Genome-Resolved Metagenomics and Denitrifying Strain Isolation Reveal New Insights into Microbial Denitrification in the Deep Vadose Zone.

The heavy use of nitrogen fertilizer in intensive agricultural areas often leads to nitrate accumulation in subsurface soil and nitrate contamination in groundwater, which poses a serious risk to public health. Denitrifying microorganisms in the subsoil convert nitrate to gaseous forms of nitrogen, thereby mitigating the leaching of nitrate into groundwater. Here, we investigated denitrifying microorganisms in the deep vadose zone of a typical intensive agricultural area in China through microcosm enrichment, genome-resolved metagenomic analysis, and denitrifying bacteria isolation. A total of 1000 metagenome-assembled genomes (MAGs) were reconstructed, resulting in 98 high-quality, dereplicated MAGs that contained denitrification genes. Among them, 32 MAGs could not be taxonomically classified at the genus or species level, indicating that a broader spectrum of taxonomic groups is involved in subsoil denitrification than previously recognized. A denitrifier isolate library was constructed by using a strategy combining high-throughput and conventional cultivation techniques. Assessment of the denitrification characteristics of both the MAGs and isolates demonstrated the dominance of truncated denitrification. Functional screening revealed the highest denitrification activity in two complete denitrifiers belonging to the genus Pseudomonas. These findings greatly expand the current knowledge of the composition and function of denitrifying microorganisms in subsoils. The constructed isolate library provided the first pool of subsoil-denitrifying microorganisms that could facilitate the development of microbe-based technologies for nitrate attenuation in groundwater.

eDNA reveals spatial homogenization of fish diversity in a mountain river affected by a reservoir cascade.

One of the main reasons for the decline in global freshwater biodiversity can be attributed to alterations in hydrological conditions resulting from dam construction. However, the majority of current research has focused on single or limited numbers of dams. Here, we carried out a seasonal fish survey, using environmental DNA (eDNA) method, on the Wujiang River mainstream (Tributaries of the Yangtze River, China) to investigate the impact of large-scale cascade hydropower development on changes in fish diversity patterns. eDNA survey revealed that native fish species have decreased in contrast to alien fish. There was also a shift in fish community structure, with declines of the dominant rheophilic fish species, an increase of the small-size fish species, and homogenization of species composition across reservoirs. Additionally, environmental factors, such as temperature, dissolved oxygen and reservoir age, had a significant effect on fish community diversity. This study provides basic information for the evaluation of the impact of cascade developments on fish diversity patterns.

Short daylight photoperiod alleviated alarm substance-stimulated fear response of zebrafish.

Photoperiod has been well-documented to be involved in regulating many activities of animals. However, whether photoperiod takes part in mood control, such as fear response in fish and the underlying mode(s) of action remain unclear. In this study, adult zebrafish males and females (Danio rerio) were exposed to different photoperiods, Blank (12 h light: 12 h dark), Control (12 h light: 12 h dark), Short daylight (SD, 6 h light: 18 h dark) and Long daylight (LD, 18 h light: 6 h dark) for 28 days. After exposure, fear response of the fish was investigated using a novel tank diving test. After alarm substance administration, the onset to higher half, total duration in lower half and duration of freezing in SD-fish were significantly decreased, suggesting that short daylight photoperiod is capable of alleviating fear response in zebrafish. In contrast, comparing with the Control, LD didn't show significant effect on fear response of the fish. Further investigation revealed that SD increased the levels of melatonin (MT), serotonin (5-HT) and dopamine (DA) in the brain while decreased the plasma level of cortisol comparing to the Control. Moreover, the expressions of genes in MT, 5-HT and DA pathways and HPI axis were also altered consistently. Our data indicated that short daylight photoperiod might alleviate fear response of zebrafish probably through interfering with MT/5-HT/DA pathways and HPI axis.

Transarterial chemoembolization plus lenvatinib versus transarterial chemoembolization plus sorafenib as first-line treatment for hepatocellular carcinoma with portal vein tumor thrombus: A prospective randomized study.

