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This study integrates pharmacology databases with bulk RNA-seq and scRNA-seq to reveal the latent anti-PDAC capacities of BBR. Target genes of BBR were sifted through TargetNet, CTD, SwissTargetPrediction, and Binding Database. Based on the GSE183795 dataset, DEG analysis, GSEA, and WGCNA were sequentially run to build a disease network. Through sub-network filtration acquired PDAC-related hub genes. A PPI network was established using the shared genes. Degree algorithm from cytoHubba screened the key cluster in the network. Analysis of differential mRNA expression and ROC curves gauged the diagnostic performance of clustered genes. CYBERSORT uncovered the potential role of the key cluster on PDAC immunomodulation. ScRNA-seq analysis evaluated the distribution and expression profile of the key cluster at the single-cell level, assessing enrichment within annotated cell subpopulations to delineate the target distribution of BBR in PDAC. We identified 425 drug target genes and 771 disease target genes, using 57 intersecting genes to construct the PPI network. CytoHubba anchored the top 10 highest contributing genes to be the key cluster. mRNA expression levels and ROC curves confirmed that these genes showed good robustness for PDAC. CYBERSORT revealed that the key cluster influenced immune pathways predominantly associated with Macrophages M0, CD8 T cells, and naïve B cells. ScRNA-seq analysis clarified that BBR mainly acted on epithelial cells and macrophages in PDAC tissues. BBR potentially targets CDK1, CCNB1, CTNNB1, CDK2, TOP2A, MCM2, RUNX2, MYC, PLK1, and AURKA to exert therapeutic effects on PDAC. The mechanisms of action appear to significantly involve macrophage polarization-related immunological responses.

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Recently, owing to the effectiveness in exploiting inherent connections between data in different views, graph-based deep learning approaches have gained widespread popularity in semi-supervised multi-view tasks. Generally, the existing approaches fuse the information from different views via the linear or nonlinear weight strategies, which distinguish the importance of different views by attributing their weights between [0, 1] , i.e., some less important views are discarded since assigned with 0 and the pivotal views are not enhanced. However, these view-weighting strategies ignore the complementary information from the less important views. To address this issue, a superior-performing graph convolutional network (GCN) with self-augmented weights is proposed. The proposed self-augmented weight strategy is based on exponential series integration, which preserves the less important views and simultaneously strengthens the key views for multi-view fusion. Specifically, the designed weight strategy can adaptively preserve the complementary information from the less important views by assigning nonzero weights and strengthen the pivotal views by assigning higher weights based on exponential series integration. Besides, to further improve the model performance, an orthogonal constraint layer with a forced orthogonal weight is introduced, which is capable of making the representation more discriminative. Extensive experiments demonstrate the superiority of the proposed method.

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Cyanobacterial blooms induced by excessive loadings of nitrogen (N) and other nutrients are a severe ecological problem in aquatic ecosystems. Previous studies of N removal have primarily focused on sediment-water interface, yet the role of cyanobacterial colonies has recently been attracting more research attention. In this study, N cycling processes were quantified for cyanobacterial colonies (primarily Microcystis colonies) and their contribution to N removal was estimated for a large, shallow eutrophic lake in China, Lake Taihu. Various N cycling processes were determined via stable 15N isotope, together with 16S rRNA gene sequencing and quantitative microbial element cycling (QMEC) chip. Denitrification was found to be the most prominent process, estimated to be 36.63, 9.85, 3.35, and 3.15 times higher than dissimilatory nitrate reduction to ammonium (DNRA), nitrification, ammonium (NH4+) uptake and nitrate (NO3-) uptake rates, respectively. Denitrifiers accounted for a large part of the bacterial taxa (35.50 ± 24.65%), and the nirS gene was the most abundant among N cycling-related genes, with (2.54 ± 0.51) × 109 copies g-1Microcystis colonies. A field investigation revealed a positive correlation between the potential denitrification rate and the Chl-a concentration (mostly derived from Microcystis colonies). Based on a multiple stepwise regression model and historical data from 2007 to 2015 for Lake Taihu, the total amount of N removed via denitrification by Microcystis colonies was estimated at 171.72 ± 49.74 t yr-1; this suggests that Microcystis colonies have played an important role in N removal in Lake Taihu since the drinking water crisis in 2007. Overall, this study revealed the importance of denitrification within Microcystis colonies for N removal in eutrophic lakes, like Lake Taihu.

