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The gut microbiome of worms from composting facilities potentially harbors organisms that are beneficial to plant growth and development. In this experiment, we sought to examine the potential impacts of rhizosphere microbiomes derived from Eisenia fetida worm castings (i.e. vermicompost) on tomato (Solanum lycopersicum, L.) plant growth and physiology. Our experiment consisted of a greenhouse trial lasting 17 weeks total in which tomato plants were grown with one of three inoculant treatments: a microbial inoculant created from vermicompost (V), a microbial inoculant created from sterilized vermicompost (SV), and a no-compost control inoculant (C). We hypothesized that living microbiomes from the vermicompost inoculant treatment would enhance host plant growth and gene expression profiles compared to plants grown in sterile and control treatments. Our data showed that bacterial community composition was significantly altered in tomato rhizospheres, but fungal community composition was highly variable in each treatment. Plant phenotypes that were significantly enhanced in the vermicompost and sterile vermicompost treatments, compared to the control, included aboveground biomass and foliar δ15N nitrogen. RNA sequencing revealed distinct gene expression changes in the vermicompost treatment, including upregulation of nutrient transporter genes such as Solyc06g074995 (high affinity nitrate transporter), which exhibited a 250.2-fold increase in expression in the vermicompost treatment compared to both the sterile vermicompost and control treatments. The plant transcriptome data suggest that rhizosphere microbiomes derived from vermicompost can influence tomato gene expression and growth-related regulatory pathways, which highlights the value of RNA sequencing in uncovering molecular responses in plant microbiome studies. [ABSTRACT FROM AUTHOR]

Quantifying actual evapotranspiration (ETa) of riparian zones is important for more robust water balance analyses that will enable better planning, managing, and allocating of water resources as well as developing strategies to protect delicate riparian ecosystem functions. The ETa, sensible heat flux (H), net radiation (Rn,), soil heat flux (G), meteorological variables (air temperature, Ta; incoming shortwave radiation, Rs; wind speed, u3; relative humidity, RH," vapor pressure deficit, VPD; precipitation, etc.), and albedo were measured on an hourly time step, and leaf area index (LAI) and plant height were measured on a weekly basis for a common reed (Phragmites australis) dominated cottonwood (Populus deltoides) and peach-leaf willow (Salix amygdaloides) riparian plant community in 2009 and 2010 through extensive field campaigns conducted in the Platte River basin in central Nebraska. The two growing seasons were contrasted by warmer air temperatures, higher precipitation, and presence of flood water on the surface during the 2010 season. The seasonal variations of daily average ETa were mainly controlled by Rn and air temperature. In 2009, total ETa and precipitation were 679 mm and 280 mm, respectively, and the values were substantially greater in 2010 (982 mm and 508 mm, respectively). The seasonal daily ETa for the mixed plant community ranged from 0.5 to 8.5 mm đ¹ with a seasonal average of 3.7 mm đ¹ in 2009 and from 0.5 to 11 mm đ¹ with a seasonal average of 5.5 mm d1 in 2010. In 2010, ETa varied widely with meteorological conditions and in response to variations in phenology of the vegetation to flooding. In 2009, on a seasonal average basis, a total of 77% and 14% of the available energy was partitioned into ETa and H, respectively. In 2010, over 90% and -12% (negative due to flooding) of the available energy was partitioned into ETa and H, respectively. The research results presented here provide valuable ETa data and information for enhancing the understanding of the interactions between the surface/vegetation conditions and the surrounding microclimate and surface energy balance for mixed riparian vegetation. The results of this research should aid water managers and decision/policy makers in accounting for water use rates of phragmites-dominated cottonwood and peach-leaf willow riparian plant communities in water balance analyses to make better-informed water resources planning and management decisions. [ABSTRACT FROM AUTHOR]

Many transgender youth seek gender affirming care, such as puberty suppression, to prolong decision-making and to align their physical sex characteristics with their gender identity. During peripubertal growth, connective tissues such as tendon rapidly adapt to applied mechanical loads (e.g., exercise) yet if and how tendon adaptation is influenced by sex and gender affirming hormone therapy during growth remains unknown. The goal of this study was to understand the how pubertal suppression influences the structural and functional properties of the Achilles tendon using an established mouse model of transmasculine gender affirming hormone therapy. C57BL/6N female-born mice were assigned to experimental groups to mimic gender-affirming hormone therapy in human adolescents, and treatment was initiated prior to the onset of puberty (at postnatal day 26, P26). Experimental groups included controls and mice serially treated with gonadotropin release hormone analogue (GnRHa), delayed Testosterone (T), or GnRHa followed by T. We found that puberty suppression using GnRHa, with and without T, improved the overall tendon load capacity in female-born mice. Treatment with T resulted in an increase in the maximum load that tendon can withstand before failure. Additionally, we found that GnRHa, but not T, treatment resulted in a significant increase in cell density at the Achilles enthesis., Competing Interests: DISCLOSURES The authors declare no conflict of interest.

