Within recent decades, the development of nanotechnology has made a significant contribution to the progress of various fields of study, including the domains of medical and pharmaceutical sciences. A substantially transformed arena within the context of the latter is the development and production of various injectable parenteral formulations. Indeed, recent decades have witnessed a rapid growth of the marketed and pipeline nanotechnology-based injectable products, which is a testimony to the remarkability of the aforementioned contribution. Adjunct to the ability of nanomaterials to deliver the incorporated payloads to many different targets of interest, nanotechnology has substantially assisted to the development of many further facets of the art. Such contributions include the enhancement of the drug solubility, development of long-acting locally and systemically injectable formulations, tuning the onset of the drug’s release through the endowment of sensitivity to various internal or external stimuli, as well as adjuvancy and immune activation, which is a desirable component for injectable vaccines and immunotherapeutic formulations. The current work seeks to provide a comprehensive review of all the abovementioned contributions, along with the most recent advances made within each domain. Furthermore, recent developments within the domains of passive and active targeting will be briefly debated. Though the word “parenteral” terminologically refers to the routes of administration that avoid the alimentary canal, parenteral delivery in today’s health care system mostly involves the injection of the drug through intradermal, subcutaneous, intramuscular, intravenous and intra-arterial pathways. Adjunct to the injectable formulations, parenteral dosage forms also include biodegradable implants, transdermal patches, and ocular delivery systems . The focNotwithstanding the invasiveness, injection remains an indispensable route of delivery for a wide range of active pharmaceutical ingredients (APIs). In addition to advantages such as the rapid onset of action, possibility to administer a mixture of APIs, and convenience for hospitalized patients with special conditions , parenteral administration is associated with a wide range of benefits, such as avoiding the hostile gastrointestinal environment, possibility to deliver macromolecular APIs with low gastrointestinal absorption , circumventing the hepatic first pass metabolism, and potential to achieve an extended duration of the therapeutic effect ,3.Conventionally, injectable parenteral dosage forms can be formulated as solutions, suspensions or emulsions. The advent and development of nanotechnology, however, has introduced new opportunities to improve the efficiency and elaborate the potentials of these conventional dosage forms . A varieOne of the early grounds upon which the first injectable nanosystems were developed was to enhance the solubility of the drugs with limited hydrophilicity. Low water solubility is a major challenge restricting the clinical translation of a remarkable number of actives. According to the estimates, as high as 40% of the marketed drugs and 90% of the pipeline products suffer from low aqueous solubility issues . The BioDepending on the properties of the payload and the additional delivery considerations and requirements, various types of nanocarriers have been exploited to address low water solubility issues of different APIs. An overview of these has been presented in Polymer-based systems create a further platform to ameliorate the solubility of low water-soluble APIs. The most common polymeric systems used within this frame include the polymeric nanospheres and nanocapsules, drug-polymer conjugates, polymeric micelles and dendrimers. While hydrophobic polymers can be used to entrap or encapsulate lipophilic BCS class II compounds, direct conjugation of the hydrophobic BCS class IV APIs to the polymeric chains using cleavable bonds provides further opportunities to enhance the solubility thereof. Linear polymers conjugated to chemotherapeutic drugs have been perhaps the most fascinating candidates to improve the water solubility of these agents, while enabling their targeted delivery to the tumor site. The most common of the polymers used for this purpose include poly(ethylene glycol) (PEG), N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers, dextran, poly L-glutamic acid (PGA), and carboxymethyldextran, ,21. SeveInclusion complexion of hydrophobic drugs with cyclodextrins is another popular approach to improve their water solubility. From a structural perspective, cyclodextrins are cyclic glucosaccharides comprised of glucopyranose units connected via 1,4-linkage, and are classified as α, β and γ based on the number thereof . While hYet another interesting approach to enhance the solubility of low water-soluble APIs is their conjugation with different proteins. Albumin is one of the most popular proteins used for this purpose, to which the low water-soluble drugs can be either chemically conjugated or else adsorbed . AdditioApart from the use of nanosized carriers for solubility enhancement purposes, the field of parenteral delivery has further benefited from the advent of the nanocrystal technology. Crystalline nanosized drug particles, nanocrystals address the low solubility issues mainly through the enlargement of the surface area to volume ratio , which lNanocarriers can be formulated to tune the rate or the onset of the cargo’s release. Since these features require different formulation considerations, they will be separately discussed in the following.From the patient’s perspective, invasiveness renders parenteral injectables less favorable than their alternative non-invasively administered counterparts. Nonetheless, injection-based delivery is an indispensable route of administration for a variety of APIs. At the forefront of this approach are macromolecular drugs, such as monoclonal antibodies and recombinant proteins, many of which possess remarkably short elimination half-lives ranging from minutes to hours ,58. HencSubcutaneous or intramuscular injection of the drug solution, emulsion or suspension is a classic approach to achieve a retarded systemic penetration. The systemic release of the drug molecules in this case involves their dissolution within the tissue fluids, followed by their traverse through the interstitium to reach the blood or lymphatic capillaries . This wiDrug solutions in oily vehicles provide well-established opportunities for localized prolonged delivery of the lipophilic actives . The susAdjunct to ensuring a prolonged release of the drug from the injection site, localized injection has been also exploited to obtain an extended systemic release of the “drug-loaded nanocarriers” as an entire entity . It is wAn additional advantage of nanosystems within this frame is their ability to simultaneously prolong the release of a multitude of APIs. While conventional polymeric and lipid-based nanostructures allow for the encapsulation of a cocktail of drugs with similar lipophilic properties , incorpoActives with low water solubility can be also locally administered as nanocrystal formulations. Albeit nanocrystals lead to a rapid dissolution of the drugs when subjected to sink conditions, the presence of limited amount of fluids under the subcutaneous and intramuscular conditions can account for a sustained release behavior . For insAnother platform in prolonged drug delivery to which nanotechnology has significantly contributed is the field of in situ forming depot formulations. In principle, these systems are liquids with appropriate syringeability, which form (semi)-solid networks upon localized injection . Based oAmong various in situ forming depot systems, hydrogels have been perhaps most widely investigated. As water-insoluble polymers forming three-dimensional cross-linked networks upon injection, hydrogels are able to absorb considerable quantities of aqueous biological fluids . The degNanotechnology has contributed to the development of prolonged release hydrogel systems in two different ways. The first approach involves the development of hydrogel-nanoparticle composites, which help combine the advantages of both systems. Within this frame, the hydrogel matrix serves to protect the integrity of the nanoparticulate systems while preventing the burst release and further limiting the systemic penetration of the incorporated therapeutic cargo . NanoparA second approach reconciling the nano- and hydrogel technologies is the development of nano-scaled hydrogel, in other words nanogel, systems. Combining the advantages of both parent technologies, nanogels offer numerous advantages such as hydrophilicity, biocompatibility, versatility, flexibility, high loading capacity, controlled release properties, and high water absorptive properties of the hydrogel systems and the nanoparticle-related targeting and multifunctionalization potentials . NanogelFour different classes of polymers have been hitherto exploited for the preparation of nanogels. These include polyacrylates, poloxamer or polyethylene glycol, polypeptides and polysaccharides . One of Another platform for the development of injectable sustained release systems involves the use of amphiphilic polar lipid molecules that can self-assemble in contact with excess water to form viscose liquid crystalline formulations . DependiIn general, intravenous injection is not an appropriate route for the prolonged delivery of naked drug molecules. Nonetheless, nanocarriers have provided exciting opportunities for the development of long-acting systemically injectable formulations. It goes without saying that an extended release of the drug from systemically injected nanocarriers necessitates their prolonged presence in the systemic circulation. As the concept of long-acting and systemically targeted nanoparticles have been often inseparably investigated, more detailed information regarding the formulation of such particles will be presented under the section related to the latter. It is, however, worth noting that long systemic circulation can facilitate a prolonged release of the payload from the carriers. Alternatively, a sustained release of the drug can occur following the accumulation of the circulating nanoparticles within the target tissue. For such reasons, systemic long acting and passively targeted formulation often go hand in hand, though few studies have focused on the development of nanosized systems with the sole aim of prolonged drug delivery. Examples include the PLGA-PEG nanoparticles of low molecular weight heparin (for the treatment of venous thrombosis)  and boviOne desirable characteristic of an