Non-cytotoxic Concentration of Cisplatin Decreases Neuroplasticity-Related Proteins and Neurite Outgrowth Without Affecting the Expression of NGF in PC12 Cells.

Cisplatin is the most effective and neurotoxic platinum chemotherapeutic agent. It induces a peripheral neuropathy characterized by distal axonal degeneration that might progress to degeneration of cell bodies and apoptosis. Most symptoms occur nearby distal axonal branches and axonal degeneration might induce peripheral neuropathy regardless neuronal apoptosis. The toxic mechanism of cisplatin has been mainly associated with DNA damage, but cisplatin might also affect neurite outgrowth. Nevertheless, the neurotoxic mechanism of cisplatin remains unclear. We investigated the early effects of cisplatin on axonal plasticity by using non-cytotoxic concentrations of cisplatin and PC12 cells as a model of neurite outgrowth and differentiation. PC12 cells express NGF-receptors (trkA) and respond to NGF by forming neurites, branches and synaptic vesicles. For comparison, we used a neuronal model (SH-SY5Y cells) that does not express trkA nor responds to NGF. Cisplatin did not change NGF expression in PC12 cells and decreased neurite outgrowth in both models, suggesting a NGF/trkA independent mechanism. It also reduced axonal growth (GAP-43) and synaptic (synapsin I and synaptophysin) proteins in PC12 cells, without inducing mitochondrial damage or apoptosis. Therefore, cisplatin might affect axonal plasticity before DNA damage, NGF/trkA down-regulation, mitochondrial damage or neuronal apoptosis. This is the first study to show that neuroplasticity-related proteins might be early targets of the neurotoxic action of cisplatin and their role on cisplatin-induced peripheral neuropathy should be investigated in vivo.

ATP synthase repression in tobacco restricts photosynthetic electron transport, CO2 assimilation, and plant growth by overacidification of the thylakoid lumen.

Tobacco (Nicotiana tabacum) plants strictly adjust the contents of both ATP synthase and cytochrome b(6)f complex to the metabolic demand for ATP and NADPH. While the cytochrome b(6)f complex catalyzes the rate-limiting step of photosynthetic electron flux and thereby controls assimilation, the functional significance of the ATP synthase adjustment is unknown. Here, we reduced ATP synthase accumulation by an antisense approach directed against the essential nuclear-encoded γ-subunit (AtpC) and by the introduction of point mutations into the translation initiation codon of the plastid-encoded atpB gene (encoding the essential ß-subunit) via chloroplast transformation. Both strategies yielded transformants with ATP synthase contents ranging from 100 to <10% of wild-type levels. While the accumulation of the components of the linear electron transport chain was largely unaltered, linear electron flux was strongly inhibited due to decreased rates of plastoquinol reoxidation at the cytochrome b(6)f complex (photosynthetic control). Also, nonphotochemical quenching was triggered at very low light intensities, strongly reducing the quantum efficiency of CO(2) fixation. We show evidence that this is due to an increased steady state proton motive force, resulting in strong lumen overacidification, which in turn represses photosynthesis due to photosynthetic control and dissipation of excitation energy in the antenna bed.

Preconception counseling impact in pregnancy outcomes in patients with spondyloarthritis.

INTRODUCTION: Spondyloarthritis (SpA) is a group of chronic inflammatory diseases, often affecting women in reproductive age. These diseases can have a significant impact on the reproductive health of women. Preconception counseling and medication adjustments have shown to reduce flares and improve pregnancy outcomes in women with rheumatoid arthritis. However, in women with SpA data of the impact of preconception counselling on pregnancy outcomes is scarce. The aim of this study is to evaluate that. METHODS: In this retrospective multicentric study, data was collected from medical records of women who gave birth from 2020 to 2022. The study included 45 pregnancies, which were divided into two categories whether they received preconception consultation or not. Data was collected on patient characteristics, disease duration, medications used, and preconception counselling. Outcomes were divided into two groups: maternal and fetal outcomes. RESULTS: 30 out of 45 pregnancies (66.67%) had received preconception counselling, having a significantly lower percentage of flares occurring postpartum compared to the non-counselling group (36.6% vs 6.4%, p=0.031) and lower percentage of contraindicated medication during pregnancy (20.0 vs 0.0%, p=0.011). CONCLUSION: Preconception counselling in women with SpA can increase the likelihood of medication adjustments before pregnancy and decrease the occurrence of flares postpartum. These findings suggest that preconception counselling should be implemented in the management of pregnant women with SpA to improve pregnancy outcomes. Further studies are needed to confirm the effectiveness of preconception counselling and to determine the optimal approach.

