N-methyl-d-aspartate receptor (anti-NMDAR) encephalitis, outline the clinical presentation and make suggestions on ways to approach this rare disorder. Here we provide an overview of autoimmune conditions that result in the production of autoantibodies targeting central nervous system proteins mediating autoimmune encephalitis and offer a perspective on approaches to diagnosis and treatment.This editorial expands on a Praxis article published by Beattie and colleagues in the trainees’ section of this journal. The authors describe an interesting case of anti- N-methyl-d-aspartate receptor (anti-NMDAR) encephalitis and make suggestions for psychiatrists on how to approach such difficult clinical situations.1 The importance of this topic to psychiatrists lies in the fact that although anti-NMDAR encephalitis is primarily a neurological disorder, nearly 80% of these patients first present with psychiatric symptoms and more than 60% are first admitted to psychiatric units.2 It is estimated that nearly 75% of these individuals recover or have minimal deficits, although up to 25% endure severe functional deficits or even die, mostly owing to delays in diagnosis.3 Hence, early recognition and treatment are key to a successful outcome.In their Praxis article, Beattie and colleagues present a case of anti-4 Early cues are an important element in increasing the chance of early diagnosis of anti-NMDAR encephalitis, essential to improve outcome. Beattie and colleagues highlight the importance of ‘red flags’ to aid the diagnosis of autoimmune encephalitis. These red flags include a host of associated manifestations that could aid early diagnosis, especially when detecting for the presence of autoantibodies, central to diagnosis and treatment of anti-NMDAR encephalitis, is not feasible. This editorial provides a succinct description of the several types of autoantibodies associated with autoimmune encephalitis and highlights the difficulties in reaching the diagnosis and providing treatment.The clinical case described by Beattie and colleagues is of a woman in her mid-20s treated initially for a psychiatric disorder who later developed neurological deficits raising suspicion of anti-NMDAR encephalitis. The authors demonstrate the challenges likely to occur in clinical situations when there is the potential for being misled by the initial emergence of psychiatric symptoms as early manifestation of this disease. Following a systematic approach to clinical reasoning might help to consider the possibility of anti-NMDAR encephalitis as early as possible.5 It affects around 1.5 per million people/year.6 Autoimmune encephalitides are broadly divided into two categories according to whether the immunological mechanism involves autoantibodies targeting ‘extracellular’ or ‘intracellular’ neuronal antigens. The overall incidence has increased from 0.4/100 000 in 1995–2005 to 1.2/100 000 in 2006–2015.7 This could be explained by changes in consensus definitions, which initially focused on neuronal surface autoantibody-mediated encephalopathies and only subsequently included paraneoplastic limbic and anti-voltage-gated potassium channel antibodies.Anti-NMDAR encephalitis is the most frequently occurring of several types of autoimmune encephalitis.There is a crucial distinction between paraneoplastic syndromes associated with onconeural antibodies and other autoimmune encephalopathies. Onconeural antibodies against surface antigenic targets are in fact biomarkers with no bearing on the disease process, unlike the more common autoimmune encephalitides. Onconeural antibodies against intracellular targets represent malignancy.N-methyl-d-aspartate), AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid), GABA (gamma-aminobutyric acid), glycine receptors, and voltage-gated potassium channels (VGKC) including LGI1 (leucine-rich glioma inactivated 1) and CASPR2 (contactin-associated protein-like 2) proteins.8 Autoantibodies that target either intracellular antigens or synaptic proteins include anti-Hu, anti-Ri, anti-Yo, anti-Ma, anti-amphiphysin and anti-GAD (glutamic acid decarboxylase). The immune response that targets intracellular antigens is thought to involve CD8+ cytotoxic T-cell-mediated cell injury after binding of autoantibodies to the target intracellular antigen. These autoimmune encephalitides are often paraneoplastic manifestations of various types of cancer.10Autoantibodies that often target extracellular receptors and ion channels include those for NMDA ,9 which is analogous to psychotic manifestations observed with phencyclidine ingestion, which also acts through NMDA receptor hypofunction.10Animal studies suggest that anti-NMDA receptors passively transferred into the brains of rodents produce neurological symptoms proportionate to the surface reduction of NMDA receptors on the neurons,10 Anti-AMPAR encephalitis tends to present with psychiatric symptoms similar to anti-NMDAR encephalitis. The median age at onset of this type of encephalitis tends to be in the 40s and 50s,14 which differs from anti-NMDAR encephalitis, which mostly affects females in their 20s.8AMPA receptors are also glutamatergic ionotropic receptors that are widely expressed in the brain mediating rapid excitatory transmission.15LGI1 is a presynaptic glycoprotein involved in the regulation of AMPA and VGKC receptors. On cultured neurons, LGI1 antibodies have been shown to affect AMPA receptor localisation across the whole brain. Classic symptoms of anti-LGI1 antibody encephalitis include hyponatraemia and faciobrachial dystonic seizures with initial subtle memory loss and sleep disorders  sleep behaviour disorder, sleep reversal). This condition tends to occur at a median age of 60 years.16CASPR2 is a cell adhesion molecule that organises VGKCs in the central and peripheral nervous system. People with anti-CASPR2 antibodies develop symptoms originating in the central nervous system  and/or the peripheral nervous system (e.g. Morvan syndrome), with encephalitis characterised by confusion, amnesia and hallucinations. Onset is usually slower than for anti-NMDAR encephalitis and the median age at onset is 60 years.A and GABAB receptors result in a type of limbic encephalitis associated with severe seizures that can lead to status epilepticus, together with psychiatric manifestations such as memory loss, confusion, hallucinations and personality change.19GABA is the primary inhibitory ionotropic receptor in the brain. Antibodies targeting GABA1 subunit is the predominant antigenic target.20 Studies involving the transfer of human glycine autoantibodies to rodents have demonstrated that neural transmission at the cellular level with consecutive, altered signal cascades induces psychiatric and cognitive symptoms. Glycine-receptor antibodies have been found in ‘stiff person spectrum’ disorders such as progressive encephalomyelitis with rigidity and myoclonus and have been associated with cognitive/memory dysfunction and psychosis.11The glycine receptor is an inhibitory receptor. The α9 The latter has been associated with anti-Hu antibodies, which target an intracellular RNA-packed protein, important in memory-related synaptic plasticity.11 Anti-Ri antibodies have been linked with lung carcinoma. Typical manifestations include personality changes and neuropsychological deficits.11 Anti-Ma antibodies are often found in young males with germ cell tumours and are associated with a range of psychiatric manifestations, including obsessive–compulsive disorder, delirium, major depression, personality changes and amnesia.11 Anti-Yo antibodies are mostly found in females with breast or ovarian cancers. These antibodies most likely act through T-cell mechanisms and are associated with psychosis.22Autoantibodies that target intracellular antigens  and proteins  typically present as paraneoplastic manifestations. Neuropsychiatric symptoms often precede the diagnosis of the primary neoplasia by several months. Common symptoms include depression, irritability, hallucinations, sleep disturbance and seizures. Memory loss can occur over weeks to months, in some cases progressing to severe cognitive decline resembling a dementing illness.23 Anti-GAD antibodies target the synaptic isoform of the enzyme necessary to synthesise GABA.10 Mood changes and cognitive impairment are the most frequent symptoms.11Anti-amphiphysin antibodies are very strongly associated with breast cancer in women. These antibodies target intracellular proteins important for recycling synaptic vesicles. Animal models suggest that in rats, when anti-amphiphysin antibodies are passively transferred intrathecally they can induce anxiety behaviours.Although anti-NMDAR encephalitis is the most common of the autoimmune encephalitides, there are several other types of autoantibody targeting extracellular and intracellular antigens that can present with similar clinical characteristics. In the presence of atypical, often rapidly evolving psychiatric manifestations, especially when coexisting with neurological symptoms, an early age at onset in a female patient or a mid to late onset in both genders might raise the suspicion of encephalitis. It is important to consider the possibility of a primary neoplasia and a relatively recent diagnosis of cancer, especially if originating in the ovaries, breast, lung and germ cells.25Beattie and colleagues highlight the complexities of screening and interpreting serum/cerebrospinal fluid (CSF) anti-NMDAR autoantibody levels in patients presenting with psychiatric symptoms. Rates of false positives and false negatives in affected individuals are high, in the range of 10%.10 although results may require several weeks of processing. For extracellular antibody tests other than anti-NMDAR encephalitis, CSF is still the most sensitive and specific test, with serum offering a high rate of false-negative results.10 In the case of intracellular antibody tests, positive test results on their own might not be sufficient as definitive confirmation of a particular autoimmune aetiology. Some of these autoantibodies, for example anti-GAD65, may be associated with other disorders (e.g. type 1 diabetes) and may coexist with other autoantibodies, for example anti-GABAB.Commercial kits are now available for different types of autoantibody other than anti-NMDAR ,26 and a further recent study showed the limitations of commercial assays as diagnostic tests for autoimmune encephalitis.27 Excessive screening based on recognition of the stereotyped clinical syndromes but without sufficient prior probability is controversial. Moreover, although pairing serum with CSF testing can be helpful in correlation with clinical symptoms, it is important to recognise that in some autoimmune encephalitides (e.g. LGI1), autoantibodies are often undetectable in CSF. Furthermore, as these conditions differ epidemiologically  a targeted approach to testing is greatly advisable.A recent meta-analysis suggests that in cross-sectional studies NMDAR IgG antibodies are more common in people with psychosis than in controls28In summary, depending on the laboratory and assay used, autoantibodies can be detected outside of the canonical clinical syndromes and overinterpreting these results can cause harm. In view of this uncertainty and the possibility that immunotherapy might be effective, the recent consensus is to consider a diagnosis of ‘seronegative autoimmune encephalitis’ in the presence of clinical symptoms and absence of autoantibodies.29Diagnostic uncertainty, highly disturbed mental states, logistic difficulties in carrying out a range of essential investigations  can delay the prospective identification of these conditions, particularly in undifferentiated psychiatric services. The use of screening criteria for anti-NMDAR encephalitis can help improve the detection of autonomic dysfunction, cognitive impairment and movement disorders, but ‘red flag’ symptoms tend to overlap with those seen in functional psychiatric illness.31 Similar immunosuppressant strategies can be used for the other types of extracellular autoimmune encephalitis. Anti-LGI1 encephalitis generally responds well to first-line treatment,28 and rituximab is thought to be generally effective where the autoantibodies are of the IgG4 subtype, which predominate in anti-LGI1 and anti-CASPR2 encephalitides.10Currently there is no definitive treatment algorithm for anti-NMDAR encephalitis. Recommended initial therapy includes intravenous immunoglobulins and methylprednisolone or daily plasma exchange. For refractory illness, second-line treatments include rituximab and cyclophosphamide. The proteasome inhibitor bortezomib has shown some efficacy in highly refractory disease.28Patients with clinical symptoms due to autoantibodies targeting intracellular antigens are believed to respond poorly to immunotherapy. This is possibly related to CD8+ mediated cytotoxicity, which may cause irreversible neuronal cell injury. For these patients prompt detection and treatment of the underlying neoplasm is the best approach for symptom resolution., there is variability in the approach to diagnosis and treatment of autoimmune encephalitis often driven by the specialty of the assessing clinician rather than the clinical presentation. Hence, for suspected cases there is great need to collaborate with local neurologists to establish preferential diagnostic pathways involving regional neuroscience centres where systematic diagnostic assessment and investigations can be facilitated. Immunosuppressant strategies are considered the most effective treatment of extracellular types of autoimmune encephalitis, whereas identifying and treating the cancerous source of paraneoplastic symptoms is the most effective approach for the type of autoimmune encephalitis targetting intracellular antigens.In summaryThe presence of autoantibodies against brain receptors or proteins can result in severe yet potentially treatable autoimmune encephalitis. Detecting autoantibodies is a necessary but not always informative diagnostic step. History or suspicion of cancer should alert clinicians. Atypical psychiatric manifestations, commonly associated with neurological symptoms, raise concerns about the origin of these disorders. The clinical distinction between autoimmune encephalitis and psychiatric presentations is very challenging. The use of screening tools and preferential diagnostic pathways in collaboration with local neurologists with access to regional neuroscience centres could facilitate reaching a timely diagnosis.


