Glaucoma is the leading cause of irreversible blindness worldwide, and pathologically elevated intraocular pressure (IOP) is the major risk factor. Neuroprotection is one of the potential therapies for glaucomatous retinal damage. Intravitreal mesenchymal stem cell (MSC) transplantation provides a viable therapeutic option, and human umbilical cord- (hUC-) MSCs are attractive candidates for cell-based neuroprotection. Here, we investigated the ability of transplanted hUC-MSCs to survive and migrate within the vitreous cavity and their neuroprotective effects on chronic glaucomatous retina. For this, we developed a chronic ocular hypertension (COH) rat model through the intracameral injection of allogeneic Tenon's fibroblasts. Green fluorescent protein-transduced hUC-MSCs were then injected into the vitreous cavity one week after COH induction. Results showed that a moderate IOP elevation lasted for two months. Transplanted hUC-MSCs migrated toward the area of damaged retina, but did not penetrate into the retina. The hUC-MSCs survived for at least eight weeks in the vitreous cavity. Moreover, the hUC-MSCs were efficient at decreasing the loss of retinal ganglion cells; retinal damage was attenuated through the inhibition of apoptosis. In this study, we have developed a novel COH rat model and demonstrated the prolonged neuroprotective potential of intravitreal hUC-MSCs in chronic glaucoma. Glaucoma comprises a group of diseases causing irreversible blindness characterized by the progressive loss of retinal ganglion cells (RGCs) and their axons. Elevated intraocular pressure (IOP) is one of the most important risk factors for glaucomatous optic neuropathy. However, the common pharmacological or surgical treatments to reduce IOP do not halt progressive visual loss ; therefoThe primary obstacle limiting stem cell therapy is the identification of an appropriate cell source. Mesenchymal stem cells (MSCs) of different origins have shown the greatest potential in the treatment of optical neuropathy –6. For eTo study the mechanisms of glaucomatous damage, the widely used experimental approach is to induce elevated IOP in animals –19. InduIn the present study, we developed a novel method for inducing elevated IOP and preliminarily evaluated the reliability of this rat model of chronic glaucoma. Then, hUC-MSCs marked with GFP were transplanted intravitreally, to investigate whether transplanted hUC-MSCs could survive and migrate in the vitreous cavity. We also demonstrated long-term neuroprotective efficacy of transplanted hUC-MSCs in the novel model, through the evaluation of RGC loss, retinal damage, and apoptosis in retinal cells caused by IOP elevations.The study strictly adhered to the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research. The experimental protocols were approved by the experimental animal ethics committee of Xiamen University. Adult (8-12 weeks old) wild-type Sprague-Dawley rats were used in this study. In addition, two transgenic GFP-expressing Sprague-Dawley rats were used as donors of Tenon's fibroblasts. All rats were housed in light- and temperature-controlled conditions.In all rats, the right eye was injected intracamerally with allogeneic Tenon's fibroblasts (suspended in PBS) and used as the experimental COH eye, and the contralateral eye was injected intracamerally with PBS and served as the control group. One week after the induction of IOP elevation, all the IOP-induced rats were randomly divided into 2 groups as follows: hUC-MSCs were washed with phosphate buffer saline (PBS) and resuspended in PBS and intravitreally transplanted to the right eyes of 24 rats (COH+hUC-MSC group). PBS was intravitreally injected to the right eyes in another 18 rats that served as the COH group. All of the rats were sacrificed after 8 weeks.The GFP-Tenon's fibroblasts were isolated from the eyes of transgenic rat that ubiquitously expressed GFP. Briefly, the superficial layer of Tenon's capsule, attached to the sclera, in the eyes of the GFP-rat was isolated under an operating microscope, washed with Ca2+- and Mg2+-free Hank's solution  and digested by 0.2% dispase II . The isolated cells were incubated in humidified 95% air/5% CO2, detached with trypsin/EDTA solution and diluted 1 : 3 upon reaching 80% confluence. Cell culture was performed in Dulbecco's modified Eagle's medium , supplemented with 10% FBS and 1% penicillin/streptomycin.μL) was injected into the anterior chamber using a 30-gauge needle. Care was taken to avoid any injury to tissues inside the eye.GFP-labeled fibroblasts from passage 2 were used for cell injection. After general anesthesia, pupillary dilation of the right eye was achieved with 0.5% tropicamide. A suspension of Tenon's fibroblasts  in awake rats without anesthesia. Six measurements were consecutively performed according to instrument requirements, and the mean of the second through sixth readings was automatically calculated and then recorded. Such measurements were repeated five times on each eye at each time point. On each occasion, the IOP for each given rat was measured at the same time of day.High IOP of rats was maintained for 8 weeks, and the optic nerve of the eyeball was completely removed under anesthesia. The frozen section of optic nerve was stained with 2% toluidine blue. Optic nerve morphology changes were assessed under a bright-field microscope. For TEM, following 12 weeks of elevated IOP, optic nerve tissues were sectioned and counterstained with uranyl acetate and lead citrate. Images were acquired on a JEM-2100 electron microscope  operated at an accelerating voltage of 120 kV for morphology analysis.The use of the human umbilical cords for research was approved by the local ethics committee, and informed consent was obtained from all donors, following explanation of the research. The donors were pregnant women with no congenital diseases, hepatitis, syphilis, AIDS, or other infectious diseases, and the gestational ages were 37–40 weeks. The hUC-MSCs were isolated as previously described . BrieflyThe cells were labeled with antibodies against CD29-phycoerythrin (PE), CD44-fluorescein isothiocyanate (FITC), CD105-FITC, CD34-FITC, CD45-FITC, HLA-DR-PE, CD80-FITC, CD86-FITC, and CD31-FITC. Flow cytometry was performed using a FACS Calibur , and the data were analyzed with the Cell Quest software (BD Biosciences).μl DMEM-F12 (10% FBS) for 12 h. The GFP-gene-expressing lentiviral vectors  were added to the medium supplemented with 8 μg/ml of polybrene. Viral particles were removed after 24 h exposure. Two days later, hUC-MSCs were assessed under an inverted fluorescent microscope .To label hUC-MSCs, cells were placed in 12-well plates at a density of 105 cells per well in 500 μl, 20,000 cells/μl) was gently injected into the vitreous cavity with a 30-gauge Hamilton needle through the pars plana of the globe. The pinhole was covered up with erythromycin ointment [One week after COH induction, GFP-labeled hUC-MSCs were transplanted intravitreally into the COH eyes. After general and topical anesthesia, as described above, the hUC-MSC suspension (5 ointment .μm thickness) along the vertical meridian of the eyeballs through the optic nerve head. Tissue preparations were processed for either hematoxylin and eosin or toluidine blue or were examined under fluorescence microscope .The rats were sacrificed 60 days after hUC-MSC transplantation. The eyes were enucleated and fixed in 4% paraformaldehyde for 50 minutes at room temperature and then infiltrated with 30% sucrose/PBS for 24 h at 4°C. The whole-eye tissues were embedded in optimal cutting temperature compound. The frozen specimens were sectioned (7 μL each) into the superior colliculus at a point 6.0 mm caudal to the bregma and 1.0 mm lateral to the midline on both sides to a depth of 5.0 mm from the surface of the skull.The retrograde labeling of RGCs with Fluorogold was performed as previously described . RGCs ofSeven days after Fluorogold retrograde labeling, rats were sacrificed and the eyes were enucleated and fixed in 4% paraformaldehyde in PBS for 1 hour in the dark at 4°C. The retinas were dissected free, flat-mounted on a glass slide, and observed under the Leica fluorescence microscope. Manual RGC counting, by an investigator masked to the experimental procedures, was conducted in the superior, inferior, temporal, and nasal quadrants at distances of 1, 2, and 3 mm radially away from the optic nerve. Cells with intense dye staining and smaller or larger size than normal were considered to be microglia or macrophages and were excluded from the analysis .μm sections along the vertical meridian of the eye through the optic nerve head and stained with hematoxylin and eosin. Sections were examined under a light microscope [The cornea, lens, and vitreous of the rat were carefully removed, and the posterior eyecups were embedded in optimal cutting temperature compound. The frozen specimens of rat retinas were cut into 7 croscope .μg of total protein was dissolved in 12%-SDS gels, transferred to polyvinylidene fluoride membranes , and incubated overnight at 4°C with primary antibodies against caspase-8  and β-actin . Protein bands were detected with horseradish peroxidase-conjugated goat anti-rabbit IgG  and visualized by enhanced chemiluminescence reagents and quantified with the Quantity One software (Bio-Rad).Protein extraction and Western blots were performed as reported previously . In briep < 0.05.Data were expressed as mean ± standard deviation. All statistical analyses were performed using SPSS . One-way analysis of variance (ANOVA) was performed followed by the Student–Newman–Keuls test for multiple comparisons. Statistical significance was set at We first developed a rat COH model based on an intracameral injection of allogenic Tenon's capsule fibroblasts . To miniNext, the GFP-Tenon's fibroblasts were intracamerally injected. These cells were visible in the anterior chamber angle and the pupil at 3 and 7 days postinjection, under a Nikon AZ100 motorized zoom fluorescence microscope. Compared with the normal left eye, the iris at the edge of the pupil of the treated right eye was attached to the lens, resulting in pupil atresia, which may have further led to aqueous humor gathering in the posterior chamber. There was a very pronounced iris-bulge formation under ophthalmic operating microscope at 7 days . Two weeTo assess the reliability of the rat model, the induced elevation of IOP and the optic nerve injury were investigated. The injection of GFP-Tenon's fibroblasts into the anterior chamber (COH group) induced a prolonged and significant rise in IOP relative to the contralateral (left) eye. The increase in IOP was maintained throughout the duration of the experiment. Across animals, the mean IOP, averaged over the length of the experiment, for Tenon's fibroblast-injected eyes was 24.9 ± 6.1 mmHg, compared with 11.4 ± 0.9 mmHg for the control eyes. The peak of IOP for the injected eyes increased from 11.1 ± 1.4 mmHg to 30.9 ± 11.8 mmHg one week after the injection of Tenon's fibroblasts into the COH eyes.p < 0.05) higher mean IOPs than in