A prion-like trigger of antiviral signaling.

The MAVS protein plays a critical role in the assembly of an antiviral signaling complex on mitochondrial membranes. Hou et al. (2011) now report that virus infection induces a conformational change in MAVS, leading to the prion-like formation of functional self-aggregates that provide a sensitive trigger for antiviral signaling.

Neurodevelopmental deficits and cell-type-specific transcriptomic perturbations in a mouse model of HNRNPU haploinsufficiency.

Heterozygous de novo loss-of-function mutations in the gene expression regulator HNRNPU cause an early-onset developmental and epileptic encephalopathy. To gain insight into pathological mechanisms and lay the potential groundwork for developing targeted therapies, we characterized the neurophysiologic and cell-type-specific transcriptomic consequences of a mouse model of HNRNPU haploinsufficiency. Heterozygous mutants demonstrated global developmental delay, impaired ultrasonic vocalizations, cognitive dysfunction and increased seizure susceptibility, thus modeling aspects of the human disease. Single-cell RNA-sequencing of hippocampal and neocortical cells revealed widespread, yet modest, dysregulation of gene expression across mutant neuronal subtypes. We observed an increased burden of differentially-expressed genes in mutant excitatory neurons of the subiculum-a region of the hippocampus implicated in temporal lobe epilepsy. Evaluation of transcriptomic signature reversal as a therapeutic strategy highlights the potential importance of generating cell-type-specific signatures. Overall, this work provides insight into HNRNPU-mediated disease mechanisms and provides a framework for using single-cell RNA-sequencing to study transcriptional regulators implicated in disease.

ALS- and FTD-associated missense mutations in TBK1 differentially disrupt mitophagy.

TANK-binding kinase 1 (TBK1) is a multifunctional kinase with an essential role in mitophagy, the selective clearance of damaged mitochondria. More than 90 distinct mutations in TBK1 are linked to amyotrophic lateral sclerosis (ALS) and fronto-temporal dementia, including missense mutations that disrupt the abilities of TBK1 to dimerize, associate with the mitophagy receptor optineurin (OPTN), autoactivate, or catalyze phosphorylation. We investigated how ALS-associated mutations in TBK1 affect Parkin-dependent mitophagy using imaging to dissect the molecular mechanisms involved in clearing damaged mitochondria. Some mutations cause severe dysregulation of the pathway, while others induce limited disruption. Mutations that abolish either TBK1 dimerization or kinase activity were insufficient to fully inhibit mitophagy, while mutations that reduced both dimerization and kinase activity were more disruptive. Ultimately, both TBK1 recruitment and OPTN phosphorylation at S177 are necessary for engulfment of damaged mitochondra by autophagosomal membranes. Surprisingly, we find that ULK1 activity contributes to the phosphorylation of OPTN in the presence of either wild-type or kinase-inactive TBK1. In primary neurons, TBK1 mutants induce mitochondrial stress under basal conditions; network stress is exacerbated with further mitochondrial insult. Our study further refines the model for TBK1 function in mitophagy, demonstrating that some ALS-linked mutations likely contribute to disease pathogenesis by inducing mitochondrial stress or inhibiting mitophagic flux. Other TBK1 mutations exhibited much less impact on mitophagy in our assays, suggesting that cell-type-specific effects, cumulative damage, or alternative TBK1-dependent pathways such as innate immunity and inflammation also factor into the development of ALS in affected individuals.

Effects of ALS-associated TANK binding kinase 1 mutations on protein-protein interactions and kinase activity.