The targeted agents capmatinib and tepotinib provide a new treatment for patients with non-small cell lung cancer (NSCLC) with MET exon 14 skipping mutation (METex14). However, drug-induced pneumonitis is an uncommon but threatening adverse effect found in patients treated with both capmatinib and tepotinib. The safety of treating a patient with a MET inhibitor after drug-induced pneumonitis by another MET inhibitor remains unclear. Here, we present a case of a patient with NSCLC harboring a METex14 who was treated with a standard dose of tepotinib after advanced capmatinib-induced pneumonitis and did not present pneumonitis relapse. Tepotinib may be a safe option when medical professionals consider switching MET inhibitors after patients experience pneumonitis. Clinical Practice PointsDrug-induced pneumonitis is an uncommon but life-threatening adverse effect of target therapies. The safety of using another MET inhibitor after discontinuing the first remains unknown and such patients have rarely been reported.Given the lack of targeted therapies for MET inhibitors, rechallenge with tepotinib after capmatinib-induced pneumonitis could be an option for patients with NSCLC harboring METex14. MET exon 14 skipping mutation (METex14) occurs in approximately 3% of patients with non-small cell lung cancer (NSCLC) . TargeteDrug-induced pneumonitis, also known as interstitial lung disease (ILD), is an uncommon but life-threatening adverse effect of target therapies . While tHere, we present a case of a patient with NSCLC with METex14 who experienced pneumonitis during capmatinib treatment and was safely switched to tepotinib without pneumonitis relapse.A 69-year-old Taiwanese man without underlying lung disease and smoking history complained of right arm pain for one month. The patient was subsequently diagnosed with metastatic carcinoma based on a specimen from the operation that the patient received for treating a pathological fracture of the right humerus. NSCLC  with bone metastases was confirmed . Next-geUnfortunately, progressive exertional dyspnea and dry cough were reported after one month of capmatinib treatment. Physical examination showed bilateral basal crackles on lung auscultation. The patient’s oxygen saturation was 92% in room air, and chest radiography (CXR) and computed tomography (CT) showed increased interstitial infiltration, which was compatible with drug-induced pneumonitis/ILD . PolymerThereafter, the patient participated in a clinical trial of a novel MET inhibitor for two weeks. The patient’s pneumonitis did not relapse, but he was withdrawn from the study because of clinical progression (the metastatic tumor over the right arm progressed rapidly during treatment). No response to a novel MET inhibitor may result from the occurrence of MET resistance or no activity of this compound. Palliative chemotherapy with pemetrexed and cisplatin was immediately administered. Tepotinib, another MET inhibitor, has been reported to be active in treatment-naïve or previously treated patients with METex14 mutation. As this patient may have still benefited from MET inhibitors, tepotinib with a standard dose (one tablet of tepotinib is 225 mg and 2 tables daily is recommended dose) was also prescribed concurrently for one month and discontinued because of clinical progression (right arm tumor). There was no pneumonitis relapse within one month of tepotinib treatment .Currently, only two MET-selective inhibitors (capmatinib and tepotinib) have been approved by the U.S. Food and Drug Administration. Owing to the limited treatment options and considerable efficacy of MET inhibitors in clinical studies ,4, it isIn the current report, pneumonitis was found to occur as an uncommon adverse effect of MET inhibitor use for treating NSCLC with uncommon mutations. Therefore, the experience presented in this case report can help inform clinicians about the safety of switching MET inhibitors due to pneumonitis. Whether the dose intensity of MET inhibitors correlates with lung toxicity remains unknown. According to a previous case report, low-dose tepotinib is safe for patients with a history of grade 2 capmatinib-induced pneumonitis . HoweverThis case report had several limitations. First, no bronchoscopy study was performed to exclude other causes of pneumonitis. Furthermore, capmatinib-induced pneumonitis was diagnosed based on the rapid improvement of clinical symptoms and CXR after capmatinib discontinuation and steroid therapy initiation. Second, a longer duration of tepotinib therapy should be applied to determine whether pneumonitis relapse occurs after the long-term use of teoptinib. In this case, the patient presented pneumonitis within one month of treatment with capmatinib; therefore, we presume that pneumonitis relapse would occur sooner if an alternative MET inhibitor targeting the same molecule induces the same adverse event . NotablyWe present the case of a patient with NSCLC with a METex14 alteration who experienced capmatinib-induced pneumonitis and safely continued treatment by switching to alternative MET inhibitors, including one from a clinical trial and tepotinib. Given the lack of targeted therapies for MET inhibitors, rechallenge with tepotinib after capmatinib-induced pneumonitis could be an option for patients with NSCLC harboring METex14. Our experience suggests that tepotinib may be a safe therapeutic option for patients who have recovered from capmatinib-induced pneumonitis under close clinical surveillance.


