Liquid-assisted mechanochemical synthesis, crystallographic, theoretical and molecular docking study on HIV instasome of novel copper complexes: (µ-acetato)-bis(2,2'-bipyridine)-copper and bromidotetrakis(2-methyl-1H-imidazole)-copper bromide.

In the present work the two new Cu(II) complexes, (µ-acetato)-bis(2,2'-bipyridine)-copper [Cu(bpy)2(CH3CO2)] and bromidotetrakis(2-methyl-1H-imidazole)-copper bromide [Cu(2-methylimid)4Br]Br have been synthesized by liquid assisted mechanochemical method. The [Cu(bpy)2(CH3CO2)] complex (1) and [Cu(2-methylimid)4Br]Br complex (2) characterised by IR and UV-visible spectroscopy and the structure are confirmed by XRD diffraction studies. Complex (1) crystallized in the Monoclinic with the space group of C2/c where a = 24.312(5) Å, b = 8.5892(18) Å, c = 14.559(3) Å, α = 90°, ß = 106.177(7)° and γ = 90° and Complex (2) crystallized in the Tetragonal with the space group of P4nc, a = 9.9259(2) Å, b = 9.9259(2) Å, c = 10.9357(2) Å, α = 90°, ß = 90° and γ = 90°. The complex (1) has distorted octahedral geometry where the acetate ligand showed bidentate bridging with the central metal ion and complex (2) has slightly deformed square pyramidal geometry. The HOMO-LUMO energy gap value and the low chemical potential showed that the complex (2) is stable and difficult to polarize compare to complex (1). The molecular docking study of complexes with the HIV instasome nucleoprotein showed the binding energy values - 7.1 and - 5.3 kcal/mol for complex (1) and complex (2) respectively. The negative binding energy values showed the complexes have affinity to bind with HIV instasome nucleoproteins. The in-silico pharmacokinetic study of the complex (1) and complex (2) showed non AMES toxicity, non-carcinogens and low honey Bee toxicity but weakly inhibit Human Ether-a-go-go-related gene.

Thermal inactivation of COVID-19 specimens improves RNA quality and quantity.

The rapid spread of coronavirus disease 2019 (COVID-19), a disease caused by severe acute respiratory syndrome coronavirus 2, poses a huge demand for immediate diagnosis. Real-time reverse transcriptase-polymerase chain reaction (rRT-PCR) of nasopharyngeal (NP) and oropharyngeal (OP) swabs have been used to confirm the clinical diagnosis. To avoid the risk of viral-exposure of laboratory workers, thermal inactivation is currently recommended but has unknown effects on the accuracy of the rRT-PCR results. Thirty-six NP/OP specimens were collected from COVID-19 patients and subjected to thermal inactivation (60°C for 30 min) or the RNA extraction processes to activate the form. Here, our data showed that the concentration of extracted-RNA increases upon thermal inactivation compared to the active form (p = .028).  Significantly higher levels of RNA copy number were obtained in inactivated compared to the active samples for both N and ORF1ab genes (p = .009, p = .032, respectively). Thermal inactivation elevated concentration and copy number of extracted-RNA, possibly through viral-capsid degradation and/or nucleoprotein denaturation.

Modeling Borna Disease Virus In Vitro Spread Reveals the Mode of Antiviral Effect Conferred by an Endogenous Bornavirus-Like Element.