ideal delivery system is to release the incorporated payload at the right time and in the right place, so that the adverse effects of the drug on non-target organs or tissues as well as the required dosage can be alleviated. A potential contribution of nanotechnology to achieve the abovementioned objective is the design and development of delivery systems capable of releasing the incorporated therapeutic cargo in response to specific internal or external stimuli . The sigTemperature-sensitive nanocarriers have been designed with the main objective of releasing the incorporated payload in response to the internal or external changes of the ambient temperature. While externally induced hyperthermia is the major trigger of the drug release in such systems, internally elevated temperatures is observed in several disorders including infections, inflammation and cancer . ThermosThermosensitive polymers such as poly(N-substituted acrylamides)  and polyAnother approach for the development of thermosensitive nanocarriers can be based upon the polymeric structures that swell, rather than shrink, above their so-called upper critical solution temperature (UCST). Nanogel systems prepared with acrylamide and acrylic acid exhibit such a behavior in the presence of sodium chloride or similar salts, where the swelling above UCST triggers the release of the entrapped cargo . CompareApart from modification with thermosresponsive polymers, temperature-responsive lipid-based nanostructures such as liposomes can be also prepared using thermally sensitive lipids such as dipalmitoyl phosphocholine (DPPC), which possesses a phase transition temperature of about 41–42 °C. Above this temperature, the lipid will undergo a gel to liquid crystalline phase transition, facilitating thereby the release of the loaded cargo . The comLight is a convenient release trigger given a variety of advantages it offers. These include non-invasiveness, spatial and temporal controllability, diversity of the applicable spectrum and the inducible photochemical reactions, and possibility of remote handling ,155.A variety of strategies have been developed to render nanomaterials light responsive, the detailed discussion of which is out of the scope of this review. Herein, we seek to merely present an overview of the most important of these approaches. For more detailed information, the reader is referred to a comprehensive review by Fomina et al. . One comA major limitation of the UV and visible spectra is their limited penetration depth (about 10 mm) in the body. The application of these is thus limited to superficially  accumulated nanoparticles . Also, tHypoxia, the state of inadequate oxygen availability, is a hallmark of various disorders such as cancer, ischemia, rheumatoid arthritis, cardiomyopathy and vascular diseases . In cancDespite its association with a poor prognosis and its encouraging role in the tumor development and chemoresistance, hypoxia can serve as a specific tumor-induced trigger ensuring the site-specific release of the drug molecules from nanocarriers . The majDevelopment of pH-responsive nanocarriers has been pursued for the purpose of initiating the drug release within the organs, tissues and intracellular compartments with pH values deviating from the physiological norms . pH-RespOne of the most typical tissues of acidic nature is the tumor extracellular environment, whose slightly acidic pH (between 6.5–7.2) is a byproduct of enhanced lactic acid production and retention under the intratumoral hypoxic conditions . This unIntracellular compartments such as endosomes and lysosomes undergo rapid acidification, mainly owing to a vacuolar ATPase-mediated proton influx. Following the endocytosis of foreign particles or materials, a reduction of the pH to 5.0–6.5 and 4.0–5.0 is observed within the endosomes and lysosomes, respectively . This acDespite the diversity of the materials used for the fabrication of pH-responsive nanocarriers, the function of these systems is based on two general strategies. One mechanism involves the preparation of the nanocarriers using materials with ionizable functional groups, which due to protonation in acidic pH trigger drug release following the disruption of the nanocarrier structure . The secRedox-responsive nanocarriers often comprise chemical groups sensitive to oxidation or reduction . Among tA further application of redox-responsive nanosystems is to increase the specificity of the drug release locus to the cytosolic compartment of the tumor cells, where the GSH concentration is at least four times higher than normal . Of partSubstrate-incorporated nanocarriers provide further opportunities for the site-specific release of the APIs. Upon the biocatalytic action of the enzyme on the corresponding substrate, a programmable onset of drug activation or release can be achieved at the desirable location . ApplicaIn general, two main classes of enzymes have been exploited for the formulation of such systems; hydrolases and oxidoreductases. The former comprises different subcategories such as proteases, lipases, and glycosidases, which act upon peptides, lipids and carbohydrates, respectively . Among sSubstrates can either serve as enzyme-cleavable cross-linkers between the drug and nanoparticle ,218, or Weak electric fields  can serve as attractive exogenous stimuli to trigger drug release from the delivery systems responsive thereto. This attraction mainly lays within advantages such as ease of generation and high controllability along with the possibility of remote application and simplicity of the required equipment . HoweverConventional use of exogenous electrical fields to the contrary, abnormal electrical activities associated with seizures has been exploited as an endogenous stimulus to trigger the release of antiepileptic drugs upon demand. Wang et al. developed phenytoin sodium loaded electroresponsive nanogels modified with the brain targeting peptide angiopep-2 . DependiGiven their remote controllability and intrinsic tissue penetrability, applications of magnetic fields in drug delivery and diagnostics are abundant. Magnetic fields can be exploited as guides for targeted drug delivery and diagnostics, for the induction of local hyperthermia, for magnetic resonance imaging, and for on-command release of the therapeutic cargo from delivery systems ,229,230.3O4, or maghemite, γ-Fe2O3) with a functionalizable polymer or metal coating, or else a porous polymeric matrix with intraporous-precipitated magnetic nanoparticles [Structurally speaking, magnetic nanoparticles can comprise of a magnetic core , which is also known as reticuloendothelial system . The renA particular niche of nanoparticle-based drug delivery is revealed in the targeting of inflamed tissues with abnormal immune cell infiltration. The most renowned example is of course the tumor microenvironment, where the presence of numerous angiogenic blood vessels with defective and leaky vascular structures along with the low intratumoral lymphatic drainage prompts the intratumoral deposition of nanoparticles with appropriate sizes  . DespiteRelative to free drug molecules, nanoparticles can also facilitate drug targeting to the CNS, which in particular paves the way for the treatment of various neurodegenerative disorders . The keyA further, yet less frequently debated, potential destination for passively targeted nanoparticles is the kidneys. Though kidney is one of the organs responsible for the clearance of nanoparticles of small sizes, the substantial part of any injected nanoparticle dosage will end up in the liver and the spleen. Nonetheless, nanocarriers can be designed to provide a decent concentration of the incorporated drug in various parts of the renal tissue, which, in addition to diagnostic purposes, is indeed exploitable for the treatment of various renal or even systemic disorders including acute kidney injury, chronic kidney disease, glomerular diseases, kidney cancer and hypertension . The abiIn contrast to the above-mentioned scenarios, a long circulation time is not a prerequisite for the passive targeting of organs such as spleen. Splenic accumulation of nanoparticles is of particular interest for the passive targeting of the immunotherapeutic payloads to the splenocytes, whose activation can exert substantial therapeutic effect in many disorders . FurtherActive targeting seeks to enhance the site-specificity of drug delivery through the modification of the nanocarrier surface with moieties that possess high affinity for certain molecules  abundantly expressed in the target of interest . While aH and CL) and a variable (VH and VL) segment, with the latter being responsible for antigen recognition function [Affinity proteins possess a high selectivity in binding to certain molecular structures and are hence of great interest in active targeting approaches. Among various affinity molecules, antibodies are best known for their specific recognition of the antigenic epitope against which they have been developed. Structurally speaking, antibodies are Y-shaped glycoproteins comprising two antigen recognition domains (Fab fragment) and two identical domains with effector function (Fc fragment). Each Fab domain is in turn composed of a light (L: 24–25 kD) and a heavy (H: 55–70 kD) chain held together by disulphur bridges. Each chain has a constant  affinity proteins  with a robust three-helix structure based on a modified B-domain of staphylococcal protein A, are the most widely known types of scaffold proteins . CombinaA further category of protein scaffolds, affimers are derived from Adhiron scaffold, a synthetic thermally stable protein originally based on a cystatin consensus sequence, and is structurally related to a previously reported scaffold engineered from human stefin A . NotwithAs our knowledge of the potential binding sites for various scaffold proteins increases, it is expected that these find a more distinct spotlight as active targeting moieties for nanoparticle modification. This is in particular evident from the fact that literature has lately witnessed the emergence of various scattered studies reporting the use of different types of scaffold proteins, such as chromobodies , CentyriIn addition to antibodies and antibody mimetics, other peptides with high affinity for certain targets have been identified and exploited for nanoparticle modifications. Thanks to the development of peptide phage libraries, plasmid peptide libraries, bacterial peptide display libraries, and other novel screening technologies, numerous peptides have been identified and many have been successfully used to enable targeted nanoparticle-based drug delivery to different cells and even intracellular