Three-dimensional integration of two-dimensional field-effect transistors.

In the field of semiconductors, three-dimensional (3D) integration not only enables packaging of more devices per unit area, referred to as 'More Moore'1 but also introduces multifunctionalities for 'More than Moore'2 technologies. Although silicon-based 3D integrated circuits are commercially available3-5, there is limited effort on 3D integration of emerging nanomaterials6,7 such as two-dimensional (2D) materials despite their unique functionalities7-10. Here we demonstrate (1) wafer-scale and monolithic two-tier 3D integration based on MoS2 with more than 10,000 field-effect transistors (FETs) in each tier; (2) three-tier 3D integration based on both MoS2 and WSe2 with about 500 FETs in each tier; and (3) two-tier 3D integration based on 200 scaled MoS2 FETs (channel length, LCH = 45 nm) in each tier. We also realize a 3D circuit and demonstrate multifunctional capabilities, including sensing and storage. We believe that our demonstrations will serve as the foundation for more sophisticated, highly dense and functionally divergent integrated circuits with a larger number of tiers integrated monolithically in the third dimension.

The intermediate in a nitrate-responsive ω-amidase pathway in plants may signal ammonium assimilation status.

A metabolite of ammonium assimilation was previously theorized to be involved in the coordination of the overall nitrate response in plants. Here we show that 2-hydroxy-5-oxoproline, made by transamination of glutamine, the first product of ammonium assimilation, may be involved in signaling a plant's ammonium assimilation status. In leaves, 2-hydroxy-5-oxoproline met four foundational requirements to be such a signal. First, when it was applied to foliage, enzyme activities of nitrate reduction and ammonium assimilation increased; the activities of key tricarboxylic acid cycle-associated enzymes that help to supply carbon skeletons for amino acid synthesis also increased. Second, its leaf pools increased as nitrate availability increased. Third, the pool size of its precursor, Gln, reflected ammonium assimilation rather than photorespiration. Fourth, it was widely conserved among monocots, dicots, legumes, and nonlegumes and in plants with C3 or C4 metabolism. Made directly from the first product of ammonium assimilation, 2-hydroxy-5-oxoproline acted as a nitrate uptake stimulant. When 2-hydroxy-5-oxoproline was provided to roots, the plant's nitrate uptake rate approximately doubled. Plants exogenously provided with 2-hydroxy-5-oxoproline to either roots or leaves accumulated greater biomass. A model was constructed that included the proposed roles of 2-hydroxy-5-oxoproline as a signal molecule of ammonium assimilation status in leaves, as a stimulator of nitrate uptake by roots and nitrate downloading from the xylem. In summary, a glutamine metabolite made in the ω-amidase pathway stimulated nitrate uptake by roots and was likely to be a signal of ammonium assimilation status in leaves. A chemical synthesis method for 2-hydroxy-5-oxoproline was also developed.

Hematocrit control and thrombotic risk in patients with polycythemia vera treated with ruxolitinib in clinical practice.

Polycythemia vera (PV) is a myeloproliferative neoplasm characterized by unregulated red blood cell production resulting in elevated hemoglobin and/or hematocrit levels. Patients often have symptoms such as fatigue, pruritus, and painful splenomegaly, but are also at risk of thrombosis, both venous and arterial. Ruxolitinib, a selective Janus kinase inhibitor, is approved by the US Food and Drug Administration as second-line cytoreductive treatment after intolerance or inadequate response to hydroxyurea. Although ruxolitinib has been widely used in this setting, limited data exist in the literature on ruxolitinib treatment patterns and outcomes among patients with PV in routine clinical practice. We report a retrospective, observational, cohort study of patients treated for PV with ruxolitinib across three US centers (academic and regional practice) from December 2014-December 2019. The study included 69 patients, with a median follow-up duration of 3.7 years (95% CI, 2.9-4.4). Our data demonstrate very high rates of hematocrit control (88% of patients by three months and 89% by six months); few patients required dose adjustments or suspension. No arterial thromboses were observed; however, the follow-up duration does not allow for the generation of meaningful conclusions from this. Three patients had thrombotic events; one was in the setting of a second malignancy, one post-operative, and a third related to prolonged immobility. We also found that 28% of patients initiated ruxolitinib as a result of poorly controlled platelet counts, second only to hydroxyurea intolerance (46%) as a reason to start therapy. In clinical practice, ruxolitinib continues to be effective in controlling hematocrit levels after three and six months of treatment in patients and is associated with low thrombotic risk.

