Elevated low-density lipoprotein-cholesterol (LDL-C) is a causal factor for the development of atherosclerotic cardiovascular disease (ASCVD); accordingly, LDL-C lowering is associated with a decreased risk of progression of atherosclerotic plaques and development of complications. Currently, statins play a central role in any ASCVD management and prevention strategies, in relation to their lipid-lowering action and potentially to pleiotropic effects. After coronary artery disease, stroke is the most frequent cause of ASCVD mortality and the leading cause of acquired disability, a major public health problem. There is often a tendency to aggregate all types of stroke , which have, however, different causes and pathophysiology, what may lead to bias when interpreting the results of the studies. Survivors of a first atherothrombotic ischemic stroke are at high risk for coronary events, recurrent stroke, and vascular death. Although epidemiological studies show a weak relationship between cholesterol levels and cerebrovascular disease as a whole compared with other ASCVD types, statin intervention studies have demonstrated a decrease in the risk of stroke in patients with atherosclerosis of other territories and a decrease in all cardiovascular events in patients who have had a stroke. The Stroke Prevention by Aggressive Reduction in Cholesterol Levels (SPARCL) trial demonstrated the benefit of high doses of atorvastatin in the secondary prevention of ischemic stroke. In this review, we discuss the evidence, use and recommendations of statins in the primary and secondary prevention of stroke, and their role in other scenarios such as the acute phase of ischemic stroke, cerebral hemorrhage, cardioembolic stroke, small vessel disease, and cognitive impairment. Stroke represents a major public health issue, being a leading cause of mortality and of acquired disability in the general population. For instance, stroke is consistently ranking among the first leading diseases in the Global Burden of Diseases reports, specifically the third leading disease in the overall population in year 2019 and second after ischemic heart diseases in the population aged 50 years and over (It is well-recognized that low-density lipoprotein cholesterol (LDL-C) is a causal risk factor for the development of atherosclerosis, related to the magnitude of the elevation of LDL-C levels and for how long they remain high (cumulative exposure over time) . LowerinHowever, the etiology and mechanisms of stroke are heterogeneous and consequently the role of high LDL-C (and so of lipid-lowering interventions) may be variable depending on the type and pathogenesis of stroke . While tpost-hoc analyses. Therefore, while most primary prevention trials with statins focus on coronary artery disease or composite endpoints, conclusions on stroke are rather limited , or it is a secondary endpoint with the study design not specifically powered to address this outcome , or it is explored in  limited . This lapost-hoc analyses of the studies with pravastatin showed a reduction in the relative risk of suffering a first stroke of 9–11%, which was not statistically significant. However, a more intensive lipid-lowering strategy with a high-potency statin (atorvastatin), as used in ASCOT-LLA and CARDS (in the diabetic population), resulted in a greater reduction of LDL-C levels, and it translated into a significant reduction in the relative risk of cerebrovascular events in primary prevention of 27–48%.There are indirect or secondary data from several primary prevention studies with statins, including ALLHAT-LLT   and WOSCp < 0001) with no increase in hemorrhagic strokes and a non-significant reduction in fatal strokes of 13%.To try to overcome some limitations from individual studies , several meta-analyses have been conducted, attempting to elucidate the effect of statins on the risk of a first stroke in the primary prevention of this condition. Briel et al.  found a Most trials with statins in the secondary prevention of stroke share similar limitations with primary prevention studies, as described above; additionally, they include no or few patients with previous stroke at baseline, with the notable exceptions of the HPS (Heart Protection Study) , SPARCL The HPS was the first study to evaluate the benefit of statins, specifically simvastatin, in the secondary prevention of stroke in patients with a previous history of cerebrovascular disease . This stp = 0.03). Similarly, a relative decrease in the risk of coronary episodes of 35% was observed in patients receiving atorvastatin vs. placebo. It should also be noted that the incidence of hemorrhagic stroke was higher in the group treated with atorvastatin: 55 cases, compared to 33 cases in the placebo group, which represents an increase in the relative risk of 66%; this point raised some controversy over the results of this study. This aspect is more extensively discussed in a later section of this article. Another controversial point of SPARCL is that, from the analyses of the LDL-C levels during the follow-up, it is inferred that up to 25% of patients assigned to placebo received statin treatment at some point during the study follow-up, which, in addition to a protocol deviation bias, could have led to an underestimation of both the atorvastatin-related reduction in the risk of recurrence of ischemic stroke and the potential increased risk of hemorrhagic stroke. In a subsequent sub-analysis in which patients were categorized according to their level of LDL-C reduction  of the trial that had a longer follow-up (median 5.3 years) resulted in concordant results for both a reduction of risk of major cardiovascular events  and a no significant difference in the occurrence of intracranial hemorrhages   or transient ischemic attack (in the prior 15 days) in patients with evidence of atherosclerosis . Two tho53–2.62) .p < 0.001) with PCSK9 inhibitors vs. control  has not been adequately tested in randomized clinical trials, and observational epidemiological studies or imaging studies may not provide a robust evidence to support clinical indications. However, given the fact that in most cases these patients may be characterized as having global atherosclerotic cardiovascular risk , statin therapy may be indicated for global atherosclerotic cardiovascular disease prevention, and thus may merit statin therapy regardless of stroke considerations , 5. SpecAs discussed above, there is evidence supporting the use of statins after an ischemic stroke or a transient ischemic attack to prevent further events and, consequently, the main clinical practice guidelines recommend their use in this situation , 5. HoweThere is a limited number of published randomized clinical trials examining the role of early statin use in acute ischemic stroke patients. The FASTER (Fast Assessment of Stroke and Transient Ischemic Attack to Prevent Early Recurrence) study evaluated the effect of simvastatin 40 mg vs. placebo in patients with a transient ischemic attack or minor stroke within the previous 24 h . The triAnother additional factor to consider is the suggestion that the initiation of statin therapy during the hospitalization for an acute ischemic stroke event could have medium to long term benefits derived from a better adherence of patients to the statin therapy . FinallyCerebral small vessel disease (CSVD) is a pathologic process commonly associated with atherosclerosis and cardiovascular risk factors such as hypertension, hyperlipidemia, and aging . 25–30% There is some evidence supporting the protective effects of statins on progression of CSVD and its consequences. Most of this evidence has been related to the progression of white matter hyperintensities (WMH) and cognitive decline as markers of CSVD. Initial studies on statins and CSVD showed a potential, but not yet confirmed, benefit of these drugs on CSVD. For instance, the PROSPER (Prospective Study of Pravastatin in Elderly at Risk) trial with pravastatin showed a trend toward a lower number of new infarcts in the treatment arm but failed to demonstrate an effect on the progression of WMH volumes . The CarMost recent studies using newer MRI protocols and quantitative evaluation of WMH have shown more concordant results. In a cohort of older adults living in the community , statins were associated to a more favorable disease progression in the sub-group of lowest cognitive performance . Xiong eAll these results together, particularly those from more contemporary studies, support the hypothesis that statin therapy may have a protective effect on cognitive decline through a reduced progression of CSVD. There is however controversy about the potential benefit of statin therapy on other cognitive disorders with a different pathophysiology, such as Alzheimer's disease, although most evidence point toward a neutral effect of statins on Alzheimer's disease features , 49; somThere is a long-lasting and ongoing controversy about the relationship between statin therapy and intracerebral hemorrhage. This is based on the safety results from the randomized clinical trials SPARCL and HPS, which included patients with a prior stroke or transient ischemic attack, and which reported an increased risk of intracerebral hemorrhage among patients allocated to the statin arm compared with control , 51. ThiIn this context, current clinical guidelines state that there are insufficient data to recommend restrictions on statin use in patients with prior stroke but recommend caution in patients with spontaneous intracerebral hemorrhage, specially in the subgroup of patients with cerebral amyloid angiopathy-related lobar intracerebral hemorrhage, in which statin therapy should probably be reserved for compelling indications (very high risk of atherotrombotic events) .There is scarce evidence of the effect of statin therapy in patients with cardioembolic stroke in the absence of a known indication for statins and, consequently, clinical practice guidelines do not present any specific recommendation at this respect. Because cardioembolic stroke can be linked to atherosclerotic disease  and cardiovascular risk factors  statins might also potentially influence the outcomes in these patients. However, no randomized clinical trials addressing specifically this question have been conducted; moreover, randomized clinical trials with statins often exclude patients with cardiac sources of embolism, as occurred in SPARCL .Some evidence from observational studies in patients with cardioembolic or atrial fibrillation-related stroke suggests an association of statin therapy with a lower incidence of vascular events and mortality. A retrospective study by Choi et al. including 535 patients with a first-ever cardioembolic stroke found that statin therapy was independently associated with a reduced mortality . In the While stroke be may derived from a variety of pathologic processes different from atherosclerosis, there is often a tendency to aggregate all types of stroke  together, which have, however, different etiologies and pathophysiologic mechanisms, what may lead to bias when interpreting the results of the studies.Although epidemiological studies showed a weaker relationship between cholesterol levels and cerebrovascular disease as a whole compared with other atherosclerotic cardiovascular disease types, statin intervention trials suggest a beneficial role of statin therapy on cardiovascular, risk reduction, including stroke, particularly in the secondary prevention of stroke from atherosclerotic vascular disease. The SPARCL trial demonstrated the benefit of high doses of atorvastatin in the secondary prevention of ischemic stroke and the TST trial supports intensive lipid-lowering therapy to achieve guideline recommended LDL-C targets of at least <70 mg/dL. The effects of statins are likely mediated by LDL-C reduction, as also supported by the results from trials with other cholesterol-lowering drugs (to which no pleitropic effects similar to statins have been attributable), which have also shown stroke protection