The technical success was 100% in both groups. Conclusions: Our study confirms that transarterial embolization is a safe and effective option for the treatment of spontaneous AWHs, and it suggests that the efficacy and safety of blind embolization is comparable to non-blind.Background: Endovascular treatment of abdominal wall hematomas (AWHs) has been increasingly used when conservative treatments were not sufficiently effective, and it is often preferred to surgical interventions. The aim of our study was to evaluate the safety and technical and clinical success of percutaneous transarterial treatment of AWH and to evaluate the efficacy of blind embolization compared to targeted embolization. Materials and Methods: We retrospectively enrolled 43 patients  with spontaneous AWH who underwent digital subtraction angiography (DSA) and embolization, focusing on the presence of signs of bleeding at pre-procedural CT-Angiography (CTA) and at DSA. Furthermore, we divided patients into two groups depending on blind or targeted embolization approaches. Results: The mean age of the study population was 71 ± 12 years. CTA revealed signs of active bleeding in 31 patients (72%). DSA showed signs of active bleeding in 34 patients (79%). In nine patients (21%), blind embolization was performed. The overall technical success rate was 100%. Clinical success was achieved in 33 patients (77%), while 10 patients (23%) rebled within 96 h, and all of them were re-treated. No major peri-procedural complication was reported. The comparison between blind and targeted embolization showed no statistically significant differences for characteristics of groups and for clinical success rates (78% and 77%, respectively, − Abdominal wall hematomas (AWHs) are defined as the extravasation of blood in muscle structures that can be contained by the fascia or can diffuse into the peritoneal or retroperitoneal space. AWHs are relatively uncommon findings, but they may be a life-threatening condition, often associated with other conditions such as abdominal trauma, anticoagulation or iatrogenic injuries. The exact incidence and prevalence of AWH, however, are still unknown ,2,3,4.Traumatic AWHs are correlated with blunt or non-blunt injuries involving the abdomen; their detection is important for their strict association with potentially severe visceral and vascular injuries . IatrogeAnticoagulant and antiplatelet therapies are the most common causes of spontaneous abdominal wall hematomas, but spontaneous bleeding may also occur in patients with coagulation disorders or damage to blood vessels, such as hypertension, atherosclerosis, vasculitis and hematologic disorders .The knowledge of the vascular and topographic anatomy of the abdominal wall allow the mechanism of injury and the likelihood of hemorrhagic onset in defined regions of the abdominal wall to be understood.The abdominal wall is divided into anterior, lateral and posterior compartments. There are four anterior abdominal wall muscles per side: the rectus, the external oblique, the internal oblique and the transverse muscle .The vascular supply of these muscles is mainly provided by the deep inferior epigastric arteries (IEA), the superior epigastric arteries (SEA) and the deep circumflex iliac arteries (CIA). The IEA reach the posterior aspect of the rectus muscle where they join the SEA coming from the internal thoracic arteries. The posterior muscles are three per side: the iliac, the psoas and the erector spinae muscles. These muscles are supplied by the lumbar arteries (LA) and the iliolumbar arteries (ILA). The ILA is the first ascending branch of the posterior gluteal trunk of the internal iliac artery  Figure .AWHs of the anterior wall most often occur in the lower quadrants of the abdomen, due to the anatomic characteristics of this region. Above the arcuate line of Douglas , in fact, rectus muscles are fully enclosed both anteriorly and posteriorly by the fascial sheath that arises from the aponeuroses of the external oblique, internal oblique and transversalis muscles. This sheath protects both the superior epigastric artery and the inferior epigastric artery. Below the arcuate line, the sheath is present only anteriorly, and the epigastric vessels are placed between the transversalis fascia and the peritoneum. For these reasons, hematomas that occur above the arcuate ligament are most often contained, small and secondary to distal lesions, while hematomas located below the arcuate line of Douglas are generally bigger, can be secondary to proximal lesions and can cross the median line and sometimes reach near the posterior peritoneum and the retropubic space ,7.The diagnosis of AWHs, especially in case of posterior wall hematoma, is challenging due to heterogeneous clinical presentations, and imaging techniques are usually required. Computed tomography (CT) is essential to provide accurate information about location, size, origin, and extension of hematomas but also to exclude other acute abdominal conditions, while CT-Angiography (CTA) has a crucial role to demonstrate the presence of active bleeding within the hematoma .Depending on the patient’s condition, the underlying cause and the extent of the bleeding, the management of the AWHs can include conservative, surgical or endovascular treatment .Management of AWH remains controversial and relies currently on conservative measures such as correction of coagulation parameters, fluid repletion and blood transfusion . In hemoEndovascular treatment of the abdominal wall hematomas consists in transarterial embolization of the target vessels identified by CTA or digital subtraction angiography (DSA) using different materials such as coils, absorbable sponge or glue or a combination of these materials, and this technique is nowadays considered a fast and efficient treatment option . In the The aim of our study was to evaluate spontaneous abdominal wall hematomas, focusing on the detection of active bleeding at CTA and DSA imaging and on the safety, technical and clinical success of percutaneous endovascular treatment and to evaluate the efficacy of blind embolization compared to targeted embolization, where active bleeding was demonstrated at DSA.We retrospectively analyzed the files from our RIS (Radiology Information System) and PACS (Picture Archiving and Communication System) of all consecutive patients with abdominal wall hematoma who underwent emergency angiographic evaluation and embolization between January 2018 and November 2021.n = 3) or had recent trauma (n = 3) within the 3 weeks before the diagnosis of AWH were excluded because they were considered to have potentially traumatic or iatrogenic hematoma. Overall, the study population included 43 patients.The initial search retrieved a total of 49 patients. Patients who recently underwent surgical intervention  were obtained from the digital medical records.All patients underwent CTA before DSA. The CT examinations were performed using two different scanners: Optima 660  and Toshiba Aquilon Prime .The CT protocol consisted of a non-contrast phase, followed by an arterial phase  placed in the proximal abdominal aorta), a venous phase (acquired with a delay of 60 s) and a delayed phase (acquired with a delay of 120 s).A bolus of 80–120 mL of Iomeprol , followed by 30 mL saline flush, was administrated using an automated contrast injection system at a flow rate of 4–5 mL/s. The CT images were reformatted in both coronal and sagittal planes. The location of the hematoma was classified as an anterior or posterior abdominal wall hematoma. Signs of active bleeding  were evaluated. In patients without signs of active bleeding at CTA, it was decided to proceed with DSA evaluation and eventual embolization because of a patient’s unstable hemoglobin levels or because of the continuous need of transfusion to maintain acceptable hemoglobin levels (>8.0 g/dL).DSA and TAE were performed by interventional radiologists with at least 5 years of experience and by radiology residents attending a third or fourth year of training. DSA and endovascular treatments were performed using a Toshiba Infinix-i CAS-880A Cath Angio Lab .After skin disinfection and sterile draping, a 10-cm long 5 Fr guiding sheath was introduced via the right (preferred) or left common femoral artery. Arteriography was performed with Iomeprol  as a contrast agent, with a frame rate between two to six images per second.In patients whose pre-interventional CTA did not show active bleeding or the target vessel was not clearly identified, overview angiograms with a 4 or 5 Fr catheter placed in the abdominal aorta were performed. In all other cases, CTA-guided selective arteriography of the most likely bleeding site was immediately performed. Selective catheterization was performed with the use of 4 or 5 Fr appropriate diagnostic catheters, such as Cobra 2  or Sos-Omni , and an angled, flexible 0.035-inch hydrophilic guidewire . Super selective or distal catheterization was usually conducted with a 2.7 Fr preloaded microcatheter system, which was inserted coaxially into the diagnostic catheter.Findings considered for angiographic proof of bleeding were direct signs of active bleeding  indirect signs (vessel cut-off sign), pseudoaneurysm formation and the absence of active bleeding .