MRI-Based Prediction of Clinical Improvement Following Ventricular Shunt Placement for Normal Pressure Hydrocephalus (NPH): Development and Evaluation of an Integrated Multi-Sequence Machine Learning Algorithm.

BACKGROUND AND PURPOSE: Symptoms of normal pressure hydrocephalus (NPH) are sometimes refractory to shunt placement, with limited ability to predict improvement for individual patients. We evaluated an MRI-based artificial intelligence method to predict post-shunt NPH symptom improvement. MATERIALS AND METHODS: NPH patients who underwent magnetic resonance imaging (MRI) prior to shunt placement at a single center (2014-2021) were identified. Twelve-month post-shunt improvement in modified Rankin Scale (mRS), incontinence, gait, and cognition were retrospectively abstracted from clinical documentation. 3D deep residual neural networks were built on skull stripped T2-weighted and fluid attenuated inversion recovery (FLAIR) images. Predictions based on both sequences were fused by additional network layers. Patients from 2014-2019 were used for parameter optimization, while those from 2020-2021 were used for testing. Models were validated on an external validation dataset from a second institution (n=33). RESULTS: Of 249 patients, n=201 and n=185 were included in the T2-based and FLAIR-based models according to imaging availability. The combination of T2-weighted and FLAIR sequences offered the best performance in mRS and gait improvement predictions relative to models trained on imaging acquired using only one sequence, with AUROC values of 0.7395 [0.5765-0.9024] for mRS and 0.8816 [0.8030-0.9602] for gait. For urinary incontinence and cognition, combined model performances on predicting outcomes were similar to FLAIR-only performance, with AUROC values of 0.7874 [0.6845-0.8903] and 0.7230 [0.5600-0.8859]. CONCLUSIONS: Application of a combined algorithm using both T2-weighted and FLAIR sequences offered the best image-based prediction of post-shunt symptom improvement, particularly for gait and overall function in terms of mRS. ABBREVIATIONS: NPH = normal pressure hydrocephalus; iNPH = idiopathic NPH; sNPH = secondary NPH; AI = artificial intelligence; ML = machine learning; CSF = cerebrospinal fluid; AUROC = area under the receiver operating characteristic; FLAIR = fluid attenuated inversion recovery; BMI = body mass index; CCI = Charlson Comorbidity Index; SD = standard deviation; IQR = interquartile range.

Highly efficient removal of tetracyclines from water by a superelastic MOF-based aerogel: Mechanism quantitative analysis and dynamic adsorption.

Metal-organic frameworks (MOFs) were promising adsorbents for removing antibiotics, but the inherent poor recyclability of MOF powders limits further application. Moreover, the dominant adsorption mechanisms and their quantitative assessment are less studied. Here, ultrahigh adsorption capacities of 821.51 and 931.87 mg g-1 for tetracycline (TC) and oxytetracycline (OTC), respectively, were realised by a novel adsorbents (biochar loaded with MIL-88B(Fe), denoted as BC@MIL-88B(Fe)), which were further immobilised in a 3D porous gelatin (GA) substrate. The obtained BCM/GA200 showed superior adsorption performance under wide pH ranges and under the interference of humic acid. Moreover, it can survive >8 cycles and even maintain high adsorption efficiency in different actual water samples. Notably, BCM/GA200 can selectively remove tetracyclines in a multivariate system containing other kinds of antibiotics and from a dynamic adsorption system. Most importantly, the results of X-ray photoelectron spectroscopy, 2D Fourier transform infrared correlation spectroscopy (2D-FTIR-COS) and density functional theory techniques revealed that (1) for TC adsorption, at pH < 4.0, the contribution of complexation was 25 %-45 %, whereas pore filling and hydrogen bonding accounted for 39 %-72 % of the total uptake. At 4.0 < pH < 10.0, the contribution of complexation increased to 60 %-82 %, whereas electrostatic attraction and π-π interaction were 4 %-13 % and 2 %-10 %, respectively. (2) For OTC adsorption, complexation was dominant at 3.0 < pH < 10.0, accounting for 55 %-86 % of the total uptake, and electrostatic attraction and π-π interactions caused 3 %-10 % and 3 %-15 %, respectively. (3) At pH > 10.0, pore filling dominated TC and OTC adsorption. Finally, the reaction sequences of the main adsorption mechanisms were also probed by 2D-FTIR-COS. This work solves the poor recyclability of MOF powders and provides a mechanistic insight into antibiotic removal by MOFs.

