Exploration of a Novel Noninvasive Prenatal Testing Approach for Monogenic Disorders Based on Fetal Nucleated Red Blood Cells.

BACKGROUND: Due to technical issues related to cell-specific capture methods, amplification, and sequencing, noninvasive prenatal testing (NIPT) based on fetal nucleated red blood cells (fNRBCs) has rarely been used for the detection of monogenic disorders. METHODS: Maternal peripheral blood was collected from 11 families with hereditary hearing loss. After density gradient centrifugation and cellular immunostaining for multiple biomarkers, candidate individual fetal cells were harvested by micromanipulation and amplified by whole-genome amplification (WGA). Whole-exome sequencing/whole-genome sequencing (WGS) and Sanger sequencing were performed on the identified fNRBCs to determine the fetal genotype. The impact of single-cell and pooled WGA products on the sequencing quality and results was compared. A combined analysis strategy, encompassing whole-exome sequencing/WGS, haplotype analysis, and Sanger sequencing, was used to enhance the NIPT results. RESULTS: fNRBCs were harvested and identified in 81.8% (9/11) of families. The results of cell-based-NIPT (cb-NIPT) were consistent with those of invasive prenatal diagnosis in 8 families; the coincidence rate was 88.9% (8/9). The combined analysis strategy improved the success of cb-NIPT. The overall performance of pooled WGA products was better than that of individual cells. Due to a lack of alternative fetal cells or sufficient sequencing data, cb-NIPT failed in 3 families. CONCLUSIONS: We developed a novel fNRBC-based NIPT method for monogenic disorders. By combining multiple analysis strategies and multiple fetal cell WGA products, the problem of insufficient genome information in a single cell was remedied. Our method has promising prospects in the field of NIPT for the detection of monogenic disorders.

Neuroprotective effect of microglia against impairments of auditory steady-state response induced by anti-P IgG from SLE patients in naïve mice.

OBJECTIVE: Autoantibodies against ribosomal P proteins (anti-P antibodies) are strongly associated with the neuropsychiatric manifestations of systemic lupus erythematosus (NPSLE). The present study was designed to assess whether anti-P antibodies can induce abnormal brain electrical activities in mice and investigate the potential cytopathological mechanism. METHODS: Affinity-purified human anti-ribosomal P antibodies were injected intravenously into mice after blood-brain barrier (BBB) disruption. The auditory steady-state response (ASSR) was evaluated based on electroencephalography (EEG) signals in response to 40-Hz click-train stimuli, which were recorded from electrodes implanted in the skull of mice. Immunofluorescence staining was used to examine the morphology and density of neurons and glia in the hippocampus and cortex. The presence of apoptosis in the brain tissues was studied using the TUNEL assay. A PLX3397 diet was used to selectively eliminate microglia from the brains of mice. RESULTS: Circulating anti-P antibodies caused an enhancement of the ASSR and the activation of microglia through the disrupted BBB, while no obvious neural apoptosis was observed. In contrast, when microglia were depleted, anti-P antibodies induced a serious reduction in the ASSR and neural apoptosis. CONCLUSION: Our study indicates that anti-P antibodies can directly induce the dysfunction of auditory-evoked potentials in the brain and that microglia are involved in the protection of neural activity after the invasion of anti-P antibodies, which could have important implications for NPSLE.

Aberrant Auditory Steady-State Response of Awake Mice After Single Application of the NMDA Receptor Antagonist MK-801 Into the Medial Geniculate Body.

BACKGROUND: Systemic administration of noncompetitive N-methyl-D-aspartate receptor (NMDAR) antagonists such as MK-801 is widely used to model psychosis of schizophrenia (SZ). Acute systemic MK-801 in rodents caused an increase of the auditory steady-state responses (ASSRs), the oscillatory neural responses to periodic auditory stimulation, while most studies in patients with SZ reported a decrease of ASSRs. This inconsistency may be attributable to the comprehensive effects of systemic administration of MK-801. Here, we examined how the ASSR is affected by selectively blocking NMDAR in the thalamus. METHODS: We implanted multiple electrodes in the auditory cortex (AC) and prefrontal cortex to simultaneously record the local field potential and spike activity (SA) of multiple sites from awake mice. Click-trains at a 40-Hz repetition rate were used to evoke the ASSR. We compared the mean trial power and phase-locking factor and the firing rate of SA before and after microinjection of MK-801 (1.5 µg) into the medial geniculate body (MGB). RESULTS: We found that both the AC and prefrontal cortex showed a transient local field potential response at the onset of click-train stimulus, which was less affected by the application of MK-801 in the MGB. Following the onset response, the AC also showed a response continuing throughout the stimulus period, corresponding to the ASSR, which was suppressed by the application of MK-801. CONCLUSION: Our data suggest that the MGB is one of the generators of ASSR, and NMDAR hypofunction in the thalamocortical projection may account for the ASSR deficits in SZ.