Fluorescent Carbon dots (CDs) derived from biologically active sources have shown enhanced activities compared to their precursors. With their prominent potentiality, these small-sized (<10nm) nanomaterials could be easily synthesized from organic sources either by bottom-up or green approach. Their sources could influence the functional groups present on the CDs surfaces. A crude source of organic molecules has been used to develop fluorescent CDs. In addition, pure organic molecules were also valuable in developing practical CDs. Physiologically responsive interaction of CDs with various cellular receptors is possible due to the robust functionalization on their surface. In this review, we studied various literatures from the past ten years that reported the potential application of carbon dots as alternatives in cancer chemotherapy. The selective cytotoxic nature of some of the CDs towards cancer cell lines suggests the role of surface functional groups towards selective interaction, which results in over-expressed proteins characteristic of cancer cell lines. It could be inferred that cheaply sourced CDs could selectively bind to overexpressed proteins in cancer cells with the ultimate effect of cell death induced by apoptosis. In most cases, CDs-induced apoptosis directly or indirectly follows the mitochondrial pathway. Therefore, these nanosized CDs could serve as alternatives to the current kinds of cancer treatments that are expensive and have numerous side effects. These CDs were also involved in preparing DNA sensors to detect single gene mutation On the other hand, CDs have been utilized as initiators for low-cost, non-toxic, and one-step polymerization of pyrrole and its copolymer Several reviews emphasized carbon dots' application for cancer therapy Owing to their immense potency for various applications, CDs are continuously prepared and tested for different applications. An essential aspect of their ability to show vast potency is the methods employed for their synthesis. Therefore, discussing the methods of carbon dots synthesis is as important as discussing their applications. Under this circumstance, several studies reported synthetic methods of carbon dots 2SO4 and HNO3) The first reported synthesis of Carbon dots was achieved by electrophoretic remission of single-walled carbon nanotubes (SWCNTs) While laser ablation involves directing the laser light on a starting material which eventually leads to fragmentation of the bulk material The preparation of carbon dots by using small-sized precursors (molecules) has been reported. Generally, the method involves the use of molecules as carbon sources. The molecules undergo polymerization, dehydration, and carbonization Carbon dots from 2-amino-2-hydroxymethyl-propane-1,2-diol (TRIS), EDTA, Glycine and Cadaverine T. bispinosa peels aqueous extract O. sanctumBuchnania lannzanSynthesis of CDs is regarded as green when a less toxic starting material is used. Generally, the solvent and the precursor should exert minimal threat to the environment and the experimenter Generally, all three methods resulted in the successful production of CDs. However, higher emphasis is usually given to that method with more straightforward steps, lower energy and time consumption, and fewer safety concerns. Critically, it is evident that the top-down approach requires expensive and hazardous raw materials to produce CDs. Besides, the yields are too low and difficult to translate into large-scale production due to their production cost. Furthermore, it is not easy to control the size distribution of the CDs.In contrast, the bottom-up approach offers a much easier means of preparing the CDs. For instance, microwave-assisted synthesis provides the required energy for the synthesis to occur within a short time. However, the problem with this approach is that it might lead to environmentally unfriendly by-products that might draw serious concerns. So, straight to the point, we could say that the synthesis of CDs by the green approach employing the bottom-up method could be more favorable in terms of cost of production, eco-friendliness, and simple means of preparation.Besides the CDs synthetic method, choosing a precursor as the primary carbon source is another crucial aspect in this field. Similar to quantum dots, synthesizing carbon nanomaterials like carbon dots requires a material that could provide a carbon-rich core as it forms the nanomaterial. Carbon sources used in the synthesis includes inorganic sources like graphite rods T. bispinosa extract was used to prepare luminescent and biocompatible CDs with a size distribution between 5-10 nm for biological applications Ocimum sanctum leaves were employed to synthesize CDs having sizes between 4-7 nm 3+ sensing and fluorescent imaging As mentioned above, a crude source may constitute many secondary metabolites and proteins. CDs have been prepared from various plant extracts or directly using plant parts. Interestingly, fruit juice was used to prepare CDs. The prepared CDs have about 1.5-4.5 nm size CDs prepared from distinct organic compounds have also been reported. Citric acid has been used as the primary precursor in synthesizing CDs. If further heteroatom doping and surface functionalization is desired on the CDs, the precursor of the heteroatom is added to the reaction mixture. For instance, citric acid-derived CDs with Nitrogen doping have been synthesized by adding urea Uncaria gambir, was used to prepare CDs for tumor imaging Ortho and Para-phenylenediamine were also used as precursors in CDs synthesis et al.Furthermore, glycerol and silane prepared mitochondria imaging CDs Besides being used as surface passivation on CDs 2+ ions Polymeric precursors used for CDs synthesis includes those that were singly used as self-passivation and those used in combination with either small molecule like citric acid In summary, large molecular weight organic molecules have also shown utility in developing CDs for various applications ranging from gene delivery to tumor therapy. Although it might be presumed that the sizes of CDs sourced from small or large molecules would depend on the starting material's size, literatures has shown the contrary As conventional chemotherapy remains ambiguous regarding its swift cancer treatment with no side effects, continuous research is being made to find better alternatives. It is, nevertheless, an expensive process and remains coupled with the possibility of re-development of another tumor post-treatment. Therefore, in this section, our discussion points to the direction where carbon dots could be used as alternatives to drugs used in chemotherapy. This section revolves around the possible use of only CDs for cancer treatment.2 hydration. Such a process is required for worthwhile CO2 sequestration. CDs synthesized from MEG showed better inhibition towards CO2 hydrate formation Carbon dots were synthesized for several purposes. Although these carbon dots could be thought to possess similar behavior, they were proven otherwise. Similar to organic compounds that show various applications regarding their structural features, carbon dots could be considered nanomaterials whose structure also influences their function. However, the difference is seen in their enhanced function compared to organic compounds. For instance, monoethylene glycol (MEG) inhibits COin vitro and in vivo studies. In vitro studies are used as the minimal means of assessing the cytotoxicity of CDs. All studies reported here have proven the ability of CDs to induce cellular death. They exert such activity because they can enter the cell via interaction with receptors in cancer cells. However, such effective interactions are possible only if the CDs have surface functional groups that could bring about the desired activity.It is noteworthy that anti-cancer investigations of CDs reported generally involved two approaches, i.e., in vivo investigations using mice have shown the ability of CDs to reduce tumor weight. Interestingly, the CDs showed significantly lower side effects In addition, in vitro and in vivo anti-cancer activity. Also, selectivity could be enhanced by attaching targeting substances like monoclonal antibodies and aptamers in vitro and in vivo anti-cancer studies obtained from the literature will be summarized in the section.Ultimately, functional groups on the surface of CDs are crucial for both Table .Synthesis of CDs from specific organic compounds showed immense benefits for targeting cancer cells. For instance, transferrin derived CDs helped target transferrin receptors for cancer drug delivery @CDs was not made clear despite its relevance on the potential for its application in cancer therapy Figure : hydrothermal and microwave. Based on their synthesis method, these CDs produced from glucose showed overwhelmingly specific anti-cancer activity. CDs prepared by hydrothermal at the temperature of 200ºC and 160ºC for 5 hours showed particular anti-cancer activity against SMS-KCNR while non-toxic towards SK-N-AS and normal cell lines, HEK-293 and human mesenchymal stem cells (MSC). The anti-cancer activity was linked to the synthesis method because similar CDs prepared from microwave synthesis showed no observable toxicity towards both cancer and healthy cell lines Firstly, citric acid and ribonuclease A were used to produce CDs with cytotoxicity against human gastric cancer cell lines (MGC-808). However, information about the specificity of the RNase-Ain vitro anti-cancer study. On the contrary, not as many in vitro anti-cancer studies reported are available for in vivo studies. Nevertheless, the anti-cancer activity of CDs in vivo thus far has provided yet insightful information regarding the potential of CDs for tumor therapy. Cancer toxic RNase-A@CDs in vivo tumor imaging. Interestingly, this research reported the ability of these CDs to be excreted through urine. Therefore, if they were proven to be cytotoxic in vivo, as were proven in vitro, these CDs would have been more considerable for potential cancer therapy. More intensely, carbon dots obtained from N-hydroxyphthalimide (CD-NHF) resulted in reduced tumor growth (4T1) and metastasis in Balb/c mice.Most of the abovementioned research focuses more on the In vivo investigation was conducted by developing a model comprising ex vivo chick chorio allantoic membrane (CAM) Figure . Similar to in vitro studies, the CDs destroyed the tumor without destroying CAM, as average growth and development were noticed in the treated CAM, whereby limbs and eyes developed in vivo studies of CDs having in vitro cytotoxicity towards cancer cell lines requires more investigation because it could be possible that CDs might show potency in vitro but, in the end, would show poor in vivo activity as implied by the abovementioned research in vitro. However, they further limited the application to in vivo imaging potential. This phenomenon suggests the inability of the CDs for in vivo anti-cancer activity. Overall, in vivo studies are crucial to advance CDs for clinical applications. However, up to now, few in vivo studies have been reported. It is worth mentioning that a number in vivo studies of CDs used in Photothermal therapy have been reported The 4T1 cell line is a highly invasive and metastatic breast cancer cell line. Despite its invasiveness, the CD-NHF lowered its growth and inhibited the metastasic nature of these cell lines. Comparatively, untreated groups with 4T1 injected into them experienced additional weight and widespread lung metastasis In summary, the entire CDs possessing anti-cancer activity described above have a common source. They are either obtained from synthetic organic compounds or natural products. Therefore, it could be assumed that forming CDs with anti-cancer activity requires using a source with bioactivity similar to what the CDs are expected to be.Table .Crude organic sources also yielded CDs' cytotoxic activity towards varying cancer cells. A common ground for these sources was the phytochemicals with previously known potential anti-cancer activity. Thus, in this section, we summarized these CDs and their cytotoxic activity Simarouba glauca leaves synthesized multipurpose CDs to detect Doxycycline antibiotics. In addition, the S. glauca derived CDs were cytotoxic toward breast cancer cell lines (MCF-7). Therefore, the CDs have additional potential in cancer therapy, requiring further investigations The first noted cancer-inhibitive behavior of carbon dots was reported by Hsu and co-researchers Nerium oleander were exploited for potential anti-cancer