Endogenous retroviruses have demonstrated exaptation during long-term evolution with hosts, e.g., resulting in acquisition of antiviral effect on related extant viral infections. While empirical studies have found that an endogenous bornavirus-like element derived from viral nucleoprotein (itEBLN) in the ground squirrel genome shows antiviral effect on virus replication and de novo infection, the antiviral mechanism, dynamics, and quantitative effect of itEBLN remain unknown. In this study, we experimentally and theoretically investigated the dynamics of how an extant bornavirus, Borna disease virus 1 (BoDV-1), spreads and replicates in uninfected, BoDV-1-infected, and itEBLN-expressing cultured cells. Quantifying antiviral effect based on time course data sets, we found that the antiviral effects of itEBLN are estimated to be 75% and 34% on intercellular virus spread and intracellular virus replication, respectively. This discrepancy between intercellular virus spread and intracellular viral replication suggests that viral processes other than the replication of viral ribonucleoprotein complex (RNP) contributed to the suppression of virus spread in itEBLN-expressing cells. Because itEBLN binds to the BoDV-1 RNP, the suppression of viral RNP trafficking can be an attractive candidate explaining this discrepancy.IMPORTANCE Accumulating evidence suggests that some endogenous viral elements (EVEs), including endogenous retroviruses and endogenous nonretroviral virus elements, have acquired functions in the host as a result of long-term coevolution. Recently, an endogenous bornavirus-like element (itEBLN) found in the ground squirrel genome has been shown to have antiviral activity against exogenous bornavirus infection. In this study, we first quantified bornavirus spread in cultured cells and then calculated the antiviral activity of itEBLN on bornavirus infection. The calculated antiviral activity of itEBLN suggests its suppression of multiple processes in the viral life cycle. To our knowledge, this is the first study quantifying the antiviral activity of EVEs and speculating on a model of how some EVEs have acquired antiviral activity during host-virus arms races.

Structural Homology Between Nucleoproteins of ssRNA Viruses.

Our understanding of the viral world changed just after the first structures of icosahedral viral particles were unveiled. The structural similarities between capsid proteins of distant viral groups were not anticipated, and the findings suggested the existence of common ancestors for viruses with different host range, genomic structure and multiplication strategies. This way, diverse viruses with icosahedral particles can now be grouped based on the structural homology between their capsid proteins. In the last years, the presence of conserved folds between viral proteins in non-icosahedral viruses has also emerged. Viral particles with radically different morphologies, ranging from naked and filamentous to enveloped and pleomorphic, have shown structural homology between the nucleoproteins that bind directly to their genomes. This chapter overviews recent findings regarding the similar structure found between nucleoproteins of eukaryotic ssRNA viruses. The structural homology includes the coat proteins from all known families of flexible filamentous plant viruses, a group with monopartite (+)ssRNA genomes. Their coat proteins share a core domain with nucleoproteins of previously unrelated families of enveloped viruses that have segmented (-)ssRNA genomes. This last group consists of mostly animals viruses, including influenza virus.

Programed Assembly of Nucleoprotein Nanoparticles Using DNA and Zinc Fingers for Targeted Protein Delivery.

With a growing number of intracellular drug targets and the high efficacy of protein therapeutics, the targeted delivery of active proteins with negligible toxicity is a challenging issue in the field of precision medicine. Herein, a programed assembly of nucleoprotein nanoparticles (NNPs) using DNA and zinc fingers (ZnFs) for targeted protein delivery is presented. Two types of ZnFs with different sequence specificities are genetically fused to a targeting moiety and a protein cargo, respectively. Double-stranded DNA with multiple ZnF-binding sequences is grafted onto inorganic nanoparticles, followed by conjugation with the ZnF-fused proteins, generating the assembly of NNPs with a uniform size distribution and high stability. The approach enables controlled loading of a protein cargo on the NNPs, offering a high cytosolic delivery efficiency and target specificity. The utility and potential of the assembly as a versatile protein delivery vehicle is demonstrated based on their remarkable antitumor activity and target specificity with negligible toxicity in a xenograft mice model.

Establishment of a proteome profile and identification of molecular markers for mouse spermatogonial stem cells.

