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Daidzein has been reported to be effective in regulating lipid metabolism in animals. However, the molecular mechanisms of daidzein on adipogenesis in beef cattle are not yet reported and the results of daidzein on affecting lipid metabolism in other species have been conflicting. High‐throughput sequencing of mRNA (RNA‐Seq) technology was performed to elucidate the underlying molecular mechanisms of daidzein on adipogenesis in subcutaneous adipose tissue of finishing Xianan beef cattle. A total of 893 differentially expressed genes (DEGs) were identified by differential expression analysis, among which 405 genes were upregulated and 488 genes were downregulated. Bioinformatics analysis suggested that these DEGs were significantly enriched to the pathways related to lipid metabolism including ECM–receptor interaction, Glycolysis/Gluconeogenesis and Hedgehog signalling pathway. Daidzein significantly affected the candidate genes (Shh, Pec, Gli, Wnt6, DLK, IGFBP2, ID3 and C/EBPE) related to adipocyte differentiation. Besides, daidzein improved the ability of subcutaneous adipocytes in synthesizing triglycerides by directly using the long‐chain fatty acids and enhanced the efficiency of triglyceride synthesis of subcutaneous adipocytes in Xianan steers. In conclusion, daidzein plays a positive role not only in adipogenic differentiation, but also in triglyceride synthesis in subcutaneous adipose tissue of Xianan beef cattle. [ABSTRACT FROM AUTHOR]

An increased risk of ALS has been reported for veterans, varsity athletes, and professional football players. The mechanism underlying the increased risk in these populations has not been identified; however, it has been proposed that motor nerve injury may trigger immune responses which, in turn, can accelerate the progression of ALS. Accumulating evidence indicates that abnormal immune reactions and inflammation are involved in the pathogenesis of ALS, but the specific immune cells involved have not been clearly defined. To understand how nerve injury and immune responses may contribute to ALS development, we investigated responses of CD4+ T cell after facial motor nerve axotomy (FNA) at a presymptomatic stage in a transgenic mouse model of ALS (B6SJL SOD1G93A). SOD1G93A mice, compared with WT mice, displayed an increase in the basal activation state of CD4+ T cells and higher frequency of Th17 cells, which were further enhanced by FNA. In conclusion, SOD1G93A mice exhibit abnormal CD4+ T cell activation with increased levels of Th17 cells prior to the onset of neurological symptoms. Motor nerve injury exacerbates Th17 cell responses and may contribute to the development of ALS, especially in those who carry genetic susceptibility to this disease. [ABSTRACT FROM AUTHOR]

Abstract: Background: The production of neurotrophic factors, such as BDNF, has generally been considered an important mechanism of immune-mediated neuroprotection. However, the ability of T cells to produce BDNF remains controversial. Methods: In the present study, we examined mRNA and protein of BDNF using RT-PCR and western blot, respectively, in purified and reactivated CD4+ T cells. In addition, to determine the role of BDNF derived from CD4+ T cells, the BDNF gene was specifically deleted in T cells using the Cre-lox mouse model system. Results: Our results indicate that while both mRNA expression and protein secretion of BDNF in reactivated T cells were detected at 24h, only protein could be detected at 72h after reactivation. The results suggest a transient up-regulation of BDNF mRNA in reactivated T cells. Furthermore, in contrast to our hypothesis that the BDNF expression is necessary for CD4+ T cells to mediate neuroprotection, mice with CD4+ T cells lacking BDNF expression demonstrated a similar level of facial motoneuron survival compared to their littermates that expressed BDNF, and both levels were comparable to wild-type. The results suggest that the deletion of BDNF did not impair CD4+ T cell-mediated neuroprotection. Conclusion: Collectively, while CD4+ T cells are a potential source of BDNF after nerve injury, production of BDNF is not necessary for CD4+ T cells to mediate their neuroprotective effects. [Copyright &y& Elsevier]

Abstract: We have previously shown that immunodeficient mice exhibit significant facial motoneuron (FMN) loss compared to wild-type (WT) mice after a facial nerve axotomy. Interleukin-10 (IL-10) is known as a regulatory cytokine that plays an important role in maintaining the anti-inflammatory environment within the central nervous system (CNS). IL-10 is produced by a number of different cells, including Th2 cells, and may exert an anti-apoptotic action on neurons directly. In the present study, the role of IL-10 in mediating neuroprotection following facial nerve axotomy in Rag-2- and IL-10-deficient mice was investigated. Results indicate that IL-10 is neuroprotective, but CD4+ T cells are not the requisite source of IL-10. In addition, using real-time PCR analysis of laser microdissected brainstem sections, results show that IL-10 mRNA is constitutively expressed in the facial nucleus and that a transient, significant reduction of IL-10 mRNA occurs following axotomy under immunodeficient conditions. Dual labeling immunofluorescence data show, unexpectedly, that the IL-10 receptor (IL-10R) is constitutively expressed by facial motoneurons, but is selectively induced in astrocytes within the facial nucleus after axotomy. Thus, a non-CD4+ T cell source of IL-10 is necessary for modulating both glial and neuronal events that mediate neuroprotection of injured motoneurons, but only with the cooperation of CD4+ T cells, providing an avenue of novel investigation into therapeutic approaches to prevent or reverse motoneuron diseases, such as amyotrophic lateral sclerosis (ALS). [Copyright &y& Elsevier]