in secondary prevention trials in combination with statins. The potential role for statins in the prevention of other different stroke subtypes such as small vessel stroke or cardioembolic stroke, or in cognitive impairment, has not been adequately evaluated; however, since these clinical conditions frequently occur in patients with increased atherosclerotic cardiovascular risk, most patients with these conditions may benefit from statin treatment based on global cardiovascular risk prevention.Further studies and evidence are required, mostly in the primary prevention of stroke and among those cerebrovascular conditions where the atherosclerotic process is not the main mechanism.OMG conceived the present review. All authors reviewed the literature, contributed to the interpretation of the evidence, wrote the manuscript, and approved its submission for publication.LBR reports past participation in investigator-initiated research grants to Hospital Universitario La Paz from MSD; personal fees for consulting/advisory committee from Sanofi-Aventis, Amgen and Bayer; honoraria for lectures from Pfizer, MSD, Novartis, Sanofi-Aventis and Amgen; all outside the submitted work. AV-V reports current or past participation in investigator-initiated research grants to Imperial College London and/or European Atherosclerosis Society from Pfizer, Amgen, MSD, Sanofi-Aventis, Daiichi-Sankyo and Regeneron; personal fees for consulting/advisory committee from Bayer, Regeneron and Radcliffe Cardiology; honoraria for lectures from Amgen, Mylan and Akcea; all outside the submitted work. OMG reports current or past participation in investigator-initiated research grants to Hospital Universitario Virgen del Rocío from MSD, Astra-Zeneca and Sanofi-Aventis; personal fees for consulting/advisory committee from MSD, Ackcea, Sanofi-Aventis, Novartis and Daiichi-Sankyo; honoraria for lectures from MSD, Astra-Zeneca, Sanofi-Aventis, Daiichi-Sankyo, Novartis, Amgen, Ferrer, Esteve, Mylan-Viatris and Pfizer; all outside the submitted work.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Neuropathic pain (NP) is a chronic, debilitating, and resistant form of pain. The onset rate of NP following spinal cord injuries (SCI) is high and may reduce the quality of life more than the sensorimotor loss itself. The long-term ineffectiveness of current treatments in managing symptoms and counteracting maladaptive plasticity highlights the need to find alternative therapeutic approaches. Virtual reality (VR) is possibly the best way to administer the specific illusory or reality-like experience and promote behavioral responses that may be effective in mitigating the effects of long-established NP. This approach aims to promote a more systematic adoption of VR-related techniques in pain research and management procedures, highlighting the encouraging preliminary results in SCI. We suggest that the multisensory modulation of the sense of agency and ownership by residual body signals may produce positive responses in cases of brain-body disconnection. First, we focus on the transversal role embodiment and how multisensory and environmental or artificial stimuli modulate illusory sensations of bodily presence and ownership. Then, we present a brief overview of the use of VR in healthcare and pain management. Finally, we discus research experiences which used VR in patients with SCI to treating NP, including the most recent combinations of VR with further stimulation techniques. Pain management remains one of the biggest challenges in healthcare, mostly due to the uncertain results of drug therapies and their many undesirable side effects . Pain isNeuropathic pain (NP) is among the most prevalent forms of chronic pain in patients with SCI and appears to be particularly difficult to resolve ,6. NP maAt-level NP is perceived in the same area where the lesion occurred. In this case, pain is identified in a segmental pattern anywhere within the dermatome at the neurological level of injury or within the three dermatomes below this level. If the former condition is the case, the pain can be classified as at-level NP even if it is found in one dermatome above the neurological level of the lesion . It usuaWhat triggers maladaptive results such as NP in the reorganization process remains mostly unclear, making treatment difficult. Recently, the role of neuroimmune dysregulation has been considered central to the occurrence of NP caused by early inflammatory response to SCI. This response is necessary for tissue recovery at the injury site during the acute post-SCI stages, but may result in scars that prevent proper functional regeneration and may negatively contribute to central maladaptive neuroplasticity and chronic pain . Drugs tIn light of the above, and because in many patients pain persists despite the use of drugs, non-pharmacological approaches to chronic pain relief are increasingly sought. Such therapies are based on the principle that the pathophysiological consequences of such cortical plasticity may underlie the development of phantom sensations and pain ,13,14,15This study aims to present an overview of the role that VR could have in the management of chronic pain and PN. It focuses on the transversality of the sensory, motor, environmental, and technological factors involved. The key factors in the construction of body awareness and embodiment processes are presented in the next section to show how VR exploits these mechanisms to generate and modulate illusory sensations of bodily presence and ownership. The third section offers insights into how VR is entering clinical practice in numerous ways and then examines pain and its management through VR. The fourth section describes the specifics of patients with SCI and the use of virtual experiences in treating NP, and includes the most recent combinations of VR with further stimulation techniques.Embodiment consists of two components: the sense of ownership  and the sense of acting (understood as the feeling that our body is responsible for its movement) ,19. ProdVR can be defined as a technology that allows the creation of an artificial or virtual environment that simulates reality . The useThe brain uses representations of the body  for probably all “top-down and bottom-up” processes, all of which rely on sensory and motor signals that verify ownership of the body ,32. To bVR facilitates modulating our perceived sense of ownership of the whole body ,26 or ofIn light of the above and despite the many open questions on current understanding of the processes involved in the use of VR, it is easy to foresee its potential and ease of use. The possibility of reproducing any type of environment and situation in the healthcare and laboratory contexts, together with the ability to manipulate the user’s perceived body characteristics, can attract the interest of numerous aspects of clinical practice. The use of experimental VR procedures in a wide range of cognitive and physical rehabilitation programs yields very interesting results. VR has proven to be an effective tool in assessing cognitive functions owing to the validation of tasks carried out in VR for the assessment of memory and attention ,46,47. AOne of the greatest promises of VR consists of the possibility to systematically introduce it into clinical practice as an effective non-pharmacological tool for pain management ,56,57,58Pain-imaging studies have identified the insular cortex, secondary somatosensory cortex, and anterior cingulate cortex as part of the pain matrix, which informs the body about the intensity and location of the noxious stimulus present ,61. BaseThe embodiment effect of VR and its efficacy on pain probably share certain principles with other procedures, such as the mirror box technique. The use of mirror visual feedback (MVF) was shown to reduce pain intensity and perceived unpleasantness , probablThese early insights motivated subsequent studies that increased the use of VR (which can effectively extend the MVF technique), with all the advantages that VR possesses , such asPain reduction of the type seen in multisensory VR exposure induces or facilitates neuronal plasticity processes . NP may An analgesic response to VR combined with virtual walking, independent of the lesion level, was observed up to three months after the rehabilitation program, suggesting neuroplastic changes. Despite the non-immersive VR experience, the patients’ affected body aligned to the lower body with normal walking ability, and patients reported a good degree of body illusion . This suThese findings make it difficult to untangle the structural effect only from changes that cause pain; however, they clearly demonstrate that the analgesic effect of multiple or longer sessions is facilitated by an intact virtual sensorimotor loop. Regarding plasticity mechanisms, up-regulation of motor cortex excitability by non-invasive stimulation techniques ,95,96,97Specifically, two studies that used the combination of transcranial direct current stimulation (tDCS) and virtual walking ,99 showeRecently, two studies investigated the effect of tDCS combined with visual illusion according to sensory phenotype profiles of NP ,102. TreTheoretically, the stimulation of cortical activity using neuromodulatory techniques could promote plasticity and enhance pain reduction ,104,105.Motor imagery is a promising technique for motor rehabilitation ,106,107 However, in SCI cases the movement imagery obtained contradictory results, which prevents us from drawing certain conclusions .The critical importance of owning and sensing a virtual body is best explained using multisensory body illusions  combining visual and tactile inputs ,114,115.The use of VR in combination with coherent sensory feedback on the body has great potential for identifying and enhancing residual sensory and motor skills in patients with SCI. VR can provide an interactive simulated environment to allowing patients with somatosensory deficits to experience greater levels of bodily sensation to compensate for the lack of signals from missing limbs .Another aspect to consider is the use of immersive VR from the first- ,115 or tAnother combined treatment that has proven effective in reducing pain caused by burns  and physAttention modulates pain perception ,128,129,Furthermore, the results suggest that the greater the number of sessions, the greater the reduction in pain, which supports the need to plan long-lasting therapeutic interventions. Further comparisons and testing should be conducted to verify first- or third-person perspectives.Despite considerable variability in procedures and results, current evidence is encouraging in terms of the effectiveness of simple or combined VR procedures on chronic pain. This is a promising starting point for further verification of the immediate analgesic effect and probable long-term beneficial effects. VR therapy is a viable, non-pharmacological, and non-invasive alternative for post-SCI NP, has minimal side effects, and has been prescribed over time. Body illusions and motor imagery combined with neuromodulatory therapy constitute representative experimental paradigms to successfully induce analgesia. The sense of embodiment has demonstrated efficacy in modulating both acute and chronic pain perception irrespective of the location of the pain. VR treatment attempts to reinsert the body of patients with SCI into the flow of experience, leading to better functional outcomes. Most studies so far have focused on paradigms which empower the embodiment process in individuals with intact bodies, rather than considering bodies with sensorimotor disconnection and residual signals. Therefore, after SCI the illusory ownership evoked during the RHI does not occur when stimulation is applied to numb body parts. Body parts with normal or residual tactile sensations appear to be an alternative for remapping input in affected body parts. It may be advantageous to adapt such techniques to include somatotopically focal stimulation of non-affected body parts to boost embodiment ,132,133.


