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Background Texture quality is impotent for melon ( Cucumis melo L.) fruit. β-galactosidase (β-Gal, EC 3.2.1.23) is an important cell wall glycosyl hydrolase involved in fruit softening, However, the β-Gal gene ( BGALs ) family hasn’t been identified genome-wide in melon. Thus, it’s necessary to conduct an in-depth bioinformatic analysis on melon BGALs family and to seek out the key members who participated in melon fruit softening. Results A total of 21 BGALs members designated as CmBGAL1-CmBGAL21 were identified genome-wide in melon, clustered into A-G seven clades. Among them, three duplications CmBGAL1 : CmBGAL3 , CmBGAL19 : CmBGAL21 , and CmBGAL20 : CmBGAL21 happened . For conserved domains, besides the Glyco_hydro_35 domain (PF01301), all the members also contained the GHD domain (PF17834) except for CmBGAL12, and the Gal_Lectin (PF02140) domain existed in most CmBGALs at the C-termini. Motifs, protein secondary and tertiary structure analysis showed that the CmBGAL12 is a unique member. Moreover, protein-protein association network analysis showed that the CmBGAL12 is the only node protein. Furthermore, spatiotemporal expression pattern analysis by quantitative real-time PCR (qRT-PCR) suggested that most of CmBGAL s expressed in tissues with vigorous cell wall remodeling/disassembly. In addition, cis -acting regulatory elements analysis in promoters inferred that CmBGALs might participate in diverse responsiveness to phytohormone, biotic and abiotic signaling. Conclusions A novel clade of CmBGAL members (Clade F) related to melon fruit softening was discovered, since their expression showed a specific surge in the mature fruit of ‘HPM’ with mealy texture (softening sharply), but not in ‘HDB’ with crisp texture (softening bluntly). The homologous CmBGAL7–11 in Clade F exhibited identical spatiotemporal expression patterns may multiple genes leading to melon fruit softening.

Key messageCmCAD2 and CmCAD3 function more positively than CmCAD1 in oriental melon for lignin synthesis which is important to ensure internal water status and thus for drought tolerance.Well-lignification may be the guarantee of efficient axial water transport and barrier of lateral water flow in oriental melon tolerating drought stress, however remains to be verified. As an important enzyme in monolignol synthesis pathway, five cinnamyl alcohol dehydrogenase (CAD) genes were generally induced in melon seedlings by drought. Here we further revealed the roles of CmCAD1, 2, and 3 in lignin synthesis and for drought tolerance. Results found that overexpressing CmCAD2 or 3 strongly recovered CAD activities, lignin synthesis and composition in Arabidopsis cadc cadd, whose lignin synthesis is disrupted, while CmCAD1 functioned modestly. In melon seedlings, silenced CmCAD2 and 3 individually or collectively decreased CAD activities and lignin depositions drastically, resulting in dwarfed phenotypes. Reduced lignin, mainly composed by guaiacyl units catalyzed by CmCAD3, is mainly due to the limited lignification in tracheary elements and development of Casparion strip. While CmCAD1 and 2 exhibited catalysis to p-coumaraldehyde and sinapaldehyde, respectively. Compared with CmCAD1, drought treatments revealed higher sensitivity of CmCAD2 and/or 3 silenced melon seedlings, accompanying with lower relative water contents, water potentials and relatively higher total soluble sugar contents. Slightly up-regulated expressions of aquaporin genes together with limited lignification might imply higher lateral water loss in stems of silenced lines. In Arabidopsis, CmCAD2 and 3 transgenic lines enhanced cadc cadd drought tolerance through recovering lignin synthesis and root development, accompanying with decreased electrolyte leakage ratios and increased RWCs, thus improved survival rates. Briefly, lignin synthesized by CmCAD2 and 3 functions importantly for drought tolerance in melon.

