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BACKGROUND: Drug coated balloons (DCB) are potentially less thrombogenic than drug eluting stents (DES). AIMS: To explore the safety and the feasibility of single antiplatelet therapy in percutaneous coronary intervention with sirolimus-coated balloons. METHODS: The All-comers Sirolimus-coated Balloon European Registry (EASTBOURNE) is a prospective investigator-driven registry assessing the performance of a novel sirolimus-coated balloon (SCB) in a real-world population. This prespecified post hoc analysis aimed at comparing the outcome in patients prescribed either single antiplatelet therapy (SAPT) or dual antiplatelet therapy (DAPT); choice of antiplatelet agent and duration of the regimen were at operator's discretion in both groups. Primary endpoint was target lesion revascularization (TLR) at 12 months. Secondary endpoints were bleeding grade 3-5 according to The Bleeding Academic Research Consortium (BARC) criteria and major adverse cardiovascular events (MACE) at 12 months follow-up. RESULTS: Among 2123 patients enrolled in the study between September 2016 and November 2020, 113 patients (5.8 %) received SAPT while 1826 patients (94.1 %) received DAPT after SCB. The majority of the patients underwent DCB PCI for de novo lesions (n = 1091, 56.3 %) while 848 patients (47.7 %) had DCB revascularization for in-stent restenosis. No cases of TLR occurred in the SAPT group within one month after the index procedure, and no acute occlusive events were recorded during follow up in patients taking a single antiplatelet agent. Moreover, no differences in terms of TLR were observed between SAPT vs DAPT regimens nor in case of de novo treatment with an overall rate of TLR at 12 months of 7.7 % for SAPT and 5.6 % for DAPT (p = 0.6). The cumulative rate of MACE at 12 months was not different between SAPT and DAPT regimens (n = 12 [11.2 %] vs. n = 162 [8.9 %], p = 0.4), and results were consistent in the de novo and in-stent restenosis groups. CONCLUSIONS: Our post hoc analysis of the EASTBOURNE registry suggests that the use of single antiplatelet agent after sirolimus-DCB PCI for both de novo or in-stent restenosis lesions is safe and effective and can help to contain the risk of bleeding in a selected population. CONDENSED ABSTRACT: The manuscript aims to explore the feasibility of a single antiplatelet regimen following angioplasty using drug coated balloon with sirolimus. Among 2123 patients treated with sirolimus coated balloon (SCB), 113 patients (5.8 %) received a single antiplatelet therapy (SAPT) while 1826 patients (94.1 %) received dual antiplatelet therapy DAPT. No cases of target lesion revascularization occurred in the SAPT group within one month after the index procedure, and no acute occlusive events were recorded during follow up in patients taking a single antiplatelet agent. The cumulative rate of major adverse cardiovascular events at 12 months was not different between SAPT and DAPT regimens and results were consistent in the de novo and in-stent restenosis groups.

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Multiple sclerosis (MS) is a chronic and degenerative disease that impacts central nervous system (CNS) function. One of the major characteristics of the disease is the presence of regions lacking myelin and an oxidative and inflammatory environment. TGF-ß1 and Nrf2 proteins play a fundamental role in different oxidative/inflammatory processes linked to neurodegenerative diseases such as MS. The evidence from different experimental settings has demonstrated a TGF-ß1-Nrf2 signaling crosstalk under pathological conditions. However, this possibility has not been explored in experimental models of MS. Here, by using the cuprizone-induced demyelination model of MS, we report that the in vivo pharmacological blockage of the TGF-ß1 receptor reduced Nrf2, catalase, and TGFß-1 protein levels in the demyelination phase of cuprizone administration. In addition, ATP production, locomotor function and cognitive performance were diminished by the treatment. Altogether, our results provide evidence for a crosstalk between TGF-ß1 and Nrf2 signaling pathways under CNS demyelination, highlighting the importance of the antioxidant cellular response of neurodegenerative diseases such as MS.

