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Betalains are tyrosine-derived red-violet and yellow plant pigments known for their antioxidant activity, health-promoting properties, and wide use as food colorants and dietary supplements. By coexpressing three genes of the recently elucidated betalain biosynthetic pathway, we demonstrate the heterologous production of these pigments in a variety of plants, including three major food crops: tomato, potato, and eggplant, and the economically important ornamental petunia. Combinatorial expression of betalain-related genes also allowed the engineering of tobacco plants and cell cultures to produce a palette of unique colors. Furthermore, betalain-producing tobacco plants exhibited significantly increased resistance toward gray mold (Botrytis cinerea), a pathogen responsible for major losses in agricultural produce. Heterologous production of betalains is thus anticipated to enable biofortification of essential foods, development of new ornamental varieties, and innovative sources for commercial betalain production, as well as utilization of these pigments in crop protection.

Betalains are tyrosine‐derived red‐violet and yellow pigments, found in plants only of the Caryophyllales order. Although much progress has been made in recent years in the understanding of the betalain biosynthetic process, many questions remain open with regards to several of the proposed steps in the pathway. Most conspicuous by its absence is the characterization of the first committed step in the pathway, namely the 3‐hydroxylation of tyrosine to form l‐3,4‐dihydroxyphenylalanine (l‐DOPA). We used transcriptome analysis of the betalain‐producing plants red beet (Beta vulgaris) and four o'clocks (Mirabilis jalapa) to identify a novel, betalain‐related cytochrome P450‐type gene, CYP76AD6, and carried out gene silencing and recombinant expression assays in Nicotiana benthamiana and yeast cells to examine its functionality. l‐DOPA formation in red beet was found to be redundantly catalyzed by CYP76AD6 together with a known betalain‐related enzyme, CYP76AD1, which was previously thought to only catalyze a succeeding step in the pathway. While CYP76AD1 catalyzes both l‐DOPA formation and its subsequent conversion to cyclo‐DOPA, CYP76AD6 uniquely exhibits only tyrosine hydroxylase activity. The new findings enabled us to metabolically engineer entirely red‐pigmented tobacco plants through heterologous expression of three genes taking part in the fully decoded betalain biosynthetic pathway.

Antibody engineering technology is at the forefront of therapeutic antibody development. The primary goal for engineering a therapeutic antibody is the generation of an antibody with a desired specificity, affinity, function, and developability profile. Mature antibodies are considered antigen specific, which may preclude their use as a starting point for antibody engineering. Here, we explore the plasticity of mature antibodies by engineering novel specificity and function to a pre-selected antibody template. Using a small, focused library, we engineered AAL160, an anti-IL-1β antibody, to bind the unrelated antigen IL-17A, with the introduction of seven mutations. The final redesigned antibody, 11.003, retains favorable biophysical properties, binds IL-17A with sub-nanomolar affinity, inhibits IL-17A binding to its cognate receptor and is functional in a cell-based assay. The epitope of the engineered antibody can be computationally predicted based on the sequence of the template antibody, as is confirmed by the crystal structure of the 11.003/IL-17A complex. The structures of the 11.003/IL-17A and the AAL160/IL-1β complexes highlight the contribution of germline residues to the paratopes of both the template and re-designed antibody. This case study suggests that the inherent plasticity of antibodies allows for re-engineering of mature antibodies to new targets, while maintaining desirable developability profiles. A proof of principle approach redirects an anti-IL-1b antibody to bind the otherwise unrelated antigen, IL-17A, highlighting the plasticity of antibody scaffolds that could be manipulated for alternative binding or function.

