Effectiveness of a broad-spectrum bivalent mRNA vaccine against SARS-CoV-2 variants in preclinical studies.

Vaccines utilizing modified messenger RNA (mRNA) technology have shown robust protective efficacy against SARS-CoV-2 in humans. As the virus continues to evolve in both human and non-human hosts, risk remains that the performance of the vaccines can be compromised by new variants with strong immune escape abilities. Here we present preclinical characterizations of a novel bivalent mRNA vaccine RQ3025 for its safety and effectiveness in animal models. The mRNA sequence of the vaccine is designed to incorporate common mutations on the SARS-CoV-2 spike protein that have been discovered along the evolutionary paths of different variants. Broad-spectrum, high-titer neutralizing antibodies against multiple variants were induced in mice (BALB/c and K18-hACE2), hamsters and rats upon injections of RQ3025, demonstrating advantages over the monovalent mRNA vaccines. Effectiveness in protection against several newly emerged variants is also evident in RQ3025-vaccinated rats. Analysis of splenocytes derived cytokines in BALB/c mice suggested that a Th1-biased cellular immune response was induced by RQ3025. Histological analysis of multiple organs in rats following injection of a high dose of RQ3025 showed no evidence of pathological changes. This study proves the safety and effectiveness of RQ3025 as a broad-spectrum vaccine against SARS-CoV-2 variants in animal models and lays the foundation for its potential clinical application in the future.

Both chimpanzee adenovirus-vectored and DNA vaccines induced long-term immunity against Nipah virus infection.

Nipah virus (NiV) is a highly lethal zoonotic paramyxovirus that poses a severe threat to humans due to its high morbidity and the lack of viable countermeasures. Vaccines are the most crucial defense against NiV infections. Here, a recombinant chimpanzee adenovirus-based vaccine (AdC68-G) and a DNA vaccine (DNA-G) were developed by expressing the codon-optimized full-length glycoprotein (G) of NiV. Strong and sustained neutralizing antibody production, accompanied by an effective T-cell response, was induced in BALB/c mice by intranasal or intramuscular administration of one or two doses of AdC68-G, as well as by priming with DNA-G and boosting with intramuscularly administered AdC68-G. Importantly, the neutralizing antibody titers were maintained for up to 68 weeks in the mice that received intramuscularly administered AdC68-G and the prime DNA-G/boost AdC68-G regimen, without a significant decline. Additionally, Syrian golden hamsters immunized with AdC68-G and DNA-G via homologous or heterologous prime/boost immunization were completely protected against a lethal NiV virus challenge, without any apparent weight loss, clinical signs, or pathological tissue damage. There was a significant reduction in but not a complete absence of the viral load and number of infectious particles in the lungs and spleen tissue following NiV challenge. These findings suggest that the AdC68-G and DNA-G vaccines against NiV infection are promising candidates for further development.

Vaccines based on the fusion protein consensus sequence protect Syrian hamsters from Nipah virus infection.

Nipah virus (NiV), a bat-borne paramyxovirus, results in neurological and respiratory diseases with high mortality in humans and animals. Developing vaccines is crucial for fighting these diseases. Previously, only a few studies focused on the fusion (F) protein alone as the immunogen. Numerous NiV strains have been identified, including 2 representative strains from Malaysia (NiV-M) and Bangladesh (NiV-B), which differ significantly from each other. In this study, an F protein sequence with the potential to prevent different NiV strain infections was designed by bioinformatics analysis after an in-depth study of NiV sequences in GenBank. Then, a chimpanzee adenoviral vector vaccine and a DNA vaccine were developed. High levels of immune responses were detected after AdC68-F, pVAX1-F, and a prime-boost strategy (pVAX1-F/AdC68-F) in mice. After high titers of humoral responses were induced, the hamsters were challenged by the lethal NiV-M and NiV-B strains separately. The vaccinated hamsters did not show any clinical signs and survived 21 days after infection with either strain of NiV, and no virus was detected in different tissues. These results indicate that the vaccines provided complete protection against representative strains of NiV infection and have the potential to be developed as a broad-spectrum vaccine for human use.

The Effect of Mobile Social Media Sharing Behavior on the Subjective Well-Being and Mental Health Regulation of Internet Users.