Spermatogonial stem cells (SSCs) are undifferentiated cells that are required to maintain spermatogenesis throughout the reproductive life of mammals. Although SSC transplantation and culture provide a powerful tool to identify the mechanisms regulating SSC function, the precise signalling mechanisms governing SSC self-renewal and specific surface markers for purifying SSCs remain to be clearly determined. In the present study, we established a steady SSC culture according to the method described by Shinohara's lab. Fertile progeny was produced after transplantation of cultured SSCs into infertile mouse testis, and the red fluorescence exhibited by the culture cell membranes was stably and continuously transmitted to the offspring. Next, via advanced mass spectrometry and an optimized proteomics platform, we constructed the proteome profile, with 682 proteins expressed in SSCs. Furthermore bioinformatics analysis showed that the list contained several known molecules that are regulated in SSCs. Several nucleoproteins and membrane proteins were chosen for further exploration using immunofluorescence and RT-PCR. The results showed that SALL1, EZH2, and RCOR2 are possibly involved in the self-renewal mechanism of SSCs. Furthermore, the results of tissue-specific expression analysis showed that Gpat2 and Pld6 were uniquely and highly expressed in mouse testes and cultured SSCs. The cellular localization of PLD6 was further explored and the results showed it was primarily expressed in the spermatogonial membrane of mouse testes and cultured SSCs. The proteins identified in this study form the basis for further exploring the molecular mechanism of self-renewal in SSCs and for identifying specific surface markers of SSCs.

Effect of Placental Derived Nucleoproteins on liver regeneration in DEN-induced liver fibrosis model.

BACKGROUND: Placental Derived Nucleoproteins (PDNs) is commonly associated with the process of angiogenesis, and doesn't affect the healthy vasculature. PDNs are clinically estimated for the treatment of cancer cases and severe hepatic injuries. Thus, the pathophysiological effects of PDNs targeting liver fibrosis is a concern. OBJECTIVES: To assess the molecular, histopathological, and chemical impact of PDNs on liver regeneration in Diethylnitrosamine (DEN)-induced mice liver fibrosis. METHODS: Normal untreated reference group of ten mice and two groups of induced liver cirrhosis using the recommended weekly dose of Diethylnitrosamine in total of eleven doses, initially 20 mg/kg body weight, and then 30 mg/kg in the third week, followed by 50 mg/kg for the last eight weeks, one of them combined treatment aligned with injection with total dose of extracted PDNs 25 mg/kg, in comparison to PDNs only treated group. An autopsy was performed after 22 weeks of the initial dose of DEN in each group. Molecular characterization of Alpha smooth muscle actin, TGFß and NF-κB biomarkers for liver then liver function panel were analyzed and finally hepatopathological changes were observed using H&E stain and Sirius red stain. RESULTS: Liver enzymes, total bilirubin and total proteins in tissue in PDNs-DEN treated models were controlled in the direction of normal group and 50 % reduction of fibrosis in comparing to DEN-treated models. The cellular arrangement of fibrosis in the DEN entire groups were differentiated with high significant impact on the survival of mice. Increased levels of the biochemical markers in liver homogenate, loss of tissue architecture, and proliferation were observed in induced groups and down regulation of alpha smooth muscle actin, TGFß and NF-κB. CONCLUSION: This finding demonstrates an improvement of Liver tissue induced fibrosis using DEN combined with PDNs. This strategy is to generate an animal model with a lower occurrence of fibrosis in a short time treatment regarding liver regeneration.

Evaluation of the prophylactic and therapeutic efficacies of mucus and tissue nucleoproteins extracted from Biomphalaria alexandrina snails on schistosomiasis mansoni.