BackgroundIL-2 binds two forms of IL-2 receptor: a high affinity trimeric receptor composed of CD25, CD122, and CD132, and a low affinity dimeric receptor composed of CD122 and CD132. Binding to the dimeric receptors, expressed on effector cells, causes expansion of the effector arm of the immune system including CD8 T-cells, NK-cells and NKT-cells. Binding to the trimeric receptor, expressed on Tregs as well as on pulmonary and vascular epithelium, results in expansion of Treg cells and Vascular Leak Syndrome, both are undesired outcomes of high-dose recombinant IL-2 (Aldesleukin), approved for treatment of Melanoma and Renal-Cell-Carcinoma.MethodsFlow-cytometry analysis of immune-cell populations of C57BL/6 mice and hPBMCs. Tumor-Growth-Index of murine cancer models.ResultsAU-007, is a computationally designed human antibody that bind the CD25-binding portion on IL-2, preventing binding of IL-2 to the trimeric receptor, but not the dimeric receptor. This leads to immune effector activation while also preventing the Treg expansion via the autoinhibitory loop caused by endogenous IL-2 secreted from activated T effector cells (figure 1). AU-007 binds human IL-2 with picomolar affinity and has excellent biophysical properties with low potential for anti-drug immunogenicity (figure 2). Administration of an AU-007/low dose hIL-2 complex to non-tumor bearing C57BL/6 mice promoted proliferation of effector cells with no effect on Tregs (figure 3). Additionally, an AU-007/low dose hIL-2 complex was highly effective in inhibiting tumor progression in a syngeneic B16F10 melanoma model (figure 4). pSTAT5 analysis of hPBMCs incubated with AU-007 and hIL-2 demonstrated activation of the effector cells and inhibition of Tregs expansion (figure 5). hPBMCs activated with anti-CD3/anti-CD28 and treated with either AU-007 or an isotype control antibody but without exogenous IL-2, showed expansion of effector cells. However, while the isotype control antibody expanded also Tregs , AU-007 inhibited Tregs proliferation, indicating that AU-007 captures endogenous IL-2 and prevents the Treg expansion autoinhibitory loop caused by endogenous IL-2 secreted from activated T effector cells (figure 6).Additionally, following establishment of the IL-2 auto-secretion feedback loop in mice genetically engineered to express hIL-2 instead of murine IL-2, AU-007 treatment significantly inhibited MC38 colorectal-tumor growth for twelve days, in a manner comparable to treatment with anti-PD1 (figure 7).ConclusionsAU-007 is a human antibody that blocks the CD25-binding epitope on IL-2. It redirects endogenous IL-2 to promote effector cell expansion while simultaneously blocking the Treg expansion autoinhibitory loop, indicating its unique therapeutic profile and high potential as a novel cancer treatment. AU-007 is expected to enter clinical testing in 2021.Abstract 704 Figure 1Schematic representation of IL-2 mechanism of action and its dual role in controlling immune response. IL-2 structure consists of three binding epitope sites that interact with different forms of IL-2-R complexes with different affinities (Left Panel). IL-2R complexes expressed on different cell populations and their different affinities to IL-2 allow immunosuppression under conditions of low local concentrations of IL-2 and immune stimulation when IL-2 local concentration rises (middle panel). Au-007 utilize autocrine human IL-2 MOA to promote immune stimulation. Targeting IL-2 to different cell populations can be used to modulate the immune response toward towards immune activation. An anti-human IL-2 antibody tumor clearance while reducing IL-2's undesired interactions with endothelial CD25 expressing cells preventing IL-2 induced pulmonary edema and vascular leaking.Abstract 704 Figure 2Au-007 bind human IL-2 with high affinity and inhibits the binding to CD25 while preserving the binding to CD122. Affinity and binding site are demonstrated using Surface Plasmon Resonance. Au-007 was captured on CM5 chip and soluble hIL-2 was injected, forming a complex. Subsequently, soluble CD25 was injected followed by injection of soluble CD122 (A). SPR trace of complex formation of Ab/IL-2/IL-2R arrows indicate where Au-007 (17.069), hIL-2, CD25 and CD122 were injected (B). SPR trace and calculated binding kinetics of chip bound Au-007 with hIL-2 serving as analyte (C). Biophysical profile of Au-007. Au-007 was subjected to five freeze thaw cycles, agitation for 3 days and incubation at 40°C for 1 week. Post treatment Au-007 integrity and indicated biophysical properties were measured (D).Abstract 704 Figure 3Au-007 demonstrated in-vivo potent immune stimulating effects in a dose depended manner, with no observed effect on Tregs. C57BL/6 healthy mice were administered daily with Au-007/hIL-2 complex for four days. On day five splenocytes were isolated and immune cells populations were analyzed using flow cytometry. (A) Dosing regimen outline. (B–E) Mean values of immune cells calculated as a percentage from parent population of each experimental group (n=6 per group)Abstract 704 Figure 4Au-007 inhibits tumor growth in an I/O resistant tumor model with a tolerable profile. C57BL/6 healthy mice were inoculated with B16F10 melanoma tumor cells (day 0), at day 5 mice were randomized to experimental groups (n=10 per group) and administered daily, with single injection per day of Ab/hIL-2 mix (20 ug/1 ug respectively) or with PBS for four days. From the end of schedule administration at day 8 until experiment endpoint, mice were monitored daily for tumor volume (A) and for mean percent of body weight change for each experimental group (B).Abstract 704 Figure 5AU-007 inhibits the effect of IL-2 on Tregs while preserving its effect on Teffs and NKs. (A and B) Phosphorylated STAT5 levels of human immune cell subsets responding to various concentrations of hIL-2 with and without 200 nM AU-007. Total naïve hPBMC culture were incubated with increasing doses of hIL-2 or with increasing doses of hIL-2 + 200nM AU-007 for 15 min. Immune cells subpopulations were analyzed by flow cytometry, gating was defined based on FMOs. (C–F) Phosphorylated STAT5 levels of human immune cell subsets responding to titrated AU-007 or isotype control. Total naïve hPBMC culture were incubated with hIL-2 and with increasing doses of indicated antibody for 15 min. Data presented is an average of 3 biological repeats from 3 human PBMC donors.Abstract 704 Figure 6Au-007 can rely on endogenous IL-2 to break auto-inhibitory loop in human PBMCs. Total hPBMCs were stimulated for 24h with anti-CD3/anti-CD28 (stimulation only, green) or stimulated with anti-CD3/anti-CD28 in the presence of: 200 nM of Au-007 mAb (red) or with 200 nM of isotype control mAb (blue). No exogenous IL-2 was added. Immune cells subpopulations were analyzed by flow cytometry. Percentage of immune cell sub-populations demonstrate exclusive inhibition of Tregs (A–E). Au-007 downregulate the suppressive markers of CD4+ regulatory Tregs from panel A, as defined by significant reduction in MFI of CD25 and FoxP3 (F and G). Gating was defined based on FMOs. Data presented is an average of 3 biological repeats from 3 human PBMC donors.Abstract 704 Figure 7Au-007 captures endogenous hIL-2 and inhibits tumor growth in colorectal cancer model (MC38). Genetically modified C57BL/6 mice, engineered to express human IL-2 in the background of complete knock-out of mouse IL-2, were inoculated with MC38 colorectal tumor cells. All animals treated with Au-007 showed significant inhibition in tumor growth with no observed significant adverse effects. (A) Administration outline: PBS (black), anti-mouse-PD-1 antibody (yellow), Au-007 pre-complexed with low dose IL-2 (blue) and Au-007 alone every three days followed with a single immune kick start with IL-2 (green, IL-2 single dose is marked in red). (B) Tumor growth progression of the four groups treated. (C) Percent of body weight changes per treatment.

