Debugging and consolidating multiple synthetic chromosomes reveals combinatorial genetic interactions.

The Sc2.0 project is building a eukaryotic synthetic genome from scratch. A major milestone has been achieved with all individual Sc2.0 chromosomes assembled. Here, we describe the consolidation of multiple synthetic chromosomes using advanced endoreduplication intercrossing with tRNA expression cassettes to generate a strain with 6.5 synthetic chromosomes. The 3D chromosome organization and transcript isoform profiles were evaluated using Hi-C and long-read direct RNA sequencing. We developed CRISPR Directed Biallelic URA3-assisted Genome Scan, or "CRISPR D-BUGS," to map phenotypic variants caused by specific designer modifications, known as "bugs." We first fine-mapped a bug in synthetic chromosome II (synII) and then discovered a combinatorial interaction associated with synIII and synX, revealing an unexpected genetic interaction that links transcriptional regulation, inositol metabolism, and tRNASerCGA abundance. Finally, to expedite consolidation, we employed chromosome substitution to incorporate the largest chromosome (synIV), thereby consolidating >50% of the Sc2.0 genome in one strain.

Humoral and cellular immune memory to four COVID-19 vaccines.

Multiple COVID-19 vaccines, representing diverse vaccine platforms, successfully protect against symptomatic COVID-19 cases and deaths. Head-to-head comparisons of T cell, B cell, and antibody responses to diverse vaccines in humans are likely to be informative for understanding protective immunity against COVID-19, with particular interest in immune memory. Here, SARS-CoV-2-spike-specific immune responses to Moderna mRNA-1273, Pfizer/BioNTech BNT162b2, Janssen Ad26.COV2.S, and Novavax NVX-CoV2373 were examined longitudinally for 6 months 100% of individuals made memory CD4+ T cells, with cTfh and CD4-CTL highly represented after mRNA or NVX-CoV2373 vaccination. mRNA vaccines and Ad26.COV2.S induced comparable CD8+ T cell frequencies, though only detectable in 60-67% of subjects at 6 months. A differentiating feature of Ad26.COV2.S immunization was a high frequency of CXCR3+ memory B cells. mRNA vaccinees had substantial declines in antibodies, while memory T and B cells were comparatively stable. These results may also be relevant for insights against other pathogens.

SARS-CoV-2 vaccination induces immunological T cell memory able to cross-recognize variants from Alpha to Omicron.

We address whether T cell responses induced by different vaccine platforms (mRNA-1273, BNT162b2, Ad26.COV2.S, and NVX-CoV2373) cross-recognize early SARS-CoV-2 variants. T cell responses to early variants were preserved across vaccine platforms. By contrast, significant overall decreases were observed for memory B cells and neutralizing antibodies. In subjects ∼6 months post-vaccination, 90% (CD4+) and 87% (CD8+) of memory T cell responses were preserved against variants on average by AIM assay, and 84% (CD4+) and 85% (CD8+) preserved against Omicron. Omicron RBD memory B cell recognition was substantially reduced to 42% compared with other variants. T cell epitope repertoire analysis revealed a median of 11 and 10 spike epitopes recognized by CD4+ and CD8+ T cells, with average preservation > 80% for Omicron. Functional preservation of the majority of T cell responses may play an important role as a second-level defense against diverse variants.

A human antibody epitope map of Pfs230D1 derived from analysis of individuals vaccinated with a malaria transmission-blocking vaccine.

Pfs230 domain 1 (Pfs230D1) is an advanced malaria transmission-blocking vaccine antigen demonstrating high functional activity in clinical trials. However, the structural and functional correlates of transmission-blocking activity are not defined. Here, we characterized a panel of human monoclonal antibodies (hmAbs) elicited in vaccinees immunized with Pfs230D1. These hmAbs exhibited diverse transmission-reducing activity, yet all bound to Pfs230D1 with nanomolar affinity. We compiled epitope-binning data for seventeen hmAbs and structures of nine hmAbs complexes to construct a high-resolution epitope map and revealed that potent transmission-reducing hmAbs bound to one face of Pfs230D1, while non-potent hmAbs bound to the opposing side. The structure of Pfs230D1D2 revealed that non-potent transmission-reducing epitopes were occluded by the second domain. The hmAb epitope map delineated binary hmAb combinations that synergized for extremely high-potency, transmission-reducing activity. This work provides a high-resolution guide for structure-based design of enhanced immunogens and informs diagnostics that measure the transmission-reducing response.

