Use of Epivolve phage display to generate a monoclonal antibody with opsonic activity directed against a subdominant epitope on extracellular loop 4 of Treponema pallidum BamA (TP0326).

Introduction: Syphilis, a sexually transmitted infection caused by the spirochete Treponema pallidum (Tp), is resurging globally. Tp's repertoire of outer membrane proteins (OMPs) includes BamA (ß-barrel assembly machinery subunit A/TP0326), a bipartite protein consisting of a 16-stranded ß-barrel with nine extracellular loops (ECLs) and five periplasmic POTRA (polypeptide transport-associated) domains. BamA ECL4 antisera promotes internalization of Tp by rabbit peritoneal macrophages. Methods: Three overlapping BamA ECL4 peptides and a two-stage, phage display strategy, termed "Epivolve" (for epitope evolution) were employed to generate single-chain variable fragments (scFvs). Additionally, antisera generated by immunizing mice and rabbits with BamA ECL4 displayed by a Pyrococcus furiosus thioredoxin scaffold (PfTrxBamA/ECL4). MAbs and antisera reactivities were evaluated by immunoblotting and ELISA. A comparison of murine and rabbit opsonophagocytosis assays was conducted to evaluate the functional ability of the Abs (e.g., opsonization) and validate the mouse assay. Sera from Tp-infected mice (MSS) and rabbits (IRS) were evaluated for ECL4-specific Abs using PfTrxBamA/ECL4 and overlapping ECL4 peptides in immunoblotting and ELISA assays. Results: Each of the five mAbs demonstrated reactivity by immunoblotting and ELISA to nanogram amounts of PfTrxBamA/ECL4. One mAb, containing a unique amino acid sequence in both the light and heavy chains, showed activity in the murine opsonophagocytosis assay. Mice and rabbits hyperimmunized with PfTrxBamA/ECL4 produced opsonic antisera that strongly recognized the ECL presented in a heterologous scaffold and overlapping ECL4 peptides, including S2. In contrast, Abs generated during Tp infection of mice and rabbits poorly recognized the peptides, indicating that S2 contains a subdominant epitope. Discussion: Epivolve produced mAbs target subdominant opsonic epitopes in BamA ECL4, a top syphilis vaccine candidate. The murine opsonophagocytosis assay can serve as an alternative model to investigate the opsonic potential of vaccinogens. Detailed characterization of BamA ECL4-specific Abs provided a means to dissect Ab responses elicited by Tp infection.

Validation and the Determination of Antibody Bioactivity Using MILKSHAKE and Sundae Protocols.

To develop reproducible results, it is critical that all reagents used in an experiment be validated in an alternative or independent method. We present two such independent methods for determining the specificity of antibodies: (1) "MILKSHAKE," which can be used to validate the liability and specificity of antibodies directed against post-translationally-modified epitopes, and (2) "Sundae," which is a more complete alanine-like scanning method that can be used to better understand the binding and bioactivity of specific residues of a protein. We apply both of these methods to the interaction between an antibody and its antigen.

Epivolve: A Protocol for Site-Directed Antibodies.

Researchers can often successfully generate antibodies to predicted epitopes. Especially when the epitopes are on the surface of a protein or in a hydrophilic loop. But it is difficult to direct recombinant antibodies to bind either to- or near a specific amino acid on a protein or peptide. We have developed a unique immune-targeting strategy, that we call "Epivolve," that enables us to make site-specific antibodies (Abs). Epivolve technology leverages a highly immunogenic modified amino acid that acts as a "pseudo-hapten" immuno-target and takes advantage of Ab affinity maturation technologies to make high-affinity site-specific antibodies. Epivolve functions by the evolution of an Ab paratope to either synonymous or especially non-synonymous amino acid (aa) binding. Here we describe the use of Epivolve technology in phage display and the protocols for developing site-specific antibodies.

MILKSHAKE Western blot and Sundae ELISA: We all scream for better antibody validation.