®, Johnson & Johnson Medical N.V., New Brunswick, NJ, USA), a combination of coils and gelatin sponge or a Squidperi 18 . The choice between the embolic materials was determined by the discretion of the treating interventional radiologist and considering the vascular anatomy and the position of the microcatheter within the target vessel. The microcatheter was placed as close as possible to the bleeding site to release the embolic material, and after embolization, the adjacent vessels were catheterized selectively to exclude the presence of collateral vascular flow to the bleeding source. At the end of the procedure, the guiding sheath was left in place in case of a recurrent bleeding for 24–48 h. Other data considered for our study were the target vessel location, the procedural timing (more or less than 60 min) and the time between pre-procedural CTA and angiography.Embolization was carried out via the microcatheter using pushable or detachable coils (deployed coil diameter: 2–6 mm), a temporary embolic agent such as a re-absorbable gelatin sponge . The pre-interventional values were gathered at a maximum of 12 h before the embolization procedure. The corresponding post-interventional values were gathered within 24 h after the procedure.Targeted embolization was defined as the embolization of vessels where direct or indirect signs of active bleeding were demonstrated on the DSA study.Blind embolization was defined as the embolization of a target vessel without angiographic proof of extravasation, typically guided by CTA findings in normal-appearing vessels . TechnicClinical success was defined as an absence of clinical, laboratory or radiological signs of re-bleeding within a 96-h window after procedure. The 96-h follow-up was based on clinical and laboratory parameters and, when indicated, CTA evaluation. Clinical failure was considered in patients who presented signs of re-bleeding  and who needed to undergo a new angiographic procedure followed by embolization of bleeding vessels. Minor and major procedure-related complications were defined according to the Society of Interventional Radiology .Furthermore, we divided all patients into two groups depending on the embolization approach, blind versus targeted embolization, to analyze the differences between the two study populations and to compare clinical and technical success rates and the rate of complications between these groups and these approaches.A statistical analysis was performed using the MedCalc program . Continuous variables are presented as the mean ± SD. Categorical variables are reported as percentages. Characteristics of the study population are reported as the mean (SD), range and median. For comparative statistics, a chi-square test was performed between the blind and targeted embolization groups’ gender, clinical success rates and rates of complications.t-test. A p value of >0.05 was considered as non-statistically significant.The comparison between the mean values of age, pre-procedural hemoglobin and INR and delay between CT and DSA was assessed using an independent samples The initial search retrieved a total of 49 patients. After exclusion of patients with potentially traumatic or iatrogenic hematoma, 43 patients  with spontaneous AWH were enrolled in the study . The meaCTA revealed signs of active bleeding within the hematoma in 31 patients (72%). In 12 patients (28%), CTA did not show signs of active bleeding. In 15 patients (35%), the hematoma was localized within the anterior or lateral abdominal wall  and in the posterior abdominal wall  in 28 cases (65%) .Angiography and TAE were performed with a mean delay of 7 h and 50 min (range 1 h and 14 min–22 h and 30 min) from CT.DSA showed direct signs of active bleeding in 32 patients (74%) and indirect signs of bleeding (cut-off vessel sign) in 2 cases (5%). In 9 patients (21%), no angiographic evidence of active bleeding was found . TAE wasn = 17), lumbar artery (n = 21), iliolumbar artery (n = 17), deep circumflex iliac artery (n = 7), hypogastric artery (n = 2), deep femoral artery (n = 1), phrenic artery (n = 1) and intercostal artery (n = 2).The embolized arteries were the epigastric inferior artery  in 15 (35%) patients, coils in 5 (12%) patients, a combination of gelfoam and coils in 22 (51%) patients and a liquid embolic agent  in one case (2%). Blind embolization was performed in 9 patients out of 43 (21%). The mean time for the angiographic procedure was less than 60 min in 53% of the procedures, exceeding 60 min in 47%.Overall, a total of 69 arteries were embolized, corresponding to an average of 1.4 arteries per patient. Embolization materials used were absorbable gelatin foam  with no need for further interventions. Ten patients (23%) had evidence of recurrent bleeding within a 96 h-time window, and all of them were re-treated with a new TAE procedure: 5 patients had a recurrence within 24 h from DSA, one patient re-bled after 48 h, 2 patients rebled after 72 h and 2 patients had a longer gap with re-bleeding at 96 h. It is worthwhile to mention that one patient who had been re-treated with TAE for rebleeding within 24 h from DS, needed and underwent a third TAE procedure because he showed signs of re-bleeding 48 h later.No major peri-procedural complication was reported. Minor complications were seen in only one patient , who presented a hematoma of the femoral vascular access site, demonstrated with angiography, and that was treated with manual compression at the end of the procedure .p-value = 0.7180), showing no statistical significance between the clinical success rates of AWHs patients treated with blind and targeted embolization . In recent years, the endovascular treatment of AWH has been increasingly used for patients in which conservative treatments were not sufficiently effective, and it is often preferred to surgical interventions ,3.According to the literature, AWH is a condition more commonly observed in elderly patients, especially those with comorbidities . In a stCT angiography has gained a key role in the pre-procedural setting to identify signs of active bleeding, identifying the bleeding vessel and to help interventional radiologists plan angiography and embolization procedures  Figure . In our Blind embolization is often used for endovascular treatment of gastrointestinal bleeding when active bleeding is not identified on DSA, but, to our knowledge, currently there is a lack of published studies that compare the technical and clinical outcomes of this approach versus non-blind embolization in AWH patients. However, in literature, some cases of empiric embolization in patients with AWH without active bleeding have been sporadically reported. A systematic review performed by Touma et al. regarding the TAE of spontaneous soft tissue hematomas , collected 267 patients out of 63 studies, and 37 of those patients (13.8%) underwent empiric (or blind) embolization . In our Regarding the bleeding vessels identified at DSA and that have been subsequently embolized, in our experience, the most affected vessels were the lumbar and iliolumbar arteries  for posterior abdominal wall hematomas and the epigastric inferior artery (17 times) for anterior abdominal wall hematomas. These findings are in line with the result of Klausenitz et al. , where tIn 18 cases out of 43 (42%), TAE was performed in more than one arterial territory, and we observed that 15 of these 18 cases were patients with posterior AWH. Moreover, in 5 of these 18 cases, TAE was performed in 3 or 4 arterial territories, and 4 of these 5 cases were patients with posterior AWH. These findings suggest that posterior AWH could be more complex to treat, because more frequently there could be the need to embolize more vessels compared to anterior AWH, although definitive conclusions cannot be made, and this hypothesis should be considered cautiously.In line with other experiences ,20, evenOur study reported a technical success rate of 100% (43 out of 43) and a clinical success rate of 77% (33 out of 43). Therefore, 16 patients had evidence of re-bleeding within a 96-h interval, and they were all retreated with a new TAE procedure. The success rate of the secondary TAE treatment was 94%, since one of those 16 patients had a new episode of bleeding and needed further intervention. Overall, our results are consistent with those found in other published studies, where technical success rates range from 96% to 100% and clinical success rates range from 65% to 93% ,12,19.Furthermore, our comparison for technical and clinical success rates between patients with AWH treated with targeted TAE and those treated with blind TAE showed no statistically significant differences among them. The technical success rates were 100% in both groups, and the clinical success rates were comparable .Our study has several limitations, since it is a retrospective analysis limited to a single-center experience, and the number of patients is small, especially those treated with blind embolization, and we could not assess if patients were undergoing anticoagulant or antiplatelet drugs, since these data were not available on our digital medical records or on our RIS/PACS system. Moreover, the biggest limit of this study was the absence of a control group of patients with AWH managed with conservative treatment or open surgery, since it was not possible to identify those patients from the database of our center.The results obtained from our study confirm that TAE is a safe and effective option for the treatment of spontaneous abdominal wall hematomas. Pre-procedural CTA should always be performed, since it is essential to topographically localize the hematoma, study its characteristics, detect active bleeding and to plan the diagnostic angiography, which plays an essential role especially in cases where CT-Angiography did not show active bleeding, and eventually the subsequent embolization procedure.Moreover, our results suggest that the efficacy and safety of blind embolization is comparable to non-blind, and we believe that, in lack of angiographic proof of bleeding in case of a documented hematoma at pre-procedural CT, the choice of performing a blind embolization can be reasonable and safe and that the risk–benefit ratio could be favorable, especially in patients with risk factors or deteriorated clinical conditions. However, further studies are necessary to prospectively compare embolization treatment to conservative management and open surgery in abdominal wall hematomas and to deepen the knowledge about the role of blind or empiric embolization in those cases where no active bleeding can be identified on DSA.