activity against MCF-7 cell lines. The CDs had an average diameter of 3.6±0.7 nm and a surface covered with predominantly oxygen-rich functional groups. Anti-cancer activity of these CDs with an overall negative charge surface was studied. Compared to N. oleander leaf extracts, the CDs exhibited specific toxicity towards MCF-7 while exerting no cytotoxicity on HDF cells in vivo studies of these cytotoxic CDs were reported, carbon dots derived from ginger that was previously in vitro were further shown to be effective in inhibiting the cell lines significantly in vivo by employing nude mice Figure . Finally, CDs from crude organic extracts could also possess desirable anti-cancer potency even though the source consists of various organic compounds. Thus, suggesting the sustainable use of organic wastes in developing functional carbon dots.Moreover, CDs obtained from the leaf extract of As immense interests develop in CDs with anti-cancer potency, understanding the pathway(s) through which CDs exert such potency is crucial in order to able to develop more efficient nanotherapeutic materials. Limited data is available that investigates how CDs interact with cellular systems. Predicting the specific means through which CDs exert effective interaction with cancer cells is challenging. Despite the given situation, we studied different data that report the implication of CDs on cancer cells when exposed. Since the varying biological property of CDs was prominent to the surface functional groups they contain Nevertheless, some of the observed pathways followed in apoptosis induced by CDs could give an insight into the interaction pattern of these CDs with the cells. Furthermore, comparing the mode of interaction of traditional drugs used in chemotherapy would add to our aim of understanding how CDs interact. It is solely because the surface of CDs contains some organic compounds or surface functional groups capable of binding with the different receptors 2O2 and ROS in cancer cells which were able to trigger apoptosis. It was firmly believed that the apoptosis was induced through caspase-3 mediated pathway Majorly observed pathways found to contribute to cell death of cancers were identified to involve the generation of reactive oxygen species (ROS). A surplus amount of ROS leads to oxidative stress. As Halliwell 2O2. This resulted in the up-regulation of the p53 protein. The authors attributed the apoptosis inducement to one of the secondary metabolites in ginger, curcumin. Curcumin is capable of triggering pro-apoptotic factors Similarly, ginger-derived CDs induced intracellular apoptosis in cancer cells by elevating ROS and HBesides the apoptosis observed, there is evidence of another kind of cell death, that is, autophagy. However, apoptosis is more pronounced in the observed cell deaths due to persistence in its death even when autophagy is curbed et al.More specifically, Tiron Figure 4, CDs as xenobiotic substances enhanced the generation of ROS and H2O2. However, it leads to mitochondrial stress. Under stressful situations, the permeability of the mitochondrial membrane increases and then releases cytochrome C into the cytosol. In the cytosol, cytochrome C forms a complex with apoptosis activating factor-1 (Apaf-1) and procaspase-9. As a result, mechanical stimulation of caspase-9 was realized. Subsequently, this advances into activating apoptosis executers like caspase-3 In summary as shown in Similarly, other CDs stimulate alternative reading frame protein (ARF). Interestingly, this inhibits another responsible protein (Yes-associated protein or YAP) As helpful as these CDs seem, developing them for cancer treatment faces several limitations. The starting materials played a significant role in their activity. However, being able to tune or otherwise improve their activity as is done with conventional organic compounds remains difficult. It could be due to the complicated structure these CDs have, making it challenging to study these nanomaterials' structure-activity relationship (SAR). This is mainly due to the inability to orient the surface functional groups of CDs as desired. Since the synthesis involves the aggregation of many similar small molecules into nanosized material, there is still not enough evidence of whether the starting molecules continue to re-orient themselves in the same pattern when the same preparation condition is repeated. It could be more complicated when the crude organic source is used. Thus, understanding the detailed structure of CDs would contribute significantly to finding better cancer treatments.Figure . It suggests that the two CDs have different structures, although synthesized from the same source. Therefore, CDs also differ based on their structure, indicating possible kinds of CDs similar to organic molecules. Thus, a significant lacking intuition is the knowledge about the exact structural orientation of CDs, which is in turn essential in the CDs' activity.Consequently, this limits their further application for the therapy. For example, CDs' activity was firmly believed to correlate with their precursor molecules' activity. However, recently, glucose-derived CDs showed different activity, which correlates with the synthesis method instead of starting material. The hydrothermal method yielded CDs with anti-cancer activity, while the microwave-assisted method resulted in non-cytotoxic CDs To summarize the review, CDs synthesis could be developed quickly from renewable resources with enhanced activity through either top-down, bottom-up or green synthesis approaches. We have seen that green synthesis offers more sustainable means of CDs development. CDs have a diverse means of preparation ranging from their preparation using low molecular weight organic molecules as well as large molecular weight organic molecules. Interestingly, it was apparent that these CDs' sources influenced their function. Therefore, CDs prepared from a likely anti-cancer organic source would show anti-cancer properties with selective and enhanced anti-cancer activity. In most instances, these CDs interact with cancer cells specifically, resulting in cell death by apoptosis via the mitochondrial pathway. This intuition could provide a solution to the current challenges in cancer treatments.The future applications of CDs are now apparent. However, the production of these CDs in consideration of their future demands remains unaddressed. Although some carbon dots showed selective cytotoxicity towards some cancer cell lines and less toxicity towards normal cells, extensive study is still needed to estimate the feasibility and druggability of these CDs. Furthermore, the ability to desirably control these CDs' interaction with target proteins is crucial for improving the efficiency of selective cancer targeting. Therefore, understanding the patterns followed by precursors or molecules to carbonize and polymerize into the CDs could open the way for the prediction and engineering of surface functional groups and/or hydrophobic moieties oriented at the “right” position capable of effective interaction with the targeted cellular receptor.With the number of previously reported literatures


Arctic shrub expansion alters carbon budgets, albedo, and warming rates in high latitudes but remains challenging to predict due to unclear underlying controls. Observational studies and models typically use relationships between observed shrub presence and current environmental suitability (bioclimate and topography) to predict shrub expansion, while omitting shrub demographic processes and non-stationary response to changing climate. Here, we use high-resolution satellite imagery across Alaska and western Canada to show that observed shrub expansion has not been controlled by environmental suitability during 1984–2014, but can only be explained by considering seed dispersal and fire. These findings provide the impetus for better observations of recruitment and for incorporating currently underrepresented processes of seed dispersal and fire in land models to project shrub expansion and climate feedbacks. Integrating these dynamic processes with projected fire extent and climate, we estimate shrubs will expand into 25% of the non-shrub tundra by 2100, in contrast to 39% predicted based on increasing environmental suitability alone. Thus, using environmental suitability alone likely overestimates and misrepresents shrub expansion pattern and its associated carbon sink. Shrub encroachment trends are widespread yet complex. Here the authors demonstrate that not considering dispersal and fire leads to overestimating shrub expansion in Arctic tundra and therefore its role as carbon sink. Rapid climate warming in recent decades and associated feedbacks have led to shifts in Arctic vegetation composition and abundance4. In particular, increased tundra shrub cover has been widely observed through field surveys5, aerial photographs7, and satellite remote sensing9. Pervasive shrub expansion can heat the atmosphere through decreased albedo and increased greenhouse warming induced by atmospheric water vapor, resulting from increased evapotranspiration and regional ocean feedbacks12. Locally, shrubs can warm the soil in the winter due to the insulation of accumulated snow, which deepens the active layer and accelerates soil carbon loss compared to non-shrub tundra14. Moreover, the distribution of shrubs also affects nutrient cycling, animal populations15, and wildfire risk and associated carbon emissions17. Understanding controls of shrub expansion patterns is therefore crucial to predicting climate feedbacks and ecological consequences of the rapidly changing Arctic.The Arctic has warmed more than twice as fast as the global average and is projected to continue outpacing lower latitudes over the 21st century18. Increasing temperature has been identified as a major control of shrub expansion20. However, the influence of temperature can be attenuated or reversed by soil moisture limitation, snow distribution, and topography25. The majority of observational-based studies focus on environmental factors and attribute the heterogeneity of shrub expansion to spatial variation of environment-based suitability, i.e., the likelihood of shrub presence given environmental conditions26. Based on space-for-time substitutions, some of those studies used derived spatial environment-vegetation relationships to assess future shrub expansion21, assuming stationary relationships between species and the environment. Although this approach has been found effective in predicting species distributions when ecosystems are in dynamic equilibrium, e.g., under a relatively stable climate or over a sufficiently long time scale27, it ignores transient responses and non-stationary ecological processes, thus causing errors in projected ecosystem change30. As the Arctic tundra deviates from the historical quasi-equilibrium due to climate change, evaluating the dynamic roles of plant migration and disturbance becomes especially relevant.The area where temperature limits the growth of Arctic vegetation has been declining over the past decades32. Seed dispersal has been investigated in previous studies to estimate species range shifts34, and was found important in explaining shifts in vegetation composition at sites in alpine35, mediterranean37, and tropical biomes39. Nonetheless, the impact of seed dispersal on vegetation patterns is also compounded by suitable environmental niches and thus is not always the limiting factor42. In the Arctic, long-distance seed dispersal is a critical mechanism affecting species distribution. Genetic analysis has revealed repeated long-distance seed dispersal to a remote archipelago from multiple source regions since the last glacial retreat, while the resulting species distribution is predominantly shaped by temperature that limits environmental suitability for establishment41. In contrast to a relatively stable climate over the past several millennia, the fast-changing climate over recent decades might lead to shifts in the relative dominance of environmental suitability and seed dispersal in shaping Arctic shrub expansion.With changes in growing conditions under a warmer climate, the successful establishment of new shrub patches depends on seed dispersal. Seeds can be dispersed through many biotic and abiotic vectors, such as gravity, animals, wind, ocean currents, and drifting sea ice, which result in dispersal ranges from meters to hundreds of kilometers43. In the short term, wildfires may cause seed and seedling mortality, which could