BERT-TFBS: a novel BERT-based model for predicting transcription factor binding sites by transfer learning.

Transcription factors (TFs) are proteins essential for regulating genetic transcriptions by binding to transcription factor binding sites (TFBSs) in DNA sequences. Accurate predictions of TFBSs can contribute to the design and construction of metabolic regulatory systems based on TFs. Although various deep-learning algorithms have been developed for predicting TFBSs, the prediction performance needs to be improved. This paper proposes a bidirectional encoder representations from transformers (BERT)-based model, called BERT-TFBS, to predict TFBSs solely based on DNA sequences. The model consists of a pre-trained BERT module (DNABERT-2), a convolutional neural network (CNN) module, a convolutional block attention module (CBAM) and an output module. The BERT-TFBS model utilizes the pre-trained DNABERT-2 module to acquire the complex long-term dependencies in DNA sequences through a transfer learning approach, and applies the CNN module and the CBAM to extract high-order local features. The proposed model is trained and tested based on 165 ENCODE ChIP-seq datasets. We conducted experiments with model variants, cross-cell-line validations and comparisons with other models. The experimental results demonstrate the effectiveness and generalization capability of BERT-TFBS in predicting TFBSs, and they show that the proposed model outperforms other deep-learning models. The source code for BERT-TFBS is available at https://github.com/ZX1998-12/BERT-TFBS.

Improving resilience to high temperature in drought: water replenishment enhances sucrose and amino acid metabolisms in maize grain.

Heat stress poses a significant threat to maize, especially when combined with drought. Recent research highlights the potential of water replenishment to ameliorate grain weight loss. However, the mitigating mechanisms of heat in drought stress, especially during the crucial early grain-filling stage, remain poorly understood. We investigated the mechanism for mitigating heat in drought stress by water replenishment from the 12th to the 32nd days after silking in a controlled greenhouse experiment (Exp. I) and field trial (Exp. II). A significant reduction in grain weight was observed in heat stress compared to normal conditions. When water replenishment was applied to increase soil water content (SWC) under heat stress, the grain yield exhibited a notable increase ranging from 28.4 to 76.9%. XY335 variety was used for transcriptome sequencing to analyze starch biosynthesis and amino acid metabolisms in Exp. I. With water replenishment, the transcripts of genes responsible for trehalose 6-phosphate phosphates (TPP), alpha-trehalase (TRE), ADP-glcpyrophosphorylase, and starch synthase activity were stimulated. Additionally, the expression of genes encoding TPP and TRE contributed to an enhanced conversion of trehalose to glucose. This led to the conversion of sucrose from glucose-1-phosphate to ADP-glucose and ADP-glucose to amylopectin, ultimately increasing starch production by 45.1%. Water replenishment to boost SWC during heat stress also elevated the levels of essential amino acids in maize, including arginine, serine, tyrosine, leucine, glutamic acid, and methionine, providing valuable support to maize plants in adversity. Field trials further validated the positive impact of water replenishment on SWC, resulting in a notable increase in grain yield ranging from 7.1 to 9.2%. This study highlights the vital importance of adapting to abiotic stress and underscores the necessity of developing strategies to counteract its adverse effects on crop yield.

Deletion of a 7-amino-acid region in the porcine epidemic diarrhea virus envelope protein induces higher type I and III interferon responses and results in attenuation in vivo.

