Pyrrolysine-Inspired in Cellulo Synthesis of an Unnatural Amino Acid for Facile Macrocyclization of Proteins.

Macrocyclization has been touted as an effective strategy to enhance the in vivo stability and efficacy of protein therapeutics. Herein, we describe a scalable and robust system based on the endogenous biosynthesis of a noncanonical amino acid coupled to the pyrrolysine translational machinery for the generation of lasso-grafted proteins. The in cellulo biosynthesis of the noncanonical amino acid d-Cys-ε-Lys was achieved by hijacking the pyrrolysine biosynthesis pathway, and then, its genetical incorporation into proteins was performed using an optimized PylRS/tRNAPyl pair and cell line. This system was then applied to the structurally inspired cyclization of a 23-mer therapeutic P16 peptide engrafted on a fusion protein, resulting in near-complete cyclization of the target cyclic subunit in under 3 h. The resulting cyclic P16 peptide fusion protein possessed much higher CDK4 binding affinity than its linear counterpart. Furthermore, a bifunctional bicyclic protein harboring a cyclic cancer cell targeting RGD motif on the one end and the cyclic P16 peptide on the other is produced and shown to be a potent cell cycle arrestor with improved serum stability.

Nosocomial Outbreak of Coronavirus Disease 2019 by Possible Airborne Transmission Leading to a Superspreading Event.

BACKGROUND: Nosocomial outbreaks with superspreading of coronavirus disease 2019 due to a possible airborne transmission have not been reported. METHODS: Epidemiological analysis, environmental samplings, and whole-genome sequencing (WGS) were performed for a hospital outbreak. RESULTS: A superspreading event that involved 12 patients and 9 healthcare workers (HCWs) occurred within 9 days in 3 of 6 cubicles at an old-fashioned general ward with no air exhaust built within the cubicles. The environmental contamination by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA was significantly higher in air grilles (>2 m from patients' heads and not within reach) than on high-touch clinical surfaces (36.4%, 8 of 22 vs 3.4%, 1 of 29, P = .003). Six (66.7%) of 9 contaminated air exhaust grilles were located outside patient cubicles. The clinical attack rate of patients was significantly higher than of HCWs (15.4%, 12 of 78 exposed patients vs 4.6%, 9 of 195 exposed HCWs, P = .005). Moreover, the clinical attack rate of ward-based HCWs was significantly higher than of nonward-based HCWs (8.1%, 7 of 68 vs 1.8%, 2 of 109, P = .045). The episodes (mean ±â€…standard deviation) of patient-care duty assignment in the cubicles was significantly higher among infected ward-based HCWs than among noninfected ward-based HCWs (6.0 ±â€…2.4 vs 3.0 ±â€…2.9, P = .012) during the outbreak period. The outbreak strains belong to SARS-CoV-2 lineage B.1.36.27 (GISAID clade GH) with the unique S-T470N mutation on WGS. CONCLUSIONS: This nosocomial point source superspreading event due to possible airborne transmission demonstrates the need for stringent SARS-CoV-2 screening at admission to healthcare facilities and better architectural design of ventilation systems to prevent such outbreaks. Portable high-efficiency particulate filters were installed in each cubicle to improve ventilation before resumption of clinical service.

An outbreak of Burkholderia cepacia complex exit site infection among peritoneal dialysis patients caused by contaminated spray dressing.

Background: Wound dressing is intended to provide a physical barrier from microorganisms. Spray dressing is convenient and can be applied to wounds of various contours. In July 2020, a cluster of four Burkholderia cepacia complex (BCC) exit site infections was identified among peritoneal dialysis patients in a regional hospital in Hong Kong. In response, our hospital infection control team conducted an epidemiologic investigation. Methods: We conducted a retrospective cohort study of peritoneal dialysis patients with culture-confirmed BCC exit site infections from January 2011 to July 2020. Outbreak investigations, including case finding, molecular typing and post-outbreak surveillance, were performed. Discussion: A substantial increase in BCC exit site infections has been observed since 2013, rising from 0.23 in 2012 to 1.09 episodes per 100 patient-year in 2015, with the number of cases in the first half of 2020 already surpassing the total from 2019. The potential source had been traced to a spray dressing introduced to exit site care in December 2012. Burkholderia cepacia complex was isolated from both the unopened and in-use sprays from the same lot. Multilocus sequence typing analysis confirmed their genetic relatedness. The spray dressing was subsequently removed from exit site care. Post-outbreak surveillance over two years showed a marked and sustained decrease in BCC exit site infection. Conclusion: Water-based spray dressing can be a source of BCC causing wound infections. The use of contaminated spray dressing, especially in chronic wounds with proximity to indwelling catheters, may pose an inherent risk to patients.

Development and evaluation of an undergraduate training course for developing International Council of Nurses disaster nursing competencies in China.

