Sequence and characterization of shuttle vectors for molecular cloning in Porphyromonas, Bacteroides and related bacteria.

There is a lack of shuttle vectors to be needed for investigations into the genetics of Porphyromonas gingivalis and related species. To better understand the prevalence of candidates for such tools, we have examined multiple strains of black-pigmented anaerobes (clinical and laboratory isolates) for plasmids. As no plasmids were found in P. gingivalis strains, we have used the pYH420 plasmid, derived from P. asaccharolytica, as backbone to construct a shuttle vector in combination with pUC19 from Escherichia coli. Nucleotide sequence determination of the pYH420 plasmid revealed that it contained a gene with similarity to rep from plasmid pTS1 (isolated from Treponema denticola) as well as a homolog of mobA, a member of a gene family found on mobilizable genetic elements found in the genus Bacteroides. We constructed the pG106 and pG108 shuttle vectors using parts of the pUC19 and pYH420 vectors. This resulted in a vector with a multiple cloning site (MCS) in the lacZ gene enabling us to perform blue-white colony selection. The pG106 and pG108 shuttle vectors are electro-transformable into E. coli, P. gingivalis and B. thetaiotaomicron, where they are stable. We demonstrated that these vectors were suitable in these species for applications of molecular cloning including complementation and gene expression studies. Using the pG108 vector, we complement the hcpR mutant strain of P. gingivalis and rescued its NO2- -sensitive phenotype. We also performed a gene expression study using the P-glow BS2 fluorescent reporter gene and the ahpC promoter in B. thetaiotaomicron.

Current Perspectives Regarding Institutional Conflict of Interest : Commentary on "Institutional Conflicts of Interest in Academic Research".

Policies and processes dealing with institutional conflict of interest (iCOI) lag well behind those dealing with individual COI. To remediate this, academic institutions must develop strategies for addressing some of the unique challenges in iCOI, including: clarifying the definition of iCOI that addresses the range of individuals potentially involved; implementing a well-designed electronic database for reporting and managing iCOI across multiple leadership constituencies; and providing ongoing education to appropriate institutional officials that communicates the importance of managing iCOI.

Training NIH K award recipients: the role of the mentor.

UNLABELLED: Mentors play important roles in training new investigators. This study was designed to determine characteristics of NIH mentored K award recipients and their mentors, their interpersonal interactions, and the factors, which influence satisfaction within this relationship. METHOD: A survey of 3027 NIH mentored K recipients and 1384 mentors was conducted in 2009. Nine hundred twenty-nine (30.7%) of the K recipients and 448 (32.4%) mentors completed the survey. RESULTS: The gender of K respondents was evenly divided while the mentors were 72.1% male. The overall rating of their mentors was positive. Ideally, both thought the mentor should be important in research training; however, in actual practice, both rated the importance as lower. A total of 88.2% of recipients were satisfied with their relationship. Although the number of black K recipients was low, this group was more likely to be dissatisfied with the mentor relationship (6/29 or 20.7%) than their white counterparts. The frequency of meeting or communicating was correlated with K recipient satisfaction. CONCLUSIONS: Overall K recipients are satisfied with their mentor relationships. Although the number of black K recipient respondents was small, the higher level of mentor dissatisfaction should be further evaluated. Qualities of mentors, including the frequency of interactions and accessibility, can influence satisfaction.

Guiding the next generation of NIH investigators in responsible conduct of research: the role of the mentor.

National Institutes of Health (NIH) K award recipients and their mentors were surveyed to investigate the role of the mentor. We found that a majority of mentors provided guidance in responsible research conduct (RCR), and that most of these relationships were deemed helpful. Mentors also responded that they played a greater importance in RCR training of their mentees than the mentees reported. Our results suggest both mentors and mentees report that mentors ideally should play a more important role in RCR training than was actually the case. For conflicting interests, subjects' protection, and misconduct, approximately 50% of K recipients found the mentor to be not at all important or only somewhat important for these areas of RCR training. We conclude the mentor's role is important but not optimal based on the results of our study cohort.

Teaching authorship and publication practices in the biomedical and life sciences.