Presently, with the introduction of nanotechnology, the evolutions and applications of biosensors and/or nanobiosensors are becoming prevalent in various scientific domains such as environmental and agricultural sciences as well as biomedical, clinical, and healthcare sciences. Trends in these aspects have led to the discovery of various biosensors/nanobiosensors with their tremendous benefits to mankind. The characteristics of the various biosensors/nanobiosensors are primarily based on the nature of nanomaterials/nanoparticles employed in the sensing mechanisms. In the last few years, the identification, as well as the detection of biological markers linked with any form of diseases (communicable or noncommunicable), has been accomplished by several sensing procedures using nanotechnology vis-à-vis biosensors/nanobiosensors. Hence, this study employs a systematic approach in reviewing some contemporary developed exceedingly sensitive nanobiosensors alongside their biomedical, clinical, or/and healthcare applications as well as their potentialities, specifically for the detection of some deadly diseases drawn from some of the recent publications. Ways forward in the form of future trends that will advance creative innovations of the potentialities of nanobiosensors for biomedical, clinical, or/and healthcare applications particularly for disease diagnosis are also highlighted. The detecting of any disease  be it communicable  or noncommunicable  is one of the dominant aspects toward the improvement of the efficiency of any biomedical/clinical/healthcare process –4. AccorThe treatment and convalescence for any kind of disease primarily depend on its early-phase detention as well as the effectiveness of the diagnosis processes , 9. MicrHistorically, the development of nanoparticles (NPs) commenced with the work of Ehrlich before the initial trials by Scheffel et al. as well as the all-embracing research work by some group of senior researchers led by Prof. Speiser at the ETH Zurich toward the end of the 1960s and beginning of the 1970s, with significant devotion to its development in the 1980s, particularly for medical purposes such as the delivery of drug voyage the blood-brain barrier (3Bs) , 12. PreSupposedly, NPs are utilized as a means for delivering loaded constituents via two basic mechanisms: encapsulation (employing lipid-based/polymer-based capsules) and polymer-based components  . GeneralPresently, biosensors (BioSS) are very significant for sensing target particles with great precision, selectivity, and signal-to-noise proportion. BioSS which are technologically advanced using BMs such as enzymes or nucleic acids (DNA/RNA) which are employed as the probes for sensing the target particles are presently been given great attention by several researchers owing to their numerous dynamic advantages. Enzymes that react with definite particles rapidly and selectively as well as the DNA/RNA can combine with their corresponding categorizations precisely in nanoscale . Also, bRecently, the identification, as well as the detection of biomarker (BioMK) linked with any form of diseases, has been attained by several sensing procedures using nanotechnology (NanoTech) . Some ofConsequently, the implementation of BioSS is a beneficial approach for the protuberant detection of biological markers. Furthermore, the recent advancements in BioSS mechanism vis-à-vis nanobiosensor (NanoBioSS) have resulted in evolutionary modifications in various research fields, namely, agricultural, environmental, and biomedical/clinical/healthcare sciences as well as several other domains of human endeavours –28.Most of the preeminent applications of BioSS/NanoBioSS are found in the diverse manufacturing segments of which the biomedical/clinical/healthcare services are the primary ones , 30.  FiThe detection of diseases, retinal prostheses, cellular miRNA appearance in colorectal cancerous complications, imaging of contrast during MRIs, diagnosis of the heart, medicinal mycology, and the monitoring of health are the main momentous physiognomies or largely characterized areas well served with BioSS/NanoBioSS benefits –33. ThesDiverse irresistible diseases and infections spread such as Ebola, SARS, Hendra, Nipah, Avian influenza, and COVID-19 SARS-CoV-2) have turned into a global threat that needs extensive exertion in their proliferation to manage. As there are diverse complications related to these irresistible diseases' infections, more advance diagnostic mechanisms need to be developed for mitigation and/or eliminating the odds of infection outbreak beforehand. BioSS/NanoBioSS has stood out as one of the appealing mechanisms for giving influential statistics on these diseases and infections. The recent SARS-CoV-2 plague (pandemic), which is extremely infectious, originated from a recently known coronavirus that has adversely obstructed humanity . There h have turOver the years, several categories and subcategories of BioSS/NanoBioSS have been developed with vast applications  composites was effectively fabricated. The PtNPs-NT composites were effectively arranged and directly cast on the glassy carbon electrode (GCE) surface. With the aid of electrostatic adsorption and covalent bonding, the negative (-) l-cysteine (l-cys) and the positive (+) poly  chloride- (PDDA-) coated gold (Au) NPs were improved on the ensuing surface of the electrode, which brought additional immobilization of glucose oxidase. Manipulation of the distinctive possessions of PtNPs-NT composites resulting in the accomplishment of direct transfer of electron among the electrode and the redox-active centres of glucose oxidase and the electrode demonstrated a couple of distinct reversible redox peaks with a fast heterogeneous rate of transfer of electrons [For example, an innovative 3lectrons  The imaglectrons .It is proven that CN has the prospective properties to transform several uses and benefits where nanosized metallic and/or semiconducting mechanisms are necessary . For exaSeveral papers have reported on the uses of NanoTech vis-à-vis BioSS/NanoBioSS for biomedical/clinical/healthcare applications  , 58, envHowever, the major concentration of this facile review study is on the biomedical/clinical/healthcare applications of NanoTech vis-à-vis BioSS/NanoBioSS particularly for the detection of some deadly diseases drawn from some of the recent publications and this is being done in the subsequent section.Even with the advancements in scientific knowledge, humanity is still fronted with some challenges ensuing from both communicable and noncommunicable diseases. As stated in Introduction, the prevention and early-phase detention as well as the effectiveness of the diagnosis and treatment processes are the most appropriate means for the survival and spread of such diseases. Hence, several innovative approaches such as the use of BioSS/NanoBioSS for the detention and treatment of diseases have continued to assist in this regard , 66–68.According to several reports, presently, NanoTech innovations are felt in almost every scientific domain  and all the ensuing benefits (BioSS/NanoBioSS) are making life easier , 69–73. However, the limit of detection (LoD) on this reported NanoBioSS varies and depends on some factors such as the utilized BioMK, the nature of the disease, the procedure employed in the BioSS mechanism, and the used BMs or NPs. Consequently, BioSS/NanoBioSS are predominantly characterized based on the nature of the NMs employed in the sensing mechanisms , 23, 26.It was observed from Tables In the recent past, the evolution of BioSS/NanoBioSS has remained as one of the dynamic areas of modern material science research (NanoTech) as attested by the large numbers of research publications. In the meantime, BioSS/NanoBioSS for detecting of diseases has stimulated a great deal of attention. The recent biomedical/clinical/healthcare applications  of BioSS/NanoBioSS mechanism via the development of NanoTech present an encouraging procedure for the effective and precise detection of protein BioMK allied with various diseases. Be that as it may, this recent review study presents a facile review of some of the reported biomedical/clinical/healthcare applications and potentialities of NanoBioSS particularly for some deadly diseases, emphasizing some of the potential BioMK that could detect such diseases. As observed from most reported research publications, there are limited approaches that are concentrating in the direction for decreasing sample volumes or the duration of the analysis.Consequently, there is still a great deal of work that needs to be carried out before NanoBioSS will be broadly employed in biomedical/clinical/healthcare laboratories as a replacement for just research laboratory purposes alone. A vibrant direction of imminent research is still in the aspect of molecular diagnostics for the accomplishment of advanced permanence and sensitivity. In the interim, diagnostic validation by processing an advanced quantity of biomedical/clinical/healthcare samples coming from persons infected with various diseases is required. Also, some components such as the nature of the protein, enzyme antigen, or/and other BMs, in addition to the concerned immobilization, should be considered. It is also suggested that the commercial approach to NanoBioSS from these useful reported researches should be one of the strategic aspects that require appropriate attention especially with funding and manpower in imminent research. Nevertheless, to exclusively achieve the biomedical/clinical/healthcare potentialities of NanoBioSS, additional and more researches should be executed and NanoBioSS could be pertinent in a complex matrix and extreme settings. Future research approaches should also hypothesize and conceptualize the implementations of innovative computational procedures such as big data analytics, Internet of Things, artificial intelligence, deep learning approachability, and microchip-built devices  interconnected with NanoBioSS for various biomedical/clinical/healthcare applications vis-à-vis the detection of diseases. Consequently, biomedical/clinical/healthcare investigations that recognise these smart systems interconnected with NanoBioSS should be reinvigorated for the development of prominent future detection of diseases (diagnostics).