the contralateral (left) eyes (control group), results showed that there was no significant difference in IOP profile, across the duration of the study, between ocular hypertensive eyes that received intravitreal hUC-MSC transplantation (COH+MSC group) and COH group that did not . At passage 2 (P2), the cells displayed a parallel or whirlpool-like arrangement when they reached 80% confluence . Flow cyGFP labeling, a simple and efficient technique, was applied to track the hUC-MSCs in the vitreous cavity. The efficiency of lentiviral transduction for labeling hUC-MSCs was about 85%, as observed under an inverted fluorescent microscope. The morphologic and biologic characterizations of the infected hUC-MSCs did not change, and the cells were in good condition after transduction .To perform an in vivo assessment of the long-term survival of hUC-MSCs in the vitreous chamber after cell grafting, hUC-MSCs were labeled with the GFP-encoding lentiviral vector and monitored using a Nikon AZ100 motorized zoom microscope . Fluores2, whereas 8 weeks after induction of high IOP, RGC numbers were reduced to 1187 ± 44 cells/mm2. In eyes that received hUC-MSC transplantation, the RGC density was 1926 ± 81 cells/mm2, significantly higher than those without hUC-MSC transplantation , the inner plexiform layer (IPL), and the outer nuclear layer (ONL), and the number of RGCs had decreased by approximately 40%. In addition, the structures of the IPL and ONL were damaged by intraocular hypertension at 8 weeks in the COH model group. In contrast, the number of RGCs was significantly higher in the COH+hUC-MSC group than in the COH group. However, no differences were observed in the IPL and the ONL between the COH+hUC-MSC and COH groups, and the thicknesses of the IPL and ONL were consistent with that in the COH model group Figures . The GCLNext, to investigate whether hUC-MSCs could improve the survival of RGCs and alleviate the damage to the retinal cells, by inhibiting apoptosis signaling in the COH model, and to explore the involved mechanisms, the level of active caspase-8 in retinas was analyzed. As shown in In the present study, to test the neuroprotective effect of hUC-MSC transplantation, we first developed a rat model of COH/glaucoma by injecting cultured Tenon's fibroblasts into the anterior chamber of the eye. The results revealed that hUC-MSCs survived for at least 8 weeks that they had the ability to migrate preferentially toward the damaged site of the host retina in the vitreous cavity, and that they significantly reduced retinal damage and RGC loss. These findings suggest that there is a neuroprotective effect of hUC-MSCs in an animal model of glaucoma.Animal models are among the most useful tools for understanding disease pathology and for developing interventions against disease. To date, many rodent models have been reported to mimic the natural cause of glaucoma. In our study, rats were chosen as the animal model, in part because rats have similarities with humans in the anatomical features of the ocular anterior segment, aqueous fluid circulation, and optic nerve damage with elevated IOP . The resOur findings point to a plausible explanation for the elevated IOP in this novel model. The initial IOP elevation in this model may be the consequence of the aqueous humor drainage obstruction, due to the accumulation of fibroblasts in the anterior segment of the eye. One week after intracameral injection of fibroblasts, pupil atresia occurred, which might be the primary cause of the elevated IOP that lasted for approximately 2 months. In contrast with other methods to maintain elevated IOP , 28, 29,With the COH model established, we transplanted hUC-MSCs into the eyes and evaluated the changes in retinal morphology, RGC loss, and caspase-8 expression. Fluorescence from GFP-labeled hUC-MSCs could be detected inside the eye even 8 weeks after cell transplantation, indicating the survival of hUC-MSCs in the vitreous cavity. This result was similar to other reports showing that MSCs could survive for 5 weeks in the vitreous cavity in a glaucoma model , and thaHowever, although 2-month persistence was observed for hUC-MSCs in the vitreous, it is noteworthy that the Tenon's fibroblasts injected in the anterior ocular chamber persisted for less than 2 weeks, which is in line with other findings that transplanted MSCs persisted in the anterior chamber for only a few days . This maAnother finding in this study is the fluorescence evidence that some of the cells migrated to the retina as clusters 1-week posttransplantation, indicating that the injected hUC-MSCs had the ability to migrate preferentially toward the damaged site of the host retina, that is, in addition to the finding that the hUC-MSCs can survive a sufficiently long time within the vitreous cavity to generate a physiological effect by secreting neurotrophic factors. Together, these results imply that hUC-MSC transplantation through the vitreous cavity is feasible. This may have important implications for the development of cell-based neuroprotective therapies.Although our results demonstrated the migration of GFP-hUC-MSCs to the surface of the retina in the COH eyes, integration of hUC-MSCs with the retinal tissue could not be confirmed. In contrast to our results, a previous study reported that a limited number of transplanted adipose tissue and bone marrow-derived MSCs  did inteIntravitreally transplanted MSCs have been shown to provide a neuroprotective effect in different animal models of glaucoma. Adult bone marrow MSC transplantation increased RGC survival by about 25% at 4 weeks in a rat model of ischemia-reperfusion injury . In a COAccumulating evidence has shown that caspase signaling-dependent apoptosis plays a key role in the development and progression of glaucoma , 36. TheNevertheless, other mechanisms likely also contributed to the neuroprotective effect of hUC-MSCs. For example, the cytokines and neurotrophic factors secreted from MSCs, such as brain neurotrophic factor, ciliary neurotrophic factor, and basic fibroblast growth factor, have been shown to provide neuroprotective effects in experimental models of glaucoma , 32. TheThe eye is considered a site with relative immune privilege , and hUCOur study showed that a single injection of allogeneic cultured Tenon's fibroblasts into the anterior chamber of the eye resulted in a moderate and sustained elevation of IOP, damaged the optic nerve, and caused characteristic glaucomatous RGC loss in the rat retina. This seems consistent with some features of chronic glaucoma and may be useful for exploring the pathogenesis of chronic glaucoma. Intravitreally injected hUC-MSCs survived for a prolonged period and migrated toward the injured site of the retina in the vitreous cavity, without penetrating the inner limiting membrane into the retina. The hUC-MSC injections improved RGC survival, as well as ameliorating retinal damage, by inhibiting apoptosis caused by high IOP in this COH rat model. Further investigations are needed to enhance the ability of hUC-MSCs to integrate into the damaged retina and to understand the mechanisms underlying the neuroprotective effect of hUC-MSC transplantation.


The dielectrophoresis-based platform combined with micropipette-based atomic force microscope is demonstrated for material- and position-selective deposition.The feasibility of on-demand sorting and printing using multi-materials in the single reservoir through a (sub)-microscale nozzle in the ambient condition is presented.The online version contains supplementary material available at 10.1007/s40820-021-00760-x. Additive manufacturing–also known as 3D printing–has attracted much attention in recent years as a powerful method for the simple and versatile fabrication of complicated three-dimensional structures. However, the current technology still exhibits a limitation in realizing the selective deposition and sorting of various materials contained in the same reservoir, which can contribute significantly to additive printing or manufacturing by enabling simultaneous sorting and deposition of different substances through a single nozzle. Here, we propose a dielectrophoresis (DEP)-based material-selective deposition and sorting technique using a pipette-based quartz tuning fork (QTF)-atomic force microscope (AFM) platform DEPQA and demonstrate multi-material sorting through a single nozzle in ambient conditions. We used Au and silica nanoparticles for sorting and obtained 95% accuracy for spatial separation, which confirmed the surface-enhanced Raman spectroscopy (SERS). To validate the scheme, we also performed a simulation for the system and found qualitative agreement with the experimental results. The method that combines DEP, pipette-based AFM, and SERS may widely expand the unique capabilities of 3D printing and nano-micro patterning for multi-material patterning, materials sorting, and diverse advanced applications. The online version contains supplementary material available at 10.1007/s40820-021-00760-x. Materials patterning and materials sorting are fundamentally important for diverse materials applications; they have been empowered by modern materials technologies such as additive manufacturing and microfluidics –9. AdditDielectrophoresis (DEP), a phenomenon where a force is exerted on a dielectric material in a non-uniform electric field in a liquid medium, can be applied as a materials sorting method , 17. ManHere, we introduce an advanced selective deposition and sorting technique with DEP-empowered Pipette/QTF-AFM (DEPQA), demonstrating multi-material patterning and materials sorting through a single nozzle in the ambient condition. The pulled pipette-combined quartz tuning fork-atomic force microscope (Pipette/QTF-AFM) demonstrating in free-standing liquid meniscus channel allows efficient low-volume liquid delivery and deposition in ambient conditions. DEPQA establishes the method for making micro/nanoscale water meniscus liquid channels for accurate and efficient printing, which is based on the precise feedback control of the distance between the tip and substrate without breaking the tip apex. Moreover, the system has the capability of microscopy imaging and in situ analysis of the mechanical responses of the micro/nanoscale liquid channel using QTF signal calculations. We emphasize that this system allows not only the delivery of low-volume liquid but also the selective deposition of different species of materials: Au and silica nanoparticles contained in a single solute. First, we simulated the DEP force profiles between the apex of the pulled pipette and an Au-coated glass substrate. The selective deposition of Au and silica nanoparticles was demonstrated experimentally with the DEPQA. Moreover, we discuss the electrothermal effect of DEP including applications such as surface-enhanced Raman spectroscopy (SERS) of the selectively patterned materials and the real-time motion of the ejected nanoparticles by monitoring the amplitude and phase responses of the QTF sensor. We believe that the results provide a different approach for multi-material