Discovery of L-threonine transaldolases for enhanced biosynthesis of beta-hydroxylated amino acids.

Beta-hydroxy non-standard amino acids (ß-OH-nsAAs) have utility as small molecule drugs, precursors for beta-lactone antibiotics, and building blocks for polypeptides. While the L-threonine transaldolase (TTA), ObiH, is a promising enzyme for ß-OH-nsAA biosynthesis, little is known about other natural TTA sequences. We ascertained the specificity of the TTA enzyme class more comprehensively by characterizing 12 candidate TTA gene products across a wide range (20-80%) of sequence identities. We found that addition of a solubility tag substantially enhanced the soluble protein expression level within this difficult-to-express enzyme family. Using an optimized coupled enzyme assay, we identified six TTAs, including one with less than 30% sequence identity to ObiH that exhibits broader substrate scope, two-fold higher L-Threonine (L-Thr) affinity, and five-fold faster initial reaction rates under conditions tested. We harnessed these TTAs for first-time bioproduction of ß-OH-nsAAs with handles for bio-orthogonal conjugation from supplemented precursors during aerobic fermentation of engineered Escherichia coli, where we observed that higher affinity of the TTA for L-Thr increased titer. Overall, our work reveals an unexpectedly high level of sequence diversity and broad substrate specificity in an enzyme family whose members play key roles in the biosynthesis of therapeutic natural products that could benefit from chemical diversification.

Incorporation of a Chemically Diverse Set of Non-Standard Amino Acids into a Gram-Positive Organism.

The incorporation of non-standard amino acids (nsAAs) within proteins and peptides through genetic code expansion introduces novel chemical functionalities such as photo-crosslinking and bioconjugation. Given the utility of Bacillus subtilis in fundamental and applied science, we extended existing nsAA incorporation technology from Escherichia coli into B. subtilis , demonstrating incorporation of 20 unique nsAAs. The nsAAs we succeeded in incorporating within proteins conferred properties that included fluorescence, photo-crosslinking, and metal chelation. Here, we describe the reagents, equipment, and protocols to test for nsAA incorporation at a small scale (96-well plate and culture tube scales). We report specific media requirements for certain nsAAs, including two variants for different media conditions. Our protocol provides a consistent and reproducible method for incorporation of a chemically diverse set of nsAAs into a model Gram-positive organism.

Identifying the needs of our older community-based patients in the COVID era: A telephone survey.

Background: Older people's vulnerability during a pandemic may extend to social connectedness, access to healthcare, and information delivery. We sought to identify whether and how older community-based patients are maintaining connections and accessing information during COVID-19. Methods: We administered a telephone questionnaire to all patients (or carer/proxy answering 'on patient's behalf') who previously attended our Geriatric Medicine clinic, May-December 2019. Results: Response rate was 58.8% (151/257), carer respondents comprising 23.8% (36/151). Mean patient age was 81.8 years (SD 8.6); 59.6% were female, 15.2% lived alone. English was the preferred language for 72.9% (110/151). Almost half (46.4%, 70/151) felt COVID-related restrictions had impacted them. Thirty-eight percent (58/151) reported feelings of social isolation, most (38/58) reporting this new since COVID. Nonetheless, 92.1% (139/151) reported maintaining social connections, all with family (139/139), less often with friends (69.8%, 97/139). COVID-related information sources included television 68.9% (104/151), family/friends (54.3%), healthcare providers (24.5%), and written sources (21.2%, 32/151); 12.6% used online resources. Increasing age lowered likelihood of accessing online information, while having smartphone/computer increased. Most (82.6%) believed their healthcare needs were being met, and 76.1% had accessed their GP, 87% (100/115) in-person. Only 33.1% (50/151) agreed telehealth acceptable, more often those with smartphone/computer (OR 2.15, p=0.04). Conclusions: Interventions to reduce isolation and optimize connectedness and healthcare- despite physical distancing- are important during COVID-19. During a rapidly evolving pandemic, healthcare delivery and information provision to our older population is likely best served by a multifaceted approach which acknowledges identified preferences, practices and barriers.