Synthetic regulatory genomics uncovers enhancer context dependence at the Sox2 locus.

Sox2 expression in mouse embryonic stem cells (mESCs) depends on a distal cluster of DNase I hypersensitive sites (DHSs), but their individual contributions and degree of interdependence remain a mystery. We analyzed the endogenous Sox2 locus using Big-IN to scarlessly integrate large DNA payloads incorporating deletions, rearrangements, and inversions affecting single or multiple DHSs, as well as surgical alterations to transcription factor (TF) recognition sequences. Multiple mESC clones were derived for each payload, sequence-verified, and analyzed for Sox2 expression. We found that two DHSs comprising a handful of key TF recognition sequences were each sufficient for long-range activation of Sox2 expression. By contrast, three nearby DHSs were entirely context dependent, showing no activity alone but dramatically augmenting the activity of the autonomous DHSs. Our results highlight the role of context in modulating genomic regulatory element function, and our synthetic regulatory genomics approach provides a roadmap for the dissection of other genomic loci.

On the genetic basis of tail-loss evolution in humans and apes.

The loss of the tail is among the most notable anatomical changes to have occurred along the evolutionary lineage leading to humans and to the 'anthropomorphous apes'1-3, with a proposed role in contributing to human bipedalism4-6. Yet, the genetic mechanism that facilitated tail-loss evolution in hominoids remains unknown. Here we present evidence that an individual insertion of an Alu element in the genome of the hominoid ancestor may have contributed to tail-loss evolution. We demonstrate that this Alu element-inserted into an intron of the TBXT gene7-9-pairs with a neighbouring ancestral Alu element encoded in the reverse genomic orientation and leads to a hominoid-specific alternative splicing event. To study the effect of this splicing event, we generated multiple mouse models that express both full-length and exon-skipped isoforms of Tbxt, mimicking the expression pattern of its hominoid orthologue TBXT. Mice expressing both Tbxt isoforms exhibit a complete absence of the tail or a shortened tail depending on the relative abundance of Tbxt isoforms expressed at the embryonic tail bud. These results support the notion that the exon-skipped transcript is sufficient to induce a tail-loss phenotype. Moreover, mice expressing the exon-skipped Tbxt isoform develop neural tube defects, a condition that affects approximately 1 in 1,000 neonates in humans10. Thus, tail-loss evolution may have been associated with an adaptive cost of the potential for neural tube defects, which continue to affect human health today.

CREEPY: CRISPR-mediated editing of synthetic episomes in yeast.

Use of synthetic genomics to design and build 'big' DNA has revolutionized our ability to answer fundamental biological questions by employing a bottom-up approach. Saccharomyces cerevisiae, or budding yeast, has become the major platform to assemble large synthetic constructs thanks to its powerful homologous recombination machinery and the availability of well-established molecular biology techniques. However, introducing designer variations to episomal assemblies with high efficiency and fidelity remains challenging. Here we describe CRISPR Engineering of EPisomes in Yeast, or CREEPY, a method for rapid engineering of large synthetic episomal DNA constructs. We demonstrate that CRISPR editing of circular episomes presents unique challenges compared to modifying native yeast chromosomes. We optimize CREEPY for efficient and precise multiplex editing of >100 kb yeast episomes, providing an expanded toolkit for synthetic genomics.

SARS-CoV-2 monoclonal antibody treatment followed by vaccination shifts human memory B cell epitope recognition suggesting antibody feedback.