Knowing that an antibody's sensitivity and specificity is accurate is crucial for reliable data collection. This certainty is especially difficult to achieve for antibodies (Abs) which bind post-translationally modified proteins. Here we describe two validation methods using surrogate proteins in western blot and ELISA. The first method, which we termed "MILKSHAKE" is a modified maltose binding protein, hence the name, that is enzymatically conjugated to a peptide from the chosen target which is either modified or non-modified at the residue of interest. The surety of the residue's modification status can be used to confirm Ab specificity to the target's post-translational modification (PTM). The second method uses a set of surrogate proteins, which we termed "Sundae". Sundae consists of a set of modified maltose binding proteins with a genetically encoded target sequence, each of which contains a single amino acid substitution at one position of interest. With Sundae, Abs can be evaluated for binding specificities to all twenty amino acids at a single position. Combining MILKSHAKE and Sundae methods, Ab specificity can be determined at a single-residue resolution. These data improve evaluation of commercially available Abs and identify off-target effects for Research-Use-Only and therapeutic Abs.

New insight into Epstein-Barr virus infection using models of stratified epithelium.

Epstein-Barr virus (EBV) is a ubiquitous human pathogen that is transmitted in saliva. EBV transits through the oral epithelium to infect B cells, where it establishes a life-long latent infection. Reinfection of the epithelium is believed to be mediated by virus shed from B cells, but whether a latent reservoir can exist in the epithelia is unknown. We previously developed an in vitro organotypic model of stratified epithelium where EBV can readily replicate within the suprabasal layers of the epithelium following apical infection mediated by virus-producing B cells. Given that infected epithelial cells and cell-free virus are observed in saliva, we examined the ability of both of these to mediate infection in organotypic cultures. Epithelial-derived cell-free virus was able to infect organotypic cultures from the apical surface, but showed enhanced infection of B cells. Conversely, B cell-derived virus exhibited enhanced infection of epithelial cells. While EBV has been detected in basal cells in oral hairy leukoplakia, it is unknown whether EBV can be seen in undifferentiated primary keratinocytes in the basal layer. Undifferentiated epithelial cells expressed proposed EBV receptors in monolayer and were susceptible to viral binding and entry. Integrins, and occasionally ephrin A2, were expressed in the basal layer of gingiva and tonsil derived organotypic cultures, but the known B-cell receptors HLAII and CD21 were not detected. Following infection with cell-free virus or virus-producing B cells at either the apical or basolateral surface of preformed organotypic cultures, abundant infection was detected in differentiated suprabasal cells while more limited but readily detectable infection was observed in the undifferentiated basal cells. Together, our data has provided new insight into EBV infection in stratified epithelium.

The Epstein-Barr Virus Glycoprotein BDLF2 Is Essential for Efficient Viral Spread in Stratified Epithelium.

Epstein-Barr virus (EBV) is a ubiquitous human pathogen that infects the majority of the adult population regardless of socioeconomic status or geographical location. EBV primarily infects B and epithelial cells and is associated with different cancers of these cell types, such as Burkitt lymphoma and nasopharyngeal carcinoma. While the life cycle of EBV in B cells is well understood, EBV infection within epithelium is not, largely due to the inability to model productive replication in epithelium in vitro. Organotypic cultures generated from primary human keratinocytes can model many aspects of EBV infection, including productive replication in the suprabasal layers. The EBV glycoprotein BDLF2 is a positional homologue of the murine gammaherpesvirus-68 protein gp48, which plays a role in intercellular spread of viral infection, though sequence homology is limited. To determine the role that BDLF2 plays in EBV infection, we generated a recombinant EBV in which the BDLF2 gene has been replaced with a puromycin resistance gene. The ΔBDLF2 recombinant virus infected both B cell and HEK293 cell lines and was able to immortalize primary B cells. However, the loss of BDLF2 resulted in substantially fewer infected cells in organotypic cultures compared to wild-type virus. While numerous clusters of infected cells representing a focus of infection are observed in wild-type-infected organotypic cultures, the majority of cells observed in the absence of BDLF2 were isolated cells, suggesting that the EBV glycoprotein BDLF2 plays a major role in intercellular viral spread in stratified epithelium. IMPORTANCE The ubiquitous herpesvirus Epstein-Barr virus (EBV) is associated with cancers of B lymphocytes and epithelial cells and is primarily transmitted in saliva. While several models exist for analyzing the life cycle of EBV in B lymphocytes, models of EBV infection in the epithelium have more recently been established. Using an organotypic culture model of epithelium that we previously determined accurately reflects EBV infection in situ, we have ascertained that the loss of the viral envelope protein BDLF2 had little effect on the EBV life cycle in B cells but severely restricted the number of infected cells in organotypic cultures. Loss of BDLF2 has a substantial impact on the size of infected areas, suggesting that BDLF2 plays a specific role in the spread of infection in stratified epithelium.

