Advanced application of tea residue extracts rich in polyphenols for enhancing sludge dewaterability: Unraveling the role of pH regulation.

Tea polyphenols (TPs), as a kind of derivatives from tea waste, were employed as a novel environmentally friendly bio-based sludge conditioner in this study. The findings showed that when TPs were applied at a dosage of 300 mg g-1 DS, the sludge CST0/CST ratio significantly increased to 1.90. pH regulation was found to markedly affect the dewatering efficiency of sludge. At pH 4, the CST0/CST rose to 2.86, coupled with a reduction in the specific resistance to filtration (SRF) from 6.69 × 1013 m kg-1 to 1.43 × 1013 m kg-1 and a decrease in the moisture content (MC) from 90.57% to 68.75%. TPs formed complexes and precipitated sludge proteins, as demonstrated by changes in the extracellular polymeric substances (EPS), viscosity, zeta potential, and particles size distribution. The optimization significance of acidification treatment on sludge structure disintegration, the interaction of TPs with EPS, and the removal of sludge proteins were elucidated. The research provided an ideal approach for the integrated utilization of biomass resources from tea waste and highlighted the potential application of TPs as an environmentally friendly conditioner in sludge dewatering.

Aptameric hirudins as selective and reversible EXosite-ACTive site (EXACT) inhibitors.

Potent and selective inhibition of the structurally homologous proteases of coagulation poses challenges for drug development. Hematophagous organisms frequently accomplish this by fashioning peptide inhibitors combining exosite and active site binding motifs. Inspired by this biological strategy, we create several EXACT inhibitors targeting thrombin and factor Xa de novo by linking EXosite-binding aptamers with small molecule ACTive site inhibitors. The aptamer component within the EXACT inhibitor (1) synergizes with and enhances the potency of small-molecule active site inhibitors by many hundred-fold (2) can redirect an active site inhibitor's selectivity towards a different protease, and (3) enable efficient reversal of inhibition by an antidote that disrupts bivalent binding. One EXACT inhibitor, HD22-7A-DAB, demonstrates extraordinary anticoagulation activity, exhibiting great potential as a potent, rapid onset anticoagulant to support cardiovascular surgeries. Using this generalizable molecular engineering strategy, selective, potent, and rapidly reversible EXACT inhibitors can be created against many enzymes through simple oligonucleotide conjugation for numerous research and therapeutic applications.

Transcription factor LBD16 targets cell wall modification/ion transport genes in peach lateral root formation.

LATERAL ORGAN BOUNDARIES DOMAIN/ASYMMETRIC LEAVES2-LIKEs (LBDs/ASLs) are plant-specific transcription factors that function downstream of auxin-regulated lateral root (LR) formation. Our previous research found that PpLBD16 positively regulates peach (Prunus persica) LR formation. However, the downstream regulatory network and target genes of PpLBD16 are still largely unknown. Here, we constructed a PpLBD16 homologous overexpression line and a PpLBD16 silenced line. We found that overexpressing PpLBD16 promoted peach root initiation, while silencing PpLBD16 inhibited peach root formation. Through RNA sequencing (RNA-seq) analysis of roots from PpLBD16 overexpression and silenced lines, we discovered that genes positively regulated by PpLBD16 were closely related to cell wall synthesis and degradation, ion/substance transport, and ion binding and homeostasis. To further detect the binding motifs and potential target genes of PpLBD16, we performed DNA-affinity purification sequencing (DAP-seq) analysis in vitro. PpLBD16 preferentially bound to CCNGAAANNNNGG (MEME-1), [C/T]TTCT[C/T][T/C] (MEME-2), and GCGGCGG (ABR1) motifs. By combined analysis of RNA-seq and DAP-seq data, we screened candidate target genes for PpLBD16. We demonstrated that PpLBD16 bound and activated the cell wall modification-related genes EXPANSIN-B2 (PpEXPB2) and SUBTILISIN-LIKE PROTEASE 1.7 (PpSBT1.7), the ion transport-related gene CYCLIC NUCLEOTIDE-GATED ION CHANNEL 1 (PpCNGC1) and the polyphenol oxidase (PPO)-encoding gene PpPPO, thereby controlling peach root organogenesis and promoting LR formation. Moreover, our results displayed that PpLBD16 and its target genes are involved in peach LR primordia development. Overall, this work reveals the downstream regulatory network and target genes of PpLBD16, providing insights into the molecular network of LBD16-mediated LR development.

