Pediatric brain tumors are the most common solid tumors in children and represent a heterogenous group of diagnoses. While some are treatable with current standard of care, relapsed/refractory disease is common and some high-risk diagnoses remain incurable. A growing number of therapy options are under development for treatment of CNS tumors, including targeted therapies that disrupt key tumor promoting processes and immunotherapies that promote anti-tumor immune function. While these therapies hold promise, it is likely that single agent treatments will not be sufficient for most high-risk patients and combination strategies will be necessary. Given the central role for radiotherapy for many pediatric CNS tumors, we review current strategies that combine radiation with targeted therapies or immunotherapies. To promote the ongoing development of rational combination treatments, we highlight 1) mechanistic connections between molecular drivers of tumorigenesis and radiation response, 2) ways in which molecular alterations in tumor cells shape the immune microenvironment, and 3) how radiotherapy affects the host immune system. In addition to discussing strategies to maximize efficacy, we review principles that inform safety of combination therapies. Collectively, central nervous system (CNS) tumors are the most common solid tumors in children. These tumors represent a heterogenous group of diagnoses ranging from low grade lesions that can be observed or cured through surgical resection to aggressive tumors that are uniformly lethal. Insights into diagnosis and prognosis draw from radiographic and histopathologic features. However, these features tell only part of the story, with molecular alterations greatly impacting diagnosis, prognosis, and therapy decisions in many cases. These molecular features include mutations, copy number variations (CNVs), structural variants (SVs), epigenetic and gene expression changes. Current standard of care for pediatric brain tumors involves molecular profiling of tumor samples to facilitate more precise characterization of this heterogeneous group of tumors, and along with this comes the possibility of treating patients with more individualized regimens. For some, this means tailoring the intensity of treatment through risk stratification. For example, de-intensification of chemotherapy and radiation is being tested in the WNT medulloblastoma subgroup given favorable outcomes with current standard of care, multimodal therapy (NCT 02724579). For high-risk diseases, where standard chemotherapies have historically failed and relapse/refractory disease remains common, an understanding of the specific molecular drivers of malignancy offers the hope of improving outcomes for such patients through a more targeted approach. Ultimately, as new therapies are evaluated, assessment of response in the context of molecular features of a tumor will identify molecular determinants of response.In the quest to improve outcomes for patients with pediatric brain tumors, the armamentarium has grown to include a spectrum of therapies, including surgery, cytotoxic chemotherapy, radiation, molecularly targeted therapies, and immunotherapies. Radiation has been and continues to be standard of care for many pediatric brain tumors and in some cases, such as diffuse midline gliomas (DMG), is currently the only life-prolonging therapy . In contWe are learning that there is substantial overlap and cross talk between the molecular alterations in brain tumors, the immune microenvironment, and the response to DNA damage by radiation Figure 1Pediatric CNS tumors represent a spectrum of diagnoses, with imaging, histopathology, and molecular features contributing to an integrated diagnosis . The WorBRAF-rearrangements, gain of function BRAF mutations, and loss of function mutations in negative regulators of this pathway, such as NF1  are the most common pediatric CNS tumors overall, and portend a good overall survival (OS) of approximately 90% , 9. Someh as NF1 , 11. BRAsettings –15. GiveHIST1H3B) and 3.3 (H3F3A) that are very rarely reported in adult patients. These mutations include a substitution of lysine amino acid at position 27 with methionine (H3 K27M), which are most frequently found in DMG, and glycine at position 34 with arginine or valine (H3 G34V/R), which are found in hemispheric pHGG. Numerous other differences in the molecular drivers of pediatric HGG are well described and make these tumors distinct when comparing to adult counterparts  carry a dismal prognosis, with the primary life prolonging therapies being surgery and radiation –18. Howeterparts , 20.ALK, ROS1, and NTRK  efficacy and case reports of durable responses in patients receiving TKI therapy  exhibit some of the highest mutation rates in human cancer . In the Medulloblastoma is the most common malignant pediatric brain tumor in children and young adults. Standard of care involves multi-modal therapy including maximal safe surgical resection followed by adjuvant chemotherapy and radiation. This regimen is able to cure many patients, but is associated with acute and long-term morbidity due to intensive chemotherapy and craniospinal radiation . Recent Overall, we are beginning to unravel how best to use novel targeted and immune therapies for pediatric brain tumor treatment; however, much work remains on how to best maximize their impact, particularly as part of multi-modal approaches.TP53-dependent manner. For example, TP53 mutant or null DIPG demonstrate radio-resistance, as evidenced by in vitro assays from cell lines derived from treatment naïve biopsy specimen and in the more rapid development of disease recurrence following radiation in these patients , which characterize high risk disease in patients from SHH, Group 3, and Group 4 sub-groups. Preclinical data indicate that MYC or MYCN overexpression enhances sensitivity to WEE1 inhibition, possibly due to a vulnerability generated by MYC-induced replication stress , found within various human tumors including HGG, are associated with HR-deficiency and also increase sensitivity to PARP-inhibition in the absence of BRCA-mutation  to identify a safe dosing regimen for the CNS-penetrant PARP inhibitor veliparib and determine the safety/efficacy of combination with up front radiotherapy and maintenance TMZ . This trBRCA-mutated metastatic breast cancer (MEDIOLA) demonstrated that this combination therapy was well tolerated with a safety profile similar to individual agents and associated with objective response in 63% of patients  and CD8 T cell effector gene expression signature  expression in cancer cell lines . MechaniAs targeted treatments are developed, the safety profile of combination therapies is a major consideration. When approaching the combination of targeted therapies with radiotherapy, a useful framework accounts for both acute and late effects of each mode of treatment, with an eye toward overlapping toxicities. Consideration of overlapping toxicities has guided the development of current standard of care chemo/radiation regimens, and has informed early experience in the combination of multiple targeted therapies for pediatric brain tumors . ProactiThe use of agents targeting the RAS/MAPK pathway combined with radiotherapy has some of the most mature data in this realm. Retrospective experience is available for combination BRAF inhibitors and CNS directed radiotherapy for melanoma patients with brain metastases and may inform the use of such combinations in pediatric CNS tumors. The skin is a key organ system where overlapping toxicities of radiation and BRAF inhibitors must be considered. In patients receiving concurrent BRAF inhibitor with whole brain radiotherapy, the incidence of radiodermatitis was significantly greater in patients receiving combined therapy: reported as 44% for combination compared to 8% receiving radiation alone . StereotIn addition to injury of neighboring non-tumor tissues, acute toxicities of radiotherapy can derive from achieving tumor cell death and activation of host immune response in the tumor. A commonly encountered outcome of this treatment effect is radiographic and clinical pseudoprogression. On-target, anti-tumor response to radiation can be associated with tumor cell death, immune activation, and edema, leading to the phenomenon of pseudoprogression following radiotherapy. With respect to brain tumors, radiographic pseudoprogression is defined as increased contrast enhancement and other signs of tissue edema early following radiation therapy, which ultimately subsides without a change in disease directed therapy –90. The While the acute effects of combination therapy with targeted drugs and radiation are starting to be elucidated, late neurocognitive, neuroendocrine, and neurovascular complications remain to be discovered. Given their contemporary development, long-term side effects of targeted therapies alone are not yet well understood. As discussed above, active prospective trials evaluating the utility of MEK inhibitor for pediatric LGGs include long-term follow up assessments of vision, motor function, neurocognitive function, and quality of life. This understanding will inform the potential long-term toxicities of combination MEK inhibitors and radiotherapy. While this combination is not considered a strategy for LGG treatment, therapy for higher grade lesions may involve such combinations. When these agents are being combined with radiation, where the same organ systems (i.e. vision) can be negatively impacted by each independent strategy, care must be taken to monitor patients closely. Like the strategy for MEK inhibitors in LGG, we emphasize the importance of long-term tracking of functional outcomes in patients receiving any targeted therapy. Additionally, cranial radiotherapy carries a risk of inducing small and large vessel vasculopathy and increased stroke risk , 94. Kinvia engagement of inhibitory receptors on the T cell surface (including PD-1 and CTLA-4). Ligands for these receptors (including PD-L1 and B7) can be expressed on tumor cells, stroma, or monocytes. ICI therapies function by disrupting these signals  a much more commonly observed group of acquired mismatch repair deficits following chemotherapy treatment. In the latter group, the mechanism for acquired hypermutation was potentially due to molecular evolution of tumors under selective pressure from TMZ, as acquired mismatch repair deficits arise from this treatment  were more likely to be truncal and subsequently trigger anti-tumor cytotoxic T cells upon ICI therapy. Furthermore, recent preclinical studies in mismatch repair (MMR) deficient tumors report that TMB, and the presumed associated increase in tumor associated antigens, is not sufficient to elicit anti-tumor immunity or predict response to ICI  an understanding of the molecular determinants of anti-tumor immunity can help identify patients that may respond to immunotherapy, and 2) combination therapy will likely be indicated to boost anti-tumor immunity or overcome mechanisms of resistance for most patients. In this section, we review various immunotherapies that can be combined with radiation and mechanisms underlying radiation-induced signaling changes within tumor cells and in the microenvironment that provide rationale for such combinations. In addition to highlighting the potential combinations that maximize efficacy, we discuss key factors that will impact the safety of such combinations.Experience with immunotherapies for pediatric CNS tumors is an actively evolving field and as such, experiences with immunotherapy in adult malignancies and other pediatric cancers are pertinent. The CheckMate 143 trial was the first randomized phase III study to evaluate checkpoint blockade in patients with primary brain tumors. This study compared anti-PD-1 inhibition using nivolumab to bevacizumab in adult recurrent GBMs after standard of care surgery and radiation. This study did not find an OS difference in these two groups, but did demonstrate a side effect profile for nivolumab that was similar to those reported when used in other adult malignancies . Checkmavs. 