Impact of COVID-19 on the Research Career Advancement of Health Equity Scholars from Diverse Backgrounds.

The COVID-19 pandemic has significantly taxed scientific research and seems to have exacerbated existing inequities within the research field, particularly for early-stage investigators (ESIs). This study examines the effects of the COVID-19 pandemic on traditionally underrepresented ESIs enrolled in an NIH-supported study evaluating the effectiveness of developmental networks, grant writing coaching, and mentoring on research career advancement. The survey consisted of 24 closed-ended (quantitative) and 4 open-ended questions (qualitative) linked to a participant's ability to meet grant submission deadlines, research and professional development disruptions, stress level, career transition level, self-efficacy and management of scholarly tasks, and familial responsibilities. Results from 32 respondents (53%) suggest that COVID-19 adversely impacted the continuity of research (81%) and grant submissions (63%). On average, grant submissions were delayed by 6.69 months (i.e., greater than one grant cycle). We also conducted additional analyses characterizing nonresponse and found that there were no significant predictors of nonresponse, indicating a limited threat to the validity of our findings. The disruption caused by COVID-19 to the careers of ESIs from underrepresented groups in the biomedical workforce has been profound in the short term. The long-term consequences to the future success of these groups are unknown but is a worthwhile area of research and potential innovation.

Somatic integration from an adenoviral hybrid vector into a hot spot in mouse liver results in persistent transgene expression levels in vivo.

We have developed a hybrid vector that combines the high transduction efficiency of a gene-deleted adenoviral vector and the integration machinery of the bacteriophage-derived integrase PhiC31 for stable transduction and limited integration sites. We based our system on a two-vector system in which the transgene expression cassette is circularized from a helper-dependent vector by Flp-mediated recombination, followed by PhiC31-mediated integration. Integration of the transgene expression cassette from the adenoviral vector resulted in 5-fold higher transgene expression levels in the active PhiC31 group compared to the control group, which received a mutated and inactive version of PhiC31. We confirmed transgene integration into the previously described mpsL1 hot spot of integration by polymerase chain reaction analyses of DNA isolated from mouse livers. In addition, we cloned 40 integration sites. The hot spot mpsL1 was detected only once, and 44% of all integration events were found to be present in gene regions. With further optimization, this system represents a new tool for gene therapy protocols that may offer an alternative to gene therapy approaches based on random integrating viral vectors.

Characterization of the adenovirus fiber protein.

Entry of the adenovirus (Ad) capsids during the early stages of infection is a multistep process that includes initial attachment of the virus capsid to the cell surface followed by internalization of the virus into early endosomes. The Ad fiber protein, a complex of three apparently identical subunits, mediates the initial attachment step. In this chapter, methods for the purification and characterization of the Ad fiber protein are presented. Chromatographic methods for the isolation of the protein from infected cells can yield substantial quantities of protein for biochemical analysis. Protocols for characterization of the protein by Western blot and by indirect immunofluorescence of infected cells are also presented. The specificity of different monoclonal and polyclonal antibodies that recognize Ad fiber is also discussed. Ad fiber from a number of serotypes also contains a posttranslational modification, O-linked N-acetyl-glucosamine; methods for detection and characterization of this modification are also provided. With these tools and protocols, one can address important questions about this protein, which helps direct the tissue tropism of Ad.

Molecular analysis of chromosomal rearrangements in mammalian cells after phiC31-mediated integration.

