Unveiling the "invisible" druggable conformations of GDP-bound inactive Ras.

The prevalent view on whether Ras is druggable has gradually changed in the recent decade with the discovery of effective inhibitors binding to cryptic sites unseen in the native structures. Despite the promising advances, therapeutics development toward higher potency and specificity is challenged by the elusive nature of these binding pockets. Here we derive a conformational ensemble of guanosine diphosphate (GDP)-bound inactive Ras by integrating spin relaxation-validated atomistic simulation with NMR chemical shifts and residual dipolar couplings, which provides a quantitative delineation of the intrinsic dynamics up to the microsecond timescale. The experimentally informed ensemble unequivocally demonstrates the preformation of both surface-exposed and buried cryptic sites in Ras•GDP, advocating design of inhibition by targeting the transient druggable conformers that are invisible to conventional experimental methods. The viability of the ensemble-based rational design has been established by retrospective testing of the ability of the Ras•GDP ensemble to identify known ligands from decoys in virtual screening.

Characterization of a novel tetravalent botulism antitoxin based on receptor-binding domain of BoNTs.

Botulinum neurotoxin (BoNTs; serotypes A, B, E, and F) cause botulism disease in humans, which could be effectively treated using antitoxins. Herein, we established a novel receptor-binding domain (RBD)-based antitoxin using recombinant C terminal heavy chain (Hc) domains of BoNTs as immunogens. Immunization of horses with these recombinant Hc domains allowed the purification and digestion of IgGs from hyper-immune sera to produce high-quality and high-efficiency monovalent botulism antitoxin F(ab')2 against each BoNT (M-BATs). However, these M-BATs could not bind or neutralize other serotypes of BoNTs, and that there were no cross-protective effects among these M-BATs. This suggested the need to prepare tetravalent antitoxins to neutralize the four BoNTs simultaneously. Thus, these M-BATs were formulated into a novel tetravalent botulism antitoxin (T-BAT), in which a 10-ml volume contained 10000 IU of BoNT/A and 5000 IU of BoNT/B, BoNT/E, and BoNT/F antitoxins. The novel antitoxin preparation could prevent and treat the four mixed botulinum neurotoxins simultaneously in vivo, representing strong efficacy in an animal poisoning model. Moreover, these antibodies in T-BAT could bind the RBD, whereas conventional antitoxins based on inactivated toxins mainly bind the light chain or heavy chain translocation domain (HN) and weakly bind the important RBD in current experimental conditions. The high levels of RBD-specific novel antitoxins can efficiently bind the RBD and neutralize natural or recombinant toxins containing this RBD. The findings of the present study experimentally support the use of RBD-specific antitoxins to treat BoNT serotype A, B, E, and F-mediated botulism. This study demonstrated the concept of developing potent novel multivalent antitoxins against all BoNTs or other toxins, using the RBD of these toxins as an alternative antigen to inactivated toxins. KEY POINTS: • Antitoxins based on the receptor-binding domains of botulinum neurotoxins were made. • Novel antitoxin binds RBD; traditional antitoxin mainly binds light chain or HN domain. • A tetravalent antitoxin could prevent and treat the four mixed neurotoxins in vivo.

Exogenous Thymosin Beta 4 Suppresses IPF-Lung Cancer in Mice: Possibly Associated with Its Inhibitory Effect on the JAK2/STAT3 Signaling Pathway.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, fibrotic interstitial lung disease of unknown etiology. At present, the mortality rate of the deadly disease is still very high, while the existing treatments only delay the progression of the disease and improve the quality of life of patients. Lung cancer (LC) is the most fatal disease in the world. In recent years, IPF has been considered to be an independent risk factor for the development of LC. The incidence of lung cancer is increased in the patients with IPF and the mortality is also significantly increased in the patients inflicted with the two diseases. In this study, we evaluated an animal model of pulmonary fibrosis complicated with LC by implanting LC cells orthotopically into the lungs of mice several days after bleomycin induction of the pulmonary fibrosis in the same mice. In vivo studies with the model showed that exogenous recombinant human thymosin beta 4 (exo-rhTß4) alleviated the impairment of lung function and severity of damage of the alveolar structure by the pulmonary fibrosis and inhibited the proliferation of LC tumor growth. In addition, in vitro studies showed that exo-rhTß4 inhibited the proliferation and migration of A549 and Mlg cells. Furthermore, our results also showed that rhTß4 could effectively inhibit the JAK2-STAT3 signaling pathway and this might exert an anti-IPF-LC effect. The establishment of the IPF-LC animal model will be helpful for the development of drugs for the treatment of IPF-LC. Exogenous rhTß4 can be potentially used for the treatment of IPF and LC.