Examination of a limited number of publisher's Instructions for Authors, guidelines from two scientific societies, and the widely accepted policy document of the International Committee of Medical Journal Editors (ICMJE) provided useful information on authorship practices. Three of five journals examined (Nature, Science, and the Proceedings of the National Academy of Sciences) publish papers across a variety of disciplines. One is broadly focused on topics in medical research (New England Journal of Medicine) and one publishes research reports in a single discipline (Journal of Bacteriology). Similar elements of publication policy and accepted practices were found across the policies of these journals articulated in their Instructions for Authors. A number of these same elements were found in the professional society guidelines of the Society for Neuroscience and the American Chemical Society, as well as the ICMJE Uniform Requirements for Manuscripts Submitted to Biomedical Journals. Taken together, these sources provide the basis for articulating best practices in authorship in scientific research. Emerging from this material is a definition of authorship, as well as policy statements on duplicative publication, conflict of interest disclosure, electronic access, data sharing, digital image integrity, and research requiring subjects' protection, including prior registration of clinical trials. These common elements provide a foundation for teaching about scientific authorship and publication practices across biomedical and life sciences disciplines.

Why do we pay? A national survey of investigators and IRB chairpersons.

The principle that payment to participants should not be undue or coercive is the consensus of international and national guidelines and ethical debates; however, what this means in practice is unclear. This study determined the attitudes and practices of IRB chairpersons and investigators regarding participant payment. One thousand six hundred investigators and 1900 IRB chairpersons received an invitation to participate in a web-based survey. Four hundred and fifty-five investigators (28.3%) and 395 IRB chairpersons (18.6%) responded. The survey was designed to gather considerations that govern payment determination and practical application of these considerations in hypothetical case studies. The survey asked best answer, multiple choice, and open text questions. Short hypothetical case scenarios where presented, and participants were asked to rate factors in the study that might impact payment and then determine their recommended payment. A predictive model was developed for each case to determine factors which affected payment. Although compensation was the primary reason given to justify payment by both investigators and IRB chairpersons, the cases suggested that, in practice, payment is often guided by incentive, as shown by the impact of anticipated difficulty recruiting, inconvenience, and risk in determining payment. Payment models varied by type of study. Ranges for recommended payments by both groups for different types of procedures and studies are presented.

Who's doing the math? Are we really compensating research participants?

Although compensation for expenses to participants in research projects is considered important and the primary reason for paying, there is no evidence to support that investigators and IRB members actually calculate participant cost. Payment recommendations for six hypothetical studies were obtained from a national survey of IRB chairpersons (N = 353) and investigators (N = 495). Survey respondents also recommended payment for specific study procedures. We calculated participant cost for the six hypothetical cases both by procedures and by time involvement. A large percentage recommended only token payments for survey, registry, and medical record review studies. Most chose payment for pharmaceutical studies but the recommended payment did not compensate for calculated costs. Results suggest that compensation and reimbursement as the primary reasons for paying research participants may not match actual practice.

Scientific societies and promotion of the responsible conduct of research: codes, policies, and education.

Scientific societies have a significant opportunity to contribute to the promotion of responsible conduct of research (RCR) and to RCR education. The degree to which societies engage such opportunity spans a broad range. There are three principal ways RCR may be promoted by scientific societies. The first is through codes of conduct encouraging their membership to practice ethical research according to the tenets of these codes. The second is through specialized policies (e.g., publication practices) developed by scientific societies that help define normative behavior. Finally, societies have a role to play in creating materials and resources aimed at educating scientists and trainees in matters pertaining to proper research conduct. This article illustrates examples of each of these activities embraced by different scientific societies. The American Society for Microbiology is used as a specific example of a society that has been proactive in each of these three areas. Scientific societies need to recognize the impact they can have on promoting RCR and to expand their efforts in these three and other relevant areas. The examples provided demonstrate the components of a model for all scientific societies to follow in promoting RCR.

Genome of the opportunistic pathogen Streptococcus sanguinis.

The genome of Streptococcus sanguinis is a circular DNA molecule consisting of 2,388,435 bp and is 177 to 590 kb larger than the other 21 streptococcal genomes that have been sequenced. The G+C content of the S. sanguinis genome is 43.4%, which is considerably higher than the G+C contents of other streptococci. The genome encodes 2,274 predicted proteins, 61 tRNAs, and four rRNA operons. A 70-kb region encoding pathways for vitamin B(12) biosynthesis and degradation of ethanolamine and propanediol was apparently acquired by horizontal gene transfer. The gene complement suggests new hypotheses for the pathogenesis and virulence of S. sanguinis and differs from the gene complements of other pathogenic and nonpathogenic streptococci. In particular, S. sanguinis possesses a remarkable abundance of putative surface proteins, which may permit it to be a primary colonizer of the oral cavity and agent of streptococcal endocarditis and infection in neutropenic patients.

