Polyprotein synthesis: a journey from the traditional pre-translational method to modern post-translational approaches for single-molecule force spectroscopy.

Polyproteins, an array of protein units of similar or differential functions in tandem, have been extensively utilized by organisms, unicellular or multicellular, as concentrators of the myriad of molecular activities. Most eukaryotic proteins, two-thirds in unicellular organisms, and more than 80% in metazoans, are polyproteins. Although the use of polyproteins continues to evolve in nature, our understanding of the structure-function-property of polyproteins is still limited. Cumbersome recombinant strategies and the lack of convenient in vitro synthetic routes of polyproteins have been rate-determining factors in the progress. However, in this review we have discussed the revolutionary journey of polyprotein synthesis with a major focus on surface-based structure-function-property studies, especially using force spectroscopy at the single-molecule level.

Pattern of serum protein capillary electrophoretogram in SARS- CoV-2 infection.

BACKGROUND AND AIMS: Monoclonal/biclonalgammopathy of unknown significance (MGUS/BGUS) is observed in COVID-19. This study was conducted to determine the changes in serum protein electrophoresis (SPEP) in COVID-19. MATERIALS AND METHODS: In this descriptive (cross-sectional) study, serum inflammatory markers (CRP, IL-6 and ferritin) were measured and SPEP was carried out by capillary electrophoresis method in 35 controls and 30 moderate & 58 severe COVID-19 cases. RESULTS: Serum inflammatory markers were increased in COVID-19 cases with severity. M-band(s), ß-γ bridging and pre-albumin band(s) on SPEP were observed in 15.5, 11 & 12% of severe cases and 3, 4 & 0% moderate COVID-19 cases respectively. Area under curve (AUC) of α 1 and α 2 bands of SPEP increased significantly in severe COVID-19. CONCLUSIONS: We conclude that SPEP changes like the appearance of M-band(s) indicating MGUS(BGUS), ß- γ bridging indicating the presence of fast-moving immunoglobulins, pre-albumin band indicating the rise in serum transthyretin level and the increase in AUC of α 1 and α 2 bands indicating the rise in positive acute phase reactants occur in COVID-19. The occurrence and magnitude of these changes are higher in severe COVID-19 than that in moderate COVID-19. The diagnostic and prognostic significance of these SPEP changes are worth exploring.

Instantaneous splicing and excision of inteins to synthesize polyproteins on a substrate with tunable linkers.

Nature has adapted chimeric polyproteins to achieve superior and multiplexed functionality in a single protein. However, the hurdles in in vitro synthesis have restricted the biomimicry of and subsequent fundamental studies on the structure-function relationship of polyproteins. Recombinant expression of polyproteins and the synthesis of polyproteins via the enzyme-mediated repetitive digestion and ligation of individual protein domains have been widely practiced. However, recombinant expression often suffers from an in vitro refolding process, whereas enzyme-assisted peptide conjugation results in heterogeneous products, primarily due to enzymatic re-digestion, and prolonged and multistep reactions. Moreover, both methods incorporate enzyme-recognition residues of varying lengths as artifacts at interdomain linkers. The linkers, although tiny, regulate the spatiotemporal conformations of the polyproteins differentially and tune the folding dynamics, stability, and functions of the constituent protein. In an attempt to leave no string behind at the interdomain junctions, here, we develop a 'splice and excise' synthetic route for polyproteins on a substrate using two orthogonal split inteins. Inteins self-excise and conjugate the protein units covalently and instantaneously, without any cofactors, and incorporate a single cysteine or serine residue at the interdomain junctions.

Estimation of benzathine penicillin G demand for congenital syphilis elimination with adoption of dual HIV/syphilis rapid diagnostic tests in eleven high burden countries.

BACKGROUND: WHO recommends use of rapid dual HIV/syphilis tests for screening pregnant women (PW) during antenatal care to prevent mother-to-child transmission. Scale-up of testing implies a need to accurately forecast and procure benzathine penicillin (BPG) to treat the additionally identified PW with syphilis. METHODS: Country-reported ANC coverage, PW syphilis screening and treatment coverage values in 2019 were scaled linearly to EMTCT targets by 2030 (constant increasing slope from 2019 figures to 95% in 2030) for 11 focus countries. Antenatal syphilis screening coverage was substituted with HIV screening coverage to estimate potential contribution of rapid dual HIV/syphilis tests in identifying additional PW with syphilis. BPG demand was calculated for 2019-2030 accordingly. RESULTS: The estimated demand for BPG (in 2.4 million unit vials) using current maternal syphilis prevalence and treatment coverage will increase from a baseline of 414,459 doses in 2019 to 683,067 doses (+65%) in 2021 assuming immediate replacement of single HIV test kits with rapid dual HIV/syphilis tests for these 11 countries. Continued scale up of syphilis screening and treatment coverage to reach elimination coverage of 95% will result in an estimated demand increase of 160%, (663,969 doses) from 2019 baseline for a total demand of 1,078,428 BPG doses by 2030. CONCLUSIONS: Demand for BPG will increase following adoption of rapid dual HIV/syphilis test kits due to increases in maternal diagnoses of syphilis. To eliminate congenital syphilis, MNCH clinical programs will need to synergize with disease surveillance programs to accurately forecast BPG demand with scale up of antenatal syphilis screening to ensure adequate treatment is available for pregnant women diagnosed with syphilis.

Interdomain linkers tailor the stability of immunoglobulin repeats in polyproteins.