Recombinant Human Thymosin ß4 (rhTß4) Modulates the Anti-Inflammatory Responses to Alleviate Benzalkonium Chloride (BAC)-Induced Dry Eye Disease.

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 ß4 (rhTß4) 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% rhTß4 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% rhTß4 groups. Specifically, topical application of rhTß4 significantly increased conjunctival gobleT cells and reduced apoptotic cells in conjunctiva. Mechanically, the rhTß4 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% rhTß4 eye drops may be used as a potential therapeutic treatment for DED.

A novel human anti-TIGIT monoclonal antibody with excellent function in eliciting NK cell-mediated antitumor immunity.

TIGIT is an emerging novel checkpoint target that is expressed on both tumor-infiltrating T cells and NK cells. Some current investigational antibodies targeting TIGIT have also achieved dramatic antitumor efficacy in late clinical research. Most recently, the relevance of NK cell-associated TIGIT signaling pathway to tumors' evasion of the immune system has been clearly revealed, which endows NK cells with a pivotal role in the therapeutic effects of TIGIT blockade. In this article, we describe a novel anti-TIGIT monoclonal antibody, AET2010, which was acquired from a phage-displayed human single-chain antibody library through a cell panning strategy. With emphasis on its regulation of NK cells, we confirmed the excellent ex vivo and in vivo antitumor immunity of AET2010 mediated by the NK-92MI cells. Intriguingly, our work also revealed that AET2010 displays a lower affinity but parallel avidity and activity relative to MK7684, an investigational monoclonal antibody from MSD, implying a reasonable balance of potency and potential side effects for AET2010. Together, these results are promising and warrant further development of AET2010.

Recombinant Human Thymosin Beta-4 Protects against Mouse Coronavirus Infection.

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 (Tß4), a 43-residue peptide with an acetylated N-terminus, is widely expressed in human tissues. Tß4 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 Tß4 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 Tß4 (rhTß4). 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 rhTß4 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 rhTß4 to the treatment of human CoV diseases such as COVID-19.

Extending the Lifetime of Native GTP-Bound Ras for Site-Resolved NMR Measurements: Quantifying the Allosteric Dynamics.

Characterization of native GTP-bound Ras is important for an appreciation of its cellular signaling and for the design of inhibitors, which however has been depressed by its intrinsic instability. Herein, an effective approach for extending the lifetime of Ras⋅GTP samples by exploiting the active role of Son of Sevenless (Sos) is demonstrated that sustains the activated state of Ras. This approach, combined with a postprocessing method that suppresses residual Ras⋅GDP signals, is applied to the site-resolved NMR measurement of the allosteric dynamics of Ras⋅GTP. The observed network of concerted motions well covers the recently identified allosteric inhibitor-binding pockets, but the motions are more confined than those of Ras⋅GppNHp, advocating the use of native GTP for development of allosteric inhibitors. The Sos-based approach is anticipated to generally facilitate experiments on active Ras when native GTP is preferred.

Microsecond Timescale Dynamics of GDP-Bound Ras Underlies the Formation of Novel Inhibitor-Binding Pockets.

The recent discovery of inhibitory compounds binding to distinct pockets on GDP-bound Ras has renewed the view on the druggability of this crucial cancer driver. However, the origin of these pockets, which are not readily formed in the crystal structure in the absence of the compounds, is yet unclear. Herein, we explored the intrinsic flexibility of Ras⋅GDP on microsecond to millisecond timescales using relaxation-based NMR experiments, and identified substantial slow dynamics with τex of 34 µs at 5 °C, which maps to the regions showing a high level of correlation with those displaying conformational differences between the inhibitor-bound and free states. These findings, which have been demonstrated in both wild-type Ras and the oncogenic mutant (G12V), support the mechanism of extended conformational selection for Ras-inhibitor interactions where the small molecules redistribute the protein conformational ensemble favoring the final bound states.