Therapeutic monoclonal antibodies (mAbs) have been studied in humans, but the impact on immune memory of mAb treatment during an ongoing infection has remained unclear. We evaluated the effect of infusion of the anti-SARS-CoV-2 spike receptor binding domain (RBD) mAb bamlanivimab on memory B cells (MBCs) in SARS-CoV-2-infected individuals. Bamlanivimab treatment skewed the repertoire of memory B cells targeting Spike towards non-RBD epitopes. Furthermore, the relative affinity of RBD memory B cells was weaker in mAb-treated individuals compared to placebo-treated individuals over time. Subsequently, after mRNA COVID-19 vaccination, memory B cell differences persisted and mapped to a specific reduction in recognition of the class II RBD site, the same RBD epitope recognized by bamlanivimab. These findings indicate a substantial role of antibody feedback in regulating memory B cell responses to infection, and single mAb administration can continue to impact memory B cell responses to additional antigen exposures months later.

Effectiveness and safety of biosilicate-enhanced bleaching gels on enamel with early erosion lesion.

AIM: This study evaluated the efficacy and cytotoxicity of 35% hydrogen peroxide (HP) gel incorporated with 10% (w/w) biosilicate (BioS) on sound enamel and early-stage enamel erosion lesions. METHODS: Discs of enamel/dentin were selected, subjected to erosive cycles (0.3% citric acid, pH 2.6), and treated with (n = 8): HP (35% HP, positive control); HP_BioS [carboxymethyl cellulose (CMC) + HP + BioS]; BioS (CMC + BioS); CMC (negative control). The discs were adapted to artificial pulp chambers with the enamel exposed for bleaching, and the dentin facing toward the culture medium (Dulbecco's modified Eagle's medium [DMEM]). Bleaching was performed in three 30-min sessions at 7-day intervals. After bleaching, the diffusion product (DMEM extract + diffused HP) was pipetted onto MDPC-23 odontoblastic cell line and inoculated. Color parameters (ΔL, Δa, Δb), color change (ΔE00), and changes in whiteness index (ΔWID) were determined before (T0) and after the last bleaching session (T3). Cell viability (MTT, %), H2O2 diffusion (µg/mL), oxidative cell stress (OxS), and cell fluorescence (live/dead assay, in confocal microscopy) were assessed (ANOVA/Tukey; α = 0.05). RESULTS: No difference in ΔL, Δa, Δb, ΔE00, and ΔWID were found between HP and HP_BioS (p > 0.05). The incorporation of BioS decreased the HP diffusion into the substrates and mitigated oxidative stress in early-stage eroded enamel (p < 0.05). HP_BioS presented significantly higher cell viability compared with HP under erosion conditions. Live/dead assay indicated that BioS_HP maintained viability with larger clusters of viable cells. CONCLUSION: Incorporating BioS into HP maintained bleaching effectiveness, favored cell viability, reduced the oxidative stress, and the cytotoxicity in teeth with early-stage erosion. CLINICAL SIGNIFICANCE: BioS formulation showed promising results for reducing cytotoxicity in patients seeking tooth bleaching and presenting undetectable early-stage erosion.

The potential of conventional and bulk-fill bioactive composites to inhibit the development of caries lesions around restorations.

PURPOSE: To determine caries inhibition potential of conventional and bulk-fill bioactive composites around restorations. METHODS: Enamel and dentin blocks were prepared using a diamond saw under water irrigation, finished (SiC, 600- and 800-grit) and polished (SiC 1,200, final polish= 0.2 µm). Blocks were then selected through enamel surface microhardness, and enamel and dentin standard cavities were restored (n=10/group) with conventional bioactive composite (Beautifil II, BTF), bulk-fill bioactive composite (Activa BioACTIVE, ACT), glass-ionomer cement (Ionofil Plus, ION), conventional composite (GrandioSO, GSO), and bulk-fill composite (Admira Fusion X-TRA, ADM). Afterwards, the blocks were subjected to pH cycling: 4 hours in demineralization and 20 hours in remineralization solutions for 7 days, before being cut in the middle. One half was used to calculate the carious lesion area (ΔS) using values obtained by cross-sectional microhardness (CSMH) testing. The other half was submitted to polarized light microscopy (PLM) and scanning electron microscopy (SEM). The % of internal gap formation (GAP) of restorations' replicas were analyzed under SEM. Data were analyzed by ANOVA and Tukey test (α= 5%). RESULTS: In terms of CSMH, ION group exhibited the lowest ΔS values, with no significant difference to ADM. The composites BTF and ACT were similar to each other (P< 0.05) and to their negative controls (GSO and ADM), respectively. ION showed lower caries formation under PLM, whereas the GSO group presented a greater demineralized area. ION presented the highest % of internal GAP formation. Bioactive composites (BTF and ACT) were similar to their corresponding conventional ones (GSO and ADM) in terms of GAP formation. CLINICAL SIGNIFICANCE: The glass-ionomer cement was more effective in inhibiting the formation of caries lesions around restorations. Because of the glass-ionomer cement's limited application in high load-bearing areas, the conventional bioactive composite would be a promising clinical choice.