Acute myeloid leukemia (AML) is a molecularly and clinically heterogeneous hematologic malignancy that progresses rapidly and originates from a rare population of leukemic stem cells. Despite intensive chemotherapy followed by allogeneic stem cell transplantation and the recent approval of new targeted agents, the overall outcome of AML is still unsatisfactory, with an estimated 5-year overall survival (OS) of approximately 30% . In thisIn the last 5-10 years, knowledge regarding the role of the immune microenvironment in the pathophysiology of AML has increased significantly. A growing body of evidence indicates that, similar to most malignancies, the functional interplay between leukemic cells and bone marrow (BM) immune microenvironment comprises a distinctive hallmark of AML. AML cells interact with a variety of immune cells, which crucially influence proliferation, survival, and drug resistance, by creating an immunosuppressive microenvironment . Both inMougiakakos et al. provide a comprehensive review of the metabolic changes occurring in AML cells that lead to immune-escape. Noting that one of the cancer hallmarks is metabolic reprogramming and that metabolic adaptation is crucial for tumor development and progression, the authors discuss recent evidence indicating the role of alterations in cell metabolism for immune escape. Metabolic alterations are linked to immune-regulation and to a deep metabolic re-modeling of the microenvironment that favors an immune-suppressive phenotype. In particular, the induction of immune-regulatory cell subsets, such as regulatory T cells (Tregs) is highlighted. A correlation of these metabolic changes with oncogenic mutations is outlined and discussed by providing a comprehensive picture of potentially suitable countermeasures and their limitations.Ciciarello et al. focus on the emerging mechanisms deriving from the BM microenvironment and conferring a survival advantage to AML cells over normal hematopoietic stem cells (HSCs). In particular, the authors tackle the role of MSCs, which have, for a long time, been recognized as pivotal contributors in the setup and maintenance of the HSC niche. More recently, MSCs have emerged as crucial regulators of the immune response, mainly toward the induction of immune tolerance. Indeed, it has been demonstrated that MSCs create a protective and immune-tolerant microenvironment, which supports the survival of leukemic cells and limits response to therapies. Finally, it provides a comprehensive overview of the emerging MSC-driven mechanisms of drug resistance, including the effects of soluble factors versus cell-to-cell contact, the regulation of redox homeostasis as well as of mitochondrial metabolism, and the role of inflammatory signaling.Ocadlikova et al. summarize current knowledge about the mechanisms and effects by which chemotherapy remodels the leukemia immune microenvironment. It is well known that some chemotherapeutic agents, such as anthracyclines, may activate an anti-tumor immune response in a process known as chemotherapy-induced immunogenic cell death. Nonetheless, emerging evidence indicates that tolerogenic mechanisms and immunosuppressive pathways may be concomitantly induced. In this regard, a specific focus is dedicated to dendritic cells (DCs) which, in the presence of leukemic blasts, may be driven to acquire tolerogenic features such as the overexpression of indoleamine 2,3-dioxygensase, thus expanding Tregs. A better understanding of the whole process underlying chemotherapy-induced alterations of the immunological tumor microenvironment have important clinical implications to fully leverage the immunogenic potential of anti-leukemia agents and fine tune their application. The combination of immunogenic chemotherapy with immunotherapies may represent an interesting area of preclinical and clinical investigation.In their review, Mendez et al. summarize the most recent advances in understanding the complex interplay between the genetic and immune landscapes of AML for novel risk stratification and therapeutic approaches. Based on the growing body of evidence that places the crosstalk with the immune system at the crux of any effective therapy, ongoing and future research will reveal how AML genetics informs and alters the composition of the immune microenvironment. After discussing the most recent advances in the understanding of the effects of anti-leukemia agents on the immune landscape, the authors present the recurrent genetic mutations found in AML from an innovative cell-extrinsic perspective. Although still poorly elucidated, this article discusses the mechanisms by which recurrent genetic mutations in AML, such as NPM1, FLT3-ITD, IDH1/2, and TP53 can modulate the immune microenvironment in AML.Although the recurrent genetic mutations found in AML have been intensively studied from a cell-intrinsic perspective, leading to the genesis of multiple, recently approved, targeted therapies, there is a paucity of data on the effects of these targeted agents on the leukemia microenvironment, including the immune system. In their work, Sweeney et al. focus their review on the graft-versus-leukemia (GvL) effect in AML. Allogeneic hematopoietic stem cell transplantation  is the most potent anti-leukemic therapy in patients with AML and one of the first examples of curative immunotherapy in oncology. Preclinical and clinical advances understanding the complex mechanisms underlying GvL and graft-versus-host (GvH) effects are discussed focusing on strategies to fully exploit the anti-leukemia immunological potential of allo-SCT (GvL) whilst minimizing the toxicity of GvH.Chao et al. review the role of CD47, recently identified as a dominant macrophage inhibitory checkpoint, in myeloid malignancies and pre-clinical data supporting CD47 targeting. Initial clinical data of CD47 targeting in AML/MDS are reviewed, including the first-in-class anti-CD47 antibody, magrolimab, as a single agent and in combination with hypomethylating agents. Early clinical data highlight the importance of targeting immune checkpoints such as CD47 and the critical role for macrophages in the pathophysiology of leukemic disease. At the translational level, key questions that need to be addressed including biomarker strategies for patient selection and the identification of effective therapeutic combinations with CD47 blockade, are discussed.There is increasing evidence of the crucial role played by components of the innate immune system within the leukemia microenvironment. 1 and prognostication are still predominantly focused on cytogenetic and molecular alterations, which are historically known to influence the sensitivity of leukemic cells to conventional chemotherapy. However, based on the compelling preclinical data regarding the fundamental impact of tolerogenic tumor microenvironment mechanisms in dysregulating patients’ immune response, the prognostic significance of the immune landscape is recognized as being increasingly important in AML, revealing that immune-related genes may predict survival (In summary, the collection of review articles in this Research Topic provide a timely update on the progress made in the understanding of the biologic landscape of immunotherapy in AML. Although major strides have been made, resulting in a deeper understanding of the mechanisms underlying the role of the immune microenvironment in the pathophysiology of AML, to fully exploit and translate immunotherapy into a clinical effective modality a long list of unanswered questions remains to be answered, from the biologic perspective. Among them, the question of whether immune biomarkers capture the biological significance of the microenvironment in AML biology and response to therapy represents a major task with important implications. In many cancers, a variety of immune-specific gene expression patterns, which profile the immune microenvironment, have been recently described as predictive of the outcome in patients treated with conventional chemotherapy and, more importantly, with novel immunotherapy agents –9. In AMsurvival . Based oIn conclusion, to fully enter into the immunotherapy era and develop novel immunotherapy drugs and strategies, we need a comprehensive and continually deepening understanding of the immune-biological landscape of AML.All authors equally wrote and contributed to the manuscript. 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.


