Ccl2-Induced Regulatory T Cells Balance Inflammation Through Macrophage Polarization During Liver Reconstitution.

Inflammation is highlighted as an initial factor that helps orchestrate liver reconstitution. However, the precise mechanisms controlling inflammation during liver reconstitution have not been fully elucidated. In this study, a clear immune response is demonstrated during hepatic reconstitution. Inhibition of the hepatic inflammatory response retards liver regeneration. During this process, Ccl2 is primarily produced by type 1 innate lymphoid cells (ILC1s), and ILC1-derived Ccl2 recruits peripheral ILC1s and regulatory T cells (Tregs) to the liver. Deletion of Ccl2 or Tregs exacerbates hepatic injury and inflammatory cytokine release, accelerating liver proliferation and regeneration. The adoption of Tregs and IL-10 injection reversed these effects on hepatocyte regenerative proliferation. Additionally, Treg-derived IL-10 can directly induce macrophage polarization from M1 to M2, which alleviated macrophage-secreted IL-6 and TNF-α and balanced the intrahepatic inflammatory milieu during liver reconstitution. This study reveals the capacity of Tregs to modulate the intrahepatic inflammatory milieu and liver reconstitution through IL-10-mediated macrophage polarization, providing a potential opportunity to improve hepatic inflammation and maintain homeostasis.

Physical activity and idiopathic pulmonary fibrosis: A prospective cohort study in UK Biobank and Mendelian randomization analyses.

INTRODUCTION: The impact of physical activity on the incidence of idiopathic pulmonary fibrosis (IPF) remains less well studied. This study aimed to investigate the relationship between moderate-to-vigorous physical activity (MVPA) and the risk of developing IPF. METHODS: We analyzed data from a prospective cohort study within the UK Biobank involving 502,476 participants. Participants were categorized as meeting or not meeting the 2017 UK Physical Activity Guidelines (150 min of moderate activity or 75 min of vigorous activity per week). The cumulative incidence and hazard ratios (HRs) for IPF were analyzed using the Kaplan-Meier method, log-rank test, and Cox regression. Two-sample Mendelian randomization (MR) analyses were performed to identify potential causal links between physical activity and IPF risk. RESULTS: Over a median of 12.2 y follow-up, we identified 1,639 incident IPF cases and 395,172 controls. Individuals who met the physical activity guidelines had a significantly lower risk of IPF than those who did not meet the guidelines (adjusted HR = 0.843, 95 % confidence interval [CI] = 0.765-0.930).The cumulative incidence of IPF was lower in the meeting guideline group than in the nonmeeting guideline group (Log-rank P = 0.0019). Two-sample MR analysis revealed that a 1-standard deviation increase in moderate-to-vigorous physical activity was linked to a reduced IPF risk (odds ratio [OR] = 0.17, 95 % CIs = 0.04 to 0.81, P = 0.026). Moreover, an increase in the number of days per week of moderate physical activity was genetically correlated with decreased IPF risk (OR = 0.32, 95 % CIs = 0.15-0.70, P = 0.003). CONCLUSION: Higher levels of moderate-to-vigorous physical activity are causally associated with a significant reduction in the risk of developing IPF.

Dielectric spectroscopy technology combined with machine learning methods for nondestructive detection of protein content in fresh milk.

To quickly achieve nondestructive detection of protein content in fresh milk, this study utilized a network analyzer and an open coaxial probe to analyze the dielectric spectra of milk samples at 100 frequency points within the 2-20 GHz range, focusing on the dielectric constant ε' and the dielectric loss factor ε''. Feature variables were extracted from the full dielectric spectra using the successive projections algorithm (SPA), uninformative variables elimination (UVE), and the combined UVE-SPA method. These variables were then used to develop partial least squares regression (PLSR), support vector machine (SVM), decision tree (DT), random forest (RF), and least squares boosting (LSBOOST) models for predicting protein content. The results showed that ε' decreased monotonically with increasing frequency, while ε'' increased monotonically. The UVE-SPA method for feature extraction demonstrated superior performance, with the UVE-SPA-PLSR model being the best for predicting milk protein content, achieving the highest RC 2 = 0.998 and RP 2 = 0.989 and the lowest RMSEC = 0.019% and RMSEP = 0.032%. This study provides a theoretical reference for evaluating milk quality and developing intelligent detection equipment for natural milk.