BACKGROUND: Nurses are often called upon to play the role of first responder when disaster occurs. Yet the lack of accepted competencies and gaps in education make it difficult to recruit nurses prepared to respond to a disaster and provide assistance in an effective manner. DESIGN: Based on the International Council of Nurses (ICN) Framework of Disaster Nursing Competencies and Global Standards for the Initial Education of Professional Nurses and Midwives, a training course titled "Introduction to Disaster Nursing" was designed and implemented with 150 students. A pre-post survey design was used to assess changes in participants' self-rated disaster nursing competencies. The impact of the training course on participants' attitudes toward disaster nursing and their learning experience were also assessed. FINDINGS: All participants passed the assessments and examination with an average score of 70%. Pre- and posttraining self-ratings of the disaster nursing competencies increased from 2.09 to 3.71 (p < .001) on a Likert scale of 1 to 5, and the effect size was large, with Cohen's d higher than 0.8. No significant difference in both examination results (60% group assignments; 40% written examination) and self-rated competencies was noted between the senior year students and graduate nurse participants by Mann-Whitney U test (p value = .90). The majority of participants indicated their willingness to participate as a helper in disaster relief and saw themselves competent to work under supervision. CONCLUSIONS: The ICN Framework of Disaster Nursing Competencies was instrumental to guide the training curriculum development. This introductory training course could be incorporated into undergraduate nursing education programs as well as serve as a continuing education program for graduate nurses. CLINICAL RELEVANCE: The training program can be used for preparing generalist nurses of their nursing competencies in disaster preparedness, response and post-disaster recovery and rehabilitation.

Assessing predictive validity of the modified Braden scale for prediction of pressure ulcer risk of orthopaedic patients in an acute care setting.

AIMS AND OBJECTIVES: To assess and compare the predictive validity of the modified Braden and Braden scales and to identify which of the modified Braden subscales are predictive in assessing pressure ulcer risk among orthopaedic patients in an acute care setting. BACKGROUND: Although the Braden scale has better predictive validity, literature has suggested that it can be used in conjunction with other pressure ulcer risk calculators or that some other subscales be added. To increase the predictive power of the Braden scale, a modified Braden scale by adding body build for height and skin type and excluding nutrition was developed. DESIGN: A prospective cohort study. METHOD: A total of 197 subjects in a 106-bed orthopaedic department of an acute care hospital in Hong Kong were assessed for their risk for pressure ulcer development by the modified Braden and Braden scales. Subsequently, daily skin assessment was performed to detect pressure ulcers. Cases were closed when pressure ulcers were detected. RESULTS: Out of 197 subjects, 18 patients (9.1%) developed pressure ulcers. The area under the receiver operating characteristic curve for the modified Braden scale was 0.736 and for the Braden scale was 0.648. The modified Braden cut-off score of 19 showed the best balance of sensitivity (89%) and specificity (62%). Sensory perception (Beta = -1.544, OR=0.214, p = 0.016), body build for height (Beta = -0.755, OR = 0.470, p = 0.030) and skin type (Beta = -1.527, OR = 0.217, p = 0.002) were significantly predictive of pressure ulcer development. CONCLUSION: The modified Braden scale is more predictive of pressure ulcer development than the Braden scale. RELEVANCE TO CLINICAL PRACTICE: The modified Braden scale can be adopted for predicting pressure ulcer development among orthopaedic patients in an acute care setting. Specific nursing interventions should be provided, with special attention paid to orthopaedic patients with impaired sensory perception, poor skin type and abnormal body build for height.

Directed evolution of a genetically encoded immobilized lipase for the efficient production of biodiesel from waste cooking oil.

BACKGROUND: We have recently developed a one-step, genetically encoded immobilization approach based on fusion of a target enzyme to the self-crystallizing protein Cry3Aa, followed by direct production and isolation of the fusion crystals from Bacillus thuringiensis. Using this approach, Bacillus subtilis lipase A was genetically fused to Cry3Aa to produce a Cry3Aa-lipA catalyst capable of the facile conversion of coconut oil into biodiesel over 10 reaction cycles. Here, we investigate the fusion of another lipase to Cry3Aa with the goal of producing a catalyst suitable for the conversion of waste cooking oil into biodiesel. RESULTS: Genetic fusion of the Proteus mirabilis lipase (PML) to Cry3Aa allowed for the production of immobilized lipase crystals (Cry3Aa-PML) directly in bacterial cells. The fusion resulted in the loss of PML activity, however, and so taking advantage of its genetically encoded immobilization, directed evolution was performed on Cry3Aa-PML directly in its immobilized state in vivo. This novel strategy allowed for the selection of an immobilized PML mutant with 4.3-fold higher catalytic efficiency and improved stability. The resulting improved Cry3Aa-PML catalyst could be used to catalyze the conversion of waste cooking oil into biodiesel for at least 15 cycles with minimal loss in conversion efficiency. CONCLUSIONS: The genetically encoded nature of our Cry3Aa-fusion immobilization platform makes it possible to perform both directed evolution and screening of immobilized enzymes directly in vivo. This work is the first example of the use of directed evolution to optimize an enzyme in its immobilized state allowing for identification of a mutant that would unlikely have been identified from screening of its soluble form. We demonstrate that the resulting Cry3Aa-PML catalyst is suitable for the recyclable conversion of waste cooking oil into biodiesel.