patterning and materials sorting and a wider viewpoint for additive manufacturing and expand the applications of DEP-based research and industry.Figure -1). We used an Au nanoparticle solution  after twice removing surfactants. The solution was centrifuged for 30 min at 13,500 rpm and redispersed in deionized (DI) water. Silica nanoparticle solutions  were diluted 10 times to prevent rapid aggregation. Au and silica nanoparticle solutions were mixed together while keeping each concentration the same. To prevent the clogging phenomenon during pipetting, stabilizing polymer (polyvinylpyrrolidone) was added into the mixed nanoparticle solution.To fabricate the pipette (aperture diameter ~2 µm), a quartz tube  was stretched by a mechanical puller  and then Cr  and Au  were consecutively sputter-coated on both sides of the pipette. On the other hand, the underlying substrate was prepared using the same coating method on cover glass , which was then scratched by a buckled pipet to fabricate the metallic gap . By contEquations  and 2)  2) descrent}ε~m) , 29. In mentclass2pt{minimmentclass2pt{minimSimulation results indicate the various phase statuses of the electrodes, as shown in Fig. S1, to define the optimal geometry of the tip and sample parts. From Eq.  and is fixed at that position for either 30 or 60 s. We define the formation point of a liquid channel between the tip apex and the surface by monitoring the sudden change of amplitude and phase signals of the QTF sensor. After all processes are finished, the pipette is retracted almost 10 µm instantaneously to break the liquid channel rapidly. Note that if we retract the pipette slowly, a wire of aggregated nanoparticles and surfactants forms between the tip apex and the substrate by a streaking process such as a 3D printing schematic.DEP confirming of nanoparticles undergo the processes in Fig. S3. When the z-position of the pipette is fixed near 750 nm on the gap using the feedback of the DEPQA system, alternating current (AC) voltage is applied between surface electrodes of pipette and substrate using a function generator . Just after applying, the pipette is approached until a water meniscus forms  is within the sonication band, and so we vibrated the pipette at an amplitude of a few micrometer between experiments. While this QTF-based sonication process also slows down the clogging, it does not prevent it entirely. If some chemical treatments that prevent entirely the clogging issue could be done before DEPQA experiment, it would be very helpful to make this work more attractive and practically important. For example, removal of the aggregates that clog the orifice, application of strong DC voltage (>100 V) between inside of the pipette and the substrate far from the deposition site could help address the issue, in addition to finding the best optimum surfactants common to the two types of nanoparticles.The original solutions of nanoparticles even without any treatment prevent aggregation quite well. However, use of original solutions indicates that surfactants are also deposited at the same time and so just few particles are deposited because they could not be collected much near the orifice. Therefore, to enhance the sorting function, one has to do some proper treatment for the original solution, which is why aggregation is rather inevitable and limits the sorting capability. For example, when the removal of surfactants was executed for the Au nanoparticle solution, much more nanoparticles than the expected number in the water channel were deposited. On the other hand, centrifugation of the silica nanoparticle solution was rather challenging, so the surfactant concentration had to be reduced by dilution with deionized water. Even so, more silica nanoparticles than the expected number in the water channel were also deposited. In the mixed solution case of silica and Au nanoparticles, the silica nanoparticle solution was diluted down to the level similar to the concentration of the Au nanoparticle solution, while stabilizing polymer (polyvinylpyrrolidone) was added to the Au nanoparticle solution. Nonetheless, the well-dispersed conditions for each solution are different, and as a result, we could only obtain a few nanoparticles separated as shown in Fig. Figure S8 shows the potential obstacle to selective deposition; the electrothermal (ETE) effect . Even thIn the equations, -12, 7.5×10-11, and 2.1×10-10 W at 1 , 3, and 5 V, respectively, and assuming that the liquid channel has the similar thermal behavior with bulk liquid, For quantitative analysis, the number rates of not accurately deposited particles are defined as the number of not accurately deposited particles/number of deposited particles times 100, and the accurate deposition area of PDEP is considered as toroidal, similar to the shape of a pipette hole Below is the link to the electronic supplementary material.


BRAF V600 altered metastatic melanoma, are the incremental costs of triplet therapy of atezolizumab, vemurafenib plus cobimetinib vs vemurafenib plus cobimetinib alone cost-effective for the survival gains?In patients with In this economic evaluation, the triplet combination therapy was not cost-effective if immunotherapy was continued over a lifetime horizon at a willingness-to-pay threshold of $150 000 per quality-adjusted life-year. However, in real-world practice, in which immunotherapies are often stopped after 2 years, adding atezolizumab to vemurafenib plus cobimetinib could be cost-effective.These findings suggest that atezolizumab and vemurafenib plus cobimetinib regimen provides significant survival benefits over vemurafenib plus cobimetinib alone, and a price reduction would be encouraged to maximize the value of its survival gain. This economic evaluation assesses the cost-effectiveness of atezolizumab and vemurafenib plus cobimetinib in patients with metastatic melanoma from the US health care perspective BRAF V600 variation metastatic melanoma. However, considering high cost of this combination, it is unclear if the incremental cost is worth the additional survival benefit.In the IMspire150 trial, triplet treatment with atezolizumab and vemurafenib plus cobimetinib significantly improved progression-free survival (PFS) compared with vemurafenib plus cobimetinib alone for treatment of BRAF V600 variation metastatic melanoma from the US health care perspective.To evaluate the cost-effectiveness of atezolizumab and vemurafenib plus cobimetinib vs vemurafenib plus cobimetinib alone in patients with newly diagnosed unresectable This economic evaluation study used a 3-state partitioned survival model to assess the cost-effectiveness of the combination of atezolizumab with vemurafenib plus cobimetinib vs vemurafenib plus cobimetinib alone. The observed Kaplan-Meier curves for overall survival and PFS were digitized from the IMspire150 trial (January 2017-April 2018) and the long-term survivals (over a lifetime horizon) beyond the end of the trial were extrapolated using 7 different survival models. The cost and health preference data were collected from a literature review. This study was performed from March 2021 through June 2021.The outcomes of interest were expected life-years (LYs) gained and quality-adjusted life-years (QALYs), costs, and incremental cost-effectiveness ratio (ICER), expressed as cost per LYs and per QALYs saved.Adding atezolizumab to vemurafenib and cobimetinib provided an additional 3.267 QALYs compared with the doublet regimen of vemurafenib plus cobimetinib, at an ICER of $271 669 per QALY, which is not considered cost-effective at the willingness-to-pay threshold of $150 000 per QALY. However, the scenario analyses found that atezolizumab combined with vemurafenib plus cobimetinib could be cost-effective at 20-year  and 30-year ($98 092 per QALY) time horizons when both strategies were stopped after 2 years of treatments, and over a lifetime horizon ($122 220 per QALY) when only immunotherapy with atezolizumab was stopped after 2 years of treatment.These findings suggest that the atezolizumab and vemurafenib plus cobimetinib regimen provides significant survival benefits over vemurafenib plus cobimetinib alone, and a price reduction would be encouraged to maximize the value of its survival gain. This trial had a median (IQR) follow-up of 18.9 (10.4-23.8) months, and the median progression-free survival (PFS) was 15.1 months in the combination therapy arm of atezolizumab plus vemurafenib and cobimetinib vs 10.6 months in the doublet therapy arm with vemurafenib and cobimetinib , which met the primary PFS end point and provided a high-level of evidence for prolonged PFS.8 The objective of this study is to estimate the long-term survival and evaluate the cost-effectiveness of atezolizumab, vemurafenib, and cobimetinib compared with vemurafenib plus cobimetinib alone as first-line treatment for previously untreated patients with unresectable or metastatic BRAF V600 variation melanoma.On July 30, 2020, the Food and Drug Administration (FDA) approved atezolizumab (programmed cell death ligand 1 inhibitor) plus vemurafenib (BRAF inhibitor) and cobimetinib (MEK inhibitor) for the treatment of patients with unresectable metastatic 9 The reporting of the study followed the Consolidated Health Economic Evaluation Reporting Standards (CHEERS) reporting guideline.This study was exempted from institutional review board approval and informed consent by the University of South Carolina because this is not human participant research. We conducted this study based on recommendations of the US Second Panel on Cost-Effectiveness in Health and Medicine.10 digitizing software version 4.4 and the algorithms by Guyot et al11 and Rakap et al12 to impute patient-level time-to-event data  and PFS from the trial were first digitized using WebPlotDigitizerFrom the IMspire150 trial, we can see that the time to progression survival estimates from the Kaplan-Meier curves for triplet combination therapy group and doublet targeted therapy group did not drop to 0 at the end of the trial. For those who had not experienced the primary end point (disease progression) by the trial end, their survival times were censored. By the cutoff date , there was still a proportion of patients (approximately 25%) who were censored, suggesting a potential long-term survival benefit beyond the trial time horizon.17 The estimated hazard rates and long-term survival outcomes from 7 different survival models are shown in eFigure 3 in the 13 from each survival model are provided in eTable 1 in the 13We used 7 different survival models to extrapolate survival from the published PFS and OS curves. These included 3 standard parametric survival models , 3 mixture cure models (MCMs) to account for the potential cure rate in the population , and 1 flexible survival regression using the Royston/Parmar spline model. We extrapolated the trial survival curves over different time horizons  to fully capture the lifetime incremental benefit associated with the newly FDA-approved first-line triplet combination therapy.BRAF V600 variation melanoma. This population is