Ostwald-Ripening Induced Interfacial Protection Layer Boosts 1,000,000-Cycled Hydronium-Ion Battery.

Collapsing and degradation of active materials caused by the electrode/electrolyte interface instability in aqueous batteries are one of the main obstacles that mitigate the capacity. Herein by reversing the notorious side reactions include the loss and dissolution of electrode materials: as we applied Ostwald ripening (OR) in the electrochemical cycling of a copper hexacyanoferrate electrode in a hydronium-ion batteries, the dissolved Cu and Fe ions undergo a crystallization process that creates a stable interface layer of cross-linked cubes on the electrode surface. The layer exposed the low-index crystal planes (100) and (110) through OR-induced electrode particle growth, supplemented by vacancy-ordered (100) superlattices that facilitated ion migration. Our design stabilized the electrode-electrolyte interface considerably, achieving a cycle life of one million cycles with capacity retention of 91.6%, and a capacity retention of 91.7% after 3000 cycles for a full battery.

Boron-Doped Single-Molecule van der Waals Diode.

Single-molecule diode was the first proposed device in molecular electronics. Despite the great efforts and advances over 50 years, the reported rectification ratios, the most critical parameter of a diode, remain moderate for the single-molecule diode. Herein, we report an approach to achieve a larger rectification ratio by adopting the combined strategies of p-type boron doping, the single-layer graphene nodes, and the van der Waals layer-by-layer architecture. Measured current-voltage curves showed one of the as-fabricated single-molecule diodes hit an unprecedented large rectification ratio of 457 at ±1 V. Break junction operations and spectroscopic measurements revealed the three-atom-thick configuration of the single-molecule diodes. With the experimental and theoretical calculation results, we demonstrated the doped boron atoms induced holes to redistribute the electron density, making the asymmetric coupling at positive and negative biases, and the van der Waals interaction promoted asymmetric coupling and significantly boosted diode performance.

Ag+-Induced Assembly of Pt Clusters for Photocatalytic Hydrogen Production.

Cluster-assembled nanowires provide a unique strategy for the preparation of high-performance nanostructures. However, existing preparations are limited by complex processes and harsh reaction conditions. Here, Ag+ ions were utilized as a novel structure-directing agent to generate the self-assembly of Pt clusters to form ultrafine nanowires with a diameter of less than 5 nm. Electrospray ionization mass spectrometry (ESI-MS) and extended X-ray absorption fine structure (EXAFS) characterizations demonstrated that every Ag+ bridged two [Pt3(CO)3(µ2-CO)3]n2- clusters through coordination and formed a sandwich-like structure of [Pt3(CO)3(µ2-CO)3]nAg[Pt3(CO)3(µ2-CO)3]m3-. As a result, multiple sandwich-like structures of [Pt3(CO)3(µ2-CO)3]nAg[Pt3(CO)3(µ2-CO)3]m3- were established by Ag+ to form Pt nanowire superstructures {[Pt3(CO)6]nAg[Pt3(CO)6]mAg[Pt3(CO)6]x}∞ (abbreviated as Ag-Pt NWS). Our results demonstrate that the Pt nanowire superstructures showed promising cocatalytic performance for photocatalytic H2 production with the involvement of Ag+, which promises a desirable way to develop advanced functional nanomaterials.

Chronic dietary exposure to glyphosate-induced connexin 43 autophagic degradation contributes to blood-testis barrier disruption in roosters.

Glyphosate (GLY) is the most universally used herbicide worldwide and its application has caused extensive pollution to the ecological environment. Increasing evidence has revealed the multi-organ toxicity of GLY in different species, but its male reproductive toxicity in avian species remains unknown. Thus, in vivo and in vitro studies were conducted to clarify this issue. Data firstly showed that chronic GLY exposure caused testicular pathological damage. Intriguingly, we identified and verified a marked down-regulation gap junction gene Connexin 43 (Cx43) in GLY-exposed rooster testis by transcriptome analysis. Cx43 generated by Sertoli cells acts as a key component of blood-testis barrier (BTB). To further investigate the cause of GLY-induced downregulation of Cx43 to disrupt BTB, we found that autophagy activation is revealed in GLY-exposed rooster testis and primary avian Sertoli cells. Moreover, GLY-induced Cx43 downregulation was significantly alleviated by ATG5 knockdown or CQ administration, respectively, demonstrating that GLY-induced autophagy activation contributed to Cx43 degradation. Mechanistically, GLY-induced autophagy activation and resultant Cx43 degradation was due to its direct interaction with ER-α. In summary, these findings demonstrate that chronic GLY exposure activates autophagy to induce Cx43 degradation, which causes BTB damage and resultant reproductive toxicity in roosters.