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BACKGROUND: Multiple sclerosis (MS) is an irreversible progressive CNS pathology characterized by the loss of myelin (i.e. demyelination). The lack of myelin is followed by a progressive neurodegeneration triggering symptoms as diverse as fatigue, motor, locomotor and sensory impairments and/or bladder, cardiac and respiratory dysfunction. Even though there are more than fourteen approved treatments for reducing MS progression, there are still no cure for the disease. Thus, MS research is a very active field and therefore we count with different experimental animal models for studying mechanisms of demyelination and myelin repair, however, we still lack a preclinical MS model assembling demyelination mechanisms with relevant clinical-like signs. RESULTS: Here, by inducing the simultaneous demyelination of both callosal and cerebellar white matter fibers by the double-site injection of lysolecithin (LPC), we were able to reproduce CNS demyelination, astrocyte recruitment and increases levels of proinflammatory cytokines levels along with motor, locomotor and urinary impairment, as well as cardiac and respiratory dysfunction, in the same animal model. Single site LPC-injections either in corpus callosum or cerebellum only, fails in to reproduce such a complete range of MS-like signs. CONCLUSION: We here report that the double-site LPC injections treatment evoke a complex MS-like mice model. We hope that this experimental approach will help to deepen our knowledge about the mechanisms of demyelinated diseases such as MS.

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BACKGROUND: Studies evaluating the safety and efficacy of drug coating balloons (DCB) for the treatment of lesions in large coronary vessel are limited. AIMS: Our study aimed to evaluate the performance of a sirolimus DCB in large coronary arteries. METHODS: We analyzed all the procedures included in the EASTBOURNE Registry (NCT03085823) enrolling patients with a clinical indication to percutaneous coronary intervention performed by a sirolimus DCB according to investigator judgment. In the present analysis, a cut-off of 2.75 mm was used to define large coronary arteries. Primary endpoint of the study was clinically driven target lesion revascularization (TLR) at 24 months whereas secondary endpoint included procedural success, myocardial infarction (MI), cardiac death and total mortality. RESULTS: Among the 2123 patients and 2440 lesions enrolled in the EASTBOURNE study between 2016 and 2020, 757 patients/810 lesions fulfilled the criteria for the present analysis. Mean reference vessel diameter was 3.2 ± 0.3 mm with mean lesion length of 22 ± 7 mm. Procedural success was high (96%) and at 2-year follow up the device showed a good efficacy with a TLR rate of 9%. There were 34 deaths (4.5%), 30 MIs (4%) and 8 BARC type 3-5 bleedings (1.1%). In-stent restenosis (629 lesions) and de novo lesions (181) were associated with 11% and 4% rates of TLR at 2 years, respectively (p = 0.003). CONCLUSIONS: Clinical performance of a sirolimus DCB in large coronary artery vessels shows promising signals at 2-year follow up, both in de novo and in-stent restenosis lesions.

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INTRODUCTION AND OBJECTIVES: The PEACE study (Performance of a sirolimus-eluting balloon strategy in acute and chronic coronary syndromes) investigated for the first time whether a sirolimus-coated balloon (SCB) (Magic Touch, Concept Medical, India) is associated with different outcomes depending on whether it is used in acute coronary syndromes (ACS) or chronic coronary syndromes (CCS). METHODS: This was a post-hoc analysis from the all-comers EASTBOURNE Registry (NCT03085823). Out of 2083 patients enrolled, an SCB was used to treat 968 (46.5%) ACS and 1115 (53.5%) CCS patients. The primary endpoint was target lesion revascularization at 12 months, while secondary endpoints were angiographic success and major adverse cardiovascular events. RESULTS: Baseline demographics, mean reference vessel diameter and mean lesion length were comparable between ACS and CCS. Predilatation was more commonly performed in ACS (P=.007). SCB was inflated at a standard pressure in both groups with a slight trend toward longer inflation time in ACS. Angiographic success was high in both groups (ACS 97.4% vs CCS 97.7%, P=.820) with limited bailout stenting. Similarly, at 12 months the cumulative incidence of target lesion revascularization (ACS 6.6% vs CCS 5.2%, P=.258) was comparable between ACS and CCS. Conversely, a higher rate of major adverse cardiovascular events in acute presenters was mainly driven by myocardial infarction recurrencies (ACS 10.4% vs CCS 8.3%, P=.009). In-stent restenosis showed a higher proportion of target lesion revascularization and major adverse cardiovascular events than de novo lesions, independently of the type of presentation at the index procedure. CONCLUSIONS: This SCB shows good performance in terms of acute and 1-year outcomes independently of the clinical presentation.

