A Multi-Epitope Vaccine Designed Against Blood-Stage of Malaria: An Immunoinformatic And Structural Approach

Malaria is a complex disease caused by genus Plasmodiumis parasites and is the leading cause of morbidity and mortality worldwide. The most severe form of malaria disease is caused by Plasmodium falciparum. A combination of different approaches is needed to control malaria, and on the other hand, resistance to first-line drugs and insecticides makes the need for an effective vaccine more mandatory than ever. Erythrocyte parasites have the most clinical symptoms, so designing the potential vaccine for this stage of infection could be very helpful. In this research, we used various bioinformatics tools to design an effective antibody-inducing multi-epitope vaccine against the blood-stage of malaria infection. For this purpose, we selected the malaria PfGARP protein as the target here. The predicted B and HTL epitopes and flagellin molecule (as an adjuvant) were connected with suitable linkers and the final construct vaccine was designed. The various properties of this construct, including physicochemical properties, 3D structures, molecular docking, molecular simulations, and in silico cloning were then carried out. Based on preliminary findings, our designed fusion construct could be proposed as a novel potential vaccine candidate against Malaria. However, in vitro and in vivo studies are essential for further validation.

Assessment of L5–S1 anterior lumbar interbody fusion stability in the setting of lengthening posterior instrumentation constructs: a cadaveric biomechanical study

OBJECTIVE Excessive stress and motion at the L5–S1 level can lead to degenerative changes, especially in patients with posterior instrumentation suprajacent to L5. Attention has turned to utilization of L5–S1 anterior lumbar interbody fusion (ALIF) to stabilize the lumbosacral junction. However, questions remain regarding the effectiveness of stand-alone ALIF in the setting of prior posterior instrumented fusions terminating at L5. The purpose of this study was to assess the biomechanical stability of an L5–S1 ALIF with increasing lengths of posterior thoracolumbar constructs. METHODS Seven human cadaveric spines (T9–sacrum) were instrumented with pedicle screws from T10 to L5 and mounted to a 6 degrees-of-freedom robot. Posterior fusion construct lengths (T10–L5, T12–L5, L2–5, and L4–5) were instrumented to each specimen, and torque-fusion level relationships were determined for each construct in flexion-extension, axial rotation, and lateral bending. A stand-alone L5–S1 ALIF was then instrumented, and L5–S1 motion was measured as increasing pure moments (2 to 12 Nm) were applied. Motion reduction was calculated by comparing L5–S1 motion across the ALIF and non-ALIF states. RESULTS The average motion at L5–S1 in axial rotation, flexion-extension, and lateral bending was assessed for each fusion construct with and without ALIF. After adding ALIF to a posterior fusion, L5–S1 motion was significantly reduced relative to the non-ALIF state in all but one fused surgical condition (p < 0.05). Longer fusions with ALIF produced larger L5–S1 motions, and in some cases resulted in motions higher than native state motion. CONCLUSIONS Posterior fusion constructs up to L4–5 could be appropriately stabilized by a stand-alone L5–S1 ALIF when using a nominal threshold of 80% reduction in native motion as a potential positive indicator of fusion. The results of this study allow conclusions to be drawn from a biomechanical standpoint; however, the clinical implications of these data are not well defined. These findings, when taken in appropriate clinical context, can be used to better guide clinicians seeking to treat L5–S1 pathology in patients with prior posterior thoracolumbar constructs.

Genome-wide identification and expression profiling of duplicated flavonoid 3'-hydroxylase gene family in Carthamus tinctorius L.

Flavonoid 3′-hydroxylase (F3’H) enzyme is essential in determining the flavonoids B-ring hydroxylation pattern. It is mainly implicated in the biosynthetic pathway of cyaniding-based anthocyanins, flavonols, and flavan-3-ols. However, the evolution and regulatory mechanism of these important flavonoid hydroxylases have not been systematically investigated in safflower (Carthamus tinctorius L.). In this study, we identified 22 duplicatedCtF3'H-encoding genes from safflower through genome-wide prediction and conservation analysis. Phylogenetic analysis revealed the pattern of conservation and divergence of CtF3'Hs encoding proteins and their homologs from different plant species. The distribution of conserved protein motifs and cis-regulatory units suggested several structural components that could be crucial in deciphering the final function of CtF3'H proteins. Furthermore, the results of RNA-seq and qRT-PCR assay in different flowering tissues suggested differential expression level of CtF3’H genes during flower development. Based on the unique homology of CtF3’H5 with flavonoid 3’ hydroxylases from other plant species, further validation of CtF3’H5 was carried out. The transient expression of CtF3’H5 in onion epidermal cells implied that the subcellular localization of the fusion construct containing CtF3’H5 and GFP was predominantly detected in the plasma membrane. Similarly, the prokaryotic expression and western blot hybridization of CtF3’H5 demonstrated the detection of a stable 50.3kD target protein. However, more efforts are needed to further extend the functional validation of CtF3’H5 in safflower. This study provides a fundamental gateway for future functional studies and understanding the genetic evolution of F3'Hs in plants.