Acute frailty services: results of a national day of care survey.

INTRODUCTION: Older people living with frailty are at high risk of emergency hospital admission and often have complex care needs which may not be adequately met by conventional models of acute care. This has driven the introduction of adaptations to acute care pathways designed to improve outcomes in this patient group. The identification of differences in the organisational approach to frailty may highlight opportunities for quality improvement. METHODS: The Society for Acute Medicine Benchmarking audit is a national service evaluation which uses a single day-of-care methodology to record patient and organisational level data. All acute hospitals in the United Kingdom are eligible to participate. Emergency admissions referred to acute medical services between 00:00 and 23:59 on Thursday 23rd June 2022 were recorded. Information on the structure and operational design of acute frailty services was collected. The use of a validated frailty assessment tool, clinical frailty scale within the first 24 h of admission, assessment by an acute frailty service and clinical outcomes were reported in patients aged 70 year and above. A mixed effect generalised linear model was used to determine factors associated same-day discharge without overnight stay in patients with frailty. RESULTS: A total of 152 hospitals participated. There was significant heterogeneity in the operational design and staffing model of acute frailty services. The presence of an acute frailty unit was reported in 57 (42.2%) hospitals. The use of validated frailty assessment tools was reported in 117 (90.0%) hospitals, of which 107 (91.5%) used the clinical frailty scale. Patient-level data were recorded for 3604 patients aged 70 years and above. At the patient level, 1626 (45.1%) were assessed using a validated tool during the admission process. Assessment by acute frailty services was associated with an increased likelihood of same-day discharge (adjusted OR 1.55, 95%CI 1.03- 2.39). CONCLUSION: There is significant variation in the provision of acute frailty services. Frailty-related policies and services are common at the organisational level but implemented inconsistently at the patient level. Older people with frailty or geriatric syndromes assessed by acute frailty services were more likely to be discharged without the need for overnight bed-based admission.

Acute care models for older people living with frailty: a systematic review and taxonomy.

BACKGROUND: The need to improve the acute care pathway to meet the care needs of older people living with frailty is a strategic priority for many healthcare systems. The optimal care model for this patient group is unclear. METHODS: A systematic review was conducted to derive a taxonomy of acute care models for older people with acute medical illness and describe the outcomes used to assess their effectiveness. Care models providing time-limited episodes of care (up to 14 days) within 48 h of presentation to patients over the age of 65 with acute medical illness were included. Care models based in hospital and community settings were eligible. Searches were undertaken in Medline, Embase, CINAHL and Cochrane databases. Interventions were described and classified in detail using a modified version of the TIDIeR checklist for complex interventions. Outcomes were described and classified using the Core Outcome Measures in Effectiveness Trials (COMET) taxonomy. Risk of bias was assessed using RoB2 and ROBINS-I. RESULTS: The inclusion criteria were met by 103 articles. Four classes of acute care model were identified, acute-bed based care, hospital at home, emergency department in-reach and care home models. The field is dominated by small single centre randomised and non-randomised studies. Most studies were judged to be at risk of bias. A range of outcome measures were reported with little consistency between studies. Evidence of effectiveness was limited. CONCLUSION: Acute care models for older people living with frailty are heterogenous. The clinical effectiveness of these models cannot be conclusively established from the available evidence. TRIAL REGISTRATION: PROSPERO registration (CRD42021279131).

Using Test Question Templates to teach physiology core concepts.