Abstract: We have previously shown that CD4+ T helper (Th) 2 cells, but not Th1 cells, participate in the rescue of mouse facial motoneurons (FMN) from axotomy-induced cell death. Recently, a number of other CD4+ T cell subsets have been identified in addition to the Th1 and Th2 effector subsets, including Th17, inducible T regulatory type 1 (Tr1), and naturally thymus-born Foxp3+ regulatory (Foxp3+ Treg) cells. These subsets regulate the nature of a T cell-mediated immune response. Th1 and Th17 cells are pro-inflammatory subsets, while Th2, Tr1, and Foxp3+ Treg cells are anti-inflammatory subsets. Pro-inflammatory responses in the central nervous system are thought to be neurodestructive, while anti-inflammatory responses are considered neuroprotective. However, it remains to be determined if another CD4+ T cell subset, other than the Th2 cell, develops after peripheral nerve injury and participates in FMN survival. In the present study, we used FACS analysis to determine the temporal frequency of Th1, Th17, Th2, Tr1 and Foxp3+ Treg CD4+ T cell subset development in C57BL/6 wild type mice after facial nerve transection at the stylomastoid foramen in the mouse. The results indicate that all of the known CD4+ T cell subsets develop and expand in number within the draining lymph node, with a peak in number primarily at 7 days postoperative (dpo), followed by a decline at 9 dpo. In addition to the increase in subset frequency over time, FACS analysis of individual cells showed that the level of cytokine expressed per cell also increased for interferon-γ (IFN-γ), interleukin (IL)-10 and IL-17, but not IL-4. Additional control double-cytokine labeling experiments were done which indicate that, at 7dpo, the majority of cells indeed have committed to a specific phenotype and express only 1 cytokine. Collectively, our findings indicate for the first time that there is no preferential activation and expansion of any single CD4+ T cell subset after peripheral nerve injury but, rather, that both pro-inflammatory and anti-inflammatory CD4+ T cells develop. [Copyright &y& Elsevier]

Acquired aplastic anemia is an autoimmune-mediated bone marrow failure syndrome. The mechanism by which such an autoimmune reaction is initiated is unknown. Whether and how the genetic lesions detected in patients cause autoimmune bone marrow failure have not yet been determined. We found that mice with spontaneous deletion of the TGFβ-activated kinase-1 gene in a small subset of hematopoietic cells developed bone marrow failure which resembled the clinical manifestations of acquired aplastic anemia patients. Bone marrow failure in such mice could be reversed by depletion of CD4 + T lymphocytes or blocked by knockout of interferon-γ, suggesting a Th1-cell-mediated autoimmune mechanism. The onset and progression of bone marrow failure in such mice were significantly accelerated by the inactivation of tumor necrosis factor-α signaling. Tumor necrosis factor-α restricts autoimmune bone marrow failure by inhibiting type-1 T-cell responses and maintaining the function of myeloid-derived suppressor cells. Furthermore, we determined that necroptosis among a small subset of mutant hematopoietic cells is the cause of autoimmune bone marrow failure because such bone marrow failure can be prevented by deletion of receptor interacting protein kinase-3 Our study suggests a novel mechanism to explain the pathogenesis of autoimmune bone marrow failure., (Copyright© Ferrata Storti Foundation.)

The authors have previously demonstrated a neuroprotective mechanism of facial motoneuron (FMN) survival after facial nerve transection that is dependent on CD4(+)T helper 2 (Th2) cell interactions with peripheral antigen presenting cells, as well as central nervous system (CNS) resident microglia. Pituitary adenylyl cyclase activating polypeptide is expressed by injured FMN and increases Th2-associated chemokine expression in cultured murine microglia. Collectively, these data suggest a model involving CD4(+) Th2 cell migration to the facial motor nucleus after injury via microglial expression of Th2-associated chemokines. In this study, the authors tested the hypothesis that Th2-associated chemokine expression occurs in the facial motor nucleus after facial nerve axotomy at the stylomastoid foramen. Initial microarray analysis of Th2-associated and Th1-associated chemokine mRNA levels was accomplished after facial nerve axotomy in wild type (WT) and presymptomatic mutant superoxide dismutase 1 (mSOD1) [model of familial amyotrophic lateral sclerosis (ALS)] mice. Based on that initial microarray analysis, the Th2-associated chemokine, CCL11, and Th1-associated chemokine, CXCL11, were further analyzed by RT-PCR. The results indicate that facial nerve injury predominantly increases Th2-associated chemokine, but not Th1-associated chemokine mRNA levels in the mouse facial motor nucleus. Interestingly, no differences were detected between WT and mSOD1 mice for CCL11 and CXCL11 after injury. These data provide a basis for further investigation into Th2-associated chemokine expression in the facial motor nucleus after FMN injury, which may lead to more specifically targeted therapeutics in motoneuron diseases, such as ALS.

