Among the challenges to the 21st-century health care industry, one that demands special mention is the transport of drugs/active pharmaceutical agents across the blood–brain barrier (BBB). The epithelial-like tight junctions within the brain capillary endothelium hinder the uptake of most pharmaceutical agents. With an aim to understand more deeply the intricacies of cell-penetrating and targeted peptides as a powerful tool for desirable biological activity, we provide a critical review of both CPP and homing/targeted peptides as intracellular drug delivery agents, especially across the blood–brain barrier (BBB). Two main peptides have been discussed to understand intracellular drug delivery; first is the cell-penetrating peptides (CPPs) for the targeted delivery of compounds of interest (primarily peptides and nucleic acids) and second is the family of homing peptides, which specifically targets cells/tissues based on their overexpression of tumour-specific markers and are thus at the heart of cancer research. These small, amphipathic molecules demonstrate specific physical and chemical modifications aimed at increased ease of cellular internalisation. Because only a limited number of drug molecules can bypass the blood–brain barrier by free diffusion, it is essential to explore all aspects of CPPs that can be exploited for crossing this barrier. Considering siRNAs that can be designed against any target RNA, marking such molecules with high therapeutic potential, we present a synopsis of the studies on synthetic siRNA-based therapeutics using CPPs and homing peptides drugs that can emerge as potential drug-delivery systems as an upcoming requirement in the world of pharma- and nutraceuticals. There are considerable breakthrough discoveries on the structure and development of synthetic and natural drugs that have gained importance in science and clinical studies. Often, scientists from around the globe create compounds that possess the potential to revolutionise health sciences. However, not all of them produce significant results when administered. Brain tumours and associated cancers are often fatal to patients because treatment such as chemotherapy is complicated due to extensive intratumoral heterogeneity that renders poor penetration of the drugs through the blood–brain barrier (BBB), causing less bioavailability of drugs and a lack of selective tumour targeting . Thus, tA very superficial drug classification involves two terms in daily use: (i) extracellular and (ii) intracellular drug delivery. The latter has captured the attention of medicine because the introduction of various biomolecules into the cytosol (or a targeted intracellular compartment) is a powerful tool for the manifestation of desired biological activities. For a long time, transport across the lipid bilayer remained the major challenge. Micelles provided a solution to this long-encountered transportation hurdle as they could transport a series of different molecules ranging from hydrophobic drugs and proteins to genes . TranspoDespite the number of advantages that this system possesses, one cannot completely rule out the advantages of the lipid-mediated mechanism, which practically started the science of intracellular molecular delivery . Hence, Additional classes of targeting peptides are the ‘homing peptides’. These classes of peptides have become helpful in cancer research, especially for brain tumours, as they are known to target specific cells/tissues based on their overexpression of tumour-antigens or specific markers . This arTwo decades ago, the concept of peptide-transduction domains (PTDs) emerged with the observation of transcription factors that could move to and from the cell membrane as well as from one cell to another . The 198The Pep- and MPG families of small peptides are instances of such amphipathic cell-penetrating molecules that can form conjugates with proteins and nucleic acids, respectively, and can aid in obtaining the desired results . CPPs caCPPs are designed to successfully deliver macromolecules into the cytosol; thus, they are used as delivery systems rather than therapeutic agents . CPPs maAt physiologically low temperatures, when a positively charged CPP interacts with the negatively charged phospholipid bilayer of a membrane, it may skip adherence to the lipid bilayer and be translocated without the aid of energy . This diCPPs at low levels are classically internalised via endocytic pathways, and the macromolecules used as therapeutic peptides often enter cells and become entrapped inside endosomes. Endosomal escape represents a major hurdle for the usage of CPPs as delivery systems and/or therapeutics ,27. The Cell toxicity caused by strong peptide–lipid