How is physical attractiveness associated with depression in China: A moderated mediation analysis.

Rewarding beauty has never fallen out of vogue in human history. Attractive people enjoy many social and economic advantages. This study aims to investigate the relationship between physical attractiveness and depression and its potential mechanism among Chinese residents. A population-based data including 12,172 Chinese adults from 3 waves of CFPS (2012, 2016, 2020) were used. Moderated-mediation models were utilized to assess the mediation effect of unfair experiences and the moderation effect of socioeconomic-status (SES), in the linkage between physical attractiveness and depression. Firstly, our results demonstrated that physical attractiveness is negatively associated with depression. Secondly, the effect of unfair experiences as a mediator between physical attractiveness and depression is statistically significant. Lastly, socioeconomic status (SES) performs a crucial moderating role. Among Chinese residents, the mitigating effect of physical attractiveness on unfair experiences and depression would be stronger among those with a lower SES compared to those with a higher SES.

Comparative evaluation of protective immunity against Francisella tularensis induced by subunit or adenovirus-vectored vaccines.

Tularemia is a highly contagious disease caused by infection with Francisella tularensis (Ft), a pathogenic intracellular gram-negative bacterium that infects a wide range of animals and causes severe disease and death in people, making it a public health concern. Vaccines are the most effective way to prevent tularemia. However, there are no Food and Drug Administration (FDA)-approved Ft vaccines thus far due to safety concerns. Herein, three membrane proteins of Ft, Tul4, OmpA, and FopA, and a molecular chaperone, DnaK, were identified as potential protective antigens using a multifactor protective antigen platform. Moreover, the recombinant DnaK, FopA, and Tul4 protein vaccines elicited a high level of IgG antibodies but did not protect against challenge. In contrast, protective immunity was elicited by a replication-defective human type 5 adenovirus (Ad5) encoding the Tul4, OmpA, FopA, and DnaK proteins (Ad5-Tul4, Ad5-OmpA, Ad5-FopA, and Ad5-DnaK) after a single immunization, and all Ad5-based vaccines stimulated a Th1-biased immune response. Moreover, intramuscular and intranasal vaccination with Ad5-Tul4 using the prime-boost strategy effectively eliminated Ft lung, spleen and liver colonization and provided nearly 80% protection against intranasal challenge with the Ft live vaccine strain (LVS). Only intramuscular, not intranasal vaccination, with Ad5-Tul4 protected mice from intraperitoneal challenge. This study provides a comprehensive comparison of protective immunity against Ft provided by subunit or adenovirus-vectored vaccines and suggests that mucosal vaccination with Ad5-Tul4 may yield desirable protective efficacy against mucosal infection, while intramuscular vaccination offers greater overall protection against intraperitoneal tularemia.

Development and evaluation of a tetravalent botulinum vaccine.

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.

Recombinant human thymosin beta-4 (rhTß4) improved scalp condition and microbiome homeostasis in seborrheic dermatitis.

Seborrheic dermatitis (SD) is a recurrent common inflammatory skin disease that affects all ethnic groups in all regions worldwide. However, no specific treatment or preventive measure is yet available. Identifying effective treatments with acceptable safety and tolerability is desirable. In this study, scalp microbiota alterations were measured in SD, showing significantly greater abundance of Malassezia and Staphylococcus and diminished fungal and bacterial diversity compared with healthy controls. We investigated the benefit of a 4-week treatment with 0.5 mg ml-1 recombinant human thymosin ß4 (rhTß4) gel or 2% ketoconazole lotion on the scalp condition of 71 patients with SD compared with 21 healthy individuals. Clinical assessment (Adherent Scalp Flaking Score, and the Maximum Erythema Area) and physiological conditions (transepidermal water loss, hydration, and sebum secretion) were evaluated. The rhTß4 treatment provided significantly greater efficacy than ketoconazole and a sustained effect in the treatment of scalp SD. More importantly, rhTß4 dramatically improved the microbiome homeostasis and prompted a shift of scalp microflora towards healthy composition, helping symptoms and ameliorating physiological conditions more effectively and durably than ketoconazole. Our research demonstrated the scalp microbe dysbiosis of SD and highlighted rhTß4 as a promising therapeutic strategy in the prevention and treatment of SD.