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Biomolecular condensates regulate a wide range of cellular functions from signaling to RNA metabolism 1, 2 , yet, the physiologic conditions regulating their formation remain largely unexplored. Biomolecular condensate assembly is tightly regulated by the intracellular environment. Changes in the chemical or physical conditions inside cells can stimulate or inhibit condensate formation 3-5 . However, whether and how the external environment of cells can also regulate biomolecular condensation remain poorly understood. Increasing our understanding of these mechanisms is paramount as failure to control condensate formation and dynamics can lead to many diseases 6, 7 . Here, we provide evidence that matrix stiffening promotes biomolecular condensation in vivo . We demonstrate that the extracellular matrix links mechanical cues with the control of glucose metabolism to sorbitol. In turn, sorbitol acts as a natural crowding agent to promote biomolecular condensation. Using in silico simulations and in vitro assays, we establish that variations in the physiological range of sorbitol, but not glucose, concentrations, are sufficient to regulate biomolecular condensates. Accordingly, pharmacologic and genetic manipulation of intracellular sorbitol concentration modulates biomolecular condensates in breast cancer - a mechano-dependent disease. We propose that sorbitol is a mechanosensitive metabolite enabling protein condensation to control mechano-regulated cellular functions. Altogether, we uncover molecular driving forces underlying protein phase transition and provide critical insights to understand the biological function and dysfunction of protein phase separation.

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Osteoporosis is a disease characterized by impairment of bone microarchitecture that causes high socioeconomic impacts in the world because of fractures and hospitalizations. Although dual-energy X-ray absorptiometry (DXA) is the gold standard for diagnosing the disease, access to DXA in developing countries is still limited due to its high cost, being present only in specialized hospitals. In this paper, we analyze the performance of Osseus, a low-cost portable device based on electromagnetic waves that measures the attenuation of the signal that crosses the medial phalanx of a patient's middle finger and was developed for osteoporosis screening. The analysis is carried out by predicting changes in bone mineral density using Osseus measurements and additional common risk factors used as input features to a set of supervised classification models, while the results from DXA are taken as target (real) values during the training of the machine learning algorithms. The dataset consisted of 505 patients who underwent osteoporosis screening with both devices (DXA and Osseus), of whom 21.8% were healthy and 78.2% had low bone mineral density or osteoporosis. A cross-validation with k-fold = 5 was considered in model training, while 20% of the whole dataset was used for testing. The obtained performance of the best model (Random Forest) presented a sensitivity of 0.853, a specificity of 0.879, and an F1 of 0.859. Since the Random Forest (RF) algorithm allows some interpretability of its results (through the impurity check), we were able to identify the most important variables in the classification of osteoporosis. The results showed that the most important variables were age, body mass index, and the signal attenuation provided by Osseus. The RF model, when used together with Osseus measurements, is effective in screening patients and facilitates the early diagnosis of osteoporosis. The main advantages of such early screening are the reduction of costs associated with exams, surgeries, treatments, and hospitalizations, as well as improved quality of life for patients.

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BACKGROUND: Drug-coated balloons (DCB) represent 1 of the most promising innovations in interventional cardiology and may represent a valid alternative to drug-eluting stents. Currently, some sirolimus-coated balloons (SCB) are being investigated for several coronary artery disease applications. OBJECTIVES: This study sought to understand the role of a novel SCB for the treatment of coronary artery disease. METHODS: EASTBOURNE (All-Comers Sirolimus-Coated Balloon European Registry) is a prospective, multicenter, investigator-driven clinical study that enrolled real-world patients treated with SCB. Primary endpoint was target lesion revascularization (TLR) at 12 months. Secondary endpoints were procedural success, myocardial infarction (MI), all-cause death, and major adverse clinical events (a composite of death, MI, and TLR). All adverse events were censored and adjudicated by an independent clinical events committee. RESULTS: A total population of 2,123 patients (2,440 lesions) was enrolled at 38 study centers in Europe and Asia. The average age was 66.6 ± 11.3 years, and diabetic patients were 41.5%. De novo lesions (small vessels) were 56%, in-stent restenosis (ISR) 44%, and bailout stenting occurred in 7.7% of the patients. After 12 months, TLR occurred in 5.9% of the lesions, major adverse clinical events in 9.9%, and spontaneous MI in 2.4% of the patients. The rates of cardiac/all-cause death were 1.5% and 2.5%, respectively. The primary outcome occurred more frequently in the ISR cohort (10.5% vs 2.0%; risk ratio: 1.90; 95% CI: 1.13-3.19). After multivariate Cox regression model, the main determinant for occurrence of the primary endpoint was ISR (OR: 5.5; 95% CI: 3.382-8.881). CONCLUSIONS: EASTBOURNE, the largest DCB study in the coronary field, shows the safety and efficacy of a novel SCB in a broad population of coronary artery disease including small vessels and ISR patients at mid-term follow-up. (The All-Comers Sirolimus-Coated Balloon European Registry [EASTBOURNE]; NCT03085823).