compartments . A few eLectin is a general term referring to proteins or glycoproteins with affinity for sugar moieties available in various glycoconjugates. Not only can lectins serve the purpose of site-specific nanoparticle-based drug targeting, they can also prolong the nanoparticle/drug residence in the target site, establish close contact between the nanoparticle/drug and the target cell membrane surface, and enhance the trans-epithelial drug transport rate through specific cellular interactions . Even thAs potential binding sites for lectins require the superficial expression of a good number of sugar moieties, lectin targeting is often plausible for mucus secreting tissues such as the oral cavity , gastroiThe above-debated notion can be reversed to target endogenous lectins or carbohydrate receptors by decorating the nanoparticles with appropriate sugar or carbohydrate moieties, also known as glycosylation. Glycans can be classified both as affinity molecules and ligands for receptor targeting depending on the nature of the molecular target. Since the previous section dealt with the introduction of the lectin-based targeting, however, the glycan-based targeting will be elaborated here. Two strategies have been hitherto used for glycan-based targeting; the first involves the decoration of nanoparticles with various sugar moieties, among which glucosyl , mannosyAptamers are affinity molecules of oligonucleotide nature. They are single-stranded DNA or RNA oligonucleotides of 6–30 KD  with unique three-dimensional structures that endows them high affinity and specificity for certain target molecules. Aptamers are produced through SELEX  technology. Despite the diversity of the available SELEX methods, they are all based on the repetition of four different steps: incubation of the target of interest with the available library of random aptamers, elution of the bound sequences, amplification of the bound sequences, and the separation of the single-stranded oligonucleotides .To this day, numerous aptamers have been developed through SELEX technology and used to facilitate active targeting of various organic and inorganic nanoparticles to different types of cancer cells, ,356,357 Nanoparticle decoration with the ligands of overly expressed receptors on the target of interest has provided a further popular platform for active targeting to various healthy or diseased destinations. Ligands used for targeting purposes need to have substantially high affinity and specificity for the receptor of interest, and relatively simple conjugation chemistry. For instance, abundant expression of transferrin receptors , folate A less commonly debated domain to which injectable nanoparticles have assisted is the field of vaccination. The contribution of nanoparticles to this arena goes well beyond their ability to load, protect, target and deliver the immunotherapeutic cargos to the immune cells of interest, particularly APCs . Those cIt is widely known that the specific size range of nanomaterials promotes their recognition as “foreign” and “non-self” by the cells of the innate immunity . Once onAlternatively, many nanoparticles have been shown to behave in a pathogen-mimicking manner, interacting with various pattern recognition receptors (PRRs). For instance, some nanoparticulate systems are believed to induce the self-oligomerization of Nod-like Receptor (NLR) family members and the subsequent activation of the inflammasomes , thereby triggering the autocleavage of caspase 1 and the subsequent production of IL-18 and IL-1β . NLRP3 aA further platform of nanoparticles’ contribution to the field of immunotherapy involves the design of biomimetic nanoparticulate artificial APCs (aAPCs). These particles are tagged with moieties that enable the activation of both the first and the second activating signal on the T cells . BrieflyThe advent and development of nanotechnology has made a significant contribution to the development of various aspects of injectable parenteral delivery. While the possibility of active and passive targeting might be considered the major niche of nanotechnology within this arena, nanoparticles have indeed substantially assisted further facets of the art. Beginning with the development stage, nanotechnology has enabled the formulation of numerous low water-soluble APIs as aqueous-based injectables, whose administration as such would be otherwise challenging, if not totally impossible. The nanoparticle platform can be also exploited for the development of locally or systemically injectable long-acting formulations, which will for sure improve the patient compliance. From the biointeraction and biodistribution perspectives, nanoparticles can be modified to have substantial or minimal encounter with the immune system depending on the desired objective. A significant interaction will be of great benefit for immunotherapeutic and vaccination purposes, whereas a minimum exposure is desired for long-acting or targeted formulations. Eventually, passive and active targeting approaches can facilitate the accumulation of the cargo within the desired destination, increasing thereby the ratio of the off-target to on-target drug and improving the patient’s benefit. This can be of course significantly favored by the possibility of a triggered drug release through the design of stimuli-responsive nanocarriers.The question that presents itself at this stage is to what extent a translation of such nanoformulations from the laboratory to the market would be possible. Notwithstanding the considerable number of the hitherto marketed nanopharmaceuticals see , the ratThe first level involves the material characterization, scale-up and production. Many of the nanoformulations reported in the literature have been prepared using non-FDA approved materials, and through processes that sometimes involve harsh chemicals and relatively unsafe organic solvents . While tFrom the manufacturing perspective, development of nanopharmaceuticals often requires sophisticated processes involving size reduction , purification , stabilization , sterilization, and so forth . ConventThe second level includes the limitations associated with the availability of standardized characterization protocols, particularly under the GMP conditions. In fact, nanoparticle characterization techniques and protocols in terms of particle size and surface charge measurements, determination of drug loading, localization and release, and assessment of in vitro and in vivo cytotoxicity require fundamental validation and standardization, a detailed discussion of which has been presented elsewhere ,404,405.Last but not least, it is essential to heed that, in many cases, nanotechnology comprises a disease-driven approach for drug delivery and targeting. Accordingly, a lack of in-depth knowledge regarding the disease heterogeneity in patients can lead to lower clinical efficiency of many nanoformulations compared to their preclinical performance . This, oNotwithstanding the above-debated limitations, nanotechnology has indeed rapidly grown within the past few decades. Within this context, the extensively debated contributions of nanotechnology to the field of injectable parenterals have led to a significant increase in the number of the marketed and pipeline injectable nanoproducts. With the growth of our understanding about the disease pathophysiology, development of personalized medicine, advancements in dosage form manufacturing, standardization of the characterization techniques and protocols, and development of detailed regulatory guidelines for nanomedicines, the future decades will hopefully witness the introduction of further contributions of nanotechnology to the platform of injectable formulations.


Research into the underlying parameters and mechanisms involved in the emergence and maintenance of major depressive disorder (MDD) has elicited the development of new treatment approaches and improved existing ones, for instance, pharmacotherapy or psychotherapy. Even though there has been significant progress on different levels and in different fields, the efforts to gain a better understanding of MDD have faced constant challenges. High relapse rates and lack of treatment responsiveness leave much room for improvement and call for new insights to reduce the disease burden in MDD—for affected individuals as well as for societies.Regarding the development of new treatments of depression, ketamine and esketamine, represent such an approach from the pharmacological field and has a rapid anti-depressive effect. In the current issue of European Archives of Psychiatry and Clinical Neuroscience, Leal and colleagues present results from an open-label pilot study  showing The development of new treatment modalities, such as ketamine, and the alteration of existing ones to better target-specific symptom profiles emphasizes the need for individualized pharmacological and psychotherapeutic care in MDD. Establishing criteria for optimized treatment allocation remain the goal of extensive research.Within this context, Imai et al.  implemennegative and positive valence, cognitive systems, social processes, arousal and regulatory systems and sensorimotor systems do not only represent a diagnostic framework beyond symptoms, but also the basis for the development of process-oriented treatments.The Research Domain Criteria (RDoC) approach aims to establish the classification of mental disorders based on neurobiological processes and integrate them with the associated behavioral correlates, thus providing a transdiagnostic, dimensional and process-oriented perspective . The domTwo contributions in the current volume pursue the RDoC-perspective. Chen et al.  investigNevertheless, previous research on the effectiveness of different types of MDD treatments, such as ADM or psychotherapy, have often relied upon general outcome parameters, ignoring the underlying processes and their temporal dynamics. This applies especially to psychotherapy research, where RCTs on treatment protocols for disorders have dominated the research landscape. Unsatisfactory answers regarding the superiority of a certain approach and the question “What works for whom?” have spurred on the differentiation of psychotherapeutic approaches.In particular, cognitive behavioral therapy (CBT) underwent this differentiating development, for example, with the establishment of mindfulness-based CBT, schema therapy, and the Cognitive Behavioral System of Psychotherapy (CBASP), which specifically targets the demands of patients suffering from persistent depressive disorder (PDD).CBASP focuses on biographical experiences of maltreatment during childhood and the resulting interpersonal deficits, which