Development and evaluation of candidate subunit vaccine and novel antitoxin against botulinum neurotoxin serotype E.

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 dose- and 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(ab')2 from pepsin-digested serum IgG of rEHc-inoculated horses. The protective effect of the F(ab')2 antitoxin was determined in vitro and in vivo. The results showed that our F(ab')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.

Immunological characterisation and immunoprotective efficacy of functional domain antigens of botulinum neurotoxin serotype A.

Botulinum neurotoxins (BoNTs) are highly toxic proteins that mediate their effects by binding to neuronal receptors and block the neutralizing ability of therapeutic antibodies. Vaccination is currently the most effective strategy to prevent botulism. In this study, a series of recombinant functional domain antigens of BoNT/A were prepared and identified, and their immunoprotective efficacies were explored and compared. Our results showed that all antigens produced strong humoral immune responses, although their protective effects against the toxin were different. Only the Hc and HN-L antigens produced strong protective effects and afforded complete immunoprotection. In addition, the combined vaccine groups showed that there was no synergistic effect on immune responses after antigen combination, suggesting that the integrity of the toxin antigen or domain is crucial to the immune effects. Studies of the dose-dependent immunoprotective effects further confirmed that the Hc domain antigen afforded more effective protective potency than the HN-L antigen, equivalent to the immune effect of the full-length toxin (Hc + HN-L combination group). Overall, our results demonstrated that the Hc domain elicited a strong protective immune response and also provided basic data and theoretical support for the development of Hc-based BoNT/A subunit vaccine. Therefore, the receptor binding domain Hc is implicated as a promising target antigen of the BoNT/A recombinant subunit vaccine as an alternative to the toxoid vaccine.

Pollen resistance to water in 80 angiosperm species: flower structures protect rain-susceptible pollen.

Flowers exhibit adaptive responses to biotic and abiotic factors. It remains unclear whether pollen susceptibility to rain damage plays a role in the evolution of floral form. We investigated flower performance in rain and compared pollen longevity in dry conditions, pure water and solutions with different sucrose concentrations in 80 flowering species from 46 families with diverse floral shapes and pollination modes. A pollen viability test showed that pollen longevity in all studied species was greatly reduced by wetting. We found that pollen of species with complete protection by flower structures was susceptible to water damage and a high proportion of resistant pollen occurred in unprotected species. Flowers whose structures expose pollen to rain may also reduce rain damage through temporal patterns of pollen presentation. This prediction was supported by our direct measurement of pollen presentation duration on rainy days. Our observations showed that variation in pollen performance in water was associated with differences in floral forms. Water-resistant pollen and extended pollen presentation duration were favored by selection via rain contact in species in which pollen was not protected from rain. These findings support the functional hypothesis that flower structures protect susceptible pollen from rain, demonstrating that rain acts as a force shaping floral form.

Development and evaluation of candidate subunit vaccine against botulinum neurotoxin serotype B.

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-His-tagged and homogeneous BHc expressed in E.coli represents a good potential candidate subunit vaccine for human use.

Probing Transient Release of Membrane-Sequestered Tyrosine-Based Signaling Motif by Solution NMR Spectroscopy.

Membrane sequestration of tyrosine-based signaling motifs of antigen receptors effectively restricts the signaling activities in resting lymphocytes. However, low level of basal signaling in resting cells is required for lymphocyte survival and antigen responsiveness, of which the molecular mechanism remains obscure. Here we probe the transient release of the cytoplasmic domain of the membrane-bound IgG heavy chain (mIgG-tail) by hydrogen exchange NMR spectroscopy, illustrating a dynamic molecular basis for its basal signaling activity. To solve the severe resonance overlap problem in the 2D spectra of mIgG-tail, a non-uniformly sampled pseudo-4D hydrogen exchange NMR experiment has been exploited to quantitatively measure site-specific hydrogen exchange rates. Our solution NMR study reveals transient solvent exposure of the ITT signaling motif that can be further enhanced by calcium ion, and provides insight into the mechanism of lymphocyte basal signaling.

A dynamic meiotic SUN belt includes the zygotene-stage telomere bouquet and is disrupted in chromosome segregation mutants of maize (Zea mays L.).

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.