The past ∼15 years have seen increasing interest in defining disciplinary core concepts. Within the field of physiology, Michael, McFarland, Modell, and colleagues have published studies that defined physiology core concepts and have elaborated many of these as detailed conceptual frameworks. With such helpful definitions now in place, attention is turning to the related issue of how to maximize student understanding of the core concepts by linking these "big ideas" to concrete student-facing resources for active learning and assessment. Our practitioner-based view begins with the recognition that in many if not most undergraduate physiology courses assessment drives learning. We have therefore linked published conceptual frameworks to Test Question Templates (TQTs), whose structure promotes transparent assessments as well as the active learning needed to prepare for such assessments. We provide examples of conceptual framework-linked TQTs for the physiology core concepts of Homeostasis, Flow Down Gradients, the Cell Membrane, and Cell-Cell Communication. We argue that this deployment of TQTs has at least two distinct benefits for the teaching and learning of core concepts. First, documenting the connections between conceptual frameworks and TQTs may clarify coverage and assessment of the core concepts for both instructors and students. Second, misconceptions about core concepts may be directly targeted and dispelled via thoughtful construction, arrangement, and iteration of TQTs. We propose that the TQT framework or similar approaches may be applied fruitfully to any sufficiently articulated physiology core concept for high school, undergraduate, or graduate students.NEW & NOTEWORTHY Our students often focus on the grades they need to advance through academic programs. How can instructors harness this understandable interest in grades to help students gain a true understanding of core concepts? The new framework of Test Question Templates (TQTs) shows promise in linking student priorities like test scores to instructor priorities like core concepts.

Hospital at home for acute medical illness: The 21st century acute medical unit for a changing population.

Recent trends across Europe show a year-on-year increase in the number of patients with acute medical illnesses presenting to hospitals, yet there are no plans for a substantial expansion in acute hospital infrastructure or staffing to address demand. Strategies to meet increasing demand need to consider the fact that there is limited capacity in acute hospitals and focus on new care models in both hospital and community settings. Increasing the efficiency of acute hospital provision by reducing the length of stay entails supporting acute ambulatory care, where patients receive daily acute care interventions but do not stay overnight in the hospitals. This approach may entail daily transfer between home and an acute setting for ongoing treatment, which is unsuitable for some patients living with frailty. Acute hospital at home (HaH) is a care model which, thanks to advances in point of care diagnostic capability, can provide a credible model of acute medical assessment and treatment without the need for hospital transfer. Investment and training to support scaling up of HaH are key strategic aims for integrated healthcare systems.

Genome-scale metabolic modeling reveals key features of a minimal gene set.

Mesoplasma florum, a fast-growing near-minimal organism, is a compelling model to explore rational genome designs. Using sequence and structural homology, the set of metabolic functions its genome encodes was identified, allowing the reconstruction of a metabolic network representing ˜ 30% of its protein-coding genes. Growth medium simplification enabled substrate uptake and product secretion rate quantification which, along with experimental biomass composition, were integrated as species-specific constraints to produce the functional iJL208 genome-scale model (GEM) of metabolism. Genome-wide expression and essentiality datasets as well as growth data on various carbohydrates were used to validate and refine iJL208. Discrepancies between model predictions and observations were mechanistically explained using protein structures and network analysis. iJL208 was also used to propose an in silico reduced genome. Comparing this prediction to the minimal cell JCVI-syn3.0 and its parent JCVI-syn1.0 revealed key features of a minimal gene set. iJL208 is a stepping-stone toward model-driven whole-genome engineering.

Frailty assessment and acute frailty service provision in the UK: results of a national 'day of care' survey.

