De Novo Synthesis of 2,2'-Bipyridines and Related Bis-azines via Cascade Coupling and Double Pyridannulation of Isocyanides.

Herein, we present a new and general protocol for the assembly of 2,2'-bipyridyls from nonpyridine substrates without using any metal catalysts or organometallic reagents. The process starts from the coupling of two 1,3-dienyl isocyanides followed by a 6π-electrocyclization/aromatization cascade featuring the simultaneous formation of two pyridine rings in a single operation. Notably, this strategy is also applicable to the construction of nonsymmetrical 2-(2-pyridyl)-quinolines/-quinoxalines. Furthermore, the aggregation-induced emission (AIE) characteristics endow our approach with great potential in biorelevant fields.

Higher perfusion of rectum carcinoma relative to tumor-free rectal wall: quantification by a new imaging biomarker diffusion-derived vessel density (DDVD).

Background: Diffusion-derived vessel density (DDVD) is a physiological surrogate of the area of micro-vessels per unit tissue area. DDVD is calculated according to: DDVD(b0b5) = Sb0/ROIarea0 - Sb5/ROIarea5, where Sb0 and Sb5 refer to the tissue signal when b is 0 or 5 s/mm2. This study applied DDVD to assess the perfusion of rectal carcinoma (RC). Methods: MRI was performed with a 3.0-T magnet. Diffusion weighted image with b-values of 0, 5 s/mm2 were acquired in 113 patients with non-mucinous RC and 15 patients with mucinous RC. Diffusion-derived vessel density ratio [DDVDr(b0b5)] was DDVD(b0b5) of RC divided by DDVD(b0b5) of tumor-free rectal wall. Results: The median value of the DDVDr(b0b5) for non-mucinous RCs was 1.430, with the majority of RCs showing a higher DDVD than the adjacent tumor-free wall [i.e., with DDVDr(b0b5) >1]. 90.3% (102/113) of non-mucinous RCs were hypervascular, 1.77% (2/113) were iso-vascular, and 7.96% (9/113) were hypovascular. The median value of the DDVDr(b0b5) for mucinous RCs was 1.660. 73.3% (11/15) of mucinous RCs were hypervascular, and 26.7% (4/15) were hypovascular. A trend (P=0.09) was noted that earlier clinical grades non-mucinous RCs had a higher DDVDr(b0b5) than those of the advanced clinical grades (2.245 for grade 0&I, 1.460 for grade II, 1.430 for grade III, 1.130 for grade IV). A non-significant trend was noted with well and moderately differentiated non-mucinous RCs had a higher DDVDr(b0b5)than that of poorly differentiated non-mucinous RCs (median: 1.460 vs. 1.320). A non-significant trend was noted with MRI-detected extramural vascular invasion (mrEMVI) positive non-mucinous RCs had a higher DDVDr(b0b5) than that of mrEMVI negative non-mucinous RCs (1.630 vs. 1.370). Conclusions: DDVD results in this study approximately agree with contrast agent dynamically enhanced CT literature data.

Value of orthogonal axial MR images in preoperative T staging of gastric cancer.

PURPOSE: To assess the value of orthogonal axial images (OAI) of MRI in gastric cancer T staging. METHODS: This retrospective study enrolled 133 patients (median age, 63 [range, 24-85] years) with gastric adenocarcinoma who underwent both CT and MRI followed by surgery. MRI lacking or incorporating OAI and CT images were evaluated, respectively. Diagnostic performance (accuracy, sensitivity, and specificity) for each T stage, overall diagnostic accuracy and rates of over- and understaging were quantified employing pathological T stage as a reference standard. The McNemar's test was performed to compare the overall accuracy. RESULTS: Among patients with pT1-pT4 disease, MRI with OAI (accuracy: 88.7-94.7%, sensitivity: 66.7-93.0%, specificity: 91.5-100.0%) exhibited superior diagnostic performance compared to MRI without OAI (accuracy: 81.2-88.7%, sensitivity: 46.2-83.1%, specificity: 85.5-99.1%) and CT (accuracy: 88.0-92.5%, sensitivity: 53.3-90.1%, specificity: 88.7-98.1%). The overall accuracy of MRI with OAI was significantly higher (83.5%) than that of MRI without OAI (67.7%) (p < .001). However, there was no significant difference in the overall accuracy of MRI with OAI and CT (78.9%) (p = .35). The over- and understaging rates of MRI with OAI (12.0, 4.5%) were lower than those of MRI without OAI (21.8, 10.5%) and CT (12.8, 8.3%). CONCLUSION: OAI play a pivotal role in the T staging of gastric cancer. MRI incorporating OAI demonstrated commendable performance for gastric cancer T-staging, with a slight tendency toward its superiority over CT.

