Use of phase plate cryo-EM reveals conformation diversity of therapeutic IgG with 50 kDa Fab fragment resolved below 6 Å.

While cryogenic electron microscopy (cryo-EM) is fruitfully used for harvesting high-resolution structures of sizable macromolecules, its application to small or flexible proteins composed of small domains like immunoglobulin (IgG) remain challenging. Here, we applied single particle cryo-EM to Rituximab, a therapeutic IgG mediating anti-tumor toxicity, to explore its solution conformations. We found Rituximab molecules exhibited aggregates in cryo-EM specimens contrary to its solution behavior, and utilized a non-ionic detergent to successfully disperse them as isolated particles amenable to single particle analysis. As the detergent adversely reduced the protein-to-solvent contrast, we employed phase plate contrast to mitigate the impaired protein visibility. Assisted by phase plate imaging, we obtained a canonical three-arm IgG structure with other structures displaying variable arm densities co-existing in solution, affirming high flexibility of arm-connecting linkers. Furthermore, we showed phase plate imaging enables reliable structure determination of Fab to sub-nanometer resolution from ab initio, yielding a characteristic two-lobe structure that could be unambiguously docked with crystal structure. Our findings revealed conformation diversity of IgG and demonstrated phase plate was viable for cryo-EM analysis of small proteins without symmetry. This work helps extend cryo-EM boundaries, providing a valuable imaging and structural analysis framework for macromolecules with similar challenging features.

Structural convergence endows nuclear transport receptor Kap114p with a transcriptional repressor function toward TATA-binding protein.

The transcription factor TATA-box binding protein (TBP) modulates gene expression in nuclei. This process requires the involvement of nuclear transport receptors, collectively termed karyopherin-ß (Kap-ß) in yeast, and various regulatory factors. In previous studies we showed that Kap114p, a Kap-ß that mediates nuclear import of yeast TBP (yTBP), modulates yTBP-dependent transcription. However, how Kap114p associates with yTBP to exert its multifaceted functions has remained elusive. Here, we employ single-particle cryo-electron microscopy to determine the structure of Kap114p in complex with the core domain of yTBP (yTBPC). Remarkably, Kap114p wraps around the yTBPC N-terminal lobe, revealing a structure resembling transcriptional regulators in complex with TBP, suggesting convergent evolution of the two protein groups for a common function. We further demonstrate that Kap114p sequesters yTBP away from promoters, preventing a collapse of yTBP dynamics required for yeast responses to environmental stress. Hence, we demonstrate that nuclear transport receptors represent critical elements of the transcriptional regulatory network.

Structural basis for a conserved neutralization epitope on the receptor-binding domain of SARS-CoV-2.

Antibody-mediated immunity plays a crucial role in protection against SARS-CoV-2 infection. We isolated a panel of neutralizing anti-receptor-binding domain (RBD) antibodies elicited upon natural infection and vaccination and showed that they recognize an immunogenic patch on the internal surface of the core RBD, which faces inwards and is hidden in the "down" state. These antibodies broadly neutralize wild type (Wuhan-Hu-1) SARS-CoV-2, Beta and Delta variants and some are effective against other sarbecoviruses. We observed a continuum of partially overlapping antibody epitopes from lower to upper part of the inner face of the RBD and some antibodies extend towards the receptor-binding motif. The majority of antibodies are substantially compromised by three mutational hotspots (S371L/F, S373P and S375F) in the lower part of the Omicron BA.1, BA.2 and BA.4/5 RBD. By contrast, antibody IY-2A induces a partial unfolding of this variable region and interacts with a conserved conformational epitope to tolerate all antigenic variations and neutralize diverse sarbecoviruses as well. This finding establishes that antibody recognition is not limited to the normal surface structures on the RBD. In conclusion, the delineation of functionally and structurally conserved RBD epitopes highlights potential vaccine and therapeutic candidates for COVID-19.

In situ structure and dynamics of an alphacoronavirus spike protein by cryo-ET and cryo-EM.