Case report: Two rare new cases of diaphragmatic bronchogenic cysts.

The bronchogenic cyst (BC) is a type of congenital anomaly that is most usually found in the mediastinum and lung, and rarely in the diaphragm. We report two cases of bronchogenic cysts originating from the left diaphragm. Case 1 involved a 50-year-old man who underwent computed tomography (CT) of the adrenal glands for hypertension, showing left adrenal changes. An adrenal CT at our hospital showed a space-occupying lesion above the left diaphragm. We performed a left-sided thoracotomy in the seventh intercostal space and found that the mass was entirely located in the diaphragm. Case 2 was a 58-year-old woman who underwent chest CT under a standard health check-up, and a subpleural lesion of approximately 4 cm was found in the posterior basal segment of the left inferior lung lobe. During thoracoscopic surgery, the mass was found to originate from the surface of the diaphragm, having no correlation with the lung tissue. The two cases of diaphragmatic bronchogenic cysts were confirmed by surgical pathology. Ectopic bronchogenic cysts arising from the diaphragm are very uncommon, and diaphragmatic bronchogenic cysts typically occur on the left side, in women, and in the angle between the vertebral column and the diaphragm. As far as we know, there are no relevant reports of intradiaphragmatic BCs similar to case 1, which was entirely located within the diaphragm. Most diaphragmatic BCs are located on the surface of the diaphragm and project toward the thoracic or abdominal cavities. Moreover, bilocular diaphragmatic bronchogenic cysts, similar to case 2, have not yet been reported.

Dual RNA-Seq Analysis Pinpoints a Balanced Regulation between Symbiosis and Immunity in Medicago truncatula-Sinorhizobium meliloti Symbiotic Nodules.

Legume-rhizobial symbiosis initiates the formation of root nodules, within which rhizobia reside and differentiate into bacteroids to convert nitrogen into ammonium, facilitating plant growth. This process raises a fundamental question: how is plant immunity modulated within nodules when exposed to a substantial number of foreign bacteria? In Medicago truncatula, a mutation in the NAD1 (Nodules with Activated Defense 1) gene exclusively results in the formation of necrotic nodules combined with activated immunity, underscoring the critical role of NAD1 in suppressing immunity within nodules. In this study, we employed a dual RNA-seq transcriptomic technology to comprehensively analyze gene expression from both hosts and symbionts in the nad1-1 mutant nodules at different developmental stages (6 dpi and 10 dpi). We identified 89 differentially expressed genes (DEGs) related to symbiotic nitrogen fixation and 89 DEGs from M. truncatula associated with immunity in the nad1-1 nodules. Concurrently, we identified 27 rhizobial DEGs in the fix and nif genes of Sinorhizobium meliloti. Furthermore, we identified 56 DEGs from S. meliloti that are related to stress responses to ROS and NO. Our analyses of nitrogen fixation-defective plant nad1-1 mutants with overactivated defenses suggest that the host employs plant immunity to regulate the substantial bacterial colonization in nodules. These findings shed light on the role of NAD1 in inhibiting the plant's immune response to maintain numerous rhizobial endosymbiosis in nodules.

Genome-wide identification of nucleotide-binding domain leucine-rich repeat (NLR) genes and their association with green peach aphid (Myzus persicae) resistance in peach.

Resistance genes (R genes) are a class of genes that are immune to a wide range of diseases and pests. In planta, NLR genes are essential components of the innate immune system. Currently, genes belonging to NLR family have been found in a number of plant species, but little is known in peach. Here, 286 NLR genes were identified on peach genome by using their homologous genes in Arabidopsis thaliana as queries. These 286 NLR genes contained at least one NBS domain and LRR domain. Phylogenetic and N-terminal domain analysis showed that these NLRs could be separated into four subfamilies (I-IV) and their promoters contained many cis-elements in response to defense and phytohormones. In addition, transcriptome analysis showed that 22 NLR genes were up-regulated after infected by Green Peach Aphid (GPA), and showed different expression patterns. This study clarified the NLR gene family and their potential functions in aphid resistance process. The candidate NLR genes might be useful in illustrating the mechanism of aphid resistance in peach.