Mobile Healthcare Social Media (MHSM) is an innovative combination of mobile devices and mobile communication technology. How can users shift from spontaneous to conscious cognitive mode, break their usage inertia, and actively adapt to the social structure of mobile health social media to improve information-sharing performance? This study uses adaptive structuring theory as the theoretical foundation to deeply analyze adaptive information-sharing behavior and its specific forms in the mHealth social media environment; it uses cognitive shift theory and social exchange theory as the theoretical framework to comprehensively explore the antecedent motivations of users engaging in adaptive information-sharing behavior and its posterior effects. Theoretically, it promotes the innovation and development of information-sharing behavior research and the further deepening of the application of adaptive structuring theory at the information behavior level. It is also conducive to bridging the digital divide and maximizing the value of health information resources. This paper takes 1000 survey data as the experimental data source for studying the influence of mobile social media sharing behavior on the subjective well-being and mental health regulation of Internet users and concludes that in practice, it is beneficial to optimize the design of information-sharing-related functions in mobile health social media, improve the effect of user information sharing in mobile health social media environment, and enhance the efficiency of information sharing so that mobile social media sharing behavior can better contribute to the subjective well-being and mental health regulation of Internet users. This paper has an obvious psychological adjustment effect on groups who use too much social media and can help them analyze why they are affected by some social media, thereby producing psychological effects.

Recombinant chimpanzee adenovirus vector vaccine expressing the spike protein provides effective and lasting protection against SARS-CoV-2 infection in mice.

SARS-CoV-2 infection is a global public health threat. Vaccines are considered amongst the most important tools to control the SARS-CoV-2 pandemic. As expected, deaths from SARS-CoV-2 infection have dropped dramatically with widespread vaccination. However, there are concerns over the duration of vaccine-induced protection, as well as their effectiveness against emerging variants of concern. Here, we constructed a recombinant chimpanzee adenovirus vectored vaccine expressing the full-length spike of SARS-CoV-2 (AdC68-S). Rapid and high levels of humoral and cellular immune responses were observed after immunization of C57BL/6J mice with one or two doses of AdC68-S. Notably, neutralizing antibodies were observed up to at least six months after vaccination, without substantial decline. Single or double doses AdC68-S immunization resulted in lower viral loads in lungs of mice against SARS-CoV-2 challenge both in the short term (21 days) and long-term (6 months). Histopathological examination of AdC68-S immunized mice lungs showed mild histological abnormalities after SARS-CoV-2 infection. Taken together, this study demonstrates the efficacy and durability of the AdC68-S vaccine and constitutes a promising candidate for clinical evaluation.

Pseudorabies virus glycoprotein gE suppresses interferon-ß production via CREB-binding protein degradation.

Cyclic GMP-AMP synthase (cGAS) is a main sensor used to detect microbial DNA in the cytoplasm, which subsequently induces the production of interferon (IFN) via the cGAS/STING/IRF3 signaling pathway, leading to an antiviral response. However, some viruses have evolved multiple strategies to escape this process. Pseudorabies virus (PRV) is a double-stranded DNA virus belonging to the Alphaherpesvirinae subfamily, which can cause serious damage to the porcine industry. Many herpesvirus components have been reported to counteract IFN production, whereas little is known of PRV. In the present study, we found that PRV glycoprotein E (gE) was involved in counteracting cGAS/STING-mediated IFN production. Ectopic expression of gE decreased cGAS/STING-mediated IFN-ß promoter activity and the level of mRNA expression. Moreover, gE targeted at or downstream of IRF3 was found to inhibit IFN-ß production. However, gE did not affect the phosphorylation, dimerization and nuclear translocation of IRF3. Furthermore, gE is located on the nuclear membrane and could subsequently degrade CREB-binding protein (CBP). MG132, a proteasome inhibitor, decreased CBP degradation and restored the IFN-ß production induced by gE. Finally, gE-deleted PRV induced a higher level of IFN-ß production and reduced CBP degradation compared to wild-type PRV. Together, these results demonstrate that PRV gE can inhibit cGAS/STING-mediated IFN-ß production by degrading CBP to interrupt the enhanced assembly of IRF3 and CBP.

Nuclear transporter karyopherin subunit alpha 3 levels modulate Porcine circovirus type 2 replication in PK-15 cells.