Background Cinnamyl alcohol dehydrogenase (CAD) is an important enzyme functions at the last step in lignin monomer synthesis pathway. Our previous work found that drought induced the expressions of CmCAD genes and promoted lignin biosynthesis in melon stems. Results Here we studied the effects of abscisic acid (ABA), hydrogen peroxide (H 2 O 2 ) and jasmonic acid (JA) to CmCADs under drought stress. Results discovered that drought-induced ABA, H 2 O 2 and MeJA were prevented efficiently from increasing in melon stems pretreated with fluridone (Flu, ABA inhibitor), imidazole (Imi, H 2 O 2 scavenger) and ibuprofen (Ibu, JA inhibitor). ABA and H 2 O 2 are involved in the positive regulations to CmCAD1 , 2 , 3 , and 5 , and JA is involved in the positive regulations to CmCAD2 , 3 , and 5 . According to the expression profiles of lignin biosynthesis genes, ABA, H 2 O 2 and MeJA all showed positive regulations to CmPAL2-like , CmPOD1-like , CmPOD2-like and CmLAC4-like . In addition, positive regulations were also observed with ABA to CmPAL1-like , CmC4H and CmCOMT , with H 2 O 2 to CmPAL1-like , CmC4H , CmCCR and CmLAC17-like , and with JA to CmCCR , CmCOMT , CmLAC11-like and CmLAC17-like . As expected, the signal molecules positively regulated CAD activity and lignin biosynthesis under drought stress. Promoter::GUS assays not only further confirmed the regulations of the signal molecules to CmCAD1~3 , but also revealed the important role of CmCAD3 in lignin synthesis due to the strongest staining of CmCAD3 promoter::GUS . Conclusions CmCADs but CmCAD4 are positively regulated by ABA, H 2 O 2 and JA under drought stress and participate in lignin synthesis.

Mitogen-activated protein kinase (MAPK) is a form of serine/threonine protein kinase that activated by extracellular stimulation acting through the MAPK cascade (MAPKKK-MAPKK-MAPK). The MAPK cascade gene family, an important family of protein kinases, plays a vital role in responding to various stresses and hormone signal transduction processes in plants. In this study, we identified 14 CmMAPKs, 6 CmMAPKKs and 64 CmMAPKKKs in melon genome. Based on structural characteristics and a comparison of phylogenetic relationships of MAPK gene families from Arabidopsis, cucumber and watermelon, CmMAPKs and CmMAPKKs were categorized into 4 groups, and CmMAPKKKs were categorized into 3 groups. Furthermore, chromosome location revealed an unevenly distribution on chromosomes of MAPK cascade genes in melon, respectively. Eventually, qRT-PCR analysis showed that all 14 CmMAPKs had different expression patterns under drought, salt, salicylic acid (SA), methyl jasmonate (MeJA), red light (RL), and Podosphaera xanthii (P. xanthii) treatments. Overall, the expression levels of CmMAPK3 and CmMAPK7 under different treatments were higher than those in control. Our study provides an important basis for future functional verification of MAPK genes in regulating responses to stress and signal substance in melon.

Background The swift transition to online teaching in medical education has presented the challenge of replicating in-class engagement and interaction essential for active learning. Despite online team-based learning (TBL) offering potential solutions through structured cooperative activities, its efficacy in virtual simulation experiment courses remains scantily researched. This study investigates the effectiveness of online TBL for teaching virtual patient experiments in a basic medical laboratory course and contrasts it with traditional offline teaching in terms of student performance and perceptions. Methods A comparative analysis involved 179 Year 3 medical students using online TBL, face-to-face TBL (FTF-TBL), and the flipped classroom (FC) approach. The learning outcomes were assessed based on experiment reports, IRAT scores, TRAT scores, and final exam performance. Students’ perceptions of both online and in-class TBL methodologies were also surveyed. Results Both online and in-class TBL groups demonstrated comparable academic outcomes and surpassed the FC group in academic performance. Students displayed a marked preference for the TBL format (whether online or in-class), valuing its enhancement of learning interest and practical knowledge application. Nevertheless, refinements in discussion efficiency, platform convenience, and student-instructor interaction were indicated as potential areas of improvement in the online setting. Conclusions Online TBL, along with its in-class counterpart, showed superior academic performance and a more positive learning experience compared to the FC group. These findings underscore the potential of online TBL in adapting to modern pedagogical challenges and enriching medical education through virtual simulation experiments.