An Aptamer That Rapidly Internalizes into Cancer Cells Utilizes the Transferrin Receptor Pathway.

Strategies to direct drugs specifically to cancer cells have been increasingly explored, and significant progress has been made toward such targeted therapy. For example, drugs have been conjugated into tumor-targeting antibodies to enable delivery directly to tumor cells. Aptamers are an attractive class of molecules for this type of drug targeting as they are high-affinity/high-specificity ligands, relatively small in size, GMP manufacturable at a large-scale, amenable to chemical conjugation, and not immunogenic. Previous work from our group revealed that an aptamer selected to internalize into human prostate cancer cells, called E3, can also target a broad range of human cancers but not normal control cells. Moreover, this E3 aptamer can deliver highly cytotoxic drugs to cancer cells as Aptamer-highly Toxic Drug Conjugates (ApTDCs) and inhibit tumor growth in vivo. Here, we evaluate its targeting mechanism and report that E3 selectively internalizes into cancer cells utilizing a pathway that involves transferrin receptor 1 (TfR 1). E3 binds to recombinant human TfR 1 with high affinity and competes with transferrin (Tf) for binding to TfR1. In addition, knockdown or knockin of human TfR1 results in a decrease or increase in E3 cell binding. Here, we reported a molecular model of E3 binding to the transferrin receptor that summarizes our findings.

Generation of a high-affinity DNA aptamer for on-site screening of toxic aristolochic acid I in herbal medicines and botanical products.

Aristolochic Acid I (AAI) is an environmental and foodborne toxin found in the Aristolochia and Asarum species of plants that are widespread all over the world. Therefore, there is an urgent need to develop a sensitive and specific biosensor for identifying AAI. Aptamers as a powerful biorecognition element provide the most viable options for solving this problem. In this study, we used library-immobilized SELEX to isolate an AAI-specific aptamer with a KD value of 86 ± 13 nM. To verify the practicability of the selected aptamer, a label-free colorimetric aptasensor was designed. This aptasensor exhibited a low detection limit of 225 nM. Besides, it had been further applied for the determination of AAI in real samples and the recoveries ranged from 97.9% to 102.4%. In the future, AAI aptamer will provide a promising tool for safety evaluation in various fields of agriculture, food, and medication.

GmNAC039 and GmNAC018 activate the expression of cysteine protease genes to promote soybean nodule senescence.

Root nodules are major sources of nitrogen for soybean (Glycine max (L.) Merr.) growth, development, production, and seed quality. Symbiotic nitrogen fixation is time-limited, as the root nodule senesces during the reproductive stage of plant development, specifically during seed development. Nodule senescence is characterized by the induction of senescence-related genes, such as papain-like cysteine proteases (CYPs), which ultimately leads to the degradation of both bacteroids and plant cells. However, how nodule senescence-related genes are activated in soybean is unknown. Here, we identified 2 paralogous NAC transcription factors, GmNAC039 and GmNAC018, as master regulators of nodule senescence. Overexpression of either gene induced soybean nodule senescence with increased cell death as detected using a TUNEL assay, whereas their knockout delayed senescence and increased nitrogenase activity. Transcriptome analysis and nCUT&Tag-qPCR assays revealed that GmNAC039 directly binds to the core motif CAC(A)A and activates the expression of 4 GmCYP genes (GmCYP35, GmCYP37, GmCYP39, and GmCYP45). Similar to GmNAC039 and GmNAC018, overexpression or knockout of GmCYP genes in nodules resulted in precocious or delayed senescence, respectively. These data provide essential insights into the regulatory mechanisms of nodule senescence, in which GmNAC039 and GmNAC018 directly activate the expression of GmCYP genes to promote nodule senescence.

A deep learning network based on CNN and sliding window LSTM for spike sorting.

