Comprehensive evaluation of T7 promoter for enhanced yield and quality in mRNA production.

The manufacturing of mRNA vaccines relies on cell-free based systems that are easily scalable and flexible compared with the traditional vaccine manufacturing processes. Typically, standard processes yield 2 to 5 g L-1 of mRNA, with recent process optimisations increasing yields to 12 g L-1. However, increasing yields can lead to an increase in the production of unwanted by-products, namely dsRNA. It is therefore imperative to reduce dsRNA to residual levels in order to avoid intensive purification steps, enabling cost-effective manufacturing processes. In this work, we exploit sequence modifications downstream of the T7 RNA polymerase promoter to increase mRNA yields whilst simultaneously minimising dsRNA. In particular, transcription performance was optimised by modifying the sequence downstream of the T7 promoter with additional AT-rich sequences. We have identified variants that were able to produce higher amounts of mRNA (up to 14 g L-1) in 45 min of reaction. These variants exhibited up to a 30% reduction in dsRNA byproduct levels compared to a wildtype T7 promoter, and have similar EGFP protein expression. The results show that optimising the non-coding regions can have an impact on mRNA production yields and quality, reducing overall manufacturing costs.

Monte Carlo simulation-guided design for size-tuned tumor spheroid formation in 3D printed microwells.

Tumor spheroid models have garnered significant attention in recent years as they can efficiently mimic in vivo models, and in addition, they offer a more controlled and reproducible environment for evaluating the efficacy of cancer drugs. In this study, we present the design and fabrication of a micromold template to form multicellular spheroids in a high-throughput and controlled-sized fashion. Briefly, polydimethylsiloxane-based micromolds at varying sizes and geometry were fabricated via soft lithography using 3D-printed molds as negative templates. The efficiency of spheroid formation was assessed using GFP-expressing human embryonic kidney 293 cells (HEK-293). After 7 days of culturing, circularity and cell viability of spheroids were >0.8 and 90%, respectively. At 1500 cells/microwell of cell seeding concentration, the spheroids were 454 ± 15 µm, 459 ± 7 µm, and 451 ± 18 µm when cultured in microwells with the diameters of 0.4, 0.6, and 0.8 µm, respectively. Moreover, the distance between each microwell and surfactant treatment before cell seeding notably impacted the uniform spheroid formation. The centrifugation was the key step to collect cells on the bottom of the microwells. Our findings were further verified using a commercial microplate. Furthermore, Monte Carlo simulation confirmed the seeding conditions where the spheroids could be formed. This study showed prominent steps in investigating spheroid formation, thereby leveraging the current know-how on the mechanism of tumor growth.

ß-Sitosterol Bioconversion in Small-Scale Devices: From Microtiter Plates to Microfluidic Reactors.

Small-scale devices are routinely used as low-cost miniaturized bioreactors due to the large number of experiments that can be conducted simultaneously under similar conditions and replicate all functions of bench-scale reactors at dramatically smaller volumes. Microtiter plates, due to the standard footprint, can be integrated with liquid handling systems and associated equipment, expanding considerably their application and use. However, care has to be taken to operate the microtiter plates in optimized mixing and oxygen transfer conditions, preventing medium evaporation in prolonged experiment runs. Recently, to increase data quality, microbioreactors have emerged as an alternative to shaken systems. These systems offer higher degree of control over key process variables and when combined with sensing technology increase dramatically the reliability of translational process data. In this chapter, we describe the production of 4-androstene-3,17-dione (androstenedione (AD)), a key pharmaceutical steroid intermediate, by Mycobacterium sp. NRRL B-3805 via the selective cleavage of the side-chain of ß-sitosterol using 24-well microtiter plates and microfluidic microbioreactors.

3D Resin-coated pressure sensor response for bite force assessment: A pilot study.