consistent with the population evaluated in the IMspire150 trial. The cost-effectiveness analysis used a 3-state partitioned survival model.19 As shown in We evaluated the cost-effectiveness of triplet combination therapy of atezolizumab with vemurafenib plus cobimetinib in patients with histologically confirmed, previously untreated metastatic 25The model’s cost inputs for drugs , costs, and cost-effectiveness , expressed as cost per LY and per QALY saved). The model calculated total expected costs for each strategy by summing across all the cost categories. Costs, LYs, and QALYs, were discounted on an annual basis of 3% beginning 1 year after therapy initiation.26 recommended a threshold of $100 000 or $150 000 per QALY for a disease like melanoma that is expensive to treat in situations in which there is no explicit resource constraint. In this study, the WTP threshold was determined at an ICER of less than $150 000/QALY over a patient’s lifetime (30 years),21 consistent with other studies of treatment of advanced melanoma. A lifetime horizon was necessary for the base case analysis, given the need to account for all important costs and health outcomes.29We calculated the base case ICER for cost-effectiveness expressed as atezolizumab and vemurafenib with cobimetinib vs vemurafenib with cobimetinib alone. Of note, in the US, there is no consensus on a willingness-to-pay (WTP) threshold value. However, Neumann et al30 so the costs were extrapolated with different treatment capping scenarios modeled as both strategies were stopped after 2 years of treatments or only immunotherapy was stopped after 2 years of treatment. Economic outcomes were estimated under time horizons of 5, 10, 15, 20, and 30 years to examine the robustness of the estimated long-term survival and cost-effectiveness model. To examine the robustness of our findings and characterize joint parameter uncertainty in all model inputs, deterministic sensitivity analysis (DSA) and probabilistic sensitivity analysis (PSA) were also conducted. Parameters included in the DSA were varied by an SD of 25% of base case estimates. The PSAs were conducted by sampling from estimated probability distributions of model parameters  and QALYs were calculated, along with the differences between strategies in expected costs and QALYs. Cost-effectiveness acceptability curves were also constructed.32In practice, the length of immunotherapy could be 2 years, as supported by the KEYNOTE-006 phase 3 trial for pembrolizumab in metastatic melanoma,All modeling analyses were performed in R statistical software version 4.1.0  and the hēRo3 platform, a web-based, open-source health economic modeling platform . This study was performed from March 2021 through June 2021.BRAF V600 variation melanoma with vemurafenib plus cobimetinib was associated with a lifetime expected cost of $1 205 321 for an expected gain of 5.733 LYs per patient. However, adding atezolizumab to this atezolizumab regimen was associated with a higher expected lifetime cost of $2 092 986 and an expected gain of 7.720 LYs per patient  and 30-year ($98 092 per QALY) time horizons. Expected costs, LYs, and QALYs increased as the time horizon increased while ICER decreased . When onThe results of DSA suggest that OS cure rate parameter for triplet regimen had the greatest influence on the ICER, followed by PFS location parameters for triplet regimen, PPS monthly cost, PFS scale parameters for triplet regimen, OS location parameters for doublet regimen, PFS shape parameters for triplet regimen, atezolizumab monthly cost, and PPS utility . The PSABRAF V600 variation melanoma. This finding implies that, for triplet therapy with atezolizumab and vemurafenib plus cobimetinib to be cost-effective, the price of this regimen needs to be substantially reduced for its incremental cost to be worth the additional benefit.This economic evaluation is the first study of a combination of an immunotherapeutic agent with a doublet targeted therapy in patients with advanced melanoma in the United States, to our knowledge. The FDA approval of the atezolizumab plus vemurafenib and cobimetinib triplet therapy for patients with advanced melanoma provided an effective treatment strategy with improved survival benefits over the targeted therapy doublet but at an additional cost. Therefore, examining the cost-effectiveness of this treatment to determine if the incremental cost is worth the additional survival benefit is necessary for the judicious use of scarce health care resources. Based on the results of the IMspire150 trial, this study found that, over a lifetime horizon, atezolizumab in combination with vemurafenib plus cobimetinib would not be cost-effective at the WTP threshold of $150 000 per QALY for treating patients with newly diagnosed unresectable locally advanced or metastatic 35 Given that, as evidence suggests, increasing drug costs contribute to inaccessibility, a substantial reduction in price or discount would enable patients to get this regimen with proven survival benefit compared with targeted therapy alone.36 Additionally, considerable reduction in price or discount of the triplet regimen in patients with advanced melanoma in the US would be consistent with the recommendation of the National Institute for Health and Care Excellence in the UK, in which it found that vemurafenib plus cobimetinib was not cost-effective.37This study’s findings are consistent with other cost-effectiveness analyses of the atezolizumab-containing regimen in different malignant diseases, such as non–small cell lung cancer and metastatic hepatocellular carcinoma. The studies consistently found that atezolizumab-containing regimens are not cost-effective, underscoring the need for a substantial price reduction or discount.30 In clinical practice, it is safe to assume that patients and treating oncologists would be willing to stop systemic immunotherapy after 2 years in the absence of disease progression and of data supporting the need for lifelong treatment continuation.In a scenario analysis, we found that atezolizumab in combination with vemurafenib plus cobimetinib could be cost-effective if oncologists stop immunotherapy at 2 years. Although there is no consensus on the optimal duration of immunotherapy in patients with melanoma, these findings suggest that the incremental cost of the triplet strategy with atezolizumab combined with vemurafenib plus cobimetinib when physicians stop treatment at 2 years would be worth the additional clinical benefit. It is important to note that the KEYNOTE-006 phase 3 trial leading to the approval of pembrolizumab in metastatic melanoma stopped pembrolizumab at 2 years, a strategy that was further supported by long-term follow up from this trial.38 that examined the cost-effectiveness of pharmacological treatments of advanced melanoma found that BRAF- and MEK-inhibitor doublet targeted therapy combinations, such as vemurafenib plus cobimetinib, compared with a BRAF monotherapy or chemotherapy, were consistently not cost-effective. This might be justified by the high level of uncertainty that vemurafenib plus cobimetinib combination from our PSA results, in which we found that the probability of vemurafenib plus cobimetinib combination being cost-effective was only 38.3%. The systematic review also found that monotherapy with programmed cell death immunotherapies, such as pembrolizumab and nivolumab, were consistently cost-effective compared with ipilimumab, a CTLA-4 inhibitor.38 Owing to its long-lasting immunotherapeutic effect even when stopped after 24 months, the addition of atezolizumab to the vemurafenib plus cobimetinib regimen might explain why we found that the triplet strategy was cost-effective over a lifetime horizon if physicians only stop treatment with either both (immunotherapy and targeted therapy) or only immunotherapy with atezolizumab after 2 years.A 2020 systematic review by Gorry et al17 in an economic evaluation in which standard parametric approaches resulted in a smaller incremental gain in QALYs and inflating ICERs, while the MCM approach led to a more accurate cost-effectiveness estimate. Finally, our findings may have implications related to future triplet combination therapy regimens currently in development in melanoma and other malignant neoplasms, for which cost-effectiveness analyses will become increasingly essential in the face of already stretched health care resources.Unlike previous economic evaluations of immunotherapy or targeted treatments of patients with advanced melanoma, this cost-effectiveness analysis accounted for the proportion of the patients cured by using MCM, assuming patients who were censored at the end of the follow-up in the IMspire150 trial are potential long-term survivors. Ignoring the proportion of the cured patients by using the standard survival models only, as is the case with previous studies, will underestimate the long-term clinical and economic outcomes by assuming the same mortality rate for all patients. This has been demonstrated by Whittington et alBRAF V600 variation. Any biases within the trial may have affected the cost and effectiveness estimates. Second, the patient-level data were not available to the public. Therefore, the survival analysis was based on digitized pseudo trial data. Third, the percentage of the population that is assumed to be cured requires empirical confirmation, and a longer follow-up is needed to substantiate the survival estimates. As a newly FDA-approved combination regimen of immunotherapy plus targeted therapy, long-term outcomes are unknown. We also assumed survival estimates would remain valid if the immunotherapy ended after 2 years. Additionally, this cost-effectiveness analysis only compared 2 treatment strategies evaluated in the IMspire150 trial. Future cost-effectiveness analyses should compare all other treatment options following a network meta-analysis that will generate relative HRs comparing the treatment alternatives directly and/or indirectly.This study has some limitations. First, the long-term survival and cost-effectiveness model essentially relied on the validity and generalizability of the IMspire150 trial, which was the first phase 3 randomized clinical trial to investigate a triplet immuno-targeted therapy regimen in patients with previously untreated metastatic or unresectable locally advanced melanoma with a In this economic evaluation, adding atezolizumab to vemurafenib plus cobimetinib was associated with survival and quality-of-life improvement but was not cost-effective at a WTP value of $150 000 per QALY over a lifetime horizon compared with vemurafenib plus cobimetinib alone for patients with newly diagnosed advanced or metastatic BRAF variation melanoma from a US health care perspective. Thus, there is a need for a substantial reduction in price for this regimen, which may be needed for its incremental cost to be worth the additional benefit. However, in real-world practice, in which immunotherapies are often stopped after 2 years of treatment, adding atezolizumab to vemurafenib plus cobimetinib could be cost-effective.