can be assigned to the RDoC-domain of social processes . In a naSuch therapy approaches, which tailor down broad, disorder-based treatments to particular target groups, recognize specific processes, such as interpersonal deficits, and consider their relationship with symptom severity. Thus, they take an important step toward a more process-based understanding of the psychopathology of MDD and its treatment.Further translation of a process-oriented, dimensional, and transdiagnostic perspective of the RDoC approach into the behavioral sphere would open up new avenues for the development of psychotherapy treatments. It could be a promising strategy to link RDoC-constructs, such as perception and understanding of self and others, cognitive control, arousal, or reward learning, with their corresponding psychological and behavioral counterparts, such as cognitive biases, emotion dysregulation, lack of motivation, or lack of reinforcing environments. These psychological and behavioral correlates could represent the target of specific intervention modules, which could be applied in personalized psychotherapy approaches rather than as a comprehensive treatment protocol for a heterogenous disorder such as MDD. Such a link not only allows for a more precisely fitting therapy, but also closes the gap between clinically relevant phenotypes and research on underlying RDoC-dimensions.Measuring the outcome of such interventions modules provides an opportunity to overcome prior shortcomings in psychotherapy research, which was often based on the effects of broad treatments on a broad range of symptoms or even questionnaire sum score levels. The idea of capturing the effects on a process level, ranging from molecular manifestations to objective behavioral markers and subjective self-reports, and accounting for process dynamics over time, for example, by ecological momentary assessment, will ensure more precise insights into what drives depression and how to address it best.By integrating a mechanism-based understanding of psychopathology and process-based psychotherapy , we can


Neurodegenerative diseases (NDDs) are incurable and debilitating conditions that result in progressive degeneration and/or death of nerve cells in the central nervous system (CNS). Identification of viable therapeutic targets and new treatments for CNS disorders and in particular, for NDDs is a major challenge in the field of drug discovery. These difficulties can be attributed to the diversity of cells involved, extreme complexity of the neural circuits, the limited capacity for tissue regeneration, and our incomplete understanding of the underlying pathological processes. Drug discovery is a complex and multidisciplinary process. The screening attrition rate in current drug discovery protocols mean that only one viable drug may arise from millions of screened compounds resulting in the need to improve discovery technologies and protocols to address the multiple causes of attrition. This has identified the need to screen larger libraries where the use of efficient high-throughput screening (HTS) becomes key in the discovery process. HTS can investigate hundreds of thousands of compounds per day. However, if fewer compounds could be screened without compromising the probability of success, the cost and time would be largely reduced. To that end, recent advances in computer-aided design, in silico libraries, and molecular docking software combined with the upscaling of cell-based platforms have evolved to improve screening efficiency with higher predictability and clinical applicability. We review, here, the increasing role of HTS in contemporary drug discovery processes, in particular for NDDs, and evaluate the criteria underlying its successful application. We also discuss the requirement of HTS for novel NDD therapies and examine the major current challenges in validating new drug targets and developing new treatments for NDDs. High-throughput screening (HTS) has increasingly been used for novel drug discovery in the field of pharmaceutics replacing the traditional “trial and error” approach to identify therapeutic targets and validate biological effects ,2,3. HTSCurrent programs for central nervous system (CNS) drug development and discovery can be subdivided into four main areas: (1) Receptor and target engagement, (2) drug “hit” identification, (3) lead identification, and (4) drug lead optimization. Active compounds resulting from HTS screens—the so-called “hits”— are the prototypes from which drug “leads” are ultimately formed through additional combinatorial and medicinal chemistry. Following the screening of several hundred thousand small-molecules, a few hundred “hits” may be identified, leading potentially to viable drug compounds . PotentiNeurodegenerative diseases (NDDs) are incurable and debilitating conditions that result in progressive degeneration and/or death of nerve cells in the CNS ,7,8. DevThe creation of successful assays includes the ability to identify the associated events that trigger and cause cell death. To this end, tests have been established to recognize biochemical events that contribute to neuronal death such as metabolic fluctuation, energy metabolism , and fraNovel diagnostic technologies for temporal analysis of the neuronal region and consistency of the cell membrane have been developed, providing assays to track neurodegeneration over time . NeuronsIn this review, we discuss the current challenges in validating new drug targets and developing new treatments for NDDs. Moreover, we review the increasing role of HTS in the drug discovery process focusing on existing platforms that mimic healthy and diseased states of the CNS. In addition, we identify the main strengths and limitations in their applications towards identifying new therapeutic targets and therapeutics for NDDs.Druggable targets are scanned either virtually utilizing virtual compound structure libraries and/or by cell-based or biochemical testing of available peptide or chemical libraries via high-throughput screening (HTS). Abbreviations: Food and Drug Administration (FDA).HTS involves in vitro, cell- or whole organism- based assays . The mosData from screens can be archived and reviewed using information management systems  or more Using cell-based assays, whole pathways can be investigated generating numerous potential points of interest, as opposed to the analysis of particular predetermined steps as in biochemical assays. Moreover, cell-based assays may provide data that cannot be obtained from a biochemical assay, such as the existence of the pharmacological activity of the screened compound at a particular receptor or the intracellular target ,34. ConsHTS is frequently accomplished using scaled down cell-based methods. Cell-based tests enable chemical libraries to be tested for molecules that exhibit a diversity of biological activities. In the pharmaceutical industry, cellular microarrays utilizing 96- or 384-well microtiter plates with 2D cell monolayer cultures are commonly used . CellulaBiochemical screening utilizes a purified target protein of interest and measures the binding of ligands or the inhibition of enzymatic activity in vitro . These aIn summary, biochemical assays have the advantage that all hits found are against a known target by design. However, in those situations, the often costly and tedious determination of the molecular mechanisms of action would be needed, even though the target is known. Furthermore, due to the degree to which the predicted target was initially validated in the disease phase, the therapeutic potential of an in vitro hit can still be inconsistent. Even following the determination of such mechanistic details, it is difficult to predict the behavior of such compounds in a more complex cellular environment, due to variability in cellular permeability and metabolism, toxicity, selectivity, and the potential off-target activity of the compound . HoweverHTS aims to decrease the costs of drug invention ,60. It iIt is crucial to extensively enhance our knowledge and understanding of CNS diseases in order to be able to develop effective therapies. Interestingly, despite the number of individuals in the US who experience CNS disorders being more than double than people who suffer from cardiovascular diseases (CVDs), the global market for CNS therapeutics constitutes less than a third of the global drug market for CVDs . TherefoThe primary explanation for this under-development of the worldwide brain drug market is that the vast majority of CNS drugs do not cross the in vivo blood-brain barrier (BBB). The BBB is a unique and highly selective vascular interface that separates the peripheral blood circulation from the neural tissue in order to maintain an optimum homeostatic microenvironment for brain function and protection ,65. HoweThe procedure involved in developing a new drug is an elaborative effort which is often a costly and lengthy process. On average, the cost of developing a new medicine is around USD 1.3 billion (2018) . HoweverCNS drugs face substantial developmental obstacles relative to non-CNS drugs, largely due to a limited understanding of the complex pathophysiology of many of the diseases they aim to treat, along with difficulties in identifying and assessing acceptable clinical endpoints. Approving a new drug for CNS diseases typically faces additional burdensome regulations. For instance, a study published by the Tufts Centre for the Study of Drug Development (Tufts CSDD) highlighted a real problem that hampers the discovery and subsequent development of CNS drugs. The study found that for the period 1995–2007, success rates for CNS drugs were less than half of non-CNS drug approval rates . Additionally, between 2000 and 2017, the time for approval, after submission of a marketing application for CNS drugs, was 38% longer than for non-CNS drugs . In 2017As a result, CNS drug research and development projects have been exposed to major layoffs and eliminations over the last decade. Although there has been recent revival of interest in CNS drug discovery, past shifts in the priorities of the pharmaceutical and biotech industries represent the well-documented reality of CNS-drug discovery projects. CNS drugs in general have higher failure rates than non-CNS drugs, both preclinically and clinically, and in certain cases, such as for the main NDD disorders, the clinical failure rate for disease-modifying medications has been 100% . CompareAlthough the last few decades have witnessed major developments in our understanding of basic neuroscience, such as neuropharmacology, most CNS pharmaceutical treatments are distinguished not by the treatment of the cause but rather