LMNA gene encoding nuclear lamin A/C are causal for more than a dozen diverse mendelian disorders. Defining the functional consequences of LMNA variants has been challenging given the pleiotropy of gene functions and potential pathogenic mechanisms. It is essential to develop trustworthy, scalable and rapidly deployable in vitro assays of function to enable timely assessment of missense variants that are being uncovered by high throughout next-generation sequencing.Rare variants in the  Nuclear lamins A and C are encoded by the LMNA gene on chromosome 1q22 and are structurally composed of coils 1A, 1B and 2 which make up the α-helical rod domain, an immunoglobulin (Ig)-fold which makes up the Ig tail, and a nuclear localization signal (NLS) which separates the coiled-coil and Ig domains4. Given the complex nature of the LMNA gene and the diverse roles played by the encoded lamin isoforms, rare pathogenic variations have been found to underlie more than a dozen heterogenous largely autosomal dominant conditions and syndromes that are collectively referred to as “laminopathies”5. It is thus not surprising that different LMNA mutations will impact different functions and roles of lamins, such as nuclear morphology, integrity, mechanosensing and mechanotransduction, direct interaction with transcription factors, altered functionality of mutant pre-lamins, altered processing of lamin and altered interaction with chromatin, which collectively help begin to explain the wide range of associated clinical phenotypes10.Nuclear lamins play multifunctional roles in cell biology. They provide structural support for the nuclear contents, help to govern nuclear sensing and mechanotransduction, and simultaneously regulate gene expression by organizing and interacting with chromatin and transcription factorsLMNA variants affecting lamin A/C can be sorted into disorders that affect specific tissues, including cardiac muscle (dilated cardiomyopathy), skeletal muscle (Emery–Dreifuss muscular dystrophy), adipose tissue , peripheral nerves (Charcot-Marie-Tooth disorder type 2B1), as well as systemic disorders (progeria syndromes)11. Over the last two decades, >600 LMNA missense variants associated with various diseases have been deposited in the ClinVar database. The sheer diversity of affected tissues, organ systems, and distinctive syndromes is exceptional for a single gene. This inherent biological complexity, in turn, poses unique challenges when attempting to ascribe pathogenicity or dysfunction to any newly discovered variant including missense variants of uncertain significance (VUSs), which are being revealed at an accelerating rate and comprise a substantial proportion of archived variants for LMNA, as they are for most other genes.Laminopathies due to rare LMNA variants to precise functional consequences that in turn might mechanistically explain the varied clinical presentations. For instance, an attribute as simple as the position of a pathogenic LMNA missense variant relative to the coding sequence for the NLS predicts tissue involvement: variants upstream of NLS are 8-times more likely to be associated with cardiomyopathy and skeletal myopathy than downstream variants, which are instead more likely to be associated with lipodystrophy and progeria12. The majority of the pathogenic mutations in LMNA identified so far have been linked to an aggressive type of inherited cardiomyopathy that includes conduction disorders, arrhythmias, ventricular dysfunction, and heart failure1. However, more work is required to increase the rigour of functional characterization and understanding of the cellular biology that links a particular rare LMNA variant to a specific clinical phenotype.For two decades investigators have endeavoured to link attributes of specific LMNA variants including VUSs, Anderson et al. in this issue of npj Genomic Medicine evaluated 178 missense variants located in different structural and functional domains of lamin A/C via a high-throughput approach13. They primarily used two mammalian overexpression models, employing human embryonic kidney (HEK) 293 cells and mouse C2C12 myoblasts. They evaluated nuclear aggregation as their key functional read-out, both because misfolding and aggregation are important mechanisms in other dominant disorders, but also because past studies of individual lamin A/C variants have provided evidence of aberrant aggregation8. Also, by demonstrating that their assay is reliable, robust and scalable, they set the stage for future applications, including potential incorporation of such an assay into the evidence-gathering pipeline for the dysfunctionality criterion within the guidelines classification of pathogenicity.To address the challenges of testing functionality of The specific read-outs of interest were lamin A aggregation in HEK 293 and C2C12 cells, and manually counted from >100 cells in triplicate, stratified by variants within either the coiled domains or Ig like domain. The authors also evaluated lamin A aggregation in induced pluripotent stem cell (iPSC)-cardiomyocytes isolated by flow cytometry to validate a cell-specific mechanism for variants associated with cardiomyopathy. The suite of experiments included co-expression of selected variants with wild-type lamin A/C to evaluate a potential dominant negative effect for variants showing dominant inheritance. They complemented this work with in silico predictions of aberrant folding and finally observed correlation between in vitro findings and clinical phenotypes.et al. showed that lamin A aggregation abnormalities are the main read-out for missense variants that are associated with skeletal and cardiac myopathies with generally good correspondence between findings across cell types. They found that 84%, 52%, 35 and 17% of variants associated with skeletal myopathies, cardiomyopathies, lipodystrophy and progeria showed abnormal aggregation, suggesting that aggregation abnormalities represent a functional phenotype that is more specific for myopathic laminopathies compared with other types of laminopathies. They also showed that aggregation-prone variants in the Ig domain are generally associated with greater protein instability and misfolding. Finally, they observed non-random concordance between these in vitro findings and clinical manifestations.From these experiments, Anderson LMNA and its relationship to disease. In vitro functional analysis is weighed heavily when gauging pathogenicity of variants, both in classical human molecular genetic research and now with guidelines like those from the American College of Medical Genetics and Genomics14. Placing priority on functional characterization may be justified if the surrogate assay actually reflects a relevant molecular or cellular process that is important for pathogenesis15. But making such a link is complicated in the case of LMNA, given the vast underlying functional pleiotropy. Nonetheless, pleiotropy itself should not be a deterrent, as it is the rule rather than the exception underlying the associations between many human genes and disease phenotypes16.The approach and findings seem to represent a step forward in making sense of the complexity of human genetic variation in 17. Furthermore, if the assay is somehow incorrect, misleading, unreliable or not really representative of the true pathogenesis, applying it prematurely in a clinical diagnostic pipeline might introduce errors into pathogenicity attribution, which benefits no one18. But these hazards have always existed in human mutation discovery when the work shifts to designing and interpreting in vitro functional assays. In the current milieu in which clinical next-generation sequencing is uncovering human missense variants at a torrential rate—many of which are VUSs—the concomitant development of reliable high throughput functional assays that can reliably upgrade (or downgrade) the pathogenicity status of a VUS is incredibly important. In the case of cardiomyopathic and skeletal myopathic variants in LMNA, the approach reported by Anderson and colleagues sketches out one possible starting point.The assays reported by Anderson et al. seem to be reproducible and scalable, and have the potential to provide useful adjunctive evidence for attributing pathogenicity to newly discovered specific missense variants. Ultimately, all in vitro functional analysis have limitations, however there would be practical advantages to timely access to a reliable result that makes sense in the context of all the other evidence, such as in silico modelling, allele frequency, co-segregation with phenotype, evolutionary conservation, etc. However, functional analysis is just one piece of evidence


Multidrug resistance protein 1 (MRP1/ABCC1) is an ATP-dependent efflux transporter, and responsible for the transport of a broad spectrum of xenobiotics, toxins, and physiological substrates across the plasma membrane. As an efflux pump, it plays a significant role in the absorption and disposition of drugs including anticancer drugs, antivirals, antimalarials, and antibiotics and their metabolites across physiological barriers in cells. MRP1 is also known to aid in the regulation of several physiological processes such as redox homeostasis, steroid metabolism, and tissue defense. However, its overexpression has been reported to be a key clinical marker associated with multidrug resistance (MDR) of several types of cancers including lung cancer, childhood neuroblastoma, breast and prostate carcinomas, often resulting in a higher risk of treatment failure and shortened survival rates in cancer patients. Aside MDR, overexpression of MRP1 is also implicated in the development of neurodegenerative and cardiovascular diseases. Due to the cellular importance of MRP1, the identification and biochemical/molecular characterization of modulators of MRP1 activity and expression levels are of key interest to cancer research and beyond. This review primarily aims at highlighting the physiological and pharmacological importance of MRP1, known MRP1 modulators, current challenges encountered, and the potential benefits of conducting further research on the MRP1 transporter. The advancement of combinatorial chemotherapy regimen to cure cancers has resulted in improved survival rates and quality of life for most cancer patients. Despite the success of this treatment modality, multidrug resistance (MDR) has limited its effectiveness. MDR is a phenomenon in which tumor cells develop resistance to a variety of drugs . CurrentThe ABC transporters are a superfamily of transporters that were initially identified in studies related to nutrient uptake in bacteria in the 1970s . P-glycoThe ATP-binding cassette (ABC) transporters play a pivotal role in the removal of hydrophobic drugs across plasma membranes. Unfortunately, some of these therapeutic agents are substrates of these transporters hence they are effluxed out leading to a decrease in intracellular drug concentration. This goes a long way to affect the bioavailability of the drug as well as their therapeutic impact on tumor cells.Various proteins in the ABC superfamily are reported to be involved in the absorption, excretion, and distribution of drugs in normal cells , howeverMDR remains a major impediment to the treatment of cancers as its resulting ineffectiveness of the drug treatment is responsible for a larger percentage (about 90%) of cancer-related deaths , hence tAn