BACKGROUND: The efficacy and safety of transarterial chemoembolization (TACE) plus lenvatinib in patients with hepatocellular carcinoma (HCC) and portal vein tumor thrombus (PVTT) have not been evaluated. METHODS: In this open-label, single-center, randomized trial (ClinicalTrials.gov identifier: NCT04127396), participants with previously untreated HCC and type I-IV PVTT were randomized 1:1 to receive TACE plus lenvatinib (arm L; orally once daily, 12 mg for body weight ≥60 kg or 8 mg for body weight <60 kg) or TACE plus sorafenib (arm S; 400 mg orally twice daily in 28-day cycles). The primary end point was time-to-progression (TTP; time from randomization to disease progression) and secondary end points included objective response rate and toxicity. Prognostic factors were evaluated using a multivariable Cox proportional hazards model. RESULTS: Between December 30, 2018 and May 31, 2020, 64 patients were randomized (arm L, n = 32; arm S, n = 32); most patients had type I/II PVTT (71.9%), and the median target tumor diameter was 9.8 cm (range, 3.8-21.8). After a median follow-up of 16.1 months, patients in arm L had a higher median TTP (4.7 vs 3.1 months; hazard ratio [HR], 0.55; 95% CI, 0.32-0.95; P = .029) and objective response rate (53.1% vs 25.0%, P = .039) versus arm S. Multivariable analysis showed that TACE plus lenvatinib was significantly associated with higher TTP versus TACE plus sorafenib (HR, 0.50; 95% CI, 0.28-0.90; P = .021). Comparable safety profiles were observed in arms L and S. CONCLUSIONS: TACE plus lenvatinib was safe, well tolerated, and had favorable efficacy versus TACE plus sorafenib in patients with advanced HCC with PVTT and large tumor burden. LAY SUMMARY: Hepatocellular carcinoma with portal vein tumor thrombus has a poor prognosis. In addition, most phase 3 trials of drugs for hepatocellular carcinoma exclude patients with major portal vein invasion, and treatment options for these patients are limited. Transarterial chemoembolization has shown promising efficacy in these patients, especially in combination with systemic treatment or radiotherapy. However, transarterial chemoembolization plus lenvatinib has not been investigated in this setting. This open-label, single-center, randomized trial showed that transarterial chemoembolization plus lenvatinib is safe, well tolerated, and has favorable efficacy versus transarterial chemoembolization plus sorafenib for the treatment of hepatocellular carcinoma with portal vein tumor thrombus.

Life Cycle Exposure to Environmentally Relevant Concentrations of Diphenyl Phosphate (DPhP) Inhibits Growth and Energy Metabolism of Zebrafish in a Sex-Specific Manner.

Due to commercial uses and environmental degradation of aryl phosphate esters, diphenyl phosphate (DPhP) is frequently detected in environmental matrices and is thus of growing concern worldwide. However, information on potential adverse effects of chronic exposure to DPhP at environmentally realistic concentrations was lacking. Here, we investigated the effects of life cycle exposure to DPhP on zebrafish at environmentally relevant concentrations of 0.8, 3.9, or 35.6 µg/L and employed a dual-omics approach (metabolomics and transcriptomics) to characterize potential modes of action. Exposure to DPhP at 35.6 µg/L for 120 days resulted in significant reductions in body mass and length of male zebrafish, but did not cause those same effects to females. Predominant toxicological mechanisms, including inhibition of oxidative phosphorylation, down-regulation of fatty acid oxidation, and up-regulation of phosphatidylcholine degradation, were revealed by integrated dual-omics analysis and successfully linked to adverse outcomes. Activity of succinate dehydrogenase and protein content of carnitine O-palmitoyltransferase 1 were significantly decreased in livers of male fish exposed to DPhP, which further confirmed the proposed toxicological mechanisms. This study is the first to demonstrate that chronic, low-level exposure to DPhP can retard growth via inhibiting energy output in male zebrafish.

Phylogenetic perspective on the relationships and evolutionary history of the Acipenseriformes.

The Acipenseriformes, as one of the earliest extant vertebrates, plays an important role in the evolution of fishes and even the whole vertebrates. Here we collected and analyzed all complete mitochondrial genomes of Acipenseriformes species. Phylogenetic analyses demonstrated that the polytomous branch included Acipenseridae and Polyodontidae formed five clades. The Polyodontidae clade and the Scaphirhynchus clade both were monophyletic group, whereas the Acipenser species and the Huso species both were polyphyletic group. The Bayesian divergence times showed that the origin time for Acipenseriformes was at 318.0 Mya, which was similar to the some previous results of 312.1 Mya, 346.9 Mya and 389.7 Mya. The result was in good consistent with the paleontological data available and the split time of the Pacific and Atlantic Oceans from the Jurassic to the Cretaceous (Laurasia splits in North America and Eurasia). The dN/dS ratios showed the evolutionary rates gradually slow down in five major Acipenseriformes clades from the Clade A (the Pacific sturgeons species) to Clade C (the genus Scaphirhynchus), which was related to the process of geographical formation.