The idiopathic inflammatory myopathies module of the Rheumatic Diseases Portuguese Register.

AIMS: To characterise the idiopathic inflammatory myopathies (IIM) module of the Rheumatic Diseases Portuguese Register (Reuma.pt/myositis) and the patients in its cohort. METHODS: Reuma.pt is a web-based system with standardised patient files gathered in a registry. This was a multicentre open cohort study, including patients registered in Reuma.pt/myositis up to January 2022. RESULTS: Reuma.pt/myositis was designed to record all relevant data in clinical practice and includes disease-specific diagnosis and classification criteria, clinical manifestations, immunological data, and disease activity scores. Two hundred eighty patients were included, 71.4% female, 89.4% Caucasian, with a median age at diagnosis and disease duration of 48.9 (33.6-59.3) and 5.3 (3.0-9.8) years. Patients were classified as having definite (N=57/118, 48.3%), likely (N=23/118, 19.5%), or possible (N=2/118, 1.7%) IIM by 2017 EULAR/ACR criteria. The most common disease subtypes were dermatomyositis (DM, N=122/280, 43.6%), polymyositis (N=59/280, 21.1%), and myositis in overlap syndromes (N=41/280, 14.6%). The most common symptoms were proximal muscle weakness (N=180/215, 83.7%) and arthralgia (N=127/249, 52.9%), and the most common clinical signs were Gottron's sign (N=75/184, 40.8%) and heliotrope rash (N=101/252, 40.1%). Organ involvement included lung (N=78/230, 33.9%) and heart (N=11/229, 4.8%) involvements. Most patients expressed myositis-specific (MSA, N=158/242, 65.3%) or myositis-associated (MAA, 112/242, 46.3%) antibodies. The most frequent were anti-SSA/SSB (N=70/231, 30.3%), anti-Jo1 (N=56/236, 23.7%), and anti-Mi2 (N=31/212, 14.6%). Most patients had a myopathic pattern on electromyogram (N=101/138, 73.2%), muscle oedema in magnetic resonance (N=33/62, 53.2%), and high CK (N=154/200, 55.0%) and aldolase levels (N=74/135, 54.8%). Cancer was found in 11/127 patients (8.7%), most commonly breast cancer (N=3/11, 27.3%). Most patients with cancer-associated myositis had DM (N=8/11, 72.7%) and expressed MSA (N=6/11) and/or MAA (N=3/11). The most used drugs were glucocorticoids (N=201/280, 71.8%), methotrexate (N=117/280, 41.8%), hydroxychloroquine (N=87/280, 31.1%), azathioprine (N=85/280, 30.4%), and mycophenolate mofetil (N=56/280, 20.0%). At the last follow-up, there was a median MMT8 of 150 (142-150), modified DAS skin of 0 (0-1), global VAS of 10 (0-50) mm, and HAQ of 0.125 (0.000-1.125). CONCLUSIONS: Reuma.pt/myositis adequately captures the main features of inflammatory myopathies' patients, depicting, in this first report, a heterogeneous population with frequent muscle, joint, skin, and lung involvements.

Predictors of cardiac involvement in idiopathic inflammatory myopathies.

Objectives: Idiopathic inflammatory myopathies (IIM) are a group of rare disorders that can affect the heart. This work aimed to find predictors of cardiac involvement in IIM. Methods: Multicenter, open cohort study, including patients registered in the IIM module of the Rheumatic Diseases Portuguese Register (Reuma.pt/Myositis) until January 2022. Patients without cardiac involvement information were excluded. Myo(peri)carditis, dilated cardiomyopathy, conduction abnormalities, and/or premature coronary artery disease were considered. Results: 230 patients were included, 163 (70.9%) of whom were females. Thirteen patients (5.7%) had cardiac involvement. Compared with IIM patients without cardiac involvement, these patients had a lower bilateral manual muscle testing score (MMT) at the peak of muscle weakness [108.0 ± 55.0 vs 147.5 ± 22.0, p=0.008] and more frequently had oesophageal [6/12 (50.0%) vs 33/207 (15.9%), p=0.009] and lung [10/13 (76.9%) vs 68/216 (31.5%), p=0.001] involvements. Anti-SRP antibodies were more commonly identified in patients with cardiac involvement [3/11 (27.3%) vs 9/174 (5.2%), p=0.026]. In the multivariate analysis, positivity for anti-SRP antibodies (OR 104.3, 95% CI: 2.5-4277.8, p=0.014) was a predictor of cardiac involvement, regardless of sex, ethnicity, age at diagnosis, and lung involvement. Sensitivity analysis confirmed these results. Conclusion: Anti-SRP antibodies were predictors of cardiac involvement in our cohort of IIM patients, irrespective of demographical characteristics and lung involvement. We suggest considering frequent screening for heart involvement in anti-SRP-positive IIM patients.