Medical students' perceptions of integrating social media into a narrative medicine programme for 5th-year clerkship in Taiwan: a descriptive qualitative study.

BACKGROUND: The growing demands in integrating digital pedagogies in learning (e.g., social media) contribute to disrupting many fields, including the medical humanities education. However, the strengths and barriers behind social media and medical humanities context are blurred and contradictive. We examined the perceptions of integrating social media - Facebook - into a narrative medicine (NM) programme for 5th -year clerkship in Taiwan. METHODS: We used purposive sampling to recruit participants. Sixteen medical students (Female/Male: 7/9) participated in four group interviews. Semi-structured focus group interviews were conducted to explore students' perceptions and experiences of the social media integrated into the NM programme. We analysed the data using a descriptive thematic analysis with a team-based approach. Data were managed and coded using ATLAS.ti version 9.0. RESULTS: We identified six main themes: (1) Positive experiences of social media integration; (2) Negative experiences of social media integration; (3) Barriers on writing and sharing NM stories in social media; (4) Barriers on reading NM stories in social media; (5) Barriers on reacting contents in social media; (6) Suggestions for future improvement. CONCLUSIONS: The study revealed the strengths and barriers from medical students' perceptions, when integrating social media into a NM programme. It is important to match students' experiences, barriers, and perceptions towards learning. Understanding participants' suggestions for future improvement are also crucial. With this knowledge, we might better develop the social media integration systems that achieve our desired outcomes based on the medical humanities education curricula.

Role of dynamic contrast-enhanced MRI in predicting severe acute radiation-induced rectal injury in patients with rectal cancer.

OBJECTIVES: To explore the potential of dynamic contrast-enhanced MRI (DCE-MRI) quantitative parameters in predicting severe acute radiation-induced rectal injury (RRI) in rectal cancer. METHODS: This retrospective study enrolled 49 patients with rectal cancer who underwent neoadjuvant chemoradiotherapy and rectal MRI including a DCE-MRI sequence from November 2014 to March 2021. Two radiologists independently measured DCE-MRI quantitative parameters, including the forward volume transfer constant (Ktrans), rate constant (kep), fractional extravascular extracellular space volume (ve), and the thickness of the rectal wall farthest away from the tumor. These parameters were compared between mild and severe acute RRI groups based on histopathological assessment. Receiver operating characteristic curve analysis was performed to analyze statistically significant parameters. RESULTS: Forty-nine patients (mean age, 54 years ± 12 [standard deviation]; 37 men) were enrolled, including 25 patients with severe acute RRI. Ktrans was lower in severe acute RRI group than mild acute RRI group (0.032 min-1 vs 0.054 min-1; p = 0.008), but difference of other parameters (kep, ve and rectal wall thickness) was not significant between these two groups (all p > 0.05). The area under the receiver operating characteristic curve of Ktrans was 0.72 (95% confidence interval: 0.57, 0.84). With a Ktrans cutoff value of 0.047 min-1, the sensitivity and specificity for severe acute RRI prediction were 80% and 54%, respectively. CONCLUSION: Ktrans demonstrated moderate diagnostic performance in predicting severe acute RRI. CLINICAL RELEVANCE STATEMENT: Dynamic contrast-enhanced MRI can provide non-invasive and objective evidence for perioperative management and treatment strategies in rectal cancer patients with acute radiation-induced rectal injury. KEY POINTS: • To our knowledge, this study is the first to evaluate the predictive value of contrast-enhanced MRI (DCE-MRI) quantitative parameters for severe acute radiation-induced rectal injury (RRI) in patients with rectal cancer. • Forward volume transfer constant (Ktrans), derived from DCE-MRI, exhibited moderate diagnostic performance (AUC = 0.72) in predicting severe acute RRI of rectal cancer, with a sensitivity of 80% and specificity of 54%. • DCE-MRI is a promising imaging marker for distinguishing the severity of acute RRI in patients with rectal cancer.