Adopting a "Both/And" Mindset to Address Relationship Violence and Sexual Misconduct (RVSM) in Institutions of Higher Education.

Michigan State University (MSU) created a long-term, values-based strategic plan to increase help-seeking and reduce the incidence of relationship violence and sexual misconduct. Creating systemic change in institutions of higher education is challenging, particularly so in the wake of massive institutional crises and betrayal, as we had at MSU. In this paper, we address the challenges and critiques of our strategic planning efforts offered by esteemed scholar-activists: Jacobson López (2023), Hirsch and Khan (2023), McMahon (2023), and Boots et al. (2023).

Creating a University Strategic Plan to Address Relationship Violence and Sexual Misconduct (RVSM): An Application of Principles-Focused Evaluation at Michigan State University.

This paper describes a multi-year initiative at Michigan State University (MSU) to change our institutional response to relationship violence and sexual misconduct (RVSM) in the aftermath of a large-scale institutional crisis. While the circumstances at MSU are unique, many universities have faced or will face moments that bring RVSM issues into the spotlight. To inform other colleges and universities, we describe how we developed a 5-year strategic plan to transform services for survivors and develop prevention programming for multiple audiences and at multiple levels of analysis. We titled this framework Know More. Do More. Support More, whereby "know more" reflects our ongoing use of campus climate surveys and data sharing to educate our community about RVSM; "do more" includes our institutional-level strategic plan for culture change; and "support more" provides guidance to our community members on how to respond to disclosures in a trauma-informed way and connect survivors to support services. We discuss the challenges and opportunities that stemmed from our choice to work "within the system" to create this model, as well as the ethical dilemmas we faced in these partnerships.

Changes in Acoustic Absorbance Pre- and Post-Cochlear Implantation.

PURPOSE: Until recently, there has been little investigation on the effects of cochlear implantation on the transmission of acoustic stimuli through the middle-ear system. Recent studies have shown that cochlear implantation decreases low-frequency acoustic absorbance, consistent with a stiffer middle-ear system postsurgery. The objectives of this study are (a) to investigate the time course of changes in acoustic absorbance post-cochlear implantation in the implanted ear and (b) to compare changes in acoustic absorbance between implanted and nonimplanted ears over time. METHOD: Seventeen adult cochlear implant (CI) recipients within 6 months of device activation participated in this study. Wideband acoustic absorbance was measured in both ears at one to six different time points from pre-implantation up to 6-month postactivation. Analyses examined (a) changes in acoustic absorbance as compared to pre-implantation and (b) differences in acoustic absorbance between implanted and nonimplanted ears over time. RESULTS: Acoustic absorbance in the implanted ear decreased postsurgery for frequencies lower than 1.5 kHz and persisted through at least 6-month postactivation. We also observed that the spectral range of decreased acoustic absorbance in the implanted ear decreased with longer time postsurgery. Differences in acoustic absorbance between implanted and nonimplanted ears occurred over a broad spectral range at the activation time point and persisted through at least 3-month postactivation, though for a narrower spectral range at the later time point. CONCLUSIONS: Cochlear implantation increased middle-ear stiffness as indicated by decreased acoustic absorbance of low-frequency acoustic power. The findings of this study are consistent with those of previous studies and may have important implications toward understanding spatial hearing and programming of acoustic components for CI-combined electric and binaural acoustic stimulation patients.