Tributyltin enhanced anxiety of adult male zebrafish through elevating cortisol level and disruption in serotonin, dopamine and gamma-aminobutyric acid neurotransmitter pathways.

Tributyltin (TBT), a widely and persistently distributed organontin, has been well documented to disrupt reproduction and behaviors in animals due to its anti-aromatase activity. TBT has been also reported to enhance anxiety in several fish species, whereas the mechanism underlying remains largely unknown. To investigate the disruption of TBT on fish anxiety and the mechanisms possibly involved, adult male zebrafish (Danio rerio) were treated with TBT (100 and 500 ng/L) for 28 days and anxiety behavior was further investigated using a novel tank dive test. Result showed that TBT treatment significantly enhanced the total time of the fish spent in the lower half, delayed the onset time to the higher half of the tank and increased the total duration of freezing of the fish, indicating an enhanced anxiety in TBT-treated fish. Accordingly, TBT sharply elevated the cortisol levels in plasma in a concentration-dependent manner, suggesting that the elevated cortisol level might be involved in the enhanced anxiety. Although the expression of crha was significantly increased and crhbp was significantly decreased in the brain of TBT-treated fish which is consistent to the elevated cortisol level, the expressions of actha and acthb were sharply down-regulated. In contrast, the expressions of genes responsible for the synthesis and action of serotonin (5-HT) (pet1, thp2 and htr1aa), dopamine (DA) (th1, slc6a3, drd2a and drd2b) and gamma-aminobutyric acid (GABA) (gad2 and gabrg2) were all significantly inhibited. The down-regulation of these pivotal genes acting in 5-HT, DA and GABA neurotransmitter systems in response to TBT corresponded well with the TBT-enhanced anxiety in fish. It was thus strongly suggested that these neurotransmitters might be also involved in TBT-enhanced anxiety in adult male zebrafish. The present study extended our understanding of the neurotoxicity of TBT on the anxiety control and behavioral modulation in fish.

Effects of Cyclic Freeze-Thaw on the Steel Bar Reinforced New-To-Old Concrete Interface.

Frost damage of concrete has significant effects on the safety and durability of concrete structures in cold regions, and the concrete structures after repair and reinforcement are still threatened by cyclic freezing and thawing. In this study, the new-to-old concrete interface was reinforced by steel bar. The shear strength of the new-to-old concrete interface was tested after the new-to-old combination was subjected to cyclic freeze-thaw. The effects of the diameter of the steel bar, the compressive strength of new concrete, the number of freeze-thaw cycles and the freezing temperatures on the shear properties of new-to-old concrete interface were studied. The results showed that, in a certain range, the shear strength of the interface was proportional to the diameter of the steel bar and the strength of the new concrete. Meanwhile, the shear strength of the reinforced interface decreased with the decreasing of the freezing temperature and the increasing of the number of freeze-thaw cycles.

[Early Mitotic Inhibitor 1 Regulates DNA Re-replication Mediated by Human Papillomavirus Subtype 16 E7 in Response to DNA Damage].

Objective To explore the molecular mechanism of human papillomavirus subtype 16(HPV-16)E7 oncogene-induced DNA re-replication in response to DNA damage. Methods Flow cytometry was performed to examine the cell cycle changes in RPE1 E7 cells stably expressing HPV-16 E7 and its control cell RPE1 Vector after DNA damage.Immunoblotting assay was used to evaluate the early mitotic inhibitor 1(Emi1)expression in RPE1 E7 and RPE1 Vector cells with or without DNA damage.The changes of the proportion of polyploidy was detected by flow cytometry in DNA-damaged RPE1 E7 cells interfered by Emi1 small interfering RNA. Results Compared with the control cells,the proportion of polyploids in RPE1 E7 cells was significantly increased in response to DNA damage(t=6.397,P=0.0031).Emi1 protein expression was significantly increased in DNA damaged RPE1 E7 cells(t=8.241,P=0.0012).The polyploid ratio of RPE1 E7 cells was significantly reduced after Emi1 was interfered by two independent small interfering RNAs(t=2.916,P=0.0434;t=3.452,P=0.0260). Conclusion In response to DNA damage,Emi1 promoted DNA re-replication caused by HPV-16 E7.