Background: Occlusal splints with sensors help in the bruxism diagnosis and monitoring, by recording the patient's bite force. The aim of this study was to evaluate the accuracy of a pressure sensor when it is covered with different thicknesses of a 3D printing resin (Anycubic 405nm Translucent Green UV Resin, Anycubic, UK). Methods: In this preliminary study, the evaluated sensor (FlexiForce A201 Sensor, Tekscan) was firstly calibrated without any type of cover material, and later tested with 3D printing resin with different thicknesses (1 mm, 1.15 mm, 1.4 mm and 1.6 mm). The load tests were performed by a force tester (MultiTest 2.5 dV, Mecmesin). Results: When the pressure sensor was covered with resin of 1mm and 1.6 mm thick specimens, a higher difference was found between the applied load and the corresponding sensor reading. Conclusion: It was concluded that it is possible to use this type of pressure sensor and that it showed better accuracy with the 1.15 mm and 1.4 mm 3D printing resin covering.

Total knee arthroplasty coronal alignment and tibial base stress-a new numerical evaluation.

Background: Total knee arthroplasty (TKA) is one of the most frequently performed orthopedic procedures. The correct positioning and alignment of the components significantly affects prosthesis survival. Considering the current controversy regarding the target of postoperative alignment of TKA, this study evaluated the tension at tibial component interface using two numerical methods. Methods: The stress of the prosthesis/bone interface of the proximal tibial component was evaluated using two numerical methods: the finite element method (FEM) and the new meshless method: natural neighbor radial point interpolation method (NNRPIM). The construction of the model was based on Zimmers NexGen LPS-Flex Mobile® prosthesis and simulated the forces by using a free-body diagram. Results: Tibiofemoral mechanical axis (TFMA) for which a higher number of nodes are under optimal mechanical tension is between 1° valgus 2° varus. For values outside the interval, there are regions under the tibial plate at risk of bone absorption. At the extremities of the tibial plate of the prosthesis, both medial and lateral, independent of the alignment, are under a low stress. In all nodes evaluated for all TFMA, the values of the effective stresses were higher in the NNRPIM when compared with the FEM. Conclusion: Through this study, we can corroborate that the optimal postoperative alignment is within the values that are currently considered of 0 ± 3° varus. It was verified that the meshless methods obtain smoother and more conservative results, which may make them safer when transposed to the clinical practice.

Better Anti-Spike IgG Antibody Response to SARS-CoV-2 Vaccine in Patients on Haemodiafiltration than on Haemodialysis.

INTRODUCTION: The antibody response to SARS-CoV-2 vaccine in haemodialysis (HD) patients is diminished compared to healthy subjects. The aim of this study was to compare the presence of reactive SARS-CoV-2 antibodies in patients with high-flux HD and on-line haemodiafiltration (HDF) three and 6 months after the second dose of SARS-CoV-2 vaccine since previous studies indicate that a sustained antibody response correlates with protection from disease. METHODS: We included 216 HD patients of which 157 had on-line HDF and 59 high-flux HD and 46 health care workers as controls and studied the presence of reactive anti-spike IgG antibodies three and 6 months after the second dose of SARS-CoV-2 vaccine. Clinical features between the patient groups were similar, but patients with on-line HDF had significantly higher Kt/V. RESULTS: The percentage of participants with reactive antibodies was significantly lower in patients compared to controls, both three and 6 months after the second dose of vaccine. Furthermore, the proportion of patients with reactive anti-spike IgG ≥1.0 6 months after the second dose of vaccine was significantly higher in patients with on-line HDF compared to in patients with high-flux HD. In logistic regression analyses adjusted for several clinical features, the variables associated with presence of reactive anti-spike IgG at 3 months after the second dose of vaccine were lower age, HDF treatment, not being obese and not having a previous solid organ transplant. The two variables with the strongest influence on the presence of reactive anti-spike IgG levels 6 months after the second dose of vaccine were treatment with on-line HDF and not having immunosuppressive therapy. CONCLUSION: This is the first study to show that on-line HDF preserves the antibody response better than high-flux HD after vaccination with SARS-CoV-2 vaccine. Treatment strategies that sustain the vaccine response are essential to apply in this vulnerable group of patients.

Etelcalcetide controls secondary hyperparathyroidism and raises sclerostin levels in hemodialysis patients previously uncontrolled with cinacalcet.