limit post-fire recruitment. On the other hand, wildfires can alter post-fire vegetation trajectories by heating the soil during the fire, cause long-term soil warming by removing surface litter, and improve seedbed nutrient availability, thus facilitating germination and seedling establishment48. For example, modeling and site-based field studies have reported both enhanced expansion and diminished recovery of shrubs from four years to two decades after wildfires at several sites in Alaska51. How wildfires affect shrub expansion across large gradients of environmental suitability and seed dispersal has barely been evaluated using observations.The dynamic trajectory of ecosystems may also be affected by disturbance. Although historically rare in Arctic ecosystems, wildfire is expected to become more intense and frequent as the climate warms3. Areas classified as shrubland include prostrate dwarf-shrub tundra and erect-shrub tundra52, dominated by species of birch (Betula spp.), alder (Alnus spp.), willow , and other dwarf evergreen and semi-deciduous shrubs. Field surveys have detected expansion of these shrub communities55. Here, shrub expansion is defined as shrub dominance in tundra originally dominated by non-woody species at a 30 m scale. We collected topographic and regionally downscaled bioclimatic variables across the domain to identify the variables most informative for observed shrub expansion (“Methods”). Based on these selected topographic and bioclimatic conditions, averaged over three decades prior to 1984, we estimated environmental suitability for shrubs in 1984 using a random forest model. The same model was then used to calculate the environmental suitability for shrubs in 2014 using 1985–2014 average bioclimatic conditions. We analyzed whether changes in environmental suitability could explain shrub expansion from 1984 to 2014. Seed-arrival probability, a measure of spatial proximity to existing shrub patches, was calculated through convolution of seed-dispersal kernels over 1984 shrub cover images. Given the variety of dispersal mechanisms, we considered both short- and long-distance dispersal kernels, and optimized the range and shape parameters to fit observed shrub expansion. The year and location of fires were obtained from a Landsat-derived annual burn scar product. We investigated individual and compound impacts of environmental suitability, seed dispersal, and fire occurrence on observed 1984–2014 shrub expansion. Based on the resulting sensitivities and the projection of bioclimatic conditions and fire from climate models, we estimated shrub expansion in 2040, 2070, and 2100, and explored the relative importance of environmental suitability change, fire, and seed dispersal on projected shrub expansion. We found observed shrub expansion did not follow the pattern of environmental suitability but can only be explained by considering seed dispersal and fire. Shrub expansion under the projected climate is likely overestimated if neglecting the limitation of seed dispersal and fire.We focused on shrub expansion from 1984 to 2014 across the northwestern region of North America covering Alaska and western Canada, i.e., the NASA Arctic-Boreal Vulnerability Experiment (ABoVE) core domain. Shrub expansion was detected using an annual dominant land-cover product derived from Landsat surface reflectance and trained over field photography and very high-resolution imageryWe first estimated environmental suitability for shrubs as of 1984. Among the 27 variables, the seven most informative variables, as identified based on variance inflation factors  than to existing shrub cover in 1984 (r = 0.92). Accounting for different initial land-cover types of the non-woody tundra and change in environmental suitability also barely contributed to explaining the pattern of shrub expansion . However, both long- and short-distance dispersal became significantly more important in facilitating shrub expansion after fire, compared to areas without fire  and r = 0.71   Fig. . We notesion Eq.  in “Meth2 more shrub cover than in 2014, with the uncertainty originating from uncertainty in the empirically derived sensitivities 9. However, because shrub growth and local seed production are expected to be controlled by environmental suitability, the low impact of environmental suitability supports seed dispersal being the dominant cause of shrub expansion across the domain. Moreover, as dispersal is estimated based on spatial proximity, our results highlight the importance of spatially connected processes. Although seed dispersal is the originating mechanism and has been recognized as a dominant spatial process controlling vegetation range shifts39, the impact of spatial proximity identified here might also be partially attributed to other spatially connected factors, such as active-layer depth, soil thermal-hydro conditions, surface litter, nutrient availability, and herbivore activities66. These factors may contribute to the spatial connectivity of shrub expansion via rates of seed germination and seedling establishment. However, these factors are unlikely to be the dominant explanation for the identified impact of spatial proximity, as they are partially related to environmental suitability via climate and topographic conditions, and they tend to exhibit smaller spatial ranges than those identified for long-distance dispersal (~40 km). Furthermore, because the remotely sensed land cover that we used cannot distinguish different shrub species while dispersal influences the expansion of each single species, the results based on the aggregation of all shrub species likely overestimate spatial proximity, thus providing conservative estimates of dispersal limitation. Field surveys and measurements are required to investigate the confounding roles of these spatial processes.Complex ecosystem processes introduce uncertainties in predicted environmental suitability, identified shrub expansion, and the relationship to seed dispersal and fire disturbance. Uncertainties related to future environmental suitability can be influenced by future bioclimatic conditions exceeding the historical ranges used to establish their relationships with environmental suitability  was downscaled from the gridded Climatic Research Unit Time-series data version 4.02 (CRU TS4.02), and the projected data (2014–2100) was downscaled from CMIP5 under the RCP8.5 scenario, which is broadly consistent with recent trends of global carbon emissions79. The elevation data were obtained from the Advanced Spaceborne Thermal Emission and Reflection Radiometer (ASTER) Global Digital Elevation Model Version 3 with a 30 m resolution80. Slope, aspect, and the topographic wetness index were derived from elevation using a terrain analysis software RichDEM81. Fire occurrence during 1985–2009 was identified using the annual product of differenced Normalized Burned Ratio (dNBR) at a 30 m resolution82, where the perimeters came from the Alaskan Interagency Coordination Center and the Natural Resources Canada fire occurrence datasets. Only fires that occurred at least 5 years prior to 2014 were considered to allow vegetation recovery. Burn area during 2015–2100 was obtained from CMIP6 projections under a SSP585 scenario. Datasets at coarse resolutions (climate and projected burn area) were resampled to a 30 m resolution using the nearest neighbor method.Shrub expansion was identified based on the Landsat-derived product of annual dominant land cover across ABoVE core domain from 1984 to 201483. This procedure ensured that the identified variables are most statistically informative in representing the bioclimatic and topographic conditions across the domain. Based on the identified variables, we applied ten species distribution models to estimate whether a pixel was shrubland. The models include generalized linear model, generalized additive model, boosted regression trees, classification tree analysis, artificial neural network, surface range envelope, flexible discriminant analysis, multiple adaptive regression splines, random forest, and maximum entropy, all applied using the biomod284 software in R85. Due to the large computation load, we trained each model using 5% of the pixels randomly selected within the target area, including both shrub and non-shrub pixels. The model accuracies were evaluated using all pixels across the entire domain. The random forest model had the highest accuracy based on the true skill statistic and the area under the receiver operating characteristic curve. Therefore, the environmental suitability, i.e., the probability of a given 30 m pixel being shrubland given its bioclimatic and topographic conditions, was calculated using only the random forest model. We assumed a relatively stable climate prior to 1984. Thus, the average bioclimatic conditions during 1955–1984 were used to train the random forest model and assess environmental suitability in 1984. Environmental suitability in 2014, 2040, 2070, and 2100 was estimated by replacing the bioclimatic conditions to the averages over the previous 30 years, respectively. We evaluated the relative importance of each variable in explaining environmental suitability. We also analyzed the response curve of environmental suitability to the variation of each variable, and the response surfaces to the covariation of the most important three variables, while setting other variables as the domain average.We considered 23 bioclimatic variables , exponential (b = 1), and fat-tailed (b  = 0.5) kernels, and has been widely used in literature86. The seed-arrival probability of a given location (s) is calculated as follows:N is the total number of shrub pixels within the maximum range; scipy.ndimage87 in Python. As seeds can arrive via multiple dispersal vectors, the seed-arrival probability results from the integral of multiple dispersal kernels with distinct ranges and shapes32. To parsimoniously account for various dispersal vectors, we considered the integral of a short-distance dispersal kernel and a long-distance dispersal kernel. For short-distance dispersal, we evaluated all combinations of 100 m The impact of seed dispersal was quantified using the probability of seed arrival at a given location, calculated using kernel convolution over the spatial pattern of shrublands. The following exponential power kernel was used to describe the relationship between seed-arrival probability and distance to parent shrub patches.Observed shrub expansion was quantified as the fraction of 30-m pixels that were not identified as shrublands in 1984, i.e., non-shrub tundra, but became shrublands in 2014 within each 4 km by 4 km gridcell. Environmental suitability and seed-arrival probabilities through short- and long-distance dispersal were aggregated by average to a 4-km scale and used to explain the spatial pattern of shrub expansion using the following multivariate linear regression.spBayes software88 in R89, for grid cells with and without fire occurrence, respectively. An exponential covariance model and the following prior distributions were used based on empirical variogram90: r was estimated for the test set. Sensitivities were calculated as the mean of 5000 posterior samples after convergence (1000 samples).In addition to the considered explanatory variables, shrub expansion may also be influenced by other unconsidered confounding factors that lead to a spatial correlation pattern independent from that induced by dispersal. To account for such spatial correlation, we also conducted a spatial regression, i.e.,Based on the estimated empirical relationships, we predicted shrub expansion by 2040, 2070, and 2100. For each 30-year period, environmental suitability was estimated using topographic conditions and the averages of projected bioclimatic conditions. Seed-arrival probability was estimated using shrub cover at the start of the period. The fire was assigned for each 30 m pixel with a probability represented by the cumulative burn area fraction, ensuring the aggregation from a 30 m scale consistent with the climate model projection at a coarser scale. Each non-shrub pixel at the start of the 30-year period was changed to shrubland at the end with a probability calculated using the estimated sensitivities to its environmental suitability, seed-arrival probability, and fire occurrence. We further quantified the uncertainty of projected shrub expansion due to the uncertainty in the estimated sensitivities. Instead of a computationally expensive bootstrapping approach, we used the lower and upper boundaries of the 95% confidence intervals for all the regression coefficients in each 30-year period, which provided an overestimate of the uncertainty range of projected shrub expansion. To disentangle the impacts of environmental suitability change, seed dispersal, and fire on the projected shrub expansion, we used synthetic scenarios where each of the three factors was turned off, i.e., environmental suitability kept the same as in 2014, zero seed-arrival probability, and no fire occurrence, respectively. The difference between the synthetic scenarios and the actual projection illustrated the contribution of the corresponding factor on the projected shrub expansion. To diagnose potential bias and spatial patterns of predicted shrub expansion using the environmental suitability-based approach in previous studies, the shrub expansion by 2100 predicted here was also compared to the prediction without considering dispersal and fire, i.e., by applying 2100 environmental suitability to the relationship established between shrub presence and environmental suitability alone in 1984.Further information on research design is available in the Supplementary Information FilePeer Review FileReporting Summary