Porcine epidemic diarrhea virus (PEDV) leads to enormous economic losses for the pork industry. However, the commercial vaccines failed to fully protect against the epidemic strains. Previously, the rCH/SX/2016-SHNXP strain with the entire E protein and the rCH/SX/2015 strain with the deletion of 7-amino-acid (7-aa) at positions 23-29 in E protein were constructed and rescued. The pathogenicity assay indicated that rCH/SX/2015 is an attenuated strain, but rCH/SX/2016-SHNXP belongs to the virulent strains. Then, the recombination PEDV (rPEDV-EΔaa23-aa29)strain with a 7-aa deletion in the E protein was generated, using the highly virulent rCH/SX/2016-SHNXP strain (rPEDV-Ewt) as the backbone. Compared with the rPEDV-Ewt strain, the release and infectivity of the rPEDV-EΔaa23-aa29 strain were significantly reduced in vitro, but stronger interferon (IFN) responses were triggered both in vitro and in vivo. The pathogenicity assay showed that the parental strain resulted in severe diarrhea (100%) and death (100%) in all piglets. Compared with the parental strain group, rPEDV-EΔaa23-aa29 caused lower mortality (33%) and diminished fecal PEDV RNA shedding. At 21 days, all surviving pigs were challenged orally with rPEDV-Ewt. No pigs died in the two groups. Compared with the mock group, significantly delayed and milder diarrhea and reduced fecal PEDV RNA shedding were detected in the rPEDV-EΔaa23-aa29 group. In conclusion, the deletion of a 7-aa fragment in the E protein (EΔaa23-aa29) attenuated PEDV but retained its immunogenicity, which can offer new ideas for the design of live attenuated vaccines and provide new insights into the attenuated mechanism of PEDV. IMPORTANCE Porcine epidemic diarrhea virus (PEDV) causes high mortality in neonatal piglets and remains a large challenge to the pork industry. Unfortunately, no safe and effective vaccines are available yet. The pathogenesis and molecular basis of the attenuation of PEDV remain unclear, which seriously hinders the development of PEDV vaccines. This study found that the rPEDV carrying EΔaa23-aa29 mutation in the E protein induced significantly higher IFN responses than the parental virus, partially attenuated, and remained immunogenic in piglets. For the first time, PEDV E was verified as an IFN antagonist in the infection context and identified as a virulence factor of PEDV. Our data also suggested that EΔaa23-aa29 mutation can be a good target for the development of live attenuated vaccines for PEDV and also provide new perspectives for the attenuated mechanism of PEDV.

9-Butyl-Harmol Exerts Antiviral Activity against Newcastle Disease Virus through Targeting GSK-3ß and HSP90ß.

The paramyxoviruses represent a large family of human and animal pathogens that cause significant health and economic burdens worldwide. However, there are no available drugs against the virus. ß-carboline alkaloids are a family of naturally occurring and synthetic products with outstanding antiviral activities. Here, we examined the antiviral effect of a series of ß-carboline derivatives against several paramyxoviruses, including Newcastle disease virus (NDV), peste des petits ruminants virus (PPRV), and canine distemper virus (CDV). Among these derivatives, 9-butyl-harmol was identified as an effective antiviral agent against these paramyxoviruses. Further, a genome-wide transcriptome analysis in combination with target validation strategies reveals a unique antiviral mechanism of 9-butyl-harmol through the targeting of GSK-3ß and HSP90ß. On one hand, NDV infection blocks the Wnt/ß-catenin pathway to suppress the host immune response. 9-butyl-harmol targeting GSK-3ß dramatically activates the Wnt/ß-catenin pathway, which results in the boosting of a robust immune response. On the other hand, NDV proliferation depends on the activity of HSP90. The L protein, but not the NP protein or the P protein, is proven to be a client protein of HSP90ß, rather than HSP90α. 9-butyl-harmol targeting HSP90ß decreases the stability of the NDV L protein. Our findings identify 9-butyl-harmol as a potential antiviral agent, provide mechanistic insights into the antiviral mechanism of 9-butyl-harmol, and illustrate the role of ß-catenin and HSP90 during NDV infection. IMPORTANCE Paramyxoviruses cause devastating impacts on health and the economy worldwide. However, there are no suitable drugs with which to counteract the viruses. We determined that 9-butyl-harmol could serve as a potential antiviral agent against paramyxoviruses. Until now, the antiviral mechanism of ß-carboline derivatives against RNA viruses has rarely been studied. Here, we found that 9-butyl-harmol exerts dual mechanisms of antiviral action, with its antiviral activities being mediated by two targets: GSK-3ß and HSP90ß. Correspondingly, the interaction between NDV infection and the Wnt/ß-catenin pathway or HSP90 is demonstrated in this study. Taken together, our findings shed light on the development of antiviral agents against paramyxoviruses, based on the ß-carboline scaffold. These results present mechanistic insights into the polypharmacology of 9-butyl-harmol. Understanding this mechanism also deepens the host-virus interaction and reveals new drug targets for anti-paramyxoviruses.