Preserving lifelong vision in glaucoma patients with minimal impact on quality of life in terms of inconvenience and side effects is the goal of all treatment guidelines. The current understanding of glaucoma is limited, and only strategies that reduce intraocular pressure (IOP) have been shown to be effective in just delaying the progression of glaucoma. However, IOP-lowering treatments come with the risk of side effects that affect adherence and patients' quality of life. Often, treatments are initiated too late due to the asymptomatic nature of the disease, and progression to severe visual impairment or blindness is inevitable.The current Special Issue addresses some aspects of basic and clinical research on neuroprotection, neurodegeneration, and medical treatment of glaucoma with an overview of the current status, including novel drugs and drug delivery systems to improve adherence as well as new approaches to potential treatment modalities in the future.Glaucoma, like many chronic diseases, has a low adherence rate, which may lead to a progression of the disease and therefore to higher costs. Meier-Gibbons and Toteberg-Harms present an overview of the published literature on the costs of glaucoma care, dividing them into indirect, associated with the consequences of an advanced disease, and direct costs, associated with diagnostic tests and treatment. The published studies focus primarily on the direct cost of glaucoma care and show great differences in the price of antiglaucomatous drugs across countries and trends of prescribing generic drugs in Europe. Despite the fact that patients appear to be better informed about their disease and there has been an improvement in medical and surgical treatment, adherence has not improved in recent decades. More studies are needed to investigate the relationship between cost and adherence and focus on strategies for improving patients' adherence.The use of generic medications for glaucoma has grown considerably in recent years. This is due to patent expiration dates for the innovators and the relative ease and short time to bring generic eye drops to the market, which does not require studies comparing branded and generic eye drops. With approval by the relevant regulatory authority, generics are required to have the same ingredient, route of administration, dosage, and be manufactured to the same quality standards as the reference medicinal product but may have different ingredients and packaging. Tatham reviews the potential pros and cons of generic drugs in terms of efficacy, cost, tolerability, and differences in formulation, adherence, and ease of use and perceptions of generic medicines among healthcare professionals and the general public. The switch to generic drugs reduces health-related costs, but there are some limitations of generic drugs that need to be addressed, such as the influence of inactive ingredients on bioavailability and thus efficacy and bottle design to improve consistency in drug delivery.Long-term glaucoma treatment with multiple benzalkonium chloride- (BAK-) preserved eye drops may lead to increased aqueous humour flare. Pakuliene et al. used laser flare photometry, a noninvasive quantitative measurement of anterior chamber protein levels, to investigate intraocular inflammation in glaucoma patients on topical treatment for more than 2 years scheduled for cataract surgery without any other ocular abnormalities. The control group included cataract subjects without glaucoma. Investigators found higher aqueous humour flare in the glaucoma patients than in the control group. Several factors were associated with the aqueous humour flare increase, including pseudoexfoliation syndrome, number of eye drops, and presence of BAK.An update of currently available IOP-lowering medications and potential new treatment targets for IOP-lowering and neuroprotective therapy are discussed by Cvenkel and Kolko along with the future trends in glaucoma therapy such as sustained drug delivery systems and drug formulations. For future personalized medicine based on genetic and other characteristics of the individual patients, it will be appropriate to identify patients with high risk of progression and treat them more vigorously.In a review of the literature on vitamin D and glaucoma, Abouzeid and Samer concluded that vitamin D metabolites may play a role in glaucoma, either through a lowering effect on IOP or a neuroprotection pathway, but the exact molecular mechanism is not known. However, the limited number of clinical studies and significant bias in the study design preclude conclusions regarding the involvement of vitamin D in primary open-angle glaucoma (POAG).Several mechanisms have been implicated in retinal ganglion cell death, and therapeutic strategies may be needed to target this to delay progressive retinal ganglion cell death in glaucoma. Mitochondrial dysfunction has been linked to optic neuropathy and brain neurodegenerative diseases. The review of Duarte focuses on how damaged mitochondria may impact the ability of neurons and glial cells to maintain homeostasis and induce sterile inflammation and neurodegeneration via mitochondrial damage-associated molecular patterns.Tsai focuses on IOP-independent strategies for neuroprotection and/or neurodegeneration, including research into neurotrophic factors, gene therapy, immune system modulation, and novel neuroregeneration pathways. However, these innovative strategies should be critically balanced and weighed against the risk of disrupting the complex central nervous system environment.Nuzzi et al. address the current new treatment strategies, including medical therapy and delivery modes, laser treatment, and minimally invasive glaucoma surgery. Furthermore, the most recent approaches to treat glaucoma in different stages are presented, including IOP-lowering effect of trans-palbebral electrostimulation of trabecular meshwork and novel techniques of cilioablation, as well as the advantages and risks of stem cell therapy, neurotrophic factors and potential therapy with stem cell-derived exosomes.An important research goal is identifying systemic risk factors that can help in predicting POAG. Pfahler et al. using nailfold capillaroscopy documented increased levels of nailfold haemorrhage, dilated capillaries, and avascular zones in POAG patients. These findings suggest that systemic microvascular dysfunction is frequent in POAG. In addition, the presence of any haemorrhage was found to be a highly significant risk factor for glaucoma.An enhanced physiological stress response to reduced oxygen supply has been documented in patients with normal tension glaucoma (NTG) compared to age-matched healthy controls, supporting the role of vascular dysfunction. Dalgaard et al. measured serum adrenaline and endothelin-1 levels and changes in distal finger temperature. In patients with NTG, a relative increase in adrenaline was found during hypoxia and a relative decrease during recovery, but not in controls. Hypoxia also induced elevated temperature in distal finger in patients with NTG only.The role and function of synucleins, a family of small proteins involved in neurodegeneration of the central nervous system, have been studied in an animal model of glaucoma. Liu et al. found that IOP elevation caused a significant loss of retinal ganglion cells in older animals, but no significant change in young rats. Beta-synuclein was significantly downregulated in young animals without any significant change in old ones. These results show a link between aging and beta-synuclein regulation. Hydrogen sulfide, a potent reductant, was effective in downregulating beta-synuclein and was neuroprotective against acute IOP elevation.Finally, Russel et al. devised a low-cost method assessing glaucoma using digital image analysis of the angle and optic nerve. Colour fundus photographs, standard optic disc OCT, and digital slit lamp gonioscopy images were used for digital image conversion and analysis. Requiring only retina digital cameras, the volumetric, geometric, and segmentational data acquired through digital image analysis correspond well with the data obtained by OCT imaging, but importantly reduce the costs.In summary, this special issue offers an overview of novel treatment strategies and future targets in the treatment of glaucoma. It also provides new findings that may be a clue for new research in this important field.


Natural products represents an important source of new lead compounds in drug discovery research. Several drugs currently used as therapeutic agents have been developed from natural sources; plant sources are specifically important. In the past few decades, pharmaceutical companies demonstrated insignificant attention towards natural product drug discovery, mainly due to its intrinsic complexity. Recently, technological advancements greatly helped to address the challenges and resulted in the revived scientific interest in drug discovery from natural sources. This review provides a comprehensive overview of various approaches used in the selection, authentication, extraction/isolation, biological screening, and analogue development through the application of modern drug-development principles of plant-based natural products. Main focus is given to the bioactivity-guided fractionation approach along with associated challenges and major advancements. A brief outline of historical development in natural product drug discovery and a snapshot of the prominent natural drugs developed in the last few decades are also presented. The researcher’s opinions indicated that an integrated interdisciplinary approach utilizing technological advances is necessary for the successful development of natural products. These involve the application of efficient selection method, well-designed extraction/isolation procedure, advanced structure elucidation techniques, and bioassays with a high-throughput capacity to establish druggability and patentability of phyto-compounds. A number of modern approaches including molecular modeling, virtual screening, natural product library, and database mining are being used for improving natural product drug discovery research. Renewed scientific interest and recent research trends in natural product drug discovery clearly indicated that natural products will play important role in the future development of new therapeutic drugs and it is also anticipated that efficient application of new approaches will further improve the drug discovery campaign. Papaver somniferum), myrrh (Commiphora species), and licorice (Glycyrrhiza glabra) have been mentioned on the clay tablets from Mesopotamia in 2600 BC; these plants are still used either alone or as one of the ingredients of herbal formulations for the treatment of various diseases. Furthermore, organic compounds from natural sources were used in the past and also used still to treat various diseases; these compounds are used in their natural form (as pure drug or phytomedicine) as well as serve as lead molecules for the development of synthetic and semisynthetic analogues with improved druggability. A number of such active constituents being in clinical application include morphine, codeine, noscapine, papaverine, quinine, artemisinin, paclitaxel, etc. [There is a long history of the usage of plant materials for treating human diseases. Several plant species such as opium  for therapeutic application, the natural products provide an important foundation as potential lead compounds for the development of new and more effective drugs through structural modification. Although natural products possess diverse and complex chemical structures, the plant secondary metabolites are seemed to exhibit greater biological friendliness and drug-likeness than those derived from purely synthetic sources. Consequently, the molecules from natural origin are supposed to be better candidates for further drug development ,5. NewmaThe natural products and related drugs exhibit a broad spectrum of pharmacological activities, and they are used for the treatment and/or prevention of most of the popular human diseases, including infectious diseases, cancers, peptic ulcers, as immunomodulators, anticoagulants, antioxidants, respiratory, digestive and cardiovascular system-related diseases, antidiabetics, etc. . After aAbout 35% of the annual global market of medicine is either from natural products or related drugs; mainly including plants (25%) followed by microorganisms 13%) and animal (3%) sources . Between% and aniThere is a great requirement to stimulate the enthusiasm of the scientific research fraternity towards the incorporation of natural products in the drug development process. However, the major concern related to natural products is the