Multimodality therapy including surgical resection is the current paradigm in treating malignant pleural mesothelioma (MPM), a thoracic surface cancer without cure. The main limitation of all surgical approaches is the lack of long-term durability because macroscopic complete resection (R1 resection) commonly predisposes to locoregional relapse. Over the years, there have been many studies that describe various intrapleural strategies that aim to extend the effect of surgical resection. The majority of these approaches are intraoperative adjuvants. Broadly, there are three therapeutic classes that employ diverse agents. The most common, widely used group of adjuvants are comprised of direct therapeutics such as intracavitary chemotherapy (± hyperthermia). By comparison, the least commonly employed intrathoracic adjuvant is the class comprised of drug-device combinations like photodynamic therapy (PDT). But the most rapidly evolving (new) class with much potential for improved efficacy are therapeutics delivered by specialized drug vehicles such as a fibrin gel containing cisplatin. This review provides an updated perspective on pleural-directed adjuncts in the management of MPM as well as highlighting the most promising near-term technology breakthroughs. Malignant pleural mesothelioma (MPM) is a rare, highly aggressive, and recalcitrant tumor arising from the mesothelial lining of the pleura. To date, there is no clinical standard  that migA focused review and update of intrapleural therapeutic modalities  that can augment surgical resection and address microscopic residual tumor foci (R1 margin) is presented here. Particular attention is dedicated to verifying the molecular mechanisms of action for each type of agent used in this class of surgical adjuvants.2) versus PD or EPP with hyperthermic intraoperative chemotherapy (HIOC) at 41.5°C  and doxorubicin (100 mg) (2 for 60 minutes at 42°C) followed by adjuvant chemoradiation (according to modern techniques and standards), there was a disappointing overall recurrence rate of 75%   , the ongn vitro) .Povidone-iodine  is a time-honored antiseptic agent commonly used throughout clinical practices for handwashing, skin cleansing, and irrigation/lavage of body cavities for disinfection , 17. Sinin vitro studies without any in vivo demonstration of PVP-I-specific effects in cancer cells. It is generally thought in human MPM cancer cell lines that PVP-I causes cellular necrosis via reactive oxygen intermediates which might contribute to stimulating anti-tumor inflammatory reactions (in vitro study showed that the necrotic phenotype (< 1% cell viability) was evident at 0.01 to 0.1% PVP-I concentration by 7.5 minutes post-exposure, without further improvement at longer exposures times up to 48 hours  PVP-I where thymic epithelial cells and MeT-5A were indiscriminately killed by cellular fixation after 30 minutes of exposure  PD procedure, 3) post-operative sequential intrapleural chemotherapy followed with IL-2 infusions, and 4) adjuvant chemoradiotherapy plus long-term subcutaneous IL-2. Median OS reached 26 months without any operative fatality. Since then, there have not been more similar-minded studies in MPM.The least explored intrapleural adjuvant strategy to date is the direct use of immunomodulatory agents that can induce anti-tumor effects. A phase II trial of 49 stage II-III MPM patients explored the feasibility and efficacy of a unique four-modality intervention, including immunotherapy . PatientOverall, this was a complicated clinical regimen. There was not an abundance of convincing preliminary data to support all elements of trial design, much less in combining them all. Consequently, it has been difficult to discern why this trial was conceived as such since there was not an easily recognized logical step-building from previous trials. In fact, there was not a specific hypothesis stated. The total length of treatment for enrolled patients was never specified (at least 4 months from study entry to the start of maintenance immunotherapy). The final outcomes were difficult to discern, especially in being able to pinpoint whether surgery, immunotherapy, chemotherapy, or radiation  was most beneficial. The role of immunotherapy as an intrapleural adjuvant remains undefined, yet should be revisited now in the context of approved frontline use of immune checkpoint inhibitors in certain scenarios of MPM .in vitro study demonstrated that IL-2 in combination with lymphokine-activated killer cells could effectively lyse the MPM cells whereas NK cells were ineffective   . Over onatients)  and the Newer strategies for the delivery of intracavitary local therapy are being developed and tested in pre-clinical models and human trials. The broad aim is to achieve durable and effective treatments against MPM by leveraging novel technologies to specifically enhance cancer cell-targeted strategies. These drug delivery depot systems  encompass a variety of form-factors such as drug-releasing gels, thin films, or nanoparticles. It is anticipated that when these approaches mature, improved clinical efficacy over intrapleural application of direct therapeutics may be attained.Building upon the clinical experience of pleural-directed drug adjuvants to improve the local effect of MPM surgical resection, therapeutic agents have been combined with delivery vehicles to maximize local drug concentrations while limiting systemic adverse events. Pre-clinical MPM studies demonstrated anti-tumor efficacy using cisplatin combined with fibrin (gel) delivered by an intracavitary injection technique , 30. TheA phase I dose escalation trial followed, in which 12 non-sarcomatoid MPM patients with mostly stages III-IV underwent extended PD procedure . CisplatHowever, many questions remain for further investigation. How the fibrin interacts with cisplatin and specifically how it forms a conjugate with cisplatin remains unknown because no mechanistic data have been presented. Whether the fibrin has any impact on the efficacy of cisplatin is unknown. Most importantly, the mechanism of cisplatin release from fibrin still needs to be investigated. These unknowns contribute to the lack of dose-dependent cisplatin tissue levels and its high variability as measured in each patient . Without this prerequisite knowledge, it remains uncertain whether this delivery strategy will be able to selectively target tumor cells/tissues and, thereby, be any more efficacious compared to direct instillation of cisplatin. The off-target effects of cisplatin-fibrin are concerning as reflected by the urine excretion of systemic-leak cisplatin and its persistence in deep tissues outside of the pleural surfaces where MPM originates.in vivo  is a polysaccharide of repeating units of D-glucuronic acid and (1-β-3) N-acetyl-D-glucosamine with physiochemical attributes useful in drug delivery strategies . Polymerin vivo . Cisplatin vivo studies in MPM have directly traced the fate of cisplatin once released from the hyaluronate film. Despite no major systemic side-effects in animals, there remains concern for treatment-related morbidity in humans considering the significant higher plasma levels seen with HYALCIS. More basic research is needed to understand the impact of the hyaluronate-cisplatin complex . Nevertheless, many facets of this technology remain obscure and could potentially predict suboptimal outcomes in future clinical settings. This methodology lacks a cancer-specific targeting mechanism. None of the  complex  to improAn alternative strategy to locoregional drug delivery can be achieved with novel synthetic polymer nanoparticle carriers (100 nm diameter) which swell upon exposure to acidic pH and subsequently release their therapeutic cargo within 24 hours  . A studyin vitro and in vivo). There were no direct data to indicate the drug was properly unloaded intracellularly, nor were there supporting results measuring tissue levels of drug after injection of eNP. It will be interesting to see this technology mature with the flexibility to load other drugs that are known to have better intrinsic efficacy against MPM. More research is needed to elucidate the mechanism of eNP drug binding and the mechanism/kinetics of drug release as it remains unknown at this time. Future studies will need to distinguish the cytotoxic effect of eNPs from the effects of physical cell swelling, in addition to how eNPs preferentially target cancer cells in vivo. Furthermore, the fate of how eNPs are metabolized in vivo will have to be traced to assess for any off-target systemic toxic side-effects.This study did not explain, however, why the paclitaxel-loaded eNP remained intact once inside cancer cells in the co-localization studies  are released over time from the net-positive charged hydrogel matrix to adjacent tissues and taken up more selectively by cancer cells [net-negative surface charge  is a unique approach to treat MPM that relies on wavelength-specific visible light generated by a laser device focused on target tissues which have accumulated light-absorbing photosensitizing agent  in the presence of oxygen. The localized interaction of these three components induces a tumoricidal photochemical reaction to produce reactive singlet oxygen leading to damage of the tumor cell wall and neovasculature . After MSince 1991, there have been 11 feasibility and safety trials, three retrospective survival studies and two prospective trials totaling 337 MPM patients who received intrapleural PDT during multimodality therapy of MPM including macroscopic complete resection . Since tWith apparent lack of superior efficacy over other local ablation strategies, more basic investigations are necessary to improve upon inherent shortcomings of this technology in the context of cancer surgery. The chest cavity geometry is a very challenging location to ensure even and consistent irradiation of light, especially when the intralipid solution cannot entirely fill up the chest during surgical procedures. More innovation is needed in devices to deliver PDT. Anticancer effects are dependent on oxygen levels in close proximity to tumor tissue, but the MPM microenvironment is characteristically hypoxic . Even moMPM remains a fatal disease in need of highly effective therapeutic agents and strategies. Macroscopic complete resection as part of multimodality care certainly can contribute to positive outcomes. However, verifying a direct effect and quantifying the relative merit of surgical resection when used in a multimodality treatment scheme has been elusive. The lack of good clinical outcomes in MPM therapy is impacted by numerous heterogeneous factors including, for example: extent of surgical resection (EPP vs. PD), tumor histology , systemic therapy , and how the interventions are employed in a sequence. Such clinical parameters add complexity to the process of study designs best equipped to demonstrate direct benefits of pleural-directed adjuncts in MPM therapy Table 2.AC, AS, DW, and KP - writing of text and table, drawing figure, and editing. CH - writing, revision and editing, concept and design. All authors contributed to the article and approved the submitted version.This work was supported by NIH Intramural Research Program with funding from National Cancer Institute - Center for Cancer Research Grant ZIA BC 011657 (to CH).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.


