The nuclear envelope (NE) plays an essential role in meiotic telomere behavior and links the cytoplasm and nucleoplasm during homologous chromosome pairing and recombination in many eukaryotic species. Resident NE proteins including SUN (Sad-1/UNC-84) and KASH (Klarsicht/ANC-1/Syne-homology) domain proteins are known to interact forming the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex that connects chromatin to the cytoskeleton. To investigate the possible cross-kingdom conservation of SUN protein functions in plant meiosis, we immunolocalized maize SUN2 using 3D microscopy of pollen mother cells from maize (Zea mays L.), a large-genome plant model with a canonical NE zygotene-stage telomere bouquet. We detected SUN2 at the nuclear periphery and found that it exhibited a distinct belt-like structure that transitioned to a half-belt during the zygotene stage and back to a full belt during and beyond the pachytene stage. The zygotene-stage half-belt SUN structure was shown by 3D immuno-FISH to include the NE-associated telomere cluster that defines the bouquet stage and coincides with homologous chromosome synapsis. Microtubule and filamentous actin staining patterns did not show any obvious belt or a retracted-like structure other than a general enrichment of tubulin staining distributed widely around the nucleus and throughout the cytoplasm. Genetic disruption of the meiotic SUN belt staining patterns with three different meiosis-specific mutants, desynaptic (dy1), asynaptic1 (as1), and divergent spindle1 (dv1) provides additional evidence for the role of the nuclear envelope in meiotic chromosome behavior. Taking into account all of the observations from this study, we propose that the maize SUN belt is directly or indirectly involved in meiotic telomere dynamics, chromosome synapsis, and possibly integration of signals and forces across the meiotic prophase nuclear envelope.
Botulinum neurotoxins (BoNTs) are among the most toxic proteins. Vaccination is an effective strategy to prevent botulism. To generate a vaccine suitable for human use, a recombinant non-His-tagged isoform of the Hc domain of botulinum neurotoxin serotype E (rEHc) was expressed in Escherichia coli and purified by sequential chromatography. The immunogenicity of rEHc was evaluated in mice and doseand time-dependent immune responses were observed in both antibody titers and protective potency. Then, the pilot-scale expression and purification of rEHc were performed, and its immunological activity was characterized. Our results showed rEHc has good immunogenicity and can elicit strong protective potency against botulinum neurotoxin serotype E (BoNT/E) in mice, indicating that rEHc is an effective botulism vaccine candidate. Further, we developed a novel antitoxin against BoNT/E by purifying F(abʹ) 2 from pepsin-digested serum IgG of rEHc-inoculated horses. The protective effect of the F(abʹ) 2 antitoxin was determined in vitro and in vivo. The results showed that our F(abʹ) 2 antitoxin can prevent botulism in BoNT/E-challenged mice and effectively alleviate the progression of paralysis caused by BoNT/E to achieve therapeutic effects. Therefore, our results provide valuable experimental data for the production of a novel antitoxin, which is a promising candidate for the treatment of BoNT/E-induced botulism. ARTICLE HISTORY
Botulinum neurotoxins (BoNTs) are potential biological weapons because of their high toxicity and mortality. Vaccination is an effective strategy to prevent botulism. The carboxyl-terminus of the heavy chain (Hc domain) is nontoxic and sufficient to generate protective immune responses against natural BoNTs in animals. To produce a vaccine suitable for human use, a recombinant non His-tagged isoform of the Hc domain of botulinum neurotoxin serotype B (BHc) was expressed in Escherichia coli and purified by sequential chromatography. The immunogenicity of recombinant E.coli-expressed BHc and the yeast-expressed mBHc antigens was explored and compared in Balb/c mice. BHc provided comparable protective potency but elicited significantly higher antibody titer and neutralization potency against BoNT/B after twice immunization, indicating that the recombinant BHc protein expressed in E.coli have better immunogenicity than the yeast-expressed mBHc. Moreover, a frequency and dose-dependent effect was observed in mice immunized with BHc subunit vaccine and the anti-BHc ELISA antibody titers correlated well with neutralizing antibody titers and protection potency. In summary, the Alhydrogel-formulated BHc subunit vaccine afforded effective protection against BoNT/B challenge. Therefore, the non-Histagged and homogeneous BHc expressed in E.coli represents a good potential candidate subunit vaccine for human use.