A scoping review on hyposalivation associated with systemic conditions: the role of physical stimulation in the treatment approaches.

BACKGROUND: Several systemic conditions can result in distinct degrees of salivary gland damage and consequent hypofunction. The development of successful management schemes is highly challenging due to the complexity of saliva. This study aimed to systematically map the literature on the physical stimulation of salivary glands for hyposalivation management and the response of individuals according to different systemic conditions causing salivary impairment. METHODS: A systematic search in the literature was performed. Two reviewers independently selected clinical trials, randomized or not, that used physical stimulation to treat hyposalivation caused by systemic conditions. Studies evaluating healthy subjects without hyposalivation were included as controls. Single-arm clinical studies or case series were also included for protocol mapping (PRISMA extension for scoping reviews). RESULTS: Out of 24 included studies, 10 evaluated healthy subjects, from which 9 tested transcutaneous electrical nerve stimulation (TENS) and 1 tested acupuncture and electroacupuncture. Fourteen studies evaluated individuals with hyposalivation: 6 applied TENS, 6 applied low-level laser therapy (LLLT), and 2 applied acupuncture, carried out in post-chemotherapy, medication use, postmenopausal women, hemodialysis patients, smokers, diabetics, Sjögren's syndrome (SS). All showed increased salivation after treatment, except for two LLLT studies in individuals with SS. CONCLUSIONS: Among the different patient groups, individuals with Sjögren's syndrome (SS) exhibited the poorest responses, while those with medication-induced hyposalivation demonstrated the most favorable treatment outcomes, independently of the management strategy for saliva stimulation. It means that physical stimulation of salivary glands holds promise as an alternative for managing hyposalivation in cases of reversible gland damage. However, to make informed decisions in current practice, it is necessary to conduct new well-designed randomized clinical trials with appropriate methodologies.

Differential expression of Acanthamoeba castellanii proteins during amoebic keratitis in rats.

Amoebic keratitis (AK) is a sight-threatening infection characterized by a severe inflammation of the cornea, caused by the free-living protozoan of the genus Acanthamoeba. Identification of amoebic proteins involved in AK pathogenesis may help to elucidate molecular mechanisms of infection and contribute to indicate diagnostic and therapeutic targets. In this study, we evaluated changes in the expression profile of Acanthamoeba proteins triggered by the invasive process, using an approach involving two-dimensional polyacrylamide gel electrophoresis (2DE PAGE), followed by mass spectrometry identification (ESI-IT-TOF LC-MSn). AK was induced by intrastromal inoculation in Wistar rats, using trophozoites from a T4 genotype, human case-derived A. castellanii strain under prolonged axenic culture. Cultures re-isolated from the lesions after two successive passages in the animals were used as biological triplicate for proteomic experiments. Analysis of the protein profile comparing long-term and re-isolated cultures indicated 62 significant spots, from which 27 proteins could be identified in the Acanthamoeba proteome database. Five of them (Serpin, Carboxypeptidase A1, Hypothetical protein, Calponin domain-containing protein, aldo/keto reductase) were exclusively found in the re-isolated trophozoites. Our analysis also revealed that a concerted modulation of several biochemical pathways is triggered when A. castellanii switches from a free-living style to a parasitic mode, including energetic metabolism, proteolytic activity, control of gene expression, protein degradation and methylation of DNA, which may be also involved in gain of virulence in an animal model of AK.

Chemically Soluble Fluoride in Na2FPO3/CaCO3-Based Toothpaste as an Indicator of Fluoride Bioavailability in Saliva during and after Toothbrushing.