20.4 months with reirradiation alone) . An initial study using the PD-1 inhibitor, pembrolizumab, in progressive DIPG, enrolled only 5 patients before being put on hold due to neurologic deterioration that appeared to be more rapid than historical controls and cautioned enrollment of subjects with late stage recurrent disease in future immunotherapy trials . This stn alone) . While tvs hypofractionated therapy is outside the scope of this review, we highlight that alternate fractionated strategies are being investigated in combination studies with immunotherapy, based on clinical and preclinical studies showing that alternate radiation strategies have distinct effects on the tumor immune environment and that sub-ablative radiation doses may have immune-priming effects , is currently evaluating the combination with radiotherapy in newly diagnosed patients with DIPG and non-brain stem HGG, as well as with re-irradiation in recurrent HGG (NCT03690869). This study is investigating alternative radiation fractionation strategies in combination with ICI. While a discussion around conventional  effects –123. Add effects . In thisvs resistance, and shed light on future combination strategies to augment PD-1 blockade. Insight gained from this trial may inform investigations of neo-adjuvant ICI in newly diagnosed patients with high-risk brain tumors.The timing of immunotherapy may also impact efficacy, as neo-adjuvant pembrolizumab, followed by maintenance therapy, significantly extended OS compared to maintenance therapy alone in patients with GBM . This suAnother immunosuppressive pathway implicated in tumor biology involves the enzyme, indoleamine 2,3 dioxygenase 1 (IDO1). IDO1 is expressed in tumor infiltrating T cells and its enzymatic activity converts tryptophan to kynurenine – a molecule which reduces cytotoxic T and NK cell activity, while promoting the expansion of immunosuppressive regulatory T cell and myeloid derived suppressor cell populations . Tumor iin vitro antigen exposure and that transfer of H3K27M specific T cells led to anti-tumor activity in mouse glioma xenografts, providing a neoantigen target  concurrent with up-front radiation or chemo/radiotherapy in newly diagnosed pediatric brain stem glioma and HGG subjects . The prin target . A a phan target . Longitun target , we highn target . Lastly,TP53-mutant SHH medulloblastoma and DIPG. A recurrent mechanism of tumor immune escape is down-regulation of surface MHC-I, which is necessary for presentation of tumor associated antigens (TAAs). The authors of this study demonstrated that mutant TP53 was sufficient to drive immune escape in mouse models of medulloblastoma and DIPG and that this immunosuppression was dependent on TP53-mediated down-regulation of MHC-I. In orthotopic tumor models, systemic TNF alpha was sufficient to restore MHC-I expression on tumor cells and enhance response to ICI in mouse models, leading to tumor regression that was associated with lasting systemic anti-tumor immunity that prevented engraftment on repeated tumor challenge. This work demonstrates that TP53 alterations may serve as key biomarker for tumor-related immunosuppression when compared to TP53 wildtype counterparts and may require combination strategies. Previous clinical studies with systemic single-agent TNF alpha demonstrated significant dose-limiting acute, systemic toxicities due to inflammatory signaling (TP53 mutated brain tumors and highlights the principle that synergistic anti-tumor effects of combination therapies might be obtainable with lower doses than those identified in studies with single agent treatments (i.e. below the maximum tolerated dose for single agents). This strategy is going to be tested in an upcoming trial combining TNF and nivolumab. Notably, radiotherapy is also sufficient to increase local TNF and enhance tumor MHC-I expression, possibly with less systemic toxicity than systemic exogenous TNF , which are not present in mammalian cells, but correspond to a pathogen associated molecular pattern (PAMP) found in bacterial and viral genetic material . Immune delivery , 145. Thvia surgically implanted catheters in 12 pediatric patients with recurrent or refractory supratentorial pHGG found that oHSV  was well tolerated and resulted in no identified peripheral blood virus shedding (8 plaque forming units with 5 Gy radiation) in a larger cohort of relapsed or refractory supratentorial pHGG and provide additional prospective information on determinant of immune and tumor response to therapy (NCT04482933).Oncolytic viruses are another avenue for local immunotherapy. These viruses exert anti-tumor activity through several possible mechanisms: direct tumor cell killing, increased tumor associated antigen presentation, and stimulation of a local pro-inflammatory environment. Multiple oncolytic viruses with tropism for CNS tumors have reached the clinic and have demonstrated safety when injected locally as a single agent. For example, oncolytic herpes simplex viruses (oHSV) have a natural tropism for neural tissue and have been modified to restrain viral replication in normal neural tissue while permitting replication in tumor cells . In precshedding . The autvia the modified oHSV. In a mouse glioma model, combination of oHSV with PD-1 and CTLA-4 blockade led to tumor regression in most mice and prevented tumor engraftment on tumor re-challenge in mice with initial tumor regression, suggesting that this combination therapy generated a lasting anti-tumor immune response . In this study, radiotherapy is initiated three to four weeks following DNX 2401 injection. An interval report describing the first eight patients on study reported no evidence of dose limiting toxicities and indicated that patients were able to discharge from the hospital three to four days post-injection  . A phasevia lumbar puncture for patients with disseminated disease at relapse. Both approaches have demonstrated safety and efficacy in preclinical models , which express the CD46 surface marker that mediate measles virus entry (NCT02962167). This trial employs local injection of virus at the time of planned surgical resection for localized recurrence or injection into the subarachnoid space l models , 158. A l models , 160. IfWhen evaluating the safety of immunotherapies in pediatric brain tumors, especially in combination with radiotherapy, treatment related inflammation and edema due to immune-mediated tumor cell death must be considered. As described above, clinical and radiographic pseudoprogression can occur in patients undergoing radiotherapy for the treatment of brain tumors, but the overall tolerability in the published experience with combined radiotherapy and ICI in pediatric CNS tumors is reassuring. However, as more patients are treated with these combinations, the incidence of these acute toxicities may become more apparent. Another consideration in evaluating response to combined immunotherapy and radiotherapy is the complexity of interpreting radiographic changes following therapy and potential pseudoprogression, which may make it challenging to ascertain disease progression versus treatment response.As far as direct CNS toxicity with immune-based therapies, the greatest amount of literature is available for ICI. Anti-CTLA4 therapy is associated with auto-immune hypophysitis in 13% of patients, more so than PD-1 or PD-L1 blockade. Auto-immune thyroiditis is also reported in patient receiving ICI . FortunaTP53 mutations are likely associated with greater immune suppression, while tumors with MAPK pathway activation are associated with a more immunogenic environment. As a result, different patients likely require different levels of immunotherapy to achieve therapeutic response. This highlights the need to prospectively investigate immunotherapy on clinical trials where correlative studies, such as pre- and post-treatment biopsies, can be performed to provide hypothesis generating data on determinants of response. Such data will aid in identifying novel drug combinations that can overcome the immunosuppressive environment.As investigations around immunotherapy for pediatric CNS tumors continues, efforts are underway to identify biomarkers that predict response to agents like ICI. However, much remains unknown about the molecular determinants of immune environment and subsequently on the potential response to immunotherapies. As summarized above, some understanding is beginning to emerge. For example, tumors harboring Given crosstalk between the mechanisms underlying various cancer therapies and the ongoing need for better therapies for many CNS tumors affecting children and young adults, it is vital to continue exploring novel combination strategies of radiation, targeted agents, and immune-based therapies Figure 1BQ: conceptualization; writing, original draft; writing, review and editing. CK: conceptualization; writing, review and editing. SM: conceptualization; writing, review and editing. All authors contributed to the article and approved the submitted version.CK’s work is supported the Kortney Rose Foundation.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.


