Disruption of fatty acid amide hydrolase activity prevents the effects of chronic stress on anxiety and amygdalar microstructure.

Hyperactivation of the amygdala following chronic stress is believed to be one of the primary mechanisms underlying the increased propensity for anxiety-like behaviors and pathological states; however, the mechanisms by which chronic stress modulates amygdalar function are not well characterized. The aim of the current study was to determine the extent to which the endocannabinoid (eCB) system, which is known to regulate emotional behavior and neuroplasticity, contributes to changes in amygdalar structure and function following chronic stress. To examine the hypothesis, we have exposed C57/Bl6 mice to chronic restraint stress, which results in an increase in fatty acid amide hydrolase (FAAH) activity and a reduction in the concentration of the eCB N-arachidonylethanolamine (AEA) within the amygdala. Chronic restraint stress also increased dendritic arborization, complexity and spine density of pyramidal neurons in the basolateral nucleus of the amygdala (BLA) and increased anxiety-like behavior in wild-type mice. All of the stress-induced changes in amygdalar structure and function were absent in mice deficient in FAAH. Further, the anti-anxiety effect of FAAH deletion was recapitulated in rats treated orally with a novel pharmacological inhibitor of FAAH, JNJ5003 (50 mg per kg per day), during exposure to chronic stress. These studies suggest that FAAH is required for chronic stress to induce hyperactivity and structural remodeling of the amygdala. Collectively, these studies indicate that FAAH-mediated decreases in AEA occur following chronic stress and that this loss of AEA signaling is functionally relevant to the effects of chronic stress. These data support the hypothesis that inhibition of FAAH has therapeutic potential in the treatment of anxiety disorders, possibly by maintaining normal amygdalar function in the face of chronic stress.

Saliva "Treat-and-Heat" Triplex Reverse Transcription Loop-Mediated Isothermal Amplification Assay for SARS-CoV-2.

The demand for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) molecular diagnostics that are faster, cheaper, and simpler to run than nasopharyngeal-based reverse transcription quantitative PCR (RT-qPCR) tests remains unmet in many parts of the world. In the Philippines, geographical and economic access to quality diagnostic testing remains out of reach for many communities. We describe the preclinical development of a fluorescence-based reverse transcription loop-mediated isothermal amplification test that uses drooled saliva as the biospecimen. Six treat-and-heat ("direct") procedures that inactivate the virus and release the target RNA were compared. Using duplexed As1e and E1 primers, protocols derived from Ben-Assa et al. (2020) using proteinase K or from Rabe and Cepko (2020) using TCEP (Tris(2-carboxyethyl)phosphine hydrochloride)/EDTA provided reliable RNA amplification. The TCEP/EDTA-based method in particular showed improvement in robustness in duplex vs. singleplex format. Inclusion of human ß-actin primers provided a triplex test with an internal amplification control that could be distinguished from SARS-CoV-2 amplicons based on melt curve analysis. After including the dUTP/uracil-DNA glycosylase system and implementing laboratory procedures to avoid cross-contamination, false positive amplification was acceptably rare. The duplex or triplex tests are predicted to reliably detect patient salivary viral loads >100 copies/µL and to yield equivocal results between 10 and 100 copies/µL. These viral loads, corresponding to RT-qPCR C t ∼29-32, are expected to identify the majority of infected and, particularly, of infectious patients. If clinically validated, the test would provide additional testing capacity requiring only a fraction of the time, cost, and infrastructure of the current nasopharyngeal swab-based RT-qPCR test, thereby improving access to testing for more Filipinos.

Loop-Mediated Isothermal Amplification Detection of SARS-CoV-2 and Myriad Other Applications.

As the second year of the COVID-19 pandemic begins, it remains clear that a massive increase in the ability to test for SARS-CoV-2 infections in a myriad of settings is critical to controlling the pandemic and to preparing for future outbreaks. The current gold standard for molecular diagnostics is the polymerase chain reaction (PCR), but the extraordinary and unmet demand for testing in a variety of environments means that both complementary and supplementary testing solutions are still needed. This review highlights the role that loop-mediated isothermal amplification (LAMP) has had in filling this global testing need, providing a faster and easier means of testing, and what it can do for future applications, pathogens, and the preparation for future outbreaks. This review describes the current state of the art for research of LAMP-based SARS-CoV-2 testing, as well as its implications for other pathogens and testing. The authors represent the global LAMP (gLAMP) Consortium, an international research collective, which has regularly met to share their experiences on LAMP deployment and best practices; sections are devoted to all aspects of LAMP testing, including preanalytic sample processing, target amplification, and amplicon detection, then the hardware and software required for deployment are discussed, and finally, a summary of the current regulatory landscape is provided. Included as well are a series of first-person accounts of LAMP method development and deployment. The final discussion section provides the reader with a distillation of the most validated testing methods and their paths to implementation. This review also aims to provide practical information and insight for a range of audiences: for a research audience, to help accelerate research through sharing of best practices; for an implementation audience, to help get testing up and running quickly; and for a public health, clinical, and policy audience, to help convey the breadth of the effect that LAMP methods have to offer.