Prognostic significance of the CRP/Alb and neutrophil to lymphocyte ratios in hepatocellular carcinoma patients undergoing TACE and RFA.

BACKGROUND: The C-reactive protein (CRP)/albumin (Alb) ratio (CAR) is a basic inflammatory factor that has been related to poor survival of patients with various tumors. Our research retrospectively examined the relationship between the CAR and the prognosis of hepatocellular carcinoma (HCC). METHODS: This study included 172 patients with HCC who were treated with transcatheter arterial chemoembolization (TACE) and radiofrequency ablation (RFA). RESULTS: The CAR was weakly related to the neutrophil/lymphocyte ratio (NLR, r = .159, P = .037) and the lymphocyte/monocyte ratio (LMR, r = -.263, P = .001). The Glasgow Prognostic Score (GPS) (0/1-2) was related to liver cirrhosis (P = .003), tumor number (P = .02), Child-Pugh grade (P = .001), the platelet/lymphocyte ratio (PLR, P = .006), and the LMR (P = .021). Correlation analysis demonstrated that an elevated CAR was markedly correlated with the tumor size (P = .019), alpha-fetoprotein (AFP) level (P = .033), thrombosis of the portal vein (P = .004), the NLR (P = .036), and the LMR (P = .001). Multivariate analysis indicated that the prognosis of the CAR-High and NLR-High cohort (mOS = 7 months) was significantly worse than those of the CAR-High or NLR-High cohort (mOS = 15 months) and the CAR-Low and NLR-Low cohort (mOS = 26.5 months). CONCLUSIONS: Combination of the NLR and the CAR represents a convenient, quick, and noninvasive biological marker that could improve prognostic prediction in patients with HCC.

Ilexgenin A exerts anti-inflammation and anti-angiogenesis effects through inhibition of STAT3 and PI3K pathways and exhibits synergistic effects with Sorafenib on hepatoma growth.

Recently, we reported that Ilexgenin A exhibits anti-cancer activities and induces cell arrest. Here, we investigated the effect of Ilexgenin A on the inflammation, angiogenesis and tumor growth of hepatocellular carcinoma (HCC). Our current study revealed that Ilexgenin A significantly inhibited the inflammatory cytokines TNF-α and IL-6 levels and downregulated pro-angiogenic factor VEGF production and transcription in HepG2 cells. The underlying mechanism for Ilexgenin A effects appears to be through inhibiting STAT3 and PI3K pathways. Furthermore, we found that not only Ilexgenin A inhibited STAT3 and PI3K pathways in HepG2 cells but also blocked these signaling pathways in HUVECs. Most importantly, by employing two HCC xenografts models - HepG2 and H22, we showed that Ilexgenin A reduced tumor growth and exhibited synergy effect with Sorafenib. ELISA assay, histological analysis and immunohistochemistry examination revealed that the expression of VEGF and MVD was significantly decreased after the treatment with Ilexgenin A and the combination. Moreover, Ilexgenin A could enhance caspase-3/7 activity in vitro and transmission electron microscope indicated that the combination induced evident apoptosis of tumor cells and caused the structural changes of mitochondria in vivo. Although no apparent adverse effects occurred during the treatment period, Sorafenib monotherapy elicited hepatotoxicity for specific expression in the increased level of AST and the ratio of AST/ALT. However, the combination could remedy this adverse effect. In conclusion, the results described in the present study identifies Ilexgenin A as a promising therapeutic candidate that modulates inflammation, angiogenesis, and HCC growth.

GRIM-19, a gene associated with retinoid-interferon-induced mortality, affects endometrial receptivity and embryo implantation.

