Large-scale snake genome analyses provide insights into vertebrate development.

Snakes are a remarkable squamate lineage with unique morphological adaptations, especially those related to the evolution of vertebrate skeletons, organs, and sensory systems. To clarify the genetic underpinnings of snake phenotypes, we assembled and analyzed 14 de novo genomes from 12 snake families. We also investigated the genetic basis of the morphological characteristics of snakes using functional experiments. We identified genes, regulatory elements, and structural variations that have potentially contributed to the evolution of limb loss, an elongated body plan, asymmetrical lungs, sensory systems, and digestive adaptations in snakes. We identified some of the genes and regulatory elements that might have shaped the evolution of vision, the skeletal system and diet in blind snakes, and thermoreception in infrared-sensitive snakes. Our study provides insights into the evolution and development of snakes and vertebrates.

Genomic adaptations for arboreal locomotion in Asian flying treefrogs.

SignificanceTo adapt to arboreal lifestyles, treefrogs have evolved a suite of complex traits that support vertical movement and gliding, thus presenting a unique case for studying the genetic basis for traits causally linked to vertical niche expansion. Here, based on two de novo-assembled Asian treefrog genomes, we determined that genes involved in limb development and keratin cytoskeleton likely played a role in the evolution of their climbing systems. Behavioral and morphological evaluation and time-ordered gene coexpression network analysis revealed the developmental patterns and regulatory pathways of the webbed feet used for gliding in Rhacophorus kio.

Intergrative metabolomic and transcriptomic analyses reveal the potential regulatory mechanism of unique dihydroxy fatty acid biosynthesis in the seeds of an industrial oilseed crop Orychophragmus violaceus.

BACKGROUND: Orychophragmus violaceus is a potentially important industrial oilseed crop due to the two 24-carbon dihydroxy fatty acids (diOH-FA) that was newly identified from its seed oil via a 'discontinuous elongation' process. Although many research efforts have focused on the diOH-FA biosynthesis mechanism and identified the potential co-expressed diacylglycerol acyltranferase (DGAT) gene associated with triacylglycerol (TAG)-polyestolides biosynthesis, the dynamics of metabolic changes during seed development of O. violaceus as well as its associated regulatory network changes are poorly understood. RESULTS: In this study, by combining metabolome and transcriptome analysis, we identified that 1,003 metabolites and 22,479 genes were active across four stages of seed development, which were further divided into three main clusters based on the patterns of metabolite accumulation and/or gene expression. Among which, cluster2 was mostly related to diOH-FA biosynthesis pathway. We thus further constructed transcription factor (TF)-structural genes regulatory map for the genes associated with the flavonoids, fatty acids and diOH-FA biosynthesis pathway in this cluster. In particular, several TF families such as bHLH, B3, HD-ZIP, MYB were found to potentially regulate the metabolism associated with the diOH-FA pathway. Among which, multiple candidate TFs with promising potential for increasing the diOH-FA content were identified, and we further traced the evolutionary history of these key genes among species of Brassicaceae. CONCLUSION: Taken together, our study provides new insight into the gene resources and potential relevant regulatory mechanisms of diOH-FA biosynthesis uniquely in seeds of O. violaceus, which will help to promote the downstream breeding efforts of this potential oilseed crop and advance the bio-lubricant industry.

Contrastive voxel clustering for multiscale modeling of brain network.