Reports on insertional mutagenesis due to integration of gene therapy vectors into the host genome have raised concerns about the genetic manipulation of somatic cells. Previously, it was demonstrated that integrase phiC31 derived from a Streptomyces phage mediates site-specific integration into the host genome of mammalian cells in vitro and in vivo by recombining the attB recognition site in an episomal plasmid and one or more pseudoattP sites in the host chromosomes. In the present study we investigated whether cryptic phiC31 recognition sites in the host genome may result in chromosomal rearrangements. Of 69 independent integration events analyzed in human cells, 6 (8.7%) integrated into human chromosome 19 (19q13.31) and 10 (14.49%) integrated into human chromosome 12 (12q22). Most importantly, of all integration sites analyzed, 15% were found to contain an integrated transgene that was flanked by DNA sequences originating from two different chromosomes. To confirm chromosomal translocations we performed a polymerase chain reaction analysis of chromosomal DNA flanking the transgene and also performed limited studies to determine the genotype of single-cell clones. Although the mechanism responsible for chromosomal translocations needs to be further characterized, we speculate that cryptic phiC31 attachment sites flanking the transgene and cryptic phiC31 attachment sites in the host genome recombine with each other.

Expressed murine and human CDR-H3 intervals of equal length exhibit distinct repertoires that differ in their amino acid composition and predicted range of structures.

Immunoglobulin junctional diversity is concentrated in the third complementarity-determining region of the heavy chain (CDR-H3), which often plays a dominant role in antigen binding. The range of CDR-H3 lengths in mouse is shorter than in human, and thus the murine repertoire could be presumed to be a subset of the human one. To test this presumption, we analyzed 4751 human and 2170 murine unique, functional, published CDR-H3 intervals. Although tyrosine, glycine, and serine were found to predominate in both species, the human sequences contained fewer tyrosine residues, more proline residues, and more hydrophobic residues (p<0.001, respectively). While changes in amino acid utilization as a function of CDR-H3 length followed similar trends in both species, murine and human CDR-H3 intervals of identical length were found to differ from each other. These differences reflect both divergence of germline diversity and joining gene sequence and somatic selection. Together, these factors promote the production of a rather uniform repertoire in mice of tyrosine-enriched CDR-H3 loops with stabilized hydrogen bond-ladders versus a much more diverse repertoire in human that contains CDR-H3 loops sculpted by the presence of intra-chain disulfide bonds due to germline-encoded cysteine residues as well as the enhanced presence of somatically generated proline residues that preclude hydrogen bond ladder formation. Thus, despite the presumed need to recognize a similar range of antigen epitopes, the murine CDR-H3 repertoire is clearly distinct from its human counterpart in its amino acid composition and its predicted range of structures. These findings represent a benchmark to which CDR-H3 repertoires can be compared to better characterize and understand the shaping of the CDR-H3 repertoire over evolution and during immune responses. This information may also be useful for the design of species-specific CDR-H3 sequences in synthetic antibody libraries.

Nuclear localization of KLF4 is associated with an aggressive phenotype in early-stage breast cancer.

PURPOSE: The Krüppel-like transcription factor KLF4/GKLF induces both malignant transformation and a slow-growth phenotype in vitro. Although KLF4 expression is increased in most cases of breast cancer, it was unknown whether these cases represent a distinct subtype with a different clinical outcome. EXPERIMENTAL DESIGN: We examined expression of KLF4 by immunostaining 146 cases of human primary infiltrating ductal carcinoma of the breast. Staining patterns were correlated with clinical outcome and with established prognostic factors. RESULTS: Subcellular localization exhibited case-to-case variation. Tumors with high nuclear staining and low cytoplasmic staining were termed type 1. For patients with early-stage disease (i.e., stage I or IIA), type 1 staining was associated with eventual death because of breast cancer (hazard ratio, 2.8; 95% confidence interval, 1.23-6.58; P = 0.011). The association was stronger in patients with early-stage cancer and small primary tumors (i.e., < or =2.0 cm in diameter; hazard ratio, 4.3; 95% confidence interval, 1.75-10.62; P < 0.001). For patients with early-stage disease, multivariate analysis indicated that type 1 staining was independently associated with outcome (adjusted hazard ratio 2.6; 95% confidence interval, 1.10-6.05; P = 0.029). Type 1 staining was also associated with high histological grade (P = 0.032), increased expression of Ki67 (P = 0.016), and reduced expression of BCL2 (P = 0.032). In vitro, KLF4 was localized within the nucleus of transformed RK3E epithelial cells, consistent with a nuclear function of this transcription factor during induction of malignant transformation. CONCLUSIONS: The results suggest that localization of KLF4 in the nucleus of breast cancer cells is a prognostic factor and identify KLF4 as a marker of an aggressive phenotype in early-stage infiltrating ductal carcinoma.