BACKGROUND: The incorporation of acute frailty services into the acute care pathway is increasingly common. The prevalence and impact of acute frailty services in the UK are currently unclear. METHODS: The Society for Acute Medicine Benchmarking Audit (SAMBA) is a day of care survey undertaken annually within the UK. SAMBA 2019 (SAMBA19) took place on Thursday 27th June 2019. A questionnaire was used to collect hospital and patient-level data on the structure and organisation of acute care delivery. SAMBA19 sought to establish the frequency of frailty assessment tool use and describe acute frailty services nationally. Hospitals were classified based on the presence of acute frailty services and metrics of performance compared. RESULTS: A total of 3218 patients aged ≥70 admitted to 129 hospitals were recorded in SAMBA19. The use of frailty assessment tools was reported in 80 (62.0%) hospitals. The proportion of patients assessed for the presence of frailty in individual hospitals ranged from 2.2 to 100%. Bedded Acute Frailty Units were reported in 65 (50.3%) hospitals. There was significant variation in admission rates between hospitals. This was not explained by the presence of a frailty screening policy or presence of a dedicated frailty unit. CONCLUSION: Two fifths of participating UK hospitals did not have a routine frailty screening policy: where this existed, rates of assessment for frailty were variable and most at-risk patients were not assessed. Responses to positive results were poorly defined. The provision of acute frailty services is variable throughout the UK. Improvement is needed for the aspirations of national policy to be fully realised.

Response to winter pressures in acute services: analysis from the Winter Society for Acute Medicine Benchmarking Audit.

BACKGROUND: There is increased demand for urgent and acute services during the winter months, placing pressure on acute medicine services caring for emergency medical admissions. Hospital services adopt measures aiming to compensate for the effects of this increased pressure. This study aimed to describe the measures adopted by acute medicine services to address service pressures during winter. METHODS: A survey of acute hospitals was conducted during the Society for Acute Medicine Benchmarking Audit, a national day-of-care audit, on 30th January 2020. Survey questions were derived from national guidance. Acute medicine services at 93 hospitals in the United Kingdom completed the survey, evaluating service measures implemented to mitigate increased demand, as well as markers of increased pressure on services. RESULTS: All acute internal medicine services had undertaken measures to prepare for increased demand, however there was marked variation in the combination of measures adopted. 81.7% of hospitals had expanded the number of medical inpatient beds available. 80.4% had added extra clinical staff. The specialty of the physicians assigned to provide care for extra inpatient beds varied. A quarter of units had reduced beds available for providing Same Day Emergency Care on the day of the survey. Patients had been waiting in corridors within the emergency medicine department in 56.3% of units. CONCLUSION: Winter pressure places considerable demand on acute services, and impacts the delivery of care. Although increased pressure on acute hospital services during winter is widely recognised, there is considerable variation in the approach to planning for these periods of increased demand.

Nurse staffing levels within acute care: results of a national day of care survey.

INTRODUCTION: The relationship between nurse staffing levels and patient safety is well recognised. Inadequate provision of nursing staff is associated with increased medical error, as well as higher morbidity and mortality. Defining what constitutes safe nurse staffing levels is complex. A range of guidance and planning tools are available to inform staffing decisions. The Society for Acute Medicine (SAM) recommend a 'nurse-to-bed'ratio of greater than 1:6. Whether this standard accurately reflects the pattern and intensity of work on the Acute Medical Unit (AMU) is unclear. METHODS: Nurse staffing levels in AMUs were explored using the Society for Acute Medicine Benchmarking Audit 2019 (SAMBA19). Data from 122 acute hospitals were analysed. Nurse-to-bed ratios were calculated and compared. Estimates of the total nursing time available within the acute care system were compared to estimates of the time required to perform nursing activities. RESULTS: The total number of AMU beds across all 122 units was 4997. The mean daytime nurse-to-bed ratio was 1:4.3 and the mean night time nurse-to-bed ratio was 1:5.2. The SAM standard of a nurse to bed ratio of greater than 1:6 was achieved in 99 units (81.9%) during daytime hours and achieved by 74 units (60.6%) at night. The estimated time required to deliver direct clinical care was 35,698 h. A deficit of 4128 h (11.5% of time required) was estimated, representing the time difference between the total number of nursing hours available with current staffing and the estimated time needed for direct clinical care across all participating units. CONCLUSION: This UK-wide study suggests a significant proportion of AMUs do not meet the recommenced SAM staffing levels, particularly at night. A difference was observed between the total number of nursing hours within the acute care system and the estimated time required to perform direct nursing activities. This suggests a workforce shortage of nurses within acute care at the system level.

Integrative characterization of the near-minimal bacterium Mesoplasma florum.