Joint Efforts of Replicative Helicase and SSB Ensure Inherent Replicative Tolerance of G-Quadruplex.

G-quadruplex (G4) is a four-stranded noncanonical DNA structure that has long been recognized as a potential hindrance to DNA replication. However, how replisomes effectively deal with G4s to avoid replication failure is still obscure. Here, using single-molecule and ensemble approaches, the consequence of the collision between bacteriophage T7 replisome and an intramolecular G4 located on either the leading or lagging strand is examined. It is found that the adjacent fork junctions induced by G4 formation incur the binding of T7 DNA polymerase (DNAP). In addition to G4, these inactive DNAPs present insuperable obstacles, impeding the progression of DNA synthesis. Nevertheless, T7 helicase can dismantle them and resolve lagging-strand G4s, paving the way for the advancement of the replication fork. Moreover, with the assistance of the single-stranded DNA binding protein (SSB) gp2.5, T7 helicase is also capable of maintaining a leading-strand G4 structure in an unfolded state, allowing for a fraction of T7 DNAPs to synthesize through without collapse. These findings broaden the functional repertoire of a replicative helicase and underscore the inherent G4 tolerance of a replisome.

Physiologic 18F-FDG Uptake in Brown Adipose Tissue and Lactating Breast in a Patient with Hodgkin Lymphoma.

We present the case of a young woman with Hodgkin lymphoma exhibiting physiologic 18F-FDG uptake in brown adipose tissue and lactating breast on consecutive 18F-FDG PET/CT scans. Both entities are common imaging interpretation pitfalls and should be recognized in oncologic 18F-FDG PET/CT practice. We review the imaging features and differential diagnosis of these 2 entities and discuss the radiation safety precautions during breastfeeding.

Discrete RNA-DNA hybrid cleavage by the EXD2 exonuclease pinpoints two rate-limiting steps.

EXD2 is a recently identified exonuclease that cleaves RNA and DNA in double-stranded (ds) forms. It thus serves as a model system for investigating the similarities and discrepancies between exoribonuclease and exodeoxyribonuclease activities and for understanding the nucleic acid (NA) unwinding-degradation coordination of an exonuclease. Here, using a single-molecule fluorescence resonance energy transfer (smFRET) approach, we show that despite stable binding to both substrates, EXD2 barely cleaves dsDNA and yet displays both exoribonuclease and exodeoxyribonuclease activities toward RNA-DNA hybrids with a cleavage preference for RNA. Unexpectedly, EXD2-mediated hybrid cleavage proceeds in a discrete stepwise pattern, wherein a sudden 4-bp duplex unwinding increment and the subsequent dwell constitute a complete hydrolysis cycle. The relatively weak exodeoxyribonuclease activity of EXD2 partially originates from frequent hybrid rewinding. Importantly, kinetic analysis and comparison of the dwell times under varied conditions reveal two rate-limiting steps of hybrid unwinding and nucleotide excision. Overall, our findings help better understand the cellular functions of EXD2, and the cyclic coupling between duplex unwinding and exonucleolytic degradation may be generalizable to other exonucleases.

Different Intermolecular Interactions Drive Nonpathogenic Liquid-Liquid Phase Separation and Potentially Pathogenic Fibril Formation by TDP-43.

The liquid-liquid phase separation (LLPS) of proteins has been found ubiquitously in eukaryotic cells, and is critical in the control of many biological processes by forming a temporary condensed phase with different bimolecular components. TDP-43 is recruited to stress granules in cells and is the main component of TDP-43 granules and proteinaceous amyloid inclusions in patients with amyotrophic lateral sclerosis (ALS). TDP-43 low complexity domain (LCD) is able to de-mix in solution, forming the protein condensed droplets, and amyloid aggregates would form from the droplets after incubation. The molecular interactions regulating TDP-43 LCD LLPS were investigated at the protein fusion equilibrium stage, when the droplets stopped growing after incubation. We found the molecules in the droplet were still liquid-like, but with enhanced intermolecular helix-helix interactions. The protein would only start to aggregate after a lag time and aggregate slower than at the condition when the protein does not phase separately into the droplets, or the molecules have a reduced intermolecular helix-helix interaction. In the protein condensed droplets, a structural transition intermediate toward protein aggregation was discovered involving a decrease in the intermolecular helix-helix interaction and a reduction in the helicity. Our results therefore indicate that different intermolecular interactions drive LLPS and fibril formation. The discovery that TDP-43 LCD aggregation was faster through the pathway without the first protein phase separation supports that LLPS and the intermolecular helical interaction could help maintain the stability of TDP-43 LCD.