Enhancement of gene delivery to cancer cells by a retargeted adenovirus.

The inefficiency of in vivo gene transfer using currently available vectors reflects a major hurdle in cancer gene therapy. Both viral and non-viral approaches that improve gene transfer efficiency have been described, but suffer from a number of limitations. Herein, a fiber-modified adenovirus, carrying the small peptide ligand on the capsid, was tested for the delivery of a transgene to cancer cells. The fiber-modified adenovirus was able to mediate the entry and expression of a beta-galactosidase into cancer cells with increased efficiency compared to the unmodified adenovirus. Particularly, the gene transfer efficiency was improved up to 5 times in OVCAR3 cells, an ovarian cancer cell line. Such transduction systems hold promise for delivering genes to transferrin receptor overexpressing cancer cells, and could be used for future cancer gene therapy.

Functional Implications in Apoptosis by Interferon Inducible Gene Product 1-8D, the Binding Protein to Adenovirus Preterminal Protein.

Adenovirus (Ad) precursor to the terminal protein (pTP) plays an essential roles in the viral DNA replication. Ad pTP serves as a primer for the synthesis of a new DNA strand during the initiation step of replication. In addition, Ad pTP forms organized spherical replication foci on the nuclear matrix (NM) and anchors the viral genome to the NM. Here we identified the interferon inducible gene product 1-8D (Inid) as a pTP binding protein by using a two-hybrid screen of a HeLa cDNA library. Of the clones obtained in this assay, nine were identical to the Inid, a 13-kDa polypeptide that shares homology with genes 1-8U and Leu-13/9-27, most of which have little known functions. The entire open reading frame (ORF) of Inid was cloned into the tetracycline inducible expression vector in order to determine the biological functions related with adenoviral infection. When Inid was introduced to the cells along with adenoviruses, fifty to sixty percent of Ad-infected cells expressing Inid had rounded morphology, which was suggestive of apoptosis. Results from the terminal deoxynucleotidyl transferase (TdT) and DNA fragmentation assays confirmed that Inid induces apoptosis in Ad-infected or in uninfected cells. The Inid binding to pTP may target the cell for apoptotic destruction as a host defense mechanism against the viral infection.

A direct comparison of two nonviral gene therapy vectors for somatic integration: in vivo evaluation of the bacteriophage integrase phiC31 and the Sleeping Beauty transposase.

In this study we performed a head-to-head comparison of the integrase phiC31 derived from a Streptomyces phage and the Sleeping Beauty (SB) transposase, a member of the TC1/mariner superfamily of transposable elements. Mouse liver was cotransfused with a vector containing our most robust human coagulation factor IX expression cassette and the appropriate recombinase recognition site and either a phiC31- or a SB transposase-expressing vector. To analyze transgene persistence and to prove somatic integration in vivo we induced cell cycling of mouse hepatocytes and found that the transgene expression levels dropped by only 16 to 21% and 56 to 66% in mice that received phiC31 and SB, respectively. Notably, no difference in the toxicity profile was detected in mice treated with either recombinase. Moreover we observed that with the integrase-mediated gene transfer, transgene expression levels were dependent on the remaining noncoding vector sequences, which also integrate into the host genome. Further analyses of a hot spot of integration after phiC31-mediated integration revealed small chromosomal deletions at the target site and that the recombination process was not dependent on the orientation in which the phiC31 recognition site attached to the pseudo-recognition sites in the host genome. Coupled together with ongoing improvements in both systems this study suggests that both nonviral vector systems will have important roles in achieving stable gene transfer in vivo.