Effect of Vascular Senescence on the Efficacy and Safety of Warfarin: Insights from Rat Models and a Prospective Cohort Study.

Warfarin, with its narrow therapeutic range, requires the understanding of various influencing factors for personalized medication. Vascular senescence, marked by vascular stiffening and endothelial dysfunction, has an unclear effect on the efficacy and safety of warfarin. Based on previous studies, we hypothesized that vascular senescence increases the risk of bleeding during warfarin therapy. This study aimed to explore these effects using animal models and clinical cohorts. We established rat models of vascular senescence and calcification using d-galactose, vitamin D, and nicotine. After validating the models, we examined changes in the international normalized ratio (INR) at fixed warfarin doses (0.20 and 0.35 mg/kg). We found that vascular senescence caused significantly elevated INR values and increased bleeding risk. In the prospective clinical cohort study (NCT06428110), hospitalized warfarin patients with standard dose adjustments were divided into vascular senescence and control groups based on ultrasound and computed tomography diagnosis. Using propensity score matching to exclude the influence of confounding factors, we found that the vascular senescence group had lower steady-state warfarin doses and larger dose adjustments, with a higher probability of INR exceeding the therapeutic range. The vascular senescence group tended to experience more bleeding or thromboembolic/ischemic events during 1 year of follow-up, while there was no statistical difference. In conclusion, vascular senescence leads to unstable INR values and increases higher bleeding risk during warfarin therapy, highlighting the importance of considering vascular senescence in future precision warfarin therapies. SIGNIFICANCE STATEMENT: Many factors influence warfarin efficacy; however, the effect of vascular senescence remains unclear. This study aimed to investigate the effects of vascular senescence on the efficacy and safety of warfarin. Through both rat models and clinical cohort studies, our findings indicated that vascular senescence may compromise the stability of warfarin, presenting challenges in maintaining its efficacy and safety.

Next-generation CRISPR technology for genome, epigenome and mitochondrial editing.

The application of rapidly growing CRISPR toolboxes and methods has great potential to transform biomedical research. Here, we provide a snapshot of up-to-date CRISPR toolboxes, then critically discuss the promises and hurdles associated with CRISPR-based nuclear genome editing, epigenome editing, and mitochondrial editing. The technical challenges and key solutions to realize epigenome editing in vivo, in vivo base editing and prime editing, mitochondrial editing in complex tissues and animals, and CRISPR-associated transposases and integrases in targeted genomic integration of very large DNA payloads are discussed. Lastly, we discuss the latest situation of the CRISPR/Cas9 clinical trials and provide perspectives on CRISPR-based gene therapy. Apart from technical shortcomings, ethical and societal considerations for CRISPR applications in human therapeutics and research are extensively highlighted.

AAPM task group report 288: Recommendations for guiding radiotherapy event narratives.

Incident reporting and learning systems provide an opportunity to identify systemic vulnerabilities that contribute to incidents and potentially degrade quality. The narrative of an incident is intended to provide a clear, easy to understand description of an incident. Unclear, incomplete or poorly organized narratives compromise the ability to learn from them. This report provides guidance for drafting effective narratives, with particular attention to the use of narratives in incident reporting and learning systems (IRLS). Examples are given that compare effective and less than effective narratives. This report is mostly directed to organizations that maintain IRLS, but also may be helpful for individuals who desire to write a useful narrative for entry into such a system. Recommendations include the following: (1) Systems should allow a one- or two-sentence, free-text synopsis of an incident without guessing at causes; (2) Information included should form a sequence of events with chronology; and (3) Reporting and learning systems should consider using the headings suggested to guide the reporter through the narrative: (a) incident occurrences and actions by role; (b) prior circumstances and actions; (c) method by which the incident was identified; (d) equipment related details if relevant; (e) recovery actions by role; (f) relevant time span between responses; (g) and how individuals affected during or immediately after incident. When possible and appropriate, supplementary information including relevant data elements should be included using numerical scales or drop-down choices outside of the narrative. Information that should not be included in the narrative includes: (a) patient health information (PHI); (b) conjecture or blame; (c) jargon abbreviations or details without specifying their significance; (d) causal analysis.