interactions may be harmful to the cell but may help the CPP to escape the endosomal membrane. Thus, key properties may be harnessed for the selective benefit of CPPs. Endosomes undergo the high synthesis of bis(monoacylglycerol)phosphate (BMP) in the late phase, which makes them more acidic on maturation. This shift in pH driven by a proton pump facilitates some CPPs to undergo alterations in their three-dimensional structure and helps them to cross endosomal membranes ,31,32.In some studies, it has been shown that a six-polyethylene glycol unit (PEG-P6-GFWFG) TAT used as a CPP, in combination with hydrophobic endosomal escape domains (EEDs), significantly downregulated cellular toxicity while sustaining cell-penetrating capabilities . HistidiIn 2014, Qian et al. proposed that the cyclic peptide cFΦR4, commonly used for stability, could be strategically internalised through endocytosis, and thereafter escape from endosomes . Along sThe CPPs that can successfully overcome endosomal entrapment usually employ the following mechanisms to escape endosomes: (a) Budding: This proposal has recently gained prominence as it is very likely that high concentrations of CPPs can lead to the formation of smaller vesicles by cutting off from the original endosome that readily degrades and releases their content . (b) MemClearly, there is no ‘rule of thumb’ to overcome this challenge of endosomal entrapment. It is an uphill task to achieve a win–win situation of not breaking the cell membrane while breaking free from the endosomal membrane. To achieve endosomal selectivity, several factors that affect cellular uptake and translocation across the plasma membrane should be employed. The uptake of cargo depends first on the composition of the lipid and protein content of the plasma membrane and second on the concentration and physiochemical properties of the peptide and its cargo. CPPs with a high positive charge, e.g., more arginine domain with the guanidinium group as well as amphipathic peptides, are better suited for direct cellular uptake than endocytosis. Generally, at high concentrations, direct transportation occurs by temporarily destabilising the plasma membrane and, at low concentrations, endocytosis is observed. Transportan, a primary amphipathic CPP, and arginine-rich CPPs at low concentrations are mainly endocytosed, while rapid cytoplasm entry occurs at higher concentrations. Similarly, at higher concentrations, CPPs like R8, R9, and TAT are taken up via vesicular structures like clathrin or endosomes and, at low concentrations, uptake mainly occurs via nonendocytic nucleation zones or direct transportation across the plasma membrane. But there are many alterations to these observations. Penetratin at low concentrations leads to direct translocation, while at high quantities, endocytosis prevails . Thus, CA recent paper by Nadal-Bufí et al. presented the strategies that form the basis of future directions towards disabling the endosomal entrapment of CPPs and releasing therapeutic peptides to their targeted site . First, Targeting peptides face another problem of proteolytic degradation within the cellular compartments. Nanomaterial-based drug delivery systems have been proven to cross the BBB either by carrier-mediated transcytosis (CMT), adsorptive-mediated transcytosis (AMT), or receptor-mediated transcytosis (RMT) and have often offered protection to proteolysis. Lipid-based nanoparticles (NPs) with surface modifications via transferrin, lactoferrin, glucose, and glutathione polyethylene (PEG) are more effective in BBB permeability. PEGylation of gold and silica NPs have also been shown to increase biocompatibility. Polymeric-based NPs such as chitosan, hydroxyl polyamidoamine (PAMAM), and poly  (PLGA) have better physical and chemical properties and are highly resistant to degradation .The modification of peptide sequences including amino acid incorporation within the backbone or the non-canonical side chains, enantio/retro-enantio isomerisation, and the cyclisation of N and C-termini further enhances protease resistance. Peptides modified with such changes are termed ‘peptidomimetics’. N and C termini modifications prevent exoprotease-mediated hydrolysis. Backbone changes like isosteric replacement of amide bonds, carbon skeleton extension or amide alkylation, N-methylation and α-methylation, or the addition of β or γ amino acid residues impart protection from endoproteases. These changes increase lipophilicity by reducing hydrogen bond formation, thereby enabling peptides to cross biological barriers. The D-enantiomeric amino acid is usually a ‘retro-enantio isomer’ that displays side chain topology like that of its native L-form with inverted amide