Overexpression of AHL proteins enhances root hair production by altering the transcription of RHD6-downstream genes.

AT-HOOK MOTIF NUCLEAR LOCALIZED (AHL) proteins occur in all sequenced plant species. They bind to the AT-rich DNA sequences in chromosomes and regulate gene transcription related to diverse biological processes. However, the molecular mechanism underlying how AHL proteins regulate gene transcription is poorly understood. In this research, we used root hair production as a readout to study the function of two Arabidopsis AHL proteins, AHL17, and its closest homolog AHL28. Overexpression of AHL17 or AHL28 greatly enhanced root hair production by increasing the transcription of an array of genes downstream of RHD6. RHD6 is a key transcription factor that regulates root hair development. Mutation of RHD6 completely suppressed the overproduction of root hairs by blocking the transcription of AHL17-activated genes. The overexpression of AHL17 or AHL28, however, neither affected the transcription of RHD6 nor the accumulation of RHD6 protein. These two AHL proteins also did not directly interact with RHD6. Furthermore, we found that three members of the Heat Shock Protein70 family, which have been annotated as the subunits of the plant Mediator complex, could form a complex with both AHL17 and RHD6. Our research might reveal a previously unrecognized mechanism of how AHL proteins regulate gene transcription.

Molecular characterization and expression changes of cytoglobin genes in response to hypoxia in a Tibetan schizothoracine fish, Schizopygopsis pylzovi.

Schizopygopsis pylzovi, an endemic fish of the subfamily Schizothoracinae, is comparatively well adapted to dissolved oxygen fluctuations in the aqueous environments of the Qinghai-Tibetan Plateau. Here, we cloned the complete cDNA of cytoglobin 1 and 2 (Cygb1 and Cygb2) from S. pylzovi and then investigated transcriptional changes of both genes in the selected tissues in response to hypoxia. Both the two genes had the standard exon-intron structure of vertebrate Mb genes but lacked an exon at downstream of the H helix (HC11.2) as seen in mammals. We applied severe hypoxia (4 h at PO2 = 3.6% saturation) and moderate hypoxia (72 h at PO2 = 36.0% saturation) to adult S. pylzovi. Under severe hypoxia, the Cygb1 mRNA levels decreased significantly in the liver, kidney, and brain, but increased significantly in the heart, while the Cygb2 mRNA levels downregulated significantly in the muscle and liver. But, the transcriptional activity of Cygb1 in muscle and that of Cygb2 in the kidney, brain, and heart remained almost unchanged. Under moderate hypoxia, the transcriptional activities of both genes in muscle and brain were turned down quickly after onset hypoxia, while in the liver, kidney, and heart, the transcriptional activities of both genes showed a short-term upregulation in different time periods of hypoxia exposure. Our data suggest that both the Cygb1 and Cygb2 in S. pylzovi are hypoxia-induced genes, and the responses of the transcription regulation of Cygb1 and Cygb2 genes to hypoxia are tissue specific and also depend on the hypoxia regime, which are different from that of other fish species.

Transcriptome Analysis Provides Insights Into the Adaptive Responses to Hypoxia of a Schizothoracine Fish (Gymnocypris eckloni).

The schizothoracine fish endemic to the Qinghai-Tibetan Plateau are comparatively well adapted to aquatic environments with low oxygen partial pressures. However, few studies have used transcriptomic profiling to investigate the adaptive responses of schizothoracine fish tissues to hypoxic stress. This study compared the transcriptomes of Gymnocypris eckloni subjected to 72 h of hypoxia (Dissolved oxygen, DO = 3.0 ± 0.1 mg/L) to those of G. eckloni under normoxia (DO = 8.4 ± 0.1 mg/L). To identify the potential genes and pathways activated in response to hypoxic stress, we collected muscle, liver, brain, heart, and blood samples from normoxic and hypoxic fish for RNA-Seq analysis. We annotated 337,481 gene fragments. Of these, 462 were differentially expressed in the hypoxic fish as compared to the normoxic fish. Under hypoxia, the transcriptomic profiles of the tissues differed, with muscle the most strongly affected by hypoxia. Our data indicated that G. eckloni underwent adaptive changes in gene expression in response to hypoxia. Several strategies used by G. eckloni to cope with hypoxia were similar to those used by other fish, including a switch from aerobic oxidation to anaerobic glycolysis and the suppression of major energy-requiring processes. However, G. eckloni used an additional distinct strategy to survive hypoxic environments: a strengthening of the antioxidant system and minimization of ischemic injury. Here, we identified several pathways and related genes involved in the hypoxic response of the schizothoracine fish. This study provides insights into the mechanisms used by schizothoracine fish to adapt to hypoxic environments.