INTRODUCTION: There is scarce clinical experience with etelcalcetide in patients with secondary hyperparathyroidism uncontrolled with cinacalcet. The effect of etelcalcetide on serum sclerostin levels remains to be clarified. MATERIALS AND METHODS: Prospective cohort study in prevalent hemodialysis patients with uncontrolled sHPT under cinacalcet for at least 3 months, mean parathyroid hormone (PTH)>800pg/mL and calcium (Ca)>8.3mg/dL. Etelcalcetide 5mg IV/HD was initiated after cinacalcet washout. Levels of PTH, Ca, and phosphorus (Pi) followed monthly for 6 months. Plasma sclerostin levels measured before etelcalcetide treatment and after 6 months. RESULTS: Thirty-four patients were enrolled, 19 (55.9%) male gender. Mean age 60.7 (± 12.3) years; median time on HD 82.5 (7-296) months and median cinacalcet dose was 180mg/week (Interquartile Range: 180-270). Serum Ca, Pi and PTH levels showed a significant reduction after etelcalcetide treatment from 8.8mg/dL, 5.4mg/dL and 1005pg/mL to 8.1mg/dL (p=0.08), 4.9mg/dL (p=0.01) and 702pg/mL (p<0.001), respectively. Median etelcalcetide dose remained at 5mg/HD. Plasma sclerostin concentration increased from 35.66pmol/L (IQR11.94-54.58) to 71.05pmol/L (IQR54.43-84.91) (p<0.0001). CONCLUSION: Etelcalcetide improved sHPT control in this group of patients, previously under cinacalcet treatment, and significantly increased plasma sclerostin concentration. The impact of etelcalcetide treatment on sclerostin levels is a novel finding.

Maximizing mRNA vaccine production with Bayesian optimization.

Messenger RNA (mRNA) vaccines are a new alternative to conventional vaccines with a prominent role in infectious disease control. These vaccines are produced in in vitro transcription (IVT) reactions, catalyzed by RNA polymerase in cascade reactions. To ensure an efficient and cost-effective manufacturing process, essential for a large-scale production and effective vaccine supply chain, the IVT reaction needs to be optimized. IVT is a complex reaction that contains a large number of variables that can affect its outcome. Traditional optimization methods rely on classic Design of Experiments methods, which are time-consuming and can present human bias or based on simplified assumptions. In this contribution, we propose the use of Machine Learning approaches to perform a data-driven optimization of an mRNA IVT reaction. A Bayesian optimization method and model interpretability techniques were used to automate experiment design, providing a feedback loop. IVT reaction conditions were found under 60 optimization runs that produced 12 g · L-1 in solely 2 h. The results obtained outperform published industry standards and data reported in literature in terms of both achievable reaction yield and reduction of production time. Furthermore, this shows the potential of Bayesian optimization as a cost-effective optimization tool within (bio)chemical applications.

Biocatalysis in Continuous-Flow Microfluidic Reactors.

The implementation of continuous-flow transformations in biocatalysis has received remarkable attention in the last few years. Flow microfluidic reactors represent a crucial technological tool that has catalyzed this trend by promising tremendous improvement in biocatalytic processes across a host of different levels, including bioprocess development, intensification of reactions, implementation of new methods of reaction screening, and enhanced reaction scale-up. However, the full realization of this promise requires a synergy between these biocatalytic reaction features and the design and operation of microfluidic reactors. Here an overview on the different applications of flow biocatalysis is provided according to the format of the enzyme used: free vs immobilized form. Until now, flow biocatalysis has been implemented on a case-by-case approach but challenges and limitations are discussed in order to be overcome, and making continuous-flow microfluidic reactors as universal tool a reality.

mRNA vaccines manufacturing: Challenges and bottlenecks.

Vaccines are one of the most important tools in public health and play an important role in infectious diseases control. Owing to its precision, safe profile and flexible manufacturing, mRNA vaccines are reaching the stoplight as a new alternative to conventional vaccines. In fact, mRNA vaccines were the technology of choice for many companies to combat the Covid-19 pandemic, and it was the first technology to be approved in both United States and in Europe Union as a prophylactic treatment. Additionally, mRNA vaccines are being studied in the clinic to treat a number of diseases including cancer, HIV, influenza and even genetic disorders. The increased demand for mRNA vaccines requires a technology platform and cost-effective manufacturing process with a well-defined product characterisation. Large scale production of mRNA vaccines consists in a 1 or 2-step in vitro reaction followed by a purification platform with multiple steps that can include Dnase digestion, precipitation, chromatography or tangential flow filtration. In this review we describe the current state-of-art of mRNA vaccines, focusing on the challenges and bottlenecks of manufacturing that need to be addressed to turn this new vaccination technology into an effective, fast and cost-effective response to emerging health crises.