Lupus serum IgG induces microglia activation through Fc fragment dependent way and modulated by B-cell activating factor.

BACKGROUND: Neuropsychiatric manifestations are frequent in patients with systemic lupus erythematosus (SLE), yet the etiology and pathogenesis of brain damage in SLE remains unclear. Because the production of autoantibodies, formation and deposition of immunocomplexes are major serological characteristics of SLE, the elevated level of serum immunoglobulin may contribute to brain tissue injury of SLE. To testify this, in this study, we examined whether immunoglobulin G (IgG) in the serum of SLE patients affects the cellular functions in central nervous system and the potential mechanism. METHODS: In vivo intracerebral injection of SLE-serum in mouse was used to activate microglia and the production of pro-inflammatory cytokine was assessed by ELISA. Sera was divided into IgG and IgG depleted fractions, while IgG was further divided into Fc and Fab fragments to examine which part has an effect on microglia. Flow cytometry, immunofluorescence and quantitative PCR (qPCR) were used to verify the synergistic effect of B-cell activating factor (BAFF) on IgG stimulation of microglia. RESULTS: We found that IgG in lupus sera can induce M1 activation of brain microglia following intraventricular injection into normal mice, and BAFF facilitates this process. In vitro, we identified that IgG bound to microglia through Fc rather than Fab fragments, and BAFF up-regulated the expression of Fc receptors (FcγR) on the surface of microglia, consequently, promote IgG binding to microglia. CONCLUSION: Our results suggest that lupus serum IgG causes inflammatory responses of microglia by involving the Fc signaling pathway and the activity could be up-regulated by BAFF. Accordingly, disruption of the FcγR-mediated signaling pathway and blockade of microglia activation may be a therapeutic target in patients with neuropsychiatric lupus erythematosus.

Emotional arousal modifies auditory steady state response in the auditory cortex and prefrontal cortex of rats.

Emotional state has been shown to influence cognitive performance. However, the influence of mood on auditory processing is not fully understood. The auditory steady state response (ASSR) is the entrainment of neural activities elicited by periodic auditory stimulation, which is commonly used to evaluate the sensory and cognitive functions of brain. It has been shown that ASSR at 40 Hz is impaired at some psychotic disorders, such as schizophrenia and bipolar disorder. The primary goal of this study is to investigate the effect of emotional arousal on ASSR. To this end, we performed simultaneous recordings of local field potential (LFP) in response to 40 Hz click-train stimuli in the primary auditory cortex (A1) and medial prefront cortex (mPFC) of rats. During the electrophysiological recording, a negative mood was induced by means of the foot shocks occurred randomly in the inter-stimulus intervals. We found that both the power and phase-locking of ASSR in A1 were significantly increased under arousal condition, and phase-locking of ASSR in mPFC was also increased. The enhanced ASSRs were accompanied by an increase in coherence between A1 and mPFC. Our results suggest that A1-to-mPFC information transfer is enhanced under arousal state and the functional connectivity between mPFC and A1 may contribute to the emotional modulation of auditory process.

Research on the Application of Polymer Materials in Contemporary Ceramic Art Creation.

Ceramicized polymer composites are prepared by adding various additives into the polymer matrix, such as functional clay fillers, porcelain-forming additives, crosslinking agents, flame retardants, reinforcement agents, etc. In recent years, polymer materials have been widely used in the preparation of ceramic materials. Moreover, the addition of polymer materials in ceramic materials results in increased bending in the ceramic body, and its mechanical strength has been greatly improved; this advantage has led many contemporary ceramists to use polymer materials in the creation of ceramic works, providing ceramic creation more space for operation. The introduction of polymer materials into ceramic materials brings more possibilities than traditional ceramic creation based on the tests of toughness, strength, and yield of the ceramic body. This article investigates ceramic raw materials with high-polymer material ceramic function, high-polymer materials for modern pottery to convey the artistic expression of porcelain texture, as well as the use of high-polymer materials in gel-powered three-dimensional (3D) printing to refine the injection molding process, all of which solve the difficulties of creating delicate artworks in modern ceramic art creation. This paper mainly adopts the research method of recording and comparing the numerical value of adding ceramic materials into polymer materials and the physical shape after firing in ceramic creation, to form a relatively stable numerical value and firing curve for a certain type of ceramic creation form. In this regard, the integration of modern ceramic creation and polymer materials makes ceramic works a relatively special style in contemporary art, increasing its cultural connotation and visual tension.