bonds. Such retro-enantio isomers have reduced immunogenicity and are resistant to proteolytic degradation. Cyclical peptides have better biological activity than linear peptides as the cyclical configuration is mostly favoured for high peptide affinity due to better binding of the target protein .One class of tumour-homing peptides (THPs) includes 3–15 residues and long, receptor-specific peptide molecules wherein the target receptors can vary from intracellular to cell-surface bound receptor molecules . THPs caUnlike the CPPs that can be internalised by diverse cell lineages, THPs show receptor-mediated, endocytic cellular internalisation and are hence increasingly relevant for lineage- or tumour-specific drug targeting . EvidentThe brain, like all other vital organs of the body, needs nutrients and gases to function properly. It is substantially protected by three coatings of meninges protecting the BBB from overexposure to potassium, glutamate, and glycine, which, at increased concentrations, can be neurotoxic . Armed wSeveral ways of transport are known that enable drug molecules, lipid-soluble small molecules, weak bases, and electrically neutral solutes to diffuse passively across the BBB, as shown in  65,66].,66.65,66CPPs are the peptide-based drug delivery system that holds promising and attainable prospects to deliver drugs to the brain. These small synthetic peptide shuttles  enable the influx of a varied range of small molecules across the BBB. These natural peptides are derived from various sources, namely, HIV proteins , the rabies virus (RVG-29), phage receptors , venom neurotoxins , and neurotropic endogenous peptides . Incidentally, although these compounds are highly pathogenic or toxic, they are reported to be non-toxic to neuronal cells .Despite the well-studied ability of CPPs to enter mammalian cells, it is only a few fragmentary studies that mention their transcellular aspects . A studyMost of the earlier known CPPs are either covalent or non-covalent peptide-based delivery systems. Carrier peptides have many limitations: (i) lack of biocompatibility and bioavailability; (ii) may be toxic and antigenic; (iii) lack chemical fixation; (iv) may lose specificity to the target site; and (v) may get degraded by endosomes or proteasomes. In this context, MPG and Pep families of cell-penetrating small peptides have been successfully applied to the delivery of different cargoes (siRNA and peptides) both in vitro and in vivo, especially delivering therapeutics across the BBB. Listed are those CPPs that can act as a substitute for a covalent and non-covalent strategy for the delivery of drugs across the BBB.Numerous novel shuttle peptides have been explored but efficient transport to the brain must be improvised, and researchers are still seeking the perfect approach to allow drugs to pass through the BBB. Lipoproteins seem to possess a significant ability as delivery systems to cross the BBB; for example, apolipoprotein B (ApoB), and apolipoprotein E (ApoE) equivalents were found to infiltrate the BBB ,78. Analvirus-derived peptides have revolutionised targeted drug delivery across the BBB. A rabies virus glycoprotein (RVG-29) readily docks to the nicotinic acetylcholine (nAChR) receptor located on the endothelial cell lining and neuronal cells, thus facilitating its penetration across the BBB  imidazolinium chloride bound to the peptide moiety nAChRs penetrated the BBB to deliver siRNA into FVB mouse brains  with lipt growth .PAMAM–PEG–angiopep/DNA NPs are dendrimer nanoparticles that were combined with apolipoprotein A-I (ApoA-I) and NL4-peptide, shown to be efficient carriers across the BBB. PAMAM is a surface primary amino group and has shown clathrin- and caveolae-mediated endocytosis of the nanocarriers comprising angiopep peptides. Intravenous injection of dendrimer nanoparticles of PAMAM–PEG–angiopep loaded with pEGFP plasmid was given to mice. Compared to the control groups of PAMAM/DNA NPs, gene expression was observed in all four regions of the mouse brains for the PAMAM–PEG–angiopep/DNA NPs, although cationic dendrimers showed haemolytic activity and cell cytotoxicity . A combiPeptide-based drug delivery systems across the BBB have pros and cons such as low alteration in the BBB integrity, specific targeting and reduced toxicity, and some concerns associated with serum stability. NPs, shuttle peptides, liposomes, exosomes, and dendrimers conjugated with CPPs have shown much-enhanced permeability across the BBB. Although advances have been achieved with CPPs to cross the BBB, it has been shown that in many cases, CPPs selectively cross the BBB, which does not qualify the peptides as having effective BBB-penetrating ability. The differential