Porcine epidemic diarrhea (PED) is a highly contagious swine disease caused by porcine epidemic diarrhea virus (PEDV). PED causes enteric disorders with an exceptionally high fatality in neonates, bringing substantial economic losses in the pork industry. The trimeric spike (S) glycoprotein of PEDV is responsible for virus-host recognition, membrane fusion, and is the main target for vaccine development and antigenic analysis. The atomic structures of the recombinant PEDV S proteins of two different strains have been reported, but they reveal distinct N-terminal domain 0 (D0) architectures that may correspond to different functional states. The existence of the D0 is a unique feature of alphacoronavirus. Here we combined cryo-electron tomography (cryo-ET) and cryo-electron microscopy (cryo-EM) to demonstrate in situ the asynchronous S protein D0 motions on intact viral particles of a highly virulent PEDV Pintung 52 strain. We further determined the cryo-EM structure of the recombinant S protein derived from a porcine cell line, which revealed additional domain motions likely associated with receptor binding. By integrating mass spectrometry and cryo-EM, we delineated the complex compositions and spatial distribution of the PEDV S protein N-glycans, and demonstrated the functional role of a key N-glycan in modulating the D0 conformation.

Efficacy of Er:YAG laser for the peri-implantitis treatment and microbiological changes: a randomized controlled trial.

The aims of this study were to identify the microbiological changes in the periodontal pockets following an Er:YAG laser (ERL) irradiation and mechanical debridement to compare the effectiveness of ERL irradiation to mechanical debridement for peri-implantitis treatment through randomized controlled trials. Twenty-three patients with peri-implantitis lesions were treated in either a test group, ERL set at energy level of 100 mJ/pulse, frequency of 10 Hz, pulse duration was 100 µs, and irradiated by three passages, or a control group, with mechanical debridement using an ultrasonic scaler. An examiner measured the following clinical parameters at different stages (a baseline and at 3- and 6-month post-treatment): probing depth (PD), bleeding on probing (BOP), marginal bone loss (MBL), and anaerobic bacteria counts. Linear regression, with generalized estimation equations, was used to compare the clinical parameters and anaerobic bacterial counts at different stages and between groups. The anaerobic bacterial counts significantly decreased within the control group during the follow-ups. At the 6-month follow-up, both groups showed a significant reduction in PD (test group: mean difference of 0.84 mm; control group: mean difference of 0.41 mm), and the test group showed a significantly higher PD reduction on the buccal site (1.31 mm) compared to that of the control group (0.25 mm). Both ERL and mechanical debridement treatments led to significant improvements in PD. When mechanical debridement therapy was used, significant anaerobic bacterial count reductions were observed. Future treatment of peri-implantitis should involve a combination of both of these therapies.

Vaccination with SARS-CoV-2 spike protein lacking glycan shields elicits enhanced protective responses in animal models.

A major challenge to end the pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is to develop a broadly protective vaccine that elicits long-term immunity. As the key immunogen, the viral surface spike (S) protein is frequently mutated, and conserved epitopes are shielded by glycans. Here, we revealed that S protein glycosylation has site-differential effects on viral infectivity. We found that S protein generated by lung epithelial cells has glycoforms associated with increased infectivity. Compared to the fully glycosylated S protein, immunization of S protein with N-glycans trimmed to the mono-GlcNAc-decorated state (SMG) elicited stronger immune responses and better protection for human angiotensin-converting enzyme 2 (hACE2) transgenic mice against variants of concern (VOCs). In addition, a broadly neutralizing monoclonal antibody was identified from SMG-immunized mice that could neutralize wild-type SARS-CoV-2 and VOCs with subpicomolar potency. Together, these results demonstrate that removal of glycan shields to better expose the conserved sequences has the potential to be an effective and simple approach for developing a broadly protective SARS-CoV-2 vaccine.

Sub-3 Å Cryo-EM Structures of Necrosis Virus Particles via the Use of Multipurpose TEM with Electron Counting Camera.