Purpose: Body image is a major psychosocial concern for all cancer patients but can affect the adolescent and young adult (AYA) population in distinct ways. Similarly, the prospect of infertility and the fertility preservation process can create additional stress during cancer treatment. Discussions regarding infertility inherently implicate the body and its reproductive function, but downstream effects on self-perception have not been previously described. The aim of this study was to explore the experiences of AYAs as they considered their risk of infertility and options for fertility preservation (FP), specifically the ways in which this impacted body image and FP decision-making., Methods: AYA cancer patients ( n  = 27) aged 12-25 years whose cancer and treatment conferred risk of infertility were recruited through electronic health record query at an NCI-Designated Comprehensive Cancer Center. Participants completed semi-structured interviews, which were recorded, transcribed, and deductively coded for themes related to information needs, knowledge of treatment effects on fertility, and reproductive concerns after cancer. Emergent, inductive themes related to body image were identified., Results: Body image concerns, related to both physical appearance and body functioning emerged. Common concerns included anticipating change as it pertains to the body and its functions, physical discomfort, fear of judgment, and meeting expectations of the body. While these themes are broad in nature, they have been previously explored in relation to body image in general and their emergence in the oncofertility space provides guidance for further optimization of infertility and fertility preservation discussions., Conclusions: AYA cancer patients experience a multitude of body image related disturbances when faced with the possibility of infertility and fertility preservation. In identifying and exploring these themes, future opportunities for improving oncofertility practice and discussions among AYAs with a focus on body image positivity are called upon.

Reproductive late effects after hematopoietic stem cell transplant can have a significant impact on cancer survivors' quality of life. Potential late effects include gonadal insufficiency, genital graft-versus-host disease, uterine injury, psychosexual dysfunction, and an increased risk of breast and cervical cancer in patients treated with total body irradiation. Despite guidelines, screening and treatment are not standardized among at-risk patients. Provider barriers include lack of knowledge of at-risk therapies and evidenced-based guidelines. Patient barriers include a reluctance to report symptoms and lack of awareness of treatment options. System barriers include inefficient implementation of screening tools and poor dissemination of guidelines to providers who serve as the medical home for survivors. This review guides the clinician in identifying and managing reproductive late effects after hematopoietic stem cell transplant to improve outcomes., (© 2023 Wiley Periodicals LLC.)

Objective: To investigate associations between reproductive endocrinology and infertility (REI) providers' prior training and current knowledge, skills, attitudes, and behaviors regarding fertility preservation and family building for transgender and gender-diverse (T/GD) patients., Design: The survey was distributed to members of the Society for Reproductive Endocrinology and Infertility, the REI-physician-focused professional body within the American Society for Reproductive Medicine, with additional participants recruited through snowball sampling., Results: Participants (n = 206) reported on training in T/GD care; 51% endorsed prior training. Most participants (93%) believed T/GD individuals were as fit for parenthood as cisgender individuals. Prior training was associated with an increased likelihood of offering T/GD health resources and more frequent consultations with specialist colleagues.Common barriers to providing care indicated by respondents included cost, delays in gender-affirming care, and lack of knowledge of the potential impact of hormonal interventions. Common facilitators included education and training, prior experience, and affordability of services., Conclusions: Most REI providers believed T/GD individuals are fit for parenthood and agreed that prior training facilitates care for T/GD patients. The lack of provider knowledge emerged as a barrier to care. Although training helped facilitate some components of care, systemic barriers such as the cost and variability of patient population characteristics/experiences are important considerations when serving T/GD individuals., (© 2023 Published by Elsevier Inc. on behalf of American Society for Reproductive Medicine.)