MRI-detected tumor deposits in cT3 and cT4 rectal cancer following neoadjuvant chemoradiotherapy.

OBJECTIVES: To evaluate the identification of tumor deposits (TDs) and the prognostic significance of an MRI tumor regression grade for TDs in patients with rectal cancer treated with neoadjuvant chemoradiotherapy (nCRT). METHODS: Ninety-one patients with cT3 or cT4 rectal cancer who underwent surgery following nCRT between August 2014 and June 2020 were retrospectively analyzed. Changes in pre-nCRT MRI-detected TDs (mrTDs) were described as mrTD regression grade. The diagnostic performance of post-nCRT MRI-detected TDs (ymrTDs) was compared with histopathological reference standard. The correlation between ymrTDs, mrTD regression grade, and disease-free survival (DFS) was assessed. RESULTS: The sensitivity and specificity of ymrTDs were 88.00% and 89.39%, respectively. The area under the receiver operating characteristic curve was 0.887 (95% confidence interval [CI]: 0.803-0.944). The 3-year DFS of patients with positive ymrTDs was significantly lower than of the negative group (44.83% vs 82.73%, p < 0.001). The 3-year DFS was 33.33% for patients with poor regression of mrTDs following nCRT and 55.56% for those with moderate regression, compared to 69.23% in good responders and 83.97% in patients without mrTDs (p < 0.001). On multivariable Cox regression, mrTD regression grade was the only independent MRI factor associated with DFS (p = 0.042). CONCLUSIONS: Diagnostic performance of ymrTDs was moderate. The mrTD regression grade was independently correlated with DFS, which may have a prognostic implication for treatment and follow-up. CLINICAL RELEVANCE STATEMENT: Patients with poor regression of MRI-detected tumor deposits may benefit from more aggressive treatments, such as chemoradiation therapy plus induction or consolidation chemotherapy. KEY POINTS: • MRI provides a preoperative and noninvasive way to visualize tumor deposits (TDs) after neoadjuvant chemoradiotherapy (nCRT). • Post-nCRT MRI-detected TDs are a poor prognostic marker in cT3 and cT4 rectal cancer patients. • The regression of MRI-detected TDs after nCRT is associated with an improved disease-free survival.

Cryo-EM of α-tubulin isotype-containing microtubules revealed a contracted structure of α4A/ß2A microtubules.

Microtubules are hollow α/ß-tubulin heterodimeric polymers that play critical roles in cells. In vertebrates, both α- and ß-tubulins have multiple isotypes encoded by different genes, which are intrinsic factors in regulating microtubule functions. However, the structures of microtubules composed of different tubulin isotypes, especially α-tubulin isotypes, remain largely unknown. Here, we purify recombinant tubulin heterodimers composed of different mouse α-tubulin isotypes, including α1A, α1C and α4A, with the ß-tubulin isotype ß2A. We further assemble and determine the cryo-electron microscopy (cryo-EM) structures of α1A/ß2A, α1C/ß2A, and α4A/ß2A microtubules. Our structural analysis demonstrates that α4A/ß2A microtubules exhibit longitudinal contraction between tubulin interdimers compared with α1A/ß2A and α1C/ß2A microtubules. Collectively, our findings reveal that α-tubulin isotype composition can tune microtubule structures, and also provide evidence for the "tubulin code" hypothesis.

A direct spino-cortical circuit bypassing the thalamus modulates nociception.