success rate during the various drug development stages, mainly in the case of a random selection of the sources. In order to increase the success rate, the use of a well-designed strategy is necessary for the selection and shortlisting of the candidate species for investigation. A well-documented knowledge of phytomedicines, as in the traditional system of medicine or ethnopharmacological knowledge of herbal medicines, may be helpful to overcome the problems of low success rate and decrease the cost, and time of natural product development, which are considered to be the main associated factors ,19. The The new chemical entities (NCEs) can be discovered from one of the four major natural sources including plants, marine, animals, and microorganisms (fungi and bacteria). In addition to these natural sources, the new compounds can also be prepared by using synthetic chemistry and combinatorial chemistry. Among the natural sources mentioned above, plants  source is of considerable importance and the present article is describing the approaches in the context of this source, with the main emphasis given to the bioactivity-guided fractionation approach. The natural compounds obtained from botanical sources may belong to one of the classes including, biologically active compounds directly used as therapeutic agents, lead compounds  for development of more potent analogues, compounds whose structures may provide novel pharmacophore that can be converted to druggable compounds or chemical entities to be used as a marker for standardization of crude plant extracts. Additionally, plant extracts can be used to develop herbal formulations .Generally, the compounds isolated from natural sources possess some unique structural characteristics including, a greater number of oxygen atoms, more chiral centers, higher steric complexity, and molecular rigidity, a greater number of hydrogen bond acceptors and donors, and low aromatic ring atoms to total heavy atoms ratio. Moreover, there is a broader range of molecular properties including partition coefficient, molecular mass, and diversity of ring systems . Owing tIn the process of drug discovery from plant sources, the first and one of the most important steps involves the selection of plant candidates for extraction/isolation of active principles and screening for biological activities. According to Fabricant and Farnsworth 2001), out of approximately 250,000 available species of higher plants, only 15% were subjected to phytochemical screening and about 6% of the plant species were evaluated for their biological properties [01, out oAndrographis paniculta, which was used for the treatment of dysentery in ethnomedicine. Moreover, a number of active constituents, including berberine, morphine, and picroside from Berberis aristate, Papaver somniferum, and Picrorrhiza kurroa were isolated through this approach. In this approach, the candidate plants are being selected on the basis of observation, description, and even some experimental evaluation. It may involve the study of botany, chemistry, pharmacology, biochemistry, archaeology, anthropology, and the historical background of the plant [The discovery of new biologically active compounds using this approach of plant selection depends on the empirical experiences related to the use of the plants. The approach is based on the ethnomedicinal usage history of the plants, for example, andrographolide was isolated from the plant he plant .In this approach, mainly the plants are selected randomly from the local/national regions, and screening of the selected plants for target bioassays is performed. In addition to that, any of the target chemical classes of compounds such as flavonoids, alkaloids, polysaccharides, etc., may also be screened. This approach is used for focused as well as general screenings and provides a good chance of success. It is simple to select the plant candidates through this approach, however, disadvantageous in the sense that it does not provide any prior information regarding the biological activity of the selected species.Countries such as China and India have a rich heritage of well-documented records of traditional/herbal medicines, which is based on a codified system of medicines from botanical sources. The codified system approach is newer than the ethnomedicinal practice. It differs from ethnomedicinal practices in the following three accounts; firstly, these codified systems made up the empirical practices on the strong conceptual fundamentals of pharmacology and human physiology, while the latter relies mainly on the empirical experiences. Secondly, the concept of pharmaceutical formulations was more developed in the traditional codified system as compared to ethnomedicinal practices, where the products were used mainly as crude extracts such as decoction and juices. The term standardization was common in the traditional system of medicine. Lastly, the ethnomedicinal practices are generally controlled by a small fraction of the community and are localized in nature; on the other hand, the traditional system is much institutionalized.Bacopa monnieri, artemisinin from Artemesia alba, boswellic acid from Boswellia serrata, and reserpine from Rauwolfia serpentine used as a memory enhancer, antimalarial, anti-inflammatory, and antihypertensive agents, respectively [Some of the important examples of natural products discovered by adopting the approach based on the codified system of medicine include bacosides from ectively . ComparaAuthentication of the collected raw materials is the basic starting point in the development of natural products. The authentication of plant materials may be achieved through the application of one or more of the methods involving taxonomic, macroscopic, microscopic, chromatographic, spectroscopic, chemometric, immunoassays, and DNA fingerprinting analysis. Depending on the type of adulterants and closeness of the chemical constituents, a simple method such as examining the organoleptic properties may be enough to authenticate certain drugs, while a highly sophisticated method may be required by some other drugs. Therefore, it is up to researchers to select a suitable procedure for the material of interest.The first step of authentication of medicinal plants involves identification of the botanical origin and the scientific binomial name is to be determined .Macroscopic identification is performed by comparing the organoleptic properties, including color, odor, taste, size, shape, fracture characteristics, surface properties, and texture of the plant material with standard reference material .Microscopic method is generally used to differentiate and identify very similar medicinal plants. The method is fast, convenient, and involves the determination of internal structural features at tissue and cellular levels using a microscope. For this purpose usually ordinary light microscope is enough, however, a polarized and fluorescence microscope may also be used to increase the accuracy of the detection ,52,53.Chromatographic techniques including thin-layer chromatography (TLC), high-performance thin-layer chromatography (HPTLC), high-performance liquid chromatography (HPLC), and capillary electrophoresis (CE) are extremely useful for qualitative and quantitative analysis of natural products. Herbal medicines possessing volatile principles are analyzed by gas chromatographic technique (GC) ,55. TLC DNA barcoding may offer reliable information for authentication and quality control of medicinal plants. DNA barcoding offers species-level identification of plants using a short DNA region (DNA barcode). Consequently, this technique is widely applied in research and industry for molecular identification and solves a variety of problems related to taxonomy and population genetics, prevention of illegal wildlife collection and trade, and quality assurance of food and medicinal products. In the field of medicinal plants, initially, DNA barcoding was used as a tool for identification only, however, in recent years, it has made remarkable progress and is widely applied for quality assurance with respect to authentication of a number of herbal products [Ocimum tenuiflorum L. (Lamiaceae) as an example and the method emphasized plant sampling, DNA extraction, barcode region, purification, amplification, and sequence reference database [As the genetic make-up of each plant species is unique and remains unaffected by environmental factors, age, and other conditions; products ,57,58. Rdatabase ,60.Camptotheca acuminata and Taxus brevifolia, respectively [Galanthus nivalis and arteether, an antimaerial agent derived from artemisinin [As a result of increasing interest in the plant kingdom as a potential source of new therapeutic agents, several techniques have been developed for the extraction and isolation of natural products. Recently, biological activity-guided fractionation and isolation linked with chromatographic separation techniques have been extensively applied. In this approach, the fractionation of extract is based on biological activity rather than a class of compound of interest which involves step-by-step separation of the plant extract. On the basis of physicochemical properties and screening for biological activity, further fractionation and screening are followed. In the first round, all the fractions are screened for biological activity, and only the fractions possessing significant activity are further processed until the achievement of the pure isolate, responsible for target biological activity. The chemical characterization and structural elucidation are performed after the identification of the active isolates ,61,62. Tectively . Other nThe bioactivity-guided fractionation of natural products is a relatively new technique. In the experimental processes, mainly two approaches are used to isolate either known or unknown compounds which may be used as drugs or lead structures that may provide platforms in developing new analogues with better druggability. However, based on the circumstances other approaches can also be used. These two approaches are as follows.This approach is used when the selected plants are known for their biological activities from traditional or ethnopharmacological knowledge. The active compounds responsible for the target activity are isolated from the crude plant material as described in Initially, at least three fractions of extracts, for example, 100% aqueous, 100% ethanolic, and 50% aqueous −50% ethanol extracts are collected and evaluated for target biological activity in the primary screening.The most active extract(s) are extracted into sub-fractions in the sequence of decreasing polarity of a solvent such as butanol, chloroform, and hexane. The sub-fractions are further evaluated for biological activity.The most active sub-fraction(s) obtained at stage 2 are subjected to chromatographic separation to isolate the compounds of interest. Each compound is purified through appropriate purification techniques such as column chromatography, preparative HPLC, etc., and screened for target biological activity. Chemical structures of the compounds exhibiting optimum biological activity are elucidated by using modern techniques such as NMR spectroscopy, Mass spectrometry, LC-MS, etc.This approach is mainly used for the plants selected by random selection strategy and the biological activity of the selected plant is not known. The extraction/fractionation, isolation, and biological screening processes used in this approach have been summarized in In this stage, the extraction of the plant material and fractionation of the extracts take place simultaneously. The extractions are performed in the solvent systems of increasing polarity and fractions are collected in a sequential manner using petroleum ether, chloroform, ethyl acetate, ethanol, and water, for instance. All the fractions are screened for target biological activity.secondary screening is mainly oriented to the evaluation of the mechanism of action and may involve in-vitro screening at molecular levels.The fractions from the stage 1 experiment possessing the highest biological activity are selected, and the compounds responsible for specific biological activity are isolated by following the techniques mentioned in the previous approach. The isolated compounds are analyzed for structure elucidation by using modern techniques such as LC-MS, NMR spectroscopy, FT-IR spectroscopy, and Mass spectrometry. As shown in In both approaches, the extraction of the plant material is performed by using a range of polar and non-polar solvents. However, their method of extraction and fractionation remained largely invariable. In general, the chemical classes of constituents present in the extract or fraction