During this global pandemic, cryo-EM has made a great impact on the structure determination of COVID-19 proteins. However, nearly all high-resolution results are based on data acquired on state-of-the-art microscopes where their availability is restricted to a number of centers across the globe with the studies on infectious viruses being further regulated or forbidden. One potential remedy is to employ multipurpose microscopes. Here, we investigated the capability of 200 kV multipurpose microscopes equipped with a direct electron camera in determining the structures of infectious particles. We used 30 nm particles of the grouper nerve necrosis virus as a test sample and obtained the cryo-EM structure with a resolution as high as ∼2.7 Šfrom a setting that used electron counting. For comparison, we tested a high-end cryo-EM (Talos Arctica) using a similar virus (Macrobrachium rosenbergii nodavirus) to obtain virtually the same resolution. Those results revealed that the resolution is ultimately limited by the depth of field. Our work updates the density maps of these viruses at the sub-3Šlevel to allow for building accurate atomic models from de novo to provide structural insights into the assembly of the capsids. Importantly, this study demonstrated that multipurpose TEMs are capable of the high-resolution cryo-EM structure determination of infectious particles and is thus germane to the research on pandemics.

Identification of fidelity-governing factors in human recombinases DMC1 and RAD51 from cryo-EM structures.

Both high-fidelity and mismatch-tolerant recombination, catalyzed by RAD51 and DMC1 recombinases, respectively, are indispensable for genomic integrity. Here, we use cryo-EM, MD simulation and functional analysis to elucidate the structural basis for the mismatch tolerance of DMC1. Structural analysis of DMC1 presynaptic and postsynaptic complexes suggested that the lineage-specific Loop 1 Gln244 (Met243 in RAD51) may help stabilize DNA backbone, whereas Loop 2 Pro274 and Gly275 (Val273/Asp274 in RAD51) may provide an open "triplet gate" for mismatch tolerance. In support, DMC1-Q244M displayed marked increase in DNA dynamics, leading to unobservable DNA map. MD simulation showed highly dispersive mismatched DNA ensemble in RAD51 but well-converged DNA in DMC1 and RAD51-V273P/D274G. Replacing Loop 1 or Loop 2 residues in DMC1 with RAD51 counterparts enhanced DMC1 fidelity, while reciprocal mutations in RAD51 attenuated its fidelity. Our results show that three Loop 1/Loop 2 residues jointly enact contrasting fidelities of DNA recombinases.

A Carbohydrate-Binding Protein from the Edible Lablab Beans Effectively Blocks the Infections of Influenza Viruses and SARS-CoV-2.

The influenza virus hemagglutinin (HA) and coronavirus spike (S) protein mediate virus entry. HA and S proteins are heavily glycosylated, making them potential targets for carbohydrate binding agents such as lectins. Here, we show that the lectin FRIL, isolated from hyacinth beans (Lablab purpureus), has anti-influenza and anti-SARS-CoV-2 activity. FRIL can neutralize 11 representative human and avian influenza strains at low nanomolar concentrations, and intranasal administration of FRIL is protective against lethal H1N1 infection in mice. FRIL binds preferentially to complex-type N-glycans and neutralizes viruses that possess complex-type N-glycans on their envelopes. As a homotetramer, FRIL is capable of aggregating influenza particles through multivalent binding and trapping influenza virions in cytoplasmic late endosomes, preventing their nuclear entry. Remarkably, FRIL also effectively neutralizes SARS-CoV-2, preventing viral protein production and cytopathic effect in host cells. These findings suggest a potential application of FRIL for the prevention and/or treatment of influenza and COVID-19.

Roles of TREM2 in degeneration of human nucleus pulposus cells via NF-κB p65.