[Experimental study on acute respiratory distress syndrome and analysis of relevant factors in rabbits subjected to thoracic blast trauma].

OBJECTIVE: To reproduce acute respiratory distress syndrome (ARDS) model in rabbit induced by chest blast injury and to analyze the pathogenesis and causes of early death in order to provide the basis for the early diagnosis of lung blast injury and its early warning system to facilitate an early treatment. METHODS: Sixty healthy New Zealand white rabbits were divided into six groups according to the different explosion distance with the random number table method. The survival rate and its resulting pathological changes were observed and patho physiological indexes and lung fluid content were determined at sequential time points post explosion. RESULTS: Shock wave pressure less than 1 210.5 mm Hg (1 mm Hg=0.133 kPa, group A, B) resulted in limited injury to the lung within grade 2 as assessed with the abbreviated injury scale (AIS). The rabbits in these groups recovered soon and survived without any complication. Shock pressure higher than 2 036.1 mm Hg (group D, E) caused severe injuries to the lung, including deep laceration , disruption of lung hilus and large hematoma in the lung, and the injury severity of lungs was assessed above grade 5 as assessed with AIS. All rabbits died within 1 hour post explosion. The groups described above failed to meet the demand of an ARDS model for the present study. Shock wave pressure at 1 917.3 mm Hg (group C) produced extensive contusion from grade 4 to grade 5 as assessed with AIS. The rabbits survived in poor general condition, and arterial partial pressure of oxygen (PaO(2)) lowered within 6 hours . Pathological examination showed extensive and constant multi focal bleeding involving more than four lobes. The alveolar wall was edematous, with partial rupture and alveolar fusion in lung tissues was observed in the group C. Alveoli were filled with inflammatory cells, and hyaline membrane was formed occasionally . Compared with control group, the wet to dry weight ratio (W/D) in lungs increased obviously (6.46±0.24 vs. 3.98±0.19, P<0.01) in group C within 6 hours postinjury. The contents of tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) in plasma and bronchoalveolar lavage fluid (BALF) were also increased distinctly compared with the control group [TNF-α (ng/L) in plasma: 328.89±6.26 vs. 62.12±2.98, TNF-α (ng/L) in BALF: 164.87±4.59 vs. 29.51±1.12; IL-6 (ng/L) in plasma: 128.51±4.13 vs. 19.32±1.53, IL-6 (ng/L) in BALF: 94.97±1.14 vs. 22.72±0.19, all P<0.05]. CONCLUSION: In an airtight environment, rabbit ARDS model can be reproduced successfully by blast injury with 1 917.3 mm Hg explosion pressure; TNF-α and IL-6 are involved in the pathogenesis and development of ARDS in blast injury. Pneumothorax as a result of lung rupture is the chief reason for early death and dysfunction of circulatory system is also an important reason in producing early death.

Effect of locomotion on the auditory steady state response of head-fixed mice.

OBJECTIVES: Electroencephalographic (EEG) examinations of the auditory steady-state response (ASSR) can non-invasively probe cortical function to generate the gamma-band (40 Hz) oscillation, which is increasingly applied to the neurophysiological studies on the rodent models of psychiatric disorders. Though, it has been well established that the brain activities are significantly modulated by the behavioural state (such as locomotion), how the ASSR is affected remains unclear. METHODS: We investigated the effect of locomotion by recording local field potential (LFP) evoked by 40-Hz click-train from multiple brain areas: auditory cortex (AC), medial geniculate body (MGB), hippocampus (HP) and prefrontal cortex (PFC), in head-fixed mice free to run on a treadmill. Comparisons were conducted on the LFPs during spontaneous movement and stationary conditions. RESULTS: We found that in both the auditory (AC and MGB) and non-auditory areas (HP and PFC), locomotion reduced the initial negative deflection of LFP (early response during 0-100 ms from stimulus onset), and had no significant effect on the ASSR phase-locking to the late stimulus (100-500 ms). CONCLUSIONS: Our results suggest that different neural mechanisms contribute to the early response and ASSR, and the ASSR is a more robust biomarker to investigate the pathogenesis of neuropsychiatric disorders.