Construction of the fluorescence sensing platform with a bifunctional Cu@MOF nanozyme for determination of alkaline phosphatase and its inhibitor.

In this work, a novel and sensitive fluorescence sensing system for alkaline phosphatase (ALP) was constructed using a bifunctional copper metal-organic framework (Cu@MOF) nanozyme, which had excellent oxidase-mimetic activity and fluorescence properties. Owing to the presence of 2-amino-1,4-benzenedicarboxylic acid (1,4-BDC-NH2) ligand, Cu@MOF displays excellent fluorescence performance at 444 nm. Additionally, Cu2+ endows the oxidase-like activity of Cu@MOF, which could trigger p-phenylenediamine (PPD) to be oxidized to a brown product (PPDox) and quench the photoluminescence of Cu@MOF through the inner filtration effect (IFE). As the preferential affinity of ATP for Cu2+, the catalytic activity of Cu@MOF was significantly reduced once ATP was added, thus PPD could not be oxidized and fluorescence was recovered. In the presence of ALP, ATP was hydrolyzed to adenosine and Pi, which allowed Cu@MOF to regain its catalytic activity and continued to catalyze the generation of PPDox. The fluorescence of Cu@MOF was therefore weakened once again. The ALP activity was directly proportional to the degree of decrease in fluorescence intensity. Thus, this novel fluorescence sensing strategy had a linear range of 0.5-60 U/L and the limit of detection was 0.14 U/L. The established sensing method could also be used to for ALP inhibitors screening, and achieved satisfactory results in determining the level of ALP activity in human serum.

Continuous Strain Regulation of Palladium-Gold at the Atomic Level.

Revealing the effect of surface structure changes on the electrocatalytic performance is beneficial to the development of highly efficient catalysts. However, precise regulation of the catalyst surface at the atomic level remains challenging. Here, we present a continuous strain regulation of palladium (Pd) on gold (Au) via a mechanically controllable surface strain (MCSS) setup. It is found that the structural changes induced by the strain setup can accelerate electron transfer at the solid-liquid interface, thus achieving a significantly improved performance toward hydrogen evolution reaction (HER). In situ X-ray diffraction (XRD) experiments further confirm that the enhanced activity is attributed to the increased interplanar spacing resulting from the applied strain. Theoretical calculations reveal that the tensile strain modulates the electronic structure of the Pd active sites and facilitates the desorption of the hydrogen intermediates. This work provides an effective approach for revealing the relationships between the electrocatalyst surface structure and catalytic activity.

Molecular Insights into the Effect of Cholesterol on the Binding of Bicarbonate Ions in Band 3 Protein.

Band 3, or anion exchanger 1 (AE1), is one of the indispensable transmembrane proteins involved in the effective respiratory process of the human body and is primarily responsible for the exchange of bicarbonate and chloride anions across the plasma membrane of erythrocyte. However, the molecular mechanism of ion transport of Band 3 is not completely understood, yet. In this work, we systematically investigate the key binding sites of bicarbonate ions in Band 3 and the impact of cholesterol (CHOL) in lipid bilayers on bicarbonate ion binding using all-atom molecular dynamics (MD) simulations. We examine the dynamics of interactions of bicarbonate ions with Band 3 in the microsecond time scale and calculate the binding free energy of the anion in Band 3. The results indicate that the residue R730 of Band 3 is the most probable binding site for bicarbonate ions. CHOL enhances the bicarbonate ion binding by influencing the conformational stability of Band 3 and compressing the volume of the Band 3 cavity. These findings provide some insights into the bicarbonate ion binding in Band 3 and are helpful for understanding the anion exchange of Band 3.

Identification of the immune repertoire of γδ T-cell receptors in psoriasis.

