[Deletion of Aldh4a1 Leads to Impaired Sperm Maturation in Mice].

ALDH4A1, a member of the aldehyde dehydrogenase superfamily, is a key enzyme in the mitochondrial proline metabolism pathway. Recent studies have shown that mutations in aldh4a1 lead to reduced fertility and reproductive premature aging of male nematodes. However, the effect of ALDH4A1 on fertility of male mice has not been studied. In this study, we used CRISPR-Cas9 technology to construct a knockout mouse model of Aldh4a1 for the first time to explore the effect of this gene on the reproduction of male mice. The results showed that compared with WT male mice, Aldh4a1^(-/-) male mice were fertile, had normal spermatogenesis but defect in sperm maturation in the epididymis documented by impaired motility, increased morphological abnormalities and increased spontaneous acrosome reaction. In addition, transmission electron microscopy showed vacuoles in the sperm mitochondria, and fracture in the neck of sperms and vacuoles in these mice. These results revealed that ALDH4A1 plays a vital role in the structure of sperm flagellum and the process of sperm maturation in mice.

Research Note: Development of a sandwich ELISA for determining plasma growth hormone concentrations in goose.

Growth hormone (GH) is required for normal postnatal development in poultry; however, no immunoassay exists to assess its levels in geese plasma, hindering the study of endocrine regulation in this species. We developed a sandwich ELISA to determine the GH concentrations in the plasma of geese. Recombinant goose GH was produced using a eukaryotic expression system and purified for use as the reference standard in ELISA and the antigen for producing the polyclonal antibodies in rabbits. Rabbit anti-goose GH polyclonal antibody was used to coat the wells of the ELISA plate, and its biotinylated form served as the detection antibody. An avidin-conjugated horseradish peroxidase was used to bind the detection antibody and catalyze the chromogenic reaction of 3,3,5,5-tetramethylbenzidine and H2O2. A sigmoidal curve was fitted to the optical density and the log of the standard GH concentration using the four-parameter logistic model. The sensitivity of the assay was less than 0.156 ng/mL. The intra- and interassay coefficients of variation were less than 9 and 13%, respectively. The response curve of the serially diluted plasma samples from geese exhibited a good parallel relationship with that observed for the reference standards. The assay effectively detected differences in GH concentrations in plasma samples from geese at various physiological stages; thus, it will be useful for future study of their growth and metabolism.

[Hashimoto's encephalopathy presenting with isolated cerebellar ataxia in 13 children].

Objective: To analyze the clinical characteristics, treatment and prognosis of Hashimoto's encephalopathy presenting with isolated cerebellar ataxia in children. Methods: A retrospective analysis was performed on the clinical features, laboratory tests, neuroelectrophysiological examination, imaging, treatment and outcomes of 13 patients with Hashimoto's encephalopathy presenting with isolated cerebellar ataxia, who were admitted to the Department of Pediatric Neurology of Guangzhou Women and Children's Medical Center from January 2016 to May 2021. Results: Among the 13 cases, 6 were males and 7 were females. The onset age was 2.6 (2.0,3.3) years, 9 children had precursor infection or vaccination before the first course of disease. All the 13 children had gait abnormalities or unsteady sitting, 10 had intentional tremor, 6 had dysarthria, 3 had body tremor, 2 had nystagmus, 3 had fatigue, 3 had hypotonia, 2 had vomiting and 1 had irritability. Thyroglobulin antibody (TgAb) was 500.0 (298.9,587.2) kU/L and thyroid peroxidase antibody (TPOAb) was 621.9 (449.6,869.4) kU/L in 13 cases. Autoantibodies were positive in 9 cases, and cerebrospinal fluid leukocytosis was seen in 4 cases. Regarding electroencephalography result, 4 cases had background slowing and 1 case had occasional sharp waves. Among the 3 patients who had relapses, 1 had cerebellar atrophy shown on cranial magnetic resonance imaging (MRI) during the recurrence. All the patients received intravenous immunoglobulin (IVIG) and intensive methylprednisolone therapy during the first onset, followed by the disappearance of the symptoms, 1 patient had repeated episodes which was decreased after immunosuppressive treatment with Rituximab.Followed up for 25.0 (22.5,33.3) months after the last episode, 12 achieved complete remission and 1 had a wide base gait. Conclusions: Trunk ataxia is the common symptom of Hashimoto's encephalopathy presenting with isolated cerebellar ataxia in children.Children with cerebellar ataxia should be tested for TgAb and TPOAb to detect Hashimoto's encephalopathy, avoiding missed diagnosis and treatment delays; IVIG and intensive steroid therapy is effective, and immunosuppressive therapy for patients with multiple relapses could reduce the recurrence.