The relationship between the concentration of chemically soluble fluoride found in toothpaste and that present in saliva, during and after brushing, was evaluated as an indicator of potentially bioavailable fluoride in toothpaste. Ten adult participants brushed their teeth with the assigned toothpastes: group I: fresh sample of a fluoride toothpaste: Na2FPO3/CaCO3, 1,378 µg F/g of total soluble fluoride (TSF); groups II-IV: aged samples of toothpaste presenting TSF concentrations of 1,160, 900, and 597 µg F/g, respectively; group V: non-F placebo toothpaste. The volunteers brushed their teeth for 1 min with 0.7 g of the toothpaste, all toothbrushing residues (TR) produced were collected, the mouth was rinsed with water, and saliva samples were collected up to 120 min. Total fluoride (TF) and TSF concentrations were determined in TR and in saliva samples using a fluoride ion-selective electrode. TSF concentration (µg F/mL) in TR was determined as an indicator of fluoride bioavailability during toothbrushing and the areas under curves of saliva fluoride concentration versus time (area under the curve, AUC = µg F/mL × min) were calculated as an indicator of fluoride bioavailability after toothbrushing. A significant correlation was found between the TSF concentrations in the toothpastes and the variables TR (r = 0.850; p = 0.0001) and AUC (r = 0.445; p = 0.004). For TF no significant correlation was found for TR (r = -0.099; p = 0.542) and AUC (r = -0.018; p = 0.912). The findings suggest that TSF concentration chemically found in Na2FPO3/CaCO3-based toothpaste could estimate how much fluoride would be bioavailable in saliva when the teeth are brushed.

Effects of activated charcoal powder combined with toothpastes on enamel color change and surface properties.

AIM: To evaluate the effects of activated charcoal powder (COAL) combined with regular (RT) or whitening (WT) toothpastes on enamel color and surface in comparison to carbamide peroxide (CP). METHODS: Dental blocks (n = 10/group) were randomly divided into COAL, COAL/RT, COAL/WT, CP, CP/RT, CP/WT, RT, WT, and CONT (without treatment). Simulated toothbrushing and whitening treatments were followed by colorimetric (ΔE00 , L*, a*, b*), surface roughness (Ra), and enamel topography assays. ΔE00 was submitted to two-way ANOVA and Tukey test. Color coordinates and Ra were tested with three-way repeated measures ANOVA (α = 5%). RESULTS: COAL exhibited greater ΔE00 than CONT (P = .048), but it did not enhance ΔE00 promoted by RT or WT (P > .05). COAL alone increased Ra (P < .001) and altered enamel topography. COAL did not increase Ra caused by RT and WT (P > .05). CP exhibited the highest ΔE00 (P < .05), but it raised Ra and changed enamel topography to a less extent than COAL. CONCLUSION: Even though charcoal powder did not increase enamel Ra when combined with toothpastes, the topography was negatively impacted by COAL. Also, COAL was unable to enhance the color change of RT and WT, or reach the effectiveness of CP. CLINICAL SIGNIFICANCE: The use of activated charcoal-based product, claimed as a natural whitener, before brushing with toothpastes is not only ineffective to change the color of teeth, but also it might result in alterations on the enamel surface. Whitening with CP, instead, was effective during the same period of treatment, which still represents a more appropriate technique to whiten teeth.

Inhibition of neuronal nitric oxide synthase activity does not alter vasopressin secretion in septic rats.