Cisplatin-induced nephrotoxicity and targets of nephroprotection: an update.

Cisplatin is a highly effective antitumor agent whose clinical application is limited by the inherent nephrotoxicity. The current measures of nephroprotection used in patients receiving cisplatin are not satisfactory, and studies have focused on the investigation of new possible protective strategies. Many pathways involved in cisplatin nephrotoxicity have been delineated and proposed as targets for nephroprotection, and many new potentially protective agents have been reported. The multiple pathways which lead to renal damage and renal cell death have points of convergence and share some common modulators. The most frequent event among all the described pathways is the oxidative stress that acts as both a trigger and a result. The most exploited pathways, the proposed protective strategies, the achievements obtained so far as well as conflicting data are summarized and discussed in this review, providing a general view of the knowledge accumulated with past and recent research on this subject.

Sex differences in axial spondyloarthritis: data from a Portuguese spondyloarthritis cohort.

BACKGROUND: Axial spondyloarthritis (axSpA), particularly ankylosing spondylitis was historically considered a male's disease and has been under-recognized in women. Emerging evidence reveals sex differences in pathophysiology, disease presentation and therapeutic efficacy. OBJECTIVE: To identify differences between sexes in a Portuguese cohort of patients with axSpA regarding clinical manifestations, disease activity, functional capacity, patient related outcomes and presence of sacroiliitis on x-ray or magnetic resonance imaging. METHODS: Patients with ≥18 years fulfilling the ASAS- Assessment of Spondyloarthritis International Society classification criteria for axSpA registered in the electronic Rheumatic Diseases Portuguese Register (Reuma.pt) were included in this multicentric cross-sectional study. Sociodemographic data, clinical features and imaging were collected from the first record in Reuma.pt. These variables were compared between sexes using Mann-Whitney test and Chi-Square test. Variables with a significant association with variable sex were considered in the multiple variable analysis to adjust the sex effect on the outcome variables. Statistical analysis was performed with R version 4.0.2 and p <0.05 was considered statistically significant. RESULTS: A total of 1995 patients were included, 1114 (55.9%) men and 881 (44.1%) women. Men had an earlier disease onset (25.1 vs 28.4, p <0.001), were younger at diagnosis (26.9 vs 30.4, p<0.001) and were more frequently smokers (32.1% vs 15.7%, p <0.001). Comparing to women, men had worse Bath Ankylosing Spondylitis Metrological Index scores (4.0 vs 3.4, p<0.001), higher levels of C-Reactive Protein (10.5 vs 6.9 mg/L, p <0.001) and were more often Human Leukocyte Antigen-B27 positive (67.8% vs 54%, p <0.001). In contrast, women more frequently had inflammatory bowel disease (8.8% vs 4.9%, p =0.004), higher levels of erythrocyte sedimentation rate (25.0 vs 21.0mm/h, p=0.003) and worse patient-related outcomes- Bath Ankylosing Spondylitis Disease Activity Index (5.7 vs 4.5, p<0.001), Patient Global Assessment (60.0 vs 50.0, p <0.001) and fatigue (6.2 vs 5.0, p <0.001). DISCUSSION: In this large multicentric study from a Portuguese axSpA cohort, we confirmed sex differences in patients with axSpA. This work brings awareness to these differences, resulting in less underdiagnosis and misdiagnosis, optimizing treatment strategies, and improving outcomes in axSpA.

Molecular cloning, overexpression, purification, crystallization and preliminary X-ray diffraction analysis of a purine nucleoside phosphorylase from Bacillus subtilis strain 168.