Longitudinal Analysis of Antibody Responses to the mRNA BNT162b2 Vaccine in Patients Undergoing Maintenance Hemodialysis: A 6-Month Follow-Up.

Background: Patients on hemodialysis (HD) are at higher risk for COVID-19, overall are poor responders to vaccines, and were prioritized in the Portuguese vaccination campaign. Objective: This work aimed at evaluating in HD patients the immunogenicity of BTN162b2 after the two doses induction phase, the persistence of specific antibodies along time, and factors predicting these outcomes. Methods: We performed a prospective, 6-month long longitudinal cohort analysis of 156 HD patients scheduled to receive BTN162b2. ELISA quantified anti-spike IgG, IgM, and IgA levels in sera were collected every 3 weeks during the induction phase (t0 before vaccine; t1, d21 post first dose; and t2 d21 post second dose), and every 3-4 months during the waning phase (t3, d140, and t4, d180 post first dose). The age-matched control cohort was similarly analyzed from t0 to t2. Results: Upon exclusion of participants identified as previously exposed to SARS-CoV-2, seroconversion at t1 was lower in patients than controls (29 and 50%, respectively, p = 0.0014), while the second vaccine dose served as a boost in both cohorts (91 and 95% positivity, respectively, at t2, p = 0.2463). Lower response in patients than controls at t1 was a singularity of the participants ≤ 70 years (p = 2.01 × 10-05), associated with immunosuppressive therapies (p = 0.013), but not with lack of responsiveness to hepatitis B. Anti-spike IgG, IgM, and IgA levels decreased at t3, with IgG levels further waning at t4 and resulting in >30% seronegativity. Anti-spike IgG levels at t1 and t4 were correlated (ρ = 0.65, p < 2.2 × 10-16). Conclusions: While most HD patients seroconvert upon 2 doses of BNT162b2 vaccination, anti-spike antibodies levels wane over the following 4 months, leading to early seroreversion in a sizeable fraction of the patients. These findings warrant close monitoring of COVID-19 infection in vaccinated HD patients, and advocate for further studies following reinforced vaccination schedules.

On the mechanical response of the actomyosin cortex during cell indentations.

A mechanical model is presented to analyze the mechanics and dynamics of the cell cortex during indentation. We investigate the impact of active contraction on the cross-linked actin network for different probe sizes and indentation rates. The essential molecular mechanisms of filament stretching, cross-linking and motor activity, are represented by an active and viscous mechanical continuum. The filaments behave as worm-like chains linked either by passive rigid linkers or by myosin motors. In the first example, the effects of probe size and loading rate are evaluated using the model for an idealized rounded cell shape in which properties are based on the results of parallel-plate rheometry available in the literature. Extreme cases of probe size and indentation rate are taken into account. Afterward, AFM experiments were done by engaging smooth muscle cells with both sharp and spherical probes. By inverse analysis with finite element software, our simulations mimicking the experimental conditions show the model is capable of fitting the AFM data. The results provide spatiotemporal dependence on the size and rate of the mechanical stimuli. The model captures the general features of the cell response. It characterizes the actomyosin cortex as an active solid at short timescales and as a fluid at longer timescales by showing (1) higher levels of contraction in the zones of high curvature; (2) larger indentation forces as the probe size increases; and (3) increase in the apparent modulus with the indentation depth but no dependence on the rate of the mechanical stimuli. The methodology presented in this work can be used to address and predict microstructural dependence on the force generation of living cells, which can contribute to understanding the broad spectrum of results in cell experiments.

A 3D trabecular bone homogenization technique.

