Saliva microbiome, dietary, and genetic markers are associated with suicidal ideation in university students.

Here, salivary microbiota and major histocompatibility complex (MHC) human leukocyte antigen (HLA) alleles were compared between 47 (12.6%) young adults with recent suicidal ideation (SI) and 325 (87.4%) controls without recent SI. Several bacterial taxa were correlated with SI after controlling for sleep issues, diet, and genetics. Four MHC class II alleles were protective for SI including DRB1*04, which was absent in every subject with SI while present in 21.7% of controls. Increased incidence of SI was observed with four other MHC class II alleles and two MHC class I alleles. Associations between these HLA alleles and salivary bacteria were also identified. Furthermore, rs10437629, previously associated with attempted suicide, was correlated here with SI and the absence of Alloprevotella rava, a producer of an organic acid known to promote brain energy homeostasis. Hence, microbial-genetic associations may be important players in the diathesis-stress model for suicidal behaviors.

Fermentation revival in the classroom: investigating ancient human practices as CUREs for modern diseases.

A course-based undergraduate research experience (CURE) was designed to integrate key microbiological principles and techniques into an authentic research experience in a classroom setting and was implemented in an undergraduate microbiology laboratory course. Students conducted a 6-week study in order to determine the identity and quantity of unique probiotic species from various types of kefir. This course module followed an inquiry-based pedagogical approach in which students use the scientific process to investigate an unknown question with no predetermined outcome. During each lab, relevant microbiological topics and laboratory concepts were presented. Students then performed various laboratory techniques, reinforcing the lecture material with hands-on experience. In addition, students participated in reflection through group presentation of their results, bioinformatic analysis and literature review. Based on data collected from pre- and post-study survey responses, both student knowledge and attitudes towards the topics covered improved due to participation in this CURE. Importantly, this CURE can be implemented at many levels of education, requiring only minimal resources and common laboratory equipment.

The student-centered classroom: the new gut feeling.

A student-centered, interactive course-based undergraduate research experience (CURE) was implemented in a microbiology course in order to provide an authentic research experience and to stimulate student interest and improve understanding of fermentation, probiotics, the human microbiome and related topics. Students were immersed in the scientific process as they used fundamental techniques to investigate the probiotic composition of a fermented milk beverage, kefir-an unknown question with no predetermined outcomes. In order to assess the benefits and effect of this learning experience on the students, pre- and post-study surveys were administered using Qualtrics. Post-study, 93% of participants agreed that fermented foods are beneficial to human health (compared to 52% pre-study), and notably, 100% of participants indicated that they plan to apply this material in both their personal and professional lives and would suggest consuming probiotics or fermented products to alleviate gastrointestinal issues. As evidenced by demographic data, this CURE is suitable for implementation at both large and small institutions with diverse student populations. Collectively, these data indicate that this collaborative, discovery-based learning experience is a powerful educational tool, encouraging students to make real-life connections between microbiology, medicine and their own health.

Eco-oncology: Applying ecological principles to understand and manage cancer.

Cancer is a disease of single cells that expresses itself at the population level. The striking similarities between initiation and growth of tumors and dynamics of biological populations, and between metastasis and ecological invasion and community dynamics suggest that oncology can benefit from an ecological perspective to improve our understanding of cancer biology. Tumors can be viewed as complex, adaptive, and evolving systems as they are spatially and temporally heterogeneous, continually interacting with each other and with the microenvironment and evolving to increase the fitness of the cancer cells. We argue that an eco-evolutionary perspective is essential to understand cancer biology better. Furthermore, we suggest that ecologically informed therapeutic approaches that combine standard of care treatments with strategies aimed at decreasing the evolutionary potential and fitness of neoplastic cells, such as disrupting cell-to-cell communication and cooperation, and preventing successful colonization of distant organs by migrating cancer cells, may be effective in managing cancer as a chronic condition.

Isolation gowns as a potential work hazard.