Rationale: Hepatocellular carcinoma (HCC) is a challenging disease due to its heterogenous etiology. Several breakthroughs have occurred in treatment of HCC, associated with an enormous number of patent publications for a variety of HCC treatment modalities. As patents can provide valuable information for academic research and commercial development, this study aims to unravel the cutting-edge therapies for HCC by using patents as an indicator. The outcome from this analysis may offer meaningful insights for respective policymaking, strategic plan and research and development (R&D) prioritization.Methods: Derwent Innovation platform was employed to collect the sample data of patents related to HCC treatment technologies worldwide as of December 31, 2019. Data inclusion, screening and exclusion were according to the rules of preferred reporting items for systematic reviews and meta-analyses (PRISMA). Technologies were classified based on Barcelona Clinic Liver Cancer (BCLC) staging system and recent clinical publications. Patent citation network analysis was carried out to identify and understand HCC therapeutic technology flow.Results: A dataset of 2543 patent documents and 528 patent families was generated. 11 technological categories were classified. Numerous researches were focalized on refinements in technologies and innovations within the field of HCC therapy, and the major achievements are technology advancement on molecular target therapy, chemotherapy, locoregional therapy, combination therapy and immunotherapy with demonstrated clinical benefits. In patent citation network, Notch pathway investigation, antibody drug conjugate (ADC) technology development and drug eluting beads trans artery chemoembolization (DEB-TACE) advancement are the major technological communities involving patents with the greatest future exploratory potential.Conclusion: Numerous emerging technologies have been identified in this study, in which exploring novel therapeutic targets in molecular target therapy, more localized and visible locoregional therapy and combination of immunotherapy with target therapy or other traditional therapies are highlighted as the future trends in treating HCC. Liver cancer is the sixth most commonly diagnosed cancer and the fourth leading cause of cancer death globally Given that HCC pathogenesis is complicated and heterogeneous, multidisciplinary team with diversified treatment modalities is essential to make decision by considering the tumor size, extent of tumor burden, functional status of the liver and patient performance status All in all, HCC is a challenging disease to treat due to its unique, complicated and heterogeneous etiology. Constantly, there is a medical unmet need of more effective and tolerated treatment options for diversified HCC characteristics. In the past years, there were many efforts on researching, developing and evaluating novel HCC therapeutic methods and technologies, which also led to abundant patent publications on HCC treatment technologies. These patents are worth noting not only as a legal shield of protecting interests of original researchers and developers by blocking competitors to copy innovations but in terms of the fact that new therapies usually publish patent documents before accessing to market. The implication of patents as an early signal of marketed products is emphasized in medical sector due to its lengthy, costly and risky process of R&D. However, patent documents represent a potentially useful but often underused resource To address this knowledge gap, HCC therapeutic technologies were systemically reviewed from the perspective of patents by categorizing the technologies into distinctive groups mainly based on BCLC guideline and other relative clinical papers as well st line therapy for advanced HCC, setting up the new gold standard for the coming years As shown in Figure https://clarivate.com/products/derwent-innovation/), a well-known patent database that is recognized worldwide. Data inclusion, screening and exclusion were conducted according to the rules of preferred reporting items for systematic reviews and meta-analyses (PRISMA)  . To avoiMolecular targeted therapy, as an emerging treatment modality for HCC, has been paid significant attentions in the technical invention. There are 126 patent families (filed in 743 patent documents) about molecular targeted therapy for treating HCC, accounting for 24% of all , blocking the signaling pathway of cancer cell growth and division (WO2007041379), and changing protein activities within the cancer cell (WO2007075567). Some small molecules have been invented targeting factors involved in angiogenesis, such as VEGF (WO2013025944). Several drugs have been developed to inhibit epidermal growth factor receptor (EGFR) (WO2012087943), while others act on pathways that are existing targets of other drugs, such as mitogen active protein kinase (MEK) (JP06431770) and tyrosine-protein kinase Met (c-Met) (WO2007075567). Patent WO2007075567 described an invention to use triazolopyridazine compounds as protein tyrosine kinase modulators, particularly to inhibit c-Met activity and modulate c-Met expression in tumor cell in order to prevent tumor cell proliferation or disorder related to c-Met. As HCC has been shown with increased expression of hepatocyte growth factor receptor, the drugs that target to c-Met may have therapeutic efficacy against HCC tumor cells 6, blockiCytotoxic chemotherapy is the secondary hot spot in HCC patent research, which has 93 patent families (filed in 550 patent documents) being published, accounting for 18% of total patents Figure . TraditiThere are devices developed to remove chemotherapeutic agents  from the blood coming from the tumor, in order to prevent contaminating the circulatory system (US4820261). Many patents claimed to use nanoparticle carriers loaded with anti-cancer agents to achieve more localized treatment (WO2008109163). In addition, back to 1995, electrochemotherapy was developed to introduce anti-cancer agents directly into cancer cells by increasing the permeability of cell walls through the use of electric pulses (US5468223). This technique is also called irreversible electroporation (IRE), which was further developed by Scherman Yves Leon as an individual from France in 2005, who used two electrode needles with two physical forces to enhance the penetration of anti-cancer agents into tumor cells (FR2872055). Hepatic artery infusion chemotherapy (HAIC) for local delivery of therapeutic agent on treating liver cancer was filed for patent by Abbott Cardiovascular Systems (WO2014117075) in the US, Japan, China and Europe from 2014-2016. HAIC is expected to provide a stronger antitumor effect and lower incidence of systemic adverse events due to the increased local concentrations in the tumor and reduced systemic distribution of anticancer drugs compared to traditional systemic chemotherapy In this study, patents in terms of cytotoxic chemotherapy also cover the use of doxorubicin (WO2000042832), method to shield bone marrow during chemotherapy (WO1999038442), stent as an intra-lumenal drug delivery device (WO2003103743), and thermoactivated drugs release (WO2006102471). Therefore, the growing interest of novel forms of chemotherapy for the treatment of HCC is mainly due to the technological advances that allow a targeted release of higher concentrated anticancer drugs.For HCC patients with cirrhosis who are not eligible for surgical therapies, locoregional therapy provides minimally invasive procedures, including ablation, bland embolization, TACE, trans artery embolization (TAE), DEB-TACE and radiation therapy, and these technologies have been filed for patents, accounting 29% of all in this study  , microwave ablation (MWA) , high-intensity focused ultrasound (HIFU) , electroablation  and laser ablation (US20030097152). Among them, electroablation has gained the most researchers' attentions.In general, RFA is the most commonly used and preferred ablation treatment for HCC, which delivers electromagnetic pulse to cause tumor necrosis by heat injury Although TACE and TAE have been widely used on HCC clinical setting, their total patent families only accounts 5% of all inventions Figure . They coConventional TACE involves the injection of selected chemotherapeutic agent mixed with lipiodol (a contrast medium made from poppy oil), followed by injection of embolic material via the hepatic artery The first patent related to TACE in our study was filed in 2001, discussing about catheter and perfusion system in arteries WO2001003755). After that, the majority of patents were concentrated on DEB-TACE, using different types of materials as a carrier to deliver anticancer agent to tumor as well as to embolize the tumor blood supplies, and provide a localized drug release to the tumor. Based on our patent dataset, these carriers/beads could be made of hydrogel (WO2009073193), silicon (WO2002067998), magnetic nanoparticles and embolism composite (CN102652729), polymers (WO2004071495), polyvinyl alcohol nano-fiber particles (CN108187127) or gelatin sponges (WO2016093412). The growing interest on DEB-TACE may be due to the limitation of conventional TACE, which doesn't have a standardized protocol about the choice, dosage, concentration, rate of injection of chemo drugs, and optimal retreatment strategy 55. AfterRadiotherapy is another local regional therapy that has been proven with promising therapeutic results. In fact, TACE and ablation have their own limitations. For instance, HCC with portal vein thrombosis (PVT) is a contraindication to TACE, while HCC with bleeding diathesis is a contraindication to RFA and MWA in vivo imaging targeting the enzyme aldehyde dehydrogenase was invented in 2010 (WO2010048144). Charged particle therapy is an advanced technique of EBRT to overcome dosimetry challenges, such as radium particle therapy (EP1644049), neutron capture enhanced particle therapy (NCEPT/NCT) , and radioactive nanoparticles for NCT .Historically, conventional EBRT has had a limited role in treating HCC because there is 8.4% risk of causing radiation-induced liver disease (RILD) when the radiation dose is higher than 50 Gy Other than the approach to give radiation from outside to inside patient's body like EBRT, scientists/researchers have also invented SIRT to offer radiation from inside in a localized manner. SIRT in treating HCC involves the injection of microspheres with radioisotopes or radioactive complex through hepatic arteries, which supply more than 80% of the total blood to the tumor In recent years, there are increased clinical evidences of efficacy of radiotherapy in HCC either used alone or in combination with other therapies Immunotherapy alone occupies 8% of the total patent families, excluding the ones being claimed as combination therapy Figure . The numHCC is an immunogenic cancer, because it is usually developed from chronic liver hepatitis accompanied with liver cirrhosis, due to viral or non-viral pathogenesis. Such inflammation is associated with high tumor immunogenicity Thus far, two checkpoint inhibitors have been developed, which are cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) inhibitors and programmed cell death protein 1 pathway (PD-1/PD-L1) inhibitors. Accordingly, human anti-CTLA-4 antibodies (US20030086930), human antibodies to PD-1  and anti-PD-L1 antibody  were found in our patent dataset.Since HCC tumor expresses insufficient antigens to activate T-cells, the first immunotherapy patent in our dataset is from Japan dated back to 1999, which is about replicating defect recombinant retrovirus containing a vector structure to command the antigen expression (JP11262397). In 2016/17, new technologies were developed to deliver an antigen to the cytosol of an immune cell (WO2016070136), and intratumorally deliver particles containing a tumor antigen (WO2017181128). Furthermore, many works intending to activate and regulate T-cells have been done, for instance, using recombinant replication-deficient cytomegalovirus to generate a long-term, repeatedly stimulated T cell-based immune response (WO2011093858), generating antigen-specific CD8+ regulatory T cells (WO2012149282), converting the negative signal of TGFβ for T cell proliferation into a T cell activation signal (WO2014172584), promoting the formation, expansion and recruitment of T-cells in an antigen-specific manner (WO2016198932), using antigen-presenting cell-mimetic scaffolds to manipulate T-cells (WO2018013797), regulating T-cells production by nano-pulse stimulation (WO2018106672) and disrupting tumor microenvironment to regulate (chimeric antigen receptor) CAR-T cells (US20190287656). Additionally, in order to counteract cytokine dysregulation in HCC, inventors have also worked on modifying cytokines as anti-tumor medicine (CN1853730), producing cytokine in a cell to increase tumor specific immune response (CN102264760) and preparing tetrameric cytokines with improved pharmacokinetics by the dock-and-lock technology (US20130109073).Currently, clinical exploration on the use of monotherapy with immuno-oncologic agents in HCC treatment is on the rise. Two anti-PD-1 antibodies to treat advanced HCC have been approved by FDA so far, which are nivolumab and pembrolizumab in vivo or in ex vivoGene therapy is defined as curing a genetic condition by delivering therapeutic nucleic acids into human cells, which can be performed either in vivo gene therapy, the risk of adverse effects is high, as nucleic acids are directly delivered into patients' body through intravenous, intra-arterial, intra-portal and intra-tumoral administration In the area of gene therapy, the most challenging and important research question in the past decades was that what delivery systems should be used to introduce therapeutic nucleic acids, siRNA or suicide genes. In 2011, the invention (WO2011043719) provided replicating-competent adenoviral vector systems carrying one or more inserted heterologous gene that could offer enhanced transfection efficiency and specificity for gene delivery. Adenoviral vector is an efficient system for HCC hepatocyte targeted gene therapy by affecting