Nociceptive signals are usually transmitted to layer 4 neurons in somatosensory cortex via the spinothalamic-thalamocortical pathway. The layer 5 corticospinal neurons in sensorimotor cortex are reported to receive the output of neurons in superficial layers; and their descending axons innervate the spinal cord to regulate basic sensorimotor functions. Here, we show that a subset of layer 5 neurons receives spinal inputs through a direct spino-cortical circuit bypassing the thalamus, and thus define these neurons as spino-cortical recipient neurons (SCRNs). Morphological studies revealed that the branches from spinal ascending axons formed a kind of disciform structure with the descending axons from SCRNs in the basilar pontine nucleus (BPN). Electron microscopy and calcium imaging further confirmed that the axon terminals from spinal ascending neurons and SCRNs made functional synaptic contacts in the BPN, linking the ascending sensory pathway to the descending motor control pathway. Furthermore, behavioral tests indicated that the spino-cortical connection in the BPN was involved in nociceptive responses. In vivo calcium imaging showed that SCRNs responded to peripheral noxious stimuli faster than neighboring layer 4 cortical neurons in awake mice. Manipulating activities of SCRNs could modulate nociceptive behaviors. Therefore, this direct spino-cortical circuit represents a noncanonical pathway, allowing a fast sensory-motor transition of the brain in response to noxious stimuli.

The distinct initiation sites and processing activities of TTLL4 and TTLL7 in glutamylation of brain tubulin.

Mammalian brain tubulins undergo a reversible posttranslational modification-polyglutamylation-which attaches a secondary polyglutamate chain to the primary sequence of proteins. Loss of its erasers can disrupt polyglutamylation homeostasis and cause neurodegeneration. Tubulin tyrosine ligase like 4 (TTLL4) and TTLL7 were known to modify tubulins, both with preference for the ß-isoform, but differently contribute to neurodegeneration. However, differences in their biochemical properties and functions remain largely unknown. Here, using an antibody-based method, we characterized the properties of a purified recombinant TTLL4 and confirmed its sole role as an initiator, unlike TTLL7, which both initiates and elongates the side chains. Unexpectedly, TTLL4 produced stronger glutamylation immunosignals for α-isoform than ß-isoform in brain tubulins. Contrarily, the recombinant TTLL7 raised comparable glutamylation immunoreactivity for two isoforms. Given the site selectivity of the glutamylation antibody, we analyzed modification sites of two enzymes. Tandem mass spectrometry analysis revealed their incompatible site selectivity on synthetic peptides mimicking carboxyl termini of α1- and ß2-tubulins and a recombinant tubulin. Particularly, in the recombinant α1A-tubulin, a novel region was found glutamylated by TTLL4 and TTLL7, that again at distinct sites. These results pinpoint different site specificities between two enzymes. Moreover, TTLL7 exhibits less efficiency to elongate microtubules premodified by TTLL4, suggesting possible regulation of TTLL7 elongation activity by TTLL4-initiated sites. Finally, we showed that kinesin behaves differentially on microtubules modified by two enzymes. This study underpins the different reactivity, site selectivity, and function of TTLL4 and TTLL7 on brain tubulins and sheds light on their distinct role in vivo.

Pathway and mechanism of tubulin folding mediated by TRiC/CCT along its ATPase cycle revealed using cryo-EM.

The eukaryotic chaperonin TRiC/CCT assists the folding of about 10% of cytosolic proteins through an ATP-driven conformational cycle, and the essential cytoskeleton protein tubulin is the obligate substrate of TRiC. Here, we present an ensemble of cryo-EM structures of endogenous human TRiC throughout its ATPase cycle, with three of them revealing endogenously engaged tubulin in different folding stages. The open-state TRiC-tubulin-S1 and -S2 maps show extra density corresponding to tubulin in the cis-ring chamber of TRiC. Our structural and XL-MS analyses suggest a gradual upward translocation and stabilization of tubulin within the TRiC chamber accompanying TRiC ring closure. In the closed TRiC-tubulin-S3 map, we capture a near-natively folded tubulin-with the tubulin engaging through its N and C domains mainly with the A and I domains of the CCT3/6/8 subunits through electrostatic and hydrophilic interactions. Moreover, we also show the potential role of TRiC C-terminal tails in substrate stabilization and folding. Our study delineates the pathway and molecular mechanism of TRiC-mediated folding of tubulin along the ATPase cycle of TRiC, and may also inform the design of therapeutic agents targeting TRiC-tubulin interactions.