Schistosomiasis is a neglected tropical disease with considerable morbidity. The lone effective drug, praziquantel (PZQ), is showing emergence of drug resistance hence, searching for new supportive treatment is crucial. This study aimed to evaluate the efficacy of mucus and nucleoproteins (NPs) extracted from Biomphalaria alexandrina (B. alexandrina) snails on miracidia, cercariae and Schistosoma mansoni (S. mansoni) adults in vitro and assess their experimental in vivo effect through parasitological, histopathological, and biochemical parameters. The in vivo study included 90 male Swiss albino mice. Mice were grouped into 9 groups; G1-G5 were infected and treated with; GI: PZQ, GII: mucus, GIII: combined PZQ and mucus, GIV: NPs, GV: combined PZQ and NPs. Control groups; C1: Non infected non treated (negative control), C2: Infected non treated (positive control), C3: Non infected mucus treated and C4: Non infected NPs treated. The in vitro study proved that the mucus had a better lethal effect on cercariae than miracidia, while NPs had better lethal effect on miracidia. The mucus lethal effect on adults surpassed the NPs as 100% and 60%, respectively. The in vivo study proved that the combined NPs or mucus with PZQ added to the effect of individual PZQ resulting in 100% total worm burden (TWB) reduction. As regard oxidative stress markers, the lowest level of nitric oxide (NO) was shown with combined PZQ and NPs. While, the highest glutathione (GSH) level was produced by individual PZQ. The study concluded that mucus and NPs of B. alexandrina had cercaricidal, miracidicidal and anti-schistosomal effect in vitro and that their combination could be considered a contribution to PZQ potentiality in vivo.

Structural studies of protein-nucleic acid complexes: A brief overview of the selected techniques.

Protein-nucleic acid complexes are involved in all vital processes, including replication, transcription, translation, regulation of gene expression and cell metabolism. Knowledge of the biological functions and molecular mechanisms beyond the activity of the macromolecular complexes can be determined from their tertiary structures. Undoubtably, performing structural studies of protein-nucleic acid complexes is challenging, mainly because these types of complexes are often unstable. In addition, their individual components may display extremely different surface charges, causing the complexes to precipitate at higher concentrations used in many structural studies. Due to the variety of protein-nucleic acid complexes and their different biophysical properties, no simple and universal guideline exists that helps scientists chose a method to successfully determine the structure of a specific protein-nucleic acid complex. In this review, we provide a summary of the following experimental methods, which can be applied to study the structures of protein-nucleic acid complexes: X-ray and neutron crystallography, nuclear magnetic resonance (NMR) spectroscopy, cryogenic electron microscopy (cryo-EM), atomic force microscopy (AFM), small angle scattering (SAS) methods, circular dichroism (CD) and infrared (IR) spectroscopy. Each method is discussed regarding its historical context, advancements over the past decades and recent years, and weaknesses and strengths. When a single method does not provide satisfactory data on the selected protein-nucleic acid complex, a combination of several methods should be considered as a hybrid approach; thus, specific structural problems can be solved when studying protein-nucleic acid complexes.

Apoptotic stress induces Bax-dependent, caspase-independent redistribution of LINC complex nesprins.

The canonical function of Bcl-2 family proteins is to regulate mitochondrial membrane integrity. In response to apoptotic signals the multi-domain pro-apoptotic proteins Bax and Bak are activated and perforate the mitochondrial outer membrane by a mechanism which is inhibited by their interaction with pro-survival members of the family. However, other studies have shown that Bax and Bak may have additional, non-canonical functions, which include stress-induced nuclear envelope rupture and discharge of nuclear proteins into the cytosol. We show here that the apoptotic stimuli cisplatin and staurosporine induce a Bax/Bak-dependent degradation and subcellular redistribution of nesprin-1 and nesprin-2 but not nesprin-3, of the linker of nucleoskeleton and cytoskeleton (LINC) complex. The degradation and redistribution were caspase-independent and did not occur in Bax/Bak double knockout (DKO) mouse embryo fibroblasts (MEFs). Re-expression of Bax in Bax/Bak DKO MEFs restored stress-induced redistribution of nesprin-2 by a mechanism which requires Bax membrane localization and integrity of the α helices 5/6, and the Bcl-2 homology 3 (BH3) domain. We found that nesprin-2 interacts with Bax in close proximity to perinuclear mitochondria in mouse and human cells. This interaction requires the mitochondrial targeting and N-terminal region but not the BH3 domain of Bax. Our results identify nesprin-2 as a Bax binding partner and also a new function of Bax in impairing the integrity of the LINC complex.

Proteomic analysis of haem-binding protein from Arabidopsis thaliana and Cyanidioschyzon merolae.