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BACKGROUND: Genetic variants in COL4A2 are less common than those of COL4A1 and their fetal clinical phenotype has not been well described to date. We present a fetus from China with an intronic variant in COL4A2 associated with a prenatal diagnosis of severe cerebral encephalomalacia and subdural hemorrhage. METHODS: Whole exome sequencing (WES) was applied to screen potential genetic causes. Bioinformatic analysis was performed to predict the pathogenicity of the variant. In in vitro experiment, the minigene assays were performed to assess the variant's effect. RESULTS: In this proband, we observed ventriculomegaly, subdural hemorrhage, and extensive encephalomalacia that initially suggested cerebral hypoxic-ischemic and/or hemorrhagic lesions. WES identified a de novo heterozygous variant c.549 + 5G > A in COL4A2 gene. This novel variant leads to the skipping of exon 8, which induces the loss of 24 native amino acids, resulting in a shortened COL4A2 protein (p.Pro161_Gly184del). CONCLUSION: Our study demonstrated that c.549 + 5G > A in COL4A2 gene is a disease-causing variant by aberrant splicing. This finding enriches the variant spectrum of COL4A2 gene, which not only improves the understanding of the fetal neurological disorders associated with hypoxic-ischemic and hemorrhagic lesions from a clinical perspective but also provides guidance on genetic diagnosis and counseling.

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RPFdb (http://www.rpfdb.org or http://sysbio.gzzoc.com/rpfdb/) is a comprehensive repository dedicated to hosting ribosome profiling (Ribo-seq) data and related content. Herein, we present RPFdb v3.0, a significant update featuring expanded data content and improved functionality. Key enhancements include (i) increased data coverage, now encompassing 5018 Ribo-seq datasets and 2343 matched RNA-seq datasets from 496 studies across 34 species; (ii) implementation of translation efficiency, combining Ribo-seq and RNA-seq data to provide gene-specific translation efficiency; (iii) addition of pausing score, facilitating the identification of condition-specific triplet amino acid motifs with enhanced ribosome enrichment; (iv) refinement of open reading frame (ORF) annotation, leveraging RibORF v2.0 for more sensitive detection of actively translated ORFs; (v) introduction of a resource hub, curating advances in translatome sequencing techniques and data analytics tools to support a panoramic overview of the field; and (vi) redesigned web interface, providing intuitive navigation with dedicated pages for streamlined data retrieval, comparison and visualization. These enhancements make RPFdb a more powerful and user-friendly resource for researchers in the field of translatomics. The database is freely accessible and regularly updated to ensure its continued relevance to the scientific community.

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INTRODUCTION: The prognostic value of baseline variant allele frequency (VAF) in circulating tumor DNA (ctDNA) of colorectal cancer liver metastases (CRLM) patients after curative resection was rarely investigated. METHODS: A single-center prospective study was performed to investigate the prognostic impact of baseline VAF in ctDNA and matched tumor tissues of CRLM patients after curative resection between May 2019 and May 2021 by the Illumina NovoSeq 6000 platform. The relationship of the tumor burden score (TBS) and the VAF in ctDNA and matched tumor tissues was evaluated by the Pearson correlation method. The survival curves of recurrence-free survival (RFS) and overall survival (OS) were plotted. Factors associated with RFS were calculated using Cox regression analysis, and an integrated prognostic model using significant baseline variables was proposed. RESULTS: There were 121 patients with baseline ctDNA and matched tumor tissues enrolled in the study. A total of 417 mutations spanning 20 genes were identified in baseline tumor tissues of 119/121 (98.3 %) cases. The overall mutations in tumor tissues were completely covered by ctDNA in 52 of 121(43.0 %) patients. Baseline VAF in ctDNA but not in tumor tissues was significantly correlated to TBS of CRLM (R = 0.36, p < 0.001). Significantly longer RFS but not OS was observed in patients with lower VAF in ctDNA compared to those with higher one (p < 0.001 and p = 0.33 respectively). Multivariate Cox regression analysis showed higher VAF in baseline ctDNA was an independent risk factor for RFS. An integrated prognostic model including baseline metastasis location and VAF in ctDNA outperformed the traditional CRS model in predicting RFS. CONCLUSION: Baseline VAF in ctDNA but not in tumor tissues influenced RFS of CRLM patients after curative resection.