Background Lipoxygenases (LOXs) play significant roles in abiotic stress responses, and identification of LOX gene promoter function can make an important contribution to elucidating resistance mechanisms. Here, we cloned the CmLOX08 promoter of melon ( Cucumis melo ) and identified the main promoter regions regulating transcription in response to signalling molecules and abiotic stresses. Results The 2054-bp promoter region of CmLOX08 from melon leaves was cloned, and bioinformatic analysis revealed that it harbours numerous cis -regulatory elements associated with signalling molecules and abiotic stress. Five 5′-deletion fragments obtained from the CmLOX08 promoter—2054 (LP1), 1639 (LP2), 1284 (LP3), 1047 (LP4), and 418 bp (LP5)—were fused with a GUS reporter gene and used for tobacco transient assays. Deletion analysis revealed that in response to abscisic acid, salicylic acid, and hydrogen peroxide, the GUS activity of LP1 was significantly higher than that of the mock-treated control and LP2, indicating that the − 2054- to − 1639-bp region positively regulates expression induced by these signalling molecules. However, no deletion fragment GUS activity was induced by methyl jasmonate. In response to salt, drought, and wounding treatments, LP1, LP2, and LP4 promoted significantly higher GUS expression compared with the control. Among all deletion fragments, LP4 showed the highest GUS expression, indicating that − 1047 to − 1 bp is the major region regulating promoter activity and that the − 1047 to − 418-bp region positively regulates expression induced by salt, drought, and wounding, whereas the − 1284 to − 1047-bp region is a negative regulatory segment. Interestingly, although the GUS activity of LP1 and LP2 was not affected by temperature changes, that of LP3 was significantly induced by heat, indicating that the − 1284- to − 1-bp region is a core sequence responding to heat and the − 2054- to − 1284-bp region negatively regulates expression induced by heat. Similarly, the − 1047- to − 1-bp region is the main sequence responding to cold, whereas the − 2054- to − 1047-bp region negatively regulates expression induced by cold. Conclusions We cloned the CmLOX08 promoter and demonstrated that it is a signalling molecule/stress-inducible promoter. Furthermore, we identified core and positive/negative regulatory regions responding to three signalling molecules and five abiotic stresses.

Methylmalonic acid is a surrogate biomarker of mitochondrial dysfunction and oxidative stress. Serum soluble α-Klotho, as a key anti-aging factor, is regarded as one of the biomarkers of aging. The correlation between Methylmalonic Acid (MMA) and Alpha-Klotho (α-Klotho) remains uncertain. This study aims to explore the relationship between MMA and Alpha-Klothoα-Klotho in American adults. Based on the availability of complete biochemical assays for MMA and α-Klotho, we restricted our analyses to the 2011-2014 cycles of the National Health and Nutrition Examination Survey (NHANES), which included 5,216 eligible participants with comprehensive laboratory data. Due to the limited amount of data, there may be a selection bias. In this study, MMA serves as the independent variable while α-Klotho functions as the dependent variable. MMA is a categorical variable, while α-Klotho is a categorical variable. The covariates examined include sociodemographic factors, lifestyle choices, and various systemic diseases. Logistic regression was used to assess the associations between covariates and different independent groups. To explore the relationship between serum MMA levels and α-Klotho, we employed three models: Model 1 adjusted for age, gender, race/ethnicity, education, Marital status, Poverty Income Ratio (PIR), smoking, Body Mass Index (BMI) and Physical activity (PA). Model 2 included all variables from Model 1 plus Cardiovascular diseases (CVD), Hypertension, Diabetes Mellitus (DM). Considering the collinearity problem, Model 3 adjusted for Marital status, PIR, Educations, Smoke, PA, CVD, Hypertension, DM, Glycated Hemoglobin A1c (HbA1c), High-Density Lipoprotein (HDL), Vitamin B12 (VitB12), Estimated Glomerular Filtration Rate (eGFR). A total of 5216 participants were included in the study. Among them, 29.4% (1531 participants) had α-Klotho levels below 704.00 pg/mL, while 70.6% (3685 participants) had levels of 704 pg/mL or greater. Compared to individuals with low serum MMA levels Q1 (<120 nmol/L), the adjusted odds ratios (OR) for α-Klotho with MMA levels in Q2 (120-175 nmol/L), Q3 (175-250 nmol/L), and Q4 (≥250 nmol/L) were 0.81 (95% CI: 0.72 to 0.91, p = 0.001), 0.81 (95% CI: 0.71 to 0.93, p = 0.003), and 0.80 (95% CI: 0.68 to 0.94, p = 0.007), respectively.The correlation between MMA levels and α-Klotho was linearly correlated (non-linearity: P = 0.62). Our findings indicate a significant association between higher MMA levels and lower serum α-Klotho concentrations, It suggests that mitochondrial dysfunction may play a significant role in the aging process. Further research is necessary to validate these findings. This relationship warrants further investigation to clarify its implications for aging and health outcomes.