GRIM-19 is associated with apoptosis, abnormal proliferation, immune tolerance and malignant transformation, and it also plays an important role in early embryonic development. Although the homologous deletion of GRIM-19 causes embryonic lethality in mice, the precise role of GRIM-19 in embryo implantation has not been elucidated. Here we show that GRIM-19 plays an important role in endometrial receptivity and embryo implantation. Day 1 to Day 6 pregnant mouse uteri were collected. Immunohistochemistry studies revealed the presence of GRIM-19 on the luminal epithelium and glandular epithelium throughout the implantation period in pregnant mice. The protein and mRNA levels of GRIM-19 were markedly decreased on Day 4 of pregnancy in pregnant mice, but there was no change in GRIM-19 levels in a group of pseudopregnant mice. Overexpression of GRIM-19 decreased the adhesion rate of RL95-2-BeWo co-cultured spheroids and increased apoptosis. Furthermore, STAT3 and IL-11 mRNA and protein levels were reduced by overexpressing GRIM-19, but protein and mRNA levels of TNF-α were increased. These findings indicate the involvement of GRIM-19 in the embryo implantation process by regulating adhesion, apoptosis and immune tolerance.

Successful cryopreservation of whole sheep ovary by using DMSO-free cryoprotectant.

PURPOSE: The study aims to assess the protective effects of dimethyl sulfoxide (DMSO)-free solution based on trehalose on the cryopreservation of a whole sheep ovary and evaluate its use as an efficient cryoprotectant. METHOD: Twenty-one ovaries collected from 6- to 8-month-old non-pregnant female sheep were randomly distributed into three groups, namely, a fresh group, a DMSO-free group, and a DMSO group. The morphology, cell apoptosis (by hematoxylin and eosin (HE) staining and terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) assay), and mRNA transcript of Bcl-2-associated X protein (BAX) and cold inducible RNA-binding protein (CIRP) (by real-time PCR) of the thawed sheep ovaries and fresh controls were tested to establish a criterion for appraising the results of the cryopreservation. RESULTS: (i) The histological assessment indicated that the structure of the DMSO-free ovaries remained largely intact and comparable to those of the fresh control groups; whereas, significant damage was observed in the ovaries of the DMSO group (P < 0.05). (ii) The TUNEL assay and mRNA transcript of the BAX assessment showed that the apoptosis parameter in the fresh group was the lowest among all the groups (P < 0.05), and the parameter in the DMSO-free group was significantly lower than that in the DMSO group (P < 0.05). (iii) The level of the CIRP transcripts increased the most in the DMSO-free group followed by the DMSO group and the fresh control group (P < 0.05). CONCLUSIONS: These results indicate that a DMSO-free cryoprotectant solution, especially a trehalose cryoprotectant, is an efficient cryoprotectant and has a beneficial effect on the cryopreservation of whole sheep ovaries.

Deletion of TRIM32 protects mice from anxiety- and depression-like behaviors under mild stress.

Chronic stress causes a variety of psychiatric disorders such as anxiety and depression, but its mechanism is not well understood. Tripartite motif-containing protein 32 (TRIM32) was strongly associated with autism spectrum disorder, attention deficit hyperactivity disorder, anxiety and obsessive compulsive disorder based on a study of copy number variation, and deletion of TRIM32 increased neural proliferation and reduced apoptosis. Here, we propose that TRIM32 is involved in chronic stress-induced affective behaviors. Using a chronic unpredictable mild stress mouse depression model, we studied expression of TRIM32 in brain tissue samples and observed behavioral changes in Trim32 knockout mice. The results showed that TRIM32 protein but not its mRNA was significantly reduced in hippocampus in a time-dependent manner within 8 weeks of chronic stress. These stress-induced affective behaviors and reduction of TRIM32 protein expression were significantly reversed by antidepressant fluoxetine treatment. In addition, Trim32 knockout mice showed reduced anxiety and depressive behaviors and hyperactivities compared with Trim32 wild-type mice under normal and mild stress conditions. We conclude that TRIM32 plays important roles in regulation of hyperactivities and positively regulates the development of anxiety and depression disorders induced by chronic stress.

The protective role of antifreeze protein 3 on the structure and function of mature mouse oocytes in vitrification.