non-dividing hepatocytes, because liver normally has less than 1% of dividing hepatocytes The technology development pace in the field of HCC therapeutics has not been as much as with other cancers, although huge milestones have been made in gene therapy during the past decades. However, gene therapy has developed in a much slower pace, when compared to molecular targeted therapy Figure , with onRegarding all aforementioned HCC therapeutic options, there may be a ceiling of effects for using monotherapy, especially for advanced HCC, as for cancer treatment, there is no such “one fits all” solution. Thus, combination therapy became a hotspot to enhance the treatment efficacy. In the present study, the amount of patent publications related to combination therapy accounts for 13% of all HCC treatment patents Figure . During In this study, around 46% of combination therapies are about combining immunotherapy with other types of therapy as immunomodulatory approaches. The basic mechanism behind is to positively modulate the tumor microenvironment (TME) aiming at counterbalancing the strong immune suppressive setting. Among them, this present study shows that the research gravity is moving more towards molecular targeted therapy in combination with immunotherapy, such as antibodies binding to human CSF-1R plus immunotherapy (CN104271158), mTOR inhibitor plus anti-CD20 antibody (JP06042801), anti-IL-10 antibody or antigen binding fragment plus CpG-C oligonucleotide (CN107949399), and anti-PD-L antibody plus DNA-PK inhibitor (WO2018178040). This trend is aligning with the hotspot in clinical research, as recent clinical studies have demonstrated the more potent anti-tumor effects by combination of an anti-PD-1 antibody and an anti-angiogenesis agent Radiotherapy is used for unresectable HCC, which can induce tumor apoptosis and necrosis, as well as releasing tumor associated antigens and neoantigens at the same time Despite the enhancement of immune response, there are also abundant other combination regimens showing in the patent database, such as chemotherapy plus radiotherapy , molecular targeted therapy plus TACE/TARE , RFA plus cancer drugs , miRNA plus radiation/chemotherapy (WO2016149580), hyperbaric oxygen with histone deacetylase inhibitors plus glycolytic therapies  and alcohol ablation plus ultrasound (CN110101859). Altogether, emerging strategies with novel agents combing with the existing therapeutic approaches bring higher possibility to improve HCC patients' therapeutic outcomes.Patents related to transplantation, resection and biological therapy have the smallest proportions, less than 3% in terms of each type of therapy Figure . AlthougFigure In many studies, patent citations have been employed to explore technology transfer and technology flow Figure In general, this citation map shows that the major clusters do not have connections with each other, and they all belong to the type of technologically concentrated community. This is different from our previous studies In order to reflect the evolution of network clusters, the 26 clusters with more than five patents were extracted and divided into three time periods, 1990-1999, 2000-2009, and 2010-2019, as illustrated in Fig. When patents have a high out-degree in citation network, it means that these patents are mainly cited by subsequent patents, because they contain either fundamental or innovative technologies, which others in the field are trying to imitate WO2007145840 in cluster 1 is the invention that for the first time identified antibody that specifically binds to a non-ligand binding region of the extracellular domain of a human Notch receptor therapeutically effective against cancer. In the field of HCC therapy, research on signaling pathways are of interest because of their specific anti-cancer characteristics. For example, sorafenib is the approved therapy for advanced HCC by targeting ras/raf signaling pathway All the patents in cluster 1 are related to Notch pathway belonging to OncoMed Pharmaceuticals, Inc. These inventions are the scientific basis of their two molecular targeting clinical pipelines, Tarextumab and Brontictuzumab that are being developed in collaboration with GlaxoSmithKline In the second largest cluster 2, the most influential node represents patent of US20140004078, which is the invention of antibody drug conjugates (ADC) technology. The monoclonal antibody and therapeutic agent are linked by an acid cleavable linkage to increase cancer therapeutic efficacy (US20140004078). For many years, it has been a scientific goal in the field of specifically targeted drug therapy to use monoclonal antibodies for the specific delivery of toxic agents to human cancers. Conjugates of tumor-associated antibodies and suitable toxic agents have been developed but have had mixed success in the therapy of cancer. During the development of this ADC technology, the most critical challenge was believed to be the linker between the antibody and drug for retention of good anti-tumor activity. With that, inventors have developed novel linkers for a variety of drugs or cancer toxic agents, such as disulfide linkage and ester linkage In addition, we have found another influential invention in cluster 2, which is a method using ultrafiltration and diafiltration to prepare stable and high concentrated formulations of immunoglobulins, antibodies and antigen binding antibody fragment . While the standard mode of antibody administration is intravenous infusion, several side effects such as rash, hypotension or urticaria have limited the antibody infusion rate Overall, abovementioned three biggest nodes in cluster 2 have highlighted the two essential elements in the field of using ADC to effectively treat cancer, which are AD linkage and antibody concentration. With these achievements, many specific technologies in this field have been invented by citing these two inventions. If we look into the small nodes with higher in-degree in cluster 2  that have cited the biggest nodes, it is obvious that the technology flows to anti-Trop-2-SN-38 immunoconjugates with a CL2A linker with specified dosage to maximize efficacy for cancer treatment with minimal side effects. While anti-Trop-2 is the antibody, SN-38 is one of the potent camptothecin derivatives that is potent antitumor agent.All the patents in cluster 2 belong to Immunomedics, which is a pharmaceutical company focusing on human monoclonal antibody and ADC for treatment of cancer. From cluster 2, we can see that antibody-SN-38 immunoconjugates are their focus in recent years, also could be their future R&D development direction. One of the clinical pipelines from Immunomedics, sacituzumab govitecan (IMMU-132) is an anti-Trop-2-SN-38 ADC for cancer. Phase I/II study of IMMU-132 in patients with epithelial cancers including HCC is undergoing now WO2004071495 in cluster 3 is the invention of polyvinyl alcohol-based polymer particles loaded with doxorubicin for the purpose of embolization of vessels supplying malignant hyper vascularized tumors, accompanied with a local, controlled, sustained delivery of doxorubicin to the tumors. This patent is the fundamental technology for the development of DEB-TACE that is one of the key clinical pipelines from Biocompatible UK company. This invention paved the way for DEB-TACE, compensating the limitations of conventional TACE, with numerous advantages including standardized procedure and reduced systemic exposure to doxorubicin. However, there is no head to head comparison in clinical trial to show if DEB-TACE overperforms cTACE in terms of HCC patients' survival.From cluster 3, we can see that the DEB-TACE technology flows to imaginable radiopaque polymer  and beads in smaller size (JP2017025071). These patents are also from Biocompatible UK. Thus, it is not difficult to see that this company's strategy on DEB-TACE tends to make visible and smaller drug eluting beads. Several company sponsored clinical studies regarding to these new generations of DEB-TACE have also been published in recent years, including Lencioni et al 2018 and Aliberti et al 2017 Based on the technology citation network, the major clusters do not have connections with each other, implicating a strong technology barrier among HCC therapeutic technology categories. This interesting discovery may be attributed to the unique, complicated and heterogeneous etiology of HCC leading to the needs of therapy diversification, utilizing distinctive technologies, which could barely have overlapping technology development. National borders have also been shown as a negative impact on knowledge flows. As geographical distance has a significant impact on knowledge spillovers Therefore, one assignee can't involve into multiple technologies. Company strategy teams can see a full picture of technology development trend and gain early insight into innovation competitors from this patent analysis. While combination therapy is one of the future trends in treating HCC, we recommend making investment onto advancement of combination therapy clinical evidence by cooperating with other companies, who have alternative therapeutic technologies. Such action also can break boundaries among various dispersed technology clusters in order to achieve the best clinical outcome in treating HCC. For big company with sufficient funds, acquisition and merge of other companies is an efficient strategy to integrate multiple technologies into one company, so that one stop window can be provided to HCC patients and related health care providers.In addition, diversified HCC therapeutic modalities are recommended to get approval from government due to the heterogeneous HCC characteristics in order to achieve personalized therapy for individual patient. In the battle of fighting with this changeling disease, the emerging cutting-edge therapies are demanded to be covered by government reimbursement in order to fulfill affordability.From researcher's perspective, molecular target therapy for advanced HCC was the dominant technology in concerned field with largest amount of inventions, which implicates the importance of exploring novel therapeutic targets and pathways by R&D teams. The trend of research and technology development in treating HCC tends to be more localized, penetrated, degradable (if using beads as drug carrier) and imageable therapies for the purpose of reducing toxicity and boosting efficacy at the same time, especially in chemotherapy and locoregional therapy developments, and there are increased clinical evidence on locoregional therapy nowadays leading the recommendations of it on HCC guidelines. Immunotherapy grows rapidly in the recent years, but it must be noted that there is a technology lacking on reducing potential toxicities of immunotherapy from this patient analysis, while it has been reported that immune checkpoint inhibitors may cause serious toxicities. Therefore, reducing such toxicities could be a future important focus in immunotherapy research for safely treating HCC. Regarding to gene therapy, research investment is recommended to be located at exploring delivery system, such as nanoparticle that demands clinical trials before using on HCC patients.It is always difficult to determine a single target gene, because HCC is polygenic and multifactorial. Therefore, combination therapy might be more effective than standalone therapy. Investment and researches on studying emerging strategies with novel agents combing with the existing therapeutic approaches can bring higher possibility to improve HCC patients' therapeutic outcomes. We predict that the research gravity and company strategies will move towards molecular targeted therapy or other traditional therapies in combination with immunotherapy in the coming five years. Moving into 2010-2019, we see the protagonist in HCC therapy has been changed to target therapy, immunotherapy and DEB-TACE. Combination use of ADC may be carried on in a near future. Notch-based strategies will be investigated continually, and combination with other approved treatment approaches will provide alternative treatment options to aggressive cancer types, such as HCC.This study still has some limitations. Some recent emerging but important patents may be disregarded when patents are analyzed in citation network, because latest patents are often cited less frequently. Thus, it is believed that continuous tracking of the inventions in the next several years remains the optimal approach to verify emerging favorable technologies. Another limitation is due to the nature of patent, where not that all R&D work on HCC therapy are fully reflected in patent-based analysis, although patent is generally considered as a key indicator of innovation and potential commercialization. In addition, many of the patents in this study are not specifically claimed for HCC, but also for other cancer types. This could be a potent cofounding factor in this research.At present, there are a huge number of patents in the HCC field. This research mainly explores patents in the therapeutic field. The final patents included are designed to ensure accuracy. Noteworthy, there are enormous patents related to HCC, which include the relative surgery devices, fundamental experiments, biomarkers, vaccines etc. Although they cannot reflect HCC therapy development directly, they do indirectly contribute to the evolvement of HCC therapeutic field. In the future, we will continue to explore patent research in the entire related field. Overall, this HCC treatment patent landscape analysis still provides the first overview and significant supplementary knowledge about the previous and recent technological and clinical developments on HCC therapy.By the end, this study is highly descriptive in nature by focusing on a specific therapeutic field. Future works are highly demanded from explanatory perspective by approaches of different statistical modeling techniques, such as special analysis, agent-based simulation etc. Furthermore, we are planning to add more attributes to transform this descriptive research into explanatory research or even predictive research.Supplementary figures.Click here for additional data file.