Chloroplast biogenesis involves the coordinated expression of the plastid and nuclear genomes, requiring information to be sent from the nucleus to the developing chloroplasts and vice versa. Although it is well known how the nucleus controls chloroplast development, it is still poorly understood how the plastid communicates with the nucleus. Currently, haem is proposed as a plastid-to-nucleus (retrograde) signal that is involved in various physiological regulations, such as photosynthesis-associated nuclear genes expression and cell cycle in plants and algae. However, components that transduce haem-dependent signalling are still unidentified. In this study, by using haem-immobilized high-performance affinity beads, we performed proteomic analysis of haem-binding proteins from Arabidopsis thaliana and Cyanidioschyzon merolae. Most of the identified proteins were non-canonical haemoproteins localized in various organelles. Interestingly, half of the identified proteins were nucleus proteins, some of them have a similar function or localization in either or both organisms. Following biochemical analysis of selective proteins demonstrated haem binding. This study firstly demonstrates that nucleus proteins in plant and algae show haem-binding properties. This article is part of the theme issue 'Retrograde signalling from endosymbiotic organelles'.

H-NS is the major repressor of Salmonella Typhimurium Pef fimbriae expression.

Fimbriae play an important role in adhesion and are therefore essential for the interaction of bacteria with the environments they encounter. Most of them are expressed in vivo but not in vitro, thus making difficult the full characterization of these fimbriae. Here, we characterized the silencing of plasmid-encoded fimbriae (Pef) expression, encoded by the pef operon, in the worldwide pathogen Salmonella Typhimurium. We demonstrated that the nucleoid-associated proteins H-NS and Hha, and their respective paralogs StpA and YdgT, negatively regulate at pH 5.1 and pH 7.1 the transcription of the pef operon. Two promoters, PpefB and PpefA, direct the transcription of this operon. All the nucleoid-associated proteins silence the PpefB promoter and H-NS also targets the PpefA promoter. While Hha and YdgT are mainly considered as acting primarily through H-NS to modulate gene transcription, our results strongly suggest that Hha and YdgT silence pef transcription at acidic pH either by interacting with StpA or independently of H-NS and StpA. We also confirmed the previously described post-transcriptional repression of Pef fimbriae by CsrA titration via the fim mRNA and CsrB and CsrC sRNA. Finally, among all these regulators, H-NS clearly appeared as the major repressor of Pef expression. These results open new avenues of research to better characterize the regulation of these bacterial adhesive proteins and to clarify their role in the virulence of pathogens.

Novel application of Influenza A virus-inoculated chorioallantoic membrane to characterize a NP-specific monoclonal antibody for immunohistochemistry assaying.

Monoclonal antibodies (MAbs) are widely applied in disease diagnoses. Herein, we report a MAb, WF-4, against Influenza A virus nucleoprotein (NP), its broad response with Influenza A virus, and its application in an immunohistochemistry (IHC) assay. WF-4 was screened by immunofluorescence assay (IFA). The results showed that its reactivity with baculovirus-expressed full-length recombinant NP (rNP) in Western blot (WB), indicating its IHC applicability. Fifteen Influenza A virus (reference subtypes H1 to H15) infected chicken embryonated chorioallantoic membranes (CAM), fixed by formalin, were all detectable in the WF-4-based IHC assay. Also, the reactivity of the IHC test with NP from experimentally inoculated H6N1 and from all recent outbreaks of H5 subtype avian Influenza A virus (AIV) field cases in Taiwan showed positive results. Our data indicate that CAM, a by-product of Influenza A virus preparation, is helpful for Influenza A virus-specific MAb characterization, and that the WF-4 MAb recognizes conserved and linear epitopes of Influenza A virus NP. Therefore, WF-4 is capable of detecting NP antigens via IHC and may be suitable for developing various tests for diagnosis of Influenza A virus and, especially, AIV infection.

Structure and oligomerization state of the C-terminal region of the Middle East respiratory syndrome coronavirus nucleoprotein.