Purpose: As a secondary report to elucidate the diverse spectrum of oncofertility practices for childhood cancer around the globe, we present and discuss the comparisons of oncofertility practices for childhood cancer in limited versus optimum resource settings based on data collected in the Repro-Can-OPEN Study Part I & II., Methods: We surveyed 39 oncofertility centers including 14 in limited resource settings from Africa, Asia, and Latin America (Repro-Can-OPEN Study Part I), and 25 in optimum resource settings from the USA, Europe, Australia, and Japan (Repro-Can-OPEN Study Part II). Survey questions covered the availability of fertility preservation and restoration options offered in case of childhood cancer as well as their degree of utilization., Results: In the Repro-Can-OPEN Study Part I & II, responses for childhood cancer and calculated oncofertility scores showed the following characteristics: (1) higher oncofertility scores in optimum resource settings than in limited resource settings for ovarian and testicular tissue cryopreservation; (2) frequent utilization of gonadal shielding, fractionation of anticancer therapy, oophoropexy, and GnRH analogs; (3) promising utilization of oocyte in vitro maturation (IVM); and (4) rare utilization of neoadjuvant cytoprotective pharmacotherapy, artificial ovary, in vitro spermatogenesis, and stem cells reproductive technology as they are still in preclinical or early clinical research settings., Conclusions: Based on Repro-Can-OPEN Study Part I & II, we presented a plausible oncofertility best practice model to help optimize care for children with cancer in various resource settings. Special ethical concerns should be considered when offering advanced and innovative oncofertility options to children., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Osteoarthritis (OA) is the most common degenerative disease of the connective tissue of the human musculoskeletal system. Despite its widespread prevalence, there are many limitations in its diagnosis and treatment. OA diagnosis currently relies on the presence of clinical symptoms, sometimes accompanied by changes in joint X-rays or MRIs. Biomarkers help not only to diagnose early disease progression but also to understand the process of OA in many ways. In this article, we briefly summarize information on articular joints and joint tissues, the pathogenesis of OA and review the literature about biomarkers in the field of OA, specifically inflammatory cytokines/chemokines, proteins, miRNA, and metabolic biomarkers found in the blood, synovial fluid and in extracellular vesicles.

Lymph node metastasis is the most important prognostic factor for breast cancer patients. In addition to the number of nodes involved and the largest metastatic focus, extranodal extension (ENE) is also used to subclassify breast cancer patients into different risk groups. More recently, pathologists are required to report the size/extent of ENE per the new CAP guideline, as it seems to be associated with more axillary nodal burden and/or a worse prognosis. Although the definition of ENE is largely understood and agreed upon among pathologists around the world, evaluation and reporting for the size of ENE are not. To understand current practice, we conducted an international survey among pathologists who are interested in breast pathology. A total of 70 pathologists responded. The results showed that (1) 98% of the participants reported the presence or absence of ENE and 61% also reported the size of ENE in millimeter (mm). (2) There was no uniform method of measuring the size of ENE; 47% measured the largest dimension regardless of orientation, while 30% measured the largest perpendicular distance from the capsule. (3) The most common factors affecting the accuracy in diagnosis of ENE are the presence of lymphovascular invasion (LVI), lack of capsule integrity, and the presence of fatty hilar or fatty replacement of a lymph node. (4) 71% felt that the H&E stain is adequate to evaluate ENE, deeper levels and IHC analysis for vascular and cytokeratin markers can be helpful if needed. (5) 75% agreed that there is an urgent need to standardize the measurement and reporting for ENE. Our survey highlights the variation in ENE evaluation and the need for its standardization in breast cancer patients with axillary node metastasis., Competing Interests: Competing Interest All authors have no competing interests in regarding this manuscript., (Copyright © 2022 Elsevier GmbH. All rights reserved.)

This article addresses issues related to clinical and research approaches to oncofertility for adolescent and young adult (AYA) sexual and gender minority (SGM) cancer patients. Limited attention has been dedicated to understanding the extent to which oncofertility services are appropriately and equitably delivered to AYAs with diverse orientations toward sexual orientation, gender identity, and future family. Unresolved challenges to conducting research with this vulnerable population perpetuate a lack of adequate knowledge about SGM AYA oncofertility needs. Therefore, the purpose of this paper is to inform considerations of sex, gender identity, and sexual orientation for investigations that include SGM AYAs. In order to improve the knowledge base and clinical services for this population, we discuss (1) challenges to sampling this population; (2) categorization and survey logic (e.g., skip patterns) in light of fluid sexual orientation and gender identities; and (3) clinical implications of accurately assessing sex and gender for oncofertility research and practice. We also recommend strategies for producing inclusive and accurate assessments of sexual and gender identity categories in both research and clinical encounters with SGM AYAs. Incorporating "queer insights" into empirical research - that is, positioning queer theory at the center of oncofertility study design - is suggested as a future direction for oncofertility research and practice.