PURPOSE: Bone is a hierarchical material that can be characterized from the microscale to macroscale. Multiscale models make it possible to study bone remodeling, inducing bone adaptation by using information of bone multiple scales. This work proposes a computationally efficient homogenization methodology useful for multiscale analysis. This technique is capable to define the homogenized microscale mechanical properties of the trabecular bone highly heterogeneous medium. METHODS: In this work, a morphology- based fabric tensor and a set of anisotropic phenomenological laws for bone tissue was used, in order to define the bone micro-scale mechanical properties. To validate the developed methodology, several examples were performed in order to analyze its numerical behavior. Thus, trabecular bone and fabricated benchmarks patches (representing special cases of trabecular bone morphologies) were analyzed under compression. RESULTS: The results show that the developed technique is robust and capable to provide a consistent material homogenization, indicating that the homogeneous models were capable to accurately reproduce the micro-scale patch mechanical behavior. CONCLUSIONS: The developed method has shown to be robust, computationally less demanding and enabling the authors to obtain close results when comparing the heterogeneous models with equivalent homogenized models. Therefore, it is capable to accurately predict the micro-scale patch mechanical behavior in a fraction of the time required by classic homogenization techniques.

Large-scale opacification of a hydrophilic/hydrophobic intraocular lens.

PURPOSE: To determine the prevalence and risk factors related to the opacification of the LS-502-1 intraocular lens. METHODS: Cross-sectional study including patients submitted to cataract surgery between January 2010 and March 2012, with implantation of the LS-502-1 intraocular lens. Past medical history was registered and a complete ophthalmologic evaluation, that included best-corrected visual acuity, slit-lamp examination and fundoscopy, was performed. Anterior segment photographs were taken whenever intraocular lens opacification was present. RESULTS: One hundred and sixty-nine eyes of 154 patients were included, mean age 78.5 ± 7.9 years. The average follow-up after intraocular lens implantation was 65.6 ± 10.0 months. Intraocular lens opacification was seen in 53.3% (n = 90) and presented as one of four different patterns: peripheral (15.6%, n = 14), central (4.4%, n = 4), diffuse (71.1%, n = 64) and superficial white deposits (8.9%, n = 8). There was no statistically significant association with systemic or ophthalmic conditions. In patients with bilateral implantation, intraocular lens opacification in one eye was significantly related to intraocular lens opacification in the fellow eye. A significant variability in opacification was found across intraocular lens serial numbers: the odds ratio for opacification in intraocular lens with serial number beginning with 200003 was 6.0 when comparing with the remaining lenses. CONCLUSION: The opacification prevalence of the LS-502-1 intraocular lens was 53.3%, which is the highest ever described for any intraocular lens model. Our results suggest that this occurrence is secondary to an interaction between unknown patient variables and problems related to intraocular lens manufacturing and storage procedures.

Combining radial point interpolation meshless method with a new homogenization technique for trabecular bone multiscale structural analyses.

PURPOSE: Bone tissue is a dynamic tissue, possessing different functional requirements at different scales. This layered organization indicates the existence of a hierarchical structure, which can be characterized to distinguish macro-scale from micro-scale levels. Structurally, both scales can be linked by the use of classic multiscale homogenization techniques. Since in bone tissue each micro-scale domain is distinct form its neighbour, applying a classic multiscale homogenization technique to a complete bone structure could represent an inadmissible computational cost. Thus, this work proposes a homogenization methodology that is computationally efficient, presenting a reduced computational cost, and is capable to define the homogenized microscale mechanical properties of the trabecular bone highly heterogeneous medium. METHODS: The methodology uses the fabric tensor concept in order to define the material principal directions. Then, using an anisotropic phenomenological law for bone tissue correlating the local apparent density with directional elasticity moduli, the anisotropic homogenized material properties of the micro-scale patch are fully defined. To validate the developed methodology, several numerical tests were performed, measuring the sensitivity of the technique to changes in the micro-patch size and preferential orientation. RESULTS: The results show that the developed technique is robust and capable to provide a consistent material homogenization. Additionally, the technique was combined with two discrete numerical techniques: the finite element method and radial point interpolation meshless method. CONCLUSIONS: Structural analyses were performed using real trabecular patches, showing that the proposed methodology is capable to accurately predict the micro-scale patch mechanical behavior in a fraction of the time required by classic homogenization techniques.

Prediction of Stress-Strain Curves Based on Hydric Non-Destructive Tests on Sandstones.