Middle East respiratory syndrome coronavirus (MERS-CoV) is a human pathogen responsible for a severe respiratory illness that emerged in 2012. Structural information about the proteins that constitute the viral particle is scarce. In order to contribute to a better understanding of the nucleoprotein (N) in charge of RNA genome encapsidation, the structure of the C-terminal domain of N from MERS-CoV obtained using single-crystal X-ray diffraction is reported here at 1.97 Šresolution. The molecule is present as a dimer in the crystal structure and this oligomerization state is confirmed in solution, as measured by additional methods including small-angle X-ray scattering measurements. Comparisons with the structures of the C-terminal domains of N from other coronaviruses reveals a high degree of structural conservation despite low sequence conservation, and differences in electrostatic potential at the surface of the protein.

Nucleoprotein vaccine induces cross-protective cytotoxic T lymphocytes against both lineages of influenza B virus.

PURPOSE: The influenza B virus diverges into two antigenically distinct lineages: B/Yamagata and B/Victoria. Influenza B is the dominant circulating virus during some influenza seasons, and recent data demonstrated that influenza A and B infection similarly cause severe clinical symptoms in hospitalized patients. Nucleoprotein (NP) is a good target for a universal influenza vaccine. This study investigated whether NP epitope variation within two lineages affects the dominant cytotoxic T lymphocyte (CTL) responses induced by vaccination and the resultant protective immunity. MATERIALS AND METHODS: The NP of B/Yamagata/16/1988, the representative strain of the Yamagata lineage, includes a dominant CTL epitope, FSPIRITFL, while B/Shangdong/7/1997 from the Victoria lineage has one amino acid difference in this sequence, FSPIRVTFL. Two recombinant replication-deficient adenovirus (rAd)-vectored vaccines expressing either NP were prepared (rAd/B-NP(I) and rAd/B-NP(V), respectively) and administered to BALB/c mice intranasally. To examine the efficacy of vaccination, antibody responses, CTL responses, and morbidity/mortality after challenge were measured. RESULTS: Both vaccines induce similar antibody and CD8 T-cell responses cross-reacting to both epitopes, and also confer cross-protection against both lineages regardless of amino acid difference. CONCLUSION: The rAd-vectored vaccine expressing the NP could be developed as universal influenza B vaccine which provides broader protection.

Distinctive Nuclear Features of Dinoflagellates with A Particular Focus on Histone and Histone-Replacement Proteins.

Dinoflagellates are important eukaryotic microorganisms that play critical roles as producers and grazers, and cause harmful algal blooms. The unusual nuclei of dinoflagellates "dinokaryon" have led researchers to investigate their enigmatic nuclear features. Their nuclei are unusual in terms of their permanently condensed nucleosome-less chromatin, immense genome, low protein to DNA ratio, guanine-cytosine rich methylated DNA, and unique mitosis process. Furthermore, dinoflagellates are the only known group of eukaryotes that apparently lack histone proteins. Over the course of evolution, dinoflagellates have recruited other proteins, e.g., histone-like proteins (HLPs), from bacteria and dinoflagellates/viral nucleoproteins (DVNPs) from viruses as histone substitutes. Expression diversity of these nucleoproteins has greatly influenced the chromatin structure and gene expression regulation in dinoflagellates. Histone replacement proteins (HLPs and DVNPs) are hypothesized to perform a few similar roles as histone proteins do in other eukaryotes, i.e., gene expression regulation and repairing DNA. However, their role in bulk packaging of DNA is not significant as low amounts of proteins are associated with the gigantic genome. This review intends to summarize the discoveries encompassing unique nuclear features of dinoflagellates, particularly focusing on histone and histone replacement proteins. In addition, a comprehensive view of the evolution of dinoflagellate nuclei is presented.