Improvement of goose embryonic and muscular developments by wider angle egg turning during incubation and the regulatory mechanisms.

Egg turning during incubation plays important roles in achieving high hatching performance and gosling quality. The objective of this study was to improve embryonic and muscular developments so to achieve better gosling quality by wider egg turning angles during incubation, and to unravel the associated regulatory molecular mechanisms. In each of three consecutive incubations, 1,728 goose eggs were divided into 3 groups that were set in the same type of commercial incubators with turning angles adjusted differently to 50°, 60°, and 70°, respectively. On average of the 3 tests, incubation with wider 70° turning angle reduced the post-18-day embryo mortality, promoted embryonic growth and development, improved the hatchability and gosling quality. On embryonic day of 29, gene mRNA expression levels of the hypothalamic growth hormone-releasing hormone (GHRH), pituitary growth hormone (GH), and liver insulin-like growth factor 1 (IGF-1) were higher in the 70° turning group than in the 50° or 60° groups. Wider angle turning also increased mRNA expression levels of the muscle development regulatory genes such as MYF5, MyoD, Myogenin (MyoG), and MRF4. Changes in expression of the above genes, together with the upregulation of the Pax3 and Pax7 genes in leg muscles, well explained the enhancement of the muscular growth and development when eggs were incubated by wider turning angles. These results also extended our understanding of the impacts and mechanisms of egg turning during incubation on hatching performance and gosling quality.

Identification of miRNAs, mRNAs, lncRNAs, and circRNAs associated with hepatocellular carcinoma recurrence after interferon treatment.

To study the molecular mechanism of interferon-alpha (IFN-α) in the treatment of hepatocellular carcinoma (HCC) and the molecular markers that can predict the therapeutic effect, differentially expressed (DE)-miRNAs, -mRNAs, -lncRNAs, and -circRNAs were screened between 12 samples collected from 4 patients who had not received treatment (control), 4 patients who had received recombinant human interferon a-2b treatment (case1), and 4 patients who had relapsed after receiving recombinant human interferon a-2b treatment (case2). Enrichment analyses were performed to determine the principal functions of the DE-RNAs. We also constructed protein-protein interactions (PPI) and competing endogenous RNA (ceRNA) networks. In addition, a series-cluster analysis was performed to analyze changes in gene expression across different groups of HCC. Furthermore, the expression of the genes were verified in the Cancer Genome Atlas (TCGA) database. A total of 36 union DE-miRNAs, 175 union DE-mRNAs, 65 union DE-lncRNAs, and 52 union DE-circRNAs were obtained between the control vs case1, and case2 vs case1 groups. DE-mRNAs were mainly involved in the mitochondrial inner membrane. DE-circRNAs were mainly enriched in the Golgi apparatus. ceRNA network contained 68 DE-mRNAs, 26 DE-miRNAs, 45 DE-lncRNAs, and 23 DE-circRNAs. A total of 24 DE-miRNAs, 175 DE-mRNAs, 65 DE-lncRNAs, and 52 DE-circRNAs were classified into eight profiles, respectively. A total of 26 genes showed a significant correlation with prognosis of HCC (p < 0.05). Some genes may be used to predict the efficacy of IFN-α in the treatment of HCC. The results may lay a foundation for investigating the different sensitivities of IFN-α in the treatment of HCC.

Impairment of testes development in Yangzhou ganders by augmentation of leptin receptor signaling.