extensive survey of existing literature was conducted using Google Scholar and PubMed to analyze the role of ABC transporters in the development of MDR in cancer, with a focus on the MRP1 transporter, its modulators, challenges that limit their efficacy as well as potential strategies that can be explored in overcoming MRP1 mediated MDR in cancer cells. Scientific literature reviewed was not refined by journal type, authors, or publishing date. We also considered results from our previous research works on the MRP1 transporter and cross-referenced published literature to identify other appropriate and related resources.via active transport  using ATP hydrolysis as the energy source  of muscle cells , ABCB (11 members), ABCC (13 members), ABCD (4 members), ABCE (1 member), ABCF (3 members), ABCG (5 members) . ABC traThe ABCC subfamily comprises thirteen members. This family of transporters is primarily associated with the export of amphiphilic anions which includes conjugates of lipophilic compounds with glutathione . However2–terminal region and also predicted to have a third membrane-spanning domain in addition to the two other membrane-spanning domains known as the MSD0  which did not overexpress P-glycoprotein (P-gp) (a well-studied ABC transporter) . This trStructural analysis of MRP1 reveals two nucleotide-binding domains (NBDs) and two membrane-spanning domains (MSDs) similar to most ABC transporters. The MSDs consist of membrane-spanning domains (MSD1 and MSD2) with each MSD-containing six transmembrane α-helices . Also, MStudies have shown that MSD0 facilitates interactions between the transporter and other protein partners . StructuRecently, several retrospective analyses of chemotherapy results have reported high expression profiles of MRP1 aside from Breast Cancer Resistance Protein (BCRP) and P-gp . Yet, MRABC transporters play a critical role in maintaining normal cellular function and cellular balance. However, cancer cells in their intelligence take advantage of the efflux activity of these transporters by overexpressing these transporters to reduce the antitumor effect of chemotherapeutic drugs. As a result, overexpression of ABC transporters contributes to multidrug resistance in several tumor cells. Studies have shown that ABC transporters like MRP1 play a critical role in the development of multidrug resistance, thus it is of great clinical interest to identify pathways or drugs that can reduce the negative impact due to their overexpression. One feasible approach is to completely shut off these efflux pumps, however, due to their pivotal role in maintaining physiological homeostasis, this option may negatively influence normal cellular balance and function . For insvia biochemical modulation. In this approach, pathways can be biochemically modified by therapeutic agents to enhance the therapeutic potency of anticancer drugs as well as reduce their toxic side effects to normal cells  levels, thereby reducing the activity of glutathione-s-transferase (GST) the enzyme responsible for glutathione conjugation of xenobiotics. Thus, 3ATA was able to sensitize human small cell lung carcinoma cells (GLC4/ADR) to antineoplastic drugs, doxorubicin, and vincristine (Catharanthus roseus (L.) G. Don), used in the treatment of leukemia, lymphoma, rhabdomyosarcoma (soft tissue tumors), neuroblastoma (cancer that forms in nerve tissue) and other carcinomas is also reported to be a well-known substrate of MRP1. Similar activity is also demonstrated by the plant alkaloid and topoisomerase II inhibitor, etoposide  as a therapeutic tool has gained a lot of interest in recent years. It has also gained a lot of attention in the field of oncology where aside from acting as biomarkers for fingerprinting various diseases (circulating miRNAs), they are also reported to play key roles in the development of therapeutic interventions with the added advantage of silencing a wide spectrum of genes . Thus, t4 (LTC4) transport in the presence of GSH . SNPs are defined as polymorphisms that are caused by a point mutation  resulting in varying alleles having alternative bases at a precise position of nucleotide within a locus . The dif


Children born small for gestational age (SGA), and failing to catch-up growth in their early years, are a heterogeneous group, comprising both known and undefined congenital disorders. Care for these children must encompass specific approaches to ensure optimal growth. The use of recombinant human growth hormone (rhGH) is an established therapy, which improves adult height in a proportion of these children, but not with uniform magnitude and not in all of them. This situation is complicated as the underlying cause of growth failure is often diagnosed during or even after rhGH treatment discontinuation with unknown consequences on adult height and long-term safety. This review focuses on the current evidence supporting potential benefits from early genetic screening in short SGA children. The pivotal role that a Next Generation Sequencing panel might play in helping diagnosis and discriminating good responders to rhGH from poor responders is discussed. Information stemming from genetic screening might allow the tailoring of therapy, as well as improving specific follow-up and management of family expectations, especially for those children with increased long-term risks. Finally, the role of national registries in collecting data from the genetic screening and clinical follow-up is considered. Growth failure is a frequent reason for referral to a pediatric endocrinologist, and short stature in children born small for gestational age (SGA) is among the indications for the use of recombinant human growth hormone (rhGH).Children with birth weight and/or length two standard deviations (SDs) below the mean, compared with reference standards for gestational age and gender, are defined as SGA. SGA children may or may not have a history of intra-uterine growth retardation (IUGR). SGA patients, as well as those with IUGR, have been extensively investigated and several conditions have been associated with fetal growth restrictions , which aAbout 90% of SGA newborns show catch-up growth in the first 2 years of life, their height normalizing by 4 years of age , 4.In 2001, rhGH was approved for use in short SGA children, older than 2 years in the USA and older than 4 years in Europe, based on trials demonstrating increased height velocity and improved height gain in children born SGA compared with untreated children. The effects of rhGH administration in the short-term were evaluated by a meta-analysis including 4 independent, randomized, controlled, multicenter studies conducted over 2 years in short pre-pubertal children born SGA . Treated2 per day) may improve height gain and adult height, and that the combined GH/gonadotropin-releasing hormone a treatment at the onset of puberty, may be a successful strategy to improve short stature in SGA adolescents with a height <140 cm at the onset of puberty  , confirmIn 2009, a meta-analysis of randomized, controlled trials (four RCTs including 391 children) on the impact of rhGH therapy on AH in short SGA children concluded that AH of the rhGH-treated group significantly exceeded controls by 0.9 SDs (mean height gain 1.5 SDs in treated vs 0.25 SDs in untreated SGA subjects) . Once agDespite the efforts to identify variables predictive of good response to long-term rhGH therapy and the development of prediction models –18, to dClinical trials identified younger chronological and bone age at the start of treatment with rhGH as major determinants of height gain in SGA children. No other considered parameters correlated significantly with the height gain SDs from the start of rhGH treatment until AH . InteresThe polymorphic deletion in exon 3 of the GH receptor (d3-GHR) has been investigated as a potential predictive marker of growth response in SGA patients, as previously shown in children with other rhGH-treated growth disorders . HoweverIGF1 and IGF1R genes may be potential candidate markers, only one polymorphism in the promoter region of the IGF1 gene has been associated with growth in SGA children, and its exact contribution in clinical practice remains to be elucidated  , Silver-IGF1, IGF1R, and SHOX mutations have been demonstrated in both short SGA and ISS children, although in a smaller percentage of cases , especially those without an increased risk for metabolic and cardiovascular disease in later life. SGA and ISS share the same challenges for elucidating the genetic/epigenetic cause(s) of these conditions and for finding successful treatment(s). Their similarity is supported by case studies, showing that skeletal dysplasia accounts for more than 20% of short stature in both SGA and ISS children . Moreoveof cases . Both reof cases . The genThe identification of good responders is, therefore, a priority to successfully treat SGA patients, as well as ISS patients.ELAC2, CEP57, and HNRHPH1 genes by exome sequencing, the potential involvement of chromosomal regions such as 5q.35 and 22q.11 by SNP array analysis, and different patterns of methylation in specific regions regulating the GNAS gene  by genome-wide methylation analysis.The diagnostic capabilities of medical genetics are constantly evolving, and great strides forward have been made in understanding the genetic profile underlying many SGA children. Extensive research has contributed to this progress, and, in highlighting a few key papers, we acknowledge that the few cited articles are far from an exhaustive list. A representative review of what has been achieved so far, is reported by Peeters et al. , who demACAN) gene mutation  and Whole Exome Sequencing (WES) have revealed the association of an SGA phenotype with syndromic conditions not previously elucidated or whose heterogeneous phenotype-genotype correlation did not allow a proper diagnosis in a proportion of cases –40. It sPotentially, genetic screening strategies Figure 1National registries play a fundamental role in collecting data on both efficacy and safety of rhGH during active treatment and long after , 52. UnfIn conclusion, rhGH is currently the best available therapy to improve adult height in SGA children who do not spontaneously catch-up their growth.Management of rhGH therapy in SGA children should go beyond prescribing the appropriate dose, IGF1 monitoring and the timing of treatment initiation - it should also include:- Pre-arranged safety screenings, possibly standardized and internationally recognized;- Promotion of a continuous diagnostic effort, independently dictated by patient age and duration of rhGH therapy;- Detailed collection of efficacy and safety data by a network of interconnected registries designed to identify good and bad responders to rhGH therapy.The author confirms being the sole contributor of this work and has approved it for publication.This article has been financially supported by Scientific Seminars International Foundation , to cover publishing cost through an educational grant received from Merck Healthcare KGaA, Darmstadt, Germany. The sponsor has had no role in data selection or in manuscript preparation.CG received honoraria from Novo Nordisk for consultancies as an expert board member; honoraria from Merck Serono as a medical advisor; honoraria from Ferring for article authorship. CG has no non-financial relationships  that may potentially influence the writing of this manuscript.