Synthetic auxotrophy remains stable after continuous evolution and in coculture with mammalian cells.

Understanding the evolutionary stability and possible context dependence of biological containment techniques is critical as engineered microbes are increasingly under consideration for applications beyond biomanufacturing. While synthetic auxotrophy previously prevented Escherichia coli from exhibiting detectable escape from batch cultures, its long-term effectiveness is unknown. Here, we report automated continuous evolution of a synthetic auxotroph while supplying a decreasing concentration of essential biphenylalanine (BipA). After 100 days of evolution, triplicate populations exhibit no observable escape and exhibit normal growth rates at 10-fold lower BipA concentration than the ancestral synthetic auxotroph. Allelic reconstruction reveals the contribution of three genes to increased fitness at low BipA concentrations. Based on its evolutionary stability, we introduce the progenitor strain directly to mammalian cell culture and observe containment of bacteria without detrimental effects on HEK293T cells. Overall, our findings reveal that synthetic auxotrophy is effective on time scales and in contexts that enable diverse applications.

Personal protective equipment related skin reactions in healthcare professionals during COVID-19.

Since the outbreak of COVID-19 pandemic, clinicians have had to use personal protective equipment (PPE) for prolonged periods. This has been associated with detrimental effects, especially in relation to the skin health. The present study describes a comprehensive survey of healthcare workers (HCWs) to describe their experiences using PPE in managing COVID-19 patients, with a particular focus on adverse skin reactions. A 24-hour prevalence study and multi-centre prospective survey were designed to capture the impact of PPE on skin health of hospital staff. Questionnaires incorporated demographics of participants, PPE type, usage time, and removal frequency. Participants reported the nature and location of any corresponding adverse skin reactions. The prevalence study included all staff in intensive care from a single centre, while the prospective study used a convenience sample of staff from three acute care providers in the United Kingdom. A total of 108 staff were recruited into the prevalence study, while 307 HCWs from a variety of professional backgrounds and demographics participated in the prospective study. Various skin adverse reactions were reported for the prevalence study, with the bridge of the nose (69%) and ears (30%) being the most affected. Of the six adverse skin reactions recorded for the prospective study, the most common were redness blanching (33%), itchiness (22%), and pressure damage (12%). These occurred predominantly at the bridge of the nose and the ears. There were significant associations (P < .05) between the adverse skin reactions with both the average daily time of PPE usage and the frequency of PPE relief. The comprehensive study revealed that the use of PPE leads to an array of skin reactions at various facial locations of HCWs. Improvements in guidelines are required for PPE usage to protect skin health. In addition, modifications to PPE designs are required to accommodate a range of face shapes and appropriate materials to improve device safety.

Recombineering and MAGE.

Recombination-mediated genetic engineering, also known as recombineering, is the genomic incorporation of homologous single-stranded or double-stranded DNA into bacterial genomes. Recombineering and its derivative methods have radically improved genome engineering capabilities, perhaps none more so than multiplex automated genome engineering (MAGE). MAGE is representative of a set of highly multiplexed single-stranded DNA-mediated technologies. First described in Escherichia coli, both MAGE and recombineering are being rapidly translated into diverse prokaryotes and even into eukaryotic cells. Together, this modern set of tools offers the promise of radically improving the scope and throughput of experimental biology by providing powerful new methods to ease the genetic manipulation of model and non-model organisms. In this Primer, we describe recombineering and MAGE, their optimal use, their diverse applications and methods for pairing them with other genetic editing tools. We then look forward to the future of genetic engineering.

CD8+ lymphocytes but not B lymphocytes are required for protection against chronic hepatitis E virus infection in chickens.