The aim of this study was to determine the cellular and molecular mechanisms of leptin (LEP) and the leptin receptor (LEPR) in testicular development of prepubertal ganders. In an in vivo animal experiment, active immunization against LEPR severely depressed prepubertal testicular development by significantly reducing testicular weights at 200 and 227 days of age. The number of elongated spermatids in the seminiferous tubules was also significantly decreased by immunization with LEPR at ages of 200 and 227 days. Inhibition of testicular development by LEPR immunization was associated with decreases in LHR, StAR, 3ß-HSD, CYP11A1, CYP17A1, and PRLR mRNA expression levels in testicular tissue, which resulted in a significant decrease in testosterone synthesis. In the in vitro experiments, the addition of LEP combined with anti-LEPR antibodies strengthened LEPR signal transduction, and inhibited significantly testosterone production in cultured Leydig cells isolated from prepubertal gander testes. The mRNA expression of LHR, StAR, 3ß-HSD, CYP11A1, CYP17A1 also decreased significantly after treatment with LEP combined with anti-LEPR antibodies in cultured Leydig cells. These results suggest that anti-LEPR antibodies strengthen LEPR signaling transduction in the presence of LEP, and immunization against LEPR inhibited testes development and testosterone secretion in prepubertal ganders.

[Clinical characteristics, treatment and prognosis of myelin oligodendrocyte glycoprotein antibody-associated optic neuritis in children].

Objective: To investigate the clinical characteristics, treatment and prognosis of myelin oligodendrocyte glycoprotein antibody-associated optic neuritis (MOG-ON) in pediatric patients. Methods: Clinical data, laboratory examination, the initial best corrected visual acuity (BCVA), fundus, neuroelectrophysiological results, MRI imaging, treatment and prognosis of children diagnosed with MOG-ON from 2016 to 2019 were retrospectively analyzed. Results: A total of 29 eyes from 16 children were involved, with a male/female ratio of 1∶1, onset age of (7.0±2.9) years. Seven of 16 patients had prodromal infection, with a unilateral/bilateral ratio of 3∶13, and 2 cases had recurrent optic neuritis. Before treatment, BCVA of 19 eyes (65.5%) was ≤0.1, among them, 4 had no sense of light, 5 had light sense, 5 with sense of hands in front of eyes, and 5 with sense of fingers in front of eyes. There were 10 eyes (34.5%) with BCVA of 0.1-0.5. After treatment, there were 4 eyes (13.8%), 5 eyes (17.2%) and 20 eyes (69.0%) in groups with BCVA of 0.1-0.5, 0.5-1.0, and>1.0, respectively. Twelve of 16 patients had optic papillitis in fundus examination during acute phase. The latency was prolonged and the amplitude was decreased in P100 wave of all the children. Thirteen out of 16 children showed swelling and thickening of optic nerve in MRI T2WI. MRI images exhibited intracranial demyelinating lesions in 12 of 16 children and long segment spinal cord lesions in 3 of 16 children. Thirteen of 16 patients showed effective results after intravenous methylprednisolone (IVMP) and intravenous immunoglobulin (IVIG) treatment. There was no relapse after administration of mycophenolate mofetil in 2 recurrent children. No progression after administration of rituximab was found in 1 child with corticosteroid insensitivity. The average follow-up time was (16±9) months and no recurrence occurred. Ten of 16 patients had full recovery, 4 had significant improvement, and 2 showed no significant improvement. Conclusions: There is no significant gender difference in the incidence of pediatric MOG-ON. Bilateral involvement and severe visual impairment are common in acute phase. Most patients have good response to IVMP combined with IVIG treatment and hence have a good prognosis. Only a few of them have neurological sequelae.

[Clinical features and prognosis of pediatric myelin oligodendrocyte glycoprotein antibody associated acute disseminated encephalomyelitis].