Despite the known importance of this neuron-immune axis in CNS function and pathology, until recently it was not clear whether this signaling axis played a role in macrophage chemotaxis and SGN survival following cochlear injury. In this review, we will explore the importance of innate immunity in neurodegenerative disease development, specifically focusing on the regulation of the CX3CL1-CX3CR1 axis, and present evidence for a role of FKN signaling in cochlear neuroprotection.Immune system dysregulation is increasingly being attributed to the development of a multitude of neurodegenerative diseases. This, in large part, is due to the delicate relationship that exists between neurons in the central nervous system (CNS) and peripheral nervous system (PNS), and the resident immune cells that aid in homeostasis and immune surveillance within a tissue. Classically, the inner ear was thought to be immune privileged due to the presence of a blood-labyrinth barrier. However, it is now well-established that both vestibular and auditory end organs in the inner ear contain a resident  population of macrophages which are the phagocytic cells of the innate-immune system. Upon cochlear sterile injury or infection, there is robust activation of these resident macrophages and a predominant increase in the numbers of macrophages as well as other types of leukocytes. Despite this, the source, nature, fate, and functions of these immune cells during cochlear physiology and pathology remains unclear. Migration of local macrophages and infiltration of bone-marrow-derived peripheral blood macrophages into the damaged cochlea occur through various signaling cascades, mediated by the release of specific chemical signals from damaged sensory and non-sensory cells of the cochlea. One such signaling pathway is CX The immune system is the body’s defense mechanism against pathogens and tissue injury. The immune system consists of an intricate network of cells and molecules that work together to protect the body from damage. These cells include lymphoid cells, such as T-cells, B-cells, and NK cells, and myeloid cells, such as monocytes, dendritic cells, macrophages, and granulocytes. Most immune cells, including T/B-cells, monocytes, and macrophages can be found under physiological conditions circulating in the blood. These cells have surface receptors that act in surveillance of the body searching for chemical signals that are released from damaged cells and tissues Chaplin, . These “Classically, the study of neurodegenerative diseases has focused on the pathological progression of the disease, specifically targeting toxic protein aggregation leading to neuronal dysfunction and death, rather than the underlying disease mechanism  and its cognate receptor (CX3CR1). FKN is a unique transmembrane protein that is constitutively expressed by neurons and endothelial cells . Finally, we will postulate further work that needs to be conducted to determine the mechanisms by which macrophages and FKN signaling mediate neuroprotection during cochlear damage, to harness these protective capabilities clinically.First discovered by Elie Metchnikoff in the late 19th century, macrophages are the main effector cells of innate immunity that are derived from precursor monocytes Tauber, . The maiCsf1) which is a cytokine required for differentiation of hematopoietic stem cells into macrophages and labels yolk sac-derived macrophages. Alternatively, fetal liver-derived macrophages differentiate from the cluster of differentiation molecule 11b (CD11b) expressing progenitor monocytes. Yolk sac-derived macrophages originate around mouse embryonic day 7.5 (E7.5) and travel to the otocyst around mouse E10.5. Fetal liver-derived macrophages travel to the developing cochlea around mouse E14.5  mice revealed drastically reduced density of macrophages in the spiral ligament and spiral ganglion, suggesting that these macrophages predominantly originate from the yolk sac  content of cochlear endolymph, creating an environment that is likely toxic for macrophage survival. Under normal conditions, resident macrophages in the cochlea display site-specific morphology. Apical macrophages are characterized by dendritic morphology with long and thin processes (pseudopodia). This dendritic morphology is characteristic of macrophages in surveillance mode , stria vascularis (SVA), spiral ligament, spiral limbus, basilar membrane, and spiral ganglion  on POU class 4 transcription factor 3 (Pou4f3) expressing inner hair cells (IHCs) and outer hair cells (OHCs) to selectively ablate hair cells upon diphtheria toxin (DT) administration , with the X representing the presence of an amino acid  is unique in that it only contains one analog, FKN  signaling, where ADAM10 cleavage is constitutive in unstimulated cells  high expressing monocytes have low levels of CX3CR1, and CCR2 low expressing monocytes express high levels of CX3CR1 , CD14highCD16−) which express low levels of CX3CR1, and non-classical monocytes (CD14lowCD16+) which express high levels of CX3CR1 , also known as CX3CR1GFP/GFP or CX3CR1 KO  on astrocytes  in the substantia nigra pars compacts (SNpc) of the midbrain, resulting in severe motor, balance, and coordination deficits. In PD, loss of dopaminergic neurons is precipitated by intraneuronal aggregation of α-synuclein, commonly referred to as Lewy bodies  death and increased levels of proinflammatory interleukin 1 beta (IL-1β). These results suggest that in a DR mouse model, intact FKN signaling is neuroprotective towards RGCs by reducing microglia activity and diminishing proinflammatory mediator production. Furthermore, in a 2016 study by Mendiola et al. (Diabetic retinopathy (DR) is a common microvascular disorder associated with diabetes and a leading cause of blindness worldwide  is a genetic mutation disease characterized by the progressive degeneration of photoreceptors and is a main cause of blindness worldwide, specifically in children  and MS. CX3CR1 has two single nucleotide polymorphisms (SNPs) in humans: V249I and T280M , Stojkovic et al.  on all cells as a model to disrupt FKN signaling to understand its function in macrophage chemotaxis in the injured cochlea  and CX3CR1 heterozygous  mice with aminoglycoside kanamycin to induce ototoxicity. The number of macrophages increased after kanamycin-induced hair cell death however, the degree of increase was not significantly different between CX3CR1 heterozygous and KO mice, and rather was found to be higher in the spiral ligament in certain cochlear frequency locations of the CX3CR1 global KO mice , resulted in a significant loss of OHCs in the 5–16 kHz range of the cochlea. This loss of OHCs correlated to elevated ABR thresholds in CX3CR1 global KO animals in the same tonotopic cochlear regions  potentials which is necessary for the release of excitatory glutamate from IHC. This, in turn, inhibits action potential generation in afferent cochlear SGN fibers, and therefore hearing is lost  when compared to animals with intact FKN signaling. Additionally, in this study, they also used a model of Kanamycin/Furosemide-induced ototoxicity  and distortion product otoacoustic emissions (DPOAE) thresholds) without causing sensory hair cell death. Interestingly, despite the complete recovery of ABR and DPOAE thresholds, they found that suprathreshold ABR Wave 1 amplitudes never fully recovered. This was supported by histological analysis showing permanent loss of IHC ribbon synapses, and progressive SGN death. Clinically, the loss of IHC ribbon synapses causing SGN death has profound effects on sound localization and understanding speech in noisy environments . Such noise trauma resulted in a rapid degeneration of ~50% of IHC-ribbon synapses in the mid-basal and basal regions of the cochlea and an increase in macrophage numbers in the IHC synaptic region despite any evident hair cell death. The noise-damaged synapses gradually repaired in mice with intact FKN signaling, however the absence of CX3CR1 impaired such spontaneous synaptic repair that correlated with reduced ABR Wave 1 input-output function (amplitudes). Altogether, this work suggests that FKN signaling also plays a vital role in the spontaneous repair of damaged ribbon synapses. However, further work needs to be done to determine the exact roles of macrophages and FKN signaling in ribbon synapse degeneration and repair in order to develop novel immuno-therapies for hidden-hearing loss. In the final section, we will look at some of the work being done examining the therapeutic benefits of FKN overexpression on a variety of neurodegenerative diseases.The idea of “hidden hearing loss” was first described by Kujawa and Liberman  in 2009,3CR1, this signaling axis has the potential to be an intriguing therapeutic target for any disease involving chronic neuroinflammation. In this section, we will investigate some of the work being done on targeting the FKN signaling to treat neurodegenerative diseases.The previous sections outlined the multitude of studies surrounding the role FKN signaling has in modulating the immune response during neurodegenerative disease development and progression. Due to the role of FKN signaling in microglia/macrophage activation, as well as FKN’s exclusivity for its receptor CXvia AAV serotype 2 (AAV2) delivery led to a rescue in cognitive function, as seen through novel recognition tasks and radial arm water maze behavior, in the same mouse model of tauopathy. This suggests exogenous FKN expression can rescue pathology associated with tauopathy. Furthermore, a 2011 study by Pabon et al. (3CR1 receptor agonists, to target the FKN signaling axis could prove to be a viable treatment option for a variety of neurodegenerative diseases.In 2013, Nash et al. , they fon et al. , where An et al. . Finallyn et al.  showed t3CR1 SNPs, there is diminished recruitment of peripheral macrophages and dysregulated production of proinflammatory mediators, leading to unregulated phagocytosis and SGN death. If accurate, these hypotheses would support the viability of exogenous FKN as a therapeutic mechanism to treat inner ear pathology. However, to fully determine the role of FKN signaling in cochlear physiology and pathology, the following questions need to be carefully addressed:The study of immune dysfunction continues to accumulate evidence for a primary role in the development and progression of neurodegenerative diseases. Specifically, interest in immune signaling pathways has grown, due to their involvement in tissue development, tissue homeostasis, and immune surveillance. Dysfunction in these communication pathways can result in neurotoxicity, resulting from unregulated immune activation. Evidence in several neurodegenerative diseases, including AD, PD, MS, and diseases of the retina suggests FKN signaling is responsible for dampening microglia activation, resulting in neuroprotection. Emerging research also suggests FKN signaling plays an important role in inner ear neuroprotection. However, the question remains, what is the specific role of macrophages and FKN signaling in cochlear pathology as well as other neurological conditions? We hypothesize that FKN signaling plays dual roles during cochlear pathology, as a chemokine and a checkpoint molecule. As a chemokine, FKN signaling mediates cochlear sensory and neuron protection by recruitment of anti-inflammatory and pro-healing macrophages from the circulation to the site of damage, which may be context or injury dependent. As a check point molecule, FKN signaling modulates the cochlear inflammatory state by regulating the inflammatory and phagocytic phenotype of resident and circulating macrophages. These functions may be inclusive or exclusive . However3CR1. Therefore, determining the effects of presence and absence of FKN ligand in the normal and damaged cochlea and in oto- and neuroprotection and the factors that regulate the endogenous levels of soluble and membrane-bound FKN in the injured cochlea are equally vital.What is the role of the