Spike sorting plays an essential role to obtain electrophysiological activity of single neuron in the fields of neural signal decoding. With the development of electrode array, large numbers of spikes are recorded simultaneously, which rises the need for accurate automatic and generalization algorithms. Hence, this paper proposes a spike sorting model with convolutional neural network (CNN) and a spike classification model with combination of CNN and Long-Short Term Memory (LSTM). The recall rate of our detector could reach 94.40% in low noise level dataset. Although the recall declined with the increasing noise level, our model still presented higher feasibility and better robustness than other models. In addition, the results of our classification model presented an accuracy of greater than 99% in simulated data and an average accuracy of about 95% in experimental data, suggesting our classifier outperforms the current "WMsorting" and other deep learning models. Moreover, the performance of our whole algorithm was evaluated through simulated data and the results shows that the accuracy of spike sorting reached about 97%. It is noteworthy to say that, this proposed algorithm could be used to achieve accurate and robust automated spike detection and spike classification.

Effects of Valine and Urea on Carbon and Nitrogen Accumulation and Lignin Content in Peach Trees.

Nitrogen availability and uptake levels can affect nutrient accumulation in plants. In this study, the effects of valine and urea supplementation on the growth of new shoots, lignin content, and carbon and the nitrogen metabolism of 'Ruiguang 39/peach' were investigated. Relative to fertilization with urea, the application of valine inhibited shoot longitudinal growth, reduced the number of secondary shoots in autumn, and increased the degree of shoot lignification. The application of valine also increased the protein level of sucrose synthase (SS) and sucrose phosphate synthase (SPS) in plant leaves, phloem, and xylem, thereby increasing the soluble sugar and starch content. It also resulted in an increase in nitrate reductase (NR), glutamine synthase (GS), and glutamate synthase (GOGAT) protein levels, with an increase in plant contents of ammonium nitrogen, nitrate nitrogen, and soluble proteins. Although urea application increased the protein level of carbon- and nitrogen-metabolizing enzymes, the increase in plant growth reduced the overall nutrient accumulation and lignin content per unit tree mass. In conclusion, the application of valine has a positive effect on increasing the accumulation of carbon and nitrogen nutrients in peach trees and increasing the lignin content.

Applications and future of aptamers that achieve rapid-onset anticoagulation.

In this short Perspective, we discuss the history of, and recent progress toward, the development of aptamers that can serve as rapid onset anticoagulants during cardiopulmonary bypass (CPB), extracorporeal membrane oxygenation (ECMO), and catheter-based diagnostic and interventional procedures, several million of which are performed each year worldwide. Aptamer anticoagulants provide potent and antidote-controllable anticoagulation and have low immunogenicity. New methods of aptamer isolation and engineering have not only improved the quality of aptamers, but also accelerated their development. Unfortunately, no aptamer identified to date can produce an anticoagulant effect as potent as that produced by unfractionated heparin (UFH), the standard anticoagulant for CPB. We have suggested several possible strategies to amplify the anticoagulant potency of existing aptamer anticoagulants.

Mixed-surface polyamidoamine polymer variants retain nucleic acid-scavenger ability with reduced toxicity.

Nucleic acid-binding polymers can have anti-inflammatory properties and beneficial effects in animal models of infection, trauma, cancer, and autoimmunity. PAMAM G3, a polyamidoamine dendrimer, is fully cationic bearing 32 protonable surface amines. However, while PAMAM G3 treatment leads to improved outcomes for mice infected with influenza, at risk of cancer metastasis, or genetically prone to lupus, its administration can lead to serosal inflammation and elevation of biomarkers of liver and kidney damage. Variants with reduced density of cationic charge through the interspersal of hydroxyl groups were evaluated as potentially better-tolerated alternatives. Notably, the variant PAMAM G3 50:50, similar in size as PAMAM G3 but with half the charge, was not toxic in cell culture, less associated with weight loss or serosal inflammation after parenteral administration, and remained effective in reducing glomerulonephritis in lupus-prone mice. Identification of such modified scavengers should facilitate their development as safe and effective anti-inflammatory agents.

CMTM6 and CMTM4 as two novel regulators of PD-L1 modulate the tumor microenvironment.