The urea cycle is a liver-based pathway enabling disposal of nitrogen waste. Urea cycle disorders (UCDs) are inherited metabolic diseases caused by deficiency of enzymes or transporters involved in the urea cycle and have a prevalence of 1:35,000 live births. Patients present recurrent acute hyperammonaemia, which causes high rate of death and neurological sequelae. Long-term therapy relies on a protein-restricted diet and ammonia scavenger drugs. Currently, liver transplantation is the only cure. Hence, high unmet needs require the identification of effective methods to model these diseases to generate innovative therapeutics. Advances in both induced pluripotent stem cells (iPSCs) and genome editing technologies have provided an invaluable opportunity to model patient-specific phenotypes in vitro by creating patients’ avatar models, to investigate the pathophysiology, uncover novel therapeutic targets and provide a platform for drug discovery. This review summarises the progress made thus far in generating 2- and 3-dimensional iPSCs models for UCDs, the challenges encountered and how iPSCs offer future avenues for innovation in developing the next-generation of therapies for UCDs. Six enzymes and two transporters have been identified as causing primary UCDs  derived vectors (NTC02991144)10 or mRNA encapsulated lipid nanoparticles (NTC04442347)11. An early phase clinical trial of AAV gene therapy for ornithine transcarbamylase deficiency in adults has recently shown promise as an alternative disease-modifying therapy12. These diseases are rare, which precludes an optimal understanding of the complex pathophysiology of the disease, especially in the brain17. Indeed, the spectrum of clinical severity of the same UCD can be highly variable between individuals19. Yet, despite a common cerebral toxicity as a result of hyperammonaemia, there are pathophysiological specificities, which remain only partially understood, such as arginase deficiency and dysmyelination20, argininosuccinic aciduria and oxidative stress24, or Lysinuric Protein Intolerance and impaired phagocytosis25. Some mouse models fail to recapitulate the human phenotype, such as the hypomorphic Spfash mouse modelling ornithine transcarbamylase deficiency (OTCD), where a high residual ureagenesis capacity prevents hyperammonaemia, UCDs’ cardinal feature, under standard diet. In the Spfash mouse, hyperammonaemia is observed with a protein-enriched diet meaning that only partial OTCD is recapitulated27. In addition, traditional cell lines usually derived from immortalised cancer cell lines, have their own limitations with metabolic alterations, leading to a need for alternative modelling methods28. The parallel emergence of induced pluripotent stem cells (iPSCs) and CRISPR/Cas9 editing have provided the opportunity to model these disorders in vitro giving the potential to uncover novel mechanisms of action and new therapeutics, whilst overcoming some of the limitations posed by other models. iPSCs are derived from differentiated adult somatic cells and are characterised by their ability to propagate indefinitely and their capacity to differentiate into any cell type of the human body31. This review aims to discuss the ongoing progress and remaining limitations that have occurred in modelling these rare disorders using iPSCs.Urea cycle disorders (UCDs) encompass a set of rare inherited disorders characterised by defects in one of the urea cycle enzymes or transporters that contribute to detoxify nitrogen waste i.e., ammonia. Ammonia is produced by the deamination of amino acids and is a highly neurotoxic molecule. The urea cycle pathway is fully expressed in the liver Fig. , precise34. In addition, immortalised cancer cell lines are often immature, have dysfunctional apoptotic pathways and have limited genotypic variability35. Primary hepatocytes are considered the gold standard and provide a more biologically relevant model, recapitulating several features of a healthy liver cell in vivo37. However, these cells are challenging to isolate, have limited proliferative capacity, limited viability in vitro and rapidly lose hepatic function, preventing them being maintained in culture long-term41. Foetal and adult human stem cells can as well be used although they can be technically challenging to isolate and culture43. Therefore, in the last few years, iPSCs have provided an alternative strategy to model inherited metabolic diseases, particularly UCDs 51. In vivo animal models can be labour intensive and expensive, and the resultant animal may fail to recapitulate several features of the human phenotype. In addition, the translation of drug trials to humans from animal models can be unsuccessful due to species-specific biological responses, contributing to the translational “valley of death” and high failure rates in the approval of novel therapies52. With iPSCs, mutliple cell lines derived from the same patient can be cultured simultaneously in a much smaller space, with each line having the capacity to be differentiated into several tissue types, which can also reduce the time and labour required. iPSCs are therefore more easily scalable and accessible than animal models. Experiments can be compared across different tissue types to cells that are all derived from the same genetic background. Furthermore, pharmacology and toxicology assessments can be made patient-specific to avoid possible adverse side effects.Utilising iPSCs can also have advantages over in vivo models, particularly when considering the ethical principles of the three Rs , which oversee animal research  identify pathogenicity of variant of uncertain significance (VUS), (ii) study disease mechanisms, (iii) develop and screen therapeutic strategies in vitro, with a potential for in vivo applications and iPSC transplantation to treat animal models Table . As the 54. In this study, patient iPSC lines were genetically modified via CRISPR/Cas9 to constitutively express human codon optimised CPS1 from the AAVS1 safe harbour site and then differentiated into hepatocytes. The study aimed to assess the therapeutic potential of this approach. Despite successful differentiation, interestingly, the corrected cells failed to metabolise ammonia any more effectively than the patient cell lines. This unexpected outcome was proposed to be due to transgene promoter methylation and resultant transcriptional silencing in the undifferentiated cells. This study emphasises the requirement for thorough quality control and functional assessment of iPSCs prior to use for downstream experiments.This disease has been investigated using patient-derived iPSCs, enabling the results to be compared to the clinical phenotype of the affected individuals56 , a membrane channel protein that facilitates transit of water, glycerol, and urea, in OTCD iPSC-derived hepatocytes. In healthy control, AQP9 overexpression enabled increased ureagenesis and a phenotype closer to the in vivo environment. OTCD iPSC-derived hepatocytes recapitulated the hepatic manifestation of the patient phenotype with reduced ureagenesis and OTC activity. This phenotype was corrected after lentiviral gene therapy57.Some studies develop iPSC lines from peripheral blood mononuclear cells (PBMCS), such as with neonatal-onset OTCD patients56 Table . These sASS1 gene have been described60. The identification of variant of uncertain significance (VUS) is a large-scale problem in the era of high-throughput genomics61. The use of patients’ iPSCs can provide a unique model to perform functional studies to assess pathogenicity of a mutation with the additional possibility of screening personalised therapeutics. iPSCs carrying the disease-specific mutations can be artificially created and then used for functional studies as highlighted in citrullinaemia type I (CTLN1)62. Yoshitoshi-Uebayashi et al. differentiated iPSCs from a citrullinaemic patient and isogenic controls into functional hepatocytes, but failed to fully recapitulate a UCD phenotype, potentially due to incomplete hepatocyte differentiation. Baseline ammonia and urea levels between patient and control lines were not different but an ammonia challenge assay exhibited significantly reduced ureagenesis in ASS1-/- cells. Interestingly, the study highlighted significantly increased levels of several tricarboxylic acid (TCA) cycle-related metabolites such as pyruvate, succinate and aspartate, shedding light on aspartate accumulation caused by ASS deficiency and diversion towards TCA cycle63.More than 130 private mutations of the 64. The liver organoids for both wild-type and patient cells demonstrated similar architecture and appropriate hepatocyte differentiation confirmed by expression of hepatocyte markers, e.g., HNF4A and CK19 and functional assays, e.g., albumin production, low density lipoprotein (LDL) uptake and glycogen storage. Transcriptomic analysis was similar between wild-type and patient 3D cultures indicating that ASS1 mutation did not interfere with differentiation ability. Functionally, the CTLN1 organoids had significantly increased ammonia, with overexpression of wild-type ASS1 restoring arginine levels to wild-type levels and partially rescuing ureagenesis. These data demonstrated that hepatic organoids can recapitulate several features of the disease phenotype that can be alleviated with therapies targeting gene function64.Akbari et al. differentiated iPSCs from a CTLN1 patient into endoderm-derived hepatic organoids, following a 2 week-differentiation protocol and prolonged expansion over a year21. Hence ASLD is a challenging disease to treat as liver-targeted gene therapy corrects ureagenesis but does not alleviate other phenotypic symptoms, especially within the brain65 or endothelial cells66. ASLD iPSCs have been differentiated into other tissues than hepatocytes for investigating ASLD pathophysiology, to address the consequences of arginine and downstream metabolite nitric oxide (NO) deficiencies. Kho et al. demonstrated that ASLD can present as endothelial-dependent hypertension. The authors used multiple models, in addition to clinical studies, including iPSC-derived endothelial cells67. ASLD iPSCs did not differentiate as efficiently into endothelial cells as the controls, indicating that ASL participates in vasculogenesis via NO production. ASLD iPSC-derived endothelial cells had decreased levels of intracellular NO. The cells, when injected into immunodeficient (NOD/SCID/IL2Rγ − ) mice, demonstrated a decreased ability to establish blood capillaries and arterioles in vivo. The data, combined with their results from a human clinical study, mouse model and in vitro cell models, demonstrated that endothelial dysfunction is the main cause of hypertension in ASLD. This shows the ability of iPSCs to be used to model disease and validate findings from other in vitro and in vivo models67. Jin et al. investigated the effect of ASLD in human osteoblasts, using ASLD iPSCs compared to two isogenic controls68. The ASLD-iPSC derived osteoblasts had decreased mineralisation capacity, significantly reduced expression of glycolytic genes such as SLC2A1, PKM and LDHA, or GLUT1 protein suggesting that ASLD leads to the deficient glycolysis. Treatment with NO donor S-nitroso-N-acetylpenicillamine (SNAP) during osteoblast differentiation led to an upregulation of the glycolytic genes. These findings that ASLD-iPSCs have a decreased ability to differentiate into osteoblasts and impaired glycolysis was confirmed in two additional animal models and murine osteoblastic cell lines, highlighting the reliability of iPSCs in investigating pathophysiology and identifying new therapeutic targets68.Argininosuccinate lyase deficiency (ASLD) is an atypical UCD due to a systemic phenotype affecting multiple organs69. Such studies provide a strategy for restoring gene function by editing paving the way for genetically modified cell therapies to treat UCDs. Arginase-1 deficiency has also been studied using iPSC-derived hepatocyte-like cells, as well as iPSC-derived macrophages. Utilising PiggyBac technology alongside CRISPR/Cas9, exons 7 and 8 of ARG1 were reincorporated into iPSCs derived from an arginase-1 deficient mouse model70, a knockout model where mutant animals die at about 2 weeks of age, in order to assess whether ARG1 function could be restored. Successful gene repair was confirmed, but ureagenesis remained suboptimal compared to adult hepatocytes, probably