Resting-state functional magnetic resonance imaging (fMRI) provides an efficient way to analyze the functional connectivity between brain regions. A comprehensive understanding of brain functionality requires a unified description of multi-scale layers of neural structure. However, existing brain network modeling methods often simplify this property by averaging Blood oxygen level dependent (BOLD) signals at the brain region level for fMRI-based analysis with the assumption that BOLD signals are homogeneous within each brain region, which ignores the heterogeneity of voxels within each Region of Interest (ROI). This study introduces a novel multi-stage self-supervised learning framework for multiscale brain network analysis, which effectively delineates brain functionality from voxel to ROIs and up to sample level. A Contrastive Voxel Clustering (CVC) module is proposed to simultaneously learn the voxel-level features and clustering assignments, which ensures the retention of informative clustering features at the finest voxel-level and concurrently preserves functional connectivity characteristics. Additionally, based on the extracted features and clustering assignments at the voxel level by CVC, a Brain ROI-based Graph Neural Network (BR-GNN) is built to extract functional connectivity features at the brain ROI-level and used for sample-level prediction, which integrates the functional clustering maps with the pre-established structural ROI maps and creates a more comprehensive and effective analytical tool. Experiments are performed on two datasets, which illustrate the effectiveness and generalization ability of the proposed method by analyzing voxel-level clustering results and brain ROIs-level functional characteristics. The proposed method provides a multiscale modeling framework for brain functional connectivity analysis, which will be further used for other brain disease identification. Code is available at https://github.com/yanliugroup/fmri-cvc.

Systemic delivery of oncolytic herpes virus using CAR-T cells enhances targeting of antitumor immuno-virotherapy.

Recent studies have indicated that combining oncolytic viruses with CAR-T cells in therapy has shown superior anti-tumor effects, representing a promising approach. Nonetheless, the localized delivery method of intratumoral injection poses challenges for treating metastatic tumors or distal tumors that are difficult to reach. To address this obstacle, we employed HSV-1-infected CAR-T cells, which systemically delivery HSV into solid tumors. The biological function of CAR-T cells remained intact after loading them with HSV for a period of three days. In both immunocompromised and immunocompetent GBM orthotopic mouse models, B7-H3 CAR-T cells effectively delivered HSV to tumor lesions, resulting in enhanced T-cell infiltration and significantly prolonged survival in mice. We also employed a bilateral subcutaneous tumor model and observed that the group receiving intratumoral virus injection exhibited a significant reduction in tumor volume on the injected side, while the group receiving intravenous infusion of CAR-T cells carrying HSV displayed suppressed tumor growth on both sides. Hence, CAR-THSV cells offer notable advantages in the systemic delivery of HSV to distant tumors. In conclusion, our findings emphasize the potential of CAR-T cells as carriers for HSV, presenting significant advantages for oncolytic virotherapy targeting distant tumors.

18ß-glycyrrhetinic acid alleviates radiation-induced skin injury by activating the Nrf2/HO-1 signaling pathway.

Radiation-induced skin injury is a common side effect of radiotherapy, but there are few therapeutic drugs available for prevention or treatment. In this study, we demonstrate that 18ß-Glycyrrhetinic acid (18ß-GA), a bioactive component derived from Glycyrrhiza glabra, substantially reduces the accumulation of reactive oxygen species (ROS) and inhibits apoptosis in HaCaT cells after ionizing radiation (IR), thereby mitigating radiation-induced skin injury. Mechanistically, 18ß-GA promotes the nuclear import of Nrf2, leading to activation of the Nrf2/HO-1 signaling pathway in response to IR. Importantly, Nrf2 silencing increases cell apoptosis and reverse the protective effect of 18ß-GA on radiation-induced skin injury. Furthermore, 18ß-GA preserves skin tissue structure after irradiation, inhibits inflammatory cell infiltration, and alleviates radiation dermatitis. In conclusion, our results suggest that 18ß-GA reduces intracellular ROS production and apoptosis by activating the Nrf2/HO-1 signaling pathway, leading to amelioration of radiation dermatitis.

Roles of very long-chain fatty acids in compound leaf patterning in Medicago truncatula.

