD1. We show that TBC1D31 is essential for ciliogenesis. Mechanistically, upon G‐protein‐coupled receptor (GPCR)‐cAMP stimulation, PKA phosphorylates OFD1 at ser735, thus promoting OFD1 proteolysis through the praja2‐UPS circuitry. This pathway is essential for ciliogenesis. In addition, a non‐phosphorylatable OFD1 mutant dramatically affects cilium morphology and dynamics. Consistent with a role of the TBC1D31/praja2/OFD1 axis in ciliogenesis, alteration of this molecular network impairs ciliogenesis in vivo in Medaka fish, resulting in developmental defects. Our findings reveal a multifunctional transduction unit at the centrosome that links GPCR signalling to ubiquitylation and proteolysis of the ciliopathy protein OFD1, with important implications on cilium biology and development. Derangement of this control mechanism may underpin human genetic disorders.

Multiplexed detection of bacterial pathogens based on a cocktail of dual-modified phages

Rapid, quantitative, and sensitive assays for the multiplexed detection of bacterial pathogens are urgently needed for public health. Here, we report the generation of dual-modified phage sensors for the simultaneous detection of multiple pathogenic bacteria. The M13KE phage was dual modified to display the targeting peptide on the minor coat protein pIII (∼5 copies) and the streptavidin-binding (StrB) peptide on the major coat protein pVIII (∼2700 copies). The targeting peptide specifically recognizes the target bacteria, and the StrB peptide acts as the efficient signal amplification and transduction unit upon binding with fluorescently tagged streptavidin. The bright fluorescence emitted from individual target bacteria can be clearly distinguished from the background via both the flow cytometry and fluorescence microscopy. Three different dual-modified phages targeting E. coli O157:H7, Salmonella Typhimurium, and Pseudomonas aeruginosa were constructed, and high specificity was verified via a large excess of other non-target bacteria. Using a 40 mL sample volume, the target bacteria detection limit was approximately 102 cells/mL via flow cytometry measurement in the presence of other non-target bacteria. By combining these three dual-modified phages into a cocktail, simultaneous detection and quantification of three target bacterial pathogens was demonstrated with good linearity. The strategy of constructing dual-modified phage represents a promising tool in the detection of bacterial pathogens.

Promoter usage regulating the surface density of CAR molecules may modulate the kinetics of CAR-T cells in vivo

Although chimeric antigen receptor T (CAR-T) cell therapy achieves high remission rates, challenges (e.g., toxicity management and relapse prevention) remain. The major risks are cytokine release syndrome and related neurological toxicity. The influence of the CAR surface density on the efficacy/safety of CAR-T cell therapy and the factors determining CAR density were not elucidated comprehensively. Here, we discovered that the use of the MND promoter increased the transduction rate and reduced the CAR surface density. Additionally, MND-driven CAR-T cells had prolonged antileukemia activity in a mouse model. In an initial dual-armed anti-CD19 CAR-T cell pilot study (ClinicalTrials.gov: NCT03840317), eight and six subjects were infused with MND and EF1α promoter-driven autologous CAR-T cells (3 × 105 CAR-T cells/kg), respectively. MND subjects developed mild fever and lower interferon gamma (IFN-γ) concentrations than in the EF1A19 group. All but one subject in each cohort reached minimal residual disease (MRD)-negative complete remission after the first month of evaluation. These results represent the first comprehensive study on the promoter-driven modulation of CAR-T cell functionality. These findings encourage further evaluation of the potential of the MND promoter to drive CAR-T cells as a broadly applicable cellular product for anticancer immunotherapy.

ViPSN: A Vibration-Powered IoT Platform

In this article, we introduce a vibration-powered sensing node (ViPSN), a programmable Internet-of-Things (IoT) platform for the development of vibration-powered or motion-powered sensing and transmitting systems. It leverages the exploitation and utilization of ambient vibration energy by using a piezoelectric transducer. The roles and relations of six necessary modules, including energy generation unit (EGU), energy transduction unit (ETU), energy enhancement unit (EEU), energy management unit (EMU), energy user unit (EUU), and edge demonstration unit (EDU) are discussed in detail. In particular, an enhanced EMU is proposed by making necessary complements to an extensively used off-the-shelf integrated circuit (IC) solution for piezoelectric transducers. It provides more comprehensive energy storage indicating signals, such that the sensing, computing, and transmitting tasks can be carried out more robustly by keeping a good awareness of the remaining energy. Owing to the enhanced EMU design, vibration energy in various forms, such as intermittent and transient ones, can be more effectively harvested and utilized. The performance of ViPSN is evaluated, in terms of its lifetime and Quality of Service (QoS), under different vibration scenarios. The inclusive design and affiliated opensource project of ViPSN help build a new ecosystem for the research and development of vibration- or motion-powered IoT systems.

Review of Current Guided Wave Ultrasonic Testing (GWUT) Limitations and Future Directions.

Damage is an inevitable occurrence in metallic structures and when unchecked could result in a catastrophic breakdown of structural assets. Non-destructive evaluation (NDE) is adopted in industries for assessment and health inspection of structural assets. Prominent among the NDE techniques is guided wave ultrasonic testing (GWUT). This method is cost-effective and possesses an enormous capability for long-range inspection of corroded structures, detection of sundries of crack and other metallic damage structures at low frequency and energy attenuation. However, the parametric features of the GWUT are affected by structural and environmental operating conditions and result in masking damage signal. Most studies focused on identifying individual damage under varying conditions while combined damage phenomena can coexist in structure and hasten its deterioration. Hence, it is an impending task to study the effect of combined damage on a structure under varying conditions and correlate it with GWUT parametric features. In this respect, this work reviewed the literature on UGWs, damage inspection, severity, temperature influence on the guided wave and parametric characteristics of the inspecting wave. The review is limited to the piezoelectric transduction unit. It was keenly observed that no significant work had been done to correlate the parametric feature of GWUT with combined damage effect under varying conditions. It is therefore proposed to investigate this impending task.