Australian Parkinson's Genetics Study (APGS): pilot (n=1532).

PURPOSE: Parkinson's disease (PD) is a neurodegenerative disorder associated with progressive disability. While the precise aetiology is unknown, there is evidence of significant genetic and environmental influences on individual risk. The Australian Parkinson's Genetics Study seeks to study genetic and patient-reported data from a large cohort of individuals with PD in Australia to understand the sociodemographic, genetic and environmental basis of PD susceptibility, symptoms and progression. PARTICIPANTS: In the pilot phase reported here, 1819 participants were recruited through assisted mailouts facilitated by Services Australia based on having three or more prescriptions for anti-PD medications in their Pharmaceutical Benefits Scheme records. The average age at the time of the questionnaire was 64±6 years. We collected patient-reported information and sociodemographic variables via an online (93% of the cohort) or paper-based (7%) questionnaire. One thousand five hundred and thirty-two participants (84.2%) met all inclusion criteria, and 1499 provided a DNA sample via traditional post. FINDINGS TO DATE: 65% of participants were men, and 92% identified as being of European descent. A previous traumatic brain injury was reported by 16% of participants and was correlated with a younger age of symptom onset. At the time of the questionnaire, constipation (36% of participants), depression (34%), anxiety (17%), melanoma (16%) and diabetes (10%) were the most reported comorbid conditions. FUTURE PLANS: We plan to recruit sex-matched and age-matched unaffected controls, genotype all participants and collect non-motor symptoms and cognitive function data. Future work will explore the role of genetic and environmental factors in the aetiology of PD susceptibility, onset, symptoms, and progression, including as part of international PD research consortia.

Ligand-Directed GPCR Antibody Discovery.

Developing affinity reagents recognizing and modulating G-protein coupled receptors (GPCR) function by traditional animal immunization or in vitro screening methods is challenging. Some anti-GPCR antibodies exist on the market, but the success rate of development is still poor compared with antibodies targeting soluble or peripherally anchored proteins. More importantly, most of these antibodies do not modulate GPCR function. The current pipeline for antibody development primarily screens for overall affinity rather than functional epitope recognition. We developed a new strategy utilizing natural ligand affinity to generate a library of antibody variants with an inherent bias toward the active site of the GPCR. Instead of using phage libraries displaying antibodies with random CDR sequences at polymorphism sites observed in natural immune repertoire sequences, we generated focused antibody libraries with a natural ligand encoded within or conjugated to one of the CDRs or the N-terminus. To tailor antibody binding to the active site, we limited the sequence randomization of the antibody in regions holstering the ligand while leaving the ligand-carrying part unaltered in the first round of randomization. With hits from the successful first round, the second round of randomization of the ligand-carrying part was then performed to eliminate the bias of the ligand. Based on our results on three different GPCR targets, the proposed pipeline will enable the rapid generation of functional antibodies (both agonists and antagonists) against high-value targets with poor function epitope exposures including GPCR, channels, transporters as well as cell surface targets whose binding site is heavily masked by glycosylation.

MILKSHAKE: novel validation method for antibodies to post-translationally modified targets by surrogate Western blot.

Antibody (Ab) validation is the procedure in which an Ab is thoroughly assayed for sensitivity and specificity in a given application. Validation of Abs against post-translationally modified (PTM) targets is particularly challenging because it requires specifically prepared antigen. Here we describe a novel validation method using surrogate proteins in a Western blot. The surrogate protein, which we termed 'MILKSHAKE,' is a modified maltose binding protein enzymatically conjugated to a peptide from the chosen target that is either modified or nonmodified at the residue of interest. The certainty of the residue's modification status can be used to confirm Ab specificity. This method also allows for Ab validation even in the absence or limited availability of treated cell lysates.

Comparative performance of bio-based coatings formulated with cellulose, chitin, and chitosan nanomaterials suitable for fruit preservation.