Molecular evolution of myoglobin in the Tibetan Plateau endemic schizothoracine fish (Cyprinidae, Teleostei) and tissue-specific expression changes under hypoxia.

Myoglobin (Mb) is an oxygen-binding hemoprotein that was once thought to be exclusively expressed in oxidative myocytes of skeletal and cardiac muscle where it serves in oxygen storage and facilitates intracellular oxygen diffusion. In this study, we cloned the coding sequence of the Mb gene from four species, representing three groups, of the schizothoracine fish endemic to the Qinghai-Tibetan Plateau (QTP), then conducted molecular evolution analyses. We also investigated tissue expression patterns of Mb and the expression response to moderate and severe hypoxia at the mRNA and protein levels in a representative of the highly specialized schizothoracine fish species, Schizopygopsis pylzovi. Molecular evolution analyses showed that Mb from the highly specialized schizothoracine fish have undergone positive selection and one positively selected residue (81L) was identified, which is located in the F helix, close to or in contact with the heme. We present tentative evidence that the Mb duplication event occurred in the ancestor of the schizothoracine and Cyprininae fish (common carp and goldfish), and that the Mb2 paralog was subsequently lost in the schizothoracine fish. In S. pylzovi, Mb mRNA is expressed in various tissues with the exception of the intestine and gill, but all such tissues, including the liver, muscle, kidney, brain, eye, and skin, expressed very low levels of Mb mRNA (< 8.0%) relative to that of the heart. The trace levels of Mb expression in non-muscle tissues are perhaps the major reason why non-muscle Mb remained undiscovered for so long. The expression response of the Mb gene to hypoxia at the mRNA and protein levels was strikingly different in S. pylzovi compared to that found in the common carp, medaka, zebrafish, and goldfish, suggesting that the hypoxia response of Mb in fish may be species and tissue-specific. Notably, severe hypoxia induced significant expression of Mb at the mRNA and protein levels in the S. pylzovi heart, which suggests Mb has a major role in the supply of oxygen to the heart of Tibetan Plateau fish.

Identification, molecular evolution of toll-like receptors in a Tibetan schizothoracine fish (Gymnocypris eckloni) and their expression profiles in response to acute hypoxia.

Hypoxia plays an important role in regulating a variety of physiological responses as well as in pathological situations, but to date the roles of Toll-like receptors (TLRs) in fish in response to hypoxia are still poorly understood. Here, we sequenced the transcriptome of G. eckloni and identified the members of TLR family by scanning transcriptome, and then investigated the expression profiles of a complete set of TLRs in G. eckloni in response to acute hypoxia (4 h at DO = 0.3 ± 0.1 mg/L). The de novo-assembled transcriptome consisted of a total of 162,235 transcripts, further clustered into 110,231 unigenes. Based on the transcriptome, a total of 18 TLRs were identified in G. eckloni, and of them three TLRs (TLR5, TLR8 and TLR22) possessed two distinct paralogous genes. The duplicated genes of TLR22 were discovered for the first time in cyprinid fish, but did not origin from a recent duplication event. Of them TLR22b may be specific for schizothoracine fish, at least for G. eckloni. Phylogenetic analysis supported the classification of TLRs into six families as in other vertebrates but was partly different from the previous study. The sliding window analysis showed strong signals of positive selection in TLR2, TLR 4, TLR 5a, TLR 7, TLR 19, TLR 20, TLR 21, TLR 22a and TLR 22b, but most codons under positive selection were located in the putative LRR regions. The mRNA expression of most TLRs in head kidney, spleen and gill decreased significantly or remained unchanged under acute hypoxia, whereas acute hypoxia increased expressions of TLR2 and TLR3 in head kidney, of TLR8a, TLR12 and TLR19 in spleen, and of TLR1 in gill, suggesting tissues-specific expressions of TLRs play important roles in mediating innate immune responses for host defense against tissue damages or physiological changes induced by hypoxia.