Purine nucleoside phosphorylase (PNP; EC 2.4.2.1) is a key enzyme of the purine-salvage pathway. Its ability to transfer glycosyl residues to acceptor bases is of great biotechnological interest owing to its potential application in the synthesis of nucleoside analogues used in the treatment of antiviral infections and in anticancer chemotherapy. Although hexameric PNPs are prevalent in prokaryotes, some microorganisms, such as Bacillus subtilis, present both hexameric and trimeric PNPs. The hexameric PNP from B. subtilis strain 168, named BsPNP233, was cloned, expressed and crystallized. Crystals belonging to different space groups (P32(1), P2(1)2(1)2(1), P6(3)22 and H32) were grown in distinct conditions with pH values ranging from 4.2 to 10.5. The crystals diffracted to maximum resolutions ranging from 2.65 to 1.70 Å.

Effectiveness of commercial inhibitors against subtype F HIV-1 protease.

Subtype F wild type HIV protease has been kinetically characterized using six commercial inhibitors (amprenavir, indinavir, lopinavir, nelfinavir, ritonavir and saquinavir) commonly used for HIV/AIDS treatment, as well as inhibitor TL-3 and acetyl-pepstatin. We also obtained kinetic parameters for two multi-resistant proteases (one of subtype B and one of subtype F) harboring primary and secondary mutations selected by intensive treatment with ritonavir/nelfinavir. This newly obtained biochemical data shows that all six studied commercially available protease inhibitors are significantly less effective against subtype F HIV proteases than against HIV proteases of subtype B, as judged by increased K(i) and biochemical fitness (vitality) values. Comparison with previously reported kinetic values for subtype A and C HIV proteases show that subtype F wild type proteases are significantly less susceptible to inhibition. These results demonstrate that the accumulation of natural polymorphisms in subtype F proteases yields catalytically more active enzymes with a large degree of cross-resistance, which thus results in strong virus viability.

Treatment of Persistent Cutaneous Atrophy After Corticosteroid Injection With Fat Graft.

Subcutaneous fat atrophy and hypopigmentation are potential adverse side effects of local corticosteroid injection that may resolve spontaneously within 1-2 years. This report shows that fat grafting provides a simple, effective and safe correction of corticosteroid induced cutaneous atrophy with very satisfying esthetic and functional results.

Biologic therapy use and pregnancy outcomes in women with immune-mediated inflammatory rheumatic diseases.

INTRODUCTION: Concerns about the side effects and interactions of biologic drugs with reproduction and pregnancy have been always an issue between experts. The safety of these therapies during conception and/or pregnancy is not fully understood. The aim of this study was to assess the exposure to biologic drugs before and/or during conception/pregnancy and the risk of adverse pregnancy outcomes in women with rheumatic diseases. METHODS: We conducted a cohort study of pregnancies reported in women with immune-mediated rheumatic diseases registered at the Rheumatic Diseases Portuguese Registry (Reuma.pt) and exposed to biologic drugs. Data concerning fetal and maternal outcomes (live birth, spontaneous abortion, neonatal and intrauterine death, intrauterine growth restriction, premature delivery, congenital malformations, neonatal lupus, voluntary or medical interruption of pregnancy, disease flares and need for treatment with other drugs) was extracted. RESULTS: In total, 69 pregnancies from 56 females were analysed, the majority with the diagnosis of spondyloarthritis or rheumatoid arthritis. In almost half of the cases (n=32, 46.4%) the biologic was stopped for pregnancy planning, in 31 cases (44.9%) it was stopped when pregnancy was diagnosed and in 6 pregnancies (8.7%) biologic therapy was maintained, at least until the 2nd trimester. There were 76.8% of live births and 22% of spontaneous abortions. Congenital anomalies were reported in 2 newborns. CONCLUSIONS: In half cases, it was decided to stop biologic therapy in the family planning period. Using biologic disease-modifying anti-rheumatic drugs before and/or during pregnancy doesn't seem to affect the overall maternal and fetal outcomes. Pregnancy planning and treatment options should be discussed and a shared decision should be established between physician and patient.

Structural characterization of B and non-B subtypes of HIV-protease: insights into the natural susceptibility to drug resistance development.

