Unravelling bacterial virulence factors in yeast: From identification to the elucidation of their mechanisms of action.

Pathogenic bacteria employ virulence factors (VF) to establish infection and cause disease in their host. Yeasts, Saccharomyces cerevisiae and Saccharomyces pombe, are useful model organisms to study the functions of bacterial VFs and their interaction with targeted cellular processes because yeast processes and organelle structures are highly conserved and similar to higher eukaryotes. In this review, we describe the principles and applications of the yeast model for the identification and functional characterisation of bacterial VFs to investigate bacterial pathogenesis. The growth inhibition phenotype caused by the heterologous expression of bacterial VFs in yeast is commonly used to identify candidate VFs. Then, subcellular localisation patterns of bacterial VFs can provide further clues about their target molecules and functions during infection. Yeast knockout and overexpression libraries are also used to investigate VF interactions with conserved eukaryotic cell structures (e.g., cytoskeleton and plasma membrane), and cellular processes (e.g., vesicle trafficking, signalling pathways, and programmed cell death). In addition, the yeast growth inhibition phenotype is also useful for screening new drug leads that target and inhibit bacterial VFs. This review provides an updated overview of new tools, principles and applications to study bacterial VFs in yeast.

Exploring the role of impedance spectroscopy in assessing 405 nm laser-induced inactivation of saccharomyces cerevisiae.

Impedance spectroscopy was employed to assess the electrical properties of yeast following 405 nm laser irradiation, exploring the effects of visible, non-ionizing laser-induced inactivation as a more selective and safer alternative for photoinactivation applications compared to the use of DNA targeting, ionizing UV light. Capacitance and impedance spectra were obtained for yeast suspensions irradiated for 10, 20, 30, and 40 min using 100 and 200 mW laser powers. Noticeable differences in capacitance spectra were observed at lower frequencies (40 Hz to 1 kHz), with a significant increase at 40 min for both laser powers. ß-dispersion was evident in the impedance spectra in the frequency range of 10 kHz to 10 MHz. The characteristic frequency of dielectric relaxation steadily shifted to higher frequencies with increasing irradiation time, with a drastic change observed at 40 min for both laser powers. These changes signify a distinct alteration in the physical state of yeast. A yeast spot assay demonstrated a decrease in cell viability with increasing laser irradiation dose. The results indicate a correlation between changes in electrical properties, cell viability, and the efficacy of 405 nm laser-induced inactivation. Impedance spectroscopy is shown to be an efficient, non-destructive, label-free method for monitoring changes in cell viability in photobiological effect studies. The development of impedance spectroscopy-based real-time studies in photoinactivation holds promise for advancing our understanding of light-cell interactions in medical applications.

Histiocytoid Sweet Syndrome Presenting in Two Sisters With Deficiency of Deaminase Type 2.

ABSTRACT: Deficiency of adenosine deaminase type 2 (DADA2) is an autosomal recessive monogenic autoinflammatory syndrome that is classically characterised by polyarteritis nodosa, systemic vasculitis and stroke. The spectrum of disease manifestations has broadened to encompass a range of cutaneous, vascular and haematological manifestations. We report a novel association in two sisters with heterozygous p.R169G/p.M309l mutations in ADA2 with low serum ADA2 activity who both presented similarly with clinical and histological features consistent with histiocytoid Sweet syndrome.

Identifying Leptospira interrogans putative virulence factors with a yeast protein expression screen.

Leptospirosis is a zoonotic disease caused by pathogenic Leptospira spp., with global implications primarily in tropical countries. However, the mechanisms of leptospiral pathogenesis are still not fully known and not all virulence factors (VFs) have been identified. Budding yeast, Saccharomyces cerevisiae is a popular eukaryotic model which has been used to identify bacterial VFs that target the conserved eukaryotic cellular processes. In this study, we screened for putative VFs of L. interrogans, one of the dominant species causing leptospirosis, by expressing candidate VFs in budding yeast and examining their impact on yeast growth in a high-throughput format. From an initial selection of 288 L. interrogans ORFs, we screened 226 candidate VFs in a yeast growth inhibition assay and identified nine putative VFs in four categories (adhesion, enzymatic, host structure interaction, and immunogenicity). Notably, LIC10280 was highly toxic even when expressed at low copies. We also observed specific subcellular localization for several putative VFs. This study shows that there are still potential L. interrogans VFs that await discovery. KEY POINTS: • High-throughput cloning and expression of leptospiral proteins in yeast. • Heterologous expression of nine leptospiral proteins inhibited yeast growth. • An uncharacterized protein LIC10280 maybe a putative VF for further validation.