Isocyanide Heterodimerization-Triggered Three-Component Reaction: Diversity-Oriented Synthesis of Quinoxalines.

Known isocyanide-based multicomponent reactions (IMCRs) are usually triggered by addition of the isocyano group to the strong electrophilic component. We herein report a new isocyanide-based three-component reaction, in which an unprecedented quinoxaline-based zwitterionic intermediate is generated from the chemoselective heterodimerization of weak electrophilic ortho-diisocyanoarenes and common isocyanides. This reactive zwitterion could react in situ with various trapping agents to furnish a range of structurally diverse quinoxalines.

Somatosensory neurons express specific sets of lincRNAs, and lincRNA CLAP promotes itch sensation in mice.

Somatosensory neurons are highly heterogeneous with distinct types of neural cells responding to specific stimuli. However, the distribution and roles of cell-type-specific long intergenic noncoding RNAs (lincRNAs) in somatosensory neurons remain largely unexplored. Here, by utilizing droplet-based single-cell RNA-seq (scRNA-seq) and full-length Smart-seq2, we show that lincRNAs, but not coding mRNAs, are enriched in specific types of mouse somatosensory neurons. Profiling of lincRNAs from single neurons located in dorsal root ganglia (DRG) identifies 200 lincRNAs localized in specific types or subtypes of somatosensory neurons. Among them, the conserved cell-type-specific lincRNA CLAP associates with pruritus and is abundantly expressed in somatostatin (SST)-positive neurons. CLAP knockdown reduces histamine-induced Ca2+ influx in cultured SST-positive neurons and in vivo reduces histamine-induced scratching in mice. In vivo knockdown of CLAP also decreases the expression of neuron-type-specific and itch-related genes in somatosensory neurons, and this partially depends on the RNA binding protein MSI2. Our data reveal a cell-type-specific landscape of lincRNAs and a function for CLAP in somatosensory neurons in sensory transmission.

m6A-modified lincRNA Dubr is required for neuronal development by stabilizing YTHDF1/3 and facilitating mRNA translation.

Long intergenic noncoding RNAs (lincRNAs) are crucial regulators in numerous biological processes. However, the functions and mechanisms of m6A-modified lincRNAs in neuronal development remain unclear. Here, we report an m6A-modified lincRNA, Dppa2 upstream binding RNA (Dubr), abundantly expressed at the early developmental stage of dorsal root ganglion (DRG) and cerebral cortex. Silencing Dubr impairs axon elongation of DRG neurons and axon projection and migration of cortical neurons, whereas lacking m6A modification of Dubr fully loses its functions. Mechanically, Dubr interacts with m6A-binding proteins, the YTHDF1/3 complex, through its m6A motifs to protect YTHDF1/3 from degradation via the proteasome pathway. Furthermore, Tau and Calmodulin are regulated by YTHDF1/3 and m6A-modified Dubr. Overexpression of YTHDF1/3 not only rescues the reduced Tau and Calmodulin but also restores axon elongation of DRG neurons by Dubr knockdown. This study uncovers a critical role of m6A-modified lincRNA in neuronal development by regulating the degradation of RNA-binding protein.

Activation of Wnt/ß-catenin signaling by Zeb1 in endothelial progenitors induces vascular quiescence entry.