OBJECTIVES: Isolation gowns are used as a barrier to bacterial transmission from patient to provider and vice versa. If an isolation gown is ineffective, the patient and provider have a potential breach of safety and increased infection risk. This study compared the bacterial permeability of differently rated, commonly uses isolation gowns to assess their effectiveness in preventing simulated bacterial transmittance, and thus contamination, from patient to provider. METHODS: Serial dilutions of Staphylococcus epidermidis in sterile saline were applied to a simulated skin surface. Unrated and Levels 1 through 4 non-sterile isolation gowns contacted the solution, simulating patient contact. Both sides of the contaminated gowns were then cultured on blood agar by rolling a sterile swab across the gown and evenly inoculating the culture plate. Colony counts from inside and outside of the gowns were compared. Separately, S. epidermidis was placed on a sample of each gown and scanning electron microscopy was used to visualize the contaminated gowns' physical structure. RESULTS: Mean bacterial transmittance from outside of the gown (i.e. patient contact side) to inside of the gowns (i.e. provider clothing or skin side) based on gown rating was as follows: unrated: 50.4% (SD 9.0%); Level 1: 39.7% (SD 11.2%); Level 2: 16.3% (SD 10.3%); Level 3: 0.3% (SD 0.8%); Level 4: 0.0% (SD 0.0%). Scanning electron microscope imaging of unrated, Level 1, and Level 2 gowns revealed gown pore sizes much larger than the bacteria. The Welch one-way analysis of variance statistic showed significant difference dependent on gown-level rating. CONCLUSIONS: Unrated, Level 1, and Level 2 isolation gowns do not provide effective bacterial isolation barriers when bacteria like S. epidermidis make contact with one side of the gown material. Not studied, but implied, is that unrated and lower rated isolation gowns would be as or even more physically permeable to virus particles, which are much smaller than bacteria.

Successful Integration of Face-to-Face Bootcamp Lab Courses in a Hybrid Online STEM Program.

The Microbiology and Cell Science program at the University of Florida compressed two standard 16-week lab courses into five-day versions of the course, which are referred to as bootcamp labs. The bootcamp labs have the same objectives, activities, and assessments as their traditional counterparts. Development of the bootcamp labs was part of a larger effort to increase access to the major, and more broadly STEM, by offering a 2+2 hybrid online transfer program. The results of this mixed-methods study include a direct comparison between bootcamp and traditional lab format as an approach for delivery of a face-to-face lab course. The bootcamp lab cohort has a greater diversity of students, with more women and underrepresented minorities in STEM than the traditional semester-long cohorts. Students in the bootcamp labs have comparable grade outcomes and learning gains to students in traditional lab format. Regression analysis identified GPA, but not lab format, as the most significant predictor of success for students enrolled in lab courses. Qualitative results suggest that the bootcamp format may be a better way than traditional formats to teach microbiology lab. In summary, the results demonstrate that a bootcamp version of a face-to-face microbiology course is just as effective as the traditional semester-long version. This work has broader implications as it supports the bootcamp lab approach as a model in STEM education for increasing access and for overcoming a major barrier to online STEM programs: face-to-face delivery of key lab courses.

Break Scrub to Take That Phone Call?

BACKGROUND: The American College of Surgeons reports that 60% of the hundreds of thousands of surgical site infections occurring annually are preventable. The practice of surgeons taking phone calls while remaining sterile in the operating field is often accomplished by interposing a sterile disposable towel between the phone and their glove. After completing the call, surgeons resume operating. The purpose of our study was to test the conceptual idea of whether bacteria transmit from an inanimate object, such as a telephone, to the gloves of a surgeon through a sterile disposable towel. STUDY DESIGN: Glo Germ (Glo Germ Co), an ultraviolet light-enhanced particle powder sized to mimic bacteria, was placed on an inanimate surface and held with a sterile disposable operating room towel covering a sterile surgical glove. The glove was then inspected for Glo Germ using an ultraviolet light. Additionally, 18 operating room telephones were cultured and then held with a Sterile Disposable OR Towel (Medline Industries Inc) covering a sterile surgical glove. The surgical gloves were then cultured to determine if bacteria had transmitted from the telephone through the towel and onto the sterile glove. RESULTS: The Glo Germ powder readily transmitted through the towel to the gloves. Median colony-forming units (CFU) on the cultured telephones for the 17 samples was 10, ranging from 1 to 35 CFUs. Of these 17 samples, 47% had transmission from the telephone to the glove, which was significantly greater than 0% (95% CI 26% to 69%, p < 0.001). CONCLUSIONS: Sterile disposable operating room towels do not provide an effective barrier between bacteria present on operating room telephones and the otherwise sterile gloves of a surgeon.