Lineage 1 PRRSVs infection induces hemorrhagic injury in intestines of piglets: Effects on complement and coagulation cascades.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes a highly transmissible disease of significant concern in the pig industry. Previous studies have demonstrated that the XM-2020 strain (a lineage 1.8 PRRSV IA/2012/NADC30) can induce special hemorrhagic injury in the small intestines. However, the specific mechanism underlying this injurious effect remains incompletely understood. In this study, we examined the pathogenic properties of XM-2020 and YC-2020 strains (a lineage 1.5 PRRSV IA/2014/NADC34) in piglets. Animal pathogenic tests revealed that with either Lineage 1 PRRSVs strains XM-2020 or YC-2020 demonstrated pronounced intestinal hemorrhage and suppression of peripheral immunological organs, comparing to JXA1 infection. Transcriptome analysis of diseased small intestines unveiled that PRRSV infection stimulated oxidative and inflammatory reactions. Remarkably, we also observed activation of the complement system alongside a notable down-regulation of complement and coagulation cascade pathways in the Lineage 1 PRRSVs infection group. Based on these findings, we propose that the primary mechanism driving the hemorrhagic injury of the small intestine caused by Lineage 1 PRRSVs is the suppression of complement and coagulation cascades resulting from immunosuppression. This discovery deepens our understanding of the pathogenicity of PRRSV in the small intestine and provides promising ways out for the development of innovative strategies aimed at controlling PRRSV.

Emerging perfluoroalkyl substances retard skeletal growth by accelerating osteoblasts senescence via ferroptosis.

Due to the persistent nature and significant negative impacts of perfluorooctanoic acid (PFOA) on human health and other organisms, the emergence of new PFOA alternatives, such as perfluoro (2-methyl-3-oxhexanoic) acid (GenX) and perfluoro-3,6,9-trioxyundecanoic acid (PFO3TDA), have drawn significant attention. However, the toxic effects of PFOA and its substitutes on bones remain limited. In this study, we administered different concentrations of PFOA, GenX, and PFO3TDA via gavage to 3-week-old male BALB/C mice for four weeks. X-ray and micro-CT scans revealed shortening of the femur and tibia and significant reduction in bone density. Additionally, PFOA, GenX, and PFO3TDA promoted osteoblast senescence and impaired osteogenic capabilities. This was characterized by a decrease in the expression of osteogenesis-related genes (OCN, ALP, Runx2, etc.) and an increase in the expression of aging and inflammation-related factors (p16INK4a, P21, MMP3, etc). Furthermore, RNA sequencing revealed activation of the ferroptosis pathway in PFOA-treated osteoblasts, characterized by notable lipid peroxidation and excessive iron accumulation. Finally, by inhibiting the ferroptosis pathway with ferrostatin-1 (Fer-1), we effectively alleviated the senescence of MC3T3-E1 cells treated with PFOA, GenX, and PFO3TDA, and improved their osteogenic capabilities. Therefore, our study provides a new therapeutic insight into the impact of PFOA and its substitutes on bone growth and development.

[Determination of 11 nutrients in liquid milk by ultra-performance liquid chromatography-tandem mass spectrometry].

OBJECTIVE: To establish an ultra-performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS) method for simultaneous determination of 11 nutritional components(thiamine, riboflavin, nicotinamide, nicotinic acid, pantothenic acid, pyridoxine, pyridoxal, pyridoxamine, biotin, choline, L-carnitine) in liquid milk. METHODS: Milk samples were shaken with 20 mmol/L ammonium formate solution and heated in a water bath at 100 ℃ for 30 min, then incubated with papain and acid phosphatase at 45 ℃ for 16 h, the lower liquid was collected after centrifugation for analysis. UPLC separation was performed on an ACQUITY~(TM) HSS T3(3.0 mm×150 mm, 1.8 µm) column, 2 mmol/L ammonium formate(containing 0.1% formic acid) solution and acetonitrile(containing 0.1% formic acid) were used as mobile phase. Quantitative detection was performed by internal standard method. RESULTS: 11 nutritional components can be effectively separated and detected in 12 min, and the linear correlation coefficients(R~2) were all above 0.995. The limits of detection(LODs) were between 0.05 and 0.50 µg/L, and the limits of quantification(LOQs) were between 0.20 and 1.25 µg/L. The recovery rates of three-level addition were 85.6%-119.3%, and the precision RSDs were between 3.68% and 7.82%(n=6). Based on the detection of 60 liquid milk samples from 5 different animals, it was found that the contents of 11 nutrients in liquid milk from different milk sources were significantly different, but pyridoxine could not be detected. CONCLUSION: The method can quantitatively detect 11 water-soluble nutrients, including free and bound forms, by effective enzymolysis. It is sensitive, reproducible and can meet the needs of quantitative detection.