Simultaneous Mechanical and Fluorescence Detection of Helicase-Catalyzed DNA Unwinding.

Helicases are ubiquitous molecular motor proteins that utilize the energy derived from the hydrolysis of nucleoside triphosphates (NTPs) to transiently convert the duplex form of nucleic acids to single-stranded intermediates for many biological processes. These enzymes play vital roles in nearly all aspects of nucleic acid metabolism, such as DNA repair and RNA splicing. Understanding helicase's functional roles requires methods to dissect the mechanisms of motor proteins at the molecular level. In the past three decades, there has been a large increase in the application of single-molecule approaches to investigate helicases. These techniques, such as optical tweezers and single-molecule fluorescence, offer capabilities to monitor helicase motions with unprecedented spatiotemporal resolution, to apply quantitative forces to probe the chemo-mechanical activities of these motors and to resolve helicase heterogeneity at the single-molecule level. In this chapter, we describe a single-molecule method that combines optical tweezers with confocal fluorescence microscopy to study helicase-catalyzed DNA unwinding. Using Bloom syndrome protein (BLM), a multifunctional helicase that maintains genome stability, as an example, we show that this method allows for the simultaneous detection of displacement, force and fluorescence signals of a single DNA molecule during unwinding in real time, leading to the discovery of a distinct bidirectional unwinding mode of BLM that is activated by a single-stranded DNA binding protein called replication protein A (RPA). We provide detailed instructions on how to prepare two DNA templates to be used in the assays, purify the BLM and RPA proteins, perform single-molecule experiments, and acquire and analyse the data.

Bloom Syndrome Helicase Compresses Single-Stranded DNA into Phase-Separated Condensates.

Bloom syndrome protein (BLM) is a conserved RecQ family helicase involved in the maintenance of genome stability. BLM has been widely recognized as a genome "caretaker" that processes structured DNA. In contrast, our knowledge of how BLM behaves on single-stranded (ss) DNA is still limited. Here, we demonstrate that BLM possesses the intrinsic ability for phase separation and can co-phase separate with ssDNA to form dynamically arrested protein/ssDNA co-condensates. The introduction of ATP potentiates the capability of BLM to condense on ssDNA, which further promotes the compression of ssDNA against a resistive force of up to 60 piconewtons. Moreover, BLM is also capable of condensing replication protein A (RPA)- or RAD51-coated ssDNA, before which it generates naked ssDNA by dismantling these ssDNA-binding proteins. Overall, our findings identify an unexpected characteristic of a DNA helicase and provide a new angle of protein/ssDNA co-condensation for understanding the genomic instability caused by BLM overexpression under diseased conditions.

Stochastically multimerized ParB orchestrates DNA assembly as unveiled by single-molecule analysis.

The tripartite ParABS system mediates chromosome segregation in a wide range of bacteria. Dimeric ParB was proposed to nucleate on parS sites and spread to neighboring DNA. However, how properly distributed ParB dimers further compact chromosomal DNA into a higher-order nucleoprotein complex for partitioning remains poorly understood. Here, using a single-molecule approach, we show that tens of Bacillus subtilis ParB (Spo0J) proteins can stochastically multimerize on and stably bind to nonspecific DNA. The introduction of CTP promotes the formation and diffusion of the multimeric ParB along DNA, offering an opportunity for ParB proteins to further forgather and cluster. Intriguingly, ParB multimers can recognize parS motifs and are more inclined to remain immobile on them. Importantly, the ParB multimer features distinct capabilities of not only bridging two independent DNA molecules but also mediating their transportation, both of which are enhanced by the presence of either CTP or parS in the DNA. These findings shed new light on ParB dynamics in self-multimerization and DNA organization and help to better comprehend the assembly of the ParB-DNA partition complex.