The tumor microenvironment (TME) plays crucial roles in regulating tumor occurrence, progress, metastasis and drug resistance. However, it remains largely elusive how the components of TME are regulated to govern its functions in tumor biology. Here, we discussed how the two novel functional proteins, chemokine-like factor (CKLF)-like MARVEL transmembrane domain-containing 6 (CMTM6) and CMTM4, which involved in the post-translational regulation of PD-L1, modulate the TME functions. The roles of CMTM6 and CMTM4 in regulating TME components, including immune cells and tumor cells themselves were discussed in this review. The potential clinical applications of CMTM6 and CMTM4 as biomarkers to predict therapy efficacy and as new or combined immunotherapy targets are also highlighted. Finally, the current hot topics for the biological function of CMTM6/4 and several significant research directions for CMTM6/4 are also briefly summarized in the review.

Coupling sludge-based biochar and electrolysis for conditioning and dewatering of sewage sludge: Effect of char properties.

The addition of sludge-based biochar during electrochemical pretreatment of sewage sludge, as an efficient hybrid technology, is potentially to be applied in sludge deep-dewatering. The chars functioned as conductors, catalysts and skeleton particles could enhance the sludge dewaterability and increase the calorific value of the dewatered sludge cake. However, the effect of synthesis conditions on the char properties and further on the dewatering performance is still unknown. Herein, the sludge-based particle electrodes (SPEs) under three main synthesis conditions, including liquid-solid ratio, pyrolysis temperature and time, were prepared. The sludge-based biochars (i.e., SPE-400, SPE-600, and SPE-800 pyrolyzed under 400, 600 and 800 °C, respectively) were characterized and utilized as three-dimensional electrodes during sludge electrolysis. The increased pyrolysis temperature (within 400-800 °C) resulted in the enrichment of metallic ions and increment of specific surface area and pore volume of SPE, which led to the increased catalysis and adsorption sites for viscous proteins (PNs). Particularly, the pores of SPE-800 provided more drainage channels as skeleton builders. Compared with raw sludge, the capillary suction time (CST) and the specific resistance of filtration (SRF) of the treated sludge with 3D-SPE-800 were reduced by 58.12% and 81.01%, respectively, but the net sludge solids yield (YN) was increased by 87.05%. The highest decrease of hydrophilic α-Helix content in PNs (from 9.93% to 7.30%) was observed when using SPE-800 as particle electrode, revealing the crucial role of char characteristics on protein reduction and subsequent dewatering enhancement. The synergistic effects of electrolysis and sludge-based biochar provided a new insight for a closed-loop pretreatment of sewage sludge in the wastewater treatment plant.

Generation of an anticoagulant aptamer that targets factor V/Va and disrupts the FVa-membrane interaction in normal and COVID-19 patient samples.

Coagulation cofactors profoundly regulate hemostasis and are appealing targets for anticoagulants. However, targeting such proteins has been challenging because they lack an active site. To address this, we isolate an RNA aptamer termed T18.3 that binds to both factor V (FV) and FVa with nanomolar affinity and demonstrates clinically relevant anticoagulant activity in both plasma and whole blood. The aptamer also shows synergy with low molecular weight heparin and delivers potent anticoagulation in plasma collected from patients with coronavirus disease 2019 (COVID-19). Moreover, the aptamer's anticoagulant activity can be rapidly and efficiently reversed using protamine sulfate, which potentially allows fine-tuning of aptamer's activity post-administration. We further show that the aptamer achieves its anticoagulant activity by abrogating FV/FVa interactions with phospholipid membranes. Our success in generating an anticoagulant aptamer targeting FV/Va demonstrates the feasibility of using cofactor-binding aptamers as therapeutic protein inhibitors and reveals an unconventional working mechanism of an aptamer by interrupting protein-membrane interactions.

Aptamer-Integrated Multianalyte-Detecting Paper Electrochemical Device.

On-site detection of multiple small-molecule analytes in complex sample matrixes would be highly valuable for diverse biosensing applications. Paper electrochemical devices (PEDs) offer an especially appealing sensing platform for such applications due to their low cost, portability, and ease of use. Using oligonucleotide-based aptamers as biorecognition elements, we here for the first time have developed a simple, inexpensive procedure for the fabrication of aptamer-modified multiplex PEDs (mPEDs), which can robustly and specifically detect multiple small molecules in complex samples. These devices are prepared via an ambient vacuum filtration technique using carbon and metal nanomaterials that yields precisely patterned sensing architecture featuring a silver pseudo-reference electrode, a gold counter electrode, and three gold working electrodes. The devices are user-friendly, and the fabrication procedure is highly reproducible. Each working electrode can be readily modified with different aptamers for sensitive and accurate detection of multiple small-molecule analytes in a single sample within seconds. We further demonstrate that the addition of a PDMS chamber allows us to achieve detection in microliter volumes of biological samples. We believe this approach should be highly generalizable, and given the rapid development of small-molecule aptamers, we envision that facile on-site multi-analyte detection of diverse targets in a drop of sample should be readily achievable in the near future.