Myocardial injury in hospitalized patients is associated with poor prognosis. This study aimed to evaluate risk factors for myocardial injury in hospitalized patients with coronavirus disease 2019 (COVID-19) and its prognostic value. We retrieved all consecutive patients who were hospitalized in internal medicine departments in a tertiary medical center from February 9 th , 2020 to August 28 th with a diagnosis of COVID-19. A total of 559 adult patients were hospitalized in the Sheba Medical Center with a diagnosis of COVID-19, 320 (57.24%) of whom were tested for troponin levels within 24-hours of admission, and 91 (28.44%) had elevated levels. Predictors for elevated troponin levels were age (odds ratio [OR], 1.04; 95% confidence interval [CI], 1.01–1.06), female sex (OR, 3.03; 95% CI 1.54–6.25), low systolic blood pressure (OR, 5.91; 95% CI 2.42–14.44) and increased creatinine level (OR, 2.88; 95% CI 1.44–5.73). The risk for death (hazard ratio [HR] 4.32, 95% CI 2.08–8.99) and a composite outcome of invasive ventilation support and death (HR 1.96, 95% CI 1.15–3.37) was significantly higher among patients who had elevated troponin levels. In conclusion, in hospitalized patients with COVID-19, elevated troponin levels are associated with poor prognosis. Hence, troponin levels may be used as an additional tool for risk stratification and a decision guide in patients hospitalized with COVID-19.

