ABSTRACTGenetic manipulation is widely employed in laboratories for purposes such as gene function characterization, the development of novel traits in transformed microorganisms, and the production of biotechnologically relevant compounds. A key step in this process is the selection of mutant strains, often facilitated by antibiotics. Tetracyclines are commonly used; however, their aqueous solutions tend to precipitate and turn brown over time, potentially compromising stability and efficacy. This necessitates frequent in situ preparation. The present study aimed to evaluate the functional stability of tetracycline hydrochloride for selecting transformed tetracycline‐resistant bacterial strains in both liquid and solid media. A tetracycline stock solution and supplemented agar plates were prepared and used to cultivate resistant and non‐resistant strains of Escherichia coli and Arcobacter butzleri. This allowed for assessment of antibiotic stability and utility over a 200‐day period. Additionally, the minimal inhibitory concentration (MIC) of tetracycline was determined for each strain. Results indicated that the tetracycline‐containing materials maintained functional stability for at least 175 days post‐preparation, effectively supporting the selection of resistant strains in genetic manipulation experiments. These findings obviate the need for the continual and time‐consuming preparation of tetracycline‐supplemented culture media, thereby enhancing the practicality of employing this antibiotic in genetic modification studies.

ABSTRACTThis study explores the locations and distribution of mutations in the exons of the TP53 gene in pulmonary large cell neuroendocrine carcinomas (LCNEC) and the corresponding expression of p53 as well as the Ki‐67 proliferation index. Real‐world data from routine diagnostics were collected retrospectively from 149 consecutive cases of pulmonary LCNEC. They included next‐generation sequencing (NGS) of the TP53 gene along with evaluation of p53 and Ki‐67 expression with immunohistochemistry (IHC). Abnormal p53 expression was defined as strong positive nuclear reaction in more than 20% of the tumor cells or complete absence of immunostaining in tumor cells. Both p53 expression and Ki‐67 proliferation index correlated with TP53 mutations. In addition, a statistically significant increase in Ki‐67 expression was found for cases with abnormal staining of p53 compared to samples with normal staining. We found that IHC staining for p53 can identify TP53 mutations in pulmonary LCNEC with an overall concordance of 76.6%. Moreover, both pathological patterns of p53 immunostaining and NGS‐detected TP53 mutations were associated with higher Ki‐67 proliferation indices.

ABSTRACTIntraoperative frozen section (FS) analysis is critical in surgical pathology, but conventional hematoxylin and eosin (H&E) staining has limitations in poorly differentiated neoplasms and resection margins. Immunohistochemistry (IHC) provides higher diagnostic specificity, yet has traditionally been too time‐consuming for intraoperative urgency. This study aimed to optimize, validate, and evaluate Fast Frozen Rapid Automated Immunohistochemistry (FFRA‐IHC) using the Q‐Stain X Autostainer to improve surgical diagnostics. After optimization with antibodies CKAE, CK5, CK7, CD45, and Synaptophysin, 44 tissue samples from patients undergoing surgery at Rigshospitalet, Copenhagen, were analyzed by FS H&E, FFRA‐IHC, and standard formalin‐fixed paraffin‐embedded (FFPE) methods. Automated FFRA‐IHC showed high diagnostic accuracy and supported immediate clinical decision‐making: of 37 tumors not classifiable by FS H&E alone, 25 (68%) were classified into specific tumor types, and all five ambiguous resection margins were resolved. A cost–benefit analysis indicated efficiency gains with reduced hands‐on time compared to manual IHC. In conclusion, FFRA‐IHC demonstrated promising results for enhancing intraoperative diagnostics, leading to its implementation in the daily workflow at our department. Future studies should expand antibody panels and assess the broader clinical impact to further improve intra‐ and perioperative care.

ABSTRACTAbnormal white blood cell (WBC) counts have been associated with ventilator‐associated pneumonia (VAP). However, the prognostic value of WBC trajectory changes in VAP patients remains understudied. This study aimed to explore the correlation between WBC trajectory changes and the 30‐day mortality rate in VAP patients. Data from VAP patients in the MIMIC‐IV database were included. Group‐based trajectory analysis was used to categorize patients based on their WBC patterns within 7 days of ICU admission. Cox proportional hazards regression analysis was conducted to assess the association between WBC trajectories and 30‐day mortality. A total of 1194 eligible VAP patients were included. Five distinct WBC trajectories were identified. Compared to the trajectory group with continuously normal WBC levels (Class 5), the group with initially high levels but subsequent decrease (Class 2) and the group with continuously increasing WBC levels (Class 3) showed independent associations with increased mortality (OR > 1, p < 0.05). Additionally, restricted cubic splines (RCS) results indicated a possible linear relationship between WBC counts on the 6th and 7th days and the 30‐day survival status of VAP patients. Within the first week of ICU admission, both the group with initially low levels but persistently increasing WBC trajectories and the group with initially high levels but continuously decreasing WBC trajectories were associated with increased 30‐day mortality in VAP patients. This finding offers insights into predicting short‐term mortality risks related to VAP and aids clinicians in early identification of high‐risk patients.

ABSTRACTThe rampant increase in antimicrobial resistance and stagnant pipeline of antibiotic development is a global concern. Biofilms further make the infections more persistent and incurable by providing a protective environment to the microbes. It demands the development of innovative therapeutics without delay. Nanomaterial‐based therapeutics is a promising approach to combat deadly bacterial infections. The distinct shape, size, and physical characteristics of nanomaterials (NMs) make them able to target biofilms through various mechanisms and thrash recalcitrant deadly infections. This comprehensive review describes (i) historical perspective of antibiotics and their role in combating microbes; (ii) different mechanisms through which bacteria develop resistance against antibiotics; (iii) different sensing mechanisms to detect bacteria; (iv) NMs to treat planktonic bacteria; (v) NMs to treat biofilms (both oral and wound biofilms); (vi) different types of NMs (e.g., metal NMs, polymeric NMs, carbon NMs) along with photothermal effects to combat microbes.

ABSTRACTThe 2021 WHO classification of thoracic tumours recommends grading pleural mesothelioma to aid prognostication. Robustness of grading and morphological characterisation is key to its clinical utility, though validation of this grading system has largely been conducted by expert thoracic pathologists. We conducted a survey inviting pathologists across a range of practices and expertise to grade digitised images of 50 epithelioid pleural mesotheliomas that had been graded by an expert in thoracic pathology. We included slides that were considered potentially problematic such as small biopsies, focal necrosis, and rare subtypes that may affect grading (small cell and deciduoid features). Using the Sectra Uniview web viewer, participants were asked to score atypia, mitotic count, and necrosis and choose from a list of cytological and architectural features. Seventy‐four pathologists anonymously participated. There was 90% agreement of consensus scores with expert opinion using the WHO 2‐tier grade and 72% for the 3‐tier nuclear grade but only 70% for nuclear atypia, 56% for mitoses, and 84% for necrosis. Both 3‐tier nuclear grade and WHO 2‐tier grading systems were significantly associated with survival. Our study affirms the overall robustness and utility of grading for pleural mesothelioma, reveals variances, and suggests the need for dedicated training.