The near-minimal bacterium Mesoplasma florum is an interesting model for synthetic genomics and systems biology due to its small genome (~ 800 kb), fast growth rate, and lack of pathogenic potential. However, fundamental aspects of its biology remain largely unexplored. Here, we report a broad yet remarkably detailed characterization of M. florum by combining a wide variety of experimental approaches. We investigated several physical and physiological parameters of this bacterium, including cell size, growth kinetics, and biomass composition of the cell. We also performed the first genome-wide analysis of its transcriptome and proteome, notably revealing a conserved promoter motif, the organization of transcription units, and the transcription and protein expression levels of all protein-coding sequences. We converted gene transcription and expression levels into absolute molecular abundances using biomass quantification results, generating an unprecedented view of the M. florum cellular composition and functions. These characterization efforts provide a strong experimental foundation for the development of a genome-scale model for M. florum and will guide future genome engineering endeavors in this simple organism.

NEWS2 to assess suspected COVID-19 in the community: a service evaluation of a primary care assessment centre.

BACKGROUND: Primary care has played a central role in the community response to the coronavirus disease-19 (COVID-19) pandemic. The use of the National Early Warning Score 2 (NEWS2) has been advocated as a tool to guide escalation decisions in the community. The performance of this tool applied in this context is unclear. AIM: To evaluate the process of escalation of care to the hospital within a primary care assessment centre (PCAC) designed to assess patients with suspected COVID-19 in the community. DESIGN AND SETTING: A retrospective service evaluation of all adult patients assessed between 30 March and 22 April 2020 within a COVID-19 primary care assessment centre within Sandwell West Birmingham CCG. METHOD: A database of patient demographics, healthcare interactions and physiological observations was constructed. NEWS2 and CRB65 scores were calculated retrospectively. The proportion of patients escalated was within risk groups defined by NHSE guidelines in place during the evaluation period was determined. RESULTS: A total of 150 patients were identified. Following assessment 13.3% (n = 20) patients were deemed to require escalation. The proportion of patients escalated with a NEWS2 greater than or equal to 3 was 46.9% (95% CI 30.8-63.6%). The proportion of patients escalated to secondary care using NHSE defined risk thresholds was 0% in the green group, 22% (n = 4) in the amber group, and 81.3% (n = 13) in the red group. CONCLUSION: Clinical decisions to escalate care to the hospital did not follow initial guidance written for the COVID-19 outbreak but were demonstrated to be safe.

In most cases, coronavirus disease-19 (COVID-19) is a mild illness that resolves on its own. Some patients develop severe disease requiring hospital treatment. Identifying which patients are likely to need hospital treatment is a challenge. Many GP practices have developed specific services designed to assess patients with suspected COVID-19 and establish whether hospital treatment is necessary. We evaluated a service providing this function in Birmingham. We examined the care pathway of 150 patients assessed within the service to established factors associated with the need for hospital assessment. We found a national decision tool designed to aid the process was a poor descriptor of what happened in practice.

The provision of hospital at home care: Results of a national survey of UK hospitals.

BACKGROUND: Hospital at home (HaH) replicates elements of hospital-based care in the community, to facilitate the safe management of a broad spectrum of acute illness in the patient's usual environment. The extent to which this model of care has been adopted in the United Kingdom is unknown. METHODS: The Society for Acute Medicine Benchmarking Audit is a day of care survey undertaken annually within the United Kingdom. Participation is open to all hospital in the United Kingdom receiving acutely unwell medical patients. A questionnaire is used to collect hospital-level data on the structure and organisation of acute care delivery. The survey included questions designed to quantify the number of hospitals that offered HaH. When present, further questions were asked to clarify the characteristics of the HaH service in terms of workforce, range of diagnostic test and interventions. This information was used to build a picture of HaH service provision on a national scale. RESULTS: A total of 130 hospitals contributed organisational data to SAMBA19. The capability to refer to a hospital at home service was recognised by 46.9% (n = 61) of units. The majority of these services, 83.3% (n = 50) were nurse-led. The capability to provide a physician review at home was reported in 23.3% (n = 14). The majority of services could provide intravenous antibiotics at home, but access to other simple interventions, such as intravenous diuretics or acute supplemental oxygen, is limited. CONCLUSION: At present, few acute hospitals for consitency in the United Kingdom have access to a hospital at home service capable of replicating essential elements of inpatient care. Our study suggests organisational change in acute care delivery and significant investment would be required to establish equal access to hospital-at-home care within the United Kingdom.