of symptoms. For example, most pain drugs (used in the CNS disease treatment) minimize the discomfort, but do not permanently influence the cause of pain. This is often acceptable for acute pain, especially when the cause is self-limiting, but when the drug is withdrawn chronic and neuropathic pain often returns. The treatment of symptoms, even if followed by severe side effects, can be very effective in psychiatric conditions, but when the drug is stopped, the symptoms usually return without a decrease in severity . The genNDD, also known as “protein-misfolding disorders”, are a heterogeneous group of diseases characterized by extensive neuronal loss, cellular toxicity, and cell proteostatic impairment. Extensive neuropathological, biochemical, and molecular genetic studies indicate that the accumulation of proteins with altered physical and chemical characteristics is a fundamental phenomenon in many forms of NDDs in the human brain, as well as in peripheral organs ,83.The precise aetiology of the majority of NDDs is highly complicated and not fully understood. Studies performed in the last few decades have shown that abnormal protein folding and deposition is a common characteristic within the different types of NDDs see . For insThe discovery and development of any new drug relies heavily on a detailed understanding of the underlying mechanisms of disease and a successful progression from the identification of candidates to the design of clinical trials . However14C]-histamine into mice has demonstrated the rate-limiting function of the BBB. Histamine is a small molecule of just 111 Da, but does not cross the BBB due to the presence of many hydrogen-bond-forming functional groups. In addition, BBB penetration is known to be inversely related to the number of hydrogen bonds  [Only a small class of drugs actually cross the BBB which includes small molecules with high lipid solubility and a low molecular weight (Mw) of <400–500 Daltons (Da). However, There are a few brain diseases that reliably react to this category of small molecules, including depression, affective disorders, chronic pain, and epilepsy ,99. The oss BBB) . Neverthoss BBB) . Except oss BBB) .S. cerevisiae to more pathophysiologically relevant patient-derived cellular models.It is widely accepted that toxic misfolded proteins potentially underlie many NDDs. However, individual targets that regulate these proteins and their detrimental downstream effects are still not fully understood nor established. Phenotypic screening is an objective approach for identifying new targets and therapeutic molecules spanning a wide range of models from primitive organisms such as The HTS of small molecules enables a rapid analysis of the effects of thousands or even millions of small molecules. Hence, it could be highly rewarding to exploit HTS in the field of drug discovery for NDDs. For example, AD is high on the list of leading causes of death in the United States and worldwide and therefore, there is a significant global market for disease-modifying medications for AD . The numDrosophila melanogaster, and Caenorhabditis elegans, have been successfully used in modelling NDDs.The ethical and financial concerns, time, and labor-intensive complexity of animal trials together with the obstacles associated with amending these models to the requirements of HTS have curtailed the wide use of such models for preliminary drug screening assays. Furthermore, the reported failure of many clinical trials addressing NDDs has raised doubts on the relevance of animal disease models to humans and implied the need for superior research tools ,105,106.Danio rerio) is a commonly used in vivo model for different NDDs [Zebrafish  against the harmful impacts of the accumulated misfolded proteins in the neurons of patients suffering from NDDs such as ALS [Drosophila melanogaster [C. elegans such as the rapid growth rate, amenability for genetic modifications, and successful application as a model of NDDs in HTS [C. elegans have a simplistic body plan, lack several defined tissues/organs such as blood, brain, and internal organs, and are evolutionarily distant from humans [Drosophila melanogaster do not have an adrenergic system [Similarly,  for HTS ,118,119.h as ALS , Huntingh as ALS , PD [122h as ALS ,124. Anonogaster ,126, whis in HTS ,128,129.s in HTS . Moreovem humans . Moreovec system . TherefoHuman embryonic stem cells (ESCs) and subsequently human induced pluripotent stem cells (iPSCs) have emerged as powerful tools due their ability to be reprogrammed into several distinctive tissue-specific cell types making them an ideal model for connecting phenotype to genotype ,134,135.Advanced 3D culture models using hydrogels, extracellular matrix (ECM) scaffolds, spheroids, organoids or organ-on-a-chip models have been developed to emulate the physiological environment and functionality of human organs which can be lost in traditional 2D models ,144,145.One of the major challenges facing the HTS in the discovery of promising “hits” for treating the NDDs is the limited capacity of the use of models to perfectly recapitulate the pathophysiological milieu of NDDs. The CNS is one of our body’s most heavily cellularized tissues, and the extracellular matrix, which occupies 20% of the CNS space, is an extremely significant additional layer of complexity. Therefore, current endeavors should indeed be targeted at developing new models and novel screening techniques that better recapitulate the in vivo physiologies . The braHTS is indispensable in the field of drug discovery for NDDs. However, the capability of HTS is not only restricted by the availability of human-relevant NDD models, but also limited by the quality and size of the library of the compounds screened in the HTS. Therefore, expanding the number of small molecules available for HTS increases the odds of discovering efficient disease-modifying drugs for the NDDs. Dynamic combinatorial chemistry (DCC) advancement, the introduction of cheminformatics to the pharmaceutical industry, along with the widespread use of artificial intelligence (AI) in the arena of drug discovery are expected to generate a huge number of compounds that may lead to more targeted drugs with activity in preliminary HTS assays ,175,176.Finally, in addition to the technical challenges and obstacles discussed previously, the elevated cost associated with the process of discovering a novel drug is one of the major factors that push the pharmaceutical industry away from the race to find novel drugs for NDDs. Interestingly, the recent advancements in exploiting AI and machine learning in pharmaceutics has shown enormous potential in making the process of novel drugs discovery cheaper and more effective . RecentlHTS informatics systems continue to embrace these new innovations and increasingly help extract HTS data more efficiently from the initial assays and subsequent platform analyses. In fact, researchers have also developed platforms to mimic multiorgan interactions that are not present in conventional tissue culture systems, using microfluidics with a co-culture system with the aim of studying the pharmacokinetics of drugs  or cell-In conclusion, NDDs are set to become a modern “silent epidemic” placing a major healthcare burden on countries with aging populations. Emerging advances in HTS combined with major developments in disease modelling and computational tools have become fundamental in tackling this unmet clinical demand and will help towards achieving more personalized treatments and effective precision medicine in the foreseeable future.


Changes in membrane potential and neural synchronization explain the ongoing and short-lasting effects of tES, while changes induced in existing proteins and new protein synthesis is required for long-lasting plastic changes (LTP/LTD). Glial cells, for decades supporting elements, are now considered constitutive part of the synapse and might contribute to the mechanisms of synaptic plasticity. This review brings into focus the neurobiological mechanisms and after-effects of tDCS and tACS from in vitro and in vivo studies, in both animals and humans, highlighting possible pathways for the development of targeted therapeutic applications.Non-invasive low-intensity transcranial electrical stimulation (tES) of the brain is an evolving field that has brought remarkable attention in the past few decades for its ability to directly modulate specific brain functions. Neurobiological after-effects of tES seems to be related to changes in neuronal and synaptic excitability and plasticity, however mechanisms are still far from being elucidated. We aim to review recent results from  In the last two decades, therapeutic efficacy of non-invasive transcranial brain stimulation techniques through low-intensity electrical fields has been demonstrated by a number of works and clinical trials providing promising results for many neurological disorders, including stroke  and epilThe basic principle of tES is very simple and based on the negative  and positive  currents and their flow into the brain . HoweverThis work aims to comprehensively summarize the neurobiological mechanisms of tES and discuss future clinical applications. In particular, we first analyzed the technical aspects of electrical stimulation techniques, and then the neurobiological after-effects of tES on the constituents of the synaptic structure, distinguishing those on membrane polarity, neural transmission, synaptic plasticity, neuronal network and connectivity, and finally the effects on glial cells and neuroinflammation.We believe that understanding the basis of the modulatory effect of tES would be particularly relevant for its clinical application in humans, where it could be used to shape the plastic properties of the brain.switching polarity) and modulates the power of oscillatory rhythms in a frequency-dependent manner by synchronizing or desynchronizing neuronal networks  or transcranial alternating current stimulation (tACS). Both techniques produce effects on cortical excitability outlasting the stimulation, up to 3 h with tDCS  and up tnetworks . For exanetworks .The association between the type of stimulation and neural response depends on many physical properties including the electrode type, length, strength, and frequency of stimulation . Low-intTACS is a non-constant current which alternates its pulses with the opposite amplitude , 39. Desstochastic resonance, consisting in the stochastic response of tACS-affected neurons to be either polarized or hyperpolarized; (2) rhythm resonance, synchronizing tACS frequency with the endogenous oscillations; (3) temporal biasing of spikes, a synergistically excitation of the