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We study a generalization of the random walk (RW) based on a deformed translation of the unitary step, inherited by the q algebra, a mathematical structure underlying nonextensive statistics. The RW with deformed step implies an associated deformed random walk (DRW) provided with a deformed Pascal triangle along with an inhomogeneous diffusion. The paths of the RW in deformed space are divergent, while those corresponding to the DRW converge to a fixed point. Standard random walk is recovered for q→1 and a suppression of randomness is manifested for the DRW with -1<γ_{q}<1 and γ_{q}=1-q. The passage to the continuum of the master equation associated to the DRW led to a van Kampen inhomogeneous diffusion equation when the mobility and the temperature are proportional to 1+γ_{q}x, and provided with an exponential hyperdiffusion that exhibits a localization of the particle at x=-1/γ_{q} consistent with the fixed point of the DRW. Complementarily, a comparison with the Plastino-Plastino Fokker-Planck equation is discussed. The two-dimensional case is also studied, by obtaining a 2D deformed random walk and its associated deformed 2D Fokker-Planck equation, which give place to a convergence of the 2D paths for -1<γ_{q_{1}},γ_{q_{2}}<1 and a diffusion with inhomogeneities controlled by two deformation parameters γ_{q_{1}},γ_{q_{2}} in the directions x and y. In both the one-dimensional and the two-dimensional cases, the transformation γ_{q}→-γ_{q} implies a change of sign of the corresponding limits of the random walk paths, as a property of the deformation employed.

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Regarding the low number of embryos that reach the blastocyst stage when cultured in vitro, this study aimed to evaluate the effects of quercetin on pre-implantation mouse (Mus musculus) embryos obtained using in vitro fertilization, especially during the passage from morula to blastocyst. Furthermore, we studied whether quercetin also affected the expression of hypoxia-inducible factor 1α (HIF-1α). The culture medium for the embryos was supplemented with quercetin, for long or short periods of time, and then the development potential, total cell number, apoptosis rates and expression of HIF-1α were studied to determine the effect of quercetin. Embryos failed to develop when cultured for long periods of time with quercetin, implying the possible toxic effects of this, alternatively antioxidant, compound. However, a short culture from morula to blastocyst significantly improved the development potential of in vitro produced embryos, increasing the final total cell number and reducing the apoptosis rate, observing similar results to those embryos cultured in low-oxygen concentrations or developed in utero. Furthermore, in embryos treated with quercetin for 2 or 4 h we found an increase in HIF-1α compared with untreated embryos. This work could imply a way to use quercetin in fertility clinics to improve the production of healthy blastocysts and, consequently, increase the success rates in assisted reproduction techniques.

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Individuals homozygous for the "Z" mutation in alpha-1 antitrypsin deficiency are known to be at increased risk for liver disease. It has also become clear that some degree of risk is similarly conferred by the heterozygous state. A lack of model systems that recapitulate heterozygosity in human hepatocytes has limited the ability to study the impact of a single Z alpha-1 antitrypsin (ZAAT) allele on hepatocyte biology. Here, we describe the derivation of syngeneic induced pluripotent stem cells (iPSCs) engineered to determine the effects of ZAAT heterozygosity in iPSC-hepatocytes (iHeps). We find that heterozygous MZ iHeps exhibit an intermediate disease phenotype and share with ZZ iHeps alterations in AAT protein processing and downstream perturbations including altered endoplasmic reticulum (ER) and mitochondrial morphology, reduced mitochondrial respiration, and branch-specific activation of the unfolded protein response in cell subpopulations. Our model of MZ heterozygosity thus provides evidence that a single Z allele is sufficient to disrupt hepatocyte homeostatic function.