Glioblastoma is a highly aggressive brain tumor. Current standard-of-care results in a marginal therapeutic outcome, partly due to acquirement of resistance and insufficient blood-brain barrier (BBB) penetration of chemotherapeutics. To circumvent these limitations, we conjugated the chemotherapy paclitaxel (PTX) to a dendritic polyglycerol sulfate (dPGS) nanocarrier. dPGS is able to cross the BBB, bind to P/L-selectins and accumulate selectively in intracranial tumors. We show that dPGS has dual targeting properties, as we found that P-selectin is not only expressed on tumor endothelium but also on glioblastoma cells. We delivered dPGS-PTX in combination with a peptidomimetic of the anti-angiogenic protein thrombospondin-1 (TSP-1 PM). This combination resulted in a remarkable synergistic anticancer effect on intracranial human and murine glioblastoma via induction of Fas and Fas-L, with no side effects compared to free PTX or temozolomide. This study shows that our unique therapeutic approach offers a viable alternative for the treatment of glioblastoma.

ObjectivesPhysicians continuously make tough decisions when discharging patients. Alerting on poor outcomes may help in this decision. This study evaluates a machine learning model for predicting 30-day mortality in emergency department (ED) discharged patients.MethodsWe retrospectively analysed visits of adult patients discharged from a single ED (1/2014–12/2018). Data included demographics, evaluation and treatment in the ED, and discharge diagnosis. The data comprised of both structured and free-text fields. A gradient boosting model was trained to predict mortality within 30 days of release from the ED. The model was trained on data from the years 2014–2017 and validated on data from the year 2018. In order to reduce potential end-of-life bias, a subgroup analysis was performed for non-oncological patients.ResultsOverall, 363 635 ED visits of discharged patients were analysed. The 30-day mortality rate was 0.8%. A majority of the mortality cases (65.3%) had a known oncological disease. The model yielded an area under the curve (AUC) of 0.97 (95% CI 0.96 to 0.97) for predicting 30-day mortality. For a sensitivity of 84% (95% CI 0.81 to 0.86), this model had a false positive rate of 1:20. For patients without a known malignancy, the model yielded an AUC of 0.94 (95% CI 0.92 to 0.95).ConclusionsAlthough not frequent, patients may die following ED discharge. Machine learning-based tools may help ED physicians identify patients at risk. An optimised decision for hospitalisation or palliative management may improve patient care and system resource allocation.

Aims: Elevated uric acid (UA) levels correlate with worse heart failure (HF) outcomes, but past studies used single UA measurements. The effect of intra-individual UA fluctuations, beyond mean levels, is unclear. This study assesses the relationship between serum UA variability and adverse clinical outcomes in HF patients. Methods: We analyzed 18,115 HF patients from the SHEBAHEART registry (2009–2025) with at least three UA measurements within three years of diagnosis. UA variability was quantified as the mean deviation (MD) from each patient’s average UA level and divided into quartiles: Q1 (≤−0.69 mg/dL), Q2–Q3 (>−0.69 and <1.53 mg/dL, reference), and Q4 (≥1.53 mg/dL). All-cause mortality was the primary outcome and HF hospitalization was secondary. Cox regression, propensity score matching, and subgroup analyses were used. Results: Over a median follow-up of 4.3 years (IQR 1.6–7.7), 36% of patients were hospitalized for HF and 65.5% died. UA variability showed a graded association with outcomes. Low variability (Q1) was linked to reduced mortality (HR 0.79, 95% CI 0.75–0.83) and HF hospitalization (HR 0.84, 95% CI 0.79–0.90), while high variability (Q4) increased mortality (HR 1.58, 95% CI 1.51–1.69) and hospitalization risk (HR 1.17, 95% CI 1.10–1.25) (all p < 0.001). These associations remained after propensity score matching and across HF subgroups. Conclusions: UA variability is a robust, independent predictor of mortality and HF hospitalization. Serial UA monitoring may enhance risk stratification in HF management.