The γδ T cells are a subpopulation of T cells that are abundantly found in the skin and mucous membranes. Their reactivity to self-antigens and ability to secrete various cytokines make them a key component in psoriasis development. Although the correlation between the immune repertoire (IR) of γδ T-cell receptors and the occurrence and severity of psoriasis remains incompletely explored, high-throughput sequencing of γδ T cells has led to a deeper understanding of IR in psoriasis. This study investigated the differences between γδ T cells in patients with psoriasis and healthy controls. The γδ T cells were identified via immunofluorescence staining and a correlation analysis was performed according to the psoriasis area and severity index (PASI) scores. The IR sequencing method was used to detect IR in the γδ T-cell receptors. The findings demonstrated more skin γδ T cells in patients with psoriasis, which were positively correlated with the PASI score. There were subtle differences in most variable (V), diversity (D) and joining (J) gene segments and VJ/VDJ combination segments between patients with psoriasis and healthy controls. However, a higher diversity of complementarity-determining region 3 (CDR3) was observed in patients with psoriasis. In summary, the IR of skin γδ T cells was significantly altered in patients with psoriasis, and the diversity in the cell's CDR3 population is a promising biomarker for assessment of psoriasis severity.

Effects of Natural Products through Inhibiting Endoplasmic Reticulum Stress on Attenuation of Idiopathic Pulmonary Fibrosis.

With ever-increasing intensive studies of idiopathic pulmonary fibrosis (IPF), significant progresses have been made. Endoplasmic reticulum stress (ERS)/unfolded protein reaction (UPR) is associated with the development and progression of IPF, and targeting ERS/UPR may be beneficial in the treatment of IPF. Natural product is a tremendous source of new drug discovery, and accumulating studies have reported that many natural products show potential therapeutic effects for IPF via modulating one or more branches of the ERS signaling pathway. Therefore, this review focuses on critical roles of ERS in IPF development, and summarizes herbal preparations and bioactive compounds which protect against IPF through regulating ERS.

Cryoprotectant-Mediated Cold Stress Mitigation in Litchi Flower Development: Transcriptomic and Metabolomic Perspectives.

Temperature is vital in plant growth and agricultural fruit production. Litchi chinensis Sonn, commonly known as litchi, is appreciated for its delicious fruit and fragrant blossoms and is susceptible to stress when exposed to low temperatures. This study investigates the effect of two cryoprotectants that counteract cold stress during litchi flowering, identifies the genes that generate the cold resistance induced by the treatments, and hypothesizes the roles of these genes in cold resistance. Whole plants were treated with Bihu and Liangli cryoprotectant solutions to protect inflorescences below 10 °C. The soluble protein, sugar, fructose, sucrose, glucose, and proline contents were measured during inflorescence. Sucrose synthetase, sucrose phosphate synthetase, antioxidant enzymes (SOD, POD, CAT), and MDA were also monitored throughout the flowering stage. Differentially expressed genes (DEGs), gene ontology, and associated KEGG pathways in the transcriptomics study were investigated. There were 1243 DEGs expressed after Bihu treatment and 1340 in the control samples. Signal transduction pathways were associated with 39 genes in the control group and 43 genes in the Bihu treatment group. The discovery of these genes may contribute to further research on cold resistance mechanisms in litchi. The Bihu treatment was related to 422 low-temperature-sensitive differentially accumulated metabolites (DAMs), as opposed to 408 DAMs in the control, mostly associated with lipid metabolism, organic oxidants, and alcohols. Among them, the most significant differentially accumulated metabolites were involved in pathways such as ß-alanine metabolism, polycyclic aromatic hydrocarbon biosynthesis, linoleic acid metabolism, and histidine metabolism. These results showed that Bihu treatment could potentially promote these favorable traits and increase fruit productivity compared to the Liangli and control treatments. More genomic research into cold stress is needed to support the findings of this study.

Venomous snakebites in children: a 10 year experience in South China.