The convergence of head-on DNA unwinding forks induces helicase oligomerization and activity transition.

Helicases are multifunctional motor proteins with the primary task of separating nucleic acid duplexes. These enzymes often exist in distinct oligomeric forms and play essential roles during nucleic acid metabolism. Whether there is a correlation between their oligomeric state and cellular function, and how helicases effectively perform functional switching remains enigmatic. Here, we address these questions using a combined single-molecule approach and Bloom syndrome helicase (BLM). By examining the head-on collision of two BLM-mediated DNA unwinding forks, we find that two groups of BLM, upon fork convergence, promptly oligomerize across the fork junctions and tightly bridge two independent single-stranded (ss) DNA molecules that were newly generated by the unwinding BLMs. This protein oligomerization is mediated by the helicase and RNase D C-terminal (HRDC) domain of BLM and can sustain a disruptive force of up to 300 pN. Strikingly, onsite BLM oligomerization gives rise to an immediate transition of their helicase activities, from unwinding dsDNA to translocating along ssDNA at exceedingly fast rates, thus allowing for the efficient displacement of ssDNA-binding proteins, such as RPA and RAD51. These findings uncover an activity transition pathway for helicases and help to explain how BLM plays both pro- and anti-recombination roles in the maintenance of genome stability.

Identification of Differentially Expressed Circular RNAs as miRNA Sponges in Lung Adenocarcinoma.

BACKGROUND: Circular RNAs (circRNAs) may function as the decoys for microRNAs (miRNAs) or proteins, the templates for translation, and the sources of pseudogene generation. The purpose of this study is to determine the diagnostic circRNAs, which are related to lung adenocarcinoma (LUAD), that adsorb miRNAs on the basis of the competing endogenous RNA (ceRNA) hypothesis. METHODS: The differentially expressed circRNAs (DEcircRNAs) in LUAD were revealed by the microarray data (GSE101586 and GSE101684) that were obtained from the Gene Expression Omnibus (GEO) database. The miRNAs that were targeted by the DEcircRNAs were predicted with the CircInteractome, and the target mRNAs of the miRNAs were found by the miRDB and the TargetScan. The ceRNA network was built by the Cytoscape. The potential biological roles and the regulatory mechanisms of the circRNAs were investigated by the Gene Ontology (GO) enrichment analysis and the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. The expression of the host genes of circRNAs was examined by the Ualcan. The survival analysis was performed by the Kaplan-Meier plotter. RESULTS: In comparison with normal lung tissues, LUAD tissues contained 7 overlapping cancer-specific DEcircRNAs with 294 miRNA response elements (MREs). Among the 7 DEcircRNAs, 3 circRNAs (hsa_circ_0072088, hsa_circ_0003528, and hsa_circ_0008274) were upregulated and 4 circRNAs (hsa_circ_0003162, hsa_circ_0029426, hsa_circ_0049271, and hsa_circ_0043256) were downregulated. A circRNA-miRNA-mRNA regulatory network, which included 33 differentially expressed miRNAs (DEmiRNAs) and 2007 differentially expressed mRNAs (DEmRNAs), was constructed. These mRNAs were enriched in the biological function of cell-cell adhesion, response to hypoxia, and stem cell differentiation and were involved in the PI3K-Akt signaling, HIF-1 signaling, and cAMP signaling pathways. CONCLUSION: Our results indicated that 7 DEcircRNAs could have diagnostic value for LUAD. Additionally, the circRNAs-mediated ceRNA network might provide a novel perspective into unraveling the pathogenesis and progression of LUAD.

Identification of Differentially Expressed and Prognostic lncRNAs for the Construction of ceRNA Networks in Lung Adenocarcinoma.