Entering the nucleus is important for Porcine circovirus type 2 (PCV2) replication. Karyopherins (KPNs) mediate the nuclear import of many cytoplasmic proteins. Our previous study showed that KPNA3 is involved in interferon production during PCV2 infection induced by Poly I:C and ISD (Interferon stimulatory DNA). However, it remains unclear whether PCV2 replication is associated with KPNA3. In the present study, knockdown of KPNA3 promoted the replication of PCV2, whereas overexpression of KPNA3 inhibited PCV2 replication in PK-15 cells. Furthermore, KPNA3 knockdown inhibited IRF3 and reduced the expression of antiviral genes including IFN-ß, ISG54, Mx1 and ISG56, while the opposite results were obtained after KPNA3 overexpression. KPNA3 knockdown also promoted p65 nuclear translocation and increased the mRNA expression of IL-10 and IL-1ß. These results suggested that KPNA3 facilitates IRF3 entry into the nucleus and the production of an antiviral response, resulting in PCV2 replication inhibition and blockage of NF-κB signal activation.

Lack of antibody-mediated cross-protection between SARS-CoV-2 and SARS-CoV infections.

BACKGROUND: The novel coronavirus (SARS-CoV-2) shares approximately 80% whole genome sequence identity and 66% spike (S) protein identity with that of SARS-CoV. The cross-neutralization between these viruses is currently not well-defined. METHODS: Here, by using the live SARS-CoV-2 virus infection assay as well as HIV-1 based pseudotyped-virus carrying the spike (S) gene of the SARS-CoV-2 (ppSARS-2) and SARS-CoV (ppSARS), we examined whether infections with SARS-CoV and SARS-CoV-2 can induce cross-neutralizing antibodies. FINDINGS: We confirmed that SARS-CoV-2 infects cells via angiotensin converting enzyme 2 (ACE2), the functional receptor for SARS-CoV, and we also found that the recombinant receptor binding domain (RBD) of the S protein of SARS-CoV effectively inhibits ppSARS-2 entry in Huh7.5 cells. However, convalescent sera from SARS-CoV and SARS-CoV-2 patients showed high neutralizing activity only against the homologous virus, with no or limited cross-neutralization activity against the other pseudotyped virus. Similar results were also observed in vaccination studies in mice. INTERPRETATION: Our study demonstrates that although both SARS-CoV and SARS-CoV-2 use ACE2 as a cellular receptor, the neutralization epitopes are not shared by these two closely-related viruses, highlighting challenges towards developing a universal vaccine against SARS-CoV related viruses. FUNDING: This work was supported by the National Key Research and Development Program of China, the National Major Project for Control and Prevention of Infectious Disease in China, and the One Belt and One Road Major Project for infectious diseases.

Delayed specific IgM antibody responses observed among COVID-19 patients with severe progression.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread rapidly worldwide since it was confirmed as the causative agent of COVID-19. Molecular diagnosis of the disease is typically performed via nucleic acid-based detection of the virus from swabs, sputum or bronchoalveolar lavage fluid (BALF). However, the positive rate from the commonly used specimens (swabs or sputum) was less than 75%. Immunological assays for SARS-CoV-2 are needed to accurately diagnose COVID-19. Sera were collected from patients or healthy people in a local hospital in Xiangyang, Hubei Province, China. The SARS-CoV-2 specific IgM antibodies were then detected using a SARS-CoV-2 IgM colloidal gold immunochromatographic assay (GICA). Results were analysed in combination with sera collection date and clinical information. The GICA was found to be positive with the detected 82.2% (37/45) of RT-qPCR confirmed COVID-19 cases, as well as 32.0% (8/25) of clinically confirmed, RT-qPCR negative patients (4-14 days after symptom onset). Investigation of IgM-negative, RT-qPCR-positive COVID-19 patients showed that half of them developed severe disease. The GICA was found to be a useful test to complement existing PCR-based assays for confirmation of COVID-19, and a delayed specific IgM antibody response was observed among COVID-19 patients with severe progression.