Nuclear factor (NF)-κB p65 plays a key role in the development of intervertebral disc degeneration (IDD). Herein, we found that messenger RNA levels of human triggering receptor expressed on myeloid cells-2 (TREM2) and NF-κB p65 were upregulated and strongly positive correlated (r2 = 0.299, P = 0.0126) in nucleus pulposus (NP) tissues of patients with IDD. To investigate the role of TREM2 in the development of IDD and whether NF-κB p65 was the underlying mechanism, whereby TREM2 played its role, we established TREM2-siRNA-transfected human degenerative NP cells and TREM2-overexpression vector-transfected human normal NP cells. Degeneration of human NP cells was assessed by measuring cell apoptosis, cell proliferation, and the secretion of tumor necrosis factor-α, interleukin (IL)-1ß, and IL-6. Protein levels of Bcl2, Bax, total NF-κB p65 in whole cell lysates, cytoplasm NF-κB p65, and nuclear NF-κB p65 were determined to evaluate underlying mechanisms. Our data elucidated that TREM2 silencing was a therapy for human degenerative NP cells through inhibiting cell apoptosis, promoting cell proliferation, suppressing production of tumor necrosis factor-α, IL 1ß, and IL-6, and the mechanisms included decreasing Bax while enhancing Bcl2, downregulating total NF-κB p65, and retarding NF-κB p65 nuclear translocation. On the contrary, upregulated TREM2 showed the opposite effects, accelerating the degeneration of human normal NP cells. Downregulating TREM2 and total NF-κB p65 and inhibiting NF-κB p65 nucleus translocation were also confirmed in NP tissue samples of four IDD rats. We concluded that TREM2 functioned as a promoter in the degeneration of human NP cells. Downregulating TREM2 may be a novel therapeutic strategy for human IDD.

Aggregatibacter (Actinobacillus) actimycetemcomitans leukotoxin and human periodontitis - A historic review with emphasis on JP2.

Aggregatibacter (Actinobacillus) actimycetemcomitans (Aa) is a gram-negative bacterium that colonizes the human oral cavity and is causative agent for localized aggressive (juvenile) periodontitis (AgP). In the middle of 1990s, a specific JP2 clone of belonging to the cluster of serotype b strains of Aa with highly leukotoxicity (leukotoxin, LtxA) able to kill human immune cells was isolated. JP2 clone of Aa was strongly associated with in particularly in rapidly progressing forms of aggressive periodontitis. The JP2 clone of Aa is transmitted through close contacts. Therefore, AgP patients need intense monitoring of their periodontal status as the risk for developing severely progressing periodontitis lesions are relatively high. Furthermore, timely periodontal treatment, including periodontal surgery supplemented by the use of antibiotics, is warranted. More importantly, periodontal attachment loss should be prevented by early detection of the JP2 clone of Aa by microbial diagnosis testing and/or preventive means.

Vimentin is a potential prognostic factor for tongue squamous cell carcinoma among five epithelial-mesenchymal transition-related proteins.

We aimed to investigate the association of the expression levels of five epithelial-mesenchymal transition (EMT)-related proteins (Snail, Twist, E-cadherin, N-cadherin, and Vimentin) with tumorigenesis, pathologic parameters and prognosis in tongue squamous cell carcinoma (TSCC) patients by immunohistochemistry of tissue microarray. The expression levels of Snail, E-cadherin, N-cadherin and Vimentin were significantly different between the tumor adjacent normal and tumor tissues. In tumor tissues, lower E-cadherin and higher N-cadherin levels were associated with a higher grade of cell differentiation, advanced stage of disease, and lymph node metastasis. However, higher Vimentin expression was associated with poor cell differentiation and lymph node metastasis. Patients with low E-cadherin expression had poor disease-specific survival (DSS). Conversely, positive N-cadherin and higher Vimentin expression levels were associated with poor DSS and disease-free survival. Notably, our multivariate Cox regression model indicated that high Vimentin expression was an adverse prognostic factor for DSS in TSCC patients, even after the adjustment for cell differentiation, pathological stage, and expression levels of Snail, Twist, E-cadherin, and N-cadherin. Snail, E-cadherin, N-cadherin, and Vimentin were associated with tumorigenesis and pathological outcomes. Among the five EMT-related proteins, Vimentin was a potential prognostic factor for TSCC patients.

Association of vitamin D receptor gene polymorphisms and periodontitis in a Taiwanese Han population.