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AIMS: The main objective of this work is to elucidate whether Quercetin (Qc) and 4-Hidroxistradiol (4OHE2 ) decrease the level of reactive oxygen species (ROS) in in vitro obtained embryos and to analyse which genes are activated under the treatments that could explain this improvement. METHODS: Oxidative stress was induced during embryo culture by H2 O2 treatment and ROS production was measured and compared with embryos treated with Qc or 4OHE2 . Gene expression was analysed by Q-PCR in control embryos obtained in utero (IU) or by IVF and compared with the levels found in embryos cultured with Qc or 4OHE2 to determine the effect of these compounds. KEY RESULTS: Qc strongly reduces ROS levels in embryos after a treatment of 4h. On the contrary, 4OHE2 had no effect in reducing ROS levels in embryos. The addition of these molecules to the culture media upregulate several hypoxia-related genes when Qc is added to the culture media, and implantation-related genes when 4OHE2 is used. CONCLUSIONS: Qc is a very strong antioxidant molecule that when used for short periods of time during culture can reduce ROS levels and improve embryo quality by activating antioxidant enzymes. 4OHE2 supplementation, despite having no effects in reducing ROS levels, acts directly in the molecular signalling implicated in the implantation process and could be also considered as a supplement for embryo culture during IVF. IMPLICATIONS: Proper supplementation of the culture media could greatly improve the quality of embryos cultured in vitro , resulting in better results in IVF clinics.

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OBJECTIVE: The aim was to evaluate the internal adaptation of composite restorations in cusp-weakened Class I preparations restored by incremental, bi-layered, and bulk-fill techniques using a microcomputed tomography analysis (micro-CT). METHODS: Sixty sound extracted human third molars were prepared with standardized cusp-weakened Class I cavity preparations and divided into six groups (n=10). Two groups were restored using an incremental oblique technique with a nanofilled (Filtek Supreme XT, 3M Oral Care, St Paul, MN, USA) and a nanohybrid composite (GrandioSO, VOCO, Cuxhaven, Germany). The other two groups were restored using a bi-layered technique with a bulk-fill flowable composite base (Filtek Bulk Fill Flowable, 3M Oral Care; X-tra Base bulk-fill flowable, VOCO) covered with the conventional composite of the same type. In the remaining groups, teeth were restored with a single-increment technique using bulk-fill full-body composites (Filtek Bulk Fill Posterior, 3M Oral Care; Admira Fusion, VOCO). All teeth were subjected to thermocycling (20,000 cycles, 5°C and 55°C) and mechanical loading (250,000 cycles, 2.5 Hz, 50 N) before analysis of internal adaptation by micro-CT. One-way analysis of variance (ANOVA) and the Games-Howell test were used to analyze the percentage and volume of internal gaps (p<0.05). RESULTS: There was found to be significantly higher internal adaptation for the nanofilled composite used incrementally or associated with a flowable base compared with the nanohybrid composite used incrementally (p=0.002). CONCLUSIONS: Bulk-fill and bi-layered techniques performed similarly in terms of internal adaptation for both types of composites. The incremental technique with the nanofilled composite demonstrated a higher internal adaptation than with the nanohybrid composite.