Genomic epitopes of Xinjiang hemorrhagic fever virus and the evaluation of its laboratory testing value / 中华检验医学杂志

Objective:To grasp the distribution of fine antigenic epitope profiles of nucleoprotein (NP) and glycoprotein (GP) fragments of Crimean-Congo hemorrhagic fever virus (CCHFV) and to clarify the value of dominant antigenic epitopes in laboratory testing of Crimean-Congo hemorrhagic fever (CCHF).Methods:In a minimal synthetic short peptide consisting of 8 amino acids was segmentally expressed by CCHFV YL04057 strain using a modified bio-peptide synthesis method from 2014 to 2021 in the laboratory of Xinjiang University, College of Life Sciences. Using CCHFV polyclonal antibody or monoclonal antibody 14B7 (IgM) or CCHFV-positive sheep serum as antibodies, the minimal antigenic epitopes (BCEs) with antigenic activity on NP and GP fragments were identified by immunoblotting, and the obtained BCEs with sequence polymorphism were spatially clustered with CCHFV from different regions using the neighbor-joining method to determine the combination mode of BCEs with geographical correlation of regional distribution, to explore its application in establishing serological diagnosis. A prokaryotic expression plasmid (pET-32a), an E. coli expression plasmid (pGEX-KG) and a prokaryotic expression plasmid with an incomplete glutathione (GST188) tag (pXXGST-ST-1) were used to construct and express six dominant antigenic epitopes of different peptide lengths on NP fragments, and an indirect Enzyme-linked immunosorbent assay (ELISA) was established. CCHF sheep serum identified by immunofluorescence assay (IFA) was used as a control, and the specificity, sensitivity and overall compliance of the recombinant proteins with different peptide lengths of antigenic epitopes with IFA assay results were statistically analyzed. Results:CCHFV, NP and GP fragments had a total of 30 antigenically active BCEs, among which the core intermediate fragment NP2 (aa 170 th-305 th), which had a concentration of antigenic epitopes in the NP fragment, has 6 BCEs, and the NP1 (aa 1 st-200 th) and NP3 (aa 286 th-482 nd) at both ends have 9 BCEs; the Gc (aa 1 st-558 th) and Gn (aa 533 th-708 th) fragments of the GP fragment have 14 BCEs and a long antigenic peptide (AP) containing 15 amino acids, and the amino acid sequence homology of the NP fragment BCEs was 97.1% and that of the GP fragment BCEs was 89.1%. There was a significant difference ( P=0.0281, P<0.05). Among the 9 BCEs with sequence polymorphism in the GP fragment, 6 combined BCEs from GnEc1, GnE2, GnE4, GcE3, GcE6 and GcAP-4 (Ap) could cluster 15 CCHFV strains from different regions of the world into 5 geographical taxa, AsiaⅠ, AsiaⅡ, AficaⅠ, AficaⅡ and Europe. The constructs expressing PET-32a-NP (full length), PGEX-KG-NP2 (aa 170 th-305 th), pGEX-KG-NP2-1 (aa 235 th-275 th), PGEX-KG-NP2-1-1 (aa 237 th-256 th), pXXGST-1-NP2-1-2 (aa 250 th-265 th) and PGEX KG-NP2-1-3 (aa 260 th-276 th), six recombinant proteins CCHFV NP rabbit polyclonal antiserum (pAb) Western Blotting reaction positive, 33 sheep sera tested by IFA XHF as a reference, the sensitivity of the assay established by indirect ELISA using the recombinant proteins constructed from two fragments of NP2 and NP2-1 as antigens. The sensitivity, specificity and overall compliance were the best, with 73.4% (11/15) and 66.7% (10/15) for sensitivity, 100% (18/18) and 94.4% (17/18) for specificity, and 87.9% (29/33) and 81.8% (27/33) for overall compliance. Conclusion:CCHFV NP and GP are distributed with a high number of BCEs with antigenic immunoreactivity, among which the dominant antigenic epitopes are of high value in the laboratory serological diagnosis of CCHF.

Toscana virus nucleoprotein oligomer organization observed in solution.