Short-term efficacy and side effects of bevacizumab or rh-endostatin combined with chemotherapy in treatment of locally advanced EGFR wild-type non-small cell lung cancer / 中国肿瘤生物治疗杂志

@# Objective: To investigate the short-term efficacy and toxicity of bevacizumab combined with DP or rh-endostatin（recombinant human vascular endostatin injection）combined with DP in locally advanced EGFR wild-type non-small cell lung cancer (NSCLC). Methods: Seventy-two patients with treatment of locally advanced EGFR wild-type NSCLC admitted to the Department of Respiratory Medicine of Zhongshan Hospital Affiliated to Guangdong Medical University from January 2014 to January 2017 were divided into bevacizumab group (34 cases) and rh-endostatin group (38 cases) according to the random number method. The former group was treated with bevacizumab combined with docetaxel and cisplatin, while the latter was treated with rh-endostatin combined with docetaxel and cisplatin. According to RECISIT 1.1 standard, the changes of lesion size before and after treatment in two groups were evaluated. Serum levels of vascular endothelial growth factor (VEGF), carcinoembryonic antigen (CEA), cytokeratin 21-1 fragment (CYFRA21-1), squamous cell carcinoma antigen (SCC) were measured. The adverse reactions during treatment were also evaluated. Results: In bevacizumab group, patients with CR, PR, SD, PD, DCR and ORR were 2 cases, 12 cases, 15 cases, 5 cases, 41.18% and 85.29%, respectively. In rh-endostatin group, patients with CR, PR, SD, PD, DCR, ORR were 2 cases, 16 cases, 14 cases, 6 cases, 47.37% and 84.21%, respectively. The DCR in rh-endostatin group was significantly higher than that in bevacizumab group (P<0.05).The serum levels of VEGF and CEAin rh-endostatin group decreased more obvious than those in bevacizumab group (all P<0.05). The incidence of gastrointestinal reaction, skin reaction and cardiac toxicity in rh-endostatin group was higher than that in bevacizumab group, while the incidence of bleeding in bevacizumab group was higher than that in rh-endostatin group (all P<0.05). Conclusion: In patients with locally advanced EGFR wild-type NSCLC, rh-endostatin combined with DP regimen is better than bevacizumab combined with DPregimen. In clinical practice, corresponding treatment regimen can be selected according to different characteristics of patients, so as to minimize the toxic reaction during treatment and avoid clinical risk.

PCV2 infection activates the cGAS/STING signaling pathway to promote IFN-ß production and viral replication in PK-15 cells.

PCV2 is a single-stranded DNA virus that we previously found to induce IFN-ß production via RIG-I and MDA-5. cGAS is known to be the most important DNA sensor for the recognition of cytosolic DNA; however, it remains unclear whether the interferon production induced by PCV2 is associated with cGAS. In the present study, PCV2 infection was found to increase the level of cGAS and STING expression, promote the release of cyclic dinucleotide cGAMP, and induce STING dimerization and translocation into the nucleus of PK-15 cells. These findings indicate that PCV2 infection activates both cGAS and STING. Furthermore, the knockdown of cGAS and STING decreased both the mRNA expression and promoter activity of IFN-ß, demonstrating that the cGAS/STING signaling pathway contributes to the production of IFN-ß. In addition, a knockdown of cGAS and STING also decreased the PCV2 viral load and infectivity. Therefore, PCV2 infection activates the cGAS/STING signaling pathway to induce IFN-ß production and the knockdown of cGAS and STING decreases viral replication in PK-15 cells. These results provide further insight into the relationship between PCV2 and host innate immunity.

Porcine circovirus type 2 inhibits inter-ß expression by targeting Karyopherin alpha-3 in PK-15 cells.

Interferon (IFN)-mediated antiviral response is an important part of host defense. Previous studies reported that porcine circovirus type 2 (PCV2) inhibits interferon production, but the mechanism is still poorly understood. In this study, PCV2 suppresses IFN-ß and IRF3 promoters and mRNA level of IFN-ß induced by ISD or Poly(I:C), but has no effect on the activation of AP-1 and NF-κB. Furthermore, PCV2 decreases the mRNA level of IFN-ß and IFN-ß promoter activity driven by STING, TBK1, IRF3, and IRF3/5D, and causes a reduction in the protein level of nuclear p-IRF3. In addition, PCV2 interrupts the interaction of KPNA3, rather than KPNA4, with p-IRF3. Overexpression of KPNA3 restores IFN-ß promoter activity. These results indicate that PCV2 disrupts the interaction of KPNA3 with p-IRF3 and blocks p-IRF3 translocation to the nucleus, thereby inhibiting IFN-ß induction in PK-15 cells.