Plant cuticles are composed of hydrophobic cuticular waxes and cutin. Very long-chain fatty acids (VLCFAs) are components of epidermal waxes and the plasma membrane and are involved in organ morphogenesis. By screening a barrelclover (Medicago truncatula) mutant population tagged by the transposable element of tobacco (Nicotiana tabacum) cell type1 (Tnt1), we identified two types of mutants with unopened flower phenotypes, named unopened flower1 (uof1) and uof2. Both UOF1 and UOF2 encode enzymes that are involved in the biosynthesis of VLCFAs and cuticular wax. Comparative analysis of the mutants indicated that the mutation in UOF1, but not UOF2, leads to the increased number of leaflets in M. truncatula. UOF1 was specifically expressed in the outermost cell layer (L1) of the shoot apical meristem (SAM) and leaf primordia. The uof1 mutants displayed defects in VLCFA-mediated plasma membrane integrity, resulting in the disordered localization of the PIN-FORMED1 (PIN1) ortholog SMOOTH LEAF MARGIN1 (SLM1) in M. truncatula. Our work demonstrates that the UOF1-mediated biosynthesis of VLCFAs in L1 is critical for compound leaf patterning, which is associated with the polarization of the auxin efflux carrier in M. truncatula.

Identification of ORF1B as a unique nonstructural protein for fowl adenovirus serotype 4.

Fowl adenovirus serotype 4 (FAdV-4), the causative agent of hepatitis-hydropericardium syndrome (HHS), is a double-stranded DNA virus. Although many structural proteins have been deeply studied, the coding potential of some other open reading frames (ORFs) and the biological functions of their products during virus infection have not been fully elucidated. Here, a unique nonstructural protein ORF1B of FAdV-4 was identified and its expression kinetics along infection was analyzed. Except that of FAdV-10, a member of the same genus as FAdV-4, FAdV-4 ORF1B shared as low homologous identity as 29.2% in amino acid sequence with the other ten counterparts. Structurally, ORF1B was mapped on the N-terminal region of the genome between 1485 nt to 1808 nt and predicted to only contain two α-helix. Confocal immunofluorescence assay with homemade rabbit polyclonal antibody demonstrated that ORF1B could be simultaneously observed with structural protein Fiber 1 in FAdV-4-infected cells. Western blot further showed that ORF1B could only be detected in the infected cells but not mature virions, suggesting ORF1B was a nonstructural protein. Subsequently, the expression level of ORF1B detected by qRT-PCR and IFA was gradually decreased along with FAdV-4 infection, suggesting ORF1B was an early gene transcript. These results will lay a solid foundation to further study the biological effect of ORF1B on the replication and pathogenicity of FAdV-4.

Allelic variation of TaABI5-A4 significantly affects seed dormancy in bread wheat.

KEY MESSAGE: We identified a pivotal transcription factor TaABI5-A4 that is significantly associated with pre-harvest sprouting in wheat; its function in regulating seed dormancy was confirmed in transgenic rice. ABI5 is a critical transcription factor in regulation of crop seed maturation, dormancy, germination, and post-germination. Sixteen copies of homologous sequences of ABI5 were identified in Chinese wheat line Zhou 8425B. Cultivars of two haplotypes TaABI5-A4a and TaABI5-A4b showed significantly different seed dormancies. Based on two SNPs between the sequences of TaABI5-A4a and TaABI5-A4b, two complementary dominant sequence-tagged site (STS) markers were developed and validated in a natural population of 103 Chinese wheat cultivars and advanced lines and 200 recombinant inbred lines (RILs) derived from the Yangxiaomai/Zhongyou 9507 cross; the STS markers can be used efficiently and reliably to evaluate the dormancy of wheat seeds. The transcription level of TaABI5-A4b was significantly increased in TaABI5-A4a-GFP transgenic rice lines compared with that in TaABI5-A4b-GFP. The average seed germination index of TaABI5-A4a-GFP transgenic rice lines was significantly lower than those of TaABI5-A4b-GFP. In addition, seeds of TaABI5-A4a-GFP transgenic lines had higher ABA sensitivity and endogenous ABA content, lower endogenous GA content and plant height, and thicker stem internodes than those of TaABI5-A4b-GFP. Allelic variation of TaABI5-A4-affected wheat seed dormancy and the gene function was confirmed in transgenic rice. The transgenic rice lines of TaABI5-A4a and TaABI5-A4b had significantly different sensitivities to ABA and contents of endogenous ABA and GA in mature seeds, thereby influencing the seed dormancy, plant height, and stem internode length and diameter.