Objective: To analyze the clinical features, outcome and prognosis of pediatric myelin oligodendrocyte glycoprotein (MOG) antibody associated acute disseminated encephalomyelitis (ADEM), and provide evidence for improving the diagnosis and treatment of this disease. Methods: This study involved 30 MOG antibody-associated ADEM patients in the Department of Neurology, Guangzhou Women and Children's Medical Center. Patients' clinical information were analyzed. Results: The mean onset age was (5.2±3.3) years old, the ration of male to female was 16∶14. Fifty percent of these patients had a history of precede infection or vaccination before onset. Encephalopathy and seizures were the most common clinical manifestations, followed by movement disorder. In addition, some patients had other positive autoantibodies. Brain Magnetic resonance imaging (MRI) showed extensive, asymmetrical, indefinite large patchy lesions in bilateral cortical and subcortical areas and the spinal cord was characterized by long segmental myelitis. In acute attack, the patients had a good response to corticosteroid combined immunoglobulin therapy. Most of these patients had a good prognosis and recurrence rate was about 20%. Conclusions: The onset age of MOG antibody-associated ADEM is around 5 years old. Encephalopathy and seizures were the most common clinical manifestations. Most patients have a good response to corticosteroid combined immunoglobulin therapy. Some patients may have a recurrent disease course.

Active immunization against AMH reveals its inhibitory role in the development of pre-ovulatory follicles in Zhedong White geese.

The aim of this study was to investigate the effects of active immunization against recombinant Anti-Müllerian hormone (AMH) protein on the ovarian follicular development, egg production, and molecular regulatory mechanisms in broody-prone Zhedong White geese. For this, a recombinant goose AMH protein was expressed using a prokaryotic expression system. Fifty incubating geese from the same genetic background were selected and equally divided into two groups. The immunization group was actively immunized against the recombinant goose AMH protein, whereas the control group was immunized against bovine serum albumin (BSA). Immunization against AMH accelerated ovarian follicular development and increased clutch sizes by one to two eggs in two consecutive laying-incubation cycles. Furthermore, immunization against AMH upregulated the mRNA transcription levels of the FSH-beta gene in the pituitary gland, and FSHR, 3beta-HSD, and Smad4 genes in the granulosa layer of pre-ovulatory follicles; however, immunization downregulated the expression of the OCLN gene in the granulosa layer of pre-ovulatory follicles, and Smad5 and Smad9 genes in the granulosa layer of SYFs. These results suggest that AMH might hinder ovarian follicular development by decreasing both pituitary FSH secretion as well as ovarian follicular sensitivity to FSH. The latter molecular mechanism could be fulfilled by regulating Smad5 or Smad9 signals in SYFs, as well as the FSHR and Smad4 signals that affect progesterone synthesis and yolk deposition in the pre-ovulatory follicles.

Cellular and molecular mechanisms of low dose prolactin potentiation of testicular development in cockerels.

The aim of this study was to determine the cellular and molecular mechanisms of prolactin (PRL) in testicular development of prepubertal cockerels. In an in vivo animal experiment, active immunization against PRL severely depressed prepubertal testicular development by significantly reducing testicular weights at both 122 and 164 d of age. The number of elongated spermatids in the seminiferous tubules was also significantly decreased by immunization with 199-residue chicken PRL (cPRL) at age 122 d. Inhibition of testicular development by cPRL immunization was associated with decreases in LH receptor (LHR), FSH receptor (FSHR), Stat5b, P450scc, steroidogenic acute regulatory (StAR) protein, and 3ß-hydroxysteroid dehydrogenase (3ß-HSD) mRNA expression levels in testicular tissue. In in vitro experiments, testosterone production by cultured Leydig cells isolated from prepubertal cockerel testes was dose-dependently enhanced by treatment with bioactive recombinant PRL, but a lesser response was seen with high concentrations of PRL. The distinct changes in testosterone production in response to high and low concentrations of added PRL were paralleled by similar patterns of change in the mRNA levels of Stat5b, LHR, P450scc, StAR, 3ß-HSD, and CYP17A1 in cultured Leydig cells, as well as protein amounts of phosphorylated Jak2 and Stat5a/b. In conclusion, low to medium doses of PRL potentiate testis development in prepubertal cockerels by enhancing testosterone secretion from Leydig cells via activation of PRLR/Stat5b signal transduction, which upregulates mRNA expression of LHR and testosterone synthesizing enzymes. However, this positive regulation was weaker in response to a high dose of PRL, which reduced PRLR/Stat5b signal transduction and the expression of genes involved in LH signaling and testosterone synthesis.