RIG-1 and MDA-5 signaling pathways contribute to IFN-ß production and viral replication in porcine circovirus virus type 2-infected PK-15 cells in vitro.

Type I Interferons (IFNs) is known for its antiviral activity; however, it is surprising that in vitro treatment of IFN-α and IFN-γ enhanced the replication of porcine circovirus type 2 (PCV2), indicating a complex relationship between interferon and PCV2. To date, it remains poorly understood how the interferon is produced during PCV2 infection and whether the interferon induced by PCV2 itself can promote viral replication. In this study, PCV2 induced the up-regulation of IFN-ß in PK-15 cells, while treatment of PCV2-infected cells with the interferon regulatory factor-3 (IRF3) inhibitor, BX795, decreased the expression of IFN-ß, whereas treatment with the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) inhibitor, BAY11-7082, did not. These findings indicate that PCV2 can induce IFN-ß production via the IRF3-mediated rather than the NF-κB-mediated signal pathway. Moreover, PCV2 increased the protein expression levels of phosphorylation-IRF3 (p-IRF3), mitochondria antiviral-signaling protein (MAVS), retinoic acid-inducible gene I (RIG-1) and melanoma differentiation-associated protein 5 (MDA-5), and the knockdown of RIG-1 and MDA-5 decreased the expression level of IFN-ß in PK-15 cells. Therefore, PCV2 induces IFN-ß production via the RIG-1/MDA-5/MAVS/IRF signaling pathway. Furthermore, the PCV2 load and PCV2 infectivity decreased after knockdown of RIG-1 and MDA-5, indicating that RIG-1 and MDA-5 signaling pathways contribute to PCV2 replication. In conclusion, PCV2 induces the production of IFN-ß via the RIG-1 and MDA-5 signaling pathways, and the IFN-ß produced during PCV2 infection facilitates viral replication. These results will help us further understand the pathogenic mechanisms of PCV2.

Co-circulation of Hantaan, Kenkeme, and Khabarovsk Hantaviruses in Bolshoy Ussuriysky Island, China.

Hemorrhagic fever with renal syndrome (HFRS) was first recognized in far eastern Asia in the 1930s, and has been highly prevalent in this region ever since. To reveal the molecular epidemiology of hantaviruses in this region, a total of 374 small mammals (eight species of rodents and one species of shrew) were captured in the Chinese part of the Bolshoy Ussuriysky Island (Heilongjiang Province). Hantavirus sequences were recovered from three striped field mice (Apodemus agrarius), 11 Maximowicz's voles (Microtus maximowiczii), and one flat-skulled shrew (Sorex roboratus). Genetic and phylogenetic analysis revealed the presence of three viruses: Hantaan virus (HTNV), Khabarovsk virus (KHAV), and Kenkeme virus (KKMV). HTNV sequences recovered from A. agrarius were closely related to those identified in Apodemus mice from the surrounding areas, while a new lineage of KHAV was present in M. maximowiczii. Additionally, while the viral sequences recovered from one flat-skulled shrew were most closely related to KKMV, their divergence to the prototype strain suggests that they represent a new viral subtype. Overall, these results suggest that Bolshoy Ussuriysky Island harbors considerable hantavirus diversity.

Closure-based density functional theory applied to interfacial colloidal fluids.

The behavior of dense colloidal fluids near surfaces can now be probed in great detail with experimental techniques like confocal microscopy. In fact, we are approaching a point where quantitative comparisons of experiment with particle-level theory, such as classical density functional theory (DFT), are appropriate. In a forward sense, we may use a known surface potential to predict a particle density distribution function from DFT; in an inverse sense, we may use an experimentally measured particle density distribution function to predict the underlying surface potential from DFT. In this paper, we tested the ability of the closure-based DFT of Zhou and Ruckenstein (J. Chem. Phys. 2000, 112, 8079-8082) to perform forward and inverse calculations on potential models commonly employed for colloidal particles and surfaces. To reduce sources of uncertainty in this initial study, Monte Carlo simulation results played the role of experimental data. The combination of Rogers-Young and modified-Verlet closures consistently performed well across the different potential models. For a reasonable range of choices of the density, temperature, and potential parameters, the inversion procedure yielded particle-surface potentials to an accuracy on the order of 0.1kT.