At present, there is no effective way to treat the consequences of spinal cord injury (SCI). SCI leads to the death of neural and glial cells and widespread neuroinflammation with persisting for several weeks after the injury. Mesenchymal stem cells (MSCs) therapy is one of the most promising approaches in the treatment of this injury. The aim of this study was to characterize the expression profile of multiple cytokines, chemokines, growth factors, and so-called neuromarkers in the serum of an SCI patient treated with autologous bone marrow-derived MSCs (BM-MSCs). SCI resulted in a significant increase in the levels of neuromarkers and proteins involved in the inflammatory process. BM-MSCs administration resulted in significant changes in the levels of neuromarkers (S100, GFAP, and pNF-H) as well as changes in the expression of proteins and growth factors involved in the inflammatory response following SCI in the serum of a patient with traumatic SCI. Our preliminary results encouraged that BM-MSCs with their neuroprotective and immunomodulatory effects could affect the repair process after injury.

Mesenchymal stem cells (MSCs) are of great interest to scientists due to their application in cell therapy of many diseases, as well as regenerative medicine and tissue engineering. Recently, there has been growing evidence surrounding the research based on extracellular vesicles (EVs), especially small EVs (sEVs)/exosomes derived from MSCs. EVs/exosomes can be secreted by almost all cell types and various types of EVs show multiple functions. In addition, MSCs-derived exosomes have similar characteristics and biological activities to MSCs and their therapeutic applications are considered as a safe strategy in cell-free therapy. The aim of this study was the characterization of MSCs isolated from the chorion (CHo-MSCs) of human full-term placenta, as well as the isolation and analysis of small EVs obtained from these cells. Accordingly, in this study, the ability of small EVs' uptake is indicated by synovial fibroblasts, osteoblasts and periosteum-derived MSCs. Improvement in the understanding of the structure, characteristics, mechanism of action and potential application of MSCs-derived small EVs can provide new insight into improved therapeutic strategies.

Purpose: Oocytes and embryos can be vitrified with and without dimethyl sulfoxide (DMSO). Objectives were to compare no vitrification (No-Vitr), vitrification with DMSO (Vitr + DMSO), and vitrification without DMSO (Vitr - DMSO) on fresh/warmed oocyte survival, induced parthenogenetic activation, parthenogenetic embryo development, and embryonic maternal imprinted gene expression., Methods: In this prospective controlled laboratory study, mature B6C3F1 female mouse metaphase II oocytes were treated as: i) No-Vitr, ii) Vitr + DMSO/warmed, and iii) Vitr - DMSO/warmed with subsequent parthenogenetic activation and culture to the blastocyst stage. Oocyte cryo-survival, parthenogenetic activation and embryo development, parthenogenetic embryo maternal imprinted gene expression were outcome measures., Results: Oocyte cryo-survival was significantly improved in Vitr + DMSO versus Vitr - DMSO at initial warming and 2 h after warming. Induced parthenogenetic activation was similar between all three intervention groups. While early preimplantation parthenogenetic embryo development was similar between control, Vitr + DMSO, Vitr - DMSO oocytes, the development to blastocysts was significantly inferior in the Vitr - DMSO oocytes group compared to the control and Vitr + DMSO oocyte groups. Finally, maternal imprinted gene expression was similar between intervention groups at both the 2-cell and blastocyst parthenogenetic embryo stage., Conclusion(s): Inclusion of DMSO in oocyte vitrification solutions improved cryo-survival and developmental potential of parthenogenetic embryos to the blastocyst stage without significantly altering maternal imprinted gene expression., (© 2021. Springer Science+Business Media, LLC, part of Springer Nature.)