The establishment of a functional vasculature requires endothelial cells to enter quiescence during the completion of development, otherwise pathological overgrowth occurs. How such a transition is regulated remains unclear. Here, we uncover a role of Zeb1 in defining vascular quiescence entry. During quiescence acquisition, Zeb1 increases along with the progressive decline of endothelial progenitors' activities, with Zeb1 loss resulting in endothelial overgrowth and vascular deformities. RNA sequencing (RNA-seq) and assay for transposase-accessible chromatin sequencing (ATAC-seq) analyses reveal that Zeb1 represses Wif1, thereby activating Wnt/ß-catenin signaling. Knockdown of Wif1 rescues the overgrowth induced by Zeb1 deletion. Importantly, local administration of surrogate Wnt molecules in the retina ameliorates the overgrowth defects of Zeb1 mutants. These findings show a mechanism by which Zeb1 induces quiescence of endothelial progenitors during the establishing of vascular homeostasis, providing molecular insight into the inherited neovascular pathologies associated with human ZEB1 mutations, suggesting pharmacological activation of Wnt/ß-catenin signaling as a potential therapeutical approach.

Quantitative assessment of the microstructure of the mesorectum with different prognostic statuses by intravoxel incoherent motion diffusion-weighed magnetic resonance imaging.

BACKGROUND: The mesorectum surrounding the rectum provides an ideal substrate for tumour spread. However, preoperative risk assessment is still an issue. This study aimed to investigate the microstructural features of mesorectum with different prognostic statuses by intravoxel incoherent motion diffusion-weighted imaging (IVIM DWI). METHODS: Patients with pathologically proven rectal adenocarcinoma underwent routine high-resolution rectal magnetic resonance imaging (MRI) and IVIM DWI sequences were acquired. The MRI-detected circumferential resection margin (mrCRM) and extramural vascular invasion (mrEMVI) were evaluated. IVIM parameters of the mesorectum adjacent to (MAT) and distant from (MDT) the tumour were measured and compared between and within the prognostic factor groups. RESULTS: The positive mrCRM (pMAT < 0.001; pMDT = 0.013) and mrEMVI (pMAT = 0.001; pMDT < 0.001) groups demonstrated higher D values in the MAT and MDT than the corresponding negative groups. Conversely, the positive mrCRM (p = 0.001) and mrEMVI (p < 0.001) groups both demonstrated lower f values in the MAT. Similarly, in the self-comparison between the MAT and MDT in the above subgroups, D showed a significant difference in all subgroups (p < 0.001 for all), and f showed a significant difference in the positive mrCRM (p = 0.001) and mrEMVI (p = 0.002) groups. Moreover, the MAT displayed a higher D* in the positive mrCRM (p = 0.014), negative mrCRM (p = 0.009) and negative mrEMVI groups (p < 0.001). CONCLUSION: The microstructure of the mesorectum in patients with rectal cancer with poor prognostic status shows changes based on IVIM parameters. IVIM parameters might be promising imaging biomarkers for risk assessment of tumour spread in mesorectum preoperatively.

Distinct neural networks derived from galanin-containing nociceptors and neurotensin-expressing pruriceptors.

Pain and itch are distinct sensations arousing evasion and compulsive desire for scratching, respectively. It's unclear whether they could invoke different neural networks in the brain. Here, we use the type 1 herpes simplex virus H129 strain to trace the neural networks derived from two types of dorsal root ganglia (DRG) neurons: one kind of polymodal nociceptors containing galanin (Gal) and one type of pruriceptors expressing neurotensin (Nts). The DRG microinjection and immunosuppression were performed in transgenic mice to achieve a successful tracing from specific types of DRG neurons to the primary sensory cortex. About one-third of nuclei in the brain were labeled. More than half of them were differentially labeled in two networks. For the ascending pathways, the spinothalamic tract was absent in the network derived from Nts-expressing pruriceptors, and the two networks shared the spinobulbar projections but occupied different subnuclei. As to the motor systems, more neurons in the primary motor cortex and red nucleus of the somatic motor system participated in the Gal-containing nociceptor-derived network, while more neurons in the nucleus of the solitary tract (NST) and the dorsal motor nucleus of vagus nerve (DMX) of the emotional motor system was found in the Nts-expressing pruriceptor-derived network. Functional validation of differentially labeled nuclei by c-Fos test and chemogenetic inhibition suggested the red nucleus in facilitating the response to noxious heat and the NST/DMX in regulating the histamine-induced scratching. Thus, we reveal the organization of neural networks in a DRG neuron type-dependent manner for processing pain and itch.