INTRODUCTION: Many studies have focused on snakebites in adults, but very few have described snakebites in children. METHODS: We reviewed the clinical characteristics and outcomes of children with venomous snakebites aged less than 15 years who presented to a regional medical centre in South China from January 2013 to December 2022. RESULTS: A total of 69 envenomed patients were analyzed in our study; 42 (60.9 per cent) patients were male, and 59 (85.5 per cent) reported lower limb bites. Most bites (89.8 per cent) occurred between April and October. Twenty-seven patients received first aid management, and 47 required admission to the general ward. Antivenom was administered to 58 patients, glucocorticoids to 43 patients, antibiotics to 48 patients, and tetanus antitoxin to 40 patients. No fatalities were reported. The most common snake identified was Trimeresurus albolabris. Four were classified as dry bites, 15 as mild, 43 as moderate, and seven as severe. The most common local signs were pain and swelling, while the most common systemic effects were haematological complications. Patients with high severity scores had significantly higher lactate dehydrogenase activities, creatine kinase isoenzyme activities, aspartate aminotransferase activities, D-dimer concentrations, prothrombin times and lower fibrinogen concentrations. In a receiver operating characteristic curve analysis of the values with the highest Youden index, the following cut-offs proved significant: lactate dehydrogenase activity > 248.1 U/L, creatine kinase isoenzyme activities > 17.5 U/L, fibrinogen concentration < 1,455 mg/L, D-dimer concentration > 437.0 µg/L, aspartate aminotransferase activity > 26.1 U/L, and prothrombin time > 15.2 seconds. DISCUSSION: This study provides insight into the epidemiology, clinical profile, and management of snakebites in children. Data from the present study were compared with those from our previous adult study. Limitations include that 50.7 per cent of our snakebites were attributed to Trimeresurus albolabris. Therefore, the results of our study may not be generalizable to all snakebites. CONCLUSION: The clinical symptoms were more severe in children than in adults in our previous study. Even though there were no fatalities, close monitoring should be performed to detect haematological and other potentially fatal complications promptly.

Unveiling Interactions of Tumor-Targeting Nanoparticles with Lipid Bilayers Using a Titratable Martini Model.

pH-responsive nanoparticles are ideal vehicles for drug delivery and are widely used in cell imaging in targeted therapy of cancer, which usually has a weakly acidic microenvironment. In this work, we constructed a titratable molecular model for nanoparticles grafted with ligands of pH-sensitive carboxylic acids and investigated the interactions between the nanoparticles and the lipid bilayer in varying pH environments. We mainly examined the effect of the grafting density of the pH-sensitive ligands of the nanoparticles on the interactions of the nanoparticles with the lipid bilayer. The results show that the nanoparticles can penetrate the lipid bilayer only when the pH value is lower than a critical value, which can be readily modulated to the specific pH value of the tumor microenvironment by changing the ligand grafting density. This work provides some insights into modulating the interactions between the pH-sensitive nanoparticles and cellular membranes to realize targeted drug delivery to tumors based on their specific pH environment.

Oxygen Vacancies Boosted Hydronium Intercalation: A Paradigm Shift in Aluminum-Based Batteries.

In aqueous aluminum-ion batteries (AAIBs), the insertion/extraction chemistry of Al3+ often leads to poor kinetics, whereas the rapid diffusion kinetics of hydronium ions (H3O+) may offer the solution. However, the presence of considerable Al3+ in the electrolyte hinders the insertion reaction of H3O+. Herein, we report how oxygen-deficient α-MoO3 nanosheets unlock selective H3O+ insertion in a mild aluminum-ion electrolyte. The abundant oxygen defects impede the insertion of Al3+ due to excessively strong adsorption, while allowing H3O+ to be inserted/diffused through the Grotthuss proton conduction mechanism. This research advances our understanding of the mechanism behind selective H3O+ insertion in mild electrolytes.

East Asian summer monsoon delivers large abundances of very-short-lived organic chlorine substances to the lower stratosphere.

Deep convection in the Asian summer monsoon is a significant transport process for lifting pollutants from the planetary boundary layer to the tropopause level. This process enables efficient injection into the stratosphere of reactive species such as chlorinated very-short-lived substances (Cl-VSLSs) that deplete ozone. Past studies of convective transport associated with the Asian summer monsoon have focused mostly on the south Asian summer monsoon. Airborne observations reported in this work identify the East Asian summer monsoon convection as an effective transport pathway that carried record-breaking levels of ozone-depleting Cl-VSLSs (mean organic chlorine from these VSLSs ~500 ppt) to the base of the stratosphere. These unique observations show total organic chlorine from VSLSs in the lower stratosphere over the Asian monsoon tropopause to be more than twice that previously reported over the tropical tropopause. Considering the recently observed increase in Cl-VSLS emissions and the ongoing strengthening of the East Asian summer monsoon under global warming, our results highlight that a reevaluation of the contribution of Cl-VSLS injection via the Asian monsoon to the total stratospheric chlorine budget is warranted.