influx property exhibited by CPPs can be attributed to their cationic nature (presence or absence of arginine residues), physicochemical properties (secondary structure at the membrane interface), and biological properties (cellular uptake ability) . The areBrain tumours and cancers are often fatal to patients because treatments such as chemotherapy suffer from poor bioavailability and reduced permeability across the BBB coupled with extensive intratumoral heterogeneity and a lack of selective tumour targeting. Peptide–drug conjugates (PDCs) are designed to link targeted peptides via a chemical linker to a therapeutic payload that can mimic an alternate antibody–drug conjugate (ADC) and expand the therapeutic potential of various drugs . In the BACE1) siRNA complexes are successfully delivered to neurons and microglial cells. Therefore, NLCs act as safe multifunctional nanocarriers with a wide receptor repertoire of the display peptide, which can effectively overcome the blood–brain barrier without toxicity and inflammation [Brain-homing peptide (BH) CNAFTPD is used to enhance the transfection efficacy of pDNA delivery across the BBB by forming biodegradable core–shell polyplexes with peptide–PEG–tris-acridine conjugates (pPAC) . Similarammation . Recentlammation .In glioblastoma (GBM), a debilitating brain tumour disease, a small, soluble peptide (BTP-7) covalently attaches to an insoluble anti-cancer drug, camptothecin (CPT), targeting the human GBM extracellular matrix (ECM) across the BBB . GliomasIn the early 1990s, Napoli and Jorgensen reported posttranscriptional gene silencing in plants by RNA interference (RNAi). RNAi cleaves double-stranded or short hairpin RNA (shRNA) into functional, small interfering RNA (siRNA) with the aid of ‘dicer’, an endogenous mammalian protein complex that mediates RNAi. siRNAs are usually short (20–25 bp), exogenous, double-stranded RNA molecules that silence gene expression either by inhibiting transcription or by the degradation of sequence-specific target mRNA . Since sCPP covalently conjugated to the siRNA (CPP-siRNA) delivery system. This method prevents separation among the CPP and its conjugate cargo both in vivo and/or in vitro. In this method, strong binding is attained between the CPP and its cargo via a cleavable linker such as the disulfide linkage, which leads to its lower molar ratio and low toxicity. This allows the components of the conjugate to be separated only in the reducing environment of the cytoplasm, e.g., by glutathione, thus avoiding its localisation in the nucleus , which are designed for an optimal balance between the peptide and siRNA. The stability of the complex depends upon the structure of the CPP as it must avoid charge neutralisation, thus preserving an overall positive charge [The second strategy is the formation of e charge . In thise charge . SpecifiA short peptide derived from rabies virus glycoprotein (RVG) and conjugated with nona-arginine peptide (RVG-9R) could bind to the acetylcholine receptor expressed by neuronal cells, resulting in specific gene silencing within the brain . A widesCPP-decorated multifunctional nano-complexes, which use amalgamations of CPPs and other carriers for siRNA delivery. These multifunctional nano-complexes possess the characteristics of multiple compounds and have a better half-life in the bloodstream. Polyethylenimine (PEI) moiety tagged with a micelle-like nanoparticle (MNP) was replaced with nona-arginine (R9), forming lipid–peptide hybrid nanoparticles (hNPs) that could readily form complexes and transfer oligonucleotides. In addition, the hNPs modified with Tat 48–60 (T-hNP) were shown to improve cellular transfection. This formulation has been reported with a better gene-silencing effect in vivo as it readily accumulates in brain tumour tissue [The third strategy is using r tissue . A stearr tissue . It was r tissue . Anotherr tissue . To facir tissue .Additional challenges are posed to therapeutics targeted to specific cell types of CNS such as astrocytes, neurons, or glia cells to cross the blood–brain barrier (BBB). Hence, early attempts to deliver RNAi in the CNS were performed by intracranial injections of lentiviral vectors encoding shRNA, but the method showed diminished efficacy. Lentiviral vectors crossed the BBB poorly. The inadequate neuro-invasion led to decreased and localised hRNA expression only around the injection site. This method also lacked temporal control and caused limited knockdown of protein expression. Another major concern regarding lentiviral vector technology and construction is its capacity to turn brain cells cancerous ,145. AnoCationic small-cell-penetrating peptides capable of crossing plasma membranes were the