BACKGROUND/PURPOSE: During the early phase of sepsis, hypotension is accompanied by increase of plasma vasopressin hormone (AVP) levels, which decline during the late phase. This hypotension is due in part to increase of nitric oxide (NO) synthesis by nitric oxide synthase (NOS) enzyme. Neuronal isoform of this enzyme (nNOS) is present in vasopressinergics neurons of hypothalamus, but its role in vasopressin secretion during sepsis is unknown. METHODS: We evaluated the role of nNOS in NO production and vasopressin secretion during sepsis. Wistar rats received 7-nitroindazole (50 mg/kg, i.p.), an inhibitor of nNOS activity, or vehicle and were submitted to septic stimulus by cecal ligation and puncture (CLP). At the time points 0, 4, 6, 18 and 24 h after sepsis induction the animals were decapitated and neurohypophysis and hypothalamus were removed for analysis of vasopressin content and NOS activity, respectively. Hematocrit, serum sodium, osmolality, proteins and plasmatic AVP were quantified. RESULTS: Mortality was not affected by 7-nitroindazole (7-NI). Sodium and plasma proteins levels decreased after CLP and the treatment anticipated the protein loss, and delayed serum sodium decrease. Septic animals treated with 7-NI showed decrease of osmolality 4 h after CLP. Nitric oxide synthase activity in hypothalamus increased at 4 and 24 h after CLP and was reduced with 7-NI. Neurohypophysis content of AVP diminished after CLP and 7-NI did not alter this parameter. Plasma AVP levels increased at 6 h and decreased 18 and 24 h after CLP. Treatment with 7-NI did not alter plasma vasopressin levels. CONCLUSION: We concluded that nNOS does not have a substantial role in vasopressin secretion during experimental sepsis.

Giardia intestinalis reshapes mucosal immunity toward a Type 2 response that attenuates inflammatory bowel-like diseases.

Diarrheal diseases are the second leading cause of death in children worldwide. Epidemiological studies show that co-infection with Giardia intestinalis decreases the severity of diarrhea. Here, we show that Giardia is highly prevalent in the stools of asymptomatic school-aged children. It orchestrates a Th2 mucosal immune response, characterized by increased antigen-specific Th2 cells, IL-25, Type 2-associated cytokines, and goblet cell hyperplasia. Giardia infection expanded IL-10-producing Th2 and GATA3+ Treg cells that promoted chronic carriage, parasite transmission, and conferred protection against Toxoplasma gondii-induced lethal ileitis and DSS-driven colitis by downregulating proinflammatory cytokines, decreasing Th1/Th17 cell frequency, and preventing collateral tissue damage. Protection was dependent on STAT6 signaling, as Giardia-infected STAT6-/- mice no longer regulated intestinal bystander inflammation. Our findings demonstrate that Giardia infection reshapes mucosal immunity toward a Type 2 response, which confers a mutualistic protection against inflammatory disease processes and identifies a critical role for protists in regulating mucosal defenses.

Modeling the role of charged residues in thermophilic proteins by rotamer and dynamic cross correlation analysis.

Discerning the determinants of protein thermostability is very important both from the theoretical and applied perspective. Different lines of evidence seem to indicate that a dynamical network of salt bridges/charged residues plays a fundamental role in the thermostability of enzymes. In this work, we applied measures of dynamic variance, like the Gini coefficients, Kullback-Leibler (KL) divergence and dynamic cross correlation (DCC) coefficients to compare the behavior of 3 pairs of homologous proteins from the thermophilic bacterium Thermus thermophilus and mesophilic Escherichia coli. Molecular dynamic (MD) simulations of these proteins were performed at 303 K and 363 K. In the characterization of their side chain rotamer distributions, the corresponding Gini coefficients and KL-divergence both revealed significant correlations with temperature. Similarly, a DCC analysis revealed a higher trend to de-correlate the movement of charged residues at higher temperatures in the thermophilic proteins, when compared with their mesophilic homologues. These results highlight the importance of dynamic electrostatic network interactions for the thermostability of enzymes.

Fracture load and failure mode of semi-direct resin composite occlusal veneers: Influence of design and mechanical cycling.