Exonic DNA sequence variants in the Tbk1 gene associate with both sporadic and familial amyotrophic lateral sclerosis (ALS). Here, we examine functional defects in 25 missense TBK1 mutations, focusing on kinase activity and protein-protein interactions. We identified kinase domain (KD) mutations that abolish kinase activity or display substrate-specific defects in specific pathways, such as innate immunity and autophagy. By contrast, mutations in the scaffold dimerization domain (SDD) of TBK1 can cause the loss of kinase activity due to structural disruption, despite an intact KD. Familial ALS mutations in ubiquitin-like domain (ULD) or SDD display defects in dimerization; however, a subset retains kinase activity. These observations indicate that TBK1 dimerization is not required for kinase activation. Rather, dimerization seems to increase protein stability and enables efficient kinase-substrate interactions. Our study revealed many aspects of TBK1 activities affected by ALS mutations, highlighting the complexity of disease pathogenicity and providing insights into TBK1 activation mechanism.

Correction of aberration-induced phase errors in phase measuring deflectometry.

Phase measuring deflectometry is a powerful measuring method of complex optical surfaces that captures the reflected fringe images associated with a displaying screen and calculates the normal vectors of the surface under test (SUT) accordingly. The captured images are usually set conjugate to the SUT, which in turn makes the screen defocused. As a result, the blurring effect caused by the defocus and aberrations of the off-axis catadioptric imaging system can severely degrade the phases solved from the blurred images. In order to correct the phase errors, the space-variant point spread functions (PSFs) are modeled using a skew-normal function. The phase bias is estimated by forward convolution between the captured images and the PSF models. Demonstrated with a highly curved aspheric surface, the measurement accuracy can be improved by three times.

Flexible one-shot geometric calibration for off-axis deflectometry.

Off-axis deflectometry is widely applied in the measurement of specular surfaces. However, the measuring accuracy depends on the reliability of geometrical calibration. Existing methods are inconvenient to be utilized due to their disadvantages of low efficiency and operational complexity. A simple geometrical calibration method is proposed by applying a flat mirror with markers, and only one image needs to be captured. A compensation process is introduced to correct the form error of the mirror. Experimental results show that the re-projection errors decrease from 0.319 pixels down to 0.12 pixels; thus the measuring efficiency and accuracy of optical surfaces can be greatly improved.

Efficient phase retrieval of two-directional phase-shifting fringe patterns using geometric constraints of deflectometry.

In the phase measuring deflectometry, two groups of fringe patterns in orthogonal directions are usually applied to establish the correspondences between the pixel pairs on the screen and camera. Usually, 16 phase-shifting fringe patterns with different spatial frequencies are required in order to calculate the absolute phases in the conventional temporal phase unwrapping algorithms. This requirement makes the measurement inefficient and not robust against environmental noise. In this paper, an efficient phase retrieval strategy is developed, which requires only six fringe patterns. The modulating information in one-direction is obtained by first using four fringe patterns, and then it is applied to assist the phase calculation in the other direction, so that only two extra fringe patterns are needed. Subsequently the phases are unwrapped by using the geometric constraints of the software configurable optical test system without additional image acquisition. The measurement time is saved by 5/8, compared to the conventional methods. In this way, the influence of the low-frequency disturbances can be suppressed in the workshop condition. Experiments demonstrate that the proposed method can reliably retrieve the absolute phases, and it is of significance to improve the measuring efficiency and stability of in situ deflectometry.

hnRNP U protein is required for normal pre-mRNA splicing and postnatal heart development and function.

We report that mice lacking the heterogeneous nuclear ribonucleoprotein U (hnRNP U) in the heart develop lethal dilated cardiomyopathy and display numerous defects in cardiac pre-mRNA splicing. Mutant hearts have disorganized cardiomyocytes, impaired contractility, and abnormal excitation-contraction coupling activities. RNA-seq analyses of Hnrnpu mutant hearts revealed extensive defects in alternative splicing of pre-mRNAs encoding proteins known to be critical for normal heart development and function, including Titin and calcium/calmodulin-dependent protein kinase II delta (Camk2d). Loss of hnRNP U expression in cardiomyocytes also leads to aberrant splicing of the pre-mRNA encoding the excitation-contraction coupling component Junctin. We found that the protein product of an alternatively spliced Junctin isoform is N-glycosylated at a specific asparagine site that is required for interactions with specific protein partners. Our findings provide conclusive evidence for the essential role of hnRNP U in heart development and function and in the regulation of alternative splicing.