De novo biosynthesis of carminic acid in Saccharomyces cerevisiae.

Carminic acid is a natural red dye extracted from the insect Dactylopius coccus. Due to its ideal dying effect and high safety, it is widely used in food and cosmetics industries. Previous study showed that introduction of polyketide synthase (OKS) from Aloe arborescens, cyclase (ZhuI) and aromatase (ZhuJ) from Streptomyces sp. R1128, and C-glucosyltransferase (UGT2) from D. coccus into Aspergillus nidulans could achieve trace amounts of de novo production. These four genes were introduced into Saccharomyces cerevisiae, but carminic acid was not detected. Analysis of the genome of A. nidulans revealed that 4'-phosphopantetheinyl transferase (NpgA) and monooxygenase (AptC) are essential for de novo biosynthesis of carminic acid in S. cerevisiae. Additionally, endogenous hydroxylase (Cat5) from S. cerevisiae was found to be responsible for hydroxylation of flavokermesic acid to kermesic acid. Therefore, all enzymes and their functions in the biosynthesis of carminic acid were explored and reconstructed in S. cerevisiae. Through systematic pathway engineering, including regulating enzyme expression, enhancing precursor supply, and modifying the ß-oxidation pathway, the carminic acid titer in a 5 L bioreactor reached 7580.9 µg/L, the highest yet reported for a microorganism. Heterologous reconstruction of the carminic acid biosynthetic pathway in S. cerevisiae has great potential for de novo biosynthesis of anthraquinone dye.

Genetic and pathogenic characterization of an NADC-34-like isolate of porcine reproductive and respiratory syndrome virus.

In this study, an NADC34-like strain of porcine reproductive and respiratory syndrome virus (PRRSV), YC-2020, was isolated from a pig farm in Yuncheng, Shanxi Province, China. Phylogenetic and molecular evolutionary analysis showed that the genome sequence of YC-2020 was very similar to those of NADC34-like PRRSV strains in the ORF2-7 region. However, it was more closely related to NADC30-like PRRSV and highly pathogenic (HP) PRRSV in the NSP2 and NSP3-9 coding regions, respectively, suggesting that recombination had occurred between viruses belonging to lineages 1 and 8. Piglets infected with YC-2020 exhibited mild clinical signs, but they had severe histopathological lesions in their lungs. These findings reveal novel genetic and pathogenic features of this isolate.

Chronic kidney disease in a giant panda (Ailuropoda melanoleuca): a case report.

BACKGROUND: Chronic kidney disease (CKD) is a common cause of morbidity and mortality in captive wildlife species. However, CKD has been rarely documented in giant pandas. CASE PRESENTATION: The following report describes a case of an eight-year-old female giant panda showing clinical signs of epistaxis, bloody diarrhea, polyuria, azotemia and anemia. The animal died despite of supportive treatments. Necropsy was performed. Grossly, both kidneys were shrunken and scarred with pallor. Subcutis edema and petechia on the epicardium of the heart were observed. The tissue samples were made into paraffin sections and stained by H.E and special staining including Periodic Acid-Schiff (PAS), von Kossa, Masson's trichrome, Phosphotungstic acid-hematoxylin (PTAH), and Congo red. Histopathology examination revealed severe chronic tubulointerstitial nephritis with marked interstitial fibrosis, glomerulosclerosis, tubular atrophy and calcification in kidneys, and acute necrotizing hemorrhagic myocarditis with calcification in heart. Other lesions included intestinal hemorrhage, hepatic fatty degeneration and necrosis with hemosiderin, and splenic hemosiderin. CONCLUSIONS: In summary, chronic kidney disease was finally diagnosed based on the association of clinical, gross, and histopathological findings. Heart failure secondary to CKD is the leading cause of death in this giant panda. The potential cause of CKD in this animal is possibly due to long term and uncontrolled hypertension. Blood pressure monitoring is essential in establishing the diagnosis and management of hypertension in giant panda.