Hepatitis E is an important global disease, causing outbreaks of acute hepatitis in many developing countries and sporadic cases in industrialized countries. Hepatitis E virus (HEV) infection typically causes self-limiting acute hepatitis but can also progress to chronic disease in immunocompromised individuals. The immune response necessary for the prevention of chronic infection is T cell-dependent; however, the arm of cellular immunity responsible for this protection is not currently known. To investigate the contribution of humoral immunity in control of HEV infection and prevention of chronicity, we experimentally infected 20 wild-type (WT) and 18 immunoglobulin knockout (JH-KO) chickens with a chicken strain of HEV (avian HEV). Four weeks postinfection (wpi) with avian HEV, JH-KO chickens were unable to elicit anti-HEV antibody but had statistically significantly lower liver lesion scores than the WT chickens. At 16 wpi, viral RNA in fecal material and liver, and severe liver lesions were undetectable in both groups. To determine the role of cytotoxic lymphocytes in the prevention of chronicity, we infected 20 WT and 20 cyclosporine and CD8+ antibody-treated chickens with the same strain of avian HEV. The CD8 + lymphocyte-depleted, HEV-infected chickens had higher incidences of prolonged fecal viral shedding and statistically significantly higher liver lesion scores than the untreated, HEV-infected birds at 16 wpi. The results indicate that CD8 + lymphocytes are required for viral clearance and reduction of liver lesions in HEV infection while antibodies are not necessary for viral clearance but may contribute to the development of liver lesions in acute HEV infection.

Antibody-Mediated Endocytosis of Polysialic Acid Enables Intracellular Delivery and Cytotoxicity of a Glycan-Directed Antibody-Drug Conjugate.

The specific targeting of differentially expressed glycans in malignant cells has emerged as an attractive anticancer strategy. One such target is the oncodevelopmental antigen polysialic acid (polySia), a polymer of α2,8-linked sialic acid residues that is largely absent during postnatal development but is re-expressed during progression of several malignant human tumors, including small-cell and non-small cell lung carcinomas, glioma, neuroblastoma, and pancreatic carcinoma. In these cancers, expression of polySia correlates with tumor progression and poor prognosis and appears to modulate cancer cell adhesion, invasiveness, and metastasis. To evaluate the potential of PolySia as a target for anticancer therapy, we developed a chimeric human polySia-specific mAb that retained low nanomolar (nmol/L) target affinity and exhibited exquisite selectivity for polySia structures. The engineered chimeric mAb recognized several polySia-positive tumor cell lines in vitro and induced rapid endocytosis of polySia antigens. To determine whether this internalization could be exploited for delivery of conjugated cytotoxic drugs, we generated an antibody-drug conjugate (ADC) by covalently linking the chimeric human mAb to the tubulin-binding maytansinoid DM1 using a bioorthogonal chemical reaction scheme. The resulting polySia-directed ADC demonstrated potent target-dependent cytotoxicity against polySia-positive tumor cells in vitro. Collectively, these results establish polySia as a valid cell-surface, cancer-specific target for glycan-directed ADC and contribute to a growing body of evidence that the tumor glycocalyx is a promising target for synthetic immunotherapies. SIGNIFICANCE: These findings describe a glycan-specific antibody-drug conjugate that establishes polySia as a viable cell surface target within the tumor glycocalyx.

Nationwide Doctor of Nursing Practice/Advanced Practice Registered Nurse Survey on Roles, Functions, and Competencies.

Educational preparation for advanced practice registered nurses (APRNs) continues to shift from the master's degree to the doctor of nursing practice (DNP). Previous analysis of the roles, functions, and competencies of APRNs by Honig, Smolowitz, and Smaldone (2011) identified differences in practice between Master's of Science in Nursing (MSN)- and DNP-prepared APRNs. The aim of this study was to use the survey instrument created by Honig et al. to survey DNP/APRNs from across the nation and gain insight into their roles, competencies, and functions. A convenience sample was drawn from DNP graduates from the 13 schools provided by the American Board of Comprehensive Care (ABCC) as eligible to sit for the ABCC examination. A total of 375 individuals from 33 states who reported that they were in practice as a nurse practitioner (NP) completed the survey. Differences between the initial study and this study indicated that DNP NPs currently provide care across settings but with less inpatient care, less subacute care, and less palliative care than was reported in the initial analysis. Pregnancy-related care was the least common clinical service provided. Overall, this research indicates that DNP NPs prepared in comprehensive care are providing direct clinical care across settings to complex patients consistent with the comprehensive care domains for the ABCC certification examination (Honig et al., 2011).