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BACKGROUND: New prevention strategies have been advocated to control the progression of HIV/AIDS, such as preexposure prophylaxis (PrEP). The aim of this study is to evaluate the potential changes in the oral and salivary conditions of HIV-uninfected subjects using PrEP. MATERIAL AND METHODS: Subjects were evaluated before beginning the medication (T0), at the first follow-up (T1), and at the second follow-up (T2). Xerostomia, presence of untreated cavitated caries, oral hygiene habits, taste, gingival and plaque index, stimulated salivary flow rate (SSFR), and salivary concentrations of calcium, glucose, urea, and total proteins were evaluated. Data obtained were analyzed using statistical tests (p<0.05). RESULTS: Forty-seven participants (41 men; 6 women) were evaluated at T0. Thirty (28 men; 2 women) and 17 men were reassessed at T1 and T2, respectively. There was no difference between the SSFR and oral and salivary conditions between T0, T1, and T2 (p>0.05), except for the salivary calcium concentration, that increased at T2 compared to T1 (p=0.02). There was significant difference between taste and xerostomia at T1 (p=0.017), and the need to drink to swallow at T2 (p=0.015). There was significant correlation between the reported amount of saliva and taste (p=0.039, r=-0.378) at T1. CONCLUSIONS: The prolonged use of PrEP seems to be associated with reports of dry mouth and worsening of taste, possibly associated with increased salivary calcium concentration.

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Nitrogen (N) is normally applied twice during sugarcane cultivation on Negros Island in the Philippines. As the first application is carried out during the early growth stage, there is a high possibility that N is not absorbed sufficiently by the sugarcane and a large amount of N may be leached with percolating water. To investigate this leaching, we monitored the vertical movement of soil water and soil solutes using GS3 dielectric moisture and electrical conductivity (EC) sensors in a sugarcane field on Negros Island. The monitoring was conducted after the first N application at different application rates (50% or 100% of the recommended rate) and timings (immediately or 30 days after planting [DAP]). The EC values increased when 100%-N was applied immediately after planting, indicating that N leached into the deeper soil layer. The monitoring results suggest that the applied N did not remain in the root zone (due to leaching) and was not replenished until the second application, when the absorption ability of the sugarcane was high. Conversely, when 100%-N was applied at 30 DAP, the applied N remained in the root zone until the second application. When 50%-N was applied at 30 DAP, the increase in EC was small compared to the 100%-N application. These results indicate that applying N immediately after planting at the recommended rate led to an insufficient nutrient supply for the sugarcane, as well as the loss of N fertiliser via leaching.

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The aim of this work was to study the evolution of fruit size and weight together with the soluble solid and total titratable acidity contents during development of Hexachlamys edulis fruit. Also, the patterns of accumulation of chlorophylls, carotenoids, phenols and antioxidant activity were analysed to define the optimal time for harvesting to obtain maximum nutraceutical characteristics. Fruits were harvested from H. edulis plants growing at the experimental field of the University of Morón (Moreno, Buenos Aires, 34°35'4.98″ SL, 58°48'52.09″ WL, 14 m.a.s.l.). Fresh fruit weight was significantly higher in Medium ripe, Ripe and Overripe fruits (40.1, 39.6 and 38.5 g, respectively) than in Unripe fruits (19.5 g). Soluble solids/total titratable acidity was significantly higher in Overripe fruits (7.3) than in Unripe, Medium ripe and Ripe fruits (3.7-4.5). Total polyphenols were maximum in Unripe fruits (905.8 mg tannic acid/100 g fresh fruit weight) decreasing during the fruit development (426.2-130.4 mg tannic acid/100 g fresh fruit weight). Also, DPPH radical scavenging activity was significantly higher in Unripe fruits (75.7%) compared with Medium ripe, Ripe and Overripe fruits (64.1-17.0%). Positive and significant correlations were observed between total polyphenol content and DPPH radical scavenging activity at each extract concentration (r = 0.74, 0.87, 0.74 and 0.60 for 1.25; 2.50; 5.00 and 12.50 mg/mL, respectively). Total carotenoid content increased during fruit development while at the same time decreased chlorophyll content. Chlorophyll b is the main chlorophyll found. Chromatographic analysis showed that lutein is the main carotenoid found in H. edulis fruits, followed by ß-cryptoxanthin and ß-carotene. As shown by the chromatograms at 280 nm, the concentration of biophenols and the complexity of the biophenol profile decreases during fruit development. Levels of polyphenols and pigments together with the antioxidant activity allow us to consider H. edulis fruit as a functional food.