Possibilities of Using De Novo Design for Generating Diverse Functional Food Enzymes.

Food enzymes have an important role in the improvement of certain food characteristics, such as texture improvement, elimination of toxins and allergens, production of carbohydrates, enhancing flavor/appearance characteristics. Recently, along with the development of artificial meats, food enzymes have been employed to achieve more diverse functions, especially in converting non-edible biomass to delicious foods. Reported food enzyme modifications for specific applications have highlighted the significance of enzyme engineering. However, using direct evolution or rational design showed inherent limitations due to the mutation rates, which made it difficult to satisfy the stability or specific activity needs for certain applications. Generating functional enzymes using de novo design, which highly assembles naturally existing enzymes, provides potential solutions for screening desired enzymes. Here, we describe the functions and applications of food enzymes to introduce the need for food enzymes engineering. To illustrate the possibilities of using de novo design for generating diverse functional proteins, we reviewed protein modelling and de novo design methods and their implementations. The future directions for adding structural data for de novo design model training, acquiring diversified training data, and investigating the relationship between enzyme-substrate binding and activity were highlighted as challenges to overcome for the de novo design of food enzymes.

Computer-Aided Designing Peptide Inhibitors of Human Hematopoietic Prostaglandin D2 Synthase Combined Molecular Docking and Molecular Dynamics Simulation.

Human hematopoietic prostaglandin D2 synthase (HPGDS) is involved in the production of prostaglandin D2, which participates in various physiological processes, including inflammation, allergic reactions, and sleep regulation. Inhibitors of HPGDS have been investigated as potential anti-inflammatory agents. For the investigation of potent HPGDS inhibitors, we carried out a computational modeling study combining molecular docking and molecular dynamics simulation for selecting and virtual confirming the designed binders. We selected the structure of HPGDS (PDB ID: 2CVD) carrying its native inhibitor compound HQL as our research target. The random 5-mer peptide library was created by building the 3-D structure of random peptides using Rosetta Buildpeptide and performing conformational optimization. Molecular docking was carried out by accommodating the peptides into the location of their native binder and then conducting docking using FlexPepDock. The two peptides RMYYY and VMYMI, which display the lowest binding energy against HPGDS, were selected to perform a comparative study. The interaction of RMYYY and VMYMI against HPGDS was further confirmed using molecular dynamics simulation and aligned with its native binder, HQL. We show the selected binders to have stronger binding energy and more frequent interactions against HPGDS than HQL. In addition, we analyzed the solubility, hydrophobicity, charge, and bioactivity of the generated peptides, and we show that the selected strong binder may be further used as therapeutic drugs.

Identification of crucial circRNAs in skeletal muscle during chicken embryonic development.

BACKGROUND: Chicken provides humans with a large amount of animal protein every year, in which skeletal muscle plays a leading role. The embryonic skeletal muscle development determines the number of muscle fibers and will affect the muscle production of chickens. CircRNAs are involved in a variety of important biological processes, including muscle development. However, studies on circRNAs in the chicken embryo muscle development are still lacking. RESULTS: In the study, we collected chicken leg muscles at 14 and 20-day embryo ages both in the fast- and slow-growing groups for RNA-seq. We identified 245 and 440 differentially expressed (DE) circRNAs in the comparison group F14vsF20 and S14vsS20 respectively. GO enrichment analysis for the host genes of DE circRNAs showed that biological process (BP) terms in the top 20 related to growth in F14vsF20 were found such as positive regulation of transcription involved in G1/S phase of mitotic cell cycle, multicellular organismal macromolecule metabolic process, and multicellular organismal metabolic process. In group S14vsS20, we also found some BP terms associated with growth in the top 20 including actomyosin structure organization, actin cytoskeleton organization and myofibril assembly. A total of 7 significantly enriched pathways were obtained, containing Adherens junction and Tight junction. Further analysis of those pathways found three crucial host genes MYH9, YBX3, IGF1R in both fast- and slow-growing groups, three important host genes CTNNA3, AFDN and CREBBP only in the fast-growing group, and six host genes FGFR2, ACTN2, COL1A2, CDC42, DOCK1 and MYL3 only in the slow-growing group. In addition, circRNA-miRNA network also revealed some key regulation pairs such as novel_circ_0007646-miR-1625-5p, novel_circ_0007646-miR-1680-5p, novel_circ_0008913-miR-148b-5p, novel_circ_0008906-miR-148b-5p and novel_circ_0001640-miR-1759-3p. CONCLUSIONS: Comprehensive analysis of circRNAs and their targets would contribute to a better understanding of the molecular mechanisms in poultry skeletal muscle and it also plays an important guiding role in the next research.