Failure of radioactive iodine in the treatment of hyperthyroidism.

BACKGROUND: Persistent or recurrent hyperthyroidism after treatment with radioactive iodine (RAI) is common and many patients require either additional doses or surgery before they are cured. The purpose of this study was to identify patterns and predictors of failure of RAI in patients with hyperthyroidism. METHODS: We conducted a retrospective review of patients treated with RAI from 2007 to 2010. Failure of RAI was defined as receipt of additional dose(s) and/or total thyroidectomy. Using a Cox proportional hazards model, we conducted univariate analysis to identify factors associated with failure of RAI. A final multivariate model was then constructed with significant (p < 0.05) variables from the univariate analysis. RESULTS: Of the 325 patients analyzed, 74 patients (22.8 %) failed initial RAI treatment, 53 (71.6 %) received additional RAI, 13 (17.6 %) received additional RAI followed by surgery, and the remaining 8 (10.8 %) were cured after thyroidectomy. The percentage of patients who failed decreased in a stepwise fashion as RAI dose increased. Similarly, the incidence of failure increased as the presenting T3 level increased. Sensitivity analysis revealed that RAI doses <12.5 mCi were associated with failure while initial T3 and free T4 levels of at least 4.5 pg/mL and 2.3 ng/dL, respectively, were associated with failure. In the final multivariate analysis, higher T4 (hazard ratio [HR] 1.13; 95 % confidence interval [CI] 1.02-1.26; p = 0.02) and methimazole treatment (HR 2.55; 95 % CI 1.22-5.33; p = 0.01) were associated with failure. CONCLUSIONS: Laboratory values at presentation can predict which patients with hyperthyroidism are at risk for failing RAI treatment. Higher doses of RAI or surgical referral may prevent the need for repeat RAI in selected patients.

Developing a competency framework for critical care to match patient need.

UNLABELLED: The competency framework developed by the critical care education group of the London Standing Conference aims to serve every grade and level of practitioner. It is neither time specific nor static. The patient is the central focus of the framework and the elements of competence reflect patient need at any critical care level [Comprehensive Critical Care: A Review of Adult Critical Care Services, The Stationary Office, London]. A group of expert nurses have developed the competency framework, with widespread consultation and collaboration. This approach intended to develop consistency for critical care education and practice. It is envisaged that this will reduce pockets of repeated activity, which places huge demands on limited resources. The critical care competency framework was developed using the method of functional analysis. A plan for the future has been identified, including continued collaboration and consultation with Trusts and Higher Educational Institutions and the development of an online manual to support the competency framework. KEY POINTS: 1. Critical care delivery has been under close scrutiny and a number of key contemporary drivers have led to the development of this competency framework. 2. The development of a consistent pan-London approach to critical care education has been identified. 3. The patient is the focus of critical care delivery and therefore patient need is central to the critical care competency framework. 4. Wider collaboration is needed with other agencies and groups to prevent the repetition of work already carried out.

Critical care competencies.

In the absence of nationally accepted critical care competencies, each educational institution providing critical care programmes is forced to define the essential competencies necessary for practice, leading to variations in expected practice and the emergence of 'postcode' competencies. This research report aims to build upon competency activity for all areas of nursing practice within critical care levels 1, 2 and 3. A functional analysis to elicit core critical care competency statements was conducted and a modified Delphi technique was used to generate consensus opinion from a pan-London purposive sample of nurses working in critical care. The functional analysis group identified four competency statements and elements of competencies. Consensus agreement of 80% was achieved with mean agreement scores that exceed 97%. A core critical care competency framework was refined and developed by expert nurses drawing on their own experience and knowledge of critical care nursing. The framework could be useful to: educationalists designing competency-based curricula; critical care managers as a tool for recruitment and retention and for education and training of staff; individual critical care nurses to facilitate continuous professional development.