NLRP2 is a suppressor of NF-ƙB signaling and HLA-C expression in human trophoblasts†,‡.

During pregnancy, fetal extravillous trophoblasts (EVT) play a key role in the regulation of maternal T cell and NK cell responses. EVT display a unique combination of human leukocyte antigens (HLA); EVT do not express HLA-A and HLA-B, but do express HLA-C, HLA-E, and HLA-G. The mechanisms establishing this unique HLA expression pattern have not been fully elucidated. The major histocompatibility complex (MHC) class I and class II transcriptional activators NLRC5 and CIITA are expressed neither by EVT nor by the EVT model cell line JEG3, which has an MHC expression pattern identical to that of EVT. Therefore, other MHC regulators must be present to control HLA-C, HLA-E, and HLA-G expression in these cells. CIITA and NLRC5 are both members of the nucleotide-binding domain, leucine-rich repeat (NLR) family of proteins. Another member of this family, NLRP2, is highly expressed by EVT and JEG3, but not in maternal decidual stromal cells. In this study, transcription activator-like effector nuclease technology was used to delete NLRP2 in JEG3. Furthermore, lentiviral delivery of shRNA was used to knockdown NLRP2 in JEG3 and primary EVT. Upon NLRP2 deletion, Tumor Necrosis Factor-α (TNFα)-induced phosphorylation of NF-KB p65 increased in JEG3 and EVT, and more surprisingly a significant increase in constitutive HLA-C expression was observed in JEG3. These data suggest a broader role for NLR family members in the regulation of MHC expression during inflammation, thus forming a bridge between innate and adaptive immune responses. As suppressor of proinflammatory responses, NLRP2 may contribute to preventing unwanted antifetal responses.

Cardiac glycosides are potent inhibitors of interferon-ß gene expression.

Here we report that bufalin and other cardiac glycoside inhibitors of the sodium-potassium ATPase (sodium pump) potently inhibit the induction of the interferon-ß (IFNß) gene by virus, double-stranded RNA or double-stranded DNA. Cardiac glycosides increase the intracellular sodium concentration, which appears to inhibit the ATPase activity of the RNA sensor RIG-I, an essential and early component in the IFNß activation pathway. This, in turn, prevents the activation of the critical transcription factors IRF3 and NFκB. Bufalin inhibition can be overcome by expressing a drug-resistant variant of the sodium pump and knocking down the pump by short hairpin RNA inhibits IFNß expression. Thus, bufalin acts exclusively through the sodium pump. We also show that bufalin inhibits tumor necrosis factor (TNF) signaling, at least in part by interfering with the nuclear translocation of NFκB. These findings suggest that bufalin could be used to treat inflammatory and autoimmune diseases in which IFN or TNF are hyperactivated.

A modification of nested PCR method for detection of Enterocytozoon hepatopenaei (EHP) in giant freshwater prawn Macrobrachium rosenbergii.

The microsporidian Enterocytozoon hepatopenaei (EHP) has become a critical threat to the global shrimp aquaculture industry, thus necessitating early detection by screening. Development of a rapid and accurate assay is crucial both for the active surveillance and for the assessment of shrimp with EHP infection. In the present study, a distinct strain of E. hepatopenaei (EHP Mr ) was found in Macrobrachium rosenbergii. The SWP1 gene analysis revealed it was a new genotype that differed with the common strain isolated from the Litopenaeus vannamei (EHP Lv ). A nested SWP-PCR method was modified to fix the bug that the original inner primers could not recognize the EHP Mr strain. The redesigned inner primers successfully amplified a product of 182 bp for both the EHP Mr strain and the EHP Lv strain. The new primers also had good specificity and high sensitivity, which may serve as an alternative for EHP genotyping. This study provided a method for detection of EHP in the biosecurity of Macrobrachium rosenbergii farming, and the developed protocol was proposed for the routine investigation and potential carrier screening, especially for molecular epidemiology.