The COVID-19 pandemic has created a period of global economic uncertainty. Financial strain, personal debt, recent job loss and housing insecurity are important risk factors for the mental health of working-age adults. Community interventions have the potential to attenuate the mental health impact of these stressors. We examined the effectiveness of community interventions for protecting and promoting the mental health of working-age adults in high-income countries during periods of financial insecurity.Eight electronic databases were systematically screened for experimental and observational studies published since 2000 measuring the effectiveness of community interventions on mental health outcomes. We included any non-clinical intervention that aimed to address the financial, employment, food or housing insecurity of participants. A review protocol was registered on the PROSPERO database (CRD42019156364) and results are reported in accordance with Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines.From 2326 studies screened, 15 met our inclusion criteria. Five categories of community intervention were identified: advice services colocated in healthcare settings; link worker social prescribing; telephone debt advice; food insecurity interventions; and active labour market programmes. In general, the evidence for effective and cost-effective community interventions delivered to individuals experiencing financial insecurity was lacking. From the small number of studies without a high risk of bias, there was some evidence that financial insecurity and associated mental health problems were amenable to change and differences by subpopulations were observed.There is a need for well-controlled studies and trials to better understand effective ingredients and to identify those interventions warranting wider implementation. The COVID-19 pandemic has created an unanticipated period of global financial insecurity.These relationships are bidirectional, reflecting both social causation and social drift.Community interventions present one means of attenuating the mental health impact of episodes of financial insecurity.While implementing interventions that act on the social determinants of mental health has been highlighted as a global priority, a substantial implementation gap exists.Previous reviews have examined the effectiveness of community interventions for improving the mental health of specific subpopulations, such as the long-term unemployed,CRD 42019156364) and results are reported in accordance with Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines.We conducted a systematic review to identify effective community interventions for improved mental health and highlight priorities for future research. A review protocol was registered on the PROSPERO database CRD 019156364An electronic search of eight academic and grey literature databases  was conducted in August 2019. The search strategy incorporated a mix of keywords, and subject headings adapted for each database. Based on our study aims, the search strategy was built around three concepts: (1) personal or household financial insecurity, including recent unemployment, precarious employment, personal debt, food and housing insecurity; (2) community interventions; and (3) mental health and well-being outcomes see . Two rev10.1136/jech-2020-215574.supp1Supplementary dataFor inclusion, studies needed to report the effectiveness of a community intervention on mental health outcomes for working-age adults (aged 18–64) in high-income countries, as defined by the Organisation for Economic Co-operation and Development,The primary outcome, mental health and well-being, was broadly defined to include psychological distress, the symptoms of common mental disorders, aspects of well-being and positive affect, and mental health service utilisation. In order to measure effectiveness, only quantitative studies employing experimental, quasiexperimental or observational designs (with either a non-exposed comparison group or before-after intervention measurement) were included. Data relevant to an economic evaluation were extracted as secondary outcomes.A data extraction framework was adapted from the Template for Intervention Description and Replication checklistTwo reviewers independently assessed individual study quality and risk of bias using the Effective Public Health Practice Project’s Quality Assessment Tool for Quantitative Studies.2326 non-duplicate titles identified were identified, 2304 from database searches and 22 from manual searches. Of these, 15 studies met our inclusion criteria . Ten stuMost studies (n=12) measured mental health outcomes using standardised screening instruments for the symptoms of anxiety and depression, stress, psychological distress, well-being and functioning . Four stFollowing data extraction, the included studies were categorised by the two reviewers into five intervention types: welfare and advice services colocated in healthcare settings (n=7),Seven studies measured the effectiveness of colocated welfare and advice services on mental health outcomes. A quasiexperimental controlled study in London rated of moderate quality reduced self-reported financial strain among participants at 3-month follow-up, compared with controls.A further six uncontrolled before-after studies, which were assessed to be of low quality, showed inconsistent results.Two studies from the UK measured the effectiveness of link workers providing referral from primary care to voluntary and community sector organisations. Both studies focused on the mechanism of referral rather than the specific social prescription that patients received. An RCT in England used trained project facilitators to encourage participation in programmes managed by local and national voluntary organisations.Two studies describing telephone debt advice services were identified, and both received a low-quality rating.A non-randomised study of food insecure, low-income households in Canada was rated low quality and compared traditional food banks to ‘alternative’ food insecurity interventions , which aim to promote empowerment, social inclusion and skill development.Three studies examined mental health outcomes following participation in active labour market programmes.Another two programmes for the recently unemployed were identified and received low-quality assessments. In Germany, participants in labour schemes who received wage and self-employment subsidies or undertook training courses showed increased life satisfaction at 12-month follow-up, compared with baseline (measured within 2 weeks of becoming unemployed).A full economic evaluation was not conducted in any of the studies meeting our inclusion criteria. Nevertheless, five studies measured the impact of services on healthcare staffing, appointments and prescribing,Interviews with healthcare workers revealed a perception that colocated welfare and advice services reduced staff workloadsThis review identified five categories of community interventions for working-age adults during periods of financial adversity. None of the included studies received a high-quality assessment and we find insufficient evidence to conclude that any one of these diverse categories of interventions is effective for improving mental health and well-being. Further, none of the studies identified conducted a full economic evaluation and there is limited evidence of an impact on costs to the health system.Three studies without a high risk of bias showed some evidence of effectiveness.This overall lack of evidence mirrors the findings of reviews examining the effect on health and social outcomes of colocated advice servicesThe studies we identified provide useful insights for intervening on health inequalities, which is particularly important in light of emerging evidence that the COVID-19 pandemic has exacerbated existing patterns of inequalities.Colocated welfare and advice services produced substantial financial gains for participants.There is some evidence that integrating community services into healthcare settings can improve mental health,A focus on universal and selective prevention during the acute stages of financial adversity means we have systematically excluded studies evaluating community interventions on specific subpopulations with recognised pre-existing challenges, such as the long-term unemployed and homeless, and people with severe mental illness. Similarly, we excluded a wider body of literature examining the effectiveness of interventions that seek to address chronic financial hardship and neighbourhood-level poverty.The evidence base for community interventions delivered to individuals experiencing personal or household financial adversity was marked by the presence of small, uncontrolled studies drawn from self-selected samples. However, our review highlights that community interventions can successfully reduce financial uncertainty, which is increasingly important given the deteriorating economic situation caused by the COVID-19 pandemic.Episodes of financial adversity, such as recent job loss, precarious employment, personal debt and housing and food insecurity, represent a crucial period for the protection and promotion of mental health in working-age adults.Community interventions are one means through which to address these social determinants of mental health.A stronger evidence base regarding the effectiveness of community interventions delivered to the general population during periods of financial insecurity is required.The evidence base for effective and cost-effective community interventions is marked by the presence of small, uncontrolled studies including potentially non-representative samples.Nevertheless, job search programmes, welfare and advice services located in healthcare settings, and link worker referral services show some evidence of effectiveness, at least for some subpopulations.The social determinants of health are amenable to change through focused community interventions and mitigating the impact of financial adversities could be an actionable pathway towards improved mental health.