Mitochondria-derived methylmalonic acid aggravates ischemia-reperfusion injury by activating reactive oxygen species-dependent ferroptosis.

Ferroptosis is a regulatory cell death process pivotal in myocardial ischemia-reperfusion (I/R) injury. However, the precise mechanism underlying myocardial ferroptosis remains less known. In this study, we investigated the pathophysiological mechanisms of methylmalonic acid (MMA) associated with ferroptosis activation in cardiomyocytes after I/R. We found an increase level of MMA in patients with acute myocardial injury after reperfusion and AC16 cells under hypoxia/reoxygenation (H/R) condition. MMA treatment was found to be associated with excessive oxidative stress in cardiomyocytes, leading to ferroptosis-related myocardial injury. In mice with I/R injury, MMA treatment aggravated myocardial oxidative stress and ferroptosis, which amplified the myocardial infarct size and cardiac dysfunction. Mechanistically, MMA promoted NOX2/4 expression to increase reactive oxygen species (ROS) production in cardiomyocytes, aggravating myocardial injury. Notably, the increased ROS further activated ferroptosis by inhibiting solute carrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4) expression. In addition, MMA decreased the ectopic nuclear distribution of nuclear factor E2-related factor 2 (NRF2) by increasing the interaction between NRF2 and kelch-like ECH-associated protein 1 (KEAP1). This impeded the activation of GPX4/SLC7A11, downstream of NRF2, activating ferroptosis and aggravating myocardial cell injury. Collectively, our study indicates that MMA activates oxidative stress and ROS generation, which induces ferroptosis to exacerbate cardiomyocyte injury in an I/R model. These findings may provide a new perspective for the clinical treatment of I/R injury and warrant further investigation.

Progress of PD-1/PD-L1 inhibitor combination therapy in immune treatment for HER2-positive tumors.

BACKGROUND: Patients with HER2-positive cancers often face a poor prognosis, and treatment regimens containing anti-HER2 have become the first-line treatment options for breast and gastric cancers. However, these approaches are faced with significant challenges in terms of drug resistance. Hence, it is crucial to explore precise treatment strategies aimed at improving survival outcomes. ADVANCEMENTS IN TREATMENT: Over the past few years, there has been rapid advancement in the realm of tumor therapy, particularly with the swift progress of immune checkpoint inhibitors, including PD-1/PD-L1 inhibitors. They exert anti-tumor effects by disrupting immune-suppressive factors within the tumor microenvironment. However, monotherapy with PD-1/PD-L1 inhibitors has several limitations. Consequently, numerous studies have explored combinatorial immunotherapeutic strategies and demonstrated highly promising avenues of development. OBJECTIVE: This article aims to review the clinical trials investigating PD-1/PD-L1 inhibitor combination therapy for HER2-positive tumors. Additionally, it provides a summary of ongoing trials evaluating the efficacy and safety of these combined treatments, with the intention of furnishing valuable insights for the clinical management of HER2-positive cancer. CONCLUSION: Combinatorial immunotherapeutic strategies involving PD-1/PD-L1 inhibitors hold considerable promise in the treatment of HER2-positive tumors. Continued research efforts and clinical trials are warranted to elucidate optimal treatment regimens that maximize therapeutic benefits while minimizing adverse effects.

Calcineurin suppresses rat H9c2 cardiomyocyteprotective autophagy under chronic intermittent hypoxia by downregulating the AMPK pathway.