AIMS: To evaluate the influence of the type of preparation and mechanical cycling on the fracture load and failure mode of semi-direct posterior resin composite restorations. METHODS: In total, 70 healthy third molars were used; 10 belonging to the control group (C - unprepared teeth) and 60 teeth prepared and restored with nanoparticle resin composite, divided into 3 groups (n = 20): O - exclusively occlusal preparation (Table Top); OV - occlusal preparation with buccal extension (Veneerlay); OVP - Occlusal preparation with chamfer on the proximal and buccal-lingual/palatal surfaces (Overlay). The preparations were performed with diamond burs with a thickness of 1 mm. The restorations were made with nanoparticulate resin composite (Filtek Z350 XT) and subsequently received additional polymerization (thermoprocessing). Cementation was performed with the use of universal adhesive system (Single Bond Universal) on dental substrate and dual resin cement (RelyX ARC). Half of the teeth in each group (n = 10) were submitted to mechanical cycling, simulating 6 months of clinical service (5 × 105 fatigue cycles), under dynamic loading of 130 N, at a frequency of 2Hz. Fracture load tests were performed in a universal testing machine with a 200 kgf load cell. Failure mode was classified using scores. Generalized linear models and Fisher Exact tests were applied to the data (significance level of 5%). RESULTS: There were no significant differences between the types of preparation (p = 0.9435), or relative to cycling (p = 0.3764). The Fisher Exact Test showed a significant association between the groups and the type of failure (p = 0.0006), with preparations O (with cycling) and OVP (with and without cycling) exhibiting most failures with restoration fractures without involvement of the dental remnant. CONCLUSIONS: Semi-direct restorations showed resistance to fracture load similar to that of healthy teeth and were capable of withstanding functional masticatory loads. The type of preparation influenced the failure mode of teeth. The Table Top and Overlay types of preparations were those had fewer catastrophic failures, suggesting that since they are more conservative preparations, they made it possible for the tooth to receive a new restorative procedure in the future in cases of failures.

Boophilin D1, a Kunitz type protease inhibitor, as a source of inhibitors for the ZIKA virus NS2B-NS3 protease.

Arboviruses are a global concern for a multitude of reasons, including their increased incidence and human mortality. Vectors associated with arboviruses include the mosquito Aedes sp., which is responsible for transmitting the Zika virus. Flaviviruses, like the Zika virus, present only one chymotrypsin-like serine protease (NS3) in their genome. Together with host enzymes, the NS2B co-factor NS3 protease complex are essential for the viral replication cycle by virus polyprotein processing. To search for Zika virus NS2B-NS3 protease (ZIKVPro) inhibitors, a phage display library was constructed using the Boophilin domain 1 (BoophD1), a thrombin inhibitor from the Kunitz family. A BoophilinD1 library mutated at positions P1-P4' was constructed, presenting a titer of 2.9x106 (cfu), and screened utilizing purified ZIKVPro. The results demonstrated at the P1-P4' positions the occurrence of 47% RALHA sequence (mut 12) and 11.8% RASWA sequence (mut14), SMRPT, or KALIP (wt) sequence. BoophD1-wt and mutants 12 and 14 were expressed and purified. The purified BoophD1 wt, mut 12 and 14, presented Ki values for ZIKVPro of 0.103, 0.116, and 0.101 µM, respectively. The BoophD1 mutant inhibitors inhibit the Dengue virus 2 protease (DENV2) with Ki values of 0.298, 0.271, and 0.379 µM, respectively. In conclusion, BoophD1 mut 12 and 14 selected for ZIKVPro demonstrated inhibitory activity like BoophD1-wt, suggesting that these are the strongest Zika inhibitors present in the BoophD1 mutated phage display library. Furthermore, BoophD1 mutants selected for ZIKVPro inhibit both Zika and Dengue 2 proteases making them potential pan-flavivirus inhibitors.

A human antibody epitope map of the malaria vaccine antigen Pfs25.

Pfs25 is a leading antigen for a malaria transmission-blocking vaccine and shows moderate transmission-blocking activity and induction of rapidly decreasing antibody titers in clinical trials. A comprehensive definition of all transmission-reducing epitopes of Pfs25 will inform structure-guided design to enhance Pfs25-based vaccines, leading to potent transmission-blocking activity. Here, we compiled a detailed human antibody epitope map comprising epitope binning data and structures of multiple human monoclonal antibodies, including three new crystal structures of Pfs25 in complex with transmission-reducing antibodies from Malian volunteers immunized with Pfs25 conjugated to EPA and adjuvanted with AS01. These structures revealed additional epitopes in Pfs25 capable of reducing transmission and expanded this characterization to malaria-exposed humans. This work informs immunogen design to focus the antibody response to transmission-reducing epitopes of Pfs25, enabling development of more potent transmission-blocking vaccines for malaria.