The Loss of TBK1 Kinase Activity in Motor Neurons or in All Cell Types Differentially Impacts ALS Disease Progression in SOD1 Mice.

DNA sequence variants in the TBK1 gene associate with or cause sporadic or familial amyotrophic lateral sclerosis (ALS). Here we show that mice bearing human ALS-associated TBK1 missense loss-of-function mutations, or mice in which the Tbk1 gene is selectively deleted in motor neurons, do not display a neurodegenerative disease phenotype. However, loss of TBK1 function in motor neurons of the SOD1G93A mouse model of ALS impairs autophagy, increases SOD1 aggregation, and accelerates early disease onset without affecting lifespan. By contrast, point mutations that decrease TBK1 kinase activity in all cells also accelerate disease onset but extend the lifespan of SOD1 mice. This difference correlates with the failure to activate high levels of expression of interferon-inducible genes in glia. We conclude that loss of TBK1 kinase activity impacts ALS disease progression through distinct pathways in different spinal cord cell types and further implicate the importance of glia in neurodegeneration.

Inborn errors of type I IFN immunity in patients with life-threatening COVID-19.

Clinical outcome upon infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ranges from silent infection to lethal coronavirus disease 2019 (COVID-19). We have found an enrichment in rare variants predicted to be loss-of-function (LOF) at the 13 human loci known to govern Toll-like receptor 3 (TLR3)- and interferon regulatory factor 7 (IRF7)-dependent type I interferon (IFN) immunity to influenza virus in 659 patients with life-threatening COVID-19 pneumonia relative to 534 subjects with asymptomatic or benign infection. By testing these and other rare variants at these 13 loci, we experimentally defined LOF variants underlying autosomal-recessive or autosomal-dominant deficiencies in 23 patients (3.5%) 17 to 77 years of age. We show that human fibroblasts with mutations affecting this circuit are vulnerable to SARS-CoV-2. Inborn errors of TLR3- and IRF7-dependent type I IFN immunity can underlie life-threatening COVID-19 pneumonia in patients with no prior severe infection.

Chromatin structure and transcription of the R1- and R2-inserted rRNA genes of Drosophila melanogaster.

About half of the rRNA gene units (rDNA units) of Drosophila melanogaster are inserted by the retrotransposable elements R1 and R2. Because transcripts to R1 and R2 were difficult to detect on blots and electron microscopic observations of rRNA synthesis suggested that only uninserted rDNA units were transcribed, it has long been postulated that inserted rDNA units are in a repressed (inactive) chromatin structure. Studies described here suggest that inserted and uninserted units are equally accessible to DNase I and micrococcal nuclease and contain similar levels of histone H3 and H4 acetylation and H3K9 methylation. These studies have low sensitivity, because psoralen cross-linking suggested few (estimated <10%) of the rDNA units of any type are transcriptionally active. Nuclear run-on experiments revealed that R1-inserted and R2-inserted units are activated for transcription at about 1/5 and 1/10, respectively, the rate of uninserted units. Most transcription complexes of the inserted units terminate within the elements, thus explaining why previous molecular and electron microscopic methods indicated inserted units are seldom transcribed. The accumulating data suggest that all units within small regions of the rDNA loci are activated for transcription, with most control over R1 and R2 activity involving steps downstream of transcription initiation.

[Effect of titanium particles on proliferation, differentiation, and cytomorphology of osteoblasts].

