The distinct binding properties between avian/human influenza A virus NS1 and Postsynaptic density protein-95 (PSD-95), and inhibition of nitric oxide production

<p>Abstract</p> <p>Background</p> <p>The NS1 protein of influenza A virus is able to bind with many proteins that affect cellular signal transduction and protein synthesis in infected cells. The NS1 protein consists of approximately 230 amino acids and the last 4 amino acids of the NS1 C-terminal form a PDZ binding motif. Postsynaptic Density Protein-95 (PSD-95), which is mainly expressed in neurons, has 3 PDZ domains. We hypothesise that NS1 binds to PSD-95, and this binding is able to affect neuronal function.</p> <p>Result</p> <p>We conducted a yeast two-hybrid analysis, GST-pull down assays and co-immunoprecipitations to detect the interaction between NS1 and PSD-95. The results showed that NS1 of avian influenza virus H5N1 (A/chicken/Guangdong/1/2005) is able to bind to PSD-95, whereas NS1 of human influenza virus H1N1 (A/Shantou/169/2006) is unable to do so. The results also revealed that NS1 of H5N1 significantly reduces the production of nitric oxide (NO) in rat hippocampal neurons.</p> <p>Conclusion</p> <p>In summary, our study indicates that NS1 of influenza A virus can bind with neuronal PSD-95, and the avian H5N1 and human H1N1 influenza A viruses possess distinct binding properties.</p

Induction of cytopathic effect and cytokines in coxsackievirus B3-infected murine astrocytes

BACKGROUND: Coxsackievirus commonly infects children and occasionally causes severe meningitis and/or encephalitis in the newborn. The underlying mechanism(s) behind the central nervous system pathology is poorly defined. METHODS: It is hypothesized that astrocytes may be involved in inflammatory response induced by CVB3 infection. Here we discuss this hypothesis in the context of CVB3 infection and associated inflammatory response in primary mouse astrocytes. RESULTS: The results showed that coxsackievirus receptor (CAR) was distributed homogeneously on the astrocytes, and that CVB3 could infect and replicate in astrocytes, with release of infectious virus particles. CVB3 induced cytopathic effect and production of proinflammatory cytokines IL-1β, TNF-α, IL-6, and chemokine CXCL10 from astrocytes. CONCLUSION: These data suggest that direct astrocyte damage and cytokines induction could be a mechanism of virus-induced meningitis and/or encephalitis

Liquid-Liquid Phase Separation Primes Spider Silk Proteins for Fiber Formation via a Conditional Sticker Domain

Many protein condensates can convert to fibrillar aggregates, but the underlying mechanisms are unclear. Liquid-liquid phase separation (LLPS) of spider silk proteins, spidroins, suggests a regulatory switch between both states. Here, we combine microscopy and native mass spectrometry to investigate the influence of protein sequence, ions, and regulatory domains on spidroin LLPS. We find that salting out-effects drive LLPS via low-affinity stickers in the repeat domains. Interestingly, conditions that enable LLPS simultaneously cause dissociation of the dimeric C-terminal domain (CTD), priming it for aggregation. Since the CTD enhances LLPS of spidroins but is also required for their conversion into amyloid-like fibers, we expand the stickers and spacers-model of phase separation with the concept of folded domains as conditional stickers that represent regulatory units

High yield production of amyloid-ß peptide enabled by a customized spider silk domain

During storage in the silk gland, the N-terminal domain (NT) of spider silk proteins (spidroins) keeps the aggregation-prone repetitive region in solution at extreme concentrations. We observe that NTs from different spidroins have co-evolved with their respective repeat region, and now use an NT that is distantly related to previously used NTs, for efcient recombinant production of the amyloid-β peptide (Aβ) implicated in Alzheimer’s disease. A designed variant of NT from Nephila clavipes fagelliform spidroin, which in nature allows production and storage of β-hairpin repeat segments, gives exceptionally high yields of diferent human Aβ variants as a solubility tag. This tool enables efficient production of target peptides also in minimal medium and gives up to 10 times more isotope-labeled monomeric Aβ peptides per liter bacterial culture than previously reported

Spidroin N-terminal domain forms amyloid-like fibril based hydrogels and provides a protein immobilization platform

Recombinant spider silks are of interest but the multimodal and aggregation-prone nature of them is a limitation. Here, the authors report on a miniature spidroin based on the N-terminal domain which forms a hydrogel at 37 degrees C which allows for ease of production and fusion protein modification to generate functional biomaterials.Recombinant spider silk proteins (spidroins) have multiple potential applications in development of novel biomaterials, but their multimodal and aggregation-prone nature have complicated production and straightforward applications. Here, we report that recombinant miniature spidroins, and importantly also the N-terminal domain (NT) on its own, rapidly form self-supporting and transparent hydrogels at 37 degrees C. The gelation is caused by NT alpha-helix to beta-sheet conversion and formation of amyloid-like fibrils, and fusion proteins composed of NT and green fluorescent protein or purine nucleoside phosphorylase form hydrogels with intact functions of the fusion moieties. Our findings demonstrate that recombinant NT and fusion proteins give high expression yields and bestow attractive properties to hydrogels, e.g., transparency, cross-linker free gelation and straightforward immobilization of active proteins at high density

Lu-177-PSMA dosimetry for kidneys and tumors based on SPECT images at two imaging time points