Enhanced serodiagnostic potential of a fusion molecule consisting of Rv1793, Rv2628 and a truncated Rv2608 of Mycobacterium tuberculosis

Serodiagnosis of tuberculosis (TB) can be rapid, reliable and cost-effective if the issue of variable antibody responses of TB patients against different Mycobacterium tuberculosis (Mtb) antigens can be overcome by developing fusion proteins containing epitopes from multiple antigens of Mtb. In this study, Mtb antigens Rv1793, Rv2628, Rv2608 and a truncated variant produced by removing non-epitopic region from N-terminal of Rv2608 (tnRv2608), and the fusion protein Rv1793-Rv2628-tnRv2608 (TriFu64), were expressed in E. coli and purified. Plasma samples from TB patients characterized by sex, age and sputum/culture positivity, were used to compare the sensitivity of the single antigens with the fusion protein. Sensitivity of Rv1793, Rv2628 and Rv2608, was 27.8%, 39% and 36.3%, respectively. Truncation of Rv2608 increased sensitivity by approximately 35% in confirmed TB cases. Sensitivity of the fusion construct, TriFu64 increased to 66% with a specificity of 100%. Importantly, tnRv2608 was better able to detect sputum and culture negative patients, and this carried through to the fusion protein. We demonstrate that fusion of Mtb proteins ensures broad sensitivity across disease types, sex and age groups in a Pakistani population.

Overproducing the BAM complex improves secretion of difficult-to-secrete recombinant autotransporter chimeras

Monomeric autotransporters have been used extensively to transport recombinant proteins or protein domains to the cell surface of Gram-negative bacteria amongst others for antigen display. Genetic fusion of such antigens into autotransporters has yielded chimeras that can be used for vaccination purposes. However, not every fusion construct is transported efficiently across the cell envelope. Problems occur in particular when the fused antigen attains a relatively complex structure in the periplasm, prior to its translocation across the outer membrane. The latter step requires the interaction with periplasmic chaperones and the BAM (β-barrel assembly machinery) complex in the outer membrane. This complex catalyzes insertion and folding of β-barrel outer membrane proteins, including the β-barrel domain of autotransporters. Here, we investigated whether the availability of periplasmic chaperones or the BAM complex is a limiting factor for the surface localization of difficult-to-secrete chimeric autotransporter constructs. Indeed, we found that overproduction of in particular the BAM complex, increases surface display of difficult-to-secrete chimeras. Importantly, this beneficial effect appeared to be generic not only for a number of monomeric autotransporter fusions but also for fusions to trimeric autotransporters. Therefore, overproduction of BAM might be an attractive strategy to improve the production of recombinant autotransporter constructs.

Fluorogenic EXO-Probe Aptamers for Imaging and Tracking Exosomal RNAs

Small extracellular vesicles (sEVs), or exosomes, play important roles in physiological and pathological cellular communication. sEVs contain both short and long non-coding RNAs that regulate gene expression and epigenetic processes. Studying the intricacies of sEV function and RNA-based communication requires tools capable of labeling sEV RNA. Here we developed a novel genetically encodable reporter system for tracking sEV RNAs comprising an sEV-loading RNA sequence, termed the EXO-Code, fused to a fluorogenic RNA Mango aptamer for RNA imaging. This fusion construct allowed the visualization and tracking of RNA puncta and colocalization with markers of multivesicular bodies; imaging RNA puncta within sEVs; and quantification of sEVs. This technology represents a useful and versatile tool to interrogate the role of sEVs in cellular communication via RNA trafficking to sEVs, cellular sorting decisions, and sEV RNA cargo transfer to recipient cells.

Does lumbar spine fusion predispose patients to future total hip replacement?