alternate nonviral strategy used for delivering siRNA molecules to the CNS ,148. TheAlternatively, more direct routes to the CNS should be explored and, in this regard, liposomes are extensively used as delivery vehicles for siRNA. Improved delivery of the liposome-encapsulated siRNA was observed with minimal siRNA degradation within the blood . CationiSystemic transit through the vasculature of the CNS may lead therapeutic drugs to encounter immune cells, causing hypersensitivity or clearance from the system. An alternate method of administering drugs or stem cells into the nasal cavity can shorten the route across the BBB ,157,158.Viral vectors or liposomes fail to deliver specifically and safely into the brain as their approaches are invasive and hindered by the immune system. Liposomes lack a long-term treatment method as they tend to elicit immune responses followed by subsequent clearance, while the use of a viral vector requires stereotaxic surgery, and the delivery is restricted to specific brain regions. These disadvantages can be overcome by the intravenous (iv) injection of modified exosomes for nucleic acid delivery into the brain. Exosomes are nanosized extracellular vesicles (30–100 nm in diameter) formed by an endocytic pathway . ExosomeAlthough the abovementioned siRNA delivery strategies have proven to be successful, some of the basic guidelines will become clearer after elaborating on the in vivo studies and clinical trials . In the Diseases of the central nervous system (CNS) are the most difficult to treat, mainly because of the obstacle of the blood–brain barrier (BBB). A vast majority of drugs fail to reach the brain because of their inability to cross the BBB. Undoubtedly, the most promising studies are those that unravel strategies to deliver CNS-active drugs and peptides targeted at the BBB. Small peptides as nanoparticles or nanocarriers can be conjugated to drugs to either form a steady link or act as pro-drugs. The last decade witnessed a plethora of uses of both small molecules and proteins in diverse therapeutic areas such as diagnostics, brain cancers, and neurodegenerative disorders. Peptides can be generally classified as ‘receptor-targeted’ , recognising membrane proteins expressed by the BBB microvessels; ‘cell-penetrating peptides’ , undergoing transcytosis through unspecific mechanisms; or ‘homing peptides’  NHQQQNPHQPPM; brain-homing peptides CNAFTPDY, CLEVSRKNC, and CLSSRLDAC), used for DNA delivery. RNA interference (RNAi) conjugated to CPPs can become a new tool to target defective genes in the brain by inducing gene silencing and has enormous clinical potential for the treatment of various neurodegenerative disorders. Most RNAi trials use small interfering RNAs (siRNAs) but, despite the enormous potential of RNAi, only 24 clinical trials have applied siRNA-CPP-mediated therapy to date. Notwithstanding their great therapeutic potential, the successful implementation of siRNAs in vivo is hampered by the low bioavailability of these hydrophilic compounds and their inability to cross the BBB . We hopeIn theory, both CPPs and homing peptides can be exploited to transform a large range of pharmaceuticals from paper to practice, but there exists a hindrance when it comes to replicating the in vitro results within physiological systems. Undeniably, despite having a generally good safety profile, some peptide conjugates may display toxicological features, causing antigenicity, cardiovascular alterations, or hemolysis. Furthermore, the life span of these peptides largely depends on their dodging capability in the presence of peptidases, endosomal/lysosomal degradation, and endosomal entrapment, making them unavailable to the target site .Despite numerous data pertaining to the basics and applications of CPPs, the efficacy of such conjugations is still debatable, primarily when it comes to the blood–brain barrier. Endogenous analysis of CPP-mediated drug targeting in cell cultures does not necessarily indicate their behaviour at the organismal level. As discussed in the text, mere penetration inside cells does not mean that a given CPP can traverse the blood–brain barrier. Such limitations necessitate further in-vivo analysis of multiple CPPs as well as additional penetrating peptides and expand the existing database. The study of CPPs should also consider the differential expression of transporter proteins, primarily solute carriers on cell surfaces since the expression levels of these transmembrane proteins are strongly associated with various normal and anomalous conditions. Certain biologically active chemical compounds might also be studied for their cell-penetration and transcellular traversing activities so that the additional