Calcineurin plays a key role in cardiovascular pathogenesis by exerting pro-apoptotic effects in cardiomyocytes. However, whether calcineurin can regulate cardiomyocyte autophagy under conditions of chronic intermittent hypoxia (CIH) remains unclear. Here, we showed that CIH induced calcineurin activity in H9c2 cells, which attenuated adenosine monophosphate-activated protein kinase (AMPK) signaling and inhibited autophagy. In H9c2 cells, autophagy levels, LC3 expression, and AMPK phosphorylation were significantly elevated under conditions of CIH within 3 days. However, after 5 days of CIH, these effects were reversed and calcineurin activity and apoptosis were significantly increased. The calcineurin inhibitor 17-Allyl-1,14-dihydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl) -1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo- [22.3.1.04,9]octacos-18- ene-2,3,10,16-tetrone (FK506) restored AMPK activation and LC3 expression and attenuated CIH-induced H9c2 cell apoptosis. In contrast, calcineurin overexpression significantly attenuated the increase in LC3 expression and enhanced H9c2 cell apoptosis under conditions of CIH. Calcineurin inhibition failed to induce autophagy or alleviate apoptosis in H9c2 cells expressing a kinase-dead K45R AMPK mutant. Autophagy inhibition abrogated the protective effects of FK506-mediated calcineurin inhibition. These results indicate that calcineurin suppresses adaptive autophagy during CIH by downregulating AMPK activation. Our findings reveal the underlying mechanism of calcineurin and autophagy regulation during H9c2 cell survival under conditions of CIH and may provide a new strategy for preventing CIH-induced cardiomyocyte damage.

Appraisal of surgical outcomes and oncological efficiency of intraoperative adverse events in robotic radical gastrectomy for gastric cancer.

BACKGROUND: Surgical quality control is a crucial determinant of evaluating the tumor efficacy. OBJECTIVE: To assess the ClassIntra grade for quality control and oncological outcomes of robotic radical surgery for gastric cancer (GC). METHODS: Data of patients undergoing robotic radical surgery for GC at a high-volume center were retrospectively analyzed. Patients were categorized into two groups, the intraoperative adverse event (iAE) group and the non-iAE group, based on the occurrence of intraoperative adverse events. The iAEs were further classified into five sublevels (ranging from I to V according to severity) based on the ClassIntra grade. Surgical performance was assessed using the Objective Structured Assessment of Technical Skill (OSATS) and the General Error Reporting Tool. RESULTS: This study included 366 patients (iAE group: n = 72 [19.7%] and non-iAE group: n = 294 [80.3%]). The proportion of ClassIntra grade II patients was the highest in the iAE group (54.2%). In total and distal gastrectomies, iAEs occurred most frequently in the suprapancreatic area (50.0% and 54.8%, respectively). In total gastrectomy, grade IV iAEs were most common during lymph node dissection in the splenic hilum area (once for bleeding [grade IV] and once for injury [grade IV]). The overall survival (OS) and disease-free survival of the non-iAE group were significantly better than those of the iAE group (Log rank P < 0.001). Uni- and multi-variate analyses showed that iAEs were key prognostic indicators, independent of tumor stage and adjuvant chemotherapy (P < 0.001). CONCLUSION: iAEs in patients who underwent robotic radical gastrectomy significantly correlated with the occurrence of postoperative complications and a poor long-term prognosis. Therefore, utilization and inclusion of ClassIntra grading as a crucial surgical quality control and prognostic indicator in the routine surgical quality evaluation system are recommended.

CXCL11-armed oncolytic adenoviruses enhance CAR-T cell therapeutic efficacy and reprogram tumor microenvironment in glioblastoma.