caused by insufficient hepatocyte differentiation. In comparison, iPSC-derived macrophages showed corrected arginase function that could be explained by a more ready differentiation into a functionally mature state71. The study demonstrated how in vitro research can be developed using iPSCs and CRISPR/Cas9 to conduct follow-up investigations in various differentiated cell types. Differentiated cells can then be transplanted in vivo to enable further maturation and long-term repopulation of hepatocytes, for instance73. The same group also used transcription activator-like effector nucleases (TALENs) to correct the same defect and again, differentiated these cells into hepatocyte-like cells. These cells were then transplanted back via intravenous infusion into 12-week-old arginase-1 deficient mice, in a tamoxifen-induced ARG1 knock-out mouse model. iPSC-transplanted arginase-1 deficient mice survived an additional week compared to untreated knock-out littermates without reaching statistically significant increase of survival. The treated mice had increased arginase-1 expression but ureagenesis was not comparable to that of wild-type controls. The inability to fully rescue the phenotype may be caused by the inability for transplanted cells to repopulate the periportal hepatocytes where the urea cycle is expressed, suboptimal hepatocyte differentiation, low engraftment or immunological rejection of transplanted cells74.Hui et al. studied CRISPR/Cas9-edited iPSCs lines from three patients with arginase-1 deficiency, in an attempt to restore arginase-1 activity. Corrected iPSCs were compared to unedited controls and exhibited restoration of arginase expression and ureagenesis from functional arginase. The three corrected lines were successfully differentiated over 21 days into hepatocytes, alongside their uncorrected counterparts and a control line. Off-target editing was evaluated by sequencing specific sites identified in silico being at risk of off-target effect. Neither insertion nor deletion had occurredSlc25a13 knockout mouse model, which recapitulated some, although not all, of the patient phenotype75.Sin et al. differentiated iPSC from a patient with citrin deficiency into hepatocytes. Functional assessment confirmed that patient cells did not exhibit ureagenesis. Genes involved in mitochondrial beta-oxidation were downregulated in the patient cells, with abnormal mitochondrial structure, leading to significantly higher levels of cellular triglycerides and lipid granule levels compared to the wild-type cells. The work was further validated by a complementary iPSCs models present intrinsic limitations, particularly for therapeutic application and clinical translation.76. This has been demonstrated in modelling UCDs, where both wild-type and mutated immature differentiated hepatocytes failed to achieve equivalent functionality with that of in vivo hepatocytes74. This means that results must be interpreted with caution for UCDs and particularly when modelling disease specificities associated with age. Furthermore, differentiation efficiency and reproducibility can vary due to differing protocols, genetic backgrounds, passage number and epigenetic memory79. iPSCs can also acquire mutations that confer a growth advantage, particularly p53 mutations, advocating for thorough genetic characterisation prior to translation to the clinic80. With CRISPR/Cas9 mediated reprogramming, off-target effects that could adversely alter the cell function need to be carefully assessed81. However, efforts have been made to improve identification of off-targets using in vitro assays, including circularisation for in vitro reporting of cleavage effects by sequencing (CIRCLE-seq)82 and selective enrichment and identification of tagged genomic DNA ends by sequencing (SITE-seq)83, as well as improving the computational platforms used to design sgRNAs84. Another approach that can reduce the number of off-target effects is base editing, whereby a catalytically inactive Cas9 is employed together with cytidine deaminases to introduce site specific DNA mutations/corrections without the requirement for a double-strand break85.One of the significant challenges faced by iPSCs research is, despite ongoing improvements in differentiation protocols, the immaturity of iPSC-derived cells, which remain closer to foetal rather than their fully differentiated counterparts86. However, there are steps to overcome this limitation with the use of co-cultures – for instance, co-culturing hepatocytes with endothelial and stromal cells aids maturation of hepatocytes87. In the context of UCDs, urea cycle activity and ammonia metabolism are confined to zone 1 located hepatocytes nearest the portal vein, as part of the regulated compartmentalisation of the hepatic lobule88. However, at present, only partial zonation is able to be achieved, which may influence how well ammonia metabolism can be modelled89. Nevertheless, these co-cultures cannot completely recapture the complexity of the in vivo environment.Another iPSC limitation of use in research is that the cell population produced may not be representative of more complex in vivo architecture and morphology. Differentiation protocols often make a single cell type, whereas in vivo hepatocytes, for instance, are in close proximity to non-parenchymal cells such as stellate cells, Kupffer cells and endothelial cells, facilitating crosstalk between parenchymal and non-parenchymal cells. This heterogeneous population, in addition to nutrients, growth factors and vasculature all work together to support the genetic expression and viability of the hepatocytes90. This is particularly a consideration for UCDs as these diseases have occasionally been associated with hepatocellular carcinoma92. There is a risk of potential tumorigenicity and malignant tumours if transplanted cells are contaminated with undifferentiated iPSCs. However, there are treatments that could potentially treat these teratomas if they occur93. There are also purification methods such as flow cytometry sorting and small chemical molecules that initiate death of undifferentiated iPSCs which could help lower the risk of tumorigenicity. However, the effectiveness of such methods has yet to be fully elucidated94. Autologous iPSC therapies effectively eliminate the potential for immune rejection. The generation of clinical grade autologous iPSC-derived cells entails high production costs associated with a rigorous quality control system. As the generation of the patient-specific tissue of interest can take several months, this timeframe might not be suitable for neonatal-onset UCD patients, which require an urgent liver replacement strategy95. Therefore, an alternative approach is to use allogeneic iPSC-derived cell sources which would enable biobanking a limited number of approved iPSCs from assorted human leukocyte antigen (HLA)-homozygous donors that would match the bulk of the population96. These cells would have all undergone regulatory clearance and have been thoroughly tested meaning that the cells could be more readily used to treat patients who require timely intervention. However, patients would be required to undergo lifelong immunosuppression treatment.A safety issue for clinical translation is the risk of teratoma formation97. Regulatory guidelines such as the US Food and Drug Administration (FDA)98, the European Medicines Agency (EMA)  and the International Society for Stem Cell Research are in place to ensure that patient safety is at the forefront101. Adhering to these guidelines, alongside potential intellectual property and patent considerations, can add substantial time and effort to the setting up and delivery of iPSCs studies in accordance with Good Clinical Practice. Additionally, scalable technology to meet regulatory standards remains a challenge for generating genetically modified iPSCs for cell therapy, as it requires special equipment alongside skilled operators104. Thus, these limitations may prevent potential therapeutic treatments from reaching patients due to prohibitive cost and time requirements.For clinical trial applications, ethical consent should be obtained freely. For children, eligibility should be considered only if there is minimal or low risk with potential for direct benefit, and the procedure is as favourable as available alternatives67 and osteoblasts68 in ASLD. In the future, iPSC differentiation towards other cell types, particularly involved in the specific pathogenesis of the central nervous system, will be of utmost interest.iPSC differentiation provides a unique opportunity to study a genetic disease in a specific genetic and epigenetic background in vitro and screen for therapeutic candidates. iPSCs have improved our knowledge of the pathophysiological mechanisms of several diseases, alongside accelerating progress in our understanding of human physiology at the cellular level. In targeting UCDs, iPSC differentiation into hepatocytes has been the primary focus as the urea cycle is only expressed in totality in this cell type. Optimisation of the hepatocyte differentiation is key in reproducing a defective ureagenesis. Differentiation in additional cell subtypes has enabled to study novel mechanistic approaches and pathophysiology, especially in endothelial cells106. This approach could be translated to drug screening using iPSCs for other compounds. Drug screening via this approach has been tried in different contexts, including using Sendai virus, and would be amenable to discover novel therapies to restore ureagenesis in UCD or to protect the brain in the context of hyperammonaemia108.iPSCs can be used as a platform for drug discovery, testing different pathways of UCDs for potential therapeutic treatment. For example, induced autophagy has recently been shown to increase ureagenesis, via the cell-penetrating autophagy-inducing Tat-Beclin-1 (TB-1) peptide in OTC and ASL deficiencies when tested in an animal model109. In addition, another study has generated an in vitro whole-organ “Bioreactor grown Artificial Liver Model” (BALM) which enables the long-term 3D culture of iPSC-derived hepatocyte-like cells. This model also allows the transduction of adeno-associated viral and lentiviral vectors, meaning this model could provide a more representative preclinical therapy testing environment110. There are also numerous techniques to help identify off-target effects associated with CRISPR/Cas9 editing, such as in vitro and cell-based assays112. These allow off-target effects to be more readily assessed and thus can be taken into account when performing differentiation experiments. Alongside this, the rapid evolution of next-generation sequencing techniques provides means to analyse transcriptomics and chromatin accessibility of both iPSCs and differentiated cell populations. Utilising different multi-omics approaches allows refinement of in-depth analysis and the discovery of novel pathophysiological mechanisms113.Advancements in both iPSCs differentiation protocols and CRISPR/Cas9 techniques are continually emerging. For instance, recently researchers have managed to cultivate four organs in a microbioreactor from iPSCs from a single donor. The organ models were positioned in separate compartments of the bioreactor and connected by a microfluidic networkUCD-derived iPSCs are progressively expanding their applications in several areas, from supporting a genetic diagnosis in the genomic era, studying pathophysiology to drug development and validation. Differentiated iPSCs creating patients’ avatars are of particular interest in proof-of-concept studies where they recapitulate key aspects of the disease in a specific disease-enabling genetic and epigenetic background, allowing novel pathophysiological insights. By providing this invaluable opportunity to analyse a genotype of interest within the genetic background of the patient, this allows functional studies in a physiologically relevant model. iPSCs offer the unique opportunity to implement personalised medicine and perform drug screens. The field still faces several challenges, particularly with optimising iPSCs differentiation and successfully translating proof-of-concept studies to effective long-term treatments whilst minimising the risk of adverse effects. While considerable efforts are in place to overcome these challenges, iPSCs present a promising avenue for UCD research and therapy development.