Purpose: Today, male and female adult and pediatric cancer patients, individuals transitioning between gender identities, and other individuals facing health extending but fertility limiting treatments can look forward to a fertile future. This is, in part, due to the work of members associated with the Oncofertility Consortium., Methods: The Oncofertility Consortium is an international, interdisciplinary initiative originally designed to explore the urgent unmet need associated with the reproductive future of cancer survivors. As the strategies for fertility management were invented, developed or applied, the individuals for who the program offered hope, similarly expanded. As a community of practice, Consortium participants share information in an open and rapid manner to addresses the complex health care and quality-of-life issues of cancer, transgender and other patients. To ensure that the organization remains contemporary to the needs of the community, the field designed a fully inclusive mechanism for strategic planning and here present the findings of this process., Results: This interprofessional network of medical specialists, scientists, and scholars in the law, medical ethics, religious studies and other disciplines associated with human interventions, explore the relationships between health, disease, survivorship, treatment, gender and reproductive longevity., Conclusion: The goals are to continually integrate the best science in the service of the needs of patients and build a community of care that is ready for the challenges of the field in the future.

Impairment of fertility and sexual/reproductive health are common after oncologic therapy, and are known to have negative impacts on romantic relationships and psychosocial well-being among childhood cancer survivors. The Pediatric Initiative Network (PIN) is an international, multidisciplinary group of providers within the Oncofertility Consortium dedicated to preserving and protecting the fertility of children and adolescents at risk for infertility due to medical conditions or treatments. The PIN and its Best Practices and Research committees meet virtually throughout the year, with one annual in-person meeting. The purpose of this "proceedings" is to highlight key discussion points from the annual PIN meeting which took place on November 11, 2019, to 1) provide a context for pediatric groups across the country on what oncofertility programs are currently doing and why, and 2) inform stakeholders of past, present and future initiatives that may be of value to them and the patient populations they serve.

Purpose: To examine potential benefits of dipeptide forms of amino acids for embryo culture by determining ability of dipeptide glycine forms to support embryo development, act as osmolytes, and reduce ammonia production., Methods: Frozen thawed 1-cell mouse embryos were cultured in media with varying osmolality with glycine and dipeptide forms of glycine and development assessed. Ammonia levels were measured in various media., Results: Dipeptide forms of glycine, alanyl- and glycyl-glycine, can support mouse embryo development in vitro. Additionally, dipeptide glycine can act as an organic osmolyte in developing embryos, permitting blastocyst formation in high osmolality media. Interestingly, as evidenced by decreased embryo development, dipeptides are not as efficient as osmolytes as their constituent individual amino acids. Dipeptide glycine produced less ammonia than glycine., Conclusion: Though dipeptides can provide osmoregulation in preimplantation embryos, efficacy may be lower than individual amino acids. The mechanism by which embryos transport and utilize dipeptide amino acids remains to be identified.

The Nhe enterotoxin from Bacillus cereus is known to induce cytotoxicity on Vero and CaCo-2 cells by ordered binding of its single components NheA, NheB, and NheC. This study aimed to elucidate functional sites on NheB by identifying the epitopes of the neutralizing monoclonal antibodies 1E11 and 2B11. The binding regions of both antibodies were determined by using recombinant NheB fragments and synthetic peptides. The antigenic site of antibody 1E11 was located within the amino acids 321 to 341 of NheB, whereas reactivity of antibody 2B11 was dependent on the presence of amino acids 122 to 150 and on conformation. Both antibodies were able to bind simultaneously to NheB and did not interfere with target cell binding as shown by immunofluorescence microscopy. A set of neutralization assays revealed that antibody 2B11 most likely interfered with the interaction between NheB and NheC both on the epithelium cell surface and in solution. In contrast, antibody 1E11 inhibited association between NheA and cell-bound NheB in a competitive manner, and effectively neutralized Nhe cytotoxicity on a variety of human cell lines. This distinct mechanism further supports that NheA is the key component during the Nhe mode of action and the C-terminal epitope recognized by antibody 1E11 points to an important functional region of NheB.

The Duopath® Cereus Enterotoxins test (Merck KGaA) is a newly developed gold-labeled lateral flow immunoassay for the detection of Bacillus cereus enterotoxins. The test uses monoclonal antibodies (MAbs) against the L(2) component of hemolysin BL (Hbl) and NheB of the non-hemolytic enterotoxin (Nhe), respectively. The inclusivity and exclusivity of the assay was tested using 44 B. cereus, B. cereus group and Bacillus spp. strains. Apart from the B. mycoides type strain the results were in full agreement with those obtained by other immunological and molecular biological methods. The detection limit of the assay was 6ng/ml for NheB and 20ng/ml for the Hbl-L(2)-component, respectively. Using artificially and naturally contaminated food samples (n=76) the assay was positive after 18-24h enrichment if at least 10(2) enterotoxin producing B. cereus/g were present. After 30h enrichment samples contaminated with as low as 1 enterotoxin producing B. cereus/g gave positive results. In addition, testing of suspected colonies for enterotoxin production is possible. The assay is easy to perform and results can be clearly read without instrumentation., (Copyright © 2010 Elsevier B.V. All rights reserved.)