BACKGROUND: Long noncoding RNAs (lncRNAs) could function as competitive endogenous RNAs (ceRNAs) to competitively adsorb microRNAs (miRNAs), thereby regulating the expression of their target protein-coding mRNAs. In this study, we aim to identify more effective diagnostic and prognostic markers for lung adenocarcinoma (LUAD). METHODS: We obtained differentially expressed lncRNAs (DElncRNAs), miRNAs (DEmiRNAs), and mRNAs (DEmRNAs) for LUAD by using The Cancer Genomes Atlas (TCGA) portal. Weighted gene coexpression network analysis (WGCNA) was performed to unveil core gene modules associated with LUAD. The Cox proportional hazards model was performed to determine the prognostic significance of DElncRNAs. The diagnostic and prognostic significance of DElncRNAs was further verified based on the receiver operating characteristic curve (ROC). Cytoscape was used to construct the ceRNA networks comprising the lncRNAs-miRNAs-mRNAs axis based on the correlation obtained from the miRcode, miRDB, and TargetScan. RESULTS: Compared with normal lung tissues, 2355 DElncRNAs, 820 DEmiRNAs, and 17289 DEmRNAs were identified in LUAD tissues. We generated 8 WGCNA core modules in the lncRNAs coexpression network, 5 modules in the miRNAs, and 12 modules in the mRNAs coexpression network, respectively. One lncRNA module (blue) consisting of 441 lncRNAs, two miRNA modules (blue and turquoise) containing 563 miRNAs, and one mRNA module (turquoise), which consisted of 15162 mRNAs, were mostly significantly related to LUAD status. Furthermore, 67 DEmRNAs were found to be tumor-associated as well as the target genes of the DElncRNAs-DEmiRNAs axis. Survival analyses showed that 6 lncRNAs (LINC01447, WWC2-AS2, OGFRP1, LINC00942, LINC01168, and AC005863.1) were significantly correlated with the prognosis of LUAD patients. Ultimately, the potential ceRNA networks including 6 DElncRNAs, 4 DEmiRNAs, and 22 DEmRNAs were constructed. CONCLUSION: Our study indicated that 6 DElncRNAs had the possibilities as diagnostic and prognostic biomarkers for LUAD. The lncRNA-mediated ceRNA networks might provide novel insights into the molecular mechanisms of LUAD progression.

Dynamics of Staphylococcus aureus Cas9 in DNA target Association and Dissociation.

Staphylococcus aureus Cas9 (SaCas9) is an RNA-guided endonuclease that targets complementary DNA adjacent to a protospacer adjacent motif (PAM) for cleavage. Its small size facilitates in vivo delivery for genome editing in various organisms. Herein, using single-molecule and ensemble approaches, we systemically study the mechanism of SaCas9 underlying its interplay with DNA. We find that the DNA binding and cleavage of SaCas9 require complementarities of 6- and 18-bp of PAM-proximal DNA with guide RNA, respectively. These activities are mediated by two steady interactions among the ternary complex, one of which is located approximately 6 bp from the PAM and beyond the apparent footprint of SaCas9 on DNA. Notably, the other interaction within the protospacer is significantly strong and thus poses DNA-bound SaCas9 a persistent block to DNA-tracking motors. Intriguingly, after cleavage, SaCas9 autonomously releases the PAM-distal DNA while retaining binding to the PAM. This partial DNA release immediately abolishes its strong interaction with the protospacer DNA and consequently promotes its subsequent dissociation from the PAM. Overall, these data provide a dynamic understanding of SaCas9 and instruct its effective applications.

Human RPA activates BLM's bidirectional DNA unwinding from a nick.

BLM is a multifunctional helicase that plays critical roles in maintaining genome stability. It processes distinct DNA substrates, but not nicked DNA, during many steps in DNA replication and repair. However, how BLM prepares itself for diverse functions remains elusive. Here, using a combined single-molecule approach, we find that a high abundance of BLMs can indeed unidirectionally unwind dsDNA from a nick when an external destabilizing force is applied. Strikingly, human replication protein A (hRPA) not only ensures that limited quantities of BLMs processively unwind nicked dsDNA under a reduced force but also permits the translocation of BLMs on both intact and nicked ssDNAs, resulting in a bidirectional unwinding mode. This activation necessitates BLM targeting on the nick and the presence of free hRPAs in solution whereas direct interactions between them are dispensable. Our findings present novel DNA unwinding activities of BLM that potentially facilitate its function switching in DNA repair.

The post-PAM interaction of RNA-guided spCas9 with DNA dictates its target binding and dissociation.