conjugation step can be eliminated.Even though modern-day medicine has tapped the potential of many physiological loopholes to the advantage of humankind, a broad area remains unexplored. A significant proportion of our physiology and metabolism is influenced by the dark matter in our system. Despite null results, unknown interactions can also lead to negative results, which are commonly referred to as side effects. For most pharmaceuticals, these side effects exceed what has been documented or studied. One way to minimise these undocumented results is to take the dark matter into consideration. This is possible owing to the CPP/homing peptide specificity. However, despite this specificity, drugs can frequently target more than one macromolecule in an organism. Additionally, drug-bound CPPs can exhibit altered behaviours that further interfere with their normal roles in biological systems, specifically, brain activity. This can be negated by using pre-conjugated drug–CPP or drug–homing peptide combinations to minimise unwanted interactions within physiological systems. The careful optimisation of this and additional techniques can aid in moving many potential pharmaceuticals from texts to tables. An underestimated yet large number of efficient drugs fail to reach the counters owing to the lack of a delivery mechanism. Potent drugs (biomolecules and nanoparticles) that can be used to cure fatal disorders of the CNS, such as Parkinson’s and Alzheimer’s disease, or treat various brain tumours, do not exhibit any significant results if not coupled with an adequate transport and translocation system. Thus, a detailed analysis of both the available and the potential drug delivery systems that can effectively cross the blood–brain barrier has emerged as a critical requirement in the world of pharma- and nutraceuticals.


CNS metastases are often terminal for cancer patients and occur at an approximately 10-fold higher rate than primary CNS tumors. The incidence of these tumors is approximately 70,000–400,000 cases annually in the US. Advances that have occurred over the past two decades have led to more personalized treatment approaches. Newer surgical and radiation techniques, as well as targeted and immune therapies, have enanled patient to live longer, thus increasing the risk for the development of CNS, brain, and leptomeningeal metastases (BM and LM). Patients who develop CNS metastases have often been heavily treated, and options for future treatment could best be addressed by multidisciplinary teams. Studies have indicated that patients with brain metastases have improved survival outcomes when cared for in high-volume academic institutions using multidisciplinary teams. This manuscript discusses a multidisciplinary approach for both parenchymal brain metastases as well as leptomeningeal metastases implemented in three academic institutions. Additionally, with the increasing development of healthcare systems, we discuss optimizing the management of CNS metastases across healthcare systems and integrating basic and translational science into our clinical care to further improve outcomes. This paper summarizes the existing therapeutic approaches to the treatment of BM and LM and discusses novel and emerging approaches to optimizing access to neuro-oncologic care while simultaneously integrating multidisciplinary teams in the care of patients with BM and LM. The treatment of brain metastases (BM) and leptomeningeal metastases (LM) includes a combination of surgical intervention, radiation therapy, systemically administered therapies, and therapies directly administered to the CNS.Surgical interventions for BM include craniotomy and emerging modalities such as laser interstitial thermal therapy (LITT), which are often utilized with large tumor metastases that produce neurologic symptoms and increase intracranial pressure; for LM, options are more limited to the placement of intraventricular reservoirs for intrathecal chemotherapy or shunt placement for communicating hydrocephalus.Radiation therapies for BM include whole-brain radiation therapy (WBRT) and stereotactic radiosurgery (SRS), of which neurocognitive toxicity remains a pressing concern; for LM, options include WBRT and craniospinal irradiation (CSI).Systemically administered therapies for BM include tyrosine kinase inhibitors (TKI), such as osimertinib and tucatinib, and immune checkpoint inhibitors (ICI), such as cytotoxic T-lymphocyte-associated antigen 4 (CTLA4) inhibitors and programmed death 1 (PD1) pathway inhibitors; for LM, there is not a standard-of-care systemic therapy, with overall prognosis remaining poor.Intrathecal (or intra-CSF) chemotherapy is an ongoing area of study for the treatment