FKN ligand in normal and damaged cochlea? To this point, FKN’s role in modulating cochlear pathology has only been investigated in terms of the presence or absence of its cognate receptor CX3CR1 expressing resident and recruited macrophages in SGN survival? CX3CR1 is expressed on both cochlear resident macrophages and on macrophages that migrate to the damaged cochlea from blood circulation. However, to what extent CX3CR1-expressing resident and/or recruited macrophages contribute towards the long-term survival of SGNs in the damaged cochlea remains to be determined.What is the precise role of CXIs FKN a chemotactic signal for resident or recruited macrophages in the injured cochlea? The FKN signaling has been explored elsewhere in the body for its role in chemotactic signaling to recruit additional immune cells to sites of tissue damage. Following cochlear trauma, there is an infiltration of circulating monocyte-derived macrophages to the site of injury. However, it is unclear if the FKN signaling axis is responsible for this migration of macrophages to the cochlea. Additionally, it is unclear if within the cochlea, FKN signaling acts as a chemotactic signal to draw additional resident macrophages from other regions of the cochlea to the site of cellular damage.3CR1 regulate cochlear inflammation and immune response to promote SGN survival following injury? It is not known if FKN signaling is directly responsible for protecting SGNs following trauma, and furthermore, if this neuroprotection is related to its regulation of proinflammatory mediator production and macrophage phagocytosis in the injured cochlea.Does CX3CR1 SNPs in acquired SNHL? Studies have shown a causal link between human CX3CR1 SNPs and increased susceptibility to various neurodegenerative diseases. However, it remains unknown if these SNPs are also associated with the development, progression, or severity of cochlear pathology and hearing loss in humans.What is the association of human CX3CR1 activity be used as a viable therapeutic target for SGN survival and ribbon synapse regeneration? Based on the number of studies indicated above on the therapeutic viability of using AAV or peptides to overexpress soluble FKN for the treatment of neurodegenerative diseases, it will be of great interest to examine the effects of overexpression of FKN as a viable treatment option for the survival of SGNs and regeneration of damaged ribbon synapses following cochlear trauma. Such putative therapeutics options may be a viable treatment for hidden-hearing loss.Can FKN overexpression or increase in CXAddressing the above questions, we will better understand the role FKN signaling has on immune regulation in the inner ear during cochlear trauma. By doing so, we can begin to develop therapeutic targets for not only prophylactic protection but post-trauma recovery. Together, understanding the role of FKN signaling in inner ear pathology has great promise for unraveling the mysteries tied to the treatment of hearing loss.AS reviewed the literature and wrote the manuscript. TK wrote and edited the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Osteoarthritis (OA) and rheumatoid arthritis (RA) are two of the most common chronic inflammatory joint diseases, for which there remains a great clinical need to develop safer and more efficacious pharmacological treatments. The pathology of both OA and RA involves multiple tissues within the joint, including the synovial joint lining and the bone, as well as the articular cartilage in OA. In this review, we discuss the potential for the development of oligonucleotide therapies for these disorders by examining the evidence that oligonucleotides can modulate the key cellular pathways that drive the pathology of the inflammatory diseased joint pathology, as well as evidence in preclinical in vivo models that oligonucleotides can modify disease progression. RA is one of the most common chronic inflammatory conditions, with an estimated prevalence of up to 2% in some ethnic groups . LargelyOA is the most common degenerative joint disorder in the world, affecting 303 million people globally . HistoriSimilarly to RA, OA results in joint pain and increased stiffness, which leads to progressive disability and a reduced quality of life, amounting to a huge socioeconomic burden. Unfortunately, since age is a significant risk factor for OA, the prevalence of OA is set to increase in developed countries with our increasingly ageing society Unfortunately, current treatment options for patients are limited. Despite a number of clinical trials, including with biologics targeting pro-inflammatory cytokines such as adalimumab and tocilizumab , there aIn this review, we discuss the potential for the development of oligonucleotide therapies in both RA and OA joint disease by examining the evidence for oligonucleotide therapies to modulate disease pathology and disease-associated cellular pathways within the multiple tissues of the joint .Studies used to write this narrative review were collected from the PubMed database. Search criteria for original articles included key words “oligonucleotide”/“siRNA”/“LNA” and “osteoarthritis” and/or “rheumatoid arthritis”, and we filtered for studies between 2000 and the present. Studies were deemed relevant if oligonucleotides were demonstrated to alleviate inflammation, pain and ameliorate OA and/or RA disease progression. The details of those studies included are summarised in Inflammation of the synovial membrane (synovitis) is a key hallmark of both RA and OA joint disease. Fibroblast-like synoviocytes (FLS), also termed synovial fibroblasts, become activated and hyperplastic in RA ,75. ThroIn attempting to develop oligonucleotide therapies to target synovitis, modulation of the activated proliferative inflammatory synovial fibroblast phenotype using antisense oligonucleotides has been documented. For example, Nakazawa et al. reported that antisense oligonucleotides targeting Notch-1 protein inhibited both basal and TNFα-induced proliferation of human synovial fibroblasts isolated from either RA or OA patient synovium , whilst As yet, few studies have reported the effect of oligonucleotides designed to target the synovium in preclinical in vivo models of either RA or OA disease ,91,92,93In bone homeostasis, receptor activator of nuclear factor kappa B (RANK)/RANKL pathway activates NF-kB-induced transcription factors that provide the balance between bone resorption and bone formation ,95. HoweIn vivo, in either a surgically induced OA model anterior cruciate ligament (ACL) transection or a collagenase-induced OA model, end-capped DKK1 antisense oligonucleotides delivered intraperitoneally (10–50 µg/kg/week for up to 8 weeks) lowered disease severity, with a reduction in bone mineral density loss, reduced serum levels of bone resorption markers osteocalcin and CTX-1 and suppressed expression of TNF-α, IL-1β, MMP3 and RANKL . Such fiCartilage degradation in OA is a key hallmark of OA incidence and of disease progression. This is driven largely by a pathological switch in the phenotype of chondrocytes . In healIn vitro, antisense oligonucleotides have been shown to modulate the inflammatory and catabolic phenotype of human OA chondrocytes. Targeted knockdown of the G-Protein Coupled Receptor (GPCR) RDC1 in human knee OA chondrocytes using antisense oligonucleotides modified the OA chondrocyte phenotype, with reduced expression of a panel of MMPs and hypertrophic markers . SimilarIn recent years, the profiling of non-coding miRNAs has identified a number of miRNAs that are dysregulated in OA diseased cartilage , and/or In addition to miRNA inhibition, miRNA oligonucleotide mimics have also been demonstrated to have potential as arthritis therapeutics in vivo. A miRNA mimic of miR-26a/26b was found to promote chondrocyte proliferation in vitro by targeting of fucosyltransferase 4 (FUT4) and to attenuate development of OA in a rat ACL model of OA when delivered by single intra-articular injection at a dose of 5 nmol, 1 week after surgical induction of OA . SimilarOne of the central challenges in the development of drugs to treat inflammatory joint diseases such as OA and RA is the effective delivery of the therapeutic to the diseased tissues of the joint. In particular, the articular cartilage represents a formidable challenge since it is avascular, and thus unmodified drug delivery into the chondrocytes occurs slowly and inefficiently . OligonuHowever, the ability to deliver therapeutics, including oligonucleotide therapeutics, directly into the joint space via intra-articular injection, thus maximising the ability of the oligonucleotides to target the diseased tissues, is an advantage. Indeed, the increasing number of studies demonstrating the efficacy of antisense oligonucleotides and miRNA mimics in reducing disease severity in experimental models of arthritis following intra-articular injection delivery is highly promising. Furthermore, delivery of the therapeutic locally to the joint limits the risk of unwanted side effects at distal sites ,111. IntIn attempting to optimise both the stability and the delivery of oligonucleotide therapeutics in the joint, a number of strategies have been employed. For example, intra-articular injection provides a route for the local delivery of viral vectors  capable of carrying oligonucleotide cargo without the risk of systemic side effects . In a muOne alternative to viral delivery systems is the use of biomaterials, such as hydrogels. Hydrogels have been successfully used to sustain oligonucleotides within the microenvironment of the joint and to facilitate slow release. For example, a hyaluronic acid hydrogel was previously utilised to facilitate the controlled release of an LNA Gapmer oligonucleotide targeting PTGS2 (COX2), the gene which promotes the expression of the inflammatory pain mediator PGE2. Notably, the hydrogel-encapsulated COX2 LNA was shown to produce gene silencing in OA chondrocytes over a 14 day period . Using aCell-penetrating peptides (CPPs) such as TAT, penetratin and antennapedia are short cationic peptide sequences that can translocate across cell membranes ,114. TheAn alternative approach is the use of nanoparticles. Although confirmation in the clinical setting is still required, such nanoparticulated drugs (nanodrugs) have recently gained recognition in drug delivery for a number of therapeutic areas ,119,120.Oligonucleotide therapeutics represent an emerging but highly promising class of therapeutics to treat inflammatory joint disease. Although yet to be successfully tested in clinical trials for arthritis treatment, data from preclinical experimental models of arthritis provide evidence that the intra-articular delivery of oligonucleotides can modify OA disease pathology, by reducing synovitis, preventing sclerotic bone formation and protecting from cartilage damage. Importantly, since oligonucleotide therapeutics are based on gene sequences, they are expected to act specifically on the target gene, and thus may be considered less likely to have off-target effects and to elicit adverse side effects. Indeed, the same basic backbone chemistry of oligonucleotides and the safety profile of oligonucleotide therapeutics in the clinic provide confidence that late-stage clinical trial failure with this class of drug may be less common than with small-molecule or monoclonal antibody entities. Furthermore, they can be rapidly and cheaply synthesised, thus greatly reducing drug development timelines. As with conventional drug delivery, approaches to optimise the stability and delivery efficacy of oligonucleotide therapies into the joint tissues remain a major area of current focus and barrier to effective disease modification. To this end, the use of nanocarriers and the GalNAc bioconjugation represent promising approaches, but further clinical studies will provide important information on the safety profile of such carriers in the clinical setting for patients with inflammatory joint disease.