The study of the mechanical behavior of building stones is traditionally supported by destructive compression tests carried out on representative specimens. However, in order to respect the monuments' integrity, the study of the mechanical behavior of stones can be based mostly on physical properties obtained from non-destructive tests (NDT). For this study, a simple and cheap NDT-water absorption under low pressure-was used to carry out fast surveys and to predict the most important design parameters of loadbearing masonry, among which are the compressive strength, strain at failure, and even elastic modulus on monument blocks. The paper presents the results of the experimental work conducted to obtain the physical properties and stress-strain curves of the sandstones tested. Supported by these results, it was possible to correlate the various parameters and develop an analytical model that predicts the stress-strain curve of the sandstones based on water absorption under low pressure tests. A good agreement is observed between the analytical model and the experimental tests.

Human Plasma Metabolomics in Age-Related Macular Degeneration: Meta-Analysis of Two Cohorts.

The pathogenesis of age-related macular degeneration (AMD), a leading cause of blindness worldwide, remains only partially understood. This has led to the current lack of accessible and reliable biofluid biomarkers for diagnosis and prognosis, and absence of treatments for dry AMD. This study aimed to assess the plasma metabolomic profiles of AMD and its severity stages with the ultimate goal of contributing to addressing these needs. We recruited two cohorts: Boston, United States (n = 196) and Coimbra, Portugal (n = 295). Fasting blood samples were analyzed using ultra-high performance liquid chromatography mass spectrometry. For each cohort, we compared plasma metabolites of AMD patients versus controls (logistic regression), and across disease stages (permutation-based cumulative logistic regression considering both eyes). Meta-analyses were then used to combine results from the two cohorts. Our results revealed that 28 metabolites differed significantly between AMD patients versus controls (false discovery rate (FDR) q-value: 4.1 × 10-2-1.8 × 10-5), and 67 across disease stages (FDR q-value: 4.5 × 10-2-1.7 × 10-4). Pathway analysis showed significant enrichment of glycerophospholipid, purine, taurine and hypotaurine, and nitrogen metabolism (p-value ≤ 0.04). In conclusion, our findings support that AMD patients present distinct plasma metabolomic profiles, which vary with disease severity. This work contributes to the understanding of AMD pathophysiology, and can be the basis of future biomarkers and precision medicine for this blinding condition.

Urine Nuclear Magnetic Resonance (NMR) Metabolomics in Age-Related Macular Degeneration.

Biofluid biomarkers of age-related macular degeneration (AMD) are still lacking, and their identification is challenging. Metabolomics is well-suited to address this need, and urine is a valuable accessible biofluid. This study aimed to characterize the urinary metabolomic signatures of patients with different stages of AMD and a control group (>50 years). It was a prospective, cross-sectional study, where subjects from two cohorts were included: 305 from Coimbra, Portugal (AMD patients n = 252; controls n = 53) and 194 from Boston, United States (AMD patients n = 147; controls n = 47). For all participants, we obtained color fundus photographs (for AMD staging) and fasting urine samples, which were analyzed using 1H nuclear magnetic resonance (NMR) spectroscopy. Our results revealed that in both cohorts, urinary metabolomic profiles differed mostly between controls and late AMD patients, but important differences were also found between controls and subjects with early AMD. Analysis of the metabolites responsible for these separations revealed that, even though distinct features were observed for each cohort, AMD was in general associated with depletion of excreted citrate and selected amino acids at some stage of the disease, suggesting enhanced energy requirements. In conclusion, NMR metabolomics enabled the identification of urinary signals of AMD and its severity stages, which might represent potential metabolomic biomarkers of the disease.

Extraocular Muscles Involvement as the Initial Presentation in Metastatic Breast Cancer.

Orbital metastasis is a rare event, and metastatic disease affecting the extraocular muscles is an even less frequent complication of solid tumors. Herein, we report an unusual case of ptosis as the initial presentation of an invasive breast cancer. A 68-year-old woman presented with III and VI partial nerve paresis, secondary to a compressive retrobulbar mass. Magnetic resonance imaging revealed an infiltrative lesion involving the extraocular muscles. Tissue biopsy yielded a result compatible with metastasis to the orbit, with immunohistochemistry analysis suggesting breast as the primary organ. Mammography identified an area of architectural distortion; stereotactic wire-guided biopsy confirmed the result of the previous orbital biopsy. A positron emission tomography scan demonstrated disseminated disease. Palliative chemotherapy with bone-modulating agents and subsequent hormonal therapy was proposed. Unfortunately, the patient did not respond to therapy and died 38 months after diagnosis.