Botulinum neurotoxins (BoNTs) are the most toxic known proteins. Naturally occurring botulism in humans is caused by botulinum serotypes A, B, E, and F. Vaccination is an effective strategy to prevent botulism. In this study, a tetravalent botulinum vaccine (TBV) that can prevent serotypes A, B, E, and F was developed using the C-terminal receptor-binding domain of BoNT (Hc) as an antigen. To develop a suitable vaccine formulation, in vitro binding experiments of antigens and aluminum adjuvant in different buffers, and in vivo experiments of TBV at different antigen concentrations, were conducted. Our results showed that the optimal vaccine formulation buffer was a pH 6.0 phosphate buffer, and the suitable antigen concentration was 40 or 80 µg/ml of each antigen. A pilot-scale TBV was then prepared and evaluated for immunogenicity and stability. The results showed that TBV could elicit strong protective efficacy against each BoNT in mice, and remain effective after two years of storage at 4ºC, indicating that the preparation was stable and highly effective. Adsorption experiments also showed that the antigens could be well adsorbed by the aluminum adjuvant after 2 years of storage. Our results provide valuable experimental data supporting the development of a tetravalent botulinum vaccine, which is a promising candidate for the prevention of botulinum serotypes A, B, E, and F.
Coronaviruses (CoVs) are enveloped and harbor an unusually large (30–32 kb) positive-strand linear RNA genome. Highly pathogenic coronaviruses cause severe acute respiratory syndrome (SARS) (SARS-CoV and SARS-CoV-2) and Middle East respiratory syndrome (MERS) (MERS-CoV) in humans. The coronavirus mouse hepatitis virus (MHV) infects mice and serves as an ideal model of viral pathogenesis, mainly because experiments can be conducted using animal-biosafety level-2 (A-BSL2) containment. Human thymosin beta-4 (Tβ4), a 43-residue peptide with an acetylated N-terminus, is widely expressed in human tissues. Tβ4 regulates actin polymerization and functions as an anti-inflammatory molecule and an antioxidant as well as a promoter of wound healing and angiogenesis. These activities led us to test whether Tβ4 serves to treat coronavirus infections of humans. To test this possibility, here, we established a BALB/c mouse model of coronavirus infection using mouse CoV MHV-A59 to evaluate the potential protective effect of recombinant human Tβ4 (rhTβ4). Such a system can be employed under A-BSL2 containment instead of A-BSL3 that is required to study coronaviruses infectious for humans. We found that rhTβ4 significantly increased the survival rate of mice infected with MHV-A59 through inhibiting virus replication, balancing the host’s immune response, alleviating pathological damage, and promoting repair of the liver. These results will serve as the basis for further application of rhTβ4 to the treatment of human CoV diseases such as COVID-19.
Dry eye disease (DED) is a multifactorial ocular disorder that interferes with daily living and reduces quality of life. However, there is no most ideal therapeutic treatment to address all the deleterious defects of DED. The purpose of this study was to investigate the ability of recombinant human thymosin β4 (rhTβ4) to promote healing in a benzalkonium chloride (BAC)-induced mice DED model and the anti-inflammatory effects involved in that process. Eye drops consisting of 0.05% and 0.1% rhTβ4 were used for treatment of DED. Tear volume and corneal staining scores were measured after 7 days. Periodic acid-Schiff staining for gobleT cells in conjunctiva, immunohistochemical staining for CD4+ T cells, TUNEL assay for apoptotic positive cells in cornea and conjunctiva, qRT-PCR and ELISA assays for multiple cytokines were performed. All clinical parameters showed improvement in both the 0.05% and 0.1% rhTβ4 groups. Specifically, topical application of rhTβ4 significantly increased conjunctival gobleT cells and reduced apoptotic cells in conjunctiva. Mechanically, the rhTβ4 groups showed significantly reduced inflammatory cytokine levels and CD4+ T cells in conjunctiva by blocking NF-κB (nuclear factor kappa B) activation, suggesting that 0.05–0.1% rhTβ4 eye drops may be used as a potential therapeutic treatment for DED.
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