Deaf intermarriage has limited effect on the prevalence of recessive deafness and no effect on underlying allelic frequency.

The idea that deaf intermarriage increases the prevalence of deafness was forcefully pushed in the late 19th century by Alexander Graham Bell, in proceedings published by the National Academy of Science. Bell's hypothesis was not supported by a 19th century study by Edward Allen Fay, which was funded by Bell's own organization, the Volta Bureau. The Fay study showed through an analysis of 4,471 deaf marriages that the chances of having deaf children did not increase significantly when both parents were deaf. In light of an apparent increase in non-complementary pairings when a modern dataset of Gallaudet alumni was compared with the 19th century Fay dataset, Bell's argument has been resurrected. This hypothesis is that residential schools for the deaf, which concentrate signing deaf individuals together, have promoted assortative mating, which in turn has increased the prevalence of recessive deafness and also the commonest underlying deafness allele. Because this hypothesis persists, even though it contradicts classical models of assortative mating, it is critically important that it be thoroughly investigated. In this study, we used an established forward-time genetics simulator with parameters and measurements collected from the published literature. Compared to mathematical equations, simulations allowed for more complex modeling, operated without assumptions of parametricity, and captured ending distributions and variances. Our simulation results affirm predictions from classical equations and show that intense assortative mating only modestly increases the prevalence of deafness, with this effect mostly completed by the third generation. More importantly, our data show that even intense assortative mating does not affect the frequency of the underlying alleles under reported conditions. These results are not locus-specific and are generalizable to other forms of recessive deafness. We explain the higher rate of non-complementary pairings measured in the contemporary Gallaudet alumni sample as compared to the Fay dataset.

Welcoming Deaf Students into STEM: Recommendations for University Science Education.

Scientists are shaped by their unique life experiences and bring these perspectives to their research. Diversity in life and cultural experiences among scientists, therefore, broadens research directions and, ultimately, scientific discoveries. Deaf individuals, for example, have successfully contributed their unique perspectives to scientific inquiry. However, deaf individuals still face challenges in university science education. Most deaf students in science, technology, engineering, and mathematics (STEM) disciplines interact with faculty who have little to no experience working with deaf individuals and who often have preconceptions or simply a lack of knowledge about deaf individuals. In addition to a lack of communication access, deaf students may also feel unwelcome in STEM, as do other underrepresented groups. In this essay, we review evidence from the literature and, where data are lacking, contribute the expert opinions of the authors, most of whom are deaf scientists themselves, to identify strategies to best support deaf students in university STEM education. We describe the journey of a hypothetical deaf student and methods for faculty to create a welcoming environment. We describe and provide recommendations for classroom seating and layout, accommodations, teaching strategies, and research mentoring. We also discuss the importance of including deaf scientists in research about deaf individuals.

"Everyone Was Nice But I Was Still Left Out": An Interview Study About Deaf Interns' Research Experiences in STEM.

Science, technology, engineering, and mathematics (STEM) undergraduate research experiences improve success, persistence, and promote a feeling of belonging to a community. Like their hearing peers, deaf STEM majors often participate in undergraduate research experiences. However, deaf students typically interact with hearing faculty lacking experience with deaf students and awareness of Deaf culture, which unintentionally impacts their research experiences. This interview study sought to understand deaf students' research experiences and their relationships with hearing mentors. Findings indicate that lack of awareness of Deaf culture and lack of communication access impact students' experiences. We make recommendations on improving deaf students' research experiences.

The Deaf Mentoring Survey: A Community Cultural Wealth Framework for Measuring Mentoring Effectiveness with Underrepresented Students.