of LM, with novel therapies demonstrating some promise.Hospital-system-wide tumor boards, also known as multidisciplinary cancer meetings (MCM), are emerging settings through which providers representing multidisciplinary neuro-oncology subspecialties meet to discuss complex cases and facilitate information sharing for the improvement of patient outcomes. These tumor boards may be in person or virtual, which remains an ongoing area of interest.Telemedicine has been a growing technology since COVID-19, with widespread potential for improving access and quality of care in the field of neuro-oncology.In this review, we discuss the clinical management of brain metastases (BM) and leptomeningeal metastases (LM), which are diseases managed by a multidisciplinary team of medical oncologists in conjunction with neuro-oncologists, radiation oncologists, and neurosurgeons . There rThe various therapeutic modalities chosen and delivered by assorted sub-specialists in the care of patients with BM and LM will be briefly reviewed. These interventions include surgical intervention, radiation therapy, systemically administered therapies, and therapies directly administered to the CNS. How individual physicians can use these therapeutic modalities within a team model and in the context of a multidisciplinary care plan developed across a healthcare system will be highlighted. Finally, the integration of basic and translational science discoveries into the clinical management of BM and LM points toward future areas of focus to continue to benefit this complex oncology patient population.The exact incidence of BM from solid tumors is not as certain as that of primary CNS tumors. It is thought there are ~70,000–400,000 new cases in the United States per annum. If correct, this makes them ~10-fold more common than primary CNS malignancies ,9. LogisUntil recently, BM was viewed as being associated with a universally dismal prognosis . We now p < 0.0009) [Surgical resection has demonstrated efficacy in prolonging survival in patients with solitary BM. The role of surgical resection is predominantly in patients with large BM that produce either neurologic symptoms or those associated with increased intracranial pressure due to mass effect. It also serves a diagnostic role, including when a patient may benefit from more detailed molecular characterization of their BM. The role of surgical resection in oligometastatic disease is less clear, although it can be considered when there is a dominant symptomatic BM in the setting of other smaller lesions. One key study was the pivotal phase 3 randomized controlled trial comparing the effectiveness of surgical resection plus post-operative whole-brain radiotherapy (WBRT) vs. WBRT alone. This trial demonstrated that surgical resection followed by radiotherapy was superior to WBRT for the treatment of a solitary BM. The median overall survival (OS) was over 15 months in the surgical group vs. over 6 months in the RT-only group ( 0.0009) . SuperioMRI-guided laser interstitial thermal therapy (LITT) is a minimally invasive surgical alternative for metastases up to 3 cm in diameter in locations where surgical resection may not be readily feasible . The tecRadiation therapy (RT) has formed the backbone of the treatment of BM. The most common approaches, WBRT and stereotactic radiosurgery (SRS), have different benefits in terms of acute and late toxicity, ease of implementation, and treatment duration, without clear differences in survival from CNS failure in oligometastatic disease (OMD). OMD has been defined as up to five metastatic lesions . ProphylWBRT has the advantages of ubiquity in being able to be implemented quickly, its ability to be administered across healthcare systems with varying resources, and the ability to treat both macro-  and micrometastatic CNS disease. A key limitation of WBRT is the neurocognitive toxicity from irradiating healthy brain tissue unaffected by tumors and the resulting deterioration of patient independence and quality of life (QOL) . In contThere have been efforts to avoid the neurocognitive toxicity of WBRT . Two recThere has been continual work on optimizing the role of SRS for BM. SRS has been shown to be both safe and efficacious for treating several “oligo” BM. Absolute tumor number, cumulative tumor volume, and tumor location are all factors that may influence the efficacy, safety, and tolerability of SRS for BM, making the discussion more complex than simply defining an optimal numeric cut-off for the number of brain metastases. In patients with 1–3 brain metastases, SRS has already replaced WBRT as the standard of care . A more For large BM and limited intracranial disease burden, post-operative SRS