Sustainable nanomaterials (SNMs) from wood, sugarcane and crab shell were prepared and used to coat selected fruits. The properties of SNMs and selected fruits were characterized and strawberry was used as an example to test antifungal activity and freshness preservation of the SNMs. The SNMs with their nano-structured morphology form strong shear-thinning dispersions for easy spraying on fruit surfaces. The fruit surface free energy was influenced by its surface morphology, predominant surface wax components, and cutin monomers. The antifungal activity of SNMs was influenced by their surface functional groups and particle size (crystals vs fibers). The coblend of wood nanocrystals (WCNCs) and chitosan nanofiber (CSNFs) exhibited the best antifungal property, which was comparable with the performance of the fungicide thiabendazole (80 mg L-1). The weight loss and color change of the WCNC/CSNF coated strawberries decreased by nearly half compared with the control samples, showing coating effectiveness on preserving fruit freshness.

Behavioral and Ovipositional Response of Diaeretiella rapae (Hymenoptera: Braconidae) to Rhopalosiphum padi and Brevicoryne brassicae in Winter Wheat and Winter Canola.

Winter canola Brassica napus L. (Brassicales: Brassicaceae) was introduced to U.S. Southern Great Plains (Kansas, Oklahoma, Texas) growers to manage some difficult-to-control grassy weeds in winter wheat Triticum aestivum L. (Poales: Poaceae). Two braconid parasitoids, Diaeretiella rapae (M'Intosh) and Lysiphlebus testaceipes (Cresson) (Hymenoptera: Braconidae) are active in this cropping landscape. Both wasps move between crops but D. rapae has a limited ability to develop in the main wheat aphid hosts, so L. testaceipes could influence D. rapae's ability to maintain itself when canola is absent in the landscape. We compared behavioral responses of naturally emerged D. rapae and wasps that were excised before emergence to odor volatiles of host plant, aphid host and aphid-infested plants using two plant/aphid combinations (wheat/Rhopalosiphum padi (L.) and canola/Brevocoryne brassicae L. (Hemiptera: Aphididae). We also compared parasitism rates of D. rapae that were naturally emerged and excised from R. padi or B. brassicae on subsequent parasitism rates of R. padi or B. brassicae hosts. Naturally emerged wasps responded more strongly to host plant and host plant + aphid odors compared to excised wasps regardless of the host origin. Neither wasp group responded to odors from aphids alone. Both wasp groups were most attracted to odors from aphid-infested host plants, regardless of the combination. D. rapae parasitism rates on canola-reared aphids were higher than on wheat-reared aphids. D. rapae parasitism rates were lower when switched from its original host to the alternate host. Results suggest that D. rapae faces challenges to maintain significant populations in the wheat/canola landscape of the Southern Great Plains, especially in years when canola is not locally present.

Rational library design by functional CDR resampling.

Successful antibody discovery relies on diversified libraries, where two aspects are implied, namely the absolute number of unique clones and the percentage of functional clones. Instead of pursuing the absolute quantity thresholded by current display technology, we have sought to maximize the effective diversity by improving functional clone percentage. With the combined effort of bioinformatics, structural biology, molecular immunology and phage display technology, we devised a bioinformatic pipeline to construct and validate libraries via combinatorial assembly of sequences from a database of experimentally validated antibodies. Furthermore, we showed that the libraries constructed as such yielded a significantly increased success rate against different antigen types and generated over 20-fold more unique hits per targets compared with libraries based on traditional degenerate nucleotide methods. Our study indicated that predefined CDR sequences with optimized CDR-framework compatibility could be a productive direction of functional library construction for in vitro antibody development.

Adverse childhood experiences, depression and mental health barriers to work among low-income women.