Tofacitinib Ameliorates Lipopolysaccharide-Induced Acute Kidney Injury by Blocking the JAK-STAT1/STAT3 Signaling Pathway.

Septic acute kidney injury (AKI) is the most common AKI syndrome in the intensive care unit (ICU), and it accounts for approximately half of AKI cases. Tofacitinib (TOFA) is a pan-Janus kinase (JAK) inhibitor that exhibits potent anti-inflammatory activity in rheumatoid arthritis. However, no study has examined the functional role of TOFA in septic AKI. In the present study, we investigated the protective effects of TOFA on septic AKI and the underlying mechanisms. A lipopolysaccharide- (LPS-) induced AKI model was established in C57BL/6 mice via an intraperitoneal injection of LPS (10 mg/kg). One hour after LPS challenge, the mice were orally administered TOFA (5, 10, or 15 mg/kg) every 6 h until sacrifice at 24 h. We found that TOFA significantly ameliorated LPS-induced renal histopathological changes and dysfunction. TOFA also suppressed the expression levels of proinflammatory cytokines (TNF-α, IL-1ß, IL-6, and IFN-γ) and the parameters of oxidative stress (MDA, GSH, SOD, and CAT) in kidney tissues. These results may be associated with the inhibitory effect of TOFA on the JAK-STAT1/STAT3 pathway, which was significantly activated by LPS challenge. TOFA treatment also inhibited LPS-induced activation of the TLR4/NF-κB pathway. In conclusion, we revealed that TOFA had a protective effect on LPS-induced AKI, and it may be a promising therapeutic agent for septic AKI.

Characterization of Oxygenated Heterocyclic Compounds and in vitro Antioxidant Activity of Pomelo Essential Oil.

PURPOSE: Citrus essential oils are widely used for aromatherapy and the alternative treatment of chronic diseases. Beyond the aroma substances, they are known to contain bioactive nonvolatile components; however, little knowledge has been gained about nonvolatiles in the essential oil of pomelo (Citrus grandis Osbeck), the largest citrus fruit. The purpose of this study was to analyze the nonvolatile oxygenated heterocyclic compounds (OHCs) of pomelo essential oils and evaluate their in vitro antioxidant activities for further development. METHODS: Cold-pressed essential oil (CPEO) and distilled essential oil (DEO) were obtained from the peel of the Liangping pomelo cultivar. High-performance liquid chromatography (HPLC) coupled with a photodiode array and fluorescence detection method was developed to identify and quantify the OHCs of the two essential oils. Ferric reducing antioxidant power and 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl 3-oxide (PTIO) radical scavenging assays were used to determine the antioxidative capabilities. RESULTS: Thirteen OHCs were identified in CPEO. Coumarins such as meranzin (2.0 mmol L-1) and furanocoumarins such as isoimperatorin (1.3 mmol L-1) composed the majority of nonvolatiles in CPEO. These OHCs were characterized by high proportion (58%) of side chain epoxides. Five OHCs, namely, auraptenol, 6',7'-dihydroxybergamottin (6',7'-DHB), imperatorin, isoimperatorin and 8-geranyloxypsoralen were first identified in pomelo CPEO. Eight OHCs were detected at trace amounts in pomelo DEO. Antioxidant assays showed that CPEO was multiple times more potent than DEO regarding the total reducing power and radical scavenging capacity. Clearance of PTIO, a stable reactive oxygen species, followed slow kinetics. CONCLUSION: Coumarins and furanocoumarins, two families of OHCs, constituted most of the nonvolatile components in CPEO. The nonvolatiles contributed significantly to the in vitro antioxidant activity of CPEO. Pomelo CPEO showed good prospects as a potential long-lasting natural antioxidant.

Aberrant Auditory Steady-State Response of Awake Mice Induced by Chronic Interferon-α Treatment.