Glioblastoma (GBM) is the most aggressive primary malignant brain cancer and urgently requires effective treatments. Chimeric antigen receptor T (CAR-T) cell therapy offers a potential treatment method, but it is often hindered by poor infiltration of CAR-T cells in tumors and highly immunosuppressive tumor microenvironment (TME). Here, we armed an oncolytic adenovirus (oAds) with a chemokine CXCL11 to increase the infiltration of CAR-T cells and reprogram the immunosuppressive TME, thus improving its therapeutic efficacy. In both immunodeficient and immunocompetent orthotopic GBM mice models, we showed that B7H3-targeted CAR-T cells alone failed to inhibit GBM growth but, when combined with the intratumoral administration of CXCL11-armed oAd, it achieved a durable antitumor response. Besides, oAd-CXCL11 had a potent antitumor effect and reprogramed the immunosuppressive TME in GL261 GBM models, in which increased infiltration of CD8+ T lymphocytes, natural killer (NK) cells, and M1-polarized macrophages, while decreased proportions of myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs) and M2-polarized macrophages were observed. Furthermore, the antitumor effect of the oAd-CXCL11 was CD8+ T cell dependent. Our findings thus revealed that CXCL11-armed oAd can improve immune-virotherapy and can be a promising adjuvant of CAR-T therapy for GBM.

Using cerebral regional oxygen saturation and amplitude-integrated electroencephalography in neonates on extracorporeal membrane oxygenation: preliminary experience from a single center.

OBJECTIVE: This study aims to evaluate the application value in neurological outcome of cerebral regional oxygen saturation (CrSO2) and amplitude-integrated electroencephalography (aEEG) monitoring during neonatal extracorporeal membrane oxygenation (ECMO) courses. METHODS: We retrospectively analyzed 18 neonates receiving veno-arterial ECMO (V-A ECMO) support at our hospital from July 2021 to December 2022. Continuous monitoring of CrSO2 and brain electrical activity was conducted using near-infrared spectroscopy (NIRS) and aEEG throughout the ECMO treatment. We collected and analyzed related clinical data. RESULTS: Among the 11 survivors, 5 were categorized as the normal group (N group) and 6 as the abnormal group (AN group) based on post-ECMO brain MRI outcomes. The N group exhibited shorter time percentage of significant CrSO2 reduction (> 25% from baseline or absolute value < 40%), better fractional tissue oxygen extraction (FTOE) rates, and more stable mean percentage changes in CrSO2 compared to the AN group. Neonates in the N group predominantly showed mildly abnormal aEEG readings, with one patient displaying disrupted sleep-wake cycles. This particular patient also had more significant CrSO2 reduction and poorer FTOE compared to others in the N group. Additionally, the Test of Infant Motor Performance (TIMP) scores indicated hypoevolutism in this patient before discharge, while others in the N group had normal TIMP scores. In the AN group, 4 exhibited moderate and 2 severe aEEG abnormalities; 5 had hypoevolutism TIMP scores, and 1 with moderate aEEG abnormalities maintained a normal TIMP score, exhibiting lesser CrSO2 reduction and improved FTOE. CONCLUSION: CrSO2 and aEEG monitoring show potential as routine assessments for neurological outcomes during neonatal ECMO. In our cohort, a tendency was observed where neonates with greater reductions in CrSO2 and more severe aEEG abnormalities experienced poorer neurological outcomes.

Comparative Analysis of Tentacle Extract and Nematocyst Venom: Toxicity, Mechanism, and Potential Intervention in the Giant Jellyfish Nemopilema nomurai.

The giant jellyfish Nemopilema nomurai sting can cause local and systemic reactions; however, comparative analysis of the tentacle extract (TE) and nematocyst venom extract (NV), and its toxicity, mechanism, and potential intervention are still limited. This study compared venom from TE and NV for their composition, toxicity, and efficacy in vitro and in vivo used RAW264.7 cells and ICR mice. A total of 239 and 225 toxin proteins were identified in TE and NV by proteomics, respectively. Pathological analysis revealed that TE and NV caused heart and liver damage through apoptosis, necrosis, and inflammation, while TE exhibited higher toxicity ex vivo and in vivo. Biochemical markers indicated TE and NV elevated creatine kinase, lactatedehydrogenase, and aspartate aminotransferase, with the TE group showing a more significant increase. Transcriptomics and Western blotting indicated both venoms increased cytokines expression and MAPK signaling pathways. Additionally, 1 mg/kg PACOCF3 (the phospholipase A2 inhibitor) improved survival from 16.7% to 75% in mice. Our results indicate that different extraction methods impact venom activities, tentacle autolysis preserves toxin proteins and their toxicity, and PACOCF3 is a potential antidote, which establishes a good extraction method of jellyfish venom, expands our understanding of jellyfish toxicity, mechanism, and provides a promising intervention.