Isolation of Natural DNA Aptamers for Challenging Small-Molecule Targets, Cannabinoids.

Aptamers are nucleic acid-based affinity reagents that are isolated via an in vitro process known as systematic evolution of ligands by exponential enrichment (SELEX). Despite their great potential for a wide range of analytical applications, there are relatively few high-quality small-molecule binding aptamers, especially for "challenging" targets that have low water solubility and/or limited moieties for aptamer recognition. The use of libraries containing chemically modified bases may improve the outcome of some SELEX experiments, but this approach is costly and yields inconsistent results. Here, we demonstrate that a thoughtfully designed SELEX procedure with natural DNA libraries can isolate aptamers with high affinity and specificity for challenging small molecules, including targets for which such selections have previously failed. We first isolate a DNA aptamer with nanomolar affinity and high specificity for (-)-trans-Δ9-tetrahydrocannabinol (THC), a target previously thought to be unsuitable for SELEX with natural DNA libraries. We subsequently isolate aptamers that exhibit high affinity and cross-reactivity to two other challenging targets, synthetic cannabinoids UR-144 and XLR-11, while maintaining excellent specificity against a wide range of non-target interferents. Our findings demonstrate that natural nucleic acid libraries can yield high-quality aptamers for small-molecule targets, and we outline a robust workflow for isolating other such aptamers in future selection efforts.

Advances and Challenges in Small-Molecule DNA Aptamer Isolation, Characterization, and Sensor Development.

Aptamers are short oligonucleotides isolated in vitro from randomized libraries that can bind to specific molecules with high affinity, and offer a number of advantages relative to antibodies as biorecognition elements in biosensors. However, it remains difficult and labor-intensive to develop aptamer-based sensors for small-molecule detection. Here, we review the challenges and advances in the isolation and characterization of small-molecule-binding DNA aptamers and their use in sensors. First, we discuss in vitro methodologies for the isolation of aptamers, and provide guidance on selecting the appropriate strategy for generating aptamers with optimal binding properties for a given application. We next examine techniques for characterizing aptamer-target binding and structure. Afterwards, we discuss various small-molecule sensing platforms based on original or engineered aptamers, and their detection applications. Finally, we conclude with a general workflow to develop aptamer-based small-molecule sensors for real-world applications.

Accelerating Post-SELEX Aptamer Engineering Using Exonuclease Digestion.

The systematic evolution of ligands by exponential enrichment (SELEX) process enables the isolation of aptamers from random oligonucleotide libraries. However, it is generally difficult to identify the best aptamer from the resulting sequences, and the selected aptamers often exhibit suboptimal affinity and specificity. Post-SELEX aptamer engineering can improve aptamer performance, but current methods exhibit inherent bias and variable rates of success or require specialized instruments. Here, we describe a generalizable method that utilizes exonuclease III and exonuclease I to interrogate the binding properties of small-molecule-binding aptamers in a rapid, label-free assay. By analyzing an ochratoxin-binding DNA aptamer and six of its mutants, we determined that ligand binding alters the exonuclease digestion kinetics to an extent that closely correlates with the aptamer's ligand affinity. We then utilized this assay to enhance the binding characteristics of a DNA aptamer which binds indiscriminately to ATP, ADP, AMP, and adenosine. We screened 13 mutants derived from this aptamer against all these analogues and identified two new high-affinity aptamers that solely bind to adenosine. We incorporated these two aptamers directly into an electrochemical aptamer-based sensor, which achieved a detection limit of 1 µM adenosine in 50% serum. We also confirmed the generality of our method to characterize target-binding affinities of protein-binding aptamers. We believe our approach is generalizable for DNA aptamers regardless of sequence, structure, and length and could be readily adapted into an automated format for high-throughput engineering of small-molecule-binding aptamers to acquire those with improved binding properties suitable for various applications.