can be predicted depending on the polarity of the solvent used. Lipophilic compounds (low polarity constituents) such as oils, fatty acids steroids, hydrocarbons, and low polarity terpenoids are extracted in non-polar solvents such as n-hexane and ether; whereas, compounds with medium polarity such as phenolics and alkaloids are usually present in ethyl acetate and chloroform extracts. The highly oxygenated and high polarity compounds such as sugars, flavonoids, glycosidic alkaloids, and small carboxylic acids are generally obtained in aqueous or methanol/ethanol extracts.Uraria crinite (L.) roots. They applied a bioactivity-guided fractionation approach in combination with NMR-based identification for isolation of the isoflavone and reported that genistein contributes to the immunomodulatory activity of the extract which may be helpful in standardization of the plant as functional food [Euphorbia dendroides, and isolated new constituents’ molecules that were not discovered by following the classical bioassay-guided fractionation approach. They used the mass spectrometric technique for dereplication of molecules prior to the isolation through molecular networking. They also implemented bioactivity score prediction to identify the biologically potential candidate molecules. The calculation of bioactivity score prediction was based on the relative abundance of the molecule in a fraction and the bioactivity level of the fractions [Tu et al. (2019) screened immunomodulatory isoflavone genistein against the production of pro-inflammatory cytokines (IL-6 and TNF-α) from the methanolic extract of nal food . Nothiasractions .Pterocarpus dalbergioides Roxb. and the butanol fraction exhibited potent anti-inflammatory and anti-hyperglycemic activities. Isoflavone genistein and phenolic acids including gallic acid and gentisic acid were isolated from the active fraction [Ficus virens bark to bioactivity-guided fractionation and a novel compound, n-octadecanyl-O-α-Dglucopyranosyl (6′→1″)-O-α-D-glucopyranoside was isolated. The compound was found to possess antihyperlipidemic activity through inhibition of HMG-Co reductase enzyme along with antioxidant potential. The new compound was further investigated by molecular docking analysis and ADME-T studies to evaluate pharmacokinetic profiles. The enzyme inhibitory potential was established through in-vivo antihyperlipidimic activity [Sclerocarya birrea. The fractions were subjected to chronological partitioning and screened from antibacterial activity against Salmonella typhi. The ethyl acetate fraction of the extract was characterized and active principles were isolated using LC-MS/LC-HRMS technique. Vidarabine was found to be mainly responsible for the antimicrobial activity of the S. birrea stem bark [Terminalia albida root. The samples were subjected to bioassay-guided isolation of phytocostituents using flash chromatography followed by characterization using NMR and HR-ESI-MS techniques. Among the 14 isolated compounds, pentolactone demonstrated the most significant activity against Plasmodium falciparum. Other compounds which exhibited antiplasmodial action include 3,4,3′-tri-O-methylellagic acid, triterpenes: arjunolic acid, arjungenin, and arjunic acid, and phenolic glycoside calophymembranside-B [In another study, a bioactivity-guided investigation was performed for fractionation of ethanolic extract of stem bark of fraction . Iqbal eactivity . Recentltem bark . In anotanside-B . In addianside-B ,72,73,74Although a majority of pharmaceutical companies are showing insignificant interest in natural product research owing to their complex phytochemistry and difficulties in their access and supply , a substLC-MS/MS is one of the most efficient techniques used for phytochemical profiling of the active fractions, which integrates good chromatographic separation efficiency with excellent characterizing capacity of mass spectrometry. Characterization of the structure of extracts, as well as pure bioactive molecules, are performed by the data obtained from a wide range of spectroscopic techniques such as FT-IR spectroscopy, NMR spectroscopy, and Mass spectrometry. In addition to these main techniques, data from X-ray diffraction, Optical rotatory dispersion, and chemical examination may also be helpful. A combination of HPLC with the above techniques has led to the development new technique useful in differentiating between known and unknown compounds and characterizing the full constituents of the natural products directly from the crude extract with a minimum amount of sample and time. The spectral data obtained from the instruments are interpreted to get information about the structure of the organic molecule under investigation.2, -NH-, OCH3, etc., and compounds including sugars, phenolics, steroids, terpenoids, and fatty acid esters. The presence of the most abundant chemical compounds or class of compounds in the extract can be identified, which helps to choose the most suitable separation method for further fractionation by either reverse or normal phase chromatography for instance [The development of efficient fractionation such as counter-current chromatography and emerging advancement in analytical techniques including NMR spectroscopy and mass spectrometry to isolate, purify and elucidate the structure of natural products is now allowing the natural product screening to fit in the timescale of high throughput screening ,79,80. Iinstance . The strGenerally, the natural products are screened for their biological activities on the basis of their reported ethnopharmacological and traditional uses. For instance, the medicinal plant traditionally used for the management of diabetes may be assayed for hypoglycemic effect and the traditional use is scientifically rationalized, once a very good ‘hit’ molecule has been achieved. However, most of the time the activity is not replicated in in-vitro screening. Most of the natural products are low-yielding, so the biological screening of such compounds can be performed by a series of bioassay methods that provide fast and sensitive results. These assays are performed by using several animal or human cell lines and microorganisms. A number of accurate and efficient instruments have been developed in this regard ,84,85,86Although biological screening of extract and pure compounds from natural sources using animal models are still used, this approach suffers from demerits, including the requirement of large quantities of samples, lengthy and difficult experimental procedures, low sensitivity, and ethical consideration. Generally, the practical yield of bioactive pure compounds from natural sources is extremely low, and it is really difficult to supply them in adequate amounts needed for animal screening. On the other hand, potential hits may be regarded as unsafe on the basis of toxic effects observed in cell-based screening, which may have been revealing good safety profiles in the animal body due to detoxification in the liver .The advancement of research in the field of life sciences has revealed a number of pathophysiological processes and mechanisms of drug actions, which led to the development of several cellular and molecular bioassay methods. A number of these bioassays fulfill the timescale requirement for HTS methods. The HTS methods could significantly minimize the amounts of test samples required for screening, needed in micrograms; permitting the assay of pure compounds isolated in very small quantities. Furthermore, the development of automation technologies, computer applications and software, and microfluid management has accelerated the identification of bioactive compounds (hits) as potential ‘lead’ molecules for target indication by making it possible to perform bioassay for thousands of samples within a short time period ,89. Incr50 and IC50 values at micro or nano mole levels is considered to be potent. A more potent analogue needed to be administered in smaller amounts and hence, help to minimize the toxicities.After determining the biological profile of the new natural products; identification of their target of action in the biological system, physiological pathways they interact with and hence, mechanism of action should be established. These parameters require new approaches of modern drug discovery to be applied. The mechanism of action of natural products and their effects on the biological target can be determined by a battery of cell-based bioassays. The SAR studies provide preliminary information regarding the mode of drug-target interaction and identification of analogues showing higher potency than the parent compound. A compound exhibiting LCIn a biological screening of drugs, the effects of solvent used on the dissociation of the drug and conformational or molecular arrangement should be taken into consideration. DMSO is one of the most commonly used solvents for this purpose, which is an aprotic solvent with intermediate polarity and capable to dissolve a variety of polar as well as non-polar compounds. However, due to the hygroscopic property of DMSO, the absorbed water sometimes reduces the solubility of non-polar compounds. Other organic solvents are usually not preferred due to their toxic effects on the test organisms or cells, in addition to poor miscibility with the assay media; however, the solvent controls are always used to cancel the toxic effects. Although, the in-vitro screening results are not always translated into in-vivo activity owing to a number of factors including dosage, solubility in a biological medium, membrane permeability, and biodegradation; a positive in-vivo activity of test compound is usually expected when it exhibited positive in-vitro results. However, the biological profile of a true drug candidate should not rely only on a single in-vivo screening, rather multiple screenings are necessary to confirm its efficacy . The strSince the last few decades, target-based screening has been considered the dominant approach in drug discovery. The starting point in this approach is a well-identified molecular target for drug interaction which has been considered to play important role in the disease. However, before the introduction of target-based screening, the chemical entities were evaluated through the phenotype screening approach, where a visible characteristic of an organism in a biological system, such as animals and cells, was investigated. The drug screening using a target-based approach is supported by advances in genomics, chemistry, and molecular biology; it provides the basis for measuring the efficacy and safety of drugs, dose setting, and selection of the patient population. The idea that rational and measurable progress would enhance the success rate and research productivity is among many factors that is responsible for the shifting of phenotype to target-based drug screening. Unfortunately, this change in approach could not greatly influence the success rate in the pharmaceutical industry. In the current scenario, owing to the scarcity of the new compounds acting on “essential” targets, there is a further revival of interest of researchers in the phenotypic approach, which relied on phenotypic measurement of responses and prior knowledge of specific drug targets or a hypothesis regarding their role in the disease is not essential. The screening approaches have been shifted to traditional phenotype and the lack of an established mechanism in this approach was considered as a deficiency, has now been observed as an opportunity. The development of genomic techniques has led to the identification of various new targets using optimized molecules as probes. Through the application of the phenotype approach, a number of new molecules have been identified as promising drugs, many without a known mode of action ,93,94,95Both the approaches have their strengths and weaknesses and experience challenges. Although the drug target and mode of action is known, the challenge of target-based drug discovery is that it may not be able to capture the whole story. The observed drug efficacy may not be a result of the mechanism originally expected because a drug generally binds to more than one target to perform its action. Another challenge encountered is the in-vitro/in-vivo disconnect, where good efficacy on the target could not be translated to similar cellular activity in in-vivo screening. Although prior information on the mechanism of action is not required in phenotypic approaches, some understanding of biology is needed and at least the biomarker that translates to human disease should be identified. Moreover, it may be a risk to proceed a compound into development without some understanding of the mechanism to determine the dose-response relationship. The