An unusual penile lesion.

We present an unusual case of an orange-brown penile lesion with a description of the histological findings. Click here for the corresponding questions to this CME article.

Evaluation of Salmonella Typhi antigen YncE alongside HlyE for the detection of typhoid fever and its carriers.

Typhoid fever is a disease caused by Salmonella Typhi that was implicated in millions of illnesses worldwide annually. Individuals that do not recover fully from typhoid fever can become asymptomatic carriers of the disease. Host antibodies against the S. Typhi antigens, HlyE (for acute typhoid) and YncE (for carriers) were previously reported to be useful biomarkers for the disease. Here, we expressed and purified recombinant HlyE and YncE antigens and tested the IgG, IgA and IgM responses in 422 sera samples retrieved from acute typhoid patients, other febrile, food handlers, and healthy individuals. The results showed that HlyE-IgG, -IgA and -IgM ELISAs have a collective sensitivity of 83% while YncE-IgG and -IgA ELISAs identified 16 possible carriers based on their antibody profiles. The identification of sensitive biomarkers for typhoid carrier detection is crucial for disease eradication.

BRAF Inhibitor-Associated Granulomatous Dermatitis: A Report of 3 Cases.

Cutaneous toxicities associated with BRAF inhibitor treatment in patients with metastatic melanoma have been well described. We present a rare association of granulomatous dermatitis in association with the BRAF inhibitor vemurafenib. Three patients with metastatic melanoma all presented with asymptomatic papular eruptions 8-21 months into vemurafenib therapy. Skin biopsies confirmed the diagnosis of granulomatous dermatitis. Other causes of granulomatous dermatitis including infectious agents and sarcoid were excluded. Treatment with potent topical and oral steroids improved the eruptions, but only after the cessation of vemurafenib did all 3 cases of granulomatous dermatitis completely resolve within 2 weeks. It is important to recognize that this association, unlike most other BRAF inhibitor-related skin toxicities, can occur many months after commencement of therapy and that vemurafenib treatment can be continued without clinically significant adverse effects.

Dermatoscopic findings of penile intraepithelial neoplasia: Bowenoid papulosis, Bowen disease and erythroplasia of Queyrat.

BACKGROUND/OBJECTIVES: The clinical diagnosis of penile intraepithelial neoplasia is challenging. No specific dermoscopic criteria for penile intraepithelial neoplasia have been described in the literature. This study aimed to describe and evaluate the dermoscopic features of penile intraepithelial neoplasia. METHODS: Clinical and dermoscopic images of 11 patients with histopathologically confirmed penile intraepithelial neoplasia were recorded and evaluated. RESULTS: The most frequent dermoscopic features were the presence of structureless areas (100%, structureless pink 72.7%) and vascular structures (81.8%), particularly dotted vessels (72.7%). Other findings included the absence of a pigment network (100%); scale (45.5%); scar-like areas (45.5%); erosions (27.3%); and pigmentation consisting of brown-grey dots and globules (27.3%). CONCLUSIONS: The dermoscopic features that characterise penile intraepithelial neoplasia are structureless pink areas and a prominent vascular pattern (mainly clustered dotted vessels). Dermoscopy is a useful tool that can aid in the diagnosis and surveillance of penile intraepithelial neoplasia.

Integrating anatomy, radiology, pathology, and surgery: An alternative approach in resecting multifocal and multicentric breast carcinoma.

The sick lobe hypothesis provides the basis for a lobar approach in radiology, pathology, and surgical treatment of breast cancer. This approach aims to remove the tumor together with the surrounding field of genetic aberrations. Detailed preoperative lobar imaging that properly maps the disease and assesses its extent guides the parenchymal resection. Integration of our knowledge of breast anatomy and pathology with the results of preoperative radiological mapping is critical in assessing the eligibility of patients with multifocal and/or multicentric breast cancer for breast conservation treatment. Through an appropriately selected incision, a multisegment resection of the diseased lobe(s) is performed, which leaves the residual parenchyma in a formation that allows dovetailing of one part into the other, like the way pieces of a jigsaw puzzle fit together. Detailed pathologic analysis of the surgical specimen provides valuable feedback to the radiologist, establishes the completeness of surgical intervention, and generates predictive information for therapeutic decisions. Our approach is a step in continuous search for ideal tailored therapy to avoid under or over-treatment of breast cancer patients.