Paediatric human immunodeficiency virus (HIV)/acquired immune deficiency syndrome (AIDS) often manifests with hearing loss (HL). Given the impact of HL, early detection is critical to prevent its associated effects. Yet, the majority of children living with HIV/AIDS (CLWHA) cannot access hearing healthcare services because of the scarcity of audiologists and expensive costs of purchasing screening equipment. Alternative solutions for early detection of HL are therefore necessary.The overall aim of this study was to assess the feasibility of using self-administered smartphone-based audiometry for early HL detection amongst CLWHA.This study was conducted at the paediatrics department of a state hospital in the Eastern Cape province, South Africa.This was a feasibility study conducted amongst twenty-seven (27) CLWHA who were in the age group of 6–12 years. The participants self-administered hearing screening tests using a smartphone-based audiometric test. The primary end-points of this study were to determine the sensitivity, specificity and test-retest reliability of self-administered hearing screening.r = 0.97) was obtained when participants self-administered the screening test.The sensitivity and specificity for self-administered screening were 82% and 94%, respectively, with positive and negative predictive values of 90% and 88%, respectively. Moreover, a strong positive test-retest reliability (Six- to 12-year-old CLWHA were able to accurately self-administer hearing screening tests using smartphone-based audiometry. These findings show that self-administered smartphone audiometry can be used for serial hearing monitoring in at-risk paediatric patients. In Cape Town, the prevalence of HL in children living with HIV/AIDS (CLWHA) aged below 14 years was estimated at 16% (n = 5/37).4 The high prevalence of HL in children at this age group is of concern given the impact that HL has on childhood development. For instance, HL during childhood negatively impacts communication skills, socio-emotional development and scholastic performance.6 The latter leads to poor academic achievement, which often translates to poor vocational outcomes and dependence on social security.6 Furthermore, HL also leads to poor socio-emotional outcomes and bullying, which result in social isolation, behavioural problems and an overall poor quality of life.10 Early detection and intervention (EDI) of HL are therefore important to prevent these negative impacts.6 This is particularly important because more than 60% of paediatric HL occurs as a result of preventable or treatable conditions.11An estimated 240 000 children are living with the human immunodeficiency virus (HIV)/acquired immune deficiency syndrome (AIDS) in South Africa (SA).12 and scarcity of audiologists.13 For instance, whilst SA population exceeds 58 million, there are only 2171 audiologists registered with the Health Profession’s Council of South Africa.13 This compounds to a 3.7:100 000 ratio of audiologist to the population served compared to 7.3:100 000 in the United States (US).14 To further compound this issue, only 23% (n = 499) of these audiologists are currently practising in state hospitals.13Although early detection leads to appropriate interventions, hearing healthcare is inaccessible to the majority of South Africans, mainly because of lack of diagnostic equipment12 Moreover, the recent global coronavirus disease 2019 (COVID-19) pandemic resulted in lockdown in different states globally that further restricted the access to hearing healthcare services. In SA, for instance, many public hospitals closed down non-emergency outpatient’s departments to prevent the further spread of COVID-19, which made it difficult for those with HL to access healthcare.This scarcity of audiologists leaves people in the rural areas of SA unable to access these services. A recent report indicates that only 22 hospitals in the Eastern Cape (EC) province have 41 audiologists to render audiological services to an estimated 6.9 million people residing in the EC province. The latter compounds an estimated 0.5:100 000 ratio of audiologist to population, notwithstanding the fact that more than 30 of these audiologists are practising in urban areas. In addition to the scarcity of professionals, the majority of these hospitals do not have the necessary equipment to facilitate EDI for HL.15 mHealth tools are cost effective, fast and efficient and can also be used by minimally trained people, which makes them ideal for use in cases where there is a lack of specialists.16 By using these tools, it may be possible for patients to self-screen and then be referred to the closest specialist or these results can be sent via store and send protocols, for further management. One application that may be used for EDI is the hearScreen®, which has been developed by the HearX Group, to screen for the presence of HL. Unfortunately, data on its reliability and clinical validity when patients self-administer the screening tests is unavailable. The latter is echoed by Mahomed-Asmail et al.,17 who argued that whilst the application may provide an alternative solution to expensive equipment, further research in its validity in children is required. As a result, alternative methods to provide hearing healthcare were needed. Therefore, this study aimed to assess the clinical validity and reliability of using self-administered smartphone applications for EDI in CLWHA.One of the models that may provide alternative solutions for early detection is mHealth, which is described as the use of mobile technologies to address healthcare challenges such as access, affordability and matching of resources.This feasibility study was part of a larger study that was carried out at the paediatrics department at a referral hospital in SA. After enrolling in this study, information related to their demographic features, hearing sensitivity, HIV status and HIV treatment was retrieved from the previous study’s records. To be included in this study, participants had to be aged between 6–12 years old and able to follow instructions to perform a hearing test. They were included in this study if their primary caregivers gave consent. Participants who were cognitively unable to follow instructions or those with known conditions that may affect concentration were excluded.Non-probability purposive sampling was used to select the participants whose audiometric information was known from the larger study. Purposive sampling was used as it allowed the researchers to select participants with HL and those without HL, which enabled the researchers to assess the sensitivity and specificity of the self-administered test. Whilst the audiometric information of the participants enrolled in the study was known, the researchers were blinded to the audiological information during data collection of this study.17Since the participants’ hearing sensitivity was already known, they were given instructions to self-administer a hearing screening test using the smartphone-based hearScreen® application. The hearing screening was developed to screen for HL at four frequencies: 500 Hz- 4000 Hz at 25 dB HL. To pass the screening, participants had to hear the tone and press the button at all four test frequencies in each ear. The screening test was carried out in an ambient room using the hearScreen on a Samsung Galaxy J5 Prime smartphone and Sennheiser HD280 Pro headphones. The hearScreen® is a clinically validated tool for hearing screening with a sensitivity and specificity of > 90% when testing is administered by a hearing professional.Prior to undergoing screening, participants were given instructions in their home language and included informing them that they “should press the green button on the screen each time they heard a beeping sound in the ear”. A conditioning was done with each participant at 1000 hertz (Hz) at 40 decibels (dB) HL prior undergoing the self-administered screening. The participants underwent screening twice, with a 30-min break between the two tests.Findings from the two self-administered tests were compared to each other to determine the test-retest reliability, sensitivity and specificity of the self-administered screening test. Moreover, self-administered results were also compared with previous audiometric information to determine correlation between the screening results with audiometric information. Statistical analysis was performed using Statistical Package for Social Sciences version 26 for Macintosh, and percentages were generated for categorical values whilst Pearson correlation coefficient assessed the test-retest reliability.This research adhered to the World Medical Association’s declaration of Helsinki for conducting research with human participants. Ethical approval was obtained from the University of KwaZulu-Natal’s Biomedical Research Ethics Committee (BREC/00001185/2020). All participants in this study provided assent and their caregivers gave informed consent.A total of twenty-seven participants with a mean age of 8.3 years (range: 6–12 years) took part in this study. Eleven participants had HL whilst 16 participants had normal hearing. A calculation was done to assess the sensitivity and specificity of the self-administered screening test in the detection of HL. Analysis of the results revealed a sensitivity of 82% and a specificity of 94% with positive and negative predictive values of 90% and 88%, respectively, when the test was self-administered by the participants.r = 1) from the normal-hearing participants whilst a strong positive reliability (r = 0.98) was obtained from the participants with HL.Further analysis using the Pearson correlation coefficient test to assess the test-retest reliability showed a true positive test-retest reliability (r = 0.97) self-administered the screening. Whilst this study is the first to document sensitivity and specificity of self-administered screening, our findings are comparable to the available literature on the studies conducted amongst adult populations and those conducted by the hearing professionals. Louw et al.16 conducted a study at two primary health care clinics in Gauteng amongst 1236 participants aged 3–97-years-old and reported a sensitivity and specificity of 81.7% and 83.1%, respectively, when using the hearScreen® for HL detection. Another local study also found a sensitivity and specificity of 75.0% and 98.7%, respectively, when using the hearScreen® application to screen for HL in school children who are in the age group of 6–12 years.15 However, unlike in the present study where the participants self-administered the screening, testing in the previous two studies16 was administered by audiologists. Nevertheless, these studies consistently reported a high sensitivity and specificity of the hearScreen® to identify HL.As far as we know, this was the first study to assess the sensitivity, specificity and test-retest reliability of self-administered smartphone-based hearing screening of CLWHA in SA. This study found a sensitivity and specificity of 82% and 94%, respectively, when the participants self-administered the smartphone-based hearing screening test. Moreover, these findings show that our participants reliably  to allow automated testing, noise monitoring capabilities, instant data capturing and cloud-based data storage, this technology has the potential to maximise human resources where audiologists spend more time on diagnostic evaluations and treatment rather than screening. In this instance, patients can self-administer the screening and only those who fail the screening are referred to audiologists for further diagnostic evaluation and management. Moreover, these findings pave the way for decentralising hearing screening where CLWHA can self-administer the screening when they access their monthly or 3-monthly follow-up care consultations, and only those who fail the screening are referred for further diagnostic evaluation and management.r = 0.97) was obtained when the participants self-administered the hearing screening tests using the hearScreen® application. As far as we know, this was the first study to determine the test-retest reliability of self-administered screening in children, and these results should be interpreted with caution given the small sample size of this study. Nevertheless, this high test-retest reliability shows that in rural contexts such as the EC province where a scarcity of audiologists exists,13 the use of mobile technology such as the hearScreen®, which has AI capabilities, can improve access to EDI. However, further large-scale research, particularly on assessing the reliability of self-administration features, is highly recommended. Further application of this mHealth-supported screening model may also assist in other areas such as ototoxicity monitoring of at-risk patients undergoing treatment with known ototoxic drugs such as anti-tuberculosis drugs and platinum-based chemotherapy.A strong positive test-retest reliability  are required because the patient does not have to be in the same room as the clinician during evaluation. Consequently, a screening/diagnostic equipment could be sent to the patient to self-administer the assessments and have the results uploaded to cloud-based storage for further analysis and treatment recommendation.The data collection was conducted when the country was under strict lockdown levels 3 and 4, during the beginning of the second wave of the COVID-19 pandemic. This imposed travel restrictions for non-emergency travel, and in addition to that, most hospitals in the country closed down non-emergency OPDs in an effort to reduce the further spread of COVID-19. These circumstances collectively reduced the number of participants who could be enrolled in this study, resulting in a small sample size in our study. Also, data collection for this study occurred in only one site, and as a result, caution should be applied when generalising these findings to the whole of the EC.The findings from this study show a high test-retest reliability when CLWHA, who are in the age group of 6–12 years, self-administered a hearing screening test. Large-scale research is recommended to determine the feasibility of using smartphone applications to decentralise hearing screening to primary healthcare clinics where majority of the CLWHA receive their follow-up care.