BackgroundPersonalized dosimetry for Lu-177-PSMA treatment requires multiple-time-point SPECT/CT scans to calculate time-integrated activity (TIA). This study evaluates two-time-point (TTP) methods for TIA calculation for kidneys and tumors.MethodsA total of 18 patients treated with 3.7-7.4 GBq Lu-177 PSMA-617 were analyzed retrospectively, including 18 sets of left and right kidneys, as well as 45 tumors. Four quantitative SPECT/CT (4TP) were acquired at 2 h, 20 h, 40 h, 60 h (n = 11), or 200 h (n = 7) after treatment, and they were fit bi-exponentially as reference. The TTP method was fitted by a mono-exponential washout function using two selected imaging time points for kidneys. For tumors, one uptake and one washout phase were modeled, assuming linear (type I) and same (type II) uptake phase between 0 h to the first time point and mono-exponential washout thereafter. Two single-time-point (STP) methods were also implemented for comparison. TIA calculated by TTP and STP methods were compared with reference to the 4TP TIA.ResultsFor the kidneys, the TTP methods using 20 h-60 h and 40 h-200 h had smaller mean absolute errors of 8.05 ± 6.05% and 4.95 ± 3.98%, respectively, as compared to other combinations of time points and STP methods. For tumors, the type I and type II TTP methods using 20h−60 h and 40–200 h had smaller mean absolute errors of 6.14 ± 5.19% and 12.22 ± 4.44%, and 8.31 ± 7.16% and 4.48 ± 7.10%, respectively, as compared to other TTP and STP methods.ConclusionThe TTP methods based on later imaging time demonstrated fewer errors than the STP methods in kidney and tumor TIA. Imaging at 20 h−60 h and 40 h−200 h could simplify the dosimetry procedures with fewer TIA estimation errors

Transforming growth factor-β1 protects mechanically injured cortical murine neurons by reducing trauma-induced autophagy and apoptosis

Transforming growth factor β1 (TGF-β1) has a neuroprotective function in traumatic brain injury (TBI) through its anti-inflammatory and immunomodulatory properties. However, the precise mechanisms underlying the neuroprotective actions of TGF-β1 on the cortex require further investigation. In this study, we were aimed to investigate the regulatory function of TGF-β1 on neuronal autophagy and apoptosis using an in vitro primary cortical neuron trauma-injury model. LDH activity was assayed to measure cell viability, and intracellular [Ca2+] was measured using Fluo-4-AM in an in vitro primary cortical neuron trauma-injury model. RNA-sequencing (RNAseq), immunofluorescent staining, transmission electron microscopy (TEM), western blot and CTSD activity detection were employed. We observed significant enrichment of DEGs related to autophagy, apoptosis, and the lysosome pathway in trauma-injured cortical neurons. TEM confirmed the presence of autophagosomes as well as autophagolysosomes. Western blot revealed upregulation of autophagy-related protein light chain 3 (LC3-II/LC3-I), sequestosome 1 (SQSTM1/p62), along with apoptosis-related protein cleaved-caspase 3 in trauma-injured primary cortical neurons. Furthermore, trauma-injured cortical neurons showed an upregulation of lysosomal marker protein (LAMP1) and lysosomal enzyme mature cathepsin D (mCTSD), but a decrease in the activity of CTSD enzyme. These results indicated that apoptosis was up-regulated in trauma- injured cortical neurons at 24 h, accompanied by lysosomal dysfunction and impaired autophagic flux. Notably, TGF-β1 significantly reversed these changes. Our results suggested that TGF-β1 exerted neuroprotective effects on trauma- injured cortical neurons by reducing lysosomal dysfunction, decreasing the accumulation of autophagosomes and autophagolysosomes, and enhancing autophagic flux

Non-invasive prediction of preeclampsia using the maternal plasma cell-free DNA profile and clinical risk factors

BackgroundPreeclampsia (PE) is a pregnancy complication defined by new onset hypertension and proteinuria or other maternal organ damage after 20 weeks of gestation. Although non-invasive prenatal testing (NIPT) has been widely used to detect fetal chromosomal abnormalities during pregnancy, its performance in combination with maternal risk factors to screen for PE has not been extensively validated. Our aim was to develop and validate classifiers that predict early- or late-onset PE using the maternal plasma cell-free DNA (cfDNA) profile and clinical risk factors.MethodsWe retrospectively collected and analyzed NIPT data of 2,727 pregnant women aged 24–45 years from four hospitals in China, which had previously been used to screen for fetal aneuploidy at 12 + 0 ~ 22 + 6 weeks of gestation. According to the diagnostic criteria for PE and the time of diagnosis (34 weeks of gestation), a total of 143 early-, 580 late-onset PE samples and 2,004 healthy controls were included. The wilcoxon rank sum test was used to identify the cfDNA profile for PE prediction. The Fisher’s exact test and Mann–Whitney U-test were used to compare categorical and continuous variables of clinical risk factors between PE samples and healthy controls, respectively. Machine learning methods were performed to develop and validate PE classifiers based on the cfDNA profile and clinical risk factors.ResultsBy using NIPT data to analyze cfDNA coverages in promoter regions, we found the cfDNA profile, which was differential cfDNA coverages in gene promoter regions between PE and healthy controls, could be used to predict early- and late-onset PE. Maternal age, body mass index, parity, past medical histories and method of conception were significantly differential between PE and healthy pregnant women. With a false positive rate of 10%, the classifiers based on the combination of the cfDNA profile and clinical risk factors predicted early- and late-onset PE in four datasets with an average accuracy of 89 and 80% and an average sensitivity of 63 and 48%, respectively.ConclusionIncorporating cfDNA profiles in classifiers might reduce performance variations in PE models based only on clinical risk factors, potentially expanding the application of NIPT in PE screening in the future