Improved gene delivery into neuroglial cells using a fiber-modified adenovirus vector.

One impediment to treating neuronal diseases is finding ways to introduce genes into specific neuroglial cell types. Here we describe the strategy for efficient gene delivery via transferrin receptor using an adenovirus bearing a peptide mimic for transferrin. The attachment of the peptide consisted of 12 amino acids on the C-terminus of adenovirus fiber protein significantly improved entry and expression of a beta-galactosidase transgene into neuroglial cells such as astrocytes, and Schwann cells. The entry of re-targeted viruses into cells depends on the attached peptide and the transferrin receptor. Furthermore, transferrin did not affect gene delivery by the engineered adenovirus, suggesting that the effectiveness of therapeutic agents targeted to the receptor would not be diminished by competition with the abundant endogenous transferrin present in the plasma. Therefore, such transduction systems hold promise for efficient delivering gene to neuroglial cells in gene therapy protocols.

Förster resonance energy transfer measurements are consistent with a helical bundle model for lipid-free apolipoprotein A-I.

Apolipoprotein (apo) A-I mutants were constructed for FRET studies to distinguish between two possible lipid-free conformers, a globular helix bundle and an elongated helical hairpin. Mutants containing a single Trp at position 50 were prepared by replacing Trps at positions 8, 72, and 108 with Phe (W@50). Two mutants were constructed from W@50 by incorporating Cys at Arg83 (W@50R83C) or Arg173 (W@50R173C) for attachment of the fluorescent probe AEDANS. Secondary structure of the mutants is very similar to wild type (wt) apo A-I, and fluorescence emission indicates that W50 is protected from solvent. Thermal stabilities of the AEDANS-labeled mutants are also similar to wt. These results indicate that no discernible changes occur in structure or stability as a result of mutations or labeling. The FRET data from W@50 to AEDANS are well-represented by a single distance distribution function with a distance of approximately 22 A for W@50R83C and approximately 19 A for W@50R173C. These distances are consistent with theoretical values calculated from a helical bundle model but not from a helical hairpin. A probability distance distribution function yields significantly small half-width values of 5.6 and 3.7 A, respectively, suggesting low conformational dynamics in both mutants. Differential scanning calorimetry (DSC) was performed on wt and a C-terminal deletion mutant, Delta(187-243), to obtain information on domain architecture. Contrary to expectations, both proteins unfold cooperatively. The results are consistent with the presence of a single folded domain within residues 1-186. These results support the presence of a discrete globular bundle conformation for lipid-free apo A-I.

The bifunctional peptidoglycan lysin of Streptococcus agalactiae bacteriophage B30.

A group B streptococcal (GBS) bacteriophage lysin gene was cloned and expressed in Escherichia coli. The purified recombinant enzyme, calculated to have a molecular mass of 49 677 Da, lysed GBS cells. The susceptibility of GBS cells to lysis by the enzyme depended upon the growth stage at which they were harvested, with early exponential phase cells most sensitive. Calcium ions enhanced the activity of the enzyme. The enzyme also lysed other beta-haemolytic streptococci, including groups A, C, E and G streptococci, but not common oral streptococci, including Streptococcus mutans. The generation of both reducing activity and N-terminal alanine residues during lysis indicated that the lysin is a bifunctional enzyme, possessing both glycosidase and endopeptidase activities. This is consistent with the presence of two conserved sequence domains, an Acm (acetylmuramidase) domain associated with lysozyme activity, and a CHAP (cysteine, histidine-dependent amidohydrolases/peptidases) domain associated with endopeptidase activity. Site-directed mutagenesis of conserved cysteine and histidine residues in the CHAP domain and conserved aspartate and glutamate residues in the Acm domain confirmed their importance for lysozyme and endopeptidase activity respectively.