Efficacy of Mechanical versus Non-Mechanical Sterile Preoperative Skin Preparation With Chlorhexidine Gluconate 4% Solution.

OBJECTIVE: To compare the efficacy of sterile preoperative skin antisepsis using either a 5-minute mechanical preparation or 5-minute non-mechanical preparation with chlorhexidine gluconate 4% solution. STUDY DESIGN: Matched design, ANOVA. ANIMALS: Healthy adult Thoroughbred horses (n = 30). METHODS: Each horse had both surgical preparation methods randomly assigned to identical sites on the left or right upper thigh. Prepared sites were sampled and cultured for bacteria after each preparation step. RESULTS: Mechanical and non-mechanical preparation techniques significantly reduced bacteria isolated from surface swab samples compared with samples taken from unprepared skin and after the preliminary rough prepared skin (P < 0.05). No difference in the number of skin-associated bacteria was detected between the mechanical and non-mechanical sterile preoperative preparation techniques (P = 0.77). Ten species of bacteria were identified by 16s PCR after final skin preparation. CONCLUSIONS: Pre-surgical skin preparation without repeated mechanical scrubbing using chlorhexidine gluconate 4% solution (total contact time, 225 seconds) is effective in reducing bacterial counts.

Development of a distance education program by a Land-Grant University augments the 2-year to 4-year STEM pipeline and increases diversity in STEM.

Although initial interest in science, technology, engineering and mathematics (STEM) is high, recruitment and retention remains a challenge, and some populations are disproportionately underrepresented in STEM fields. To address these challenges, the Microbiology and Cell Science Department in the College of Agricultural and Life Sciences at the University of Florida has developed an innovative 2+2 degree program. Typical 2+2 programs begin with a student earning an associate's degree at a local community college and then transferring to a 4-year institution to complete a bachelor's degree. However, many universities in the United States, particularly land-grant universities, are located in rural regions that are distantly located from their respective states' highly populated urban centers. This geographical and cultural distance could be an impediment to recruiting otherwise highly qualified and diverse students. Here, a new model of a 2+2 program is described that uses distance education as the vehicle to bring a research-intensive university's life sciences curriculum to students rather than the oft-tried model of a university attempting to recruit underrepresented minority students to its location. In this paradigm, community college graduates transfer into the Microbiology and Cell Science program as distance education students to complete their Bachelor of Science degree. The distance education students' experiences are similar to the on-campus students' experiences in that both groups of students take the same department courses taught by the same instructors, take required laboratory courses in a face-to-face format, take only proctored exams, and have the same availability to instructors. Data suggests that a hybrid online transfer program may be a viable approach to increasing STEM participation (as defined by enrollment) and diversity. This approach is particularly compelling as the distance education cohort has comparable grade point averages and retention rates compared to the corresponding on-campus transfer cohort.

Alterations in immunodominance of Streptococcus mutans AgI/II: lessons learned from immunomodulatory antibodies.

Streptococcus mutans antigen I/II (AgI/II) has been widely studied as a candidate vaccine antigen against human dental caries. In this report we follow up on prior studies that indicated that anti-AgI/II immunomodulatory monoclonal antibodies (MAbs) exerted their effects by destabilizing the native protein structure and exposing cryptic epitopes. We show here that similar results can be obtained by immunizing mice with truncated polypeptides out of the context of an intra-molecular interaction that occurs within the full-length molecule and that appears to dampen the functional response against at least two important target epitopes. Putative T cell epitopes that influenced antibody specificity were identified immediately upstream of the alanine-rich repeat domain. Adherence inhibiting antibodies could be induced against two discrete domains of the protein, one corresponding to the central portion of the molecule and the other corresponding to the C-terminus.

YidC1 and YidC2 are functionally distinct proteins involved in protein secretion, biofilm formation and cariogenicity of Streptococcus mutans.