BACKGROUND/PURPOSE: : Genetic polymorphisms in the vitamin D receptor (VDR) gene are related to the immune response and bone metabolism, both of which are implicated in the pathogenesis of periodontitis. This study was to investigate the association between VDR-gene polymorphisms and periodontitis among a Taiwanese Han ethnic population. MATERIALS AND METHODS: Ninety-two aggressive periodontitis (AgP), 385 chronic periodontitis (CP) and 163 healthy controls (HC) were recruited from dental clinics. Demographic characteristics and possible confounding factors were obtained using a self-reported questionnaire. The VDR rs731236 (TaqI), rs7975232 (ApaI), rs1544410 (BsmI), and rs2228570 (FokI) polymorphisms were genotyped using PCR-RFLP methods. Statistical analyses were applied to determine the associations. RESULTS: The individual VDR polymorphisms were not associated with risk of AgP and CP. The f allele of rs2228570 was related to decreased risk for AgP. Subjects with TAbF (adjusted OR = 7.2, 95% CI = 3.2-7.2, p < 0.0001) or TAbf (adjusted OR = 0.17, 95% CI = 0.05-0.48, p = 0.002) combined polymorphisms were significantly associated with AgP. Subjects with Tabf (adjusted OR = 2.6, 95% CI = 1.8-3.8, p < 0.0001), TAbF (adjusted OR = 4.4, 95% CI = 2.6-8.1, p < 0.0001), TabF (adjusted OR = 0.36, 95% CI = 0.2-0.5, p < 0.0001), or TAbf (adjusted OR = 0.45, 95% CI = 0.3-0.7, p = 0.001) combined polymorphisms were significantly associated with CP. CONCLUSION: The study indicates that VDR gene polymorphisms are associated with AgP and CP in a Taiwanese Han population.

Comparison of safety and efficiency of microendoscopic discectomy with automatic nerve retractor and with nerve hook.

This study compares the safety and efficiency of two techniques in microendoscopic discectomy (MED) for lumbar disc herniation. The two techniques are MED with automatic nerve retractor and MED with nerve hook which had been widely used for many years. The former involves a newly developed MED device which contains three parts to protect nerve roots during operation. Four hundred and twenty-eight patients underwent MED treatments between October 2010 and September 2015 were recruited and randomized to either intraoperative utilization of automatic nerve retractor (n = 315, group A) or application of nerve hook during surgery (n = 113, group B). Operation time and intraoperative bleeding volume were evaluated. Simultaneously, Visual Analogue Scales (VAS) and muscle strength grading were performed preoperatively, and 1, 2, 3 days, 1, 2 weeks, 3 and 6 months postoperatively. No dramatic difference of pain intensity was observed between the two groups before surgery and 6 months after surgery (P > 0.05). The operation time was shorter in group A (30.30 ± 1.89 min) than that in group B (59.41 ± 3.25 min). Group A (67.83 ± 13.14 ml) experienced a significant decrease in the amount of blood loss volume when compared with group B (100.04 ± 15.10 ml). There were remarkable differences of VAS score and muscle strength grading after postoperative 1, 2, 3 days, 1, 2 weeks and 3 months between both groups (P ≤ 0.05). MED with automatic nerve retractor effectively shortened operation time, decreased the amount of bleeding, down-regulated the incidence of nerve traction injury.

Matrix metalloproteinase-2, -9, and tissue inhibitor of MMP-2 gene polymorphisms in Taiwanese periodontitis patients.

BACKGROUND/PURPOSE: Matrix metalloproteinases (MMPs) and tissue inhibitor of MMPs (TIMPs) have been shown to play an important role in the pathogenesis of tissue destruction in periodontitis. The associations between single nucleotide polymorphisms (SNPs) in the promoter regions of MMP-2, MMP-9, and TIMP-2 genes and the risk of aggressive periodontitis (AgP) and chronic periodontitis (CP) were investigated in a Taiwanese population. MATERIALS AND METHODS: MMP-2 C-1306T, C-735T, T-790G, and MMP-9 C-1562T and TIMP-2 G-418C SNPs were genotyped by polymerase chain reaction-restriction fragment length polymorphism analysis in 69 patients and 129 patients with AgP and CP, respectively, and 117 periodontal healthy individuals who served as healthy controls (HC). Chi-square test and logistic regression analysis were used to investigate the possible association of genotypes with periodontitis. RESULTS: No significant differences in the distributions of the C-1306T and C-735T variants between periodontitis and HC were detected. Patients with genotype of MMP-2 -790 TT or T allele of MMP-2-790T/G as compared to genotypes of GT + GG genotypes or G allele, were less susceptible to CP [odds ratio (OR) = 0.50, 95% confidence interval (CI) = 0.25-1.00 and OR = 0.52, 95% CI = 0.28-0.96, respectively]. The frequencies of TIMP-2 G-418C gene polymorphisms in nonsmokers were statistically significantly different among AgP, CP, and HC groups (P = 0.024). The nonalcohol drinking participants with C allele of MMP-9 C-1562T as compared to T allele, were less susceptible to AgP (adjusted OR = 0.4; 95% CI, 0.18-0.90). CONCLUSION: It is suggested that MMP-2 T-790G, MMP-9 C-1562T, and TIMP-2 G-418C gene polymorphisms might be associated with periodontitis in the Taiwanese Han population.