same groups of neurons during each cycle of stimulation; (4) network entrainment of an endogenous irregular neuronal activity that necessitates an external current with sufficiently stronger amplitude; (5) imposed pattern, tACS overcomes endogenous regular oscillations and introduce a new oscillation. These mechanisms attribute the large-scale effects of tACS to two synergistic phenomena: entrainment and neuroplasticity, respectively. The first takes place when an external rhythmic system affects another one, forcing it to follow its own oscillating frequency and phase; the second, through LTP/LTD phenomena, elicits offline tACS effects by increasing or decreasing neural synchronization, as confirmed by many studies , alpha (10–12 Hz), and gamma range (40 Hz), each producing diverse neurobiological effects for modulation of different bands of neural oscillations . The effExperimental and clinical applications of transcranial magnetic stimulation (TMS) is widely and progressively increased over the past two decades. In particular, several repetitive TMS (rTMS) protocols have been proved to modulate brain functions  and human behavior , 50. ForTMS shares fundamental similarities with tES as both share neurobiological modulations at similar levels and involve rapid changes in magnetic fields . While T+ and Ca2+ channels thus causing increased presynaptic release of excitatory neurotransmitters and postsynaptic calcium influx, respectively  and physostigmine (a parasympathomimetic reversible cholinesterase inhibitor) occurred much faster while applying tACS: it was observed that the quantity of presynaptic vesicles first declined, then increased after 5 min and then returned to baseline levels after tACS  are a large family of complex proteins that regulate several functions, including neuronal survival, differentiation, synaptic function, and plasticity but also neuronal death through interaction with two types of receptors, the tyrosine kinase receptors  and the common p75NTR receptor . Most of2+ signals and activity of NMDA glutamate receptors , and LTD generation  or inhibitory (LTD-like) effect of the brain stimulation. Early LTP/LTD modifications usually last for 30–60 min after induction and reflect post-transcriptional modifications of pre-existing proteins, such as protein phosphorylation, in contrast late LTP/LTD could last hours, days, and even months and require genes and proteins expression  .In order to shed light on the pathways leading to the synthesis of new proteins, attention has been focused on the group of immediate early genes (IEGs), that are rapidly induced following neuronal activation and are thought to be involved in the maintenance of LTP , 94. AmoAbnormally high activity and hyperexcitability of some subcortical pathways, as in the case of after stroke or during central nervous system (CNS) development, may respond to tES that modulates homeostatic plasticity of the hyperexcitable tissue –99. The In addition, metaplastic changes are observed with the administration of tES . The terAberrant plasticity induced by non-invasive brain stimulation techniques has been demonstrated in many neurological and neuropsychiatric disorders including PD –108, dysRegarding tACS, both online and offline effects have reported to generate entertainment and neuroplasticity . EntertaPolarization of the brain tissue can extend beyond the area under the electrodes –121 and Functional connectivity of cortical networks increased within motor, premotor, and somatosensory areas after anodal tDCS, inducing significant intra and interhemispheric connectivity changes, as revealed by analysis of EEG frequency bands .Brain areas interact mutually creating a complex network that underlie higher brain functions and neural synchronization represents an essential system to coordinate cortico-cortical and cortico-subcortical areas , 133. A See The relevance of glial biology cannot be neglected to understand the complexity of the CNS and the comprehensive mechanisms and effects of tES. The significance is clinically appealing as glial cells create a wide neuro-glial network for rapid inter-cellular long-range signaling  and are 2+ in cortical astrocytes that is correlated to an overexpression of the glutamate NMDA receptor , an indicator of the overall brain/neural activity, and (2) a reduced neuronal reactivity (In patients with multiple sclerosis (MS) that received tDCS, MRI detected (1) increased cerebral metabolic rate of oxygen (CMROactivity .Seizures are described as a result of an increased excitability and inefficient inhibitory control in foci with altered neuronal homeostasis , 174. InApplication of tDCS is not limited to the cerebral cortex and its disorders but also for the modulation of the excitability in the cerebellum and spinal cord. Since pharmacological approaches to treat cerebellar diseases are still lacking, tES might represent a new potential therapeutic approach that is yet to be explored. The mechanisms behind the neurophysiological effects of tDCS applied over cerebellum have not been extensively researched as compared to cerebral cortex. However, it could be inferred that ionic gradient shifts, cellular activation and inhibition, modulation of neurotransmission may occur in the same way . Evidenc2+channels in the spinal motor neuron dendrites (Application of spinal tDCS is very limited but the preliminary results are extremely interesting. It has been reported that spinal anodal tDCS reduces the amplitude of laser evoked potentials of stimulated Aδ fibers  and increndrites . AltogetTo date, despite the undisputed role of tES in experimental settings in humans as a tool to “switch on/off” specific brain regions that are supposed to be involved in several higher brain functions, its translation into clinical settings is still far to be reached due to the difficulty in producing clinically significant effects in the majority of subjects/patients. This is largely due to the lack of a full comprehension of both the neurobiological bases of tES and the specific neuropathological mechanisms of disease. There are still few data on the possible clinical efficacy of prolonged/repeated protocols of stimulation that might produce persistent changes in synaptic efficacy that cannot be achieved by a short-lasting intervention. In this context, successful trials of prolonged tES protocols could eventually be translated into invasive implants of cortical electrodes for chronic stimulation. Finally, tDCS shows lack of selectivity that might influence different cortical circuits and produce side effects that counteract the effects responsible for the therapeutic action. Therefore, optimizing protocols, electrode size and intensity of stimulation should help to overcome these technical limitations that impedes a tailored approach to the patient and disease.SK: acquisition, analysis, and interpretation of data for the work and drafting the manuscript. FR: supervising and editing the manuscript and final approval of the manuscript to be submitted. VD: conception and design of the work, supervising and editing the manuscript, and final approval of the manuscript to be submitted. MP: critical supervision, manuscript editing, and final approval of the draft to be submitted. GC: conception and design of the work, analysis and interpretation of data, revising the manuscript, and final approval of the manuscript to be submitted. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Microalgae and cyanobacteria have shown significant potential for the development of the next biofuels innovation because of their own characteristics as photosynthetic microorganisms. However, it is confronted with a lot of severe challenges on the economic scaling-up of the microalgae- and cyanobacteria-based biofuels production. One of these major challenges is the lack of a reliable preventing and controlling culture system of biological contamination, which can attack the cell growth or product accumulation causing crashing effects. To increase the commercial viability of microalgae- and cyanobacteria-based biofuels production, overcoming the biological contaminations should be at the top of the priority list. Here, we highlight the importance of two categories of biological contaminations and their controlling strategies in the mass cultivations of microalgae and cyanobacteria, and outline the directions that should be exploited in the future. The worldwide increasing environmental pollution issues associated with the use of fossil fuels as well as their expected scarcity in the near future have triggered the need for exploring new alternative fuels produced from clean, efficient, and sustainable production processes, such as biofuels ,2,3,4. BBased on the source of feedstocks and bioprocess technologies, four generations of biofuels have been developed. First-generation biofuel is derived from grain crops and oleaginous plants . Second-The utilization of microalgae and cyanobacteria could provide numerous advantages in common, such as the higher growth rate and photosynthetic efficiency compared to plants, the ability to thrive in areas that cannot support agriculture, and the high degree of environmental tolerance ,14. SepaBoth microalgae and cyanobacteria biomanufacturing have proved to be a promising solution for sustainable production of biofuels, so that a large number of research and development programs in this field are in progress worldwide ,26. HoweThis review aims at updating progress on the biological contaminations and the controls in the mass cultures with microalgae and cyanobacteria for biofuels production. We emphasize here the significance of preventing and controlling both biomass-related contamination and synthetic product-related contamination emerging in the direct algae-to-biofuels production should be exploited in the future .The biological contamination is a widespread problem during mass cultivations of microalgae and cyanobacteria because the contaminants will inevitably go into the culture by water or air. Depending on the objectives, these biological contaminants can be reducible to two groups, one of which can significantly constrain or destroy the cell growth of the microalgae or cyanobacteria strain, while the other can significantly inhibit or consume the products produced or secreted by microalgae or cyanobacteria.Brachionus calyciflorus and Brachionus plicatilis were considered the most common but destructive grazer [B. calyciflorus is able to consume more than 500 cells of Chlamydomonas reinhardtii per hour per rotifer [B. plicatilis can eat about 4800 cells of Nanochloropsis sp. per hour per rotifer, thus resulting in pond crash in a few hours [At present, in general, cell