Disabled individuals, women, and individuals from cultural/ethnic minorities continue to be underrepresented in science, technology, engineering, and mathematics (STEM). Research has shown that mentoring improves retention for underrepresented individuals. However, existing mentoring surveys were developed to assess the majority population, not underrepresented individuals. We describe the development of a next-generation mentoring survey built upon capital theory and critical race theory. It emphasizes community cultural wealth, thought to be instrumental to the success of individuals from minority communities. Our survey targets relationships between deaf mentees and their research mentors and includes Deaf community cultural wealth. From our results, we identified four segregating factors: Being a Scientist, which incorporated the traditional capitals; Deaf Community Capital; Asking for Accommodations; and Communication Access. Being a Scientist scores did not vary among the mentor and mentee variables that we tested. However, Deaf Community Capital, Asking for Accommodations, and Communication Access were highest when a deaf mentee was paired with a mentor who was either deaf or familiar with the Deaf community, indicating that cultural competency training should improve these aspects of mentoring for deaf mentees. This theoretical framework and survey will be useful for assessing mentoring relationships with deaf students and could be adapted for other underrepresented groups.

Structural Basis for the Failure of the C1 Domain of Ras Guanine Nucleotide Releasing Protein 2 (RasGRP2) to Bind Phorbol Ester with High Affinity.

The C1 domain represents the recognition module for diacylglycerol and phorbol esters in protein kinase C, Ras guanine nucleotide releasing protein (RasGRP), and related proteins. RasGRP2 is exceptional in that its C1 domain has very weak binding affinity (Kd = 2890 ± 240 nm for [(3)H]phorbol 12,13-dibutyrate. We have identified four amino acid residues responsible for this lack of sensitivity. Replacing Asn(7), Ser(8), Ala(19), and Ile(21) with the corresponding residues from RasGRP1/3 (Thr(7), Tyr(8), Gly(19), and Leu(21), respectively) conferred potent binding affinity (Kd = 1.47 ± 0.03 nm) in vitro and membrane translocation in response to phorbol 12-myristate 13-acetate in LNCaP cells. Mutant C1 domains incorporating one to three of the four residues showed intermediate behavior with S8Y making the greatest contribution. Binding activity for diacylglycerol was restored in parallel. The requirement for anionic phospholipid for [(3)H]phorbol 12,13-dibutyrate binding was determined; it decreased in going from the single S8Y mutant to the quadruple mutant. The full-length RasGRP2 protein with the mutated C1 domains also showed strong phorbol ester binding, albeit modestly weaker than that of the C1 domain alone (Kd = 8.2 ± 1.1 nm for the full-length protein containing all four mutations), and displayed translocation in response to phorbol ester. RasGRP2 is a guanyl exchange factor for Rap1. Consistent with the ability of phorbol ester to induce translocation of the full-length RasGRP2 with the mutated C1 domain, phorbol ester enhanced the ability of the mutated RasGRP2 to activate Rap1. Modeling confirmed that the four mutations helped the binding cleft maintain a stable conformation.

Biological profile of the less lipophilic and synthetically more accessible bryostatin 7 closely resembles that of bryostatin 1.

The bryostatins are a group of 20 macrolides isolated by Pettit and co-workers from the marine organism Bugula neritina. Bryostatin 1, the flagship member of the family, has been the subject of intense chemical and biological investigations due to its remarkably diverse biological activities, including promising indications as therapy for cancer, Alzheimer's disease, and HIV. Other bryostatins, however, have attracted far less attention, most probably due to their relatively low natural abundance and associated scarcity of supply. Among all macrolides in this family, bryostatin 7 is biologically the most potent protein kinase C (PKC) ligand (in terms of binding affinity) and also the first bryostatin to be synthesized in the laboratory. Nonetheless, almost no biological studies have been carried out on this agent. We describe herein the total synthesis of bryostatin 7 based on our pyran annulation technology, which allows for the first detailed biological characterizations of bryostatin 7 with side-by-side comparisons to bryostatin 1. The results suggest that the more easily synthesized and less lipophilic bryostatin 7 may be an effective surrogate for bryostatin 1.

Kinetics of penetration influence the apparent potency of vanilloids on TRPV1.