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Increasing research suggests a connection between gut microbiota and depressive disorders. Targeted changes to the intestinal flora may contribute to alleviating anxiety and depression. This study aimed to identify probiotics that could attenuate stress-induced abnormal behavior and explore potential mechanisms. The administration of LR.KY16 significantly reduced stress-induced abnormal behaviors and physiological dysfunction. The mechanism may be via regulating the structure of the intestinal microbiota in mice, increasing the abundance of Akkermansia muciniphila, prompting enterochromaffin cells to secrete 5-HTP in the gut, which enters the brain through the bloodstream and promotes the synthesis of 5-HT in the brain, and then activates brain-derived neurotrophic factor (BDNF)/tyrosine kinase receptor B (TrkB) through the 5-HT1A receptor. In addition, LR.KY16 also increased the expression of claudin-7, occludin, and zonula occludens-1 (ZO-1) in the colon, inhibited microglial M1 polarization, and inhibited systemic inflammation.

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Phenolic compounds are the predominant chemical constituents in the secondary metabolites of plants and are commonly found in pears. In this study, we focused on the analysis of the phenolic composition and antioxidant activity of leaves from five pear cultivars (Cuiguan, Chaohong, Kuerle, Nanguoli, and Yali) and tea leaves (Fudingdabai as the control) using ultra-performance liquid chromatography coupled with electrospray ionization triple quadrupole mass spectrometry. The results indicated significant differences in the amount and composition of phenolic metabolites between tea and pear leaves, as well as among the five pear varieties. Only approximately one-third of the metabolites exhibited higher levels in pear leaves compared to that in tea leaves. The total phenol content in the Yali cultivar was higher than that in the other pear cultivars. Furthermore, specific phenolic metabolites with high expression were identified in the leaves of different groups. The levels of delphinidin 3-glucoside, aesculin, prunin, cosmosiin, quercetin 3-galactoside, isorhamnetin-3-O-glucoside, nicotiflorin, narcissin, chlorogenic acid, and cryptochlorogenic acid were relatively high among the five pear cultivars. (-)-Gallocatechin gallate, 6-methylcoumarin, aesculetin, hesperidin, kaempferol, and caftaric acid were identified as specific metabolic substances unique to each type of pear leaf. Most of the differential metabolites showed positive correlations and were primarily enriched in the flavonoid biosynthesis, flavone and flavonol biosynthesis, and phenylpropanoid biosynthesis pathways. DPPH (1,1-Diphenyl-2-picrylhydrazyl radical) analysis indicated that the Yali cultivar exhibited the highest antioxidant activity compared to other varieties. This systematic analysis of the differences in phenolic metabolite composition and antioxidant activity between pear and tea leaves provides a theoretical foundation for the development and utilization of pear leaf resources.

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Oxide matrix red-emitting phosphors are deemed as excellent color converters for white light emitting diodes (WLEDs) and laser diodes (LDs). Manganese-doped MgAl2O4 powder was synthesized by a solid-state reaction method at different sintering temperatures. Microstructure shows that grain size is mainly in the range of 0.2-5 µm, and grain agglomeration occurs with increased sintering temperature. XPS analysis indicates that the doped Mn ion exhibits a valence state of + 4 within the MgAl2O4 matrix. The diffraction peak of the phosphors is shifted by the sintering temperature, which affects lattice constant. Upon excitation by 300 nm ultraviolet light, the samples emit asymmetric broadband red light within the range of 620-720 nm, attributed to Mn4+ ion's transition from 2Eg to 4A2g states. With the increasing temperature, the main emission peak shifts from 677 nm to 650 nm, ascribed to the change in energy level (2Eg) resulting from the reduction of Al2O3 phase. Crystal field theory confirmed that Mn4+ ions are within a strong crystal field environment created by MgAl2O4 matrix. By affecting particle size and crystallinity, the sintering temperature influences the fluorescence lifetime of the Mn4+ ion. Notably, these red-emitting phosphors exhibits remarkable thermal stability as their emission intensity remains approximately at 58% of initial intensity even at elevated temperature (435 K). Consequently, Mn4+: MgAl2O4 red-emitting phosphors with high thermal stability render them promising candidates for WLED applications.