OBJECTIVE Hip-spine syndrome has been well studied since it was first described by Offierski and MacNab in 1983. Today, strong evidence links symptoms of hip and spine pathology to postsurgical outcomes. Recent studies have reported increased rates of hip dislocation in patients previously treated with total hip arthroplasty (THA) who had undergone lumbar fusion procedures. However, the effect of this link on native hip-joint degeneration remains an area of ongoing research. The purpose of this study was to characterize the relationship between use of lumbar fusion procedures and acceleration of hip pathology by analyzing the rate of future THA in patients with preexisting hip osteoarthritis. METHODS This population-level, retrospective cohort study was conducted by using the PearlDiver research program. The initial patient cohort was defined by the presence of diagnosis codes for hip osteoarthritis. Patients were categorized according to use of lumbar fusion after diagnosis of hip pathology. Survival curves with respect to THA were generated by comparison of the no lumbar fusion cohort with the lumbar fusion cohort. To assess the impact of fusion construct length, the lumbar fusion cohort was then stratified according to the number of levels treated (1–2, 3–7, or ≥ 8 levels). Hazard ratios (HRs) were then calculated for the risk factors of number of levels treated, patient age, and sex. RESULTS A total of 2,275,683 patients matched the authors’ inclusion criteria. Log-rank analysis showed no significant difference in the rates of THA over time between the no lumbar fusion cohort (2,239,946 patients) and lumbar fusion cohort (35,737 patients; p = 0.40). When patients were stratified according to number of levels treated, again no differences in the incidence rates of THA over the study period were determined (p = 0.30). Patients aged 70–74 years (HR 0.871, p < 0.001), 75–79 years (HR 0.733, p < 0.001), 80–84 years (HR 0.557, p < 0.001), and ≥ 85 years (HR = 0.275, p < 0.001) were less likely to undergo THA relative to the reference group (patients aged 65–69 years). CONCLUSIONS Although lumbar fusion was initially hypothesized to have a significant effect on rate of THA, lumbar fusion was not associated with increased need for future THA in patients with preexisting hip osteoarthritis. Additionally, there was no relationship between fusion construct length and rate of THA. Although lumbar fusion reportedly increases the risk of hip dislocation in patients with prior THA, these data suggest that lumbar fusion may not clinically accelerate native hip degeneration.

A synthetic nanobody targeting RBD protects hamsters from SARS-CoV-2 infection

SARS-CoV-2, the causative agent of COVID-191, features a receptor-binding domain (RBD) for binding to the host cell ACE2 protein1–6. Neutralizing antibodies that block RBD-ACE2 interaction are candidates for the development of targeted therapeutics7–17. Llama-derived single-domain antibodies (nanobodies, ~15 kDa) offer advantages in bioavailability, amenability, and production and storage owing to their small sizes and high stability. Here, we report the rapid selection of 99 synthetic nanobodies (sybodies) against RBD by in vitro selection using three libraries. The best sybody, MR3 binds to RBD with high affinity (KD = 1.0 nM) and displays high neutralization activity against SARS-CoV-2 pseudoviruses (IC50 = 0.42 μg mL−1). Structural, biochemical, and biological characterization suggests a common neutralizing mechanism, in which the RBD-ACE2 interaction is competitively inhibited by sybodies. Various forms of sybodies with improved potency have been generated by structure-based design, biparatopic construction, and divalent engineering. Two divalent forms of MR3 protect hamsters from clinical signs after live virus challenge and a single dose of the Fc-fusion construct of MR3 reduces viral RNA load by 6 Log10. Our results pave the way for the development of therapeutic nanobodies against COVID-19 and present a strategy for rapid development of targeted medical interventions during an outbreak.

Occipitocervical fusion of traumatic atlanto-occipital dissociation in a patient with autofused cervical facet joints: illustrative case

BACKGROUND Patients who survive traumatic atlanto-occipital dissociation (AOD) may present with normal neurological examinations and near-normal-appearing diagnostic images, such as cervical radiographs and computed tomography (CT) scans. OBSERVATIONS The authors described a neurologically intact 64-year-old female patient with a degenerative autofusion of her right C4–5 facet joints who presented to their center after a motor vehicle collision. Prevertebral soft tissue swelling and craniocervical subarachnoid hemorrhage prompted awareness and consideration for traumatic AOD. An abnormal occipital condyle–C1 interval (4.67 mm) on CT and craniocervical junction ligamentous injury on magnetic resonance imaging (MRI) confirmed the diagnosis of AOD. Her autofused right C4–5 facet joints were incorporated into the occipitocervical fusion construct. LESSONS Traumatic AOD can be easily overlooked in patients with a normal neurological examination and no associated upper cervical spine fractures. A high index of suspicion is needed when evaluating CT scans because normal values for craniocervical parameters are significantly different from the accepted ranges of normal on radiographs in the adult population. MRI of the cervical spine is helpful to evaluate for atlanto-occipital ligamentous injury and confirm the diagnosis. Occipitocervical fusion construct may need to be extended to incorporate spinal levels with degenerative autofusion to prevent adjacent level degeneration.