Several studies have reported the oocyte damage in mice during vitrification; however, little has been known about the protective role that antifreeze protein 3 (Afp3) plays on their cellular structure and function during vitrification. In order to observe the extracellular cryo-protective role of Afp3, four groups were divided randomly. The observations were made for changes in cytoskeleton, expression of the related genes before and after vitrification, and also for changes in the in vitro developmental potential of oocytes. The outcomes were as follows: (i) microtubules, actin filaments and chromosomal integrity were more intact in the vitrification group supplemented with additional Afp3 compared to the vitrification group. In the fresh control group and the group with additional cryoprotectant containing ethylene glycol (EG), dimethyl sulfoxide (Me2SO) and sucrose, the organelles were more intact than the other two vitrification groups. (ii) Real-time PCR analysis revealed that the relative quantification of mitotic arrest deficient 2 (Mad2) and centromere protein E (Cenp-e) were significantly higher in the vitrification group with additional Afp3, the fresh control group and the one group with additional cryoprotectant, in comparison to the vitrification group. On the contrary, the expression of cold inducible RNA-binding protein (Cirbp) and kinesin-5 motor protein (Eg5) were up-regulated in the vitrification group compared to the remaining groups. (iii) The fertilization rate and the recovery rate in the fresh control group and the group with additional cryoprotectant were higher than the other two vitrification groups; furthermore, the recovery rate and the fertilization rate in the vitrification group with Afp3 were higher than the vitrification group. However, the blastocyst formation rate in all the four groups showed no statistical significance. In conclusion, Afp3 plays a positive role in the structure and function of mice oocytes in vitrification.

Environmentally relevant estrogens impaired spermatogenesis and sexual behaviors in male and female zebrafish.

Environmental estrogens (EEs) are found extensively in natural waters and negatively affect fish reproduction. Research on the reproductive toxicity of EEs mixtures in fish at environmentally relevant concentrations is scarce. In this study, adult male zebrafish were exposed for 60 days to EES (a mixture of EEs), EE2-low (5.55 ng/L, with an estrogenic potency equal to EES), and EE2-high (11.1 ng/L). After exposure, the expression levels of vtg1, vtg3, and esr1 in the livers in EES-treated fish remained unaltered, whereas they were significantly increased in EE2-treated fish. Both EE2-high and EES exposures notably reduced the gonad somatic index and sperm count. A disrupted spermatogenesis was also observed in the testes of EE2-high- and EES-exposed fish, along with an alteration in the expression of genes associated with spermatogonial proliferation (pcna, nanog), cell cycle transition (cyclinb1, cyclind1), and meiosis (aldh1a2, cyp26a1, sycp3). Both EE2 and EES significantly lowered plasma 11-ketotestosterone levels in males, likely by inhibiting the expression level of genes for its synthesis (scc, cyp17a1 and cyp11b2), and increased 17ß-estradiol (E2) levels, possibly through upregulating the expression of cyp19a1a. A significant increase in tnfrsf1a expression and the tnfrsf1a/tnfrsf1b ratio in EE2-high and EES-treated males also suggests increased apoptosis via the extrinsic pathway. Further investigation showed that both EE2-high and EES diminished the sexual behavior of male fish, accompanied with reduced E2 levels in the brain and the expression of genes in the kisspeptin/gonadotropin-releasing hormone system. Interestingly, the sexual behavior of unexposed females paired with treated males was also reduced, indicating a synergistic effect. This study suggests that EES have a more severe impact on reproduction than EE2-low, and EEs could interfere not only with spermatogenesis in fish, but also with the sexual behaviors of both exposed males and their female partners, thereby leading to a more significant disruption in fish reproduction.

Diversifying crop rotation increases food production, reduces net greenhouse gas emissions and improves soil health.

Global food production faces challenges in balancing the need for increased yields with environmental sustainability. This study presents a six-year field experiment in the North China Plain, demonstrating the benefits of diversifying traditional cereal monoculture (wheat-maize) with cash crops (sweet potato) and legumes (peanut and soybean). The diversified rotations increase equivalent yield by up to 38%, reduce N2O emissions by 39%, and improve the system's greenhouse gas balance by 88%. Furthermore, including legumes in crop rotations stimulates soil microbial activities, increases soil organic carbon stocks by 8%, and enhances soil health (indexed with the selected soil physiochemical and biological properties) by 45%. The large-scale adoption of diversified cropping systems in the North China Plain could increase cereal production by 32% when wheat-maize follows alternative crops in rotation and farmer income by 20% while benefiting the environment. This study provides an example of sustainable food production practices, emphasizing the significance of crop diversification for long-term agricultural resilience and soil health.