Toscana virus (TOSV) is an arthropod-borne virus belonging to the Phlebovirus genus within the Bunyaviridae family. As in other bunyaviruses, the genome of TOSV is made up of three RNA segments. They are encapsidated by the nucleoprotein (N), which also plays an essential role in virus replication. To date, crystallographic structures of phlebovirus N have systematically revealed closed-ring organizations which do not fully match the filamentous organization of the ribonucleoprotein (RNP) complex observed by electron microscopy. In order to further bridge the gap between crystallographic data on N and observations of the RNP by electron microscopy, the structural organization of recombinant TOSV N was investigated by an integrative approach combining X-ray diffraction crystallography, transmission electron microscopy, small-angle X-ray scattering, size-exclusion chromatography and multi-angle laser light scattering. It was found that in solution TOSV N forms open oligomers consistent with the encapsidation mechanism of phlebovirus RNA.

Nucleoprotein supplementation enhances the recovery of rat soleus mass with reloading after hindlimb unloading-induced atrophy via myonuclei accretion and increased protein synthesis.

Hindlimb unloading results in muscle atrophy and a period of reloading has been shown to partially recover the lost muscle mass. Two of the mechanisms involved in this recovery of muscle mass are the activation of protein synthesis pathways and an increase in myonuclei number. The additional myonuclei are provided by satellite cells that are activated by the mechanical stress associated with the reloading of the muscles and eventually incorporated into the muscle fibers. Amino acid supplementation with exercise also can increase skeletal muscle mass through enhancement of protein synthesis and nucleotide supplements can promote cell cycle activity. Therefore, we hypothesized that nucleoprotein supplementation, a combination of amino acids and nucleotides, would enhance the recovery of muscle mass to a greater extent than reloading alone after a period of unloading. Adult rats were assigned to 4 groups: control, hindlimb unloaded (HU; 14 days), reloaded (5 days) after hindlimb unloading (HUR), and reloaded after hindlimb unloading with nucleoprotein supplementation (HUR + NP). Compared with the HUR group, the HUR + NP group had larger soleus muscles and fiber cross-sectional areas, higher levels of phosphorylated rpS6, and higher numbers of myonuclei and myogenin-positive cells. These results suggest that nucleoprotein supplementation has a synergistic effect with reloading in recovering skeletal muscle properties after a period of unloading via rpS6 activation and satellite cell differentiation and incorporation into the muscle fibers. Therefore, this supplement may be an effective therapeutic regimen to include in rehabilitative strategies for a variety of muscle wasting conditions such as aging, cancer cachexia, muscular dystrophy, bed rest, and cast immobilization.

AgNOR and rasp21 expression in gastric mucosal lesions caused by Helicobacter pylori infection.

AIM: To investigate AgNOR and rasp21 expression levels in gastric mucosal lesions caused by Helicobacter pylori (Hp) infection in order to gain insight into the related biological processes (i.e. tumor-like behavior) and possible underlying mechanism supporting Hp pathogenesis. METHODS: Hp infection was diagnosed in using the standard Campylobacter-like organism test along with Wathin-Starry staining. The expression of AgNOR was detected by the silver colloid staining technique. The expression of rasp21 was detected by monoclonal antibody and immunohistochemical staining using the ABC method. The study included a total of 278 patients with endoscopically- and pathologically-confirmed gastric mucosal lesions, representing chronic superficial gastritis (CSG), chronic atrophic gastritis (CAG), intestinal metaplasia, dysplasia, and gastric cancer. Among these, 146 of the patients were Hp-positive and 132 were Hp-negative. RESULTS: The Hp-positive group of patients showed significantly greater AgNOR in the gastric mucosal lesions than the Hp-negative group, with the exception of the CSG sub-group (P < 0.05 or P < 0.01). The positive rate of rasp21 expression in gastric mucosal lesions in the Hp-positive group was also significantly higher than that in the Hp-negative group, with the exception of the CSG and CAG sub-groups (P < 0.05). CONCLUSION: Hp-positive gastric mucosal lesions show biological behaviour of tumors. Hp may act as a promoter to activate the ras gene and to stimulate cell over-proliferation.