This study focuses on the interaction of the three components of the Bacillus cereus Nhe enterotoxin with particular emphasis on the functional roles of NheB and NheC. The results demonstrated that both NheB and NheC were able to bind to Vero cells directly while NheA lacked this ability. It was also shown that Nhe-induced cytotoxicity required a specific binding order of the individual components whereby the presence of NheC in the priming step as well as the presence of NheA in the final incubation step was mandatory. Priming of cells with NheB alone and addition of NheA plus NheC in the second step failed to induce toxic effects. Furthermore, in solution, excess NheC inhibited binding of NheB to Vero cells, whereas priming of cells with excess NheC resulted in full toxicity if unbound NheC was removed before addition of NheB. By using mutated NheC proteins where the two cysteine residues in the predicted beta-tongue were replaced with glycine (NheCcys-) or where the entire hydrophobic stretch was deleted (NheChr-), the predicted hydrophobic beta-tongue of NheC was found essential for binding to cell membranes but not for interaction with NheB in solution. All data presented here are compatible with the following model. The first step in the mode of action of Nhe is associated with binding of NheC and NheB to the cell surface and probably accompanied by conformational changes. These events allow subsequent binding of NheA, leading to cell lysis.

In order to meet the growing demand for fast and reliable detection of potentially toxinogenic Bacillus cereus, we developed a multiplex real-time PCR assay based on SYBR Green I with subsequent melting curve analysis. We designed and selected primers specific for genes of toxins responsible for diarrhoea (nheA, hblD and cytK1) and emesis (ces). A panel of 337 Bacillus strains was applied to the novel method on Light Cycler 2.0 with average melting temperature (T(m)) values of 73.85 degrees C (nheA), 87.01 degrees C (hblD), 78.66 degrees C (ces) and 82.19 degrees C (cytK1). An adapted version of the assay was also successfully run on Light Cycler 480 using one third (113 strains) of the total test panel. Verification of PCR results by conventional PCR as well as immunoassays and cytotoxicity tests gave an overall excellent correlation. Distinct melting peaks were only observed in B. cereus and B. cereus group strains but not in other Bacilli and Gram-positive or Gram-negative bacteria. Artificial contamination of three different food matrices with distinct bacterial counts revealed a detection limit of 10(1) CFU/g B. cereus cells after overnight enrichment. Thus, the novel multiplex real-time PCR turned out to be a reliable method for identification of B. cereus with enteropathogenic potential.

Fast and reliable methods are needed for the detection of pathogenic Bacillus cereus which should provide consistent results. Therefore, we tested a panel of 176 strains, including B. cereus strains, B. cereus group strains and other Bacillus spp. with polymerase chain reaction, immunoassays and cytotoxicity tests and assessed the consistency of the results. A screening multiplex PCR for the detection of hbl, nhe, ces and cytK1 as well as two multiplex PCRs for the differentiation of Hbl genes (hblC, hblD, hblA) and Nhe genes (nheA, nheB, nheC) was applied. All PCRs included an internal amplification control. Component specific antibody based immunoassays were used for the detection of the three components of Hbl and Nhe and the overall cytotoxicity to Vero cells and HEp-2 cells was checked. An overall excellent correlation was obtained for the results of the three, methodically independent assays and no false-negative PCR results were seen for any of the strains tested positive in immunoassays and cytotoxicity tests. The three multiplex PCRs proved to be a facile method for the identification of enterotoxinogenic B. cereus isolates.