Arg substrates has confirmed this general concept to a large extent and over numerous phyla. However, some Stramenopiles, and in particular, Diatoms (Bacillariophyta) present a notable exception. Unusually for non-fungal organisms, the nuclear genome encodes tRNAArg isoacceptors with C or U at position 20. In this case one of two nuclear-encoded cytosolic arginyl-tRNA synthetases has evolved to become insensitive to the nature of the D-loop identity element. The other, with a binding pocket that is compatible with tRNAArg-A20 recognition, is targeted to organelles that encode solely such tRNAs.Error-free protein synthesis relies on the precise recognition by the aminoacyl-tRNA synthetases of their cognate tRNAs in order to attach the corresponding amino acid. A concept of universal tRNA identity elements requires the aminoacyl-tRNA synthetases provided by the genome of an organism to match the identity elements found in the cognate tRNAs in an evolution-independent manner. Identity elements tend to cluster in the tRNA anticodon and acceptor stem regions. However, in the arginine system, in addition to the anticodon, the importance of nucleotide A20 in the tRNA D-loop for cognate enzyme recognition has been a sustained feature for arginyl-tRNA synthetase in archaea, bacteria and in the nuclear-encoded cytosolic form in mammals and plants. However, nuclear-encoded mitochondrial arginyl-tRNA synthetase, which can be distinguished from its cytosolic form by the presence or absence of signature motifs, dispenses with the A20 requirement. An examination of several hundred non-metazoan organisms and their corresponding tRNAThe online version contains supplementary material available at 10.1007/s00239-022-10053-5. Aminoacylation is the enzymatic process by which amino acids are attached to their specific tRNAs during the first step of protein biosynthesis. It requires a virtually error-free recognition at two levels; the association of one of 20 aminoacyl-tRNA synthetases with its specific amino acid and the simultaneous identification of the cognate tRNA. Heterologous aminoacylation between macromolecules from different species foretold the existence of a common sequence within a given set of tRNAs isoacceptors that is responsible for their specific enzymatic association almost 60 years ago  in tRNAArg. In contrast, Saccharomyces cerevisiae, often taken as a good model for eukaryotic systems, possesses a cytosolic arginyl-tRNA synthetase that is conspicuous in not requiring this A20 for recognition  provided by the genome of an organism match the identity elements found in the cognate tRNAs in an evolution-independent manner. In its simplest form it would be expected that cytosolic-type arginyl-tRNA synthetase has a requirement for tRNAed Igloi  for the ed Igloi . WhetherRecognition rules for non-metazoans are poorly understood but have been examined in an Apicomplexan tyrosine system from Plasmodium  taxonomic classification were assembled Igloi . SpeciesArg isoacceptors;  from species corresponding, where possible, to the source of the enzymes, were compiled (Igloi No tRNA sequence data (either cytosolic or organelle) for 61 of these species could be extracted from the databases and could not be used for identity analysis but the protein sequences were nevertheless included in the alignments to facilitate classification. In order to detect robust and systematic evidence for a molecular mechanism of tRNA recognition by alternative or unconventional co-evolution, 931 tRNAed Igloi . ExaminaThe polyphyletic group of photosynthetic organisms  contribute 103 algal species and 136 arginyl-tRNA synthetase sequences. All enzymes are of the cytosolic-type and are associated with tRNAs having A20 in all sub-cellular compartments. They may be destined for recognizing all cellular tRNAs  are represented in the databases by 51 arginyl-tRNA synthetase sequences from 26 species  have retained the algal nucleotide A20. The encoded mitochondrial tRNAArg, without exception (22 isoacceptors from 11 species) also carry the A20  databases)] Table . Both geArg isoacceptors, attention was focussed on the amino acids that have been defined crystallographically as being involved in the binding of the tRNA variable pocket (including position 20) in yeast  is replaced by a small residue  to accommodate the adenosine ring . Interestingly, the N79Q mutation, which would correspond to the Q111 position in yeast proved to be inactive with all position 20 variants, indicating that the importance of N79 relies on its associated architecture. This crucial D-loop recognition which is guided by N84 . The compilation also shows the nature of the 5∆MSTR motif found in yeast (position 408) and which is typical of nuclear-encoded mitochondrial arginyl-tRNA synthetases . In order to assess the likelihood of proteins of group 2 being involved in protein synthesis involving organelle-encoded tRNA-A20 recognition, their targeting potential was examined with five different prediction algorithms, including DEEPLOC 1.0, which has proved to be robust for mitochondrial proteins in diatoms  Protein2 clearly fulfils the requirements for A20 binding, whereas Protein1 lacks some of the essential interacting amino acids . Forty appeared to diverge from the identity framework in having cognate tRNAs without the A20 identity element required by the cytosolic arginyl-tRNA synthetase. Despite an intensive manual BLAST-supported search of genomic, transcriptome and specialized databases, numerous non-metazoan taxa were only represented by individual or few species making definitive statements regarding taxonomy-dependent identity of these unrealistic. Some proved to be questionable due to the problems of potential host contamination of environmental samples, as has been documented for other studies . Of the 26 species for which arginyl-tRNA synthetase sequences could be recovered by BLAST searches, 21 proved to have two distinct gene products which were all ascertained to originate from ancestral nuclear genes, on the basis of characteristic signature sequences Igloi . The twoArg genes coding for entities possessing C20 or U20 nucleotides took place remains a mystery. In modern-day organisms such characteristics in tRNAArg are rarely found outside Fungi and Microsporidia so that any concept regarding the origin of Diatoms would have to take the appearance of this facet of the genome into account.Although the nature of the ancestral eukaryotic host prior to endosymbiotic acquisition of the “red” plastid . For example, within the Rhizaria, the phylum Endomyxa, with the data from only three species available, may provide, at least conceptually, another example of how recognition rules could be adapted to accommodate the available tRNAs. All three species have well-defined cytosolic as well as mitochondrial enzymes (Online Resource 1). However, the tRNAThe coevolution of aminoacyl-tRNA synthetases with their tRNA partners evidently relies not only on the structural plasticity of tRNA molecules (Giegé and Eriani http://www.softberry.com/) by scanning for protein similarity using the corresponding or closely related organism-specific gene-finding parameters. For non-metazoans the available parameters are somewhat limited making N-terminal predictions, in particular, less reliable in the absence of transcriptome data. Multiple alignments were created with CLUSTALΩ  and transcriptome (tsa) NCBI and other databases were searched using TBLASTN. Accession numbers of the entries containing the corresponding arginyl-tRNA synthetase genes are given in Online Resource 3. Putative full length protein sequences were extracted from genomic hits manually by homology-based alignment FGENESH + . Where no organelle genome data is available, this clustering approach is a tool to distinguish between organelle-encoded and mis-annotated nuclear-encoded tRNAs.For tRNA sequences of non-metazoans neither the badly outdated tRNA database Supplementary file2 (PDF 337 KB)Supplementary file3 (PDF 350 KB)Below is the link to the electronic supplementary material.


The SARS-CoV-2 pandemic has challenged researchers at a global scale. The scientific community’s massive response has resulted in a flood of experiments, analyses, hypotheses, and publications, especially in the field of drug repurposing. However, many of the proposed therapeutic compounds obtained from SARS-CoV-2 specific assays are not in agreement and thus demonstrate the need for a singular source of COVID-19 related information from which a rational selection of drug repurposing candidates can be made. In this paper, we present the COVID-19 PHARMACOME, a comprehensive drug-target-mechanism graph generated from a compilation of 10 separate disease maps and sources of experimental data focused on SARS-CoV-2/COVID-19 pathophysiology. By applying our systematic approach, we were able to predict the synergistic effect of specific drug pairs, such as Remdesivir and Thioguanosine or Nelfinavir and Raloxifene, on SARS-CoV-2 infection. Experimental validation of our results demonstrate that our graph can be used to not only explore the involved mechanistic pathways, but also to identify novel combinations of drug repurposing candidates. Unprecedented in the history of science, this pandemic has elicited a worldwide, collaborative response from the scientific community. In addition to the strong focus on the epidemiology of the virus11. Such disease maps have proven valuable before in diverse areas of research such as cancer, Alzheimer's Disease, Parkinson’s Disease, and influenza15.In the field of computational biology, several initiatives have started generating disease maps that represent the current knowledge pertaining to COVID-19 mechanisms8, entity relationship graphs16, and pathways8; these disease maps represent a considerable amount of highly curated “knowledge graphs” which focus primarily on COVID-19 biology. Here, we use the term “mechanism” to describe a single, or multiple cause-and-effect relationships (i.e. a subgraph), “pathways” to refer to a well-established series of interactions resulting in cellular change or a defined product, and “models” for describing a collection of experimental data or known interactions defined in the context of a particular biological process or pathology. As of July 2020, a collection consisting of 10 models representing core knowledge about the pathophysiology of SARS-CoV-2 and its primary target, the lung epithelium, was shared with the public.When taken together with related work including cause-and-effect modeling17, interactome18, and proteome19 data), we are now in the position to challenge and validate these COVID-19 pathophysiology knowledge graphs with experimental data. This is of particular interest as validation of these knowledge graphs bears the potential to identify those disease mechanisms that are highly relevant for targeting in drug repurposing approaches.With the rapidly increasing generation of data  is not new. The major advantage of drug repurposing over conventional drug development is the massive decrease in time required for development as important steps in the drug discovery workflow have already been successfully passed for these compounds22 and the comprehensive ReFRAME library23 were used to experimentally screen for either viral proteins as targets for functional inhibition24, or for virally infected cells in phenotypic assays25. In our own work, compounds were assessed for their inhibition of virus-induced cytotoxicity using the human cell line Caco-2 and a SARS-CoV-2 isolate26. A total of 63 compounds with IC50 < 20 µM were identified, from which 90% have not yet been previously reported as being active against SARS-CoV-2. Out of the active compounds, 31 are approved drugs, 23 are in phases 1–3 and 9 are preclinical candidate molecules. The described mechanisms of action for the inhibitors included kinase signaling, PDE activity modulation, and long chain acyl transferase inhibition .Our group and many others have already begun performing assays to screen for experimental compounds and approved drugs to serve as new therapeutics for COVID-19. Dedicated drug repurposing collections, such as the Broad Institute libraryThe approach presented here integrates experimental results, the output from other informatic pipelines, as well as proprietary and public data to provide a comprehensive overview on the therapeutic efficacy of candidate