Recent research has connected childhood abuse to decreased physical and mental health for low-income women in Utah. Further, mental health has established a link to employment problems. This study conducted a secondary analysis of data collected from individuals accessing public assistance to investigate the relationships among retrospective self-reports of childhood emotional, physical and sexual abuse and prospective indicators of mental health and mental health barriers to work. Logistic regression models found strong relationships between childhood abuse and increased odds of depression and mental health barriers to work. Path models highlight the relative importance of depression for those reporting mental health as the biggest barrier to work. Recommendations for social workers, public health professionals, and program administrators are provided.

Physical and mental health correlates of adverse childhood experiences among low-income women.

The present study used secondary data gathered from a statewide random sample of 1,073 adult women enrolled in Utah's single-parent cash assistance program and logistic regression to examine associations between self-reported physical, emotional, and sexual abuse during childhood and later life physical and mental health indicators. Results demonstrated significant associations between low-income women's self-reports of physical, emotional, or sexual abuse in childhood, and current and lifetime anxiety disorder, domestic violence, current posttraumatic stress disorder, bipolar disorder, physical health or mental health issues, and any mental health diagnosis. These results build on previous research to paint a fuller picture of the associations between childhood abuse and physical and mental health for low-income women in Utah. Consistent with research by the Centers for Disease Control and Prevention, findings suggest the applicability of conceptualizing childhood abuse as a public health issue. Social workers can play an integral role in promoting and implementing broader screening practices, connecting affected individuals with long-term interventions, and applying research findings to the design and provision of services within a public health model.

Vessel co-option in primary human tumors and metastases: an obstacle to effective anti-angiogenic treatment?

Angiogenesis has been regarded as essential for tumor growth and progression. Studies of many human tumors, however, suggest that their microcirculation may be provided by nonsprouting vessels and that a variety of tumors can grow and metastasize without angiogenesis. Vessel co-option, where tumor cells migrate along the preexisting vessels of the host organ, is regarded as an alternative tumor blood supply. Vessel co-option may occur in many malignancies, but so far mostly reported in highly vascularized tissues such as brain, lung, and liver. In primary and metastatic lung cancer and liver metastasis from different primary origins, as much as 10-30% of the tumors are reported to use this alternative blood supply. In addition, vessel co-option is introduced as a potential explanation of antiangiogenic drug resistance, although the impact of vessel co-option in this clinical setting is still to be further explored. In this review we discuss tumor vessel co-option with specific examples of vessel co-option in primary and secondary tumors and a consideration of the clinical implications of this alternative tumor blood supply.

Structure-activity relationships of polybiguanides with activity against human immunodeficiency virus type 1.

Previous investigations showing that polydisperse biguanide (PDBG) molecules have activity against human immunodeficiency virus type 1 (HIV-1) also suggested a relationship between PDBG biologic activity and the lengths of hydrocarbon linkers surrounding the positively charged biguanide unit. To better define structure-activity relationships, PDBG molecules with select linker lengths were evaluated for cytotoxicity, anti-HIV-1 activity, and in vivo toxicity. Results of the in vitro experiments demonstrated that increases in linker length (and, therefore, increases in compound lipophilicity) were generally associated with increases in cytotoxicity and antiviral activity against HIV-1. However, a relationship between linker length asymmetry and in vitro therapeutic index (TI) suggested structural specificity in the mechanism of action against HIV-1. Polyethylene hexamethylene biguanide (PEHMB; biguanide units spaced between alternating ethylene and hexamethylene linkers) was found to have the highest in vitro TI (CC50/IC50) among the compounds examined. Recent improvements in PEHMB synthesis and purification have yielded preparations of PEHMB with in vitro TI values of 266 and 7000 against HIV-1 strains BaL and IIIB, respectively. The minimal toxicity of PEHMB relative to polyhexamethylene biguanide (PHMB; biguanide units alternating with hexamethylene linkers) in a murine model of cervicovaginal microbicide toxicity was consistent with considerable differences in cytotoxicity between PEHMB and PHMB observed during in vitro experiments. These structure-activity investigations increase our understanding of PDBG molecules as agents with activity against HIV-1 and provide the foundation for further preclinical studies of PEHMB and other biguanide-based compounds as antiviral and microbicidal agents.