Although a majority of HIV-1 infections in Brazil are caused by the subtype B virus (also prevalent in the United States and Western Europe), viral subtypes F and C are also found very frequently. Genomic differences between the subtypes give rise to sequence variations in the encoded proteins, including the HIV-1 protease. The current anti-HIV drugs have been developed primarily against subtype B and the effects arising from the combination of drug-resistance mutations with the naturally existing polymorphisms in non-B HIV-1 subtypes are only beginning to be elucidated. To gain more insights into the structure and function of different variants of HIV proteases, we have determined a 2.1 A structure of the native subtype F HIV-1 protease (PR) in complex with the protease inhibitor TL-3. We have also solved crystal structures of two multi-drug resistant mutant HIV PRs in complex with TL-3, from subtype B (Bmut) carrying the primary mutations V82A and L90M, and from subtype F (Fmut) carrying the primary mutation V82A plus the secondary mutation M36I, at 1.75 A and 2.8 A resolution, respectively. The proteases Bmut, Fwt and Fmut exhibit sevenfold, threefold, and 54-fold resistance to TL-3, respectively. In addition, the structure of subtype B wild type HIV-PR in complex with TL-3 has been redetermined in space group P6(1), consistent with the other three structures. Our results show that the primary mutation V82A causes the known effect of collapsing the S1/S1' pockets that ultimately lead to the reduced inhibitory effect of TL-3. Our results further indicate that two naturally occurring polymorphic substitutions in subtype F and other non-B HIV proteases, M36I and L89M, may lead to early development of drug resistance in patients infected with non-B HIV subtypes.

Caffeic Acid Phenethyl Ester (CAPE) Protects PC12 Cells from Cisplatin-Induced Neurotoxicity by Activating the NGF-Signaling Pathway.

Cisplatin is a highly effective chemotherapeutic drug that is toxic to the peripheral nervous system. Findings suggest that axons are early targets of the neurotoxicity of cisplatin. Although many compounds have been reported as neuroprotective, there is no effective treatment against the neurotoxicity of cisplatin. Caffeic acid phenethyl ester (CAPE) is a propolis component with neuroprotective potential mainly attributed to antioxidant and anti-inflammatory mechanisms. We have recently demonstrated the neurotrophic potential of CAPE in a cellular model of neurotoxicity related to Parkinson's disease. Now, we have assessed the neurotrophic and neuroprotective effects of CAPE against cisplatin-induced neurotoxicity in PC12 cells. CAPE (10 µM) attenuated the inhibition of neuritogenesis and the downregulation of markers of neuroplasticity (GAP-43, synapsin I, synaptophysin, and 200-kD neurofilament) induced by cisplatin (5 µM). This concentration of cisplatin does not affect cell viability, and it was used in order to assess the early neurotoxic events triggered by cisplatin. When a lethal dose of cisplatin was used (IC50 = 32 µM), CAPE (10 µM) increased cell viability. The neurotrophic effect of CAPE is not dependent on NGF nor is it additive to the effect of NGF, but it might involve the activation of the NGF-high-affinity receptors (trkA). The involvement of other neurotrophin receptors such as trkB and trkC is unlikely. This is the first study to demonstrate the protective potential of CAPE against the neurotoxicity of cisplatin and to suggest the involvement of trkA receptors in the neuroprotective mechanism of CAPE. Based on these findings, the beneficial effect of CAPE on cisplatin-induced peripheral neuropathy should be further investigated.

Dimethylthiourea protects against mitochondrial oxidative damage induced by cisplatin in liver of rats.

Cisplatin is one of the most effective chemotherapeutic agents. However, at higher doses liver injury may occur. The purpose of this study was to explore whether the hydroxyl radical scavenger dimethylthiourea (DMTU) protects against cisplatin-induced oxidative damage in vivo and to define the mitochondrial pathways involved in cytoprotection. Adult male Wistar rats (200-220 g) were divided into four groups of eight animals each. The control group was treated only with an intraperitoneal (i.p.) injection of saline solution (1 ml/100 g body weight). The DMTU group was given only DMTU (500 mg/kg body weight, i.p), followed by 125 mg/kg body weight, i.p. (twice a day) until sacrifice. The cisplatin group was given a single injection of cisplatin (10 mg/kg body weight, i.p.). The DMTU+cisplatin group was given DMTU (500 mg/kg body weight, i.p.), just before the cisplatin injection (10 mg/kg body weight, i.p.), followed by injections of DMTU (125 mg/kg body weight, i.p.) twice a day until sacrifice (72 h after the treatment). DMTU did not present any direct effect on mitochondria and substantially inhibited cisplatin-induced mitochondrial damage in liver, therefore preventing elevation of AST and ALT serum levels. DMTU protected against (a) decreased hepatic ATP levels; (b) lipid peroxidation; (c) cardiolipin oxidation; (d) sulfhydryl protein oxidation; (e) mitochondrial membrane rigidification; (f) GSH oxidation; (g) NADPH oxidation; (h) apoptosis. Results suggest that antioxidants, particularly hydroxyl radical scavengers, protect liver mitochondria against cisplatin-induced oxidative damage. Several mitochondrial changes were delineated and proposed as interesting targets for cytoprotective strategy.