Multi-isotype antibody responses against the multimeric Salmonella Typhi recombinant hemolysin E antigen.

The detection and measurement of different antibody isotypes in the serum provide valuable indicators of the different stages of typhoid infection. Here, the ability of S. Typhi recombinant hemolysin E (HlyE) to detect multi-isotype antibody responses in sera of patients with typhoid and paratyphoid A was investigated using an indirect antibody immunoassay. Nanogram amounts of HlyE were found to be sufficient for detection of IgG and IgA isotypes and, in a study of individuals' sera (n = 100), the immunoassay was able to distinguish between typhoid and non-typhoid sera. The overall sensitivity, specificity and efficiency of the ELISA were 70% (39/56), 100% (44/44) and 83% respectively.

Management of a Trichophyton tonsurans outbreak in a day-care center.

Trichophyton tonsurans is the leading cause of tinea capitis in the United Kingdom (UK) as well as causing tinea corporis. This organism has been linked to several outbreaks in the UK and abroad, and such outbreaks may be prolonged since T. tonsurans can be difficult to control. There remains an incomplete consensus in the literature on the optimal management of such outbreaks of this infection. Following notification that a child with T. tonsurans was identified at a day-care center in the UK, initial investigations identified nine cases of fungal infection involving children and staff over the previous 7 months. We report on the management of an outbreak of T. tonsurans tinea capitis and tinea corporis among children and staff in a day-care center. An outbreak control team with representatives from dermatology, microbiology, day-care center management, and the Health Protection Agency initiated case ascertainment by scalp inspection and brushing of all children and staff at the nursery. Two complete rounds of screening were required before the outbreak was declared over. Infection control measures included antifungal shampoo use, exclusion of identified cases for a short period, removal of shared items from the center, and enhanced decontamination of fomites. The outbreak, which lasted longer than 12 months, involved 12 children and 7 staff members. Of these, 12 cases were confirmed by positive fungal culture. T. tonsurans is difficult to manage, especially in childcare settings, but case ascertainment, appropriate treatment with oral agents, and sustained infection control measures can be effective in controlling such outbreaks.

Principles and application of antibody libraries for infectious diseases.

Antibodies have been used efficiently for the treatment and diagnosis of many diseases. Recombinant antibody technology allows the generation of fully human antibodies. Phage display is the gold standard for the production of human antibodies in vitro. To generate monoclonal antibodies by phage display, the generation of antibody libraries is crucial. Antibody libraries are classified according to the source where the antibody gene sequences were obtained. The most useful library for infectious diseases is the immunized library. Immunized libraries would allow better and selective enrichment of antibodies against disease antigens. The antibodies generated from these libraries can be translated for both diagnostic and therapeutic applications. This review focuses on the generation of immunized antibody libraries and the potential applications of the antibodies derived from these libraries.

Exploring pharmacists' perceptions of using a clinical supervision skills competency tool to reflect and develop their supervisory practices.

INTRODUCTION: To date, there are no formal self-reflection tools routinely used by pharmacists within the Australian pharmacy profession. The study involved utilizing the Clinical Supervision Skills Competency Tool (CSSCT) at a metropolitan teaching hospital in Victoria, Australia. It explored pharmacists' perceptions of the CSSCT and its impact on their ability to self-reflect and develop their supervisory practices. METHODS: The qualitative study involved adapting the Clinical Supervision Skills Review Tool (CSRT), a clinician-validated tool. Prior to tool completion, participants attended an orientation session on the CSSCT. Thematic analysis and an inductive approach was then applied to data collected from two semi-structured focus group sessions and an online survey, for those not able to attend the focus groups. RESULTS: A total of 19 pharmacists were recruited and completed the CSSCT. The three major themes regarding the CSSCT identified were: feasibility of the tool, aspects of the CSSCT, and future planning. Use of rating scales and breakdown of the supervisory competencies were perceived to be helpful. Conversely, the tool's length, pharmacists' high workloads and time pressures, were identified as potential barriers to using the tool. CONCLUSIONS: Given pharmacists internationally currently lack a formal tool to enhance their supervisory methods, the CSSCT or similar tools emerge as valuable resources for steering pharmacists towards self-reflection and goal setting. Notably, the CSSCT sheds light on previously overlooked yet critical aspects of clinical supervision in the pharmacy context, including the wellbeing and cultural sensitivity of learners.