Pertussis toxin gene: nucleotide sequence and genetic organization.

The current pertussis vaccines, although efficacious, in some instances produce undesirable side effects. Molecular engineering of pertussis toxin, the major protective antigen, could provide a safer, new generation of vaccines against whooping cough. As a first critical step in the development of such a vaccine, the complete nucleotide sequence of the pertussis toxin gene was determined and the amino acid sequences of the individual subunits were deduced. All five subunits are coded by closely linked cistrons. A promoter-like structure was found in the 5'-flanking region, suggesting that the toxin is expressed through a polycistronic messenger RNA. The order of the cistrons is S1, S2, S4, S5, and S3. All subunits contain signal peptides of variable length. The calculated molecular weights of the mature subunits are 26,024 for S1, 21,924 for S2, 21,873 for S3, 12,058 for S4, and 11,013 for S5. Subunits S2 and S3 share 70% amino acid homology and 75% nucleotide homology. Subunit S1 contains two regions of eight amino acids homologous to analogous regions in the A subunit of both cholera and Escherichia coli heat labile toxins.

Pertussis toxin S1 mutant with reduced enzyme activity and a conserved protective epitope.

Pertussis toxin (PTX) is a major virulence factor in whooping cough and can elicit protective antibodies. Amino acid residues 8 to 15 of PTX subunit S1 are important for the adenosine diphosphate-ribosyltransferase activity associated with the pathobiological effects of PTX. Furthermore, this region contains at least a portion of an epitope that elicits both toxin-neutralizing and protective antibody responses in mice. The gene encoding the S1 subunit was subjected to site-specific mutagenesis in this critical region. A mutant containing a single amino acid substitution (Arg9----Lys) had reduced enzymatic activity (approximately 0.02% of control) while retaining the protective epitope. This analog S1 molecule may provide the basis for a genetically detoxified PTX with potential for use as a component of an acellular vaccine against whooping cough.

Delivery of antigens to the MHC class I pathway using bacterial toxins.

Cytotoxic T lymphocytes (CTL) recognize antigens derived from endogenously expressed proteins presented on the cell surface in the context of major histocompatibility complex (MHC) class I molecules. Because CTL are effective in antiviral and antitumor responses, the delivery of antigens to the class I pathway has been the focus of numerous efforts. Generating CTL by immunization with exogenous proteins is often ineffective because these antigens typically enter the MHC class II pathway. This review focuses on the usefulness of bacterial toxins for delivering antigens to the MHC class I pathway. Several toxins naturally translocate into the cytosol, where they mediate their cytopathic effects, and the mechanisms by which this occurs has been elucidated. Molecular characterization of these toxins identified the functional domains and enabled the generation of modified proteins that were no longer toxic but retained the ability to translocate into the cytosol. Thus, these modified toxins could be examined for their ability to carry peptides or whole proteins into the cytosolic processing pathway. Of the toxins studied-diphtheria, pertussis, Pseudomonas, and anthrax-the anthrax toxin appears the most promising in its ability to deliver large protein antigens and its efficiency of translocation.

A novel multiplex RT-PCR probe capture hybridization (RT-PCR-ELISA) for simultaneous detection of six viroids in four genera: Apscaviroid, Hostuviroid, Pelamoviroid, and Pospiviroid.

A rapid and sensitive assay was developed for the detection and identification of viroids by standard or multiplex reverse transcription-polymerase chain reaction (RT-PCR)-probe capture hybridization (RT-PCR-ELISA). The assay was applied successfully for the detection and identification of the following six viroid species from infected tissues: Potato spindle tuber viroid (Pospiviroid), Peach latent mosaic viroid (Pelamoviroid), Apple scar skin viroid (Apscaviroid), Apple dimple fruit viroid (Apscaviroid), Pear blister canker viroid (Apscaviroid), and Hop stunt viroid (Hostuviroid). Total RNA was obtained from infected tissue by the Qiagen RNeasy kit and, then viroid cDNA was synthesized using viroid specific complementary DNA primer. To identify and differentiate the amplicons of the six viroids, each amplicon was digoxigenin (DIG)-labelled during the amplification process, and then detected by a colorimetric system using a biotinylated cDNA capture probe specific for each viroid. The results revealed that each capture probe hybridized only to its complementary DIG-labelled amplicon. Thus the six viroids can be detected and differentiated in a multiplex RT-PCR-ELISA assay. In the multiplex assay, cDNAs of six viroids were synthesized simultaneously in one tube, DIG-labelled during amplification, then a portion of the DIG-labelled amplified products was hybridized with selected capture probe. All the six viroid capture probes hybridized to their respective complementary DIG-labelled RT-PCR-amplified product. These findings are important for viroid detection and identification for studying host-viroid interactions and for management and control viroid diseases.

Mission integration preserves sponsor's values.