Ribonucleotide reductase (RR) is a unique enzyme for the reduction of NDPs to dNDPs, the building blocks for DNA synthesis and thus essential for cell proliferation. Pan-cancer profiling studies showed that RRM2, the small subunit M2 of RR, is abnormally overexpressed in multiple types of cancers; however, the underlying regulatory mechanisms in cancers are still unclear. In this study, through searching in cancer-omics databases and immunohistochemistry validation with clinical samples, we showed that the expression of MYBL2, a key oncogenic transcriptional factor, was significantly upregulated correlatively with RRM2 in colorectal cancer (CRC). Ectopic expression and knockdown experiments indicated that MYBL2 was essential for CRC cell proliferation, DNA synthesis, and cell cycle progression in an RRM2-dependent manner. Mechanistically, MYBL2 directly bound to the promoter of RRM2 gene and promoted its transcription during S-phase together with TAF15 and MuvB components. Notably, knockdown of MYBL2 sensitized CRC cells to treatment with MK-1775, a clinical trial drug for inhibition of WEE1, which is involved in a degradation pathway of RRM2. Finally, mouse xenograft experiments showed that the combined suppression of MYBL2 and WEE1 synergistically inhibited CRC growth with a low systemic toxicity in vivo. Therefore, we propose a new regulatory mechanism for RRM2 transcription for CRC proliferation, in which MYBL2 functions by constituting a dynamic S-phase transcription complex following the G1/early S-phase E2Fs complex. Doubly targeting the transcription and degradation machines of RRM2 could produce a synthetic inhibitory effect on RRM2 level with a novel potential for CRC treatment.

Aberrant activation of epidermal growth factor receptor (EGFR) signaling is closely related to the development of non-small cell lung cancer (NSCLC). However, targeted EGFR therapeutics such as tyrosine kinase inhibitors (TKIs) face the challenge of EGFR mutation-mediated resistance. Here, we showed that the reduced JmjC domain-containing 5 (JMJD5) expression is negatively associated with EGFR stability and NSCLC progression. Mechanically, JMJD5 cooperated with E3 ligase HUWE1 to destabilize EGFR and EGFR TKI-resistant mutants for proteasomal degradation, thereby inhibiting NSCLC growth and promoting TKI sensitivity. Furthermore, we identified that JMJD5 can be transported into recipient cells via extracellular vesicles, thereby inhibiting the growth of NSCLC. Together, our findings demonstrate the tumor-suppressive role of JMJD5 in NSCLC and suggest a putative therapeutic strategy for EGFR-related NSCLC by targeting JMJD5 to destabilize EGFR.

The anaerobic oxidation of methane (AOM) is an important sink of methane that plays a significant role in global warming. AOM was first found to be coupled with sulfate reduction and mediated by anaerobic methanotrophic archaea (ANME) and sulfate‐reducing bacteria (SRB). ANME, often forming consortia with SRB, are phylogenetically related to methanogenic archaea. ANME‐1 is even able to produce methane. Subsequently, it has been found that AOM can also be coupled with denitrification. The known microbes responsible for this process are Candidatus Methylomirabilis oxyfera (M. oxyfera) and Candidatus Methanoperedens nitroreducens (M. nitroreducens). Candidatus Methylomirabilis oxyfera belongs to the NC10 bacteria, can catalyze nitrite reduction through an “intra‐aerobic” pathway, and may catalyze AOM through an aerobic methane oxidation pathway. However, M. nitroreducens, which is affiliated with ANME‐2d archaea, may be able to catalyze AOM through the reverse methanogenesis pathway. Moreover, manganese (Mn4+) and iron (Fe3+) can also be used as electron acceptors of AOM. This review summarizes the mechanisms and associated microbes of AOM. It also discusses recent progress in some unclear key issues about AOM, including ANME‐1 in hypersaline environments, the effect of oxygen on M. oxyfera, and the relationship of M. nitroreducens with ANME. The anaerobic oxidation of methane (AOM) is an important sink of methane that plays a significant role in global warming. This review summarizes the mechanisms and associated microbes of AOM.