compounds, the mechanisms targeted by these candidate compounds, and a rational approach to test the drug-mechanism associations for their potential in combination therapy.Supergraph.Disparate COVID-19 disease maps focus on different aspects of COVID-19 pathophysiology. Based on comparisons of the COVID-19 knowledge graphs, we found that not a single disease map covers all aspects relevant for the understanding of the virus, host interaction and the resulting pathophysiology. Thus, we optimized the representation of integral COVID-19 pathophysiology mechanisms by integrating several public and proprietary COVID-19 knowledge graphs, disease maps, and experimental data .In addition to combining the listed models, we also performed a dedicated curation of the COVID-19 supergraph in order to annotate the mechanisms pertaining to selected targets and the biology around prioritized repurposing candidates. The resulting BEL graphs were quality controlled and subsequently loaded into a dedicated graph database system underlying the Biomedical Knowledge Miner (BiKMi), which allows for comparison and extension of biomedical knowledge graphs , we created an initial version of the nds Fig. . In ordeAt the time of the writing of this paper, six phenotypic cellular screening experiments have been shared via archive servers and journal publications  were used to identify the areas of the COVID-19 PHARMACOME responding at gene expression level to SARS-CoV-2 infection. Details of the gene expression data processing and mapping are available in the supplementary material .Two single cell sequencing data sets representing infected and non-infected cells directly derived from human samples33 feature of the gseapy Python package34. Briefly, gene symbol lists were assembled from their respective subgraph or dataset and compared against multiple pathway gene set libraries including Reactome, KEGG, and WikiPathways. To account for multiple comparisons, p values were corrected using the Benjamini-Hochberg35 method and results with p values < 0.01 were considered significantly enriched.Associated pathways for subgraphs and significant targets were identified using the Enrichr36). Survival of cells was assessed using a cell viability assay and measured by high-content imaging using the Operetta CLS platform (PerkinElmer). Details of the drug repurposing screening are described in the supplemental material.We performed phenotypic assays to screen for repurposing drugs that inhibit the replication and the cytopathic effects of virus infection. A derivative of the Broad repurposing library was used to incubate Caco-2 cells before infecting them with an isolate of SARS-CoV-2  assays was generated and mapped to the COVID-19 PHARMACOME. Figure A total number of 870 mechanisms were identified as being targeted by most of the drug repurposing candidates  > 0.25) on the COVID-19 PHARMACOME reveals a distinct pattern characterized by the high responsiveness (expressed by variation of regulation of gene expression) to the viral infection  and are targets for repurposing drugs. To this end, we mapped signals from the drug repurposing screenings to the subgraph that showed responsiveness to SARS-CoV-2 infection Fig. b. Figure40Combinatorial treatment data obtained from the results published by Bobrowski41 and Ellinger et al42 were mapped to the COVID-19 PHARMACOME. Figure We mapped existing combinatorial therapy data to the COVID-19 PHARMACOME in order to evaluate its potential in guiding rational approaches towards combination therapy using repurposing drug candidates. In drug combination therapy, the interaction between compounds can be defined as either additive , synergistic , or antagonistic Based on the association between repurposing drug candidates and the areas of the COVID-19 PHARMACOME that respond to SARS-CoV-2 infection Fig. , we hypoIn order to test our ability to predict the outcome of novel drug combinations, we selected five compounds: Remdesivir (a virus replicase inhibitor), Nelfinavir (a virus protease inhibitor), Raloxifene (a selective estrogen receptor modulator), Thioguanosine (a chemotherapy compound interfering with cell growth), and Anisomycin . These compounds were used in four different combinations  to test the potency of these drug pairings in phenotypic, cellular assays. Figure 43 and SARS-CoV-244, displayed synergistic effects when combined with high concentrations of Raloxifene. This result agrees with our predictions generated using the COVID-19 PHARMACOME in which the drug combination with the shortest distance, Raloxifene and Nelfinavir  to virus infection. Network analysis along with the overview on previous repurposing experiments provided us with the insights needed to select the optimal repurposing drug candidates for combination therapy. Experimental verification showed that this systematic approach is valid; we were able to identify two drug-target-mechanism combinations that demonstrated synergistic action of the repurposed drugs targeting different mechanisms in combinatorial treatments.We are fully aware of the fact that the COVID-19 PHARMACOME combines experimental results generated in different assay conditions. In the course of our work, we accumulated evidence that assay responses recorded using Vero E6 cells in comparison to Caco-2 cells may only partially overlap. Comparative analysis of the results of both assay systems to virus infection by means of transcriptome-wide gene expression analysis is one of the experiments we plan to perform next. However, for the identification of meaningful combinations of repurposing drugs, the current model-driven information fusion approach was shown to work well despite the putative differences between drug repurposing screening assays.Given the urgent need for treatments that work in an acute infection situation, our approach described here paves the way for systematic and rational approaches towards combination therapy of SARS-CoV-2 infections. We want to encourage all our colleagues to make use of the COVID-19 PHARMACOME, improve it, and add useful information about pharmacological findings (e.g. from candidate repurposing drug combination screenings). In addition to vaccination and antibody therapy, (combination) treatment with small molecules remains one of the key therapeutic options for combatting COVID-19. The COVID-19 PHARMACOME will therefore be continuously improved and expanded to serve integrative approaches in anti-SARS-CoV-2 drug discovery and development.Supplementary Figure 1.Supplementary Figure 2.Supplementary Information.Supplementary Table 3.Supplementary Table 4.Supplementary Table 5.Supplementary Table 6.Supplementary Table 7.


The persistence of serum-specific anti-chikungunya IgM antibodies (CHIKV-IgM) can vary after chikungunya fever (CHIK) infection. However, the factors related to its production are not yet known. We described a case series drawn up from data collected from 57 patients between 12 and 36 months after the acute phase of CHIK infection in Northeastern Brazil. CHIKV-IgM was detectable in 7/57 (12.3%) patients after 28.3 months of infection. No frequency differences in chronic musculoskeletal manifestations and underlying conditions were detected between patients with or without CHIKV-IgM. CHIKV-IgM was detected for up to 35 months in Brazilian patients after CHIK infection. Togaviridae family, transmitted by Aedes mosquitoes and causes chikungunya fever (CHIK),Chikungunya virus (CHIKV) is an alphavirus of the -It is known that in the viremia phase, CHIKV causes B-cell activation and early production of immunoglobulins, with the detection of serum-specific anti-chikungunya IgM antibodies (CHIKV-IgM) expected from the onset of infection until approximately three months after infection,,Although this antibody does not present an established pathogenic mechanism, it has been questioned whether there is a relationship between the serum persistence of this immunoglobulin and chronic joint symptoms in CHIKFrom December 2018 to July 2019, a case series was prepared from data collected from 57 patients diagnosed with CHIK, who were previously evaluated in a cross-sectional study, between 12 and 36 months after the acute phase of infection. These patients were followed-up in a specific outpatient clinic for people with chronic symptoms after CHIK infection at the Hospital das Clínicas at the Universidade Federal de Pernambuco, Northeastern Brazil. A diagnosis of CHIK was established based on clinical and epidemiological criteria and confirmed by the detection of serum CHIKV-IgM or IgG by enzyme-linked immunosorbent assay (ELISA). The study was approved by the local ethics committee, and consent forms were obtained from all patients before inclusion in the study. At the time of the evaluation, blood samples were collected and tested for CHIKV-IgM using a commercially available qualitative ELISA, following the manufacturer's instructions . All patients underwent clinical examinations by experienced rheumatologists. We compared the frequency of chronic musculoskeletal manifestations and comorbidities in the two groups of patients: CHIKV-IgM-positive and CHIKV-IgM-negative. Data were analyzed using SPSS software and Fisher’s exact test. The significance level was set at p<0.05.Between 12 and 36 months after CHIK acute infection, seven of 57 patients (12.3%) presented detectable CHIKV-IgM with a median time of 28.3 months . Six of seven patients (86%) who presented with positive CHIKV-IgM reported persistent musculoskeletal symptoms. All these patients displayed joint pain, and two experienced persistent arthritis. The descriptive characteristics of the patients with persistent detectable CHIKV-IgM are shown in Patients positive for CHIKV-IgM were compared to those without detectable antibodies with respect to the presence of chronic musculoskeletal manifestations and underlying previous disease. The descriptive characteristics of the patients are presented in CHIK was first observed in Brazil in the Northern and Northeastern regions. Northeastern Brazil was severely affected by the epidemic, and it was necessary to establish a specific outpatient clinic for CHIK at the Hospital das Clínicas at the Universidade Federal de Pernambuco due to the high frequency of chronic manifestations. This led us to assess whether the long-term progression of CHIK in Brazilian patients was different from that in patients from other affected countries. Herein, we present the serological and clinical data of patients with long-term detectable CHIKV-IgM followed in a specific outpatient clinic for people with chronic symptoms after CHIK in Northeastern Brazil.-According to initial studies, after CHIKV infection, there is a progressive reduction in the production of CHIKV-IgM, which was detected in 76.2% of patients between two and six months, 69.8% between seven and 12 months, and 55.5% between 13 and 18 months after the acute phaseIt is plausible to accept that the difference observed in the present study may only be related to the time elapsed since the acute phase of CHIK as our assessment occurred after a mean interval of 28 months, which is longer than the intervals in previous studies. Furthermore, a commercially available ELISA kit was used in the present study; this may have reduced the sensitivity for detecting such antibodies,,,,,Whether the persistence of CHIKV-IgM in the chronic phase of CHIK is related to a possible pathogenic role has not yet been fully established. Studies that only assessed individuals with persistent joint pain in the chronic phase of CHIK have demonstrated detectable CHIKV-IgM in around 63% after six months and in 11−19% after one year,In addition, some studies have controversially reported that the severity of CHIK, as well as the chronicity of symptoms, may be related to the presence of comorbidities, such as hypertension and diabetesIn summary, in contrast to most studies that have analyzed patients up to 12−18 months post-CHIKV infection, this case series detected CHIKV-IgM for up to 35 months in 12.3% of Brazilian patients. The presence of persistent joint symptoms and underlying conditions did not seem to be associated with the long-term production of CHIKV-IgM in this sample, although further investigations are required to validate these observations. This study has indicated that a positive serological test for CHIK-IgM alone should not define the diagnosis of a recent disease.