Population ageing and urbanisation are global phenomena that call for an understanding of the impacts of features of the urban environment on older adults’ cognitive function. Because neighbourhood characteristics that can potentially have opposite effects on cognitive function are interdependent, they need to be considered in conjunction. Using data from an Australian national sample of 4141 adult urban dwellers, we examined the extent to which the associations of interrelated built and natural environment features and ambient air pollution with cognitive function are explained by cardiometabolic risk factors relevant to cognitive health. All examined environmental features were directly and/or indirectly related to cognitive function via other environmental features and/or cardiometabolic risk factors. Findings suggest that dense, interconnected urban environments with access to parks, blue spaces and low levels of air pollution may benefit cognitive health through cardiometabolic risk factors and other mechanisms not captured in this study. This study also highlights the need for a particularly fine-grained characterisation of the built environment in research on cognitive function, which would enable the differentiation of the positive effects of destination-rich neighbourhoods on cognition via participation in cognition-enhancing activities from the negative effects of air pollutants typically present in dense, destination-rich urban areas. Population ageing and urbanisation are global phenomena with major consequences for almost all sectors of society ,2. The mOne of the most burdensome non-communicable diseases in people aged 60 years or over is dementia . Due to Urban environments have been identified as important contributors to cardiometabolic health  through In general, studies on environmental correlates of cognitive function examined a limited range of neighbourhood attributes and often reported mixed findings , possiblThe model in We used data from the Australian Diabetes, Obesity and Lifestyle (AusDiab) study, a three-wave population-based survey designed to examine the prevalence, incidence and determinants of diabetes in Australian adults aged 25 years and over ,21. Data2), percentage of commercial land use, non-commercial land use mix (range: 0–1), percentage of parkland, percentage of blue spaces  and annual average concentrations of nitrogen dioxide  and fine particulate matter <2.5 µm in aerodynamic diameter . Details on these measures are provided elsewhere ); and three indicators of dyslipidaemia (low-density lipoprotein [LDL] cholesterol (mg/dL), high-density lipoprotein [HDL] cholesterol (mg/dL) and triglycerides (mg/dL)). The assessment of cardiometabolic risk factors in AusDiab has been detailed elsewhere ,21.Several variables were included as potential confounders or covariates as appropriate . We then estimated the ‘direct effects’ of environmental characteristics on the cardiometabolic risk factors  by including in the regression models all environmental characteristics hypothesised to mediate the effects of the environmental exposure of interest on the response variable. As we hypothesised that adiposity (waist circumference) would be a determinant of other cardiometabolic risk factors ,32, we a2 at 5.5 ppb and PM2.5 at 6.3 μg/m3.The average age of the sample was 61 years (SD = 11). The majority of participants were of English-speaking background, female and in paid employment . Nearly 2 showed mixed total effects on cardiometabolic risk factors. In contrast, intersection density, non-commercial land use mix and PM2.5 were prevalently associated with unfavourable outcomes, while indicators of natural environment tended to show positive effects on cardiometabolic health. Taking diabetes medications moderated the total effect of the percentage of blue space on glycated haemoglobin whereby only those on medication showed a negative association . Significant total but not direct effects on cardiometabolic risk factors were observed for population density in relation to glycated haemoglobin, percentage of commercial land in relation to mean arterial pressure and non-commercial land use mix in relation to HDL cholesterol and triglycerides   . In addi2.5 and its environmental antecedents had a negative and percentage of blue space a positive, indirect effect on processing speed through waist circumference and glycated haemoglobin  had a negative indirect effect on processing speed through glycated haemoglobin. Blue space had a positive indirect effect on processing speed through glycated haemoglobin in those on diabetes medications  as depicted in the proposed conceptual model of neighbourhood environmental influences on cognitive health .2.5 and NO2 on cognitive function via cardiometabolic risk factors. These findings were expected because higher levels of these air pollutants have been previously associated with worse cognitive health outcomes in humans [2.5 and NO2 contribute to neurodegenerative processes via cardiovascular and cerebrovascular diseases, and other pathways key to dementia pathogenesis  [2.5 on cognitive function were channelled through indicators of adiposity (waist circumference), elevated blood glucose (glycated haemoglobin) and hyperlipidaemia (low HDL cholesterol), while those of NO2 were mainly explained by elevated blood glucose (glycated haemoglobin). Exposure to air pollution has been previously associated with adiposity [After adjusting for built and natural neighbourhood environmental characteristics, we observed negative indirect effects of PMn humans ,18. Alsositions) . In thisdiposity ,35, whicdiposity  that, indiposity ,37,38,392 but not PM2.5. In line with these findings, Honda et al. [2 than PM2.5 on glycated haemoglobin. Similar associations have also been observed in studies of diabetes prevalence [2 is an indicator, may be more important than particulate matter for glycaemic control [We found negative effects on cognitive function mediated by glycated haemoglobin but independent of waist circumference for NOa et al. , who estevalence ,42 sugge control ,41 and m2 and PM2.5 with walking for transport were observed in an earlier analysis of AusDiab data [2 with mean arterial pressure were found in this study, suggesting that destinations supporting engagement in utilitarian walking and other activities were not accurately captured by the land-use measures used in this study. These findings highlight the need for an accurate and comprehensive characterisation of urban neighbourhood environments, encompassing all key interrelated features, in studies of environmental determinants of cognitive function.In addition to the negative indirect effects of air pollutants on cognitive function, we observed positive direct effects, which were unexpected and likely due to using coarse measures of land use as proxies of access to destinations promoting cognition-enhancing activities . Specifically, commercial destinations that offer opportunities for cognition-enhancing activities  can be major sources of pollution generated by food preparation and high volumes of visitors and, hence, traffic ,25,43. Wiab data  and negaThis study suggests prevalently positive effects of access to parkland on cognitive function, unmediated by cardiometabolic risk factors and mediated by other environmental attributes, albeit some negative indirect effects of parkland via air pollutants and related cardiometabolic risk factors were also observed. Greenspace has been previously found to have positive effects on cognitive function in adults , whereas2.5 and NO2. The positive association with PM2.5 might have been due to natural sources, including wind-blown dust, sea salt and biogenic emissions from parkland [2.5 and NO2 concentrations could be smoke from prescribed fires [Contrary to our expectation that the positive effects of parkland on cognitive function would be in part explained by its mitigating effects on air pollution , our meaparkland . Other ped fires  and mained fires . These pWhile no direct effect of blue space on cognitive function was observed in this study, positive indirect effects through waist circumference, HDL cholesterol and glycated haemoglobin (in those on diabetes medications) were observed. These findings may be due to blue spaces promoting an active lifestyle  and physWe hypothesised that built environment indicators of densification and access to services  would show both positive and negative effects on cognitive health given that dense, complex, destination-rich environments provide opportunities for social contacts and other cognition-enhancing activities  but also increase exposures to stressors such as air pollution and noise . In suppInterestingly, street intersection density and non-commercial land use mix were positively related to cardiometabolic risk factors that did not mediate environment-cognition associations in this study  even after adjustment for air pollutants. Possible contributors to these findings might be access to unhealthy foods   and trafApart from population density and street intersection density, no other built environmental attribute was directly related to the cardiovascular risk factors associated with cognitive function in this study. However, the percentage of commercial land and land use mix were related to air pollutants which, in turn, showed negative effects on cognitive function mediated by cardiometabolic risk factors and positive effects not mediated by cardiometabolic risk factors. As explained earlier, the latter effects were likely due to air pollutants being indicators of human activities and, hence, opportunities to engage in cognition-enhancing pursuits  rather than them being beneficial to cognitive health. These findings not only highlight the need for simultaneously examining the effects of key interrelated features of urban environments on cognitive function but also the need for a more fine-grained characterisation of urban environments in terms of destinations that provide attractive opportunities for cognition-enhancing activities . This information is essential for the cognitive health impact assessment of neighbourhood