The delivery of cancer therapeutics can be limited by pharmacological issues such as poor bioavailability and high toxicity to healthy tissue. pH-low insertion peptides (pHLIPs) represent a promising tool to overcome these limitations. pHLIPs allow for the selective delivery of agents to tumors on the basis of pH, taking advantage of the acidity of the hypoxic tumor microenvironment. This review article highlights the various applications in which pHLIPs have been utilized for targeting and treating diseases in hypoxic environments, including delivery of small molecule inhibitors, toxins, nucleic acid analogs, fluorescent dyes, and nanoparticles. Toxicity to healthy tissue can limit the dose and timing of several therapeutics especially cancer therapeutics, impacting quality of life and causing delays in treatments. Therefore, the development of tumor-specific agents would represent an important step towards overcoming these limitations. To date, much of the effort to develop targeted agents with relative tumor specificity has relied on the surface expression of tumor-specific antigens or cancer-specific dependence on signaling pathways. While these approaches have all led to important and meaningful improvements in cancer therapy, by nature of their design they are limited to specific cancer types and subtypes.pH-low insertion peptides (pHLIPs) represent a potential system to specifically target tumors, and importantly do not rely on the surface expression of tumor-specific antigens. Instead, pHLIPs are a pH-based delivery system that take advantage of the acidic condition of the tumor microenvironment for selective delivery of cargo. Acidity of tumor environments arise mainly as a consequence of hypoxia. As acidity is a ubiquitous hallmark of the tumor microenvironment, pHLIPs have potential for use across a number of different tumor types. This review article outlines pHLIP function and describes the numerous ways that pHLIP has been used in experimental models to demonstrate efficacy for cancer therapy and/or imaging.In normal tissue, extracellular pH is tightly regulated to approximately 7.4, while in pathological states, such as ischemia, inflammation, and neoplasia, dysregulation of pH commonly occurs. The tumor microenvironment is largely acidic , likely pH-low insertion peptides (pHLIPs) demonstrate pH-dependent ability to insert across cell membranes  and are Cargo can be linked to the C-terminus of pHLIP using a disulfide bond or other linkers that are subject to cleavage in the intracellular reducing environment . LinkageImportantly, the mechanism of action of pHLIP confers three advantageous properties for potential cancer therapeutics. First, the pH-specificity of pHLIP confers relative selectivity in targeting tumor cells, which allows for sparing of normal tissue and therefore an expected reduction in dose-limiting toxicities. Furthermore, the pHLIP system can assist in the transport of otherwise cell-impermeable molecules across the cell membrane, ameliorating potential issues in bioavailability. Finally, pHLIP targets the tumor microenvironment on the basis of acidity, a ubiquitous feature of the tumor microenvironment, thereby distinguishing it from other attempts to develop tumor-specific reagents.In mouse models, fluorescently labeled pHLIP has shown targeting to subcutaneously injected and spontaneous tumors, as well as metastatic lesions  6]. Acc. Acc6]. pHLIP shows enhanced targeting to aggressive tumors, as evidenced by the enhanced uptake of fluorescently labeled pHLIP by tumors formed from M4A4 cells, a highly aggressive and metastatic melanoma line, as compared with tumors formed from NM2C5 cells, a less acidic and weakly metastatic melanoma cell line . The abiMany cancer therapeutics are limited by their inability to cross the blood–brain barrier, limiting potential treatment options for brain cancers and metastases. Biodistribution experiments in orthotopic and subcutaneous models of glioma show accumulation of pHLIP is limited to extracranial sites, suggesting that pHLIP is unable to cross an intact blood–brain barrier . HoweverBeyond the tumor microenvironment, a number of other pathological states involve the dysregulation of tissue pH, including inflammation . In addi50 (pH at which 50% of pHLIP are in the inserted state) of pHLIP to improve cell uptake. pHLIP-D25E-C (Asp25 is substituted with Glu), pHLIP-P20G-C (Pro substituted with Gly) have higher pH50, whereas pHLIP-R11Q-C (Arg is substituted with Gln) exhibit lower pH50 AlF-cysVar3 pHLIPAcidity is a characteristic common to tumors that occurs partly due to metabolic alterations in response to hypoxic microenvironments. In animal models, exploiting acidity for targeted tumor delivery has been achieved using pHLIP for therapeutic and imaging purposes. Conjugating molecules to the inserting end allows cytoplasmic delivery, whereas labeling of acidic cells can be achieved by conjugating to the N-terminus. Multiple variants of pHLIP with altered pharmacokinetic properties have been reported that can be manipulated for various applications. pHLIP precisely delivers small polar toxins, DNA repair inhibitors, imaging agents, nanoparticles and antisense peptide nucleic acids to acidic environments in vivo. PNAs stand out among the numerous cargos delivered by pHLIP since they are non-toxic, sequence specific DNA mimics that can regulate gene expression by targeting microRNAs and mRNAs and can be effective against oncologic targets that are otherwise undruggable. The use of pHLIPs to deliver highly potent small molecule toxins to tumors may also provide advantages over antibody-drug conjugates for the same purpose because pHLIP targeting does not depend on expression of a specific antigen but instead exploits the acidity of solid tumors.While pHLIP remains a promising technology, improving the synthetic efficiency of pHLIP cargo constructs could significantly facilitate its clinical translation. In recent years, the discovery of multiple variants of pHLIP and new conjugation approaches have expanded the possibilities of cargoes that pHLIP can deliver to the acidic and hypoxic tumor microenvironment. Ultimately, this raises new potential for the use of pHLIP in the treatment and diagnosis of cancer.


Reducing risk from pesticide applications has been gaining serious attention in the last few decades due to the significant damage to human health, environment, and ecosystems. Pesticide applications are an essential part of current agriculture, enhancing cultivated crop productivity and quality and preventing losses of up to 45% of the world food supply. However, inappropriate and excessive use of pesticides is a major rising concern. Precision spraying addresses these concerns by precisely and efficiently applying pesticides to the target area and substantially reducing pesticide usage while maintaining efficacy at preventing crop losses. This review provides a systematic summary of current technologies used for precision spraying in tree fruits and highlights their potential, briefly discusses factors affecting spraying parameters, and concludes with possible solutions to reduce excessive agrochemical uses. We conclude there is a critical need for appropriate sensing techniques that can accurately detect the target. In addition, air jet velocity, travel speed, wind speed and direction, droplet size, and canopy characteristics need to be considered for successful droplet deposition by the spraying system. Assessment of terrain is important when field elevation has significant variability. Control of airflow during spraying is another important parameter that needs to be considered. Incorporation of these variables in precision spraying systems will optimize spray decisions and help reduce excessive agrochemical applications. Tree fruits are high-value commodities worth almost $18 billion in the U.S. ,3. HowevPrecision spraying is defined as the precise spraying of pesticides based on target information such as the size, shape, structure, and density of the tree canopy as obtained from sensors such as cameras, ultrasound, and laser. Using precision spraying technologies, Esau et al.  reduced This review focuses on the importance of advanced spraying systems, limitations of current technologies, factors affecting sprayer performance, and gaps in knowledge that need to be considered in the development of advanced precision sprayer systems for tree fruit orchards. Firstly, the history and revolution of sprayers and the limitations of current spraying methods were discussed. Then, the core components and technologies for precision spraying were identified and discussed, which are centered around camera, ultrasonic, and laser-sensing technologies. The factors effecting the use of these core sensing technologies were followed, including nozzle types, spraying distance, droplet size, canopy characteristics, wind speed, temperature, and relative humidity. Finally, possible pathways to address some of the challenges were discussed and concluded.Over the past decades, orchard sprayer designs have evolved from hand-boom based horse-drawn wagons to sensor-controlled tractor-pulled sprayers, due to increased concern about chemical wastage and environmental contamination, changes in horticulture, increased demand for quality products, and development of new technologies. The revolution of sprayer design is listed in . VariousAir blast sprayers  have beeAir blast sprayers include conventional axial-fan air blast sprayers, cannon sprayers, tunnel sprayers, tower sprayers, and custom-designed or modified air blast sprayers. The axial fan air-assisted sprayer produces a large radial spray volume and is the predominant design of sprayer used in tree fruit orchards. It is poorly targeted for modern intensive tree fruit orchards and creates a significant risk of off-target contamination by spray drift and chemical wastage to the ground . Tunnel Conventional air blast sprayers designed for larger trees can waste more than 50% of pesticides to the ground and the atmosphere when spraying modern apple trees that have smaller canopies ,35,36. SWith the advancement of modern sensors and technologies, the methods utilized for measuring complex tree canopy parameters are developing rapidly. These methods aim to accurately measure geometric canopy features, canopy density, canopy leaf area density (LAD), and canopy leaf area index (LAI) . These mGeometric Canopy Dimensions: Tree geometric canopy dimensions, such as canopy height, width, and volume, are needed for calculating the canopy density, leaf area density, and canopy leaf area index. Camera sensors are mainly used to detect the LWA  and have limited use in detecting canopy volume. The main sensors used for canopy geometric characteristics measurements are ultrasonic and LiDAR. Beyaz and Dagtekin [2 = 0.95). Cheraïet et al. [2 = 0.94 and 0.89, respectively) showed the potential of using this technique to adjust the spray rate for variable-rate spraying. Chen et al. [Dagtekin  measuredDagtekin  used LiDDagtekin  also useDagtekin  measuredt et al.  used a 2n et al.  measuredCanopy Density: Tree canopy density is essential to determine the spray volume for accurate variable-rate spray applications [2) of 0.90. Mahmud et al. [ications ,50. The ications  utilizedd et al.  developeCanopy Leaf Area Density: Canopy leaf area density (LAD) is crucial for characterizing the differences in leaf area at different layers in the vertical direction and employing variable-rate control of nozzles [ nozzles . The LAD nozzles  calculat nozzles  compared nozzles  mapped f nozzles  reconstrCanopy Leaf Area Index: The canopy leaf area index (LAI) is a core parameter of vegetation structure used to represent the leaf area per unit surface area [2 of 0.92; however, the insufficient number of scans and the complexity of the data analysis for LAI estimation were considered the major limitations of this study. Comba et al. [2 of 0.82 compared with the traditional method. Indirabai et al. [2 = 0.96) between the predicted and reference LAI measurements.ace area . It is aace area  measureda et al.  measuredi et al.  estimateMeasurements of tree canopy parameters require different advanced sensor applications