Sponsorship of a healthcare organization by a religious community requires sponsors to maintain significant influence and ultimate control over mission, quality of services, and assets. To ensure the mission of its sponsor (the Religious Sisters of Mercy) permeated all its operations, St. Edward Mercy Medical Center, Fort Smith, AR, incorporated the nine Mercy values into the organization's core documents in the mid-1980s. As St. Edward's services and service area grew, the program approach to mission effectiveness appeared inadequate. St. Edward implemented a mission integration plan to ensure the inclusion of Christian values in decision making, direction setting, and strategic planning. The Mission Integration Council provides the forum and authority for planning and implementing the mission integration strategies and provides a formal process for evaluating those actions in light of the mission statement and core Mercy values.

Flattening the hierarchy. A hospital streamlines managerial layers to meet market demands.

In the early 1990s it became clear to the leaders of St. Edward Mercy Medical Center, Fort Smith, AR, that the traditional ¿functional¿ model of organization, on which their hospital was based, did not allow it to meet new market demands. A core group of managers was formed to design a new organizational model and engineer the move toward it. Analyzing the hospital's structure, the core group found that it had too many administrative layers above too many specialized departments. In 1994 the group decided to adopt a span-of-control model of organization, which would give St. Edward a higher ratio of workers per manager. In 1995 the core group streamlined the hospital's managerial layers, deciding there would be no more than five. It reduced the number of supervisory positions by 36, including one vice president's slot. No manager was fired, though some were reassigned. St. Edward's reorganization continues at present. The new structure, which has cut personnel costs, fosters more open communication and empowers its workers, leading them to think in terms of ¿us and our hospital¿ rather than ¿me and my department.¿

A regional strategy. Networking gives rural residents access to high-quality healthcare.

In 1978 the Board of Trustees at St. Edward Mercy Medical Center, Fort Smith, AR, adopted a policy that the center would increase its bed size only to meet community needs or to offer needed services. The policy choice was the first step in the development of a regional network that now serves five rural communities. Although there was some resistance to the move at first, when management formalized some of its basic assumptions and values, it became clear that establishing a regional network was right for St. Edward. It would provide economic benefits to the communities in which facilities were acquired or constructed; it would give rural residents better access to primary healthcare; and it would provide the Religious Sisters of Mercy an opportunity to extend their ministry. Networking has also allowed the facilities involved to develop economies of scale and to avoid costly duplication of certain basic services. In addition, primary care physicians in rural communities served by the network have been an important source of referrals to specialists who utilize St. Edward.

Personalized care helps facilities compete.

Catholic health facilities may believe that they provide personalized care, since this value is a part of their mission statement. In the face of shrinking inpatient services and competition for patients, however, formal efforts to provide such care are essential. Personalized care can be a primary factor in differentiating Catholic health care facilities in the marketplace. St. Edward Mercy Medical Center implemented marketing surveys to determine patients' perceptions of personalized care. Using the results, the medical center instituted employee education programs and physical improvements in order to meet patients' needs and thus maintain and improve the facility's patient market share.

A trust relationship. A medical advisory board builds physician commitment to a healthcare facility.

Today physicians and hospitals are in competition. To ensure consistent physician input and a forum for two-way communication, St. Edward Mercy Medical Center, Fort Smith, AR, has established a medical staff board. The medical staff board was organized so physicians could formally address managers' concerns without duplicating work done by other medical staff committees (e.g., executive committee, medical staff sections, hospital committees). Membership on the 24-member board was limited to the active staff. A two-year term was established, allowing for two consecutive terms to ensure continuity. The chief of staff and chief executive officer (CEO) are ex-officio members. Some of the issues of interest to physicians include how well informed operating room personnel were on current technology and procedures, how effective the emergency department could be, having been designed almost 20 years ago, and how volume purchasing affects physician familiarity with certain products. St. Edward's medical staff board has the potential to enhance the physician-hospital relationship and to serve as an effective tool in building commitment to the medical center.

Nurse specialist: an evolving need.

For too long, to advance their careers and earn higher salaries, nurses have had to veer away from clinical experience, which they knew best, into managerial and other fields. To curb this trend and respond to the need for high-quality care, St. Edward Mercy Medical Center, Fort Smith, AR, instituted the nurse specialist program, in which nurses can develop competency in multiple clinical areas with accompanying financial incentives. In developing the program, which provides a cadre of nurses to augment the regular staff wherever needed, job descriptions and the requirements for selection, recruitment, and training were developed, as was some method to evaluate the program. Requirements for participation included at least one year of hands-on experience at the medical center, good clinical skills, and adherence to the Mercy philosophy of care. Those selected were trained in five specialized clinical areas: intensive care, labor and delivery, emergency services, post-anesthesia recovery, and nursery. The program at first met resistance from other nurses and abuse from nursing supervisors. But this has been replaced by acceptance and appreciation. The nurse specialists are seen as an elite group, meeting patient and staff needs as extra pairs of caring and competent hands, with the potential to calm a stressful clinical environment.