Active secretion of a thermostable transglutaminase variant in Escherichia coli.

BACKGROUND: Streptomyces mobaraenesis transglutaminase (smTG) is widely used to generate protein crosslinking or attachment of small molecules. However, the low thermostability is a main obstacle for smTG application. In addition, it is still hard to achieve the secretory expression of active smTG in E. coli, which benefits the enzyme evolution. In this study, a combined strategy was conducted to improve the thermostability and secretory expression of active smTG in E. coli. RESULTS: First, the thermostable S. mobaraenesis transglutaminase variant S2P-S23V-Y24N-S199A-K294L (TGm1) was intracellularly expressed in pro-enzyme form in E. coli. Fusing the pro-region of Streptomyces hygroscopicus transglutaminase (proH) and TrxA achieved a 9.78 U/mL of intracellular smTG activity, 1.37-fold higher than the TGm1 fused with its native pro-region. After in vitro activation by dispase, the TGm1 with proH yielded FRAPD-TGm1, exhibiting 0.95 â„ƒ and 94.25% increases in melting temperature and half-life at 60 â„ƒ compared to FRAP-TGm1 derived from the expression using its native pro-region, respectively. Second, the TGm1 with proH was co-expressed with transglutaminase activating protease and chaperones (DnaK, DnaJ, and GrpE) in E. coli, achieving 9.51 U/mL of intracellular FRAPD-TGm1 without in vitro activation. Third, the pelB signal peptide was used to mediate the secretory expression of active TGm in E. coli, yielding 0.54 U/mL of the extracellular FRAPD-TGm1. A script was developed to shuffle the codon of pelB and calculate the corresponding mRNA folding energy. A 1.8-fold increase in the extracellular expression of FRAPD-TGm1 was achieved by the Top-9 pelB sequence derived from the coding sequences with the lowest mRNA folding energy. Last, deleting the gene of Braun's lipoprotein further increased the extracellular yield of FRAPD-TGm1 by 31.2%, reached 1.99 U/mL. CONCLUSIONS: The stabilized FRAPD-smTG here could benefit the enzyme application in food and non-food sectors, while the E. coli system that enables secretory expression of active smTG will facilitate the directed evolution for further improved catalytic properties. The combined strategy (N-terminal modification, co-expression with chaperones, mRNA folding energy optimization of signal peptide, and lipoprotein deletion) may also improve the secretory expression of other functional proteins in E. coli.

Exposure assessment and risk-based limit levels evaluation of ochratoxin A in Astragali Radix in China.

Ochratoxin A (OTA) is a mycotoxin found in a variety of foods and herbal medicines, and several governmental bodies around the world have set maximum allowable levels of OTA in different foods and herbal medicines. This study aims to evaluate the health risk of OTA in Astragali Radix (AR) in China, and to evaluate the effects of different limit levels on the risk control of OTA in AR. The concentrations of OTA in 187 samples of AR were investigated, and 61 (32.6%) samples were positive. The mean, 50th and 95th percentile values of OTA in positive samples were 56.2, 5.1 and 304.5 µg/kg, respectively. A margin of exposure (MOE) approach was applied to assess the risk. Considering other food sources, long-term consumers have a relatively high risk of OTA exposure due to the ingestion of AR. Theoretical limit levels of OTA in AR were evaluated from two dimensions by weighing the costs and the benefits. The results indicated that the limit levels that might be applied to the management of OTA contamination in AR in China could be screened out through risk-based evaluation of limit levels.

Neuronal Mitochondria Modulation of LPS-Induced Neuroinflammation.