and implementations. The following sections broadly discuss the application of different sensors for precision spraying. Machine vision systems use camera sensors , and range-sensing systems use ultrasonic and laser sensors to collect tree canopy data and carefully evaluate canopy parameters and targets. Zhang et al.  surveyedCamera-based sensing tries to mimic human perception to provide information or inputs based on processed image data to spraying systems needed for canopy characterization-based pesticide applications. Image processing includes pre-processing, segmentation, feature extraction, and classification. Image pre-processing eases the segmentation of the targeted region  from the crop images. It involves image rotation, normalization, resizing, color transformation, denoising and contrast enhancement, etc. Of these, color transformation is one of the most important for segmentation. A detailed description, including the advantages and disadvantages of different color spaces used in machine vision systems, is discussed by Cheng et al. . The conAfter image pre-processing, target area segmentation was carried out. With appropriate target area segmentation different types of targets  can be recognized for precision spraying applications. Various features are being used to differentiate target regions in crops using machine vision applications. Generally, these features are grouped into four sub-classes: morphology, visual texture, spectral, and spatial contexts ,64,65. AAfter valuable feature extraction, features are combined to improve robustness when training classifiers/models, which results in high dimensionality of features and the curse of dimensionality. A screening and elimination of unnecessary features is essential to reduce feature quantity and increase data processing speed. Different types of algorithms have been utilized to select effective features for orchard management, including stepwise discriminant analysis , quadratDeep learning (DL), an artificial intelligence approach, is a new and emerging area of machine learning that offers substantial potential for extracting complex structural information  and has A few studies have used stereo camera vision-based systems to measure tree canopy shapes and foliage densities and to detect tree crowns ,82,83. MCamera Sensor-based Spraying: Application of camera sensors for precision spraying in the tree fruit orchards was initiated when different image processing algorithms were available and easily accessible. A summary of the camera sensor applications for variable-rate spraying in orchards is listed in −1 using a sprayer developed by modification-upgrading of a trailed air-assisted sprayer , equipped with a piston pump and a 1000-liter tank. A total of 23% pesticide saving was calculated compared to spraying in the control mode. Savings were presented in this study for an average tree height of 3 m; however, the saving were not consistent for different fruit varieties with trees of varying shape and size. Suggestions were made that changing the camera position and estimating tree leaf density by scanning the trees may lead to better performance for the orchards with inconsistent tree sizes. Only color information of images was used in their study. A few years later, Xiao et al. [o et al.  captured−1, respectively, at different positions of the tree canopies. The difference of spray coverage was reported due to the variation of the spatial lag  during spraying. The study concluded that the spatial lag is influenced by the forward speed could result poor spray deposition.A real-time site-specific orchard sprayer was developed by Asaei et al.  to save Aside from tree canopy characteristics detection for spraying operation, few attempts have been investigated based on disease detection. Disease control in grapevines using a totally automatic selective spraying system was first introduced by Oberti et al. . The sysFor camera-based canopy parameter measurements, the light illumination is the process of making tree canopy clearer or brighter for capturing or acquiring images. Direct sunlight is used as a main source of light in most of the cases for field applications, and under such environments shading and high contrast transitions of illumination are a major problem. The illumination in the field usually varies with different times of a day, the position of the sun concerning the object and overcast conditions which can greatly affect image quality/characteristics . For exaUltrasonic sensor systems are another sensing technology being used to detect the tree canopy parameter to support precision spraying. These types of sensors are affordable, robust, and handy tools that can be used to map target information during spraying ,93. To d3 compared to the manual measurements. Experimenting with the same technology, ultrasonic sensors were utilized for tree volume measurement in citrus groves [2 of 0.94 (RMSE = 0.16 m−3·tree−1) and 0.94 (RMSE = 3.12 m−3·tree−1) were reported for height (2.1 to 4.3 m) and tree volume (6.3 to 54.0 m3·tree−1) measurements. Ultrasonic-sensing system was used with a differential global positioning system (DGPS) to achieve a variable-rate nitrogen application by generating a prescription map for citrus tree orchards [Ultrasonic sensors for fruit tree volume measurement have been studied widely ,98,99. As groves . Variouss groves . No signorchards  used a vorchards .Ultrasonic Sensor-based Spraying: These types of sensors regulate the spray operation based on canopy parameters measured through sound waves. A summary of ultrasonic sensor applications for site-specific spraying is presented in Balsari and Tamagnone  undertooA prototype spraying system was tested in olive, pear, and apple orchards with an electronic control system equipped with ultrasonic sensors , and repMaghsoudi et al.  utilizedDifferent trees have different canopy architectures and leaf angles, and the leaves angle could easily change with the wind . Zaman aThe major disadvantage of using an ultrasonic sensor is that the sensor has limited penetration capability, which provides measurements with low spatial resolution. They have limited detection range, and can be greatly affected by canopy detection distance, travel speed, and outdoor weather conditions, which can limit the accuracy of the canopy parameter measurements. Temperature fluctuation affects the speed of an ultrasonic sensor’s sound waves. Sound waves travel faster to and from tree canopies with temperature increases, which results tree canopies appearing closer than they really are. In addition, due to not having scanning capabilities, the ultrasonic sensors provide significantly fewer data compared to other sensing techniques including camera and LiDAR-based sensing, making the ultrasonic technology outdated in precision agriculture as more advanced technologies are already available .Laser sensing is an evolving technology mainly used in remote-sensing applications and plant phenotyping that has shown potential in tree canopy parameter detection  in the last few decades. Compared to other sensors, the major advantage of using a laser sensor for canopy volume estimation is that its electromagnetic signal can penetrate the vegetation canopy to provide canopy density information and the inner and outer structures of the tree . The las2·m−3. The study revealed the error increased with angular resolution of LiDAR and vegetation density. Li et al. [2 of 0.9738 was reported between the LiDAR measured plant volume and the tree volume measured manually. Therefore, the above studies concluded that the LiDAR-sensing system could be a powerful technique for non-destructive estimates of canopy foliage area characteristics and canopy density measurements of trees. LiDAR-sensing technologies are also widely utilized for determining spray drift and buffer zones in 3D crops [Laser scanner sensors can acquire the information and specifications of tree and fruit more precisely than ultrasonic sensors ,115,116.i et al.  used thei et al.  also mea3D crops  showing Laser Sensor-based Spraying: In the last decade, LiDAR-based laser-sensing technologies have been tested in fruit orchards due to their high precision and accuracy in real-time measurements [−1, because the necessary spray output surpassed the nozzle limit at a higher speed. The authors concluded that the measurement of different canopy foliage structures and densities need to be accounted for when evaluating the variable rate sprayer performance. urements ,123. A surements . This syAnother major limitation in using a laser sensor in this attempt was that it required two sensors for real-time detection of trees on both sides of the tree row due to the maximum radial scanning range of 180° in the laboratory-based evaluation. Considering the scanning limitations, Liu and Zhu  measuredUsing the same sensor configuration as Chen et al. , apple tBoatwright et al.  comparedIn the recent study, Chen et al.  conducte−1 is possible along with reductions of spraying time by 27–32% and labor and fuel by 28%.Manandhar et al.  conducteApart from the chemical saving, Shalal et al.  detectedChallenges are also associated with laser sensing, especially when vibrations from terrain variability are presented during scanning for tree canopy data. Inconsistencies of terrain could cause deviations of the angular orientation of the LiDAR sensor during examining/scanning , which cEstimating canopy size is a challenge because of the confounding structures and irregular shapes of trees, and variable environmental conditions in fields. Problems associated with camera vision sensors in field conditions include variable light, and distorted or blurred images due to vibrations and wind direction and velocity , which cAlthough several groups of researchers have developed prototypes to change the application flow rate based on canopy structural parameters from different sensors, precision spraying in tree fruit orchards needs further development to deal with excessive chemical usages, improper spray depositions, and losses from chemical drift . Real-tiThe effectiveness and quality of spraying depends on many factors, including spray deposition, drift, leaf absorption, and coverage volume. Spray penetration and coverage are greatly influenced by tree height, tree spread, and the continuity of canopies along rows which may easily reduce the effectiveness of the crop protection operation . In thisSeveral studies have analyzed the effect of forward speed on spray deposition and drift ,133. SalThe distance between sprayer and canopy is another important parameter that influences the amount of spray drift . Celen eThe mechanical system performing the spraying operation also affects the spray deposition and off-target losses in orchard trees. Salyani  reported2 = 0.948) with the drift potential tested with the wind tunnel for hollow-cone nozzles [The spray drift is also influenced by the type, size, and pressure of the nozzles used for spraying. Spray nozzles should be selected by following the required spray volume , nozzle  nozzles .Among the atmospheric factors, the wind speed and direction have tremendous effects on the spray depositions causing off-target agrochemical losses to the non-target receptors, including damage to the neighboring crops, water, and animals. The rate of off-target losses depends on the speed of the wind and direction of the wind flow .