other major disadvantage of this approach is that the drug action may be the result of interaction at multiple targets leading to a poor structure-activity relationship in the optimization phase. Fortunately, various technologies such as affinity purification and biochemical fraction isolation are proved to be helpful. Additionally, this approach has also enabled the researchers to apply the latest molecular technologies of proteomics, network biology, and chemical biology ,94.Comparative studies revealed that the phenotypic approaches are more successful as far as small molecules, and first-in-class medicines are concerned. In a study, Swinney and Anthony (2011)  analyzedThe discovered bioactive natural products can be used as lead compounds for the optimization of structural features to develop new and more effective analogues by applying modern medicinal chemistry approaches, including molecular modeling and combinatorial chemistry. Furthermore, the natural products present along with other compounds as a family of structurally related molecules; therefore, a number of homologues may be obtained from one source which can provide SAR-related information. Since only a small proportion of the available plants have been studied for biological activities so far, the discovery of new leads from natural sources will continue, and they will be available for biological screening to find new therapeutic agents. In the modern drug discovery of natural products, the isolated new compounds possessing acceptable bioactivity are subjected to SAR studies and molecular modeling processes to design and develop analogues with more potency, fewer toxic effects, and better pharmacokinetic profiles. The study can also reveal that interaction with certain enzymes may influence the test compounds’ biological activity. The analogues with the best druggability may be synthesized in the laboratory and evaluated by a number of in-vitro and in-vivo biological assays . The oveMolecular modeling procedures require optimized 3D structures of ligands in PDB format. Natural product databases and other databases like PUBCHEM, ZINC are reliable sources for retrieving the structures of known natural compounds in different acceptable formats like SDF., mol., mol2, PDB, etc. . StructuPreparing or downloading the 3D structures of target molecules such as proteins , receptors (examples: PPAR-α and PPAR-γ), and enzymes  from Protein Data Bank (PDB) and optimized for geometry and energy. The binding site to be defined and standard scores calculated for natural ligands present.The 3D structures of natural products are docked against the target structure by using docking software and ranked according to the binding energy. Widely used docking tools include AutoDock, AutoDock Vina, FlexX, Discovery Studio, and MDock . A pre-rAfter docking simulation, the results are analyzed and the top interactions are identified based on the energy scores. Usually, the top 10 scores are further subjected to molecular dynamics (MD) simulation studies. In this regard, two systems i.e., (I) Apo (uncomplexed) protein/receptor, (II) protein/receptor complexed with interacting compound (predicted by docking study) can be submitted to MD simulation. According to the ranking of ligands and their interactions with the target, the hit molecules having a high affinity towards the target are identified.The optimization process of hits is performed by observing better affinity towards the target by preparing various analogues of hits and the hits showing best affinity are developed and various drug-like properties viz; stability, pharmacokinetic and pharmacodynamic properties can be studied by using QSAR software ,102.Alternative to the extraction and biological assay of natural products, in modern drug discovery, a collection of a large number of compounds derived from natural sources possessing diverse chemical structures are tested through virtual screening or in-silico study. There are a number of such libraries held by academic institutions and research centers, holding up to millions of such structurally diverse compounds. Through the application of virtual screening, potential hits can be identified for a target biological activity and a good SAR can be established through a lead optimization process. In this way, virtual screening can filter down the number of compounds for the real test through bioassay ,104. HowIn natural product drug discovery, once the bioactive compound is identified, the plant extracts can be rationally designed with improved efficacy by modulating the concentrations of bioactive compounds; alternatively, the isolated compound may be used as a lead molecule for developing analogues with improved therapeutic potential. Bioactivity-guided fractionation is one of the most commonly used approaches in natural product drug discovery to identify the active molecules. As discussed above, in this approach extraction and biological screening proceed side-by-side. The fractions are collected, screened for biological activity and the process iteratively continued until the isolation and characterization of active constituents are performed ,57,58,59Hydrastis Canadensis, which were overlooked in the conventional technique [Natural products generally consist of multiple constituents which can produce their action by interacting with a number of targets. Some of these constituents may act as additives or synergists to the therapeutic effects of the other bioactive constituents. Generally, synergistic constituents display no biological activity on their own; rather, they only increase the potency of bioactive molecules in combination . Such coechnique .Historically, medicinal plants have been used for the treatment of various communicable and non-communicable diseases, and even today represent a rich source of important therapeutic agents as well as new lead compounds. A number of successful therapeutic agents have been obtained directly from plant sources or developed from naturally derived lead molecules. Revived scientific interest and research trends in plant-derived natural product drug discovery and development clearly indicated that it is one of the potential sources of new therapeutic agents in the future. In plant-based drug discovery and development research, plant metabolites are being optimized for developing potential analogues that can demonstrate desired safety and efficacy. Due to renewed interest of the medicinal chemists in natural product drug discovery, a number of new approaches accompanied by technological advancement for selection, identification, isolation, characterization, and biological screening of natural products have been developed. These new approaches could minimize the technical drawbacks associated with natural product development and address the challenges encountered in the discovery and development of new natural products owing to the complex behavior of natural products. The technological developments enabled the exploration of the profiles of complex phytoconstituents leading to the isolation or synthesis of a number of successful therapeutic drugs and novel lead compounds that can provide a core scaffold for future drugs. It is particularly important to adopt an interdisciplinary approach involving traditional and ethnopharmacological knowledge, phytochemistry, botany, analytical chemistry, suitable biological screening strategies, and modern drug development tools for successful results in this regard. In the future, the new strategies on natural product drug development will minimize the challenges and enhance the success rate; in the drug discovery process, the use of new molecules from plant origin and chemical libraries based on natural products will be increased. It may be a major contributor to new drug development and address global health challenges.


Background: Glaucoma is a chronic, vision-threatening disease, and a major cause of legal blindness. The current view is no longer limited to the progressive optic nerve injury, since growing evidence strongly support the interpretation of glaucoma as a complex neurodegenerative disease. However, the precise pathogenic mechanisms leading to the onset and progression of central nervous system (CNS) impairment, and the functional consequences of this damage, are still partially understood. The main aim of this review is to provide a complete and updated overview of the current knowledge regarding the CNS involvement in glaucoma, and the possible therapeutic perspectives.Methods: We made a careful survey of the current literature reporting all the relevant findings related to the cognitive dysfunctions occurring in glaucoma, with specific remarks dedicated on the higher-order visual function impairment and the possible employment of neuroprotective agents.Results: The current literature strongly support the interpretation of glaucoma as a multifaceted chronic neurodegenerative disease, widely affecting the CNS. The cognitive impairment may vary in terms of higher-order functions involvement and in the severity of the degeneration. Although several neuroprotective agents are currently available, the development of new molecules represents a major topic of investigation for future clinical trials.Conclusions: Glaucoma earned the right to be fully considered a neurodegenerative disease. Glaucomatous patients may experience a heterogeneous set of visual and cognitive symptoms, progressively deteriorating the quality of life. Neuroprotection is nowadays a necessary therapeutic goal and a future promising way to preserve visual and cognitive functions, thus improving patients’ quality of life. The term “glaucoma” refers to a group of optic neuropathies characterized by degeneration of retinal ganglion cells (RGCs) and their axons. Their damage lead to the progressive thinning of retinal nerve fibers layer (RNFL) and cupping of the optic nerve head (ONH). Structural changes result in characteristic visual field (VF) alterations, up to complete blindness. Glaucoma is considered a leading cause of blindness worldwide and it is estimated that the number of affected people will increase to approximately 111.82 million in 2040  which collected and ordered all the relevant information, considering the main topic of this review as expressed in the manuscript title.Multimodal imaging radically changed the diagnostic approach to posterior segment diseases. This is intended as a set of non-invasive tools assessing different morphological characteristics of the retina and the ONH. Fundus autofluorescence is able to detect the light absorption and emission properties of retinal fluorophores characterizing normal retina and pathological conditions  and AD, including extraocular muscles impairment, pupillary defects, RNFL and GCL thinning and visual alterations  techniques described widespread modifications of the white matter tissue and the brain connectivity in patients affected by glaucoma, with strong correlations with RNFL/GCL impairment and VF alterations -tacrine Lipton, . These NThis review tried to collect the main findings supporting the existence of many elements in common between neurocognitive dysfunctions in glaucoma and neurodegenerative diseases, leading to MCI/dementia. The eye and the CNS are closely interconnected, placing the basis for the onset of visual symptoms in neurodegenerative diseases and, conversely, of neurocognitive dysfunctions in ophthalmic disorders. Glaucomatous patients are characterized by a wide range of still poorly investigated cognitive dysfunctions. These alterations, governed by neuroinflammatory and neurodegenerative mechanisms, may have a strong impact on patients’ quality of life and may remarkably interfere with the proper diagnostic and therapeutic management of glaucoma. Although still representing a novel research field, the vision of glaucoma as a complex neurodegenerative disease would open new diagnostic and therapeutic routes, more focused on the involvement of the neuroretinal and the CNS structures. Further studies should be focused on a deeper assessment of CNS alterations in glaucoma and on the development of new neuroprotective therapeutic strategies.AA and EA: review design, data analysis, data interpretation, and manuscript draft. AS, DA, and FF: data acquisition and data analysis. MB and FB: data interpretation, manuscript revision, and study supervision. All authors contributed to the article and approved the submitted version.FB consultant for Alcon , Alimera Sciences , Allergan Inc. , Farmila-Thea , Bayer Shering-Pharma , Bausch and Lomb , Genentech , Hoffmann-La-Roche , Novagali Pharma , Novartis , Sanofi-Aventis , Thrombogenics , Zeiss . The remaining 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.