Microglia are the immune cells that reside in the central nervous system (CNS). Following the facial nerve injury in the mouse, microglia are activated in the facial motor nucleus, coincident with an increase in the proinflammatory cytokine interferon-gamma (IFN-γ). The authors have previously shown that maximal facial motoneuron (FMN) survival after injury depends on the CD4(+)T-cell interaction with microglia. Furthermore, it appears that the anti-inflammatory T helper (Th) 2 CD4(+) T-cell subset is required in the facial nucleus, although the mechanism of CNS recruitment is not known. Pituitary adenylyl cyclase-activating polypeptide (PACAP) is a neuropeptide that has previously been demonstrated to be expressed by injured FMN. Interestingly, PACAP has been shown to act on peripheral macrophages by inducing chemokine expression capable of recruiting Th2 cells. Whether CNS-resident microglia, a related lineage to peripheral macrophages, respond to PACAP by expressing Th2-associated chemokines is not known. In this study, fluorescence-activated cell sorting was utilized to measure the frequency of microglia positive for different chemokines after exposure to various treatments. The results indicate that PACAP increases the frequency of microglia expressing Th2-associated chemokine, CCL11, and decreases the frequency of microglia expressing Th1-associated chemokine, CXCL11. In contrast, IFN-γ decreases the frequency of microglia expressing Th2-associated chemokine, CCL11, and increases the frequency of microglia expressing Th1-associated chemokine, CXCL11. Treatment with both PACAP and IFN-γ reversed the proinflammatory effect of IFN-γ. Given the recent focus on the therapeutic value of Th2 cells in the CNS during neurode-generative disease, PACAP may be a future therapeutic target for improving neuroregeneration after injury.

Polarized Th1 cells show a stable phenotype: they become insensitive to IL-4 stimulation and lose the potential to produce IL-4. Previously, we reported that IFN-gamma played a critical role in stabilizing Th1 phenotype. However, the mechanism by which IFN-gamma stabilizes Th1 phenotype is not clear. In this study, we compared STAT6 phosphorylation in wild-type (WT) and IFN-gamma receptor knockout (IFNGR(-/-)) Th1 cells. We found a striking diminution of STAT6 phosphorylation in differentiated WT Th1 cells, but not in differentiated IFNGR(-/-) Th1 cells. The impairment of STAT6 phosphorylation in differentiated WT Th1 cells was not due to a lack of IL-4R expression or phosphorylation. Jak1 and Jak3 expression and phosphorylation were comparable in both cell types. No differential expression of suppressor of cytokine signaling 1 (SOCS1), SOCS3, or SOCS5 was observed in the two cell types. In addition, Src homology 2-containing phosphatase mutation did not affect IL-4-induced STAT6 phosphorylation in differentiated Th1 cells derived from viable motheaten (me(v)/me(v)) mice. These results led us to focus on a novel mechanism. By using a pulldown assay, we observed that STAT6 in WT Th1 cells bound less effectively to the phosphorylated IL-4R/GST fusion protein than that in IFNGR(-/-) Th1 cells. Our results suggest that IFN-gamma may suppress phosphorylation of STAT6 by inhibiting its recruitment to the IL-4R.

Objective: To resolve the problem of the accuracy and standardization of short tandem repeat-polymerase chain reaction (STR-PCR) typing in forensic practice, the authors have designed a new method of producing standard D12S391 allelic ladder., Methods: Nine different PCR amplified D12S391 allelic fragments were isolated from the gel, eluted into the distilled water and re-amplified by PCR. The purified allelic fragments were then blunt-end subcloned individually into the pUC plasmid vectors and transfected into competent E.coli DH5 alpha(TM) cells. The sequencing results confirmed that the size and the structure of the inserts were correct. The recombinant plasmids DNA with 9 inserts were then used as templates for PCR re-amplification to generate D12S391 standard ladder., Results: With the ladder, the authors studied the genetic polymorphisms of D12S391 locus in six populations (German, Japanese and Chinese south-western Han, northern Han, Weiwu'er and Hui populations), and the respective primary data in the six populations were obtained. D12S391 locus showed high polymorphism in all six populations, and its exclusion power and discrimination power are 0.609-0.786 and 0.940-0.952 respectively., Conclusion: The results demonstrate that the standard ladder generated via this method is excellent, and D12S391 locus is robust for genetic research and forensic application.

Objective: Using molecular techniques, we typed 2 short tandem repeat (STR) loci on 21 chromosome to establish the method for rapid and accurate diagnosis of trisomy 21., Methods: Genomic DNA samples from 50 individuals diagnosed previously by karyotype as trisomy 21 and 40 children with severe mental retardation (IQ < 50) suspected of trisomy 21 were analyzed for 2 short tandem repeat loci on 21 chromosome, D21S1435 and D21S2055. Typing was carried out after polymerase chain reaction (PCR) and silver staining. The trisomy was identified by the number of alleles: 3 alleles bands whose density is same, two alleles bands with one obvious higher density compared to the other and one allele band whose density is three times than the normal control., Results: All of the complete trisomy 21 were detected by this method; the parental source was easily determined., Conclusion: This method for diagnosing trisomy 21 is rapid and accurate.