−1 airflow was reported from a two-fan air-assisted sprayer when there was a crosswind speed of 5 m·s−1 with the direction of 90° that could significantly affect spray deposition. In addition, a new model of bystander and resident exposure was developed to measure spray drift deposition from orchard applications [−0.104X; intermediate: Y = 26.928 × 10−0.124X; full leaf: Y = 19.036 × 10−0.118X; where Y is spray drift deposition (% of sprayed volume) at distance X m from the last tree row.In general, all types (class-based on sizes) of droplets have the chance of being off-target depositions, but the small droplets have the highest possibility of the off-target movement. Endalew et al.  utilizedications . ObservaAlthough the effects of temperature on spraying are region-specific , there is a slight deviation of the spray drift potential observed by the researchers due to evaporation ,152. Wat−1 along with a 16 °C air temperature and a wind speed of 3 m·s−1 are considered as ideal meteorological conditions [Humidity is another region-specific factor that affects parameters by decreasing spray droplet size, especially in the lower humidity regions . Humiditnditions .Several factors influence spray coverage, including droplet size, carrier rate, and formulation . A diffeRetention of spray droplets on leaf surfaces depends both on the characteristics of the surface and of the liquid and is critical for effective spraying operation. Spray retention is significantly reduced when the velocity of spray droplets is higher due to high nozzle pressure . The droThis review has discussed the current methods of spraying in orchards, machine vision applications for precision spraying, implementation of sensor-based precision spraying systems in orchards, and limitations of different sensors and corresponding challenges. The tree fruit industry has gained considerable attention due to its high usage of agrochemicals in orchards. In the last few decades, various type of spraying systems, including handguns, boom sprayers, polar jets, tunnel air blast sprayers, and so on have been tested in orchards to try and reduce chemical usage; however, none of them was fully successful and satisfactory due to non-uniform tree canopy structures and sprayer limitations.Although various machine vision approaches have proved to be very useful and successful in weed detection, crop stress monitoring and yield prediction, etc., challenges still exist, especially in outdoor field conditions. Among the different types of sensors used in machine vision applications, camera sensors are more susceptible to outdoor lighting and weather conditions. The camera vision approaches might be best suited for spot spraying (spray based on site-specific disease or pest pressure) rather variable-rate spraying in the tree fruits. Recent developments of multispectral and hyperspectral cameras offer narrower spectral bands that can penetrate into the canopies and accurately measure the conditions (diseases or pest pressures and stresses because of nutrients) of trees. However, longer data processing time  is a major limitation. Current inventions of deep learning-based artificial intelligence systems resulted in good canopy detections and segmentations. Semantic and instance segmentation based deep learning models, including SegNet, U-Net, DeepLab v3, mask-region-based convolutional neural networks (RCNN), and so on, could be the best fit for canopy area segmentation; however, more efforts and extensive investigations are expected to develop an effective and robust system for precision spraying. Apart from camera sensors, ultrasound and laser scanner sensors can overcome light illumination problems usage sound waves and electromagnetic signals, respectively, as inputs to measure detection distance and gather canopy structure parameters. As compared to ultrasonic and camera sensors, laser scanner sensors are more accurate and reliable in collecting crop structure information, are not affected by weather conditions, and can guide the sprayer unit efficiently and effectively to apply pesticides on target.The application of laser scanners has risen during the last decade in multiple agriculture applications, from automatic tree height measurement to site-specific pesticide application. However, challenges are still presented, especially in site-specific or variable-rate spraying in tree fruit orchards. Accurate canopy information measurements require automatic adjustment of laser sensor orientation during data acquisition, especially in conditions of different sloping and uneven terrains. Therefore, a real-time terrain monitoring system may need to be incorporated along with a sensing system for precision spraying. Terrain monitoring systems can be developed using a positioning sensor and a real-time kinematic global positioning system (RTK-GPS). An inertial measurement unit (IMU) sensor can be incorporated into the spraying system to monitor the terrain conditions in real-time and adjust the laser orientation accordingly. Studies reported IMU sensors could minimize positioning errors of the laser from terrain variabilities ,112. TheLaser scanner sensors only help with measuring canopy structure information, but a successful spraying operation requires a series of tasks to be completed accurately, including maintaining constant travel speed, adjustments of the fan speed at the sprayer unit, assessment of real-time weather conditions, prediction of droplet sizes and some other factors. Auto-guidance systems that automatically steer the sprayer throughout the orchard blocks could help maintain constant travel speeds. Crossflow fan sprayers reduce off-target spray deposition because they have more appropriate nozzle positions and uniform air delivery. An automatic damper system can be added at the sprayer fan’s air inlet (back of the sprayer) to control the airflow based on tree canopy information . A matheReal-time weather assessment is also important to reduce off-target deposition. Wind can easily blow spray from targeted to non-targeted regions. The real-time control of nozzles based on weather data is not currently practical during spray operation. The future sprayer also needs to include a droplet size prediction model to determine the diameter of droplets based on the characteristics of the tree canopies during spray operations. Electrostatic sprayers have provided improved deposition of specific droplet sizes and may be a good aid to develop next-generation sprayer systems . ElectroThe future of precision spraying systems will focus on integrating a suite of robotic and sensor-guided spraying systems with fruit tree growth and structure information to ensure accurate dose of pesticide delivery and reduce off-target deposition. A wide variety of sensor-based spraying technologies are available to measure the amount of spray volume needed; however, it is also important to assess the durability of these technologies so that a variety of sensors can be added at reasonable price for the farmers. Concurrently with sensor development, site-specific disease, pests, and stress monitoring using technologies such as deep learning and remote sensing will improve the functioning of current sensor-based spraying systems. Fusing/integrating different sensors could help to add more decision-making features for more precise pesticide applications in tree fruits. Future spraying systems must also handle diverse agrochemical formulations beyond that of the standard aqueous mixtures currently used. Although tractor-based spraying systems are mostly used for agrochemical applications, an alternative UAV based application method could displace traditional sprayers. UAVs have been tested in the field crops for disease scouting, stress monitoring, and spraying, and its preliminary results for spraying is showing promise. But most of the current UAV based spraying technologies may not be suitable for spraying in large scale tree fruit orchards due to their limited payloads and variable spray deposition capability. More research and development into UAV swarms and large capability UAV similar to unmanned helicopters could result in more effective system for commercial/large scale fruit orchards. Detection of disease and stress, pest pressure are other avenues for their use in tree fruits production besides variable-rate spraying. Therefore, the integration of multiple sensing and control systems will be the key to using advanced precision sprayers in the future.The canopy geometry, canopy density, leaf area index, and leaf area density are essential for variable-rate spraying and could be measured accurately with high precision using ultrasonic and LiDAR sensors;The camera sensors-based sprayer can be a good aid for detecting position of the canopies and spot spraying; however, their performance is inferior due to environmental and sensor limitations;The sprayer with ultrasonic sensors can have high precision in the orchard where the trees are discontinuously planted or trees with low canopy density and less variation among sections. These kinds of sensors can be useful for measuring average target canopy characteristics;The LiDAR sensors guided sprayer could be suitable for many types of orchards  and provide canopy details regardless of the environmental conditions with comparatively high reliability and accuracy;Use of LiDAR sensors must need advance algorithms and microprocessors to overcome the complex filtering problem and to process large amounts of information;Fusion of different advanced sensors, application of new algorithms such as deep learning, and new research methodologies should be considered to develop the next generation advance precision sprayer for tree fruit orchards.The excessive use of agrochemicals by conventional sprayers has gained attention among orchard growers, practitioners, and ecologists. Serious efforts are required to make an advanced precision orchard spraying system commercially available in an economically and environmentally sound manner. Considering the precision, accuracy, and numerous factors of using different sensors in outdoor environments, we conclude with the following guidelines for variable-rate spraying in the tree fruit orchards:


These networks enable a neuron to configure different cellular domains for specialized functions. According to current evidence, mRNAs are mostly transported in rather small packages of one to three copies, rarely containing different transcripts. This opens up fascinating logistic problems: how are hundreds of different mRNA cargoes sorted into distinct packages and how are they coupled to and released from motor proteins to produce the observed mRNA distributions? Are all mRNAs transported by the same transport machinery, or are there different adaptors or motors for different transcripts or classes of mRNAs? A variety of often indirect evidence exists for the involvement of proteins in mRNA localization, but relatively little is known about the essential activities required for the actual transport process. Here, we summarize the different types of available evidence for interactions that connect mammalian mRNAs to motor proteins to highlight at which point further research is needed to uncover critical missing links. We further argue that a combination of discovery approaches reporting direct interactions,  FMRP was found to interact with either all KIF5 isoforms . Ample evidence exists for its direct binding to the beta-actin mRNA localization sequence (the “zipcode”)  with lower nanomolar affinity. Biochemical in vitro reconstitutions could then prove that a minimal reconstituted complex consisting of the kinesin-2 KIF3A/B/KAP3 and APC can transport RNAs over tens of micrometers . Using purified, full-length proteins and 3'UTR fragments, APC was shown to bind KAP3, a kinesin-2 adaptor, and G-rich motif-containing axonal mRNAs   screening for direct interactions with proper orthogonal validations could add valuable information to understand how mRNAs, mRNA-motor adaptors, and motor proteins directly interact : neuronal mRNA distributions are likely the result of complex dynamic processes, multiple overlapping reactions, and concentration-dependent competitive mechanisms. Revealing these processes could greatly improve the understanding of how cytoplasmic mRNA distributions are generated. As shown for a variety of cellular processes, bottom-up Developing new approaches to disentangle pleiotropic functions of transport-mRNP building blocks in neuronal models: motor proteins and RBPs have multiple and sometimes redundant functions. After a knockdown, the accumulation of secondary effects and residual activities of the latter, depending on protein stability, can obscure their true functions. Hence, fast depletion and restoration of proteins, e.g., performing auxin-induced degradation (Nishimura et al., ECR, JG, SPM, SJB, and NL wrote the manuscript. ECR designed the figure. ECR, JG, and SPM analyzed the literature. 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.