Evaluation and optimization of a clinical pharmacist driven transitions of care model for malignant hematology.

PURPOSE: To implement and optimize a pilot transitions of care model for scheduled chemotherapy admissions in patients with hematologic malignancies at our institution.Methodology: We utilized the plan-do-study-act (PDSA) quality improvement technique to prospectively measure success of interventions related to improving transitions of care processes that occurred in multiple stages including development of standardized operating procedures, electronic medical record documentation, and education to the malignant hematology multidisciplinary group. Chart review was performed retrospectively for at least nine patients per PDSA cycle. Areas of intervention addressed and measured regarding communication between the ambulatory care and acute care settings included: admission purpose, processes related to insurance benefits investigations for specialty medications required in the post-discharge setting, and plan for growth factors, prophylactic antimicrobials, and follow-up.Results and conclusions: We included 28 patients and performed a total of three PDSA cycles demonstrating specific improvements in: communication regarding status of benefits investigations performed for specialty medications prior to admission, resolution of these benefits investigations at various time points, improvement in efficient use of the electronic medical record for chemotherapy orders, and patient instructions for appropriate use of prophylactic antimicrobials. Although improvement was noted initially with prescribing of discharge antiemetics and antimicrobials, regression to baseline was noted with the third PDSA cycle.

A single-molecule sequencing assay for the comprehensive profiling of T4 DNA ligase fidelity and bias during DNA end-joining.

DNA ligases are key enzymes in molecular and synthetic biology that catalyze the joining of breaks in duplex DNA and the end-joining of DNA fragments. Ligation fidelity (discrimination against the ligation of substrates containing mismatched base pairs) and bias (preferential ligation of particular sequences over others) have been well-studied in the context of nick ligation. However, almost no data exist for fidelity and bias in end-joining ligation contexts. In this study, we applied Pacific Biosciences Single-Molecule Real-Time sequencing technology to directly sequence the products of a highly multiplexed ligation reaction. This method has been used to profile the ligation of all three-base 5'-overhangs by T4 DNA ligase under typical ligation conditions in a single experiment. We report the relative frequency of all ligation products with or without mismatches, the position-dependent frequency of each mismatch, and the surprising observation that 5'-TNA overhangs ligate extremely inefficiently compared to all other Watson-Crick pairings. The method can easily be extended to profile other ligases, end-types (e.g. blunt ends and overhangs of different lengths), and the effect of adjacent sequence on the ligation results. Further, the method has the potential to provide new insights into the thermodynamics of annealing and the kinetics of end-joining reactions.

Emergency Ambulatory Management of Low-Risk Febrile Neutropenia: Multinational Association for Supportive Care in Cancer Fits-Real-World Experience From a UK Cancer Center.

BACKGROUND: Emergency patient presentations with febrile neutropenia are a heterogeneous group. A small minority of these patients proceed to develop significant medical complications. Risk stratification using scores, such as the Multinational Association for Supportive Care in Cancer score, have been advocated to identify patients who are at low risk of adverse outcome suitable for treatment on an ambulatory care pathway. OBJECTIVES: We sought to report the experience of 100 patients presenting acutely with neutropenic fever managed in an emergency ambulatory fashion. METHODS: Patients presenting as an emergency with low-risk febrile neutropenia managed in an ambulatory setting between January 2017 and February 2019 at a tertiary cancer hospital in England were prospectively studied. Patients with a fever >38.0°C and an absolute neutrophil count <1.0 × 109/L were included. All patients with a Multinational Association for Supportive Care in Cancer score ≥21 and a National Early Warning Score ≤3 were potentially eligible for the pathway. Complications were classified as serious if the patient developed persistent hypotension, respiratory failure, intensive care unit admission, altered mental status, disseminated intravascular coagulation, renal failure requiring renal replacement therapy, electrocardiogram changes requiring antidysrhythmic treatment, and 30-day mortality. RESULTS: One hundred patients with low-risk febrile neutropenia consecutively managed in an emergency ambulatory fashion were prospectively analyzed. Eighty-one patients were female and the median age was 51 y (range 17-79 y). No patients developed serious complications. Eight (8% [95% confidence interval 4.1-15.0%]) patients had a 7-day readmission. CONCLUSION: Outpatient ambulatory care for emergency patients with low-risk febrile neutropenia can be delivered in a safe and effective fashion. Collaboration between acute care physicians and oncologists is required to develop local models based on national guidelines to facilitate individualised care for emergency oncology patients.