Authorship and publication practices: evaluation of the effect of responsible conduct of research instruction to postdoctoral trainees.

We have studied postdoctoral trainees funded by NIH F32 fellowship awards in order to test the effectiveness of responsible conduct of research (RCR) education in the areas of authorship and publication practices. We used a 3-wave telephone and on-line survey design, conducted over a period of two years, in order to test for individual change before and after completing RCR education. Overall the responses of the subjects suggested a clear awareness of standards and practices in publication. However, our results failed to suggest that RCR education in this group significantly increased the level of ethically appropriate behavioral responses measured in the study. Similarly we saw no significant effect on increasing awareness of or attention to ethical guidelines about authorship and publication practices. Our interpretation of these null findings was influenced by the significant publication experience of our cohort of subjects. We forward possible explanations for these null findings in this context. Most importantly, we do not suggest that our results argue against continued instruction in RCR education. Instead, we believe our data reinforce the importance of careful articulation of course goals and objectives with attention to the background and experience of the student audience when developing RCR curricula.

Awareness of publication guidelines and the responsible conduct of research.

We have conducted a longitudinal survey of NIH-funded F32 postdoctoral fellows to determine if mandated instruction in the responsible conduct of research (RCR) has measurable effects on awareness of, attentiveness to, and behavioral judgments about research ethics and authorship and publication. Of 418 F32 fellows participating in the study, 50% were aware of and had referred to guidelines on authorship and publication practices while 50% were either unaware of or had not referred to guidelines. Groups were similar with regard to total number of peer-reviewed publications and total number of first author publications, years of research experience, years since completing their doctoral degree, and receipt of RCR training. The equal distribution of guideline awareness and use, and group similarities with regard to career development and achievement provided us with an opportunity to consider whether awareness of and use of guidelines is associated with broader judgments about author roles and responsibilities. The findings suggest that awareness and utilization of guidelines are, at best, only modestly associated with more ethically appropriate judgments and attitudes about author roles and responsibilities among novice F32's.

Vaccination with FimA from Streptococcus parasanguis protects rats from endocarditis caused by other viridans streptococci.

The FimA protein of Streptococcus parasanguis is a virulence factor in the rat model of endocarditis, and immunization with FimA protects rats against homologous bacterial challenge. Because FimA-like proteins are widespread among the oral streptococci, the leading cause of native valve endocarditis, we evaluated the ability of this vaccinogen to protect rats when challenged by other streptococcal species. Here we report that FimA vaccination produced antibodies that cross-reacted with and protected against challenge by the oral streptococci S. mitis, S. mutans, and S. salivarius. FimA thus has promise as a vaccinogen to control infective endocarditis caused by oral streptococci.

bctA: a novel pBF4 gene necessary for conjugal transfer in Bacteroides spp.

pBF4 is a 41 kb conjugative R-plasmid that confers MLS (macrolide-lincosamide-streptogramin B) resistance in Bacteroides spp. To identify pBF4 genes governing conjugation, recombinational mutagenesis using a suicide vector carrying fragments of the pBF4 plasmid was employed. One of the six independent insertion mutants of pBF4 isolated using this method was found to be conjugation-deficient. Nucleotide sequence analysis around the insertion site on this plasmid revealed a 2.8 kb ORF that encoded a putative 110 kDa protein. A corresponding protein was observed when a 12 kb DNA fragment containing this ORF was used to program an in vitro transcription-translation system. Both the ORF and the predicted protein were novel when compared to available database sequences. This gene was designated bctA (Bacteroides conjugal transfer). Polyclonal rabbit antibodies that recognized a sub-sequence polypeptide of BctA reacted with a 55 kDa protein in Western blot analysis using a total protein extract from Bacteroides fragilis containing pBF4. The protein was not present in a B. fragilis strain containing the conjugation-deficient insertion mutant of pBF4. The 55 kDa protein was associated with the membrane fraction of B. fragilis. Although the cellular and biochemical basis of bctA-promoted conjugation remains unknown, this work demonstrates the existence of a heretofore unrecognized gene in bacterial conjugation, and the mutagenesis system used provides the means to isolate and characterize other genes involved in conjugal transfer in Bacteroides spp.