Background: Patients receiving the cytokine immunotherapy of interferon-alpha (IFN-α) frequently present with depression. This is one of the excellent models to explore the action of peripheral cytokine on central nervous system (CNS) and to study the development of depression. The auditory steady-state response (ASSR), electroencephalogram (EEG) oscillations induced by periodic acoustic stimulation, is an effective approach to evaluate the neural function in mental illness including depression. The aim of the present study was to investigate the effect of IFN-α on the cortical ASSR and its correlation with depressive-like behavior. Methods: Chronic electrodes were implanted on the skull over the auditory cortex (AC) of male C57BL/6 mice. The animals were treated with daily injection of IFN-α or saline (vehicle) for three weeks. EEGs were recorded in AC of the same mouse before and after the injection treatment to monitor the changes of ASSR induced by IFN-α. Depressive-like behavior was analyzed in the forced swim test (FST). Immunohistochemical staining was used to examine the status of neuron and glia in the hippocampus and AC. Results: Compared to pretreatment condition, injection of IFN-α significantly reduced the power of 40 Hz ASSR in the mouse AC from the second week. Such a decrease continued to the third week. The immobility times of FST were significantly increased by a 3-week treatment of IFN-α and the immobility time was negatively correlated with the power of 40 Hz ASSR. Astrocytes and microglia in the hippocampus and AC were activated by IFN-α, but the density of neuron was not significantly affected. Conclusion: Our results suggest that EEG measurement of ASSR may be used as a biomarker to monitor the CNS side effects of IFN-α treatment and to search a novel intervention with potential therapeutic implications.

Intracerebroventricular administration of lupus serum induces microglia activation and leukocyte adhesion in the cerebromicrovasculature of mice.

BACKGROUND: Central nervous system (CNS) involvement is commonly seen in the patients with system lupus erythematosus (SLE). Mechanisms underlying CNS damage in SLE remain largely unknown. Accumulating evidence suggest that activation of microglia in CNS plays an important role in the inflammatory responses in neurological diseases. The aim of this study is to examine the involvement of microglia in the CNS inflammatory responses induced by circulating serum of SLE patients. METHODS: We performed intracerebroventricular (ICV) injection of serums collected from SLE patients or healthy controls to mice, and examined phenotypic changes of microglia, the levels of cytokines, chemokine and adhesion molecules in the brain. Intravital microscopy was used to observe leukocyte rolling and adhesion in the cerebromicrovasculature. We further examined whether minocycline can block inflammatory responses induced by SLE serum. In vitro experiments were conducted to examine whether IgGs from the sera of SLE patients or healthy control can activate the primary cultured microglia. RESULTS: We found that ICV injection of SLE serum increases morphological activation of microglia in the cortex and hippocampus. Inflammatory mediators including pro-inflammatory cytokines (IL-1, IL-6 and TNF-α), chemokine (CCL2 and CCL5) and adhesion molecules (P-selectin and ICAM-1) were significantly elevated in the brains of SLE-serum-treated mice. Using intravital microscopy, we demonstrated that SLE serum promotes leukocyte rolling and adhesion. Furthermore, suppression of microglia activation by systemically using minocycline could decrease the levels of inflammatory molecular, and prevent leukocyte rolling and adhesion. The in vitro experiments revealed that IgG from SLE sera could be engulfed by microglia and stimulated the microglia to secret pro-inflammatory cytokines. CONCLUSION: Our data suggest that the activation of microglia, which promotes leukocyte adhesion to the brain microvasculature, is an important pathological mechanism of CNS involvement in SLE.

Auditory steady-state responses in primary and non-primary regions of the auditory cortex in neonatal ventral hippocampal lesion rats.

Auditory steady-state responses (ASSRs) represent the electrophysiological activity of the auditory nervous system in response to a periodic acoustic stimulus. Spectrogram analysis can reveal the frequency and phase information entrained in ASSRs. Clinically, the ASSR is used to detect abnormalities in electroencephalographs obtained from schizophrenia patients, who show reduced power and phase locking of ASSRs. The neonatal ventral hippocampal lesion (NVHL) rat is a widely used model to investigate the neurodevelopmental mechanisms of schizophrenia. It has been established that NVHL rats exhibit several schizophrenia-like behavioral and molecular abnormalities. However, no clear abnormalities in ASSRs have been reported to date. The present study compared ASSRs of adult NVHL and sham-operated rats. We inserted microelectrodes into the primary auditory cortex (A1) or posterior auditory field (PAF) and recorded the local field potential (LFP) in response to 40- and 80-Hz click train stimuli. Spectrogram analysis was performed to obtain the mean trial power (MTP) and phase-locking factor (PLF) of the click train-evoked LFPs. We found that in the control animals, A1 showed a stronger MTP and PLF of ASSR than PAF, and NVHL operation mainly impaired the ASSR in PAF. Analysis of spike activity also indicated that NVHL operation extended the duration of tone-evoked responses in PAF neurons. Our results reveal, for the first time, that NVHL may distinctly influence the neural activities of primary and non-primary fields of the auditory cortex.