Higher Incidence of Hemorrhagic Cystitis Following Haploidentical Related Donor Transplantation Compared with Matched Related Donor Transplantation.

Hemorrhagic cystitis (HC) is a common and important complication of allogeneic hematopoietic cell transplantation (HCT). Reactivation of BK virus is its most common cause. The more intense immunosuppressive regimens administered to recipients of grafts from alternative donors have been reported to account for the increased susceptibility to HC in this population. This study compares patients undergoing HCT with either a haploidentical donor or a matched related donor, all of whom received identical immunosuppression with a post-transplantation cyclophosphamide-based regimen. The incidence of HC was significantly higher in the patients receiving a haploidentical graft (P = .01). The higher incidence of HC in haploidentical graft recipients is therefore directly related to the inherent immune deficiency that follows HLA-mismatched transplantation, independent of the intensity of pharmacologic immunosuppression. This finding carries significant clinical impact for the prevention and treatment of HC in haploidentical graft recipients.

Development of oriC-Based Plasmids for Mesoplasma florum.

The near-minimal bacterium Mesoplasma florum constitutes an attractive model for systems biology and for the development of a simplified cell chassis in synthetic biology. However, the lack of genetic engineering tools for this microorganism has limited our capacity to understand its basic biology and modify its genome. To address this issue, we have evaluated the susceptibility of M. florum to common antibiotics and developed the first generation of artificial plasmids able to replicate in this bacterium. Selected regions of the predicted M. florum chromosomal origin of replication (oriC) were used to create different plasmid versions that were tested for their transformation frequency and stability. Using polyethylene glycol-mediated transformation, we observed that plasmids harboring both rpmH-dnaA and dnaA-dnaN intergenic regions, interspaced or not with a copy of the dnaA gene, resulted in a frequency of ∼4.1 × 10-6 transformants per viable cell and were stably maintained throughout multiple generations. In contrast, plasmids containing only one M. florumoriC intergenic region or the heterologous oriC region of Mycoplasma capricolum, Mycoplasma mycoides, or Spiroplasma citri failed to produce any detectable transformants. We also developed alternative transformation procedures based on electroporation and conjugation from Escherichia coli, reaching frequencies up to 7.87 × 10-6 and 8.44 × 10-7 transformants per viable cell, respectively. Finally, we demonstrated the functionality of antibiotic resistance genes active against tetracycline, puromycin, and spectinomycin/streptomycin in M. florum Taken together, these valuable genetic tools will facilitate efforts toward building an M. florum-based near-minimal cellular chassis for synthetic biology.IMPORTANCEMesoplasma florum constitutes an attractive model for systems biology and for the development of a simplified cell chassis in synthetic biology. M. florum is closely related to the mycoides cluster of mycoplasmas, which has become a model for whole-genome cloning, genome transplantation, and genome minimization. However, M. florum shows higher growth rates than other Mollicutes, has no known pathogenic potential, and possesses a significantly smaller genome that positions this species among some of the simplest free-living organisms. So far, the lack of genetic engineering tools has limited our capacity to understand the basic biology of M. florum in order to modify its genome. To address this issue, we have evaluated the susceptibility of M. florum to common antibiotics and developed the first artificial plasmids and transformation methods for this bacterium. This represents a strong basis for ongoing genome engineering efforts using this near-minimal microorganism.