Importance Acute kidney injury is associated with poor outcomes, but the clinical implication of reversible serum creatinine level fluctuations during hospitalization not necessarily defined as acute kidney injury is poorly understood. Objective To investigate the long-term outcomes of patients without previously diagnosed kidney disease who present with decreased kidney function and are subsequently discharged with apparently normal kidney function. Design, Setting, and Participants A retrospective cohort study was conducted of patients hospitalized in a large tertiary hospital in Israel between September 1, 2007, and July 31, 2022. The study included patients admitted to an internal medicine ward. Patients had not undergone dialysis during the index hospitalization, had at least 3 creatinine tests performed during hospitalization, and had a discharge estimated glomerular filtration rate (eGFR) exceeding 60 mL/min/1.73 m2. Patients with preexisting chronic kidney disease were excluded. Exposure Glomerular filtration rate was estimated from serum creatinine values using the updated 2022 Chronic Kidney Disease Epidemiology Collaboration formula, and eGFR greater than 60 mL/min/1.73 m2was regarded as normal. Exposure was defined based on the association between the first and last values determined during hospitalization. Main Outcomes and Measures All-cause mortality in the year following the index hospitalization and end-stage kidney disease (ESKD) in the 10 years following the index hospitalization. Results A total of 40 558 patients were included. Median age was 69 (IQR, 56-80) years, with 18 004 women (44%) and 22 554 men (56%). A total of 34 332 patients (85%) were admitted with a normal eGFR and 6226 (15%) with decreased eGFR. Patients with decreased eGFR on presentation had an 18% increased mortality in the year following hospitalization (adjusted hazard ratio [AHR], 1.18; 95% CI, 1.11-1.24) and a 267% increased risk of ESKD in the 10 years following hospitalization (AHR, 3.67; 95% CI, 2.43-5.54), despite having been discharged with apparently normal kidney function. The highest risk was noted in patients who presented to the hospital with an eGFR of 0 to 45 mL/min/1.73 m2. Conclusions and Relevance The findings of this cohort study suggest that patients who present with decreased kidney function and are discharged without clinically evident residual kidney disease may be at increased long-term risk for ESKD and mortality.

Background Diabetes mellitus (DM) and aortic stenosis (AS) are frequent findings in the elderly population. Data regarding the influence of DM on the outcomes of patients undergoing transcatheter aortic valve replacement (TAVR) due to AS are limited. The aim of this study was to examine the impact of DM on TAVR outcomes. Methods We investigated 443 patients with severe AS undergoing TAVR. Subjects were divided into insulin-dependent diabetic mellitus (IDDM) patients (N = 44), non-dependent insulin diabetic mellitus (NIDDM) patients (N = 114) and non-diabetics (N = 285) of whom 31 (74 %), 86 (79 %) and 209 (76 %) respectively had trans-femoral TAVR. Peri-procedural complications and outcomes were recorded according to the Valve Academic Research Consortium-2 criteria. Results Patients with IDDM as well as NIDDM demonstrated similar complication rates compared with non-diabetic patients, except for acute kidney injury (AKI) grade 3 [4 (2 %) and 3 (3 %) vs. 1 (0.4 %) respectively, p = 0.032]. Kaplan–Meier survival analysis showed that DM, regardless of the type of treatment, was not associated with increased 2 years mortality (Log-rank p value 0.44). Multivariate cox regression analysis adjusted for age, gender, coronary artery disease, DM, AKI3, hypertension, chronic renal failure and peripheral vascular disease found that AKI3 was associated with increased risk of 2 years mortality [HR = 7.35, 95 % CI 2.16–25.07, p = 0.001] whereas female gender was found as a protective factor [HR = 0.47, 95 % CI 0.28–0.8, p = 0.005], and DM was not associated with increased risk. Conclusions Following TAVR, DM patients seem to have similar peri-procedural and mid-term outcomes compared with patients without DM, while IDDM patients seem to suffer greater incidence of AKI. Further research in larger cohorts of patients is needed to validate our results.