growth-affecting biological contaminants that emerged in mass cultivations of microalgae and cyanobacteria are summed up in four forms: zooplankton, microorganisms, including prokaryote bacteria, eukaryotic fungi, and eukaryotic protozoa; other algae; and virus ,40,41. We grazer . Fischer rotifer . Lubzensew hours ,45,46.Synechocystis strain in our work [Pannonibacter phragmitetus. Physiological analysis proved that this contaminant P. phragmitetus really could grow in BG 11 medium with ethanol as the sole carbon source, and the consumption rate of ethanol reached 0.37 g per liter per day, 68% higher than the ethanol productivity of current engineered cyanobacteria [With the development of systematic metabolic engineering and synthetic biology, engineered microalgae and cyanobacteria, especially prokaryote cyanobacteria, have attracted attention as a “biosolar cell factory” for the direct conversion of carbon dioxide into biofuel products . Experimour work ,47, biolMicroalgal and cyanobacterial biomass-based biofuels belong to the third generation of biofuels, while direct converted biofuels based on microalgal and cyanobacterial cell transformation platforms are proposed as the fourth generation of biofuels; both third and fourth generations are considered as a viable alternative energy source to replace or supplement fossil fuels . All theIn order to prevent and control the biological contamination, besides the infection patterns, we should also explore the infecting sources of the biological contaminants. Whatever the materials or the structures of the photobioreactors used in mass cultivations of microalgae and cyanobacteria, biological contaminants are inevitable by large volumes of input medium, gas exchange, and the blind angles of photobioreactors. The infecting sources of biological contaminations are summarized as “AAA” as follows.7 m3 [Existing commercial mass culture systems, including open pond systems and closed photobioreactors, range in volume from <1 to >107 m3 ,55. Acco7 m3 . Apparen7 m3 .2 input and removal of excess dissolved oxygen when open ponds are used in mass culture of microalgae and cyanobacteria [2-air gas mixture. The more gas exchange during the cultivation, the greater the chance of biological contamination occurring.The exposure of culture to air is helpful for atmospheric CObacteria . HoweverPhotobioreactors with different materials and structures have different blind angles, such as the immobilized gas distributor of the membrane photobioreactors, the pipe connection of the tubular photobioreactors, and the sampling system of all kinds of bioreactors. The nutrient, salt, or the microalgae and cyanobacteria cells that remain in these blind places not only cause the contamination of biological pollutants in the next cultivation but also accelerate the bioreactors’ corrosion. In addition, the continuous cultivation mode is always applied in the practical mass culture, making the risk lying in the contamination associated with the residual contaminants attached in the blind angles even higher.2 mixed air, and the contaminants hidden at the blind angles, is also inescapable during the mass culture. Therefore, early detection and early treatment can significantly increase the chance of success for microalgae- and cyanobacterial-based biofuels bulk productions.The specific characteristics of microalgae- and cyanobacterial-based biofuels create the significant advantages as mentioned before, which, however, is a two-edged sword, and the current economically potential mass culture system has led to biological contamination being inevitable. Even the solid culture mode, like attached cultivation , supplemChlorella vulgaris over 20 days, and the biomass data was totally in agreement with the off-line measurement [Synechocystis strain [Detection of biological contaminants during the mass cultivation of microalgae and cyanobacteria is an important step before adopting possible measures for the culture system. Conventional morphologic observation methods, such as staining and microscopy, can be applied as an early and routine detection method . The waysurement . Recentlsurement . Furthers strain .Compared to the synthetic product-affecting contaminations, it is often easier to observe the phenomenon of cell growth-affecting biological contaminations, i.e., cultures gradually becoming yellow or clear. For synthetic product-affecting contaminations, not only might biological contaminants have no impact on the growth of microalgae or cyanobacteria but they also improve the photosynthetic growth. Therefore, a simple and fast detection method like an automated flow cytometry system could provide us an ideal early warning of biological contamination for cell growth-affecting biological contaminants. The detection method associated with the accumulation of the target product might be feasible for monitoring the mass cultivation process during the direct algae-to-biofuels production. In the practical algae-to-biofuels production, cell and target product monitoring detection methods should stand by before mass cultures. All the detected information and identified characterization of biological contaminants could further provide us the specific features for designing the specific control strategies.The objective of studying the biological contamination types and the corresponding infecting patterns, infecting sources, as well as the detection methods of bio-contaminants is to propose or develop reliable solutions to greatly control the biological contamination level and still prevent transmission, thus healing the entire photosynthetic biofuel productions during the mass cultivation of microalgae and cyanobacteria.Chlorella kessleri [Chemical control of microalgae and cyanobacteria cultivation systems usually serve as one feasible solution for controlling biological contaminants. As mentioned before, because of the large body water for biofuel production, hypochlorite or bleach are usually widely used to sterilize the culture medium before or during the culture processes of microalgae and cyanobacteria. Park’s results indicated that the dosage of sodium hypochlorite ranged from 0.45 to 0.6 mg Cl/L and enabled the chlorine concentration effective for inhibiting the grazing of rotifers while maintaining the growth of the microalgae kessleri . Conventkessleri ,64,65. Skessleri . Chemicakessleri . It seemkessleri ,68. TherApplying specific pathogens to control biological contaminants, which break the limitation of conventional chemical methods and overcome the effect of toxicity toward the cell of microalgae or cyanobacteria, may be the ideal contamination controlling strategy in mass cultivation. The implementation of such biological control, however, is dependent on the specific selection to the contaminants. Such selection requires detailed information about the relationships between the contaminants and the microalgae or cyanobacteria strains, which need further investigation .Spirulina platensis growth were further investigated; here, 0.003–0.006 mg/L of the botanical pesticide combination at a 1:9 ratio can inhibit rotifer regeneration within 3 days, and no influences on the Spirulina biomass and phycocyanin levels showed up [Brachionus plicatilis, thus healing the photosynthetic performance of nannochloropsis cells against the contamination damage of rotifer [Compared with the chemical reagents and specific pathogens, plant-derived pesticides may be the preferred choices for controlling biological contaminants in mass culture systems of the microalgae- and cyanobacteria-based biofuel production. According to Huang’s acute and chronic tests, celangulin, matrine, and toosendanin are considered to be good potential rotifer-control botanical pesticides in microalgal mass culture . In addihowed up . Zhang a rotifer . TherefoPhysical filtration and ultraviolet sterilization have been considered as effective and common methods for removal of biological contaminants, but only before the culture starts on the one hand. On the other hand, the removal efficiency of filtration is dependent on the cell size, which also limits the application in treating biological contaminants, such as rotifer eggs and copepod eggs. Ultrasound treatment has also been investigated for the effect on the survival of bacteria, phytoplankton, and zooplankton . The effDunaliella with extreme high-salinity conditions [Synechocystis strain was alkaline tolerant, while the golden algae Poterioochromonas strain was sensitive to alkaline stress at pH 11; thus, a high-pH culture strategy was designed and adopted to selectively inhibit or even kill the golden algae [Synechocystis strain, infection of an ethanol-consuming biocontaminant gradually ceased and devastated ethanol accumulations, and we found that a high-pH strategy maintaining culture environmental pH in an alkaline condition (pH 10–11) could successfully inhibit the contamination level and recover the photosynthetic ethanol accumulations [Microalgae and cyanobacteria are among the most primitive life forms on our planet, and they have the ability of adaptation to almost any of the most extreme habitats on Earth . Therefonditions ,75,76. Ten algae . In our ulations . In addiulations ,78,79.Biological contamination can occur in any stage of the mass cultivations for biofuel photosynthetic production in the form of chronic or acute infection. Four main categories of control approaches as shown in Photosynthetic biorefinery engineering provides a promising solution for sustainable biofuels production. Microalgae and cyanobacteria are among the most promising photosynthetic platforms. Considering economic and operation feasibilities, microalgae and cyanobacteria-based biofuels must be performed at a large scale under open systems, which, however, means potential infection and proliferation of diverse biological contaminants. To promote the concept of photosynthetic biofuel in practice, intervention strategies against biological contaminations through the outdoor mass cultivation process must be overcome and economically feasible. Developing a reliable control system for the mass cultivation by combining the specific controlling strategies coupled with early detection, which can serve to not only prevent but also control function, may be a long-term plan. For further optimization of the mass cultivation processes of microalgae and cyanobacteria for biofuels production, more robust photosynthetic platforms with commercialization potential should be constructed, which could be expected by the development of more powerful evolutionary approaches and metabolic engineering, leading to economically competitive scaled photosynthetic biofuels production.