Differences in clinico-pathological characteristics and outcomes between proteinase 3-ANCA positivity and myeloperoxidase-ANCA positivity in lupus nephritis.

BACKGROUND: Owing to the low prevalence of anti-neutrophil cytoplasmic antibodies (ANCA) in lupus nephritis (LN), there is no study about the differences between proteinase 3 (PR3)-ANCA positivity and myeloperoxidase (MPO)-ANCA positivity in LN until now. METHODS: Here we perform a retrospective study to determine whether there are differences in clinic-pathological characteristics and renal outcomes between PR3-ANCA-positive LN patients and MPO-ANCA-positive LN patients. RESULTS: A total of 26 (27.4%) PR3-ANCA-positive LN patients and 69 (72.6%) MPO-ANCA-positive LN patients (p < 0.001) were eligible for this study. Compared with PR3-ANCA-positive LN patients, MPO-ANCA-positive LN patients had significantly higher levels of serum creatinine (109.6 µmol/l vs. 74.3 µmol/l, p = 0.02), lower titers of antinuclear antibodies (ANA) (128 vs. 256, p = 0.01), and higher serum concentrations of C3 and C4 (0.54 g/l vs. 0.36 g/l, p = 0.002; 0.12 g/l vs. 0.06 g/l, p < 0.001; respectively). Furthermore, the MPO-ANCA-positive group had higher scores for chronicity index (p = 0.007), including interstitial fibrosis (p = 0.001) and tubular atrophy (p = 0.03) on biopsy specimens. The renal survival rates for MPO-ANCA-positive LN patients were 94.1% at 1 year, 83.2% at 5 years and 79.6% at 10 years; these values were worse when compared with those of the PR3-ANCA-positive group, which were 100%, 100% and 100%, respectively. CONCLUSION: MPO-ANCA-positive LN patients had more severely impaired baseline renal function and less active lupus serology. More severely chronic pathological changes, including interstitial fibrosis and tubular atrophy on renal specimens, occurred in MPO-ANCA-positive LN patients. We found that MPO-ANCA-positive LN patients had worse renal outcomes.

[Effect of monoacylglycerol lipase with proliferation of MHCC97H human liver cancer cells in vivo].

Objective: To investigate the effects of different expression of monoacylglycerol lipase (MAGL) in tumor-associated macrophages (TAMs) with the proliferation of MHCC97H human liver cancer cells in vivo and its mechanism. Methods: Human peripheral blood-derived monocyte was induced to differentiate into M2-type TAMs and was identified by flow cytometry. The co-culture model of TAMs and MHCC97H human liver cancer cells was established, and the expression of MAGL in TAMs cells was detected by qRT-PCR. The expression of MAGL in TAMs cells was detected by plasmid transfection. ELISA and qRT-PCR was used to detect the mRNA expression levels and secretion levels of inflammatory factors in TAMs cells. The subcutaneous tumor model of MHCC97H mice was constructed to observe the effect of different expression of MAGL in TAMs cells with the proliferation of MHCC97H human liver cancer cells in vivo. F-test was used for the measurement of homogeneity of variance between two independent samples. A t-test was used for homogeneity of variance, and the corrected t-test was used for non-homogeneity of variance. Results: Human peripheral blood-derived monocytes were successfully induced to differentiate into M2-type TAMs. An in vitro co-culture model was established. qRT-PCR showed that MHCC97H human liver cancer cells significantly down-regulated the expressional level of MAGL in TAMs cells. The constructed subcutaneous tumor model of mice demonstrated that up-regulation up-regulation of MAGL expression in M2-type TAMs inhibited the proliferation of MHCC97H human liver cancer cells in vivo. Furthermore, the mechanistic study illustrated that the high expression of MAGL promoted the transcription and secretion of inflammatory factors such as interleukin-1 beta, interleukin-6 and tumor necrosis factor-alpha in M2-type TAMs cells. Conclusion: The overexpression of MAGL inhibits the proliferation of MHCC97H hepatocellular carcinoma cells in vivo, and its mechanism may be associated to the release of inflammatory factors that from TAMs cells.