High-resolution structure of a Kazal-type serine protease inhibitor from the dengue vector Aedes aegypti.

Blood-feeding exoparasites are rich sources of protease inhibitors, and the mosquito Aedes aegypti, which is a vector of Dengue virus, Yellow fever virus, Chikungunya virus and Zika virus, is no exception. AaTI is a single-domain, noncanonical Kazal-type serine proteinase inhibitor from A. aegypti that recognizes both digestive trypsin-like serine proteinases and the central protease in blood clotting, thrombin, albeit with an affinity that is three orders of magnitude lower. Here, the 1.4 Šresolution crystal structure of AaTI is reported from extremely tightly packed crystals (∼22% solvent content), revealing the structural determinants for the observed inhibitory profile of this molecule.

Anti-müllerian hormone and ovarian reserve in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a chronic immune-mediated inflammatory disease that affects predominantly females during childbearing age (Lateef and Petri Best Pract Res Clin Rheumatol 27(3):435-447, 2013). Fertility in SLE patients is considered to be normal (Clowse et al. Arthritis Care Res (Hoboken) 64(5):668-674, 2012; Ekblom-Kullberg et al. Scand J Rheumatol 38:375-380, 2009) but several known factors may negatively influence fertility. Immune mechanisms are also thought to be an important cause of premature ovarian senescence, characterized by reduced ovarian reserve markers such as anti-Müllerian hormone (AMH) (Oktem et al. Obstet Gynecol Surv 70(3):196-210, 2015; Bermas and Sammaritano Fertil Res Pract 1:13, 2015; Østensen Int J Clin Rheumtol 8(1):27-37, 2013; Ulug et al. Am J Reprod Immunol 72(1):85-88, 2014; Lawrenz et al. Lupus 20(11):1193-1197, 2011). We evaluated the ovarian reserve of women in reproductive age with SLE, by measuring AMH levels and we compared it to that of non-SLE women. We also analyzed the association of SLE disease characteristics with AMH levels. AMH levels were decreased in this population of SLE women, accounting for a high proportion of women with criteria for low ovarian reserve. Age and SLE damage were associated with abnormally lower AMH levels in our SLE patients. In this way, SLE may have a negative influence on the ovarian reserve.

Multilayered membranes with tuned well arrays to be used as regenerative patches.

Membranes have been explored as patches in tissue repair and regeneration, most of them presenting a flat geometry or a patterned texture at the nano/micrometer scale. Herein, a new concept of a flexible membrane featuring well arrays forming pore-like environments to accommodate cell culture is proposed. The processing of such membranes using polysaccharides is based on the production of multilayers using the layer-by-layer methodology over a patterned PDMS substrate. The detached multilayered membrane exhibits a layer of open pores at one side and a total thickness of 38±2.2µm. The photolithography technology used to produce the molds allows obtaining wells on the final membranes with a tuned shape and micro-scale precision. The influence of post-processing procedures over chitosan/alginate films with 100 double layers, including crosslinking with genipin or fibronectin immobilization, on the adhesion and proliferation of human osteoblast-like cells is also investigated. The results suggest that the presence of patterned wells affects positively cell adhesion, morphology and proliferation. In particular, it is seen that cells colonized preferentially the well regions. The geometrical features with micro to sub-millimeter patterned wells, together with the nano-scale organization of the polymeric components along the thickness of the film will allow to engineer highly versatile multilayered membranes exhibiting a pore-like microstructure in just one of the sides, that could be adaptable in the regeneration of multiple tissues. STATEMENT OF SIGNIFICANCE: Flexible multilayered membranes containing multiple micro-reservoirs are found as potential regenerative patches. Layer-by-layer (LbL) methodology over a featured PDMS substrate is used to produce patterned membranes, composed only by natural-based polymers, that can be easily detached from the PDMS substrate. The combination of nano-scale control of the polymeric organization along the thickness of the chitosan/alginate (CHT/ALG) membranes, provided by LbL, together with the geometrical micro-scale features of the patterned membranes offers a uniqueness system that allows cells to colonize 3-dimensionally. This study provides a promising strategy to control cellular spatial organization that can face the region of the tissue to regenerate.