Evidence that the ligand binding site of TRPV1 lies on the inner face of the plasma membrane and that much of the TRPV1 itself is localized to internal membranes suggests that the rate of ligand entry into the cell may be an important determinant of the kinetics of ligand action. In this study, we synthesized a BODIPY TR-labeled fluorescent capsaicin analog (CHK-884) so that we could directly measure ligand entry. We report that CHK-884 penetrated only slowly into Chinese hamster ovary (CHO) cells expressing rat TRPV1, with a t1/2 of 30 +/- 4 min, and localized in the endoplasmic reticulum and Golgi. Although CHK-884 was only weakly potent for TRPV1 binding (Ki = 6400 +/- 230 nM), it was appreciably more potent when assayed by intracellular calcium imaging and was 3.2-fold more potent with a 1-h incubation time (37 nM) than with a 5-min incubation time. Olvanil, a highly lipophilic vanilloid, yielded an EC50 of 4.3 nM upon intracellular calcium imaging with an incubation time of 1 h, compared with an EC50 value of 29.5 nM for calcium imaging assayed at 5 min. Likewise, the antagonist 5-iodo-resiniferatoxin (5-iodo-RTX) displayed a Ki of 4.2 pM if incubated with CHO-TRPV1 cells for 2 h before addition of capsaicin compared with 1.5 nM if added simultaneously. We conclude that some vanilloids may have slow kinetics of uptake; this slow uptake may affect assessment of structure activity relations and may represent a significant factor for vanilloid drug design.

A novel diacylglycerol-lactone shows marked selectivity in vitro among C1 domains of protein kinase C (PKC) isoforms alpha and delta as well as selectivity for RasGRP compared with PKCalpha.

Although multiple natural products are potent ligands for the diacylglycerol binding C1 domain of protein kinase C (PKC), RasGRP, and related targets, the high conservation of C1 domains has impeded the development of selective ligands. We characterized here a diacylglycerol-lactone, 130C037, emerging from a combinatorial chemical synthetic strategy, which showed substantial selectivity. 130C037 gave shallow binding curves for PKC isoforms alpha, beta, gamma, delta, and epsilon, with apparent Ki values ranging from 340 nm for PKCalpha to 29 nm for PKCepsilon. When binding to isolated C1 domains of PKCalpha and -delta, 130C037 showed good affinity (Ki= 1.78 nm) only for deltaC1b, whereas phorbol 12,13-dibutyrate showed affinities within 10-fold for all. In LNCaP cells, 130C037 likewise selectively induced membrane translocation of deltaC1b. 130C037 bound intact RasGRP1 and RasGRP3 with Ki values of 3.5 and 3.8 nm, respectively, reflecting 8- and 90-fold selectivity relative to PKCepsilon and PKCalpha. By Western blot of Chinese hamster ovary cells, 130C037 selectively induced loss from the cytosol of RasGRP3 (ED50 = 286 nm), partial reduction of PKCepsilon (ED50 > 10 microm), and no effect on PKCalpha. As determined by confocal microscopy in LNCaP cells, 130C037 caused rapid translocation of RasGRP3, limited slow translocation of PKCepsilon, and no translocation of PKCalpha. Finally, 130C037 induced Erk phosphorylation in HEK-293 cells ectopically expressing RasGRP3 but not in control cells, whereas phorbol ester induced phosphorylation in both. The properties of 130C037 provide strong proof of principle for the feasibility of developing ligands with selectivity among C1 domain-containing therapeutic targets.

Role of phorbol ester localization in determining protein kinase C or RasGRP3 translocation: real-time analysis using fluorescent ligands and proteins.