Neuronal mitochondria dysfunction and neuroinflammation are two prominent pathological features increasingly realized as important pathogenic mechanisms for neurodegenerative diseases. However, little attempt has been taken to investigate the likely interactions between them. Mitofusin2 (Mfn2) is a mitochondrial outer membrane protein regulating mitochondrial fusion, a dynamic process essential for mitochondrial function. To explore the significance of neuronal mitochondria in the regulation of neuroinflammation, male and female transgenic mice with forced overexpression of Mfn2 specifically in neurons were intraperitoneally injected with lipopolysaccharide (LPS), a widely used approach to model neurodegeneration-associated neuroinflammation. Remarkably, LPS-induced lethality was almost completely abrogated in neuronal Mfn2 overexpression mice. Compared with nontransgenic wild-type mice, mice with neuronal Mfn2 overexpression also exhibited alleviated bodyweight loss, behavioral sickness, and myocardial dysfunction. LPS-induced release of IL-1ß but not TNF-α was further found greatly inhibited in the CNS of mice with neuronal Mfn2 overexpression, whereas peripheral inflammatory responses in the blood, heart, lung, and spleen remained unchanged. At the cellular and molecular levels, neuronal Mfn2 suppressed the activation of microglia, prevented LPS-induced mitochondrial fragmentation in neurons, and importantly, upregulated the expression of CX3CL1, a unique chemokine constitutively produced by neurons to suppress microglial activation. Together, these results reveal an unrecognized possible role of neuronal mitochondria in the regulation of microglial activation, and propose neuronal Mfn2 as a likely mechanistic linker between neuronal mitochondria dysfunction and neuroinflammation in neurodegeneration.SIGNIFICANCE STATEMENT Our study suggests that Mfn2 in neurons contributes to the regulation of neuroinflammation. Based on the remarkable suppression of LPS-induced neuroinflammation and neurodegeneration-associated mitochondrial dysfunction and dynamic abnormalities by neuronal Mfn2, this study centered on Mfn2-mediated neuroinflammation reveals novel molecular mechanisms that are involved in both mitochondrial dysfunction and neuroinflammation in neurodegenerative diseases. The pharmacological targeting of Mfn2 may present a novel treatment for neuroinflammation-associated diseases.

Humanized Mice for Infectious and Neurodegenerative disorders.

Humanized mice model human disease and as such are used commonly for research studies of infectious, degenerative and cancer disorders. Recent models also reflect hematopoiesis, natural immunity, neurobiology, and molecular pathways that influence disease pathobiology. A spectrum of immunodeficient mouse strains permit long-lived human progenitor cell engraftments. The presence of both innate and adaptive immunity enables high levels of human hematolymphoid reconstitution with cell susceptibility to a broad range of microbial infections. These mice also facilitate investigations of human pathobiology, natural disease processes and therapeutic efficacy in a broad spectrum of human disorders. However, a bridge between humans and mice requires a complete understanding of pathogen dose, co-morbidities, disease progression, environment, and genetics which can be mirrored in these mice. These must be considered for understanding of microbial susceptibility, prevention, and disease progression. With known common limitations for access to human tissues, evaluation of metabolic and physiological changes and limitations in large animal numbers, studies in mice prove important in planning human clinical trials. To these ends, this review serves to outline how humanized mice can be used in viral and pharmacologic research emphasizing both current and future studies of viral and neurodegenerative diseases. In all, humanized mouse provides cost-effective, high throughput studies of infection or degeneration in natural pathogen host cells, and the ability to test transmission and eradication of disease.

CD4+ effector T cells accelerate Alzheimer's disease in mice.