Interfacial colloidal sedimentation equilibrium. II. Closure-based density functional theory.

In Part I [R. E. Beckham and M. A. Bevan, J. Chem. Phys. 127, 164708 (2007)], results were presented for the sedimentation equilibrium of concentrated colloidal dispersions using confocal scanning laser microscopy experiments, Monte Carlo (MC) simulations, and a local density approximation perturbation theory. In this paper, we extended the modeling effort on those systems to include nonlocal density functional theory (DFT), which is capable of predicting the microstructure of the sediment at length scales comparable to the colloidal particle dimension. Specifically, we use a closure-based DFT formulation to predict interfacial colloidal sedimentation equilibrium density profiles. The colloid-colloid and colloid-surface interactions were modeled with DLVO screened electrostatic potentials using parameters taken directly from the experimental work. The DFT profiles were compared to the experimental and MC results from Part I. Good agreement was found for relatively dilute interfacial colloidal fluids, but agreement was less satisfactory as interfacial layering became more pronounced for conditions approaching the onset of interfacial crystallization. We also applied DFT in an inverse sense using the measured colloid density profile to extract the underlying colloid-surface potential; this can be thought of as a microscopic analog to the well-known procedure of using the macroscopic (coarse-grained) density profile to extract the osmotic equation of state. For the dilute interfacial fluid, the inverse DFT calculations reproduced the true colloid-surface potential to within 0.5kT at all elevations.

[Change of Bcl-2 expression and telomerase during apoptosis induced by oridonin on human hepatocelluar carcinoma cells].

OBJECTIVE: To investigate the change of Bcl-2 expression and telomerase during the apoptosis induced by oridonin on human hepatocelluar carcinoma BEL7402 cells. METHOD: BEL-7402 cells in culture medium were given 8,16,24,32 micromol x L(-1) different concentrations of oridonin. The cell apoptotic rate was detected by flow cytometry (FCM), morphology of cell apoptosis was observed by Hoechst 3325 staining. Bcl-2 and Bax expressions were detected by Western blotting. Reverse transcriptase polymerase chain reaction (RT-PCR) and PCR enzyme-linked immunosorbent assay (ELISA) were used to detect hTERT mRNA expression and telomerase activity. RESULT: Oridonin induced BEL-7402 cells apoptosis significantly, and the apoptosis rate was both in time-and dose-dependent manner. Marked morphological changes of cell apoptosis were observed very clearly by Hoechst 33258 staining after the cells exposed to oridonin for 60 hours; Western blotting showed that Bcl-2 expression was down-regulated and Bax expression up-regulated concurrently along with the apoptotic process, and the expression of hTERT mRNA as well as activity of telomerase were decreased concurrently. CONCLUSION: Oridonin could decrease the expression of hTERT mRNA and telomerase activity as well as down-regulation of Bcl-2 and up-regulation of Bax expression during the apoptosis of BEL-7402 cells.

Inverse density-functional theory as an interpretive tool for measuring colloid-surface interactions in dense systems.

Recent advances in optical microscopy, such as total internal reflection and confocal scanning laser techniques, now permit the direct three-dimensional tracking of large numbers of colloidal particles both near and far from interfaces. A novel application of this technology, currently being developed by one of the authors under the name of diffusing colloidal probe microscopy (DCPM), is to use colloidal particles as probes of the energetic characteristics of a surface. A major theoretical challenge in implementing DCPM is to obtain the potential energy of a single particle in the external field created by the surface, from the measured particle trajectories in a dense colloidal system. In this paper we develop an approach based on an inversion of density-functional theory (DFT), where we calculate the single-particle-surface potential from the experimentally measured equilibrium density profile in a nondilute colloidal fluid. The underlying DFT formulation is based on the recent work of Zhou and Ruckenstein [Zhou and Ruckenstein, J. Chem. Phys. 112, 8079 (2000)]. For model hard-sphere and Lennard-Jones systems, using Monte Carlo simulation to provide the "experimental" density profiles, we found that the inversion procedure reproduces the true particle-surface-potential energy to an accuracy within typical DCPM experimental limitations (approximately 0.1 kT) at low to moderate colloidal densities. The choice of DFT closures also significantly affects the accuracy.