Haemolysin BL (HBL) and non-haemolytic enterotoxin (Nhe), each consisting of three components, represent the major enterotoxins produced by Bacillus cereus. To evaluate the expression of these toxins, a set of 100 B. cereus strains was examined. Molecular biological characterization showed that 42% of the strains harboured the genes for HBL and 99% for Nhe. The production of all Nhe and HBL components were analyzed using specific antibodies and, in culture supernatants, detectable levels of HBL and Nhe were found for 100% of hbl-positive and 96% of nhe-positive strains. The concentrations of the HBL-L(2) and NheB component ranged from 0.02 to 5.6 microg mL(-1) and from 0.03 to 14.2 microg mL(-1), respectively. Comparison of the amount of NheB produced by food poisoning and food/environmental strains revealed that the median value for all food poisoning strains was significantly higher than for the food/environmental isolates. The data presented in this study provide evidence that specific and quantitative determination of the enterotoxins is necessary to evaluate the toxic potential of B. cereus. In particular, the level of Nhe seems to explain most of the cytotoxic activity of B. cereus isolates and may indicate a highly diarrheic potential.

The nonhemolytic enterotoxin (Nhe) is one of the two three-component enterotoxins which are responsible for diarrheal food poisoning syndrome caused by Bacillus cereus. To facilitate the detection of this toxin, consisting of the subunits NheA, NheB, and NheC, a complete set of high-affinity antibodies against each of the three components was established and characterized. A rabbit antiserum specific for the C-terminal part (15 amino acids) of NheC was produced using a respective synthetic peptide coupled to a protein carrier for immunization. Using purified B. cereus exoprotein preparations as immunogens, one monoclonal antibody against NheA and several antibodies against NheB were obtained. No cross-reactivity with other proteins produced by different strains of B. cereus was observed. Antibodies against the NheB component were able to neutralize the cytotoxic activity (up to 98%) of Nhe. Based on indirect enzyme immunoassays, the antibodies developed in this study were successfully used in the characterization of the enterotoxic activity of several B. cereus strains. For the first time, it could be shown that strains carrying the nhe genes usually express the complete set of the three components, including NheC. However, the amount of toxin produced varies considerably between the different strains.

A new variant of Shiga toxin 1 (Stx1), designated Stx1d, which deviates considerably more than any other known variant from Stx1 encoded by phage 933J, was identified in an Escherichia coli strain, ONT:H19, isolated from bovine feces. The complete stx(1) gene of this strain was amplified and sequenced. Nucleotide sequence homology with stx(1) from phage 933J was only 91%, resulting in the substitution of 20 amino acids in the A subunit and 7 amino acids in the B subunit of the protein. Cell culture supernatant of this strain, which was negative for stx(2) by PCR testing, was cytotoxic to Vero cells and gave positive results in two commercial enzyme-linked immunosorbent assays for Stx. PCR primers were constructed for the specific detection of the new variant. The findings of this study suggest that Stx1 is not as conserved as thought before and that there might be more variants which cannot be detected by commonly used PCR methods.

Given the societal concern about groundwater pollution from agricultural sources, public programs have been proposed or implemented to change farmer behavior with respect to nutrient use and management. However, few of these programs designed to change farmer behavior have been evaluated due to the lack of detailed data over an appropriate time frame. The Central Platte Natural Resources District (CPNRD) in Nebraska has identified an intensively cultivated, irrigated area with average groundwater nitrate-nitrogen (N) levels about double the EPA"s safe drinking water standard. The CPNRD implemented a joint education and regulatory N management program in the mid-1980s to reduce groundwater N. This analysis reports N use and management, yield, and groundwater nitrate trends in the CPNRD for nearly 3000 continuous-corn fields from 1989 to 1998, where producers faced limits on the timing of N fertilizer application but no limits on amounts. Groundwater nitrate levels showed modest improvement over the 10 years of this analysis, falling from the 1989-1993 average of 18.9 to 18.1 mg/l during 1994-1998. The availability of N in excess of crop needs was clearly documented by the CPNRD data and was related to optimistic yield goals, irrigation water use above expected levels, and lack of adherence to commercial fertilizer application guidelines. Over the 10-year period of this analysis, producers reported harvesting an annual average of 9729 kg/ha, 1569 kg/ha (14%) below the average yield goal. During 1989-1998, producers reported annually applying an average of 162.5 kg/ha of commercial N fertilizer, 15.7 kg/ha (10%) above the guideline level. Including the N contribution from irrigation water, the potential N contribution to the environment (total N available less estimated crop use) was estimated at 71.7 kg/ha. This is an estimate of the nitrates available for denitrification, volatilization, runoff, future soil N, and leaching to groundwater. On average, between 1989-1993 and 1994-1998, producers more closely followed CPNRD N fertilizer recommendations and increased their use of postemerge N applications--an indication of improved synchrony between N availability and crop uptake.