The cariogenic bacterium Streptococcus mutans has two paralogues of the YidC/Oxa1/Alb3 family of membrane protein insertases/chaperones. Disruption of yidC2 results in loss of genetic competence, decreased membrane-associated ATPase activity and stress sensitivity (acid, osmotic and oxidative). Elimination of yidC1 has less severe effects, with little observable effect on growth or stress sensitivity. To examine the respective roles of YidC1 and YidC2, a conditional expression system was developed allowing simultaneous elimination of both endogenous YidCs. The function of the YidC C-terminal tails was also investigated and a chimeric YidC1 protein appended with the C terminus of YidC2 enabled YidC1 to complement a ΔyidC2 mutant for stress tolerance, ATP hydrolysis activity and extracellular glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity. Elimination of yidC1 or yidC2 affected levels of extracellular proteins, including GtfB, GtfC and adhesin P1 (AgI/II, PAc), which were increased without YidC1 but decreased in the absence of YidC2. Both yidC1 and yidC2 were shown to contribute to S. mutans biofilm formation and to cariogenicity in a rat model. Collectively, these results provide evidence that YidC1 and YidC2 contribute to cell surface biogenesis and protein secretion in S. mutans and that differences in stress sensitivity between the ΔyidC1 and ΔyidC2 mutants stem from a functional difference in the C-termini of these two proteins.

A therapeutic anti-Streptococcus mutans monoclonal antibody used in human passive protection trials influences the adaptive immune response.

The adhesin known as Antigen I/II, P1 or PAc of the cariogenic dental pathogen Streptococcus mutans is a target of protective immunity and candidate vaccine antigen. Previously we demonstrated that immunization of mice with S. mutans complexed with anti-AgI/II monoclonal antibodies (MAbs) resulted in changes in the specificity, isotype and functionality of elicited anti-AgI/II antibodies in the serum of immunized mice compared to administration of bacteria alone. In the current study, an anti-AgI/II MAb reported in the literature to confer unexplained long term protection against S. mutans re-colonization following passive immunization in human clinical trials (MAb Guy's 13), and expressed in tobacco plants (MAb Guy's 13 plantibody), was evaluated for its potential immunomodulatory properties. Immunization of BALB/c mice with immune complexes of Guy's 13 plantibody bound to S. mutans whole cells resulted in a similar change in specificity, isotype, and functionality of elicited anti-AgI/II antibodies as had been observed for other immunomodulatory MAbs. This new information, coupled with the recently solved crystal structure of the adhesin, now provides a rational explanation and plausible mechanism of action of passively administered Guy's 13/Guy's 13 plantibody in human clinical trials, and how long-term prevention of S. mutans carriage well past the application period of the therapeutic antibody could have been achieved.

Ubiquitin C-terminal hydrolase-L1 as a biomarker for ischemic and traumatic brain injury in rats.

Ubiquitin C-terminal hydrolase-L1 (UCH-L1), also called neuronal-specific protein gene product 9.5, is a highly abundant protein in the neuronal cell body and has been identified as a possible biomarker on the basis of a recent proteomic study. In this study, we examined whether UCH-L1 was significantly elevated in cerebrospinal fluid (CSF) following controlled cortical impact (CCI) and middle cerebral artery occlusion (MCAO; model of ischemic stroke) in rats. Quantitative immunoblots of rat CSF revealed a dramatic elevation of UCH-L1 protein 48 h after severe CCI and as early as 6 h after mild (30 min) and severe (2 h) MCAO. A sandwich enzyme-linked immunosorbent assay constructed to measure UCH-L1 sensitively and quantitatively showed that CSF UCH-L1 levels were significantly elevated as early as 2 h and up to 48 h after CCI. Similarly, UCH-L1 levels were also significantly elevated in CSF from 6 to 72 h after 30 min of MCAO and from 6 to 120 h after 2 h of MCAO. These data are comparable to the profile of the calpain-produced alphaII-spectrin breakdown product of 145 kDa biomarker. Importantly, serum UCH-L1 biomarker levels were also significantly elevated after CCI. Similarly, serum UCH-L1 levels in the 2-h MCAO group were significantly higher than those in the 30-min group. Taken together, these data from two rat models of acute brain injury strongly suggest that UCH-L1 is a candidate brain injury biomarker detectable in biofluid compartments (CSF and serum).