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The continued evolution of H3 subtype avian influenza virus (AIV)-which crosses the interspecific barrier to infect humans-and the potential risk of genetic recombination with other subtypes pose serious threats to the poultry industry and human health. Therefore, rapid and accurate detection of H3 virus is highly important for preventing its spread. In this study, a method based on real-time reverse transcription recombinase-aided isothermal amplification (RT-RAA) was successfully developed for the rapid detection of H3 AIV. Specific primers and probes were designed to target the hemagglutinin (HA) gene of H3 AIV, ensuring highly specific detection of H3 AIV without cross-reactivity with other important avian respiratory viruses. The results showed that the detection limit of the RT-RAA fluorescence reading method was 224 copies/response within the 95% confidence interval, while the detection limit of the RT-RAA visualization method was 1527 copies/response within the same confidence interval. In addition, 68 clinical samples were examined and the results were compared with those of real-time quantitative PCR (RT-qPCR). The results showed that the real-time fluorescence RT-RAA and RT-qPCR results were completely consistent, and the kappa value reached 1, indicating excellent correlation. For visual detection, the sensitivity was 91.43%, the specificity was 100%, and the kappa value was 0.91, which also indicated good correlation. In addition, the amplified products of RT-RAA can be visualized with a portable blue light instrument, which enables rapid detection of H3 AIV even in resource-constrained environments. The H3 AIV RT-RAA rapid detection method established in this study can meet the requirements of basic laboratories and provide a valuable reference for the early diagnosis of H3 AIV.

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Genistein could interact with starch to slow starch digestion by forming starch-genistein complexes. However, genistein had low solubility in water, which hindered the interaction with starch and therefore the formation of the complexes. This study presented a pathway to promote the formation of starch-genistein complexes using an antisolvent method in two steps: (i) adding ethanol to the solution containing starch and genistein to increase genistein solubility, and (ii) evaporating ethanol from the solution to promote genistein interaction with starch. The complexes prepared using this antisolvent method had higher crystallinity (9.45 %), complex index (18.17 %), and higher content of resistant starch (RS) (19.04 %) compared to samples prepared in pure water or ethanol-containing aqueous solution without ethanol evaporation treatment (these samples showed crystallinity of 6.97 %-8.00 %, complex index of 9.09 %-11.4 2%, and RS of 4.45 %-14.38 %). Molecular dynamic simulation results confirmed that the changes in solution polarity significantly determined the formation of starch-genistein complexes. Findings offered a feasible pathway to efficiently promote starch interaction with genistein and in turn mitigate starch digestibility.

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Cancer cachexia is a multifactorial syndrome characterized by progressive weight loss and a disease process that nutritional support cannot reverse. Although progress has been made in preclinical research, there is still a long way to go in translating research findings into clinical practice. One of the main reasons for this is that existing preclinical models do not fully replicate the conditions seen in clinical patients. Therefore, it is important to understand the characteristics of existing preclinical models of cancer cachexia and pay close attention to the latest developments in preclinical models. The main models of cancer cachexia used in current research are allogeneic and xenograft models, genetically engineered mouse models, chemotherapy drug-induced models, Chinese medicine spleen deficiency models, zebrafish and Drosophila models, and cellular models. This review aims to revisit and summarize the commonly used animal models of cancer cachexia by evaluating existing preclinical models, to provide tools and support for translational medicine research.

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The enzymes 3-methylcrotonyl-coenzyme A (CoA) carboxylase (MCC), pyruvate carboxylase and propionyl-CoA carboxylase belong to the biotin-dependent carboxylase family located in mitochondria. They participate in various metabolic pathways in human such as amino acid metabolism and tricarboxylic acid cycle. Many human diseases are caused by mutations in those enzymes but their structures have not been fully resolved so far. Here we report an optimized purification strategy to obtain high-resolution structures of intact human endogenous MCC, propionyl-CoA carboxylase and pyruvate carboxylase in different conformational states. We also determine the structures of MCC bound to different substrates. Analysis of MCC structures in different states reveals the mechanism of the substrate-induced, multi-element synergistic activation of MCC. These results provide important insights into the catalytic mechanism of the biotin-dependent carboxylase family and are of great value for the development of new drugs for the treatment of related diseases.