OBJECTIVE: To study the effect of titanium particles on the proliferation, differentiation, and cytomorphology of osteoblasts, and to explore the possible internal relations and mechanism. METHODS: Calvarial osteoblasts were separated from 10 newborn Sprague Dawley rats by repeated enzyme digestion, and were cultured in vitro. The cells were identified by alkaline phosphatase (ALP) staining and alizarin red staining. The cells at passage 3 were cultured with titanium particles culture medium at concentrations of 0.01, 0.05, 0.1, 0.5, and 1 mg/mL (0.01, 0.05, 0.1, 0.5, and 1 mg/mL groups). The absorbance (A) values were detected by cell counting kit 8 at 7 days after cultured to compare the effect of titanium particles at different concentrations on proliferation, and median lethal concentration was screened out. The expression of collagen type I was detected by ELISA to observe the effect of titanium particles on differentiation. The osteoblasts co-cultured with titanium particles of median lethal concentration (experimental group) for 7 days, and double fluorescence staining with FITC-phalloidine and propidium iodide was performed. The cytomorphology variation of osteoblasts after swallowing titanium particles was observed under laser scanning confocal microscope. The osteoblasts at passage 3 cultured with culture medium without titanium particles served as control group. RESULTS: The cultured cells were identified as osteoblasts by ALP staining and alizarin red staining. Different concentrations of titanium particles could inhibit osteoblasts proliferation and differentiation in varying degrees, showing significant difference when compared with the control group at 7 days after culture (P < 0.05). The cell proliferation and differentiation were decreased with increased titanium particles concentration; significant differences were found between the other groups (P < 0.05) except 0.01 and 0.05 mg/mL groups (P > 0.05). The median lethal concentration of titanium particles was 0.5 mg/mL. Laser scanning confocal microscope showed cellular shrinking, microfilaments distortion, pseudopodia contraction of osteoblasts that swallowed titanium particles in the experimental group. CONCLUSION: Titanium particles can inhibit proliferation and differentiation of osteoblasts. The effect may be related to variation of cytomorphology after swallowing titanium particles.

Negative regulation of interferon-ß gene expression during acute and persistent virus infections.

The production of type I interferons (IFNs) in response to viral infections is critical for antiviral immunity. However, IFN production is transient, and continued expression can lead to inflammatory or autoimmune diseases. Thus, understanding the mechanisms underlying the negative regulation of IFN expression could lead to the development of novel therapeutic approaches to the treatment of these diseases. We report that the transcription factor IRF3 plays a central role in the negative regulation of interferon-ß (IFNß) expression during both acute and persistent (chronic) virus infections. We show that the degradation of IRF3 during acute infections, rather than the activation of transcriptional repressors, leads to the down regulation of IFNß expression. We also show that the block to IFNß expression in mouse embryonic fibroblasts that are persistently infected with Sendai virus (SeV) correlates with the absence of transcriptionally active IRF3. Remarkably, ongoing protein synthesis and viral replication are required to maintain repression of the IFNß gene in persistently infected cells, as the gene can be activated by the protein synthesis inhibitor cycloheximide, or by the antiviral drug ribavirin. Finally, we show that the SeV V protein inhibits IRF3 activity in persistently infected cells. Thus, in conjunction with the known interference with STAT1 by the SeV C protein, both IFN activation and its signaling pathways are blocked in persistently infected cells. We conclude that the transcription factor IRF3 is targeted for turnover and inactivation through distinct mechanisms from both the host cells and virus, leading to the inhibition of IFNß gene expression during acute and persistent viral infections. These observations show that IRF3 plays a critical role, not only in the activation of the IFNß gene, but also in the controlling the duration of its expression. (284 words).

Epigenetic regulation of retrotransposons within the nucleolus of Drosophila.