Effect and molecular regulatory mechanism of monochromatic light colors on the egg-laying performance of Yangzhou geese.

Photoperiodic control is essential for manipulating the reproductive performance of avian species. This study was conducted to assess the neuroendocrine mechanisms that regulate reproductive functions of Yangzhou geese when there are different monochromatic light colors from light emitter diode (LED) sources. A flock of geese was divided into four groups with white, red, blue, and green light treatments being imposed. The results indicated that peak laying rates and reproductive performance were greater in geese treated with white or red as compared with blue or green light treatments. The fertilization rate of eggs and hatchability of fertilized eggs were greater with the white or red as compared with blue or green light treatments. There was a greater abundance of OPN5, Dio2, c-Fos, and GnRH-I mRNA in the hypothalamus earlier in the treatment period and abundances of these hypothalamic factors were greater with the white or red light treatments. Abundances of pituitary LH beta and FSH beta mRNA increased at a lesser rate with the blue or green light treatments and were in greater abundances with the white or red light treatments. The lighting regimen also resulted in photo-refractoriness with there being greater abundances of GnIH, VIP, and PRL mRNA with the use of white or red light treatments. The results indicate that the use of white or red monochromatic lights while imposing a long photoperiod of 11 h daily could result in sustaining functions of the reproductive system of Yangzhou geese for considerably longer times, thus, resulting in greater egg-laying performance.

Development of a sandwich ELISA for determining plasma prolactin concentration in domestic birds.

The present study was conducted to establish a sandwich ELISA for the determination of prolactin (PRL) concentrations in the plasma of domestic fowls. The assay uses a recombinant goose PRL as the reference standard, expressed in a eukaryotic system, and as the antigen for raising a polyclonal antibody in rabbit. This rabbit anti-goose PRL polyclonal antibody was used for coating the wells of the ELISA plate, and its biotinylated form served as the detection antibody. An avidin-conjugated horseradish peroxidase was used to bind the detection antibody and to catalyze the chromogenic reaction using 3,3',5,5'-tetramethylbenzidine as the substrate. The assay showed a linear relationship between the optical density and concentration of the standard PRL in the 0 to 12.5 ng/mL range, and the assay was sensitive to a concentration as low as 0.39 ng/mL. The intra- and inter-assay CVs were <7% and 11%, respectively. The response curves of the serially diluted plasma samples from goose, duck, and chicken exhibited similar parallel relationships to that observed for the reference standards. Consistent with previous findings, the assay effectively detected differences in PRL concentration in plasma samples from chicken, duck, and goose at various reproductive stages.

Endocrine and molecular regulation mechanisms of the reproductive system of Hungarian White geese investigated under two artificial photoperiodic programs.

Hungarian White geese are regarded as good producers of meat, eggs, and feathers, but specific lighting schedules are required to improve their egg-laying performance. This study reveals the neuroendocrine regulatory mechanisms that govern the reproductive activities and egg-laying performances of Hungarian White geese. The results indicated that increasing the daily photoperiod from a short 8 h period to either 11 h or 14 h initiated reproduction. Egg-laying rates increased faster in the 14 h group, peaking (48.2%) on day 33 as compared to the peak (52.67%) reached on day 53 in the 11 h group. Changes to the plasma estradiol and progesterone concentrations produced similar patterns in the two groups. In the hypothalamus, OPN5, Dio2, c-Fos, and GnRH-I expression levels showed similar sequential increases and decreases. Changes in GnIH and VIP expression levels were the opposite to those of GnRH-I, but the levels peaked earlier under the 14 h photoperiod conditions. Pituitary LH beta and FSH beta expression levels increased at slower rates but remained significantly higher in the 11 h group than in the 14 h group. However, pituitary PRL expression increased considerably earlier and was higher in 14 h geese than in 11 h geese, which was opposite to the observed egg-laying rate patterns. An increase from a short to a relatively long photoperiod (11 h) regulated the neuroendocrine system and led to reproductive activities being sustained for a longer period, which resulted in high egg-laying performances.