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Transition metal-based nanoparticles (NPs) are emerging as potential alternatives to platinum for catalyzing the oxygen reduction reaction (ORR) in zinc-air batteries (ZAB). However, the simultaneous coexistence of single-atom moieties in the preparation of NPs is inevitable, and the structural complexity of catalysts poses a great challenge to identifying the true active site. Herein, by employing in situ and ex situ XAS analysis, we demonstrate the coexistence of single-atom moieties and iron phosphide NPs in the N, P co-doped porous carbon (in short, Fe-N4-Fe2P NPs/NPC), and identify that ORR predominantly proceeds via the atomic-dispersed Fe-N4 sites, while the presence of Fe2P NPs exerts an inhibitory effect by decreasing the site utilization and impeding mass transfer of reactants. The single-atom catalyst Fe-N4/NPC displays a half-wave potential of 0.873 V, surpassing both Fe-N4-Fe2P NPs/NPC (0.858 V) and commercial Pt/C (0.842 V) in alkaline condition. In addition, the ZAB based on Fe-N4/NPC achieves a peak power density of 140.3 mW cm-2, outperforming that of Pt/C-based ZAB (91.8 mW cm-2) and exhibits excellent long-term stability. This study provides insight into the identification of true active sites of supported ORR catalysts and offers an approach for developing highly efficient, nonprecious metal-based catalysts for high-energy-density metal-air batteries.

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Neural network (NN)-based methods are extensively used for intelligent fault diagnosis in industrial systems. Nevertheless, due to the limited availability of faulty samples and the presence of noise interference, most existing NN-based methods perform limited diagnosis performance. In response to these challenges, a self-adaptive selection graph pooling method is proposed. Firstly, graph encoders with sharing parameters are designed to extract local structure-feature information (SFI) of multiple sensor-wise sub-graphs. Then, the temporal continuity of the SFI is maintained through time-by-time concatenation, resulting in a global sensor graph and reducing the dependency on data volume from the perspective of adding prior knowledge. Subsequently, leveraging a self-adaptive node selection mechanism, the noise interference of redundant and noisy sensor-wise nodes in the graph is alleviated, allowing the networks to concentrate on the fault-attention nodes. Finally, the local max pooling and global mean pooling of the node-selection graph are incorporated in the readout module to get the multi-scale graph features, which serve as input to a multi-layer perceptron for fault diagnosis. Two experimental studies involving different mechanical and electrical systems demonstrate that the proposed method not only achieves superior diagnosis performance with limited data, but also maintains strong anti-interference ability in noisy environments. Additionally, it exhibits good interpretability through the proposed self-adaptive node selection mechanism and visualization methods.

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Structural modification is expected to be a facile way to enhance the adsorption performance of MXene. In this work, the structural modification of Ti3C2T x was carried out by a spray freezing method, and two kinds of nano-structure (spherical and flaky) of Ti3C2T x were prepared by adjusting the solution concentration of Ti3C2T x . Then the Cr(vi) adsorption capacity and removal efficiency of the spherical and flaky Ti3C2T x was investigated, respectively. It is found that flaky Ti3C2T x was produced with a Ti3C2T x concentration of 3 mg mL-1, while spherical Ti3C2T x was obtained with a concentration of 6 mg mL-1. The long diameter of flaky Ti3C2T x is about 8-10 µm, and the specific surface area is 17.81 m2 g-1. While spherical Ti3C2T x had a diameter of about 1-4 µm and a specific surface area of 17.07 m2 g-1. The optimized structure of flaky and spherical Ti3C2T x improves the maximum adsorption capacity by 97% and 33%, respectively, compared with the few-layer Ti3C2T x . The maximum adsorption capacity of flaky Ti3C2T x was 928 mg g-1, while that of spherical Ti3C2T x was 626 mg g-1. The adsorption capacity of both Ti3C2T x structures decreased with the increase of pH, and reached the maximum value at pH = 2; meanwhile, the adsorption capacity of both Ti3C2T x structures increased with the increase of Cr(vi) concentration. The adsorption of Cr(vi) on flaky Ti3C2T x was very fast, reaching equilibrium in 3 min, while spherical Ti3C2T x took 5 min. The adsorption of Cr(vi) on both Ti3C2T x structures belonged to the monolayers, heat-absorbing chemical adsorption, and the diffusion process of Cr(vi) was regulated by the external diffusion and internal diffusion of particles. Its adsorption mechanism was the combination of reductive adsorption and electrostatic adsorption.