Clinical Case Report on Treatment of Generalized Aggressive Periodontitis: 5-Year Follow-up.

Generalized aggressive periodontitis (GAgP) is a distinct type of periodontal disease associated with considerably more rapid periodontal tissue destruction than chronic periodontitis. This study presents the 5-year follow-up of a patient with GAgP. A 29-year-old man reported experiencing increasing gingival recession. He was treated using cause-related therapy, provisional splints, and flap surgery combined with allograft grafting and was followed up for 5 years. This case study shows that elimination of infectious microorganisms and meticulous long-term maintenance provide an effective treatment modality for aggressive periodontitis cases. This treatment modality can restore the masticatory function and provide the GAgP patient with improved quality of life.

Simple preparation of carbon nanofibers with graphene layers perpendicular to the length direction and the excellent li-ion storage performance.

Sulfur-containing carbon nanofibers with the graphene layers approximately vertical to the fiber axis were prepared by a simple reaction between thiophene and sulfur at 550 °C in stainless steel autoclaves without using any templates. The formation mechanism was discussed briefly, and the potential application as anode material for lithium-ion batteries was tentatively investigated. The carbon nanofibers exhibit a stable reversible capacity of 676.8 mAh/g after cycling 50 times at 0.1 C, as well as the capacities of 623.5, 463.2, and 365.8 mAh/g at 0.1, 0.5, and 1.0 C, respectively. The excellent electrochemical performance could be attributed to the effect of sulfur. On one hand, sulfur could improve the reversible capacity of carbon materials due to its high theoretical capacity; on the other hand, sulfur could promote the formation of the unique carbon nanofibers with the graphene layers perpendicular to the axis direction, favorable to shortening the Li-ion diffusion path.

Impact of removable dentures on oral health-related quality of life among elderly adults in Taiwan.

BACKGROUND: Although the use of removable dentures can improve oral function and esthetics for elderly people, compared to those who do not wear removable dentures, those wearing removable dentures could have worse oral health related-quality of life (OHRQoL). Additional information is required to assess which factors related to denture wearing influence the OHRQoL of elderly individuals. The purpose of this study is to evaluate the association between denture wearing and OHRQoL in a sample of elderly individuals in Taiwan. METHODS: The study population included 277 elderly people wearing removable dentures (mean age = 76.0 years). Using face-to-face interviews, we collected data on the participants' socio-demographic characteristics, dental care service usage (regular dental checkups, treatment during toothache, dental visits in the last year), and factors related to denture wearing (perceived oral pain, perceived loose denture, perceived oral ulcer, perceived halitosis, perceived dry mouth, and perceived total denture satisfaction scores). OHRQoL was measured using the Taiwanese version of the Geriatric Oral Health Assessment Index (GOHAI-T). The location and number of remaining natural teeth and the type of denture were also recorded. Hierarchical multiple regression analysis was performed using GOHAI-T scores as the dependent variable. RESULTS: All the predictors together accounted for 50% of the variance in GOHAI-T scores. Further, education level, number of natural teeth, denture status, perceived loose denture, perceived oral ulcer, and perceived total denture satisfaction scores had statistically significant influences on OHRQoL. When compared with other variables, factors related to denture wearing, especially perceived total denture satisfaction scores, had the greatest impact on GOHAI-T scores. CONCLUSIONS: Of the factors analyzed in this study, denture satisfaction was the strongest predictor of OHRQoL. This suggests that denture satisfaction is useful for assessing the effect of denture treatment on the OHRQoL of elderly individuals wearing removable dentures.