Short-term effects of combined freeze­thaw and saline­alkali stresses on the physiological response in highland barley (Hordeum vulgare).

Highland barley (Hordeum vulgare L.), as the dominant crop on the Qinghai-Tibetan Plateau, is a typical representative of plants adapted to extreme environmental conditions. However, the harsh environment, severe salinisation and frequent freezing and thawing in the Qinghai-Tibetan Plateau are main limiting factor for crop growth in this region. The physiological response of highland barley to salinisation and freeze-thaw stresses was studied in this paper. Under the combined stresses of 60mmol/LNaCl·60mmol/LNaHCO3 and freeze-thaw cycles (10, -5, and 10°C), the changes in the relative moisture content, relative electrical conductivity, soluble protein, malondialdehyde (MDA) and photosynthetic indices Pn and E in seedling leaves of eight groups of treatments (CK, S, A, S-A, CK (FT), S (FT), A (FT), and S-A (FT)) were analysed. Results showed that a single stress did not cause a change in the MDA content. All of the combined stresses in S-A, CK (FT), S (FT), A (FT), and S-A (FT) treatments increased the MDA content of barley seedlings, and the MDA content of S-A (FT) reached 28.438 at T2 (-5°C) µmol/g. During the freeze-thaw cycle, the cell membrane of seedlings was damaged more seriously by alkali stress, which showed a significant increase in relative conductivity. The relative moisture content value of seedlings was more than 100% because the seedlings could absorb more moisture due to mechanical injury. The protein content of osmoregulatory substances in highland barley seedlings increased with increasing stress, indicating resistance to stress. Moreover, the effect of freeze-thaw stress on photosynthesis was more significant. The changes in indices proved that an appropriate amount of salt stress could improve the resistance of the plant cell membrane. Alkali stress had a significant effect on the growth of highland barley seedlings. Freezing and thawing can aggravate the damage of saline-alkali stress to highland barley seedlings, resulting in changes in the biological membrane permeability and photosynthesis of seedlings. The fluctuation of osmoregulation substance content confirmed that highland barley seedlings had a certain degree of stress resistance. Freeze-thaw cycles will aggravate the damage of land salinisation to highland barley seedlings. To better reduce the impact and loss of land salinisation and freeze-thaw disasters on agriculture in the Qinghai-Tibetan Plateau, priority should be given to solving freeze-thaw stress in the process of grain production.

Response characteristics of highland barley (Hordeum vulgare) seedlings to the stress of salinity and artemisinin under freeze­thaw environment.

In Qinghai-Tibet Plateau, crops are commonly subjected to freeze-thaw and salt stress factors simultaneously, and allelopathy is common, which affects the growth of highland barley (Hordeum vulgare L.), the largest food crop in Tibet. In order to explore the effects of artemisinin, salt and freeze-thaw (FAS) stress on physiological characteristics of highland barley seedlings, hydroponic experiment was carried out with the addition of 20mg/L artemisinin and 150mMNaCl as well as the simulation of freeze-thaw environment. The results suggested that under combined stress, the soluble protein content in combined stresses of artemisinin, FAS increased by 97.8%, the variation of relative conductivity in FAS group was lower than that in combined salt and freeze-thaw stress (FS), the relative water content decreased significantly (P <0.05), the malondialdehyde (MDA), H2 O2 and soluble sugar content in FAS group accumulated but less than those in FS group, and the superoxide dismutase (SOD) activity in combined artemisinin and freeze-thaw stress (FA) and FAS groups decreased. In addition, after freeze-thaw treatment, photosynthesis was weakened, and internal CO2 conentration (C i ) in FAS group significantly decreased (P <0.05). This study proved that appropriate amount of artemisinin can alleviate the damage of salt and freeze-thaw stress on barley seedlings.