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BACKGROUND AND PURPOSE: Physical activity is an effective therapeutic protocol for treating chronic obstructive pulmonary disease (COPD). However, the mechanisms underlying the benefits of physical activity in COPD are not fully elucidated. EXPERIMENTAL APPROACH: In a mouse model of COPD, analysis of biological markers and lung proteomics identified the molecular pathways through which exercise ameliorates COPD. KEY RESULTS: Exercise improved pulmonary function, emphysema, small airway disease, pulmonary inflammation, glucose metabolic dysregulation, and insulin resistance in COPD mice. Proteomic analysis revealed 430 differentially expressed proteins (DEPs) between the COPD and COPD + Exercise (COPD + Ex) groups. GO analysis indicated that the enriched pathways were predominantly related to the immune response, inflammatory processes, insulin secretion, and glucose metabolic processes. GO analysis revealed IL-33 as a crucial target for the exercise-related amelioration of COPD. KEGG analysis showed that DEPs were significantly enriched in primary immunodeficiency, the intestinal immune network for IgA production, and the NF-κB signalling pathway. Exercise inhibited NF-κB activation by suppressing the CD14/TLR4/MyD88 and TNF-α/TNF-R1/TRAF2/5 pathways in COPD mice. Exercise inhibited expression of BCR, IgM, IgD, IgG, IgE, and IgA by suppressing B-cell receptor signalling. Exercise attenuated glucose metabolic dysregulation and insulin resistance through the suppression of proinflammatory mediators, including MHC I, MHC II, TNF-α, IFN-γ, and IL-1ß, while concurrently increasing insulin expression. The qRT-PCR results were consistent with the proteomic results. CONCLUSION AND IMPLICATIONS: In a mouse model, exercise improved COPD and its metabolic comorbidities through immune system regulation and inflammation suppression, offering insights into potential therapeutic targets.

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During the spring and summer of 2021, two investigations were conducted in the northern Chinese sea by using the high-performance liquid chromatography with the pigment-based chemotaxonomic tool PIGMENTUM. Results showed that the average chlorophyll a (Chl a) concentration during spring was significantly higher (p < 0.01) than that during summer. 19'-hexanoyloxyfucoxanthin-containing algae, alloxanthin-containing algae (ACA), and fucoxanthin-containing algae (FCA) were the main pigment groups in spring, whereas zeaxanthin-containing algae (ZCA) and peridinin-containing algae (PeCA) were dominated in summer. Five ecological provinces were divided, and the phytoplankton biomass (Chl a) in Province V was the lowest. Redundancy analysis showed that ACA, FCA, and PeCA were positively correlated with nutrients; in comparison, ZCA preferred high salinity. The PIGMENTUM estimates were significantly positively correlated (p < 0.01) with CHEMTAX for all phytoplankton assemblages. Nevertheless, the coefficients of determination and slope by regression analysis between two methods showed large differences for several phytoplankton groups.

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BACKGROUND: Amusement arcades are one of the most popular entertainment venues for young people, but excessive noise levels inside them could harm young people's hearing. The aim of this study is to assess the characteristics and risk of noise exposure for young people in amusement arcades and understand their knowledge of and attitudes towards safe listening. METHODS: In this study, noise measurement was conducted in five large amusement arcades to collect 40 noise samples. At the same time, questionnaires were used in each amusement arcade to investigate young players' social information, subjective perception, and cognition of noise-induced hearing loss (NIHL). RESULTS: The level of the noise samples ranged from 83.05 to 89.4 dBA. There are significant differences in noise levels between amusement arcades (P < 0.05), but there is no significant difference in noise levels between different games (P > 0.05). The subjective questionnaire survey shows that 61.9% of young people feel that the sound of video games is noisy or very noisy, but it is worth noting that 63.5% of them do not know or think that noise will not cause hearing loss, and 39.7% of people will choose to do nothing to help them endure the noise in such an environment. For safe listening, young people are more inclined to choose measures to stay in quiet areas and reduce the volume of game devices. CONCLUSIONS: The noise levels in most amusement arcades have exceeded the exposure limits recommended by the Environment Protection Agency (EPA); subjective questionnaires also indicate that internal noise levels may cause hearing loss, and young customers did not know enough about safe listening. For young people's hearing safety, these issues need more attention.

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