BACKGROUND: Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by pathological deposition of misfolded self-protein amyloid beta (Aß) which in kind facilitates tau aggregation and neurodegeneration. Neuroinflammation is accepted as a key disease driver caused by innate microglia activation. Recently, adaptive immune alterations have been uncovered that begin early and persist throughout the disease. How these occur and whether they can be harnessed to halt disease progress is unclear. We propose that self-antigens would induct autoreactive effector T cells (Teffs) that drive pro-inflammatory and neurodestructive immunity leading to cognitive impairments. Here, we investigated the role of effector immunity and how it could affect cellular-level disease pathobiology in an AD animal model. METHODS: In this report, we developed and characterized cloned lines of amyloid beta (Aß) reactive type 1 T helper (Th1) and type 17 Th (Th17) cells to study their role in AD pathogenesis. The cellular phenotype and antigen-specificity of Aß-specific Th1 and Th17 clones were confirmed using flow cytometry, immunoblot staining and Aß T cell epitope loaded haplotype-matched major histocompatibility complex II IAb (MHCII-IAb-KLVFFAEDVGSNKGA) tetramer binding. Aß-Th1 and Aß-Th17 clones were adoptively transferred into APP/PS1 double-transgenic mice expressing chimeric mouse/human amyloid precursor protein and mutant human presenilin 1, and the mice were assessed for memory impairments. Finally, blood, spleen, lymph nodes and brain were harvested for immunological, biochemical, and histological analyses. RESULTS: The propagated Aß-Th1 and Aß-Th17 clones were confirmed stable and long-lived. Treatment of APP/PS1 mice with Aß reactive Teffs accelerated memory impairment and systemic inflammation, increased amyloid burden, elevated microglia activation, and exacerbated neuroinflammation. Both Th1 and Th17 Aß-reactive Teffs progressed AD pathology by downregulating anti-inflammatory and immunosuppressive regulatory T cells (Tregs) as recorded in the periphery and within the central nervous system. CONCLUSIONS: These results underscore an important pathological role for CD4+ Teffs in AD progression. We posit that aberrant disease-associated effector T cell immune responses can be controlled. One solution is by Aß reactive Tregs.

Identification of a new amino acid mutation in the HN protein of NDV involved in pathogenicity.

The fusion (F) and haemagglutinin-neuraminidase (HN) proteins of Newcastle disease virus (NDV) are viral entry proteins and are recognized as the major virulence determinants. Previously, a lentogenic NDV virus (CE16) was derived from a mesogenic strain (CI10) through sequential passages in chick embryos. Whole-genome sequence analysis revealed that the two homologous strains shared the same F protein but differed in HN with two amino acid (aa) substitutions (A215G and T430A). To elucidate the molecular reasons for virulence attenuation, two original plasmids (HN-CI10 and HN-CE16) and two single-point mutants (G215A and A430T) reverse-mutated from HN-CE16 were constructed to analyse the known biological functions of HN. The results showed that the A430T substitution significantly weakened the haemadsorption (HAd) activity, increased the neuraminidase (NA) activity, improved the fusion-promoting activity, and enhanced the cleavage-promoting activity of HN-CE16. However, G215A failed to induce obvious functional changes. Therefore, the aa residue HN430 may play a key role in determining virulence. To test this hypothesis, further studies on A430T were conducted through reverse genetics using an infectious cDNA clone. At the viral level, the A430T-mutated virus showed dramatic promotion of viral plaque formation, propagation, and pathogenicity in vitro and in vivo. This study demonstrates a new virulence site associated with HN protein functions, viral propagation, and pathogenicity. All these findings could lay a foundation for illuminating the molecular mechanism of NDV virulence.

C1QBP inhibits proliferation of porcine circovirus type 2 by restricting nuclear import of the capsid protein.

Complement component 1 Q subcomponent-binding protein (C1QBP) has been shown to interact with the porcine circovirus type 2 (PCV2) Cap protein. Here, using yeast two-hybrid (Y2H) and co-immunoprecipitation assays, as well as laser confocal microscopy, the interaction between C1QBP and Cap was confirmed. Furthermore, overexpression of C1QBP in cells altered the intracellular location of Cap, which was observed using confocal microscopy and verified by detection of Cap in nuclear protein extracts in a Western blot assay. By inhibiting nuclear transport of Cap, overexpression of C1QBP downregulated PCV2 proliferation in PK-15 cells, as determined by quantitative polymerase chain reaction (qPCR). As C1QBP plays a similar role in a fusion of green fluorescent protein (GFP) with the Cap nuclear localisation signal (NLS) sequence, (CapNLS-GFP), we propose that the target site for C1QBP in Cap is possibly located in the NLS region. Considering all the results together, this study demonstrated that C1QBP interacts with the Cap NLS region, resulting in changes in the intracellular localisation of the Cap protein. We confirmed that overexpression of C1QBP inhibits the proliferation of PCV2, and this is possibly related to the function of C1QBP in controlling nuclear transport of Cap.