Linkers in polyproteins are considered as mere spacers between two adjacent domains. However, a series of studies using single-molecule force spectroscopy have recently reported distinct thermodynamic stability of I27 in polyproteins with varying linkers and indicated the vital role of linkers in domain stability. A flexible glycine rich linker (-(GGG)n, n ≥ 3) featured unfolding at lower forces than the regularly used arg-ser (RS) based linker. Interdomain interactions among I27 domains in Gly-rich linkers were suggested to lead to reduced domain stability. However, the negative impact of inter domain interactions on domain stability is thermodynamically counter-intuitive and demanded thorough investigations. Here, using an array of ensemble equilibrium experiments and in-silico measurements with I27 singlet and doublets with two aforementioned linkers, we delineate that the inter-domain interactions in fact raise the stability of the polyprotein with RS linker. More surprisingly, a highly flexible Gly-rich linker has no interference on the stability of polyprotein. Overall, we conclude that flexible linkers are preferred in a polyprotein for maintaining domain's independence.

Weakening of interaction networks with aging in tip-link protein induces hearing loss.

Age-related hearing loss (ARHL) is a common condition in humans marking the gradual decrease in hearing with age. Perturbations in the tip-link protein cadherin-23 that absorbs the mechanical tension from sound and maintains the integrity of hearing is associated with ARHL. Here, in search of molecular origins for ARHL, we dissect the conformational behavior of cadherin-23 along with the mutant S47P that progresses the hearing loss drastically. Using an array of experimental and computational approaches, we highlight a lower thermodynamic stability, significant weakening in the hydrogen-bond network and inter-residue correlations among ß-strands, due to the S47P mutation. The loss in correlated motions translates to not only a remarkable two orders of magnitude slower folding in the mutant but also to a proportionately complex unfolding mechanism. We thus propose that loss in correlated motions within cadherin-23 with aging may trigger ARHL, a molecular feature that likely holds true for other disease-mutations in ß-strand-rich proteins.

Tailored Polyproteins Using Sequential Staple and Cut.

Polyproteins, individual protein units joined covalently in tandem, have evolved as a promising tool for measuring the dynamic folding of biomacromolecules in single-molecule force spectroscopy. However, the synthetic routes to prepare polyproteins have been a bottleneck, and urge development of in vitro methods to knit individual protein units covalently into polyprotein. Employing two enzymes of orthogonal functionalities periodically in sequence, we synthesized monodispersed polyproteins on a solid surface. We used Sortase A (SrtA), the enzyme known for sequence specific transpeptidation, to staple protein units covalently through peptide bonds. Exploiting the sequence-specific peptide cleaving ability of TEV protease, we controlled the progress of the reaction to one attachment at a time. Finally, with unique design of the unit proteins we control the orientation of proteins in polyprotein. This simple conjugation has the potential to staple proteins with different functionalities and from different expression systems, in any number in the polyprotein and, above all, via irreversible peptide bonds. Multiple chimeric constructs can also be synthesized with interchangeable protein units.

Multiple Gingival Recession Coverage Treated with Vestibular Incision Subperiosteal Tunnel Access Approach with or without Platelet-Rich Fibrin - A Case Series.

BACKGROUND: Gingival recession involves both soft tissue and hard tissue loss. In this evolutionary era of dentistry, newer techniques have evolved for complete coverage of isolated recession defects. Since 2012, vestibular incision subperiosteal tunnel access (VISTA) technique was used with various regenerative membranes to treat multiple recession defects (MRDs). Platelet-rich fibrin (PRF) membrane, a pool of growth factors but have any added advantage to recession coverage techniques is controversial. Thus, in this case series, we compare the effect of VISTA with or without PRF-membrane for the treatment of Classes I and III MRDs. SUBJECTS AND METHODS: Four patients between of age 30 and 40 years (two patients having bilateral Class I and another two having bilateral Class III MRDs) were selected from the Department of Periodontics, ITS Dental College, Greater Noida and designated as Case I-IV simultaneously. Recession defects at antagonist sites in each patient were corrected by VISTA approach with or without PRF-membrane. Recorded clinical parameters included recession depth, recession width, pocket probing depth, and clinical attachment level (CAL) at baseline and 6 months postoperatively. RESULTS: Patients having Class I recession defects showed almost complete root coverage with VISTA technique alone and reflected no added advantage of PRF-membrane. However, patients with Class III recession defects treated with VISTA + PRF-membrane showed more reduction in recession depth and gain in CAL as compared to sites treated with VISTA only. CONCLUSION: VISTA alone is a convenient technique for treatment of Class I MRDs. Addition of PRF-membrane for Class III recession defects give better outcome in term of reduction of recession depth and gain in CAL 6 month postoperatively.

Effect of Occlusal Scheme on the Pressure Distribution of Complete Denture Supporting Tissues: An In Vitro Study.

BACKGROUND: The complete denture teeth arrangement that gives maximum denture stability, comfort, esthetics, and function have been studied since several years. Many occlusal schemes have been advised in which the lingualized occlusion, balanced occlusion, and monoplane occlusion are advocated most popularly. The purpose of this study was to compare the pressure values on the supporting tissue using three diff erent posterior occlusal schemes: Balanced occlusion, lingualized occlusion, and monoplane occlusion in simulated dentures. MATERIALS AND METHODS: The simulators used in this study, composed of the maxillary, and mandibular clear heat cure acrylic resin edentulous models. Pressures on the supporting structure under the complete denture were measured using eight strain gauges placed on the model surface on the buccal and lingual slopes of the ridges on the molar and pre-molar region. Pressure on the supporting structure was measured and signals from the sensors were amplifi ed and recorded by the multi-channel electronic strain indicator. RESULTS: The mean pressure which was obtained at each measurement point was compared by one-way ANOVA test. CONCLUSION: Overall monoplane occlusion had lesser pressure values compared to completely balanced and lingualized occlusal scheme. Lingualized occlusal scheme was found to transfer stresses from working side to non-working side to stabilize the mandibular denture.