Cas9 is an RNA-guided endonuclease that targets complementary DNA for cleavage and has been repurposed for many biological usages. Cas9 activities are governed by its direct interactions with DNA. However, information about this interplay and the mechanism involved in its direction of Cas9 activity remain obscure. Using a single-molecule approach, we probed Cas9/sgRNA/DNA interactions along the DNA sequence and found two stable interactions flanking the protospacer adjacent motif (PAM). Unexpectedly, one of them is located approximately 14 base pairs downstream of the PAM (post-PAM interaction), which is beyond the apparent footprint of Cas9 on DNA. Loss or occupation of this interaction site on DNA impairs Cas9 binding and cleavage. Consistently, a downstream helicase could readily displace DNA-bound Cas9 by disrupting this relatively weak post-PAM interaction. Our work identifies a critical interaction of Cas9 with DNA that dictates its binding and dissociation, which may suggest distinct strategies to modulate Cas9 activity.

Influence and characteristics of Bacillus stearothermophilus in ammonia reduction during layer manure composting.

Ammonia emissions is an important issue during composting because it can cause secondary pollution and a significant of nitrogen loss. Based on research adding Bacillus stearothermophilus can reduce ammonia emissions during composting because it can use sugar in organic matter fermentation to produce organic acids over 50 °C. This study conducted the batch experiments by adding different concentrations of Bacillus stearothermophilus to reduce the ammonia emissions and find out its characteristic during layer manure composting by using an aerobic composting reactor with sawdust as a bulking agent. The results show that the application of Bacillus stearothermophilus can accelerate the rate of temperature and significantly decrease pH, the warming period was 2 days in the treatment with Bacillus stearothermophilus, while it was 4 days in the treatment without Bacillus stearothermophilus. Ammonia emissions were mainly occurred in warming and high temperature period during composting. The ammonia emissions in the treatment with 8.00 g/kg initial Bacillus stearothermophilus were significantly lower than the other lower Bacillus stearothermophilus treatment and control during composting (p < 0.05), and it can significantly increase ammonium-nitrogen and nitrate-nitrogen concentration, reduce pH (p < 0.05), but the average number of Bacillus stearothermophilus copies in treatment with different initial Bacillus stearothermophilus concentration had no significant difference (p > 0.05). MiSeq System Sequencing results find that the addition of Bacillus stearothermophilus changed the bacterial community structure under warming and high-temperature periods during composting, increased the relative abundance of lactic acid bacillus and nitrification bacteria. Therefore, the reason for the low ammonia emission in 8.00 g/kg initial Bacillus stearothermophilus treatments might be not only due to the Bacillus stearothermophilus itself, but also Bacillus stearothermophilus can change the indigenous microorganism community, including increase the relative content of lactic acid Bacillus and nitrification bacteria, thus reducing the pH and promoting nitrification, and reducing ammonia emissions.

The tomato IQD gene SUN24 regulates seed germination through ABA signaling pathway.

MAIN CONCLUSION: Gene expression and functional analysis of the tomato IQD gene SUN24 revealed that it regulates seed germination through ABA signaling pathway. Ca2+ signaling plays crucial roles in diverse biological processes including ABA-mediated seed germination. The plant-specific IQ67-Domain (IQD) proteins are hypothesized to regulate Ca2+ signaling and plant development through interactions with calmodulins (CaMs). Despite a few IQD genes have been identified to regulate herbivore resistance and plant growth and development, the molecular functions of most members in this gene family are not known. In this study, we characterized the role of the tomato IQD gene SUN24 in seed germination. Using pSUN24::GUS reporter lines and by quantitative reverse transcription PCR analysis, we show that SUN24 is mainly expressed in the roots, flowers, young fruits, seeds, and other young developing tissues, and its expression is repressed by ABA treatments. Functional analysis shows that knockdown of SUN24 expression by RNA interference delays seed germination, whereas overexpression of this IQD gene promotes germination. Further gene expression analysis reveals that SUN24 negatively regulates expression of two key ABA signaling genes Solanum lycopersicum ABA-insensitive 3 (SlABI3) and SlABI5 in germinating seeds. Moreover, SUN24, targeting to microtubule and nuclear bodies, can interact with four tomato CaMs (SlCaM1, 2, 3, and 6) in yeast cells. Our results demonstrate that SUN24 regulates seed germination through ABA signaling pathway, expanding our understanding of the roles of the IQD protein family members in plant physiological processes.