Ubiquitin C-terminal hydrolase is a novel biomarker in humans for severe traumatic brain injury.

OBJECTIVE: Ubiquitin C-terminal hydrolase (UCH-L1), also called neuronal-specific protein gene product (PGP 9.3), is highly abundant in neurons. To assess the reliability of UCH-L1 as a potential biomarker for traumatic brain injury (TBI) this study compared cerebrospinal fluid (CSF) levels of UCH-L1 from adult patients with severe TBI to uninjured controls; and examined the relationship between levels with severity of injury, complications and functional outcome. DESIGN: This study was designed as prospective case control study. PATIENTS: This study enrolled 66 patients, 41 with severe TBI, defined by a Glasgow coma scale (GCS) score of < or =8, who underwent intraventricular intracranial pressure monitoring and 25 controls without TBI requiring CSF drainage for other medical reasons. SETTING: : Two hospital system level I trauma centers. MEASUREMENTS AND MAIN RESULTS: Ventricular CSF was sampled from each patient at 6, 12, 24, 48, 72, 96, 120, 144, and 168 hrs following TBI and analyzed for UCH-L1. Injury severity was assessed by the GCS score, Marshall Classification on computed tomography and a complicated postinjury course. Mortality was assessed at 6 wks and long-term outcome was assessed using the Glasgow outcome score 6 months after injury. TBI patients had significantly elevated CSF levels of UCH-L1 at each time point after injury compared to uninjured controls. Overall mean levels of UCH-L1 in TBI patients was 44.2 ng/mL (+/-7.9) compared with 2.7 ng/mL (+/-0.7) in controls (p <.001). There were significantly higher levels of UCH-L1 in patients with a lower GCS score at 24 hrs, in those with postinjury complications, in those with 6-wk mortality, and in those with a poor 6-month dichotomized Glasgow outcome score. CONCLUSIONS: These data suggest that this novel biomarker has the potential to determine injury severity in TBI patients. Further studies are needed to validate these findings in a larger sample.

Beneficial immunomodulation by Streptococcus mutans anti-P1 monoclonal antibodies is Fc independent and correlates with increased exposure of a relevant target epitope.

We showed previously that deliberate immunization of BALB/c mice with immune complexes (IC) of the cariogenic bacterium Streptococcus mutans and mAbs against its surface adhesin P1 results in changes in the specificity and isotype of elicited anti-P1 Abs. Depending on the mAb, changes were beneficial, neutral, or detrimental, as measured by the ability of the serum from immunized mice to inhibit bacterial adherence to human salivary agglutinin by a BIAcore surface plasmon resonance assay. The current study further defined changes in the host response that result from immunization with IC containing beneficial mAbs, and evaluated mechanisms by which beneficial immunomodulation could occur in this system. Immunomodulatory effects varied depending upon genetic background, with differing results in C57BL/6 and BALB/c mice. Desirable effects following IC immunization were observed in the absence of activating FcRs in BALB/c Fcer1g transgenic mice. mAb F(ab')(2) mediated desirable changes similar to those observed using intact IgG. Sera from IC-immunized BALB/c mice that were better able to inhibit bacterial adherence demonstrated an increase in Abs able to compete with an adherence-inhibiting anti-P1 mAb, and binding of a beneficial immumomodulatory mAb to S. mutans increased exposure of that epitope. Consistent with a mechanism involving a mAb-mediated structural alteration of P1 on the cell surface, immunization with truncated P1 derivatives lacking segments that contribute to recognition by beneficial immunomodulatory mAbs resulted in an improvement in the ability of elicited serum Abs to inhibit bacterial adherence compared with immunization with the full-length protein.

Biochemical, structural, and biomarker evidence for calpain-mediated cytoskeletal change after diffuse brain injury uncomplicated by contusion.