Comparing the experiences of community and hospital pharmacy learners completing a pre-registrant research training program.

BACKGROUND AND PURPOSE: Research training programs in the community pharmacy sector have not been well established. This study showcases a year-long guided research training program undertaken in hospital and community workplaces by pre-registrant pharmacists, and compares the perceived impact on learners in both sectors. EDUCATIONAL ACTIVITY AND SETTING: A two-year cohort study (2021-2022) of pre-registrant pharmacists enrolled in a research training program requiring them to undertake an individual project at their workplace over one year at either a community or hospital workplace. Outcome measures were pre-registrant perceptions of training impact and type of projects completed. FINDINGS: The results of this study demonstrate that the year-long guided research training program delivered to 403 pre-registrant pharmacists was perceived to be impactful to both community and hospital pre-registrant pharmacists and gave them the confidence to pursue further research and see research skills as an important attribute for the profession. Barriers to research included lack of time for both sectors but workplace support and lack of project ideas were especially noted in the community sector. Research project designs were mainly cross-sectional surveys or retrospective audits. SUMMARY: Programs seeking to adopt a similar model may wish to pay particular attention to supporting community pharmacy learners in providing a pre-selection of project ideas, offering training to workplace supervisors, ensuring enough academic support is given and having more check-in points/deliverables to ensure more feedback opportunities.

The application of computer-aided diagnosis in Breast Imaging Reporting and Data System ultrasound training for residents-a randomized controlled study.

Background: The consistency of Breast Imaging Reporting and Data System (BI-RADS) classification among experienced radiologists is different, which is difficult for inexperienced radiologists to master. This study aims to explore the value of computer-aided diagnosis (CAD) (AI-SONIC breast automatic detection system) in the BI-RADS training for residents. Methods: A total of 12 residents who participated in the first year and the second year of standardized resident training in Ningbo No. 2 Hospital from May 2020 to May 2021 were randomly divided into 3 groups (Group 1, Group 2, Group 3) for BI-RADS training. They were asked to complete 2 tests and questionnaires at the beginning and end of the training. After the first test, the educational materials were given to the residents and reviewed during the breast imaging training month. Group 1 studied independently, Group 2 studied with CAD, and Group 3 was taught face-to-face by experts. The test scores and ultrasonographic descriptors of the residents were evaluated and compared with those of the radiology specialists. The trainees' confidence and recognition degree of CAD were investigated by questionnaire. Results: There was no statistical significance in the scores of residents in the first test among the 3 groups (P=0.637). After training and learning, the scores of all 3 groups of residents were improved in the second test (P=0.006). Group 2 (52±7.30) and Group 3 (54±5.16) scored significantly higher than Group 1 (38±3.65). The consistency of ultrasonographic descriptors and final assessments between the residents and senior radiologists were improved (κ3 > κ2 > κ1), with κ2 and κ3 >0.4 (moderately consistent with experts), and κ1 =0.225 (fairly agreed with experts). The results of the questionnaire showed that the trainees had increased confidence in BI-RADS classification, especially Group 2 (1.5 to 3.5) and Group 3 (1.25 to 3.75). All trainees agreed that CAD was helpful for BI-RADS learning (Likert scale score: 4.75 out of 5) and were willing to use CAD as an aid (4.5, max. 5). Conclusions: The AI-SONIC breast automatic detection system can help residents to quickly master BI-RADS, improve the consistency between residents and experts, and help to improve the confidence of residents in the classification of BI-RADS, which may have potential value in the BI-RADS training for radiology residents. Trial Registration: Chinese Clinical Trial Registry (ChiCTR2400081672).

Phage display antibodies for diagnostic applications.

With major developments in molecular biology, numerous display technologies have been successfully introduced for recombinant antibody production. Even so, phage display still remains the gold standard for recombinant antibody production. Its success is mainly attributed to the robust nature of phage particles allowing for automation and adaptation to modifications. The generation of monospecific binders provides a vital tool for diagnostics at a lower cost and higher efficiency. The flexibility to modify recombinant antibodies allows great applicability to various platforms for use. This review presents phage display technology, application and modifications of recombinant antibodies for diagnostics.