environmental features that, in turn, can guide city planning policies.A strength of this study is the utilisation of data from a national cohort capturing diverse urban environments in Australia. We examined the joint linear and/or curvilinear effects of the neighbourhood built and natural environment and air pollution on cognitive function, and the potential cardiometabolic mechanisms underpinning them. By doing so, we accounted for neighbourhood self-selection to partially address reverse causality. We examined total, direct and indirect cross-sectional effects of urban environmental features on cognitive function to disentangle their potentially beneficial and harmful impacts. Study limitations include the cross-sectional nature of the available data and the employment of coarse measures of destination accessibility relevant to cognition-enhancing activities. The latter limitation has made it difficult to disentangle the positive  and negative effects  of urban densification on cognitive function and related cardiometabolic risk factors. It would have also been desirable to have data on other key covariates, such as estimates of indoor pollution levels in the home. Future research would need to address these limitations by conducting longitudinal studies able to capture changes in exposures and cognitive function  and more2.5 and NO2. Longitudinal studies with a more accurate characterisation of the built environment, the quality of natural spaces and individual activity locations (within and outside the neighbourhood) are needed to better understand how to create cities that can help preserve cognitive function in ageing populations.In line with a proposed ecological model of neighbourhood environmental influences on cognitive health, this study has found features of urban environments to be directly or indirectly related, via cardiometabolic risk factors, to cognitive function in middle-aged and older Australian adults. Dense, interconnected neighbourhoods may contribute to better cardiometabolic outcomes  and, consequently, better cognitive health by promoting active transportation. While such environments usually provide better opportunities for social contacts and other cognition-stimulating activities, they are often sources of air pollution arising from human activities and vehicular traffic that harm cardiometabolic health and, hence, cognitive function. Our findings also provide support for the positive effects of green and blue spaces on cognitive function via cardiometabolic risk factors and other mechanisms that were not examined in this study . Cognition-friendly urban environments appear to be typified by compact, interconnected neighbourhoods with good access to green and blue spaces and low average annual levels of PM


The vast majority of the human proteome is yet to be functionally characterized thus hindering ongoing investigations on potential drug resistance mechanisms and advanced treatment options. Chemical proteomics is a powerful solution for enzyme profiling and the development of next generation cancer therapeutics previously deemed undruggable by small molecules. Within this field, activity-based protein profiling (ABPP) is a specialized technology capable of discriminating enzyme interactions that occur within complex, biological environments. In a recent publication by Lovell et al, the kallikrein-related peptidase (KLK) family of serine proteases that is highly implicated in the progression of prostate cancer (PCa) was subject to ABPP to elucidate enzymatic activities in the presence of enzalutamide. This is the first report of ABPP in PCa and of activity-based chemical probes selective for individual KLKs. Further, the study reveals androgen receptor-dependent activity among KLK proteins, particularly in mediating the invasion of the bone microenvironment. ABPP can use small-molecule chemical probes to elucidate the mechanisms behind drug-target interactions or identify active sites of target proteins. These preclinical proteomics studies already have applications in the study of melphalan resistance in multiple myeloma, target discovery for dasatinib in gastric cancer, and curcumin resistance screening in colon cancer among many functional ABPP-driven investigations.2–4 From a clinical perspective, these assays may be better equipped at predicting off target effects well before entering clinical trials as protein interactions are often synergistic across multiple biological pathways.5 Chemical proteomics has aided in the characterization of enzyme activity profiles across multiple enzyme families such as proteases, phosphatases, glycosidases, oxidoreductases, and kinases further demonstrating the abundance of targeting potential from a drug development standpoint.6 Given the dynamic nature of protein interactions driving multiple disease pathways at once, functional proteomics is a vital component of drug discovery beyond the genomic and post-transcriptional level.What was once considered the undruggable proteome is a world of therapeutic potential due to functional assays powered by chemical proteomics. Among these assays, activity-based protein profiling (ABPP) is a cutting-edge technology designed to broadly classify direct enzyme activities that are dependent on the dynamic biological environments in which they exist.7 Aberrant expression of KLKs and their dysregulation are implicated in many solid tumors, including breast, ovarian, and pancreatic cancers, thus making KLKs attractive biomarkers of disease and emerging therapeutic targets.8 However, to date the development of this potential target has been hampered by the lack of technology for specific assessment of KLK activity within the complex KLK activome. Lovell et al. is the first reported design and application of activity-based probes (ABPs) capable of discriminating activities between individual KLK proteins. Further, the study is the first in PCa to investigate mechanisms of drug resistance using ABPP. Gel-based activity profiling determined that KLK2 and KLK3 are highly activated in the presence of dihydrotestosterone (DHT) and KLK14 is deactivated by DHT but rescued by the advanced PCa drug and androgen receptor (AR) antagonist, enzalutamide. The KLK14 response to DHT and enzalutamide likely proposes a functional component as to the resistance mechanisms of AR inhibitors; additional assays also show that active KLK14 is secreted from both osteoblasts and PCa cells to promote tumor cell migration. Co-culturing of osteoblasts and LNCaP cells revealed cross-reactivity of KLK14 with KLK2 and KLK3 which may further intensify osteoblast formation and metastasis. Prominently, the study found that KLK activity is dysregulated from expression as total KLK protein quantities were more abundant than quantities from ABPs. This suggests that measuring KLK proteolysis can be coupled together with routine prostate specific antigen (PSA) testing for improved PCa diagnosis and monitoring, highlighting a role for the use of ABPs to assess KLK activity as a novel PCa biomarker.A recent article by Lovell et al. presented novel findings on the largely uncharacterized activity profiles of the Kallikrein-related peptidase (KLK) family of serine proteases, a network referred to as the KLK activome and implicated in prostate cancer (PCa) growth, progression and metastasis.9 The active site-specific component of the probe consists of an electrophilic, reactive “warhead” that irreversibly binds a protein active site.10 These reactive sites are linked to molecules mimicking the active site’s natural substrate to further facilitate the covalent interaction through active site stabilization. In contrast to other inhibitors, ABPP chemical probes are typically bound to reporter tags to quantify binding interactions through either gel-based fluorescence or biotin-labeling for affinity-tagged blotting.9 Other more advanced techniques exist as well, such as in the case of isoTOP-ABPP, which utilizes isotopically labeled probes for quantification via mass spectrometry.11 Thus, using this three-part formula, the authors successfully optimized orthogonal chemical probes for the simultaneous characterization of KLK2, KLK3, and KLK14. As characterizing individual protein activities within disease pathways is complex, Lovell et al.’s findings are noteworthy, as they demonstrate that inhibiting specific KLK proteases further activates proteolysis and disease progression. The KLK enzyme family previously had no known reports of selectivity for small molecule inhibition, thus limiting its potential as a therapeutic target and biomarker candidate. The novel findings in Lovell’s study on selective KLK probes may inspire the future of ABPP-driven small molecule drug discovery not only in PCa but other malignancies as well. For instance, KLK4 has been previously published in ovarian cancer and ABPP probe development may pertain to inhibitor and small molecule optimization for this disease.13ABPP is dependent on optimized probes which typically are derived from known inhibitors or natural products specific to catalytic class.14 The possibilities of expanding the previously deemed undruggable proteome are broad given these technologies and future work will greatly aid in the optimization of therapies that are dependent on complex biological contexts.Acquired drug resistance remains a challenge for patients in the setting of castration-resistant disease who are refractory to next-generation targeted therapies. Dissecting the KLK activome has improved the understanding of its role in PCa and reveal new opportunities on how to overcome mechanisms of bone metastasis and drug resistance. Characterization of individual KLK activity, clinical utility of ABPP and related proteomic studies have strong potential to drive PCa biomarker discovery of active enzymes to supplement traditional prognostic strategies such as PSA marker screening as well as PROTAC design from a therapeutic standpoint. In fact, ABPP screens have already aided in the discovery of small molecule recruiters of E3 ligases to optimize PROTACs for the improved degradation of the bromodomain heteroprotein, BRD4.