Calpain-mediated degradation of the cytoskeletal protein alpha-II-spectrin has been implicated in the pathobiology of experimental and human traumatic brain injury (TBI). Spectrin proteolysis after diffuse/widespread TBI uncomplicated by either subtle or overt contusion and/or mass lesions, (i.e. mild to moderate TBI), has not been previously evaluated. To determine the spatiotemporal pattern and cellular localization of calpain-mediated spectrin proteolysis after diffuse/widespread TBI and the extent to which parenchymal changes in calpain-mediated spectrin proteolysis are reflected in the cerebrospinal fluid, adult rats were subjected to a moderate midline fluid percussion injury and allowed to survive for 3 hours to 7 days postinjury. Light and electron microscopic immunocytochemical and Western blot analyses were performed to identify the calpain-specific 145-kDa breakdown product of alpha-II-spectrin (SBDP145). After diffuse TBI, enhanced levels of SBDP145 immunoreactivity were observed in the neocortex, subcortical white matter, thalamus, and hippocampus, peaking between 24 and 48 hours postinjury. Immunoreactivity was localized almost exclusively to damaged axons and axonal terminal debris. Heightened levels of SBDP145 were also observed in the cerebrospinal fluid at 24 hours postinjury. These results confirm the widespread occurrence of calpain-mediated spectrin proteolysis after diffuse TBI without contusion and support the potential utility of SBDPs as biomarkers of a diffusely injured brain.

Traumatic brain injury biomarkers: from pipeline to diagnostic assay development.

In recent years, the term proteomics is often mentioned together with biomarker discovery, as proteomic studies have the capability of identifying unique and unobvious protein biomarkers from tissues or biofluids derived from animal models or human clinical samples inflicted with various diseases. Proteomics has yielded hundreds of potential biomarker candidates. However, biomarker discovery is only the beginning of a long road for generating a validated, clinically relevant, and FDA-approved biomarker assay. Many technical, financial, legal, and regulatory hurdles have to be overcome before the components can be commercially produced (1, 2). This chapter outlines in a condensed version the steps to successfully develop clinically acceptable biomarkers, given the marker of choice withstands the rigor of developmental challenges along the road.

Requirements for surface expression and function of adhesin P1 from Streptococcus mutans.

In this report, we define requirements for the successful translocation and functional maturation of the adhesin P1 of Streptococcus mutans. Conformational epitopes recognized by anti-P1 monoclonal antibodies (MAbs) were further characterized, thus facilitating the use of particular MAbs as tools to monitor the locations of various forms of the protein. We show that correct localization of P1 is dependent on structural features of the molecule itself, including a requisite A region-P region intramolecular interaction that occurs within the cell prior to secretion. P1 also was shown to be affected by several members of the protein-folding-secretion-turnover apparatus. It does not achieve a fully functional form in the absence of the trigger factor PPIase homolog RopA, and its translocation is delayed when DnaK levels are limited. In addition, dnaK message levels are differentially altered in the presence of P1 lacking the alanine-rich compared to the proline-rich repeat domains. Lastly, nonsecreted P1 lacking the P region accumulates within the cell in the absence of htrA, implying an intracellular HtrA protease function in the degradation and turnover of this particular internal-deletion polypeptide. However, the opposite effect is seen for full-length P1, suggesting a sensing mechanism and substrate-dependent alteration in HtrA's function and effect that is consistent with its known ability to switch between chaperone and protease, depending on environmental perturbations.

Use of biomarkers for diagnosis and management of traumatic brain injury patients.

BACKGROUND: Advances in the understanding of human biochemistry and physiology have provided insight into new pathways by which we can understand traumatic brain injury (TBI). Increased sophistication of laboratory techniques and developments in the field of proteomics has led to the discovery and rapid detection of new biomarkers not previously available. OBJECTIVE: To review recent advances in biomarker research for traumatic brain injury, describe the features of the ideal biomarker and to explore the potential role of these biomarkers in improving clinical management of brain injured patients. METHODS: Through a literature review of recent research on TBI biomarkers and through experience with TBI research, important elements of biomarker development are described together with potential applications to patient care. CONCLUSIONS: TBI biomarkers could have a significant impact on patient care by assisting in the diagnosis, risk stratification and management of TBI. Biomarkers could provide major opportunities for the conduct of clinical research, including confirmation of injury mechanism(s) and drug target identification. Continuing studies by the authors' group are now being conducted to elucidate more fully the relationships between new biomarkers and severity of injury and clinical outcomes in all severities of TBI patients.