The quality of such experimental models and particularly the level at which they represent cell behavior in the native tissue, is of critical importance for our understanding of cell interactions within tissues and organs. Conventionally, in vitro models are based on experimental manipulation of mammalian cells, grown as monolayers on flat, two-dimensional (2D) substrates. Despite the amazing progress and discoveries achieved with flat biology models, our ability to translate biological insights has been limited, since the 2D environment does not reflect the physiological behavior of cells in real tissues. Advances in 3D cell biology and engineering have led to the development of a new generation of cell culture formats that can better recapitulate the in vivo microenvironment, allowing us to examine cells and their interactions in a more biomimetic context. Modern biomedical research has at its disposal novel technological approaches that promote development of more sophisticated and robust tissue engineering in vitro models, including scaffold- or hydrogel-based formats, organotypic cultures, and organs-on-chips. Even though such systems are necessarily simplified to capture a particular range of physiology, their ability to model specific processes of human biology is greatly valued for their potential to close the gap between conventional animal studies and human (patho-) physiology. Here, we review recent advances in 3D biomimetic cultures, focusing on the technological bricks available to develop more physiologically relevant in vitro models of human tissues. By highlighting applications and examples of several physiological and disease models, we identify the limitations and challenges which the field needs to address in order to more effectively incorporate synthetic biomimetic culture platforms into biomedical research.Research in cell biology greatly relies on cell-based  The lack of this environmental context and structural architecture excludes physical cues for cell-cell and cell-matrix communication, critical for several cellular processes . These physical constraints also impede cells from organizing naturally and spreading vertically, forcing them to flatten out and grow as monolayers  by Gey in the 1950s, made it possible to grow cells in artificial environments, laying the foundation for cell and molecular biology  to reduce stress and unnatural cell responses and rather enable growth of different cell types to foster the development of more realistic culture systems  remain intact while the exact composition varies according to the origin of tissue/organ or the cells seeded  enhance the predictive value of preclinical studies, (ii) improve the way we study physiology and pathology and thus to address biological questions that so far necessitated animal models, and (iii) bridge the gap between current (pre-)clinical research tools and human systems by assisting and advancing drug development processes in terms of science, bioethics and economy , they are not considered ideal sources for modeling human conditions since they do not exhibit normal features, often drifting from the genetic and phenotypic profile of the tissue of origin  and to differentiate toward one or more specialized cell types (potency), representing a versatile source of cellular substitutes for a wide range of applications , mainly due to their therapeutic potential  or from organ-specific adult stem cells (ASCs) and progenitor cells , another fundamental element for effectively engineering tissue equivalents is the choice of the suitable biomaterial(s). In the classic TE paradigm, pre-engineered 3D supports/scaffolds, made from natural or synthetic materials, are used as templates for cell attachment, growth and differentiation toward functional living constructs Vacanti, . As scafA broad range of materials is now available for the fabrication of various types of substrates, the properties of which can be tailored at the micro- and nano-scale to match the requirements of specific applications  or by blending them with other biopolymers , propylene glycol diacetate (PGDA), polyvinylidene fluoride (PVDF), or co-polymers  approaches have also gained a lot of attention as an alternative fabrication route to organize cells in 3D due to their potential to provide precise spatiotemporal control over biophysical and biochemical cues necessary to reproduce a biomimetic microenvironment Murr, . AD apprFinally, a highly promising and popular method for building tissue equivalents, which has been favored by the emergence of the aforementioned AD manufacturing technology, is modular tissue engineering materials and platforms for TE applications by aiding the reconstruction and control of an environment that mimics key features of the natural niche. In the In vivo, cellular responses are influenced by various spatiotemporal biochemical signals ,—depending on the type and state of cell—, have also been found immobilized in the ECM, forming a complex functional network within the body, exerting systemic effects that go beyond their immunomodulatory role , alternatively called microphysiological in vitro models. OOCs technology has emerged from the combination of recent advances in microengineering and fluidic physics with trends in growing cells in 3D, allowing for the development of models that more faithfully recapitulate key features of specific human tissues and their interactions physiological mechanisms of the brain . This distinctive tissue structure is made up of neurons, astrocytes, oligodendrocytes, microglia, smooth muscle cells, brain epithelial and endothelial cells, and pericytes embedded in the brain extra-cellular matrix (ECM), and bears the responsibility of maintaining brain homeostasis by supporting the neuronal activity and by tightly regulating the passage of metabolites, drugs, and other solutes from the peripheral blood into the central nervous system , but also provide feedback on cellular activity, thus facilitating the study of a broad range of physiological phenomena , with which it is possible to obtain information about cell adhesion, proliferation, and differentiation over time. In fact, EIS is widely used for monitoring Transepithelial/Transendothelial Electrical Resistance (TEER), a commonly used parameter to quantitatively characterize the function and integrity of tissue barriers with fast, non-invasive measurements  doped with poly(styrene sulfonate) (PEDOT:PSS), the electrical, mechanical and biochemical properties of which we were able to fine-tune. We then integrated these tubular PEDOT:PSS scaffolds into a transistor configuration  and showed proof of principle for continuous monitoring of a simple 3D co-culture of mammalian cells over a period of 4 days. The real-time electrical readouts, cross-validated with optical analysis, enabled us to closely monitor cellular activity and even distinguish between cell adhesion and barrier tissue formation in a non-destructive, label-free manner, highlighting the added benefits of integrating in-line sensing components within engineered tissues for building more robust and sophisticated TE models  to guide phenotypic behaviors. This provides an excellent opportunity for transforming drug development routes by accelerating the process, and generating data more accurate and relevant to human systems. In addition, the use of patient-specific cells facilitates studies of rare diseases, as well as precision and personalized medicine approaches for the development of drugs and therapies optimized for specific patient biology. Smart biomaterials have now been designed to more faithfully recapitulate the chemical, mechanical and topographical properties of the complex human tissues and their microenvironment. Advanced fabrication methods have additionally enabled arrangement of tissue counterparts and environmental cues with unprecedented control and accuracy. These features have combined to generate complex biological structures of high fidelity, matching the in vivo situation. A key challenge remains however: monitoring and characterization of such models still relies predominantly on end-point, invasive assays, delaying the validation of results, particularly for drug screening, and toxicology studies. Integration of electrical components in tissue engineering platforms can help resolve this limitation. Such tools enable dynamic, non-invasive and continuous monitoring of cells, offering rapid insight into different biological events within these systems. Standardization of components such as media formulation is a non-trivial additional challenge, for maintaining the survival and function of multiple cell types, which combined with spatiotemporal delivery of tissue-specific and application-specific  environmental cues, offer a novel means for dynamically controlling and directing tissue generation and maturation in vitro. Without a doubt in vitro tissue engineered systems have shown great promise for revolutionizing many aspects of biomedical research. As more sophisticated and human relevant in vitro models appear in literature, in parallel with commercialization of platforms for hosting such models, we expect to see a paradigm shift in our understanding of human biology as well as in disease diagnosis and treatment.The implementation of TE concepts and methods into biomimetic tissue models, originally directed at regenerative medicine applications, is an accelerating trend. As summarized here, 3D C-MM and CB wrote the text and prepared the figures. RMO wrote the text and edited the manuscript and secured the funding. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


During the last 25 years we have accomplished great advances in melatonin research, regarding antioxidant or anti-inflammatory functions, oncostatic actions, glucose metabolism regulation or plant physiology, among others. Of course, we should not forget the classical, circadian-related functions of the indole, which has recently brought up new and important findings. All together these new discoveries will likely lead the way in the next decade in terms of melatonin research. This special issue collects some of these new advances focused on different aspects of the indole International Journal of Molecular Sciences has promoted the call for publication of articles focused on different aspects of melatonin research, trying to attract the attention of professionals, ranging from basic researchers to clinicians, as the interest in melatonin has grown according to the references published.During the past 25 years, research focused on melatonin has experienced a boost in either the number of laboratories involved or the research lines, and, consequently, the number of publications in the field. Thus, while in 1995 there were under 6000 publications, this has risen to more than 26,000 references at present (PubMed database). This increment in such a specific field requires the addition of a Special Issue, in an attempt to collect and funnel the most interesting topics for general public readers, researchers from other areas, or, of course, groups endeavoring towards indole investigation. For this reason, the During the last two decades, we should highlight three great discoveries in the field that has grabbed the interest of many investigators. The antioxidant properties of melatonin—directly and indirectly —accountsATP channels, concluding that the melatonin physiological rhythm exerts cardioprotection.In this Special Issue, articles have been selected to bring forth new data about some of the abovementioned topics. It is already well known that melatonin receptors play a role in mediating the cardioprotection of melatonin. Here, Prado and co-workers  show howinf), or not (N0), on the descending (repolarizing) phase of the action potential (AP). They investigated the melatonin effects on the Ninf neuronal population, thus confirming the existence of two different populations of neurons, i.e., excitability melatonin-sensitive and -non-sensitive cells . The study confirms that melatonin modulates several parameters related to the action potential in EMS neurons.Two studies focused on the modulatory effects of melatonin on the central nervous system (CNS). In the first report, Ramírez-Rodriguez and co-workers  provide Regarding cancer cells, one of the articles of this Special Issue provides new insights about how melatonin reduces proliferation and wound-healing inhibition in the highly tumorigenic B16F10 melanoma cell line. The report concludes that the profound cytoskeletal reorganization, increase in melanin synthesis, and redox modulation underlie its antiproliferative effects .Hanuszewska and colleagues went deep into the embryological stages in the goose pineal organ, and obtained detailed evidence about the 5-methoxy/hydroxyindoles synthesis . The stuAn interesting collaborative study from several clinical and academic French institutions provides here evidence that the melatonin circulating levels at day of birth are much lower—below detection—in 78% of infants born before 34 GW . This waFinally, an excellent review by Debnath and colleagues revise the current pieces of evidence of how melatonin modulates the response to stress in plants . The autAll the peer-reviewed articles included in this Special Issue are good examples of the top-quality studies currently performed, and also demonstrates that melatonin research is in a continuing forward dynamic, attracting readers, researchers, and clinicians interested in the different aspects related to indole.