R2 retrotransposable elements exclusively insert into a conserved region of the tandemly organized 28S rRNA genes. Despite inactivating a subset of these genes, R2 elements have persisted in the ribosomal DNA (rDNA) loci of insects for hundreds of millions of years. Controlling R2 proliferation was addressed in this study using lines of Drosophila simulans previously shown to have either active or inactive R2 retrotransposition. Lines with active retrotransposition were shown to have high R2 transcript levels, which nuclear run-on transcription experiments revealed were due to increased transcription of R2-inserted genes. Crosses between R2 active and inactive lines indicated that an important component of this transcriptional control is linked to or near the rDNA locus, with the R2 transcription level of the inactive parent being dominant. Pulsed-field gel analysis suggested that the R2 active and inactive states were determined by R2 distribution within the locus. Molecular and cytological analyses further suggested that the entire rDNA locus from the active line can be silenced in favor of the locus from the inactive line. This silencing of entire rDNA loci represents an example of the large-scale epigenetic control of transposable elements and shares features with the nucleolar dominance frequently seen in interspecies hybrids.

RNA from the 5' end of the R2 retrotransposon controls R2 protein binding to and cleavage of its DNA target site.

Non-LTR retrotransposons insert into eukaryotic genomes by target-primed reverse transcription (TPRT), a process in which cleaved DNA targets are used to prime reverse transcription of the element's RNA transcript. Many of the steps in the integration pathway of these elements can be characterized in vitro for the R2 element because of the rigid sequence specificity of R2 for both its DNA target and its RNA template. R2 retrotransposition involves identical subunits of the R2 protein bound to different DNA sequences upstream and downstream of the insertion site. The key determinant regulating which DNA-binding conformation the protein adopts was found to be a 320-nt RNA sequence from near the 5' end of the R2 element. In the absence of this 5' RNA the R2 protein binds DNA sequences upstream of the insertion site, cleaves the first DNA strand, and conducts TPRT when RNA containing the 3' untranslated region of the R2 transcript is present. In the presence of the 320-nt 5' RNA, the R2 protein binds DNA sequences downstream of the insertion site. Cleavage of the second DNA strand by the downstream subunit does not appear to occur until after the 5' RNA is removed from this subunit. We postulate that the removal of the 5' RNA normally occurs during reverse transcription, and thus provides a critical temporal link to first- and second-strand DNA cleavage in the R2 retrotransposition reaction.

[Comparison of internal fixation treatment of tibia intercondylar eminence fracture between absorbable screw and metallic screw].

OBJECTIVE: To compare advantage and disadvantage of internal fixation method for tibia intercondylar eminence fracture between absorbable screw and metallic screw. METHODS: From 1996 to 2002, 200 patients with fracture of tibia intercondylar eminence were divided into group A (with absorbable screw, n = 120) and group B (with metallic screw, n = 80). And the biological compatibility, biomechanics, bone union and complications were compared between two groups. RESULTS: There were no obvious differences in operating time and circumstance of recovery position between two groups. Group A was obviously better than group B in biological compatibility, biomechanics, bone union, joint function recovery. The average healing time of group A was three months, that of group B was three and half months. The good rates of joint function in two groups were 98.0% and 95.0% respectively. The occurrence rates of wound arthritis were 1.7% and 3.7%. There was significant difference between them (P < 0.01). CONCLUSION: Absorbable screw is a more ideal material of internal fixation to treat tibia intercondylar eminence fracture.

R2 retrotransposition on assembled nucleosomes depends on the translational position of the target site.

R2 retrotransposons insert into the 28S rRNA genes of insects. Integration occurs by specific cleavage of the target site and utilization of the released DNA end to prime reverse transcription of the RNA transcript. Specificity of the protein to the target site is dependent upon nucleotide sequence recognition extending from 35 bp upstream to 15 bp downstream of the cleavage site. In this report, we show that sequence recognition and cleavage by the R2 protein can occur while the target site is assembled into nucleosomes. Reconstitution of DNA fragments containing the 28S gene sequence into a set of nucleosomes with different translational frames revealed that the R2 site adopted the same rotational orientation with respect to the histone octamer. Binding and cleavage by the R2 protein were most efficient when the upstream binding site for the R2 protein was near a nucleosome end. Interaction of the R2 protein with the nucleosome disrupted the histone:DNA contacts in the 50 bp region directly bound by R2, but did not modify the remainder of the nucleosome structure.