COVID-19 pandemic is a global public health threat facing mankind. There is no specific antiviral treatment for COVID-19, and many vaccine candidates are currently under clinical trials. This study aimed to understand the perception of social media users regarding a hypothetical COVID-19 vaccine in Nigeria.we conducted a cross-sectional survey among social media users in Nigeria in August 2020 using an online questionnaire. The questionnaire includes sections on the demographic characteristics of the respondents and their perception regarding a hypothetical COVID-19 vaccine. A total of 517 respondents completed and returned the informed consent along with the questionnaire electronically. Data were coded and abstracted into Microsoft Excel spreadsheet and loaded into the STATA 14 software for final analysis.the results showed that more than half of the respondents were male 294 (56.9%). Most of the respondents 385 (74.5%) intend to take the COVID-19 vaccine when it becomes available. Among the 132 respondents that would not take the COVID-19 vaccine, the major reason for non-acceptance was unreliability of the clinical trials 49 (37.1%), followed by the belief that their immune system is sufficient to combat the virus 36 (27.3%). We found a significant association between the age of the respondents and the COVID-19 vaccine acceptance  as well as geographical location and COVID-19 vaccine acceptance .it was observed that most of the respondents were willing to take the COVID-19 vaccine. Our findings also reiterate the need to reassure the public the benefits an effective and safe COVID-19 vaccine can reap for public health. There is a need for national health authorities in Nigeria to ensure public trust is earned and all communities, including the marginalized populations, are properly engaged to ensure an optimal COVID-19 vaccine acceptance. The novel coronavirus disease 2019 (COVID-19) caused by severe acute respiratory coronavirus 2 was first discovered in Wuhan animal market, Hubei Province, China in December 2019 ,4. In NiMajor infections such as smallpox and rinderpest have been eradicated worldwide due to vaccines, while polio has almost been eradicated except for Afghanistan and Pakistan where it is still endemic -11. The Despite the immense benefits that vaccination has offered since the first discovery of the smallpox vaccine by Edward Jenner till date, saving millions of lives globally and doing so at a comparatively low cost, vaccine hesitancy has always plagued this great discovery. Vaccine hesitancy is “the reluctance or refusal to vaccinate despite the availability of vaccines” . It was Nigeria is the most populated country in Africa and has a convoluted history of vaccine hesitancy. Vaccination coverage in Nigeria has continuously dropped since its peak of 81.5% in the 1990s, and by 2013, only 25% of children under the age of 2 were fully vaccinated . The 200Study design and sampling technique: we conducted a web-based cross-sectional survey among males and females who use social media in Nigeria. We used a non-probability convenient sampling technique to recruit the respondents, who were required to fill the questionnaire within the first-one-month period stipulated for the study as used in previous studies [ studies ,19. The  studies .Sample size estimation: the survey was online-based and totally depended on the voluntary participation of all the eligible respondents, so no prior sample size was calculated, yet a total of 517 respondents took part in the study. This is further justified by the non-probability convenient sampling used in the study as well as the need for caution in findings generalization as stated in the study limitation.Study instrument and administration: the researchers developed the study questionnaire in several stages of drafts and reviews and with contributions from survey research experts. We conducted a pretest among ten respondents from the six geopolitical zones in Nigeria using the drafted questionnaire. The pretest was undertaken to examine readability, comprehensibility, and face validity. The final questionnaire comprises sections on the demographic characteristics of the respondents (independent variables), the intention to accept a COVID-19 vaccine was measured using a one-item question , another question on the reasons for not intending to take the vaccine and a question on whether they have reservations towards vaccination - with a Yes or No option (outcome variables). According to our study, having reservation towards vaccination is referred to as having doubt on the benefits vaccination other than the COVID-19 vaccination can reap for public health while willingness to take COVID-19 vaccine is referred to as the acceptance of COVID-19 vaccine and the readiness to be vaccinated when it is available.The final questionnaire was entered into an online survey system, and a link to the electronic questionnaire was shared using social media platforms, specifically WhatsApp, LinkedIn, Twitter, and Facebook, as used in a previous survey . In addiData analysis: data were coded and abstracted into the Microsoft Excel spreadsheet and loaded into the STATA 14 software for final analysis. Simple descriptive analysis, including frequencies and percentages, was computed for demographic characteristics, and the reasons for non-acceptance of the COVID-19 vaccine, intention to take vaccine and reservations toward vaccination were presented with bar charts. A Chi-square test was carried out to determine the significant level of association and the relationship between the independent variables and outcome variables with statistical significance defined at P < 0.05.Ethical consideration: no formal ethical approval was collected, however the researchers adhered strictly to all the ethical issues involved in conducting studies involving human participants such as informed consent which was obtained electronically. Confidentiality and anonymity were also ensured by not putting names or attaching any identifiable codes to the online questionnaires, and the rights of the participants to withdraw anytime from the study were also clearly stated in the online survey, if they were not comfortable in answering the questions.Socio-demographic characteristics of the respondents:Percentage distribution of respondents´ reservation toward vaccination, acceptance of COVID-19 vaccine and reasons for non-acceptance of COVID-19 vaccine: the results showed that 385 (74.5%) were willing to receive COVID-19 vaccine while 132 (25.5%) of our respondents were not willing to take the vaccine. 124 (24.0%) had reservations toward vaccination while 393 (76.0%) respondents did not have any reservations toward vaccination. The major reason for non-acceptance of the COVID-19 vaccine among our respondents is unreliability of the clinical trials 49 (37.1%), followed by the belief that their immune system is enough to combat the virus 36 (27.3%) as shown in Association between selected socio-demographic variables, having reservations toward vaccination and COVID-19 vaccine acceptance: our result revealed having reservations toward vaccination 2=0.10 P-value=0.76), Russia (54%), Poland (56%), Hungary (56%), and France (59%), according to a recent World Economic Forum´s Ipsos survey of nearly 20,000 adults on whether they will take COVID-19 vaccine or not when it is available . HoweverFinding from our study also revealed that geographical location and acceptance of the COVID-19 vaccine are significantly associated. This is evident in that respondents from the Southern part of the country are likely to take the COVID-19 vaccine compared to the Northern part of the country. This is not surprising because previous studies have reported high levels of vaccine refusal in northern part of Nigeria ,30. For Furthermore, about 25% of our respondents disagreed with taking the COVID-19 vaccine when it is available. This is higher than what was reported in China (3%), Brazil (12%), Australia (12%), India (13%), Malaysia (15%), Great Britain (15%), Saudi Arabia (16%), South Korea (16%), Peru (21%), and Canada 24%) according to a recent World Economic Forum´s Ipsos survey of nearly 20,000 adults on whether they will take COVID-19 vaccine or not when it is available % accordi.Findings from our study also revealed that the top three reasons for non-acceptance of the COVID-19 vaccine are the unreliability of clinical trials, the immune system is enough to combat COVID-19, and that the COVID-19 vaccine is not safe. This is similar to what was reported by the Ipsos survey, where the top 3 reasons were “worry about side effects”, “doubt about the vaccine effectiveness”, and “perception of not being enough at risk from COVID-19” . Also, iFindings from this study revealed 25% of our respondent will not accept the COVID-19 vaccine. Thus, there is a possible implication of influencing others with their perception of the COVID-19 vaccine leading to more people refusing the vaccine. This could lead to widespread refusal of COVID-19 vaccine due to eroded public trust with the negative information regarding the COVID-19 vaccine. The estimated reproductive number of COVID-19 for Nigeria has been determined to be 2.63 . Going bInterestingly, one of the reasons for non-acceptance of COVID-19 vaccine is the vaccine´s perceived high cost according to our findings. Access is also an important factor to be considered as regards the COVID-19 vaccine ,41, and Limitations: our study is not without its limitations. Generalization of the survey results should be avoided because the pattern of questionnaire distribution may influence the outcome. We used the social media platform, so it may omit older adults, people from lower socioeconomic classes, certain geographical locations, lower educational attainment, and those who were illiterates as well as people who did not have access to the internet. Besides, response/social bias is also a possibility which is not uncommon in self-administered questionnaire research. Finally, acceptance was assessed using a potential  vaccine, which may differ from the respondents' revealed preferences when the vaccine becomes available. Thus, future study may need to put all these gaps into consideration to ensure a far-reaching conclusion in this regard.Even though most of our respondents are willing to take the COVID-19 vaccine, our findings reiterate the need to reassure the public the benefits an effective and safe COVID-19 vaccine can reap for pandemic response. Otherwise, there is a risk of a reversal of hard-won achievement of building public trust in Nigeria´s vaccination programme, potentially compromising reaching community immunity. Public trust is important in promoting public health and plays an essential role in the public's compliance with vaccination programs and other health interventions. However, if the public trust is eroded, false information can spread, leading to the rejection of health interventions with a major threat to public health. It is also important that Nigerian government should heighten their investment in effective and clear vaccine communication and community engagement. Besides, national health authorities, stakeholders and policymakers in Nigeria need to ensure that access to the COVID-19 vaccine is equitable when it becomes available.COVID-19 pandemic is a global public health threat facing mankind;The availability of COVID-19 vaccine will reap enormous benefits to effectively contain the pandemic;There are presently many vaccine candidates under development and some of these vaccine candidates are in Phase III clinical trials.Majority of our respondents are willing to take COVID-19 vaccine when it becomes available;The top three reasons for non-acceptance of the COVID-19 vaccine among our respondents include: unreliability of clinical trials, the immune system is enough to combat COVID-19, and that the COVID-19 vaccine is not safe;Our findings reiterate the need to reassure the public the benefits an effective and safe COVID-19 vaccine can reap for public health. Earning public trust is essential for optimal COVID-19 vaccine acceptance.