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Stroke remains the leading cause of disability and death in the Philippines. Evaluating the current state of stroke care, the needed resources, and the gaps in health policies and programs is crucial to decrease stroke-related mortality and morbidity effectively. This paper aims to characterize the Philippines' stroke system of care and network using the World Health Organization health system building blocks framework. To integrate existing national laws and policies governing stroke and its risk factors dispersed across many general policies, the Philippine Department of Health (DOH) institutionalized a national policy framework for preventing and managing stroke. Despite policy reforms, government financing coverage remains limited. In terms of access to medicines, the government launched its stroke medicine access program (MAP) in 2016, providing more than 1,000 vials of recombinant tissue plasminogen activator (rTPA) or alteplase subsidized to selected government hospitals across the country. However, DOH discontinued the program due to the lack of neuroimaging machines and organized system of care to support the provision of the said medicine. Despite limited resources, stroke diagnostics and treatment facilities are more concentrated in urban settings, mostly in private hospitals, where out-of-pocket expenditures prevail. These barriers to access are also reflective of the current state of human resource on stroke where medical specialists (e.g., neurologists) serve in the few tertiary and training hospitals situated in urban settings. Meanwhile, there is no established unified national stroke registry thus, determining the local burden of stroke remains a challenge. The lack of centralization and fragmentation of the stroke cases reporting system leads to reliance on data from hospital records or community-based stroke surveys, which may inaccurately depict the country's actual stroke incidence and prevalence. Based on these gaps, specific recommendations geared toward systems approach - governance, financing, information system, human resources for health, and medicines were identified.

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Alveolar epithelial type 2 cells (AEC2s), the facultative progenitors of lung alveoli, are typically identified through the use of the canonical markers, SFTPC and ABCA3. Self-renewing AEC2-like cells have been generated from human induced pluripotent stem cells (iPSCs) through the use of knock-in SFTPC fluorochrome reporters. However, developmentally, SFTPC expression onset begins in the fetal distal lung bud tip and thus is not specific to mature AEC2s. Furthermore, SFTPC reporters appear to identify only those iPSC-derived AEC2s (iAEC2s) expressing the highest SFTPC levels. Here, we generate an ABCA3 knock-in GFP fusion reporter (ABCA3:GFP) that enables the purification of iAEC2s while allowing visualization of lamellar bodies, organelles associated with AEC2 maturation. Using an SFTPCtdTomato and ABCA3:GFP bifluorescent line for in vitro distal lung-directed differentiation, we observe later onset of ABCA3:GFP expression and broader identification of the subsequently emerging iAEC2 population based on ABCA3:GFP expression compared with SFTPCtdTomato expression. Comparing ABCA3:GFP/SFTPCtdTomato double-positive with ABCA3:GFP single-positive (SP) cells by RNA sequencing and functional studies reveals iAEC2 cellular heterogeneity with both populations functionally processing surfactant proteins but the SP cells exhibiting faster growth kinetics, increased clonogenicity, increased expression of progenitor markers, lower levels of SFTPC expression, and lower levels of AEC2 maturation markers. Over time, we observe that each population (double-positive and SP) gives rise to the other and each can serve as the parents of indefinitely self-renewing iAEC2 progeny. Our results indicate that iAEC2s are a heterogeneous population of cells with differing proliferation versus maturation properties, the majority of which can be tracked and purified using the ABCA3:GFP reporter or surrogate cell surface proteins, such as SLC34A2 and CPM.

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L-asparaginase II (ASNase) is the biopharmaceutical of choice for the treatment of acute lymphoblastic leukaemia. In this study, E. coli BL21 (DE3) transformed with the pET15b + asnB vector which expresses recombinant ASNase was used as a source to obtain this enzyme. The ideal conditions to produce ASNase would be a high level of secretion into the extracellular medium, which depends not only on the application of molecular biology techniques but also on the development of a strategy to modify cell permeability such as the addition of substances to the culture medium that stimulate destabilisation of structural components of the cell. Thus, the growth of E. coli BL21 (DE3) in modified Luria-Bertani broth, supplemented with 0.8% (w/v) glycine and 6% (v/v) n-dodecane, increased the total yield of ASNase by about 50% (15,108 IU L-1) and resulted in a 16-fold increase in extracellular enzymatic productivity (484 IU L-1 h-1), compared to production using the same medium without addition of these substances. Most of the enzyme (89%) was secreted into the culture medium 24 h after the induction step. This proposed approach presents a simple strategy to increase extracellular production of ASNase in E. coli.

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