The diacylglycerol signaling pathway, involving protein kinase C (PKC) and RasGRP, is a promising therapeutic target for both cancer and other indications. The phorbol esters, ultrapotent diacylglycerol analogues, bind to and activate PKC and RasGRP. Here, using fluorescent phorbol esters and complementary fluorescent PKC and RasGRP constructs, we determined the localization of the phorbol ester as a function of time after addition and how the resultant PKC or RasGRP3 translocation related to ligand localization. For these studies, we prepared fluorescently labeled phorbol esters of varying lipophilicities based on the BODIPY FL (green) or BODIPY 581/591 (red) fluorophores, and by using fusion constructs of green fluorescent protein or DsRed with PKC isoforms or RasGRP3 expressed in Chinese hamster ovary cells, we simultaneously compared the kinetics and pattern of localization of PKC or RasGRP3 with that of the fluorescent red or green phorbol esters. Binding assays showed that the fluorescent derivatives were potent ligands. Uptake followed a one-compartment pharmacokinetic model with a half-time of minutes to hours, depending on the ligand, and all of the fluorescent phorbol esters localized primarily to intracellular membranes, with little plasma membrane localization. The fluorescent phorbol esters induced translocation of and generally colocalized with PKCdelta or RasGRP3. However, PKCalpha and, initially, PKCdelta, translocated to the plasma membrane, in which little phorbol ester accumulated. The findings argue that the rate of uptake of phorbol esters influences the subsequent pattern of PKCdelta translocation, and that the specificity for PKCalpha translocation is dominated by factors other than the localization of the ligand.

Analysis by fluorescence resonance energy transfer of the interaction between ligands and protein kinase Cdelta in the intact cell.

The role of the protein kinase C (PKC) family of serine/threonine kinases in cellular differentiation, proliferation, apoptosis, and other responses makes them attractive therapeutic targets. The activation of PKCs by ligands in vivo varies depending upon cell type; therefore, methods are needed to screen the potency of PKCs in this context. Here we describe a genetically encoded chimera of native PKCdelta fused to yellow- and cyan-shifted green fluorescent protein, which can be expressed in mammalian cells. This chimeric protein kinase, CY-PKCdelta, retains native or near-native activity in the several biological and biochemical parameters that we tested. Binding assays showed that CY-PKCdelta and native human PKCdelta have similar binding affinity for phorbol 12,13-dibutyrate. Analysis of translocation by Western blotting and confocal microscopy showed that CY-PKCdelta translocates from the cytosol to the membrane upon treatment with ligand, that the translocation has similar dose dependence as that of endogenous PKCdelta, and that the pattern of translocation is indistinguishable from that of the green fluorescent protein-PKCdelta fusion well characterized from earlier studies. Treatment with phorbol ester of cells expressing CY-PKCdelta resulted in a dose-dependent increase in FRET that could be visualized in situ by confocal microscopy or measured fluorometrically. By using this construct, we were able to measure the kinetics and potencies of 12 known PKC ligands, with respect to CY-PKCdelta, in the intact cell. The CY-PKCdelta chimera and the in vivo assays described here therefore show potential for high throughput screening of prospective PKCdelta ligands within the context of cell type.

The lgtABCDE gene cluster, involved in lipooligosaccharide biosynthesis in Neisseria gonorrhoeae, contains multiple promoter sequences.

Biosynthesis of the variable core domain of lipooligosaccharide (LOS) in Neisseria gonorrhoeae is mediated by glycosyl transferases encoded by lgtABCDE. Changes within homopolymeric runs within lgtA, lgtC, and lgtD affect the expression state of these genes, with the nature of the LOS expressed determined by the functionality of these genes. However, the mechanism for modulating the amount of multiple LOS chemotypes expressed in a single cell is not understood. Using mutants containing polar disruptions within the lgtABCDE locus, we determined that the expression of this locus is mediated by multiple promoters and that disruption of transcription from these promoters alters the relative levels of simultaneously expressed LOS chemotypes. Expression of the lgtABCDE locus was quantified by using xylE transcriptional fusions, and the data indicate that this locus is transcribed in trace amounts and that subtle changes in transcription result in phenotypic changes. By using rapid amplification of 5' cDNA ends, transcriptional start sites and promoter sequences were identified within lgtABCDE. Most of these promoters possessed 50 to 67% homology with the consensus gearbox promoter sequence of Escherichia coli.