Changes of hemoglobin expression in response to hypoxia in a Tibetan schizothoracine fish, Schizopygopsis pylzovi.

Fishes endemic to the Qinghai-Tibetan Plateau are comparatively well adapted to aquatic environments with low oxygen partial pressures (hypoxia). Here, we cloned the complete cDNA of hemoglobin (Hb) α and ß from the Tibetan schizothoracine fish Schizopygopsis pylzovi, and then investigated changes in Hb mRNA and protein levels in spleen, liver and kidney in response to hypoxia. We applied severe hypoxia (4 h at PO2 = 0.6 kPa) and moderate hypoxia (72 h at PO2 = 6.0 kPa) to adult S. pylzovi. Changes of Hb expression under hypoxia, together with the investigations of spleen somatic index, kidney somatic index and Hb concentration in circulation, suggest that the kidney may not only serve as the erythropoietic organ, but also act as the major blood reservoir in S. pylzovi. From this perspective, the transcriptional activity of Hb in S. pylzovi, as reflected in the kidney, was turned down quickly after the onset of severe hypoxia, while under moderate hypoxia the transcriptional activity of Hb showed upregulation for a short time, but then the transcriptional machinery was turned down slowly on prolonged exposure. Notably, the changes in Hb protein levels in spleen, liver and kidney in response to severe and moderate hypoxia were not in line with the changes in mRNA levels, which are related with the blood reservoir in the kidney. Tibetan schizothoracine fish, at least S. pylzovi, show a particular response of the transcription regulation of Hb to moderate hypoxia, which is different from that of other fish species.

[Effect of aconite cake-separated moxibustion at Guanyuan (CV 4) and Mingmen (GV 4) on thyroid function in patients of Hashimoto's thyroiditis].

OBJECTIVE: To explore the effects on thyroid function in patients of Hashimoto's thyroiditis treated with aconite cake-separated moxibustion and option the better therapeutic program. METHODS: Eighty-five cases were randomly divided into a moxibustion group (42 cases) and a western medication group (43 cases). The moxibustion group was treated by aconite cake-separated moxibustion therapy with acupoints of two groups [(1) Danzhong (CV 17), Zhongwan (CV 12), Guanyuan (CV 4); (2) Dazhui (GV 14), Shenshu (BL 23), Mingmen (GV 4)] alternatively and oral administration of 25 microg Euthyrox everyday. The western medication group was oral administration of 25 microg Euthyrox everyday. Indices of thyroid function before and after treatment and clinical effect were compared between two groups. RESULTS: The clinical total effective rate and effective rate of thyroid function were 25.0% (10/40), 87.5% (35/40) in moxibustion group respectively, 7.53% (3/40) and 57.5% (23/40) in western medication group, with significant differences between two groups (both P < 0.05). Content of serum free thyroxine index (FT4) increased significantly in the moxibustion group after treatment (P < 0.01); content of serum supersensitive thyrotropin (S-TSH) in the moxibustion group was lower than that of western medication group, and contents of serum FT4 and free triiodothyronine (FT3) were higher than those of western medication group, but with no significant differences (all P > 0.05). CONCLUSION: Aconite cake-separated moxibustion at Guanyuan (CV 4) and Mingmen (GV 4) combined with oral administration of Euthyrox can improve clinical symptoms and thyroid function in patients of Hashimoto's thyroiditis, which is better than simple oral administration of Euthyrox.