Synergistic mechanism of botanical pesticide camptothecin encapsulated in a nanocarrier against fall armyworm: Enhanced stability and amplified growth suppression.

Botanical pesticides are one of the most promising alternatives to synthetic insecticides for green pest management. However, their efficacies must be further improved to meet real needs. Here we designed a nanoscale camptothecin (CPT) encapsulated in a star polycation (SPc) and determined its bioactivity against a devastating agricultural pest, Spodoptera frugiperda. The self-assembly of CPT/SPc complex was mainly driven by hydrogen bonding and Van der Waals forces to decrease the particle size from 789 to 298 nm. With the help of SPc, the contact angle of CPT decreased from 116° to 92° on maize leaves, and its retention was increased from 5.53 to 11.97 mg/cm2. The stability of SPc-loaded CPT was also improved in an alkaline environment, which is beneficial for its acting in lepidopteran insect guts. The CPT/SPc complex had stronger larvicidal activity and ovicidal activity against S. frugiperda than CPT alone, led to more complex transcriptomic changes in larvae, and had obvious adverse impacts on the activities of two digestive enzymes. Our findings demonstrated that the encapsulation of CPT by SPc-based nanodelivery system enabled better insecticidal activities against S. frugiperda, which holds great promise for the development of more efficient and sustainable pest control strategies to meet the demands of modern crop protection.

Transcriptome-Wide Identification of Dark- and Salt-Induced Senescence-Related NAC Gene Family Members in Alfalfa.

Leaves are a key forage part for livestock, and the aging of leaves affects forage biomass and quality. Preventing or delaying premature leaf senescence leads to an increase in pasture biomass accumulation and an improvement in alfalfa quality. NAC transcription factors have been reported to affect plant growth and abiotic stress responses. In this study, 48 NAC genes potentially associated with leaf senescence were identified in alfalfa under dark or salt stress conditions. A phylogenetic analysis divided MsNACs into six subgroups based on similar gene structure and conserved motif. These MsNACs were unevenly distributed in 26 alfalfa chromosomes. The results of the collinearity analysis show that all of the MsNACs were involved in gene duplication. Some cis-acting elements related to hormones and stress were screened in the 2-kb promoter regions of MsNACs. Nine of the MsNAC genes were subjected to qRT-PCR to quantify their expression and Agrobacterium-mediated transient expression to verify their functions. The results indicate that Ms.gene031485, Ms.gene032313, Ms.gene08494, and Ms.gene77666 might be key NAC genes involved in alfalfa leaf senescence. Our findings extend the understanding of the regulatory function of MsNACs in leaf senescence.

Contrast sensitivity and comfort levels with different types of polarised glasses under steady glare.

The influence of various polarised glasses on visual performance is crucial due to their widespread. This study measured the visual contrast sensitivity (CS) of dominant eyes by quick contrast sensitivity function (qCSF) procedure at 10 spatial frequencies and 3 noise levels under nonglare, steady glare, steady glare with night lenses, and steady glare with day&night lenses, respectively. Later, the second experiment measured the subjects' subjective feelings under these four viewing conditions. The results showed that there was no significant difference in the CS between the two conditions with polarised glasses and the steady glare. However, the subjects reported greater comfort with glasses than without them. These results suggest that there was an underlying bias when people rated the polarised glasses, and the qCSF procedure was a useful tool for evaluating visual performance.

Whether polarised glasses can relieve the impairment of steady glare on contrast sensitivity over multi-spatial frequency and external noise levels is unaddressed. Using the quick CSF method, we revealed that CS declines with steady glare but polarised lenses don't significantly relieve it. However, subjects reported increased comfort, suggesting a perceptual bias.