to the surgical resection cavity may be employed as a component of multimodal therapy . Over tiTraditionally, systemic chemotherapy had a limited role in the management of BM due to the CNS penetration of available agents, yet growing evidence suggests that systemically effective therapies of newer small molecules are also demonstrating responses in the CNS. The intracranial failure rate has been reduced in randomized trials, likely impacted by the low CNS penetration of the agents used. A wide range of therapeutic agents are now considered in the management of BM. The most frequently used agents are systemic targeted therapies ) and immune checkpoint inhibitors (ICI).BRAF V600E mutation. Retrospective cohort data have demonstrated the efficacy and acceptable safety profiles of another TKI, cabozantinib, for the treatment of renal cell carcinoma (RCC) BM [One example within the targeted therapy class of systemic therapy is the TKI osimertinib, a third-generation agent for the treatment of epidermal growth factor receptor (EGFR) mutant non-small cell lung cancer (NSCLC). Osimertinib has demonstrated efficacy in the treatment of T790M-positive advanced NSCLC BM . Another(RCC) BM .Drugs within the ICI class of systemic therapies used for the treatment of BM often target the cytotoxic T-lymphocyte-associated antigen 4 (CTLA4) and programmed death 1 (PD1) pathways. Theoretical benefits of ICI therapies include their lower toxicity profiles compared to traditional chemotherapeutic agents as well as their potential for efficacy both outside of and within the CNS . CombinaLeptomeningeal spread is increasingly observed in advanced metastatic cancer, with a relative increase as systemic treatments and disease control have substantially improved over the last decade. LM occurs in approximately 5% of advanced solid tumors, yet autopsy series reveal a prevalence of up to 20% for asymptomatic or undiagnosed LM ,44. The Recently, progress has been made in the understanding of patients at risk for and the pathogenesis of LM ,52,53. TThe role of surgical intervention in LM is limited to the placement of an intraventricular reservoir  to facilitate the delivery of intrathecal chemotherapy or the placement of a shunt in patients with communicating hydrocephalus due to flow obstruction by metastatic cells in the cerebrospinal fluid ,58. One Radiation has been used to treat LM, although practice patterns vary between institutions . DiffereThe benefits of CSI have traditionally been limited by significant potential CNS and extra-CNS toxicity. However, in comparison to standard photon radiation techniques, the application of protons for CSI may be a means to overcome some of the toxicities, particularly myelosuppression and radiation esophagitis. Proton RT is a type of radiation treatment . A recenp = 0.003) [The role of systemic therapies in the treatment of LM is evolving. One study compared the efficacy of systemically administered high-dose methotrexate (HD-MTX) with IT MTX for patients with LM, finding that systemic MTX may be superior for the cytologic clearing of CSF and for OS in patients  . However= 0.003) .Following up on prior data demonstrating the benefit of combination therapy with tucatinib and capecitabine plus trastuzumab for parenchymal BM, a randomized phase II study is currently ongoing to further explore this treatment combination in LM (NCT03501979). Furthermore, a phase II trial of ANG1005, a novel taxane derivative designed to penetrate the blood–brain and blood–CSF barriers more effectively, showed that 79% of patients with LM had intracranial disease control with a median OS of 8 months . DespiteThe direct administration of antineoplastic agents into the CSF is a means to circumvent the blood–CSF barrier . TherapeIntrathecal (or intra-CSF) chemotherapy may be delivered via surgically implanted ventricular Ommaya reservoirs or via lumbar puncture . The theOf particular interest has been the use of antibodies for specific targets, such as HER2(+). These have proven to be very well tolerated and associated with favorable survival outcomes ,75. One The integrated, interdisciplinary team management of BM and LM necessitates seamless cross-coordination between different subspecialty providers. One such platform that has enabled this includes hospital-system-wide tumor boards, also known as multidisciplinary cancer meetings (MCM), specifically directed for CNS metastases. These can bring together experts from medical oncology, neurosurgery, neuro-oncology, radiation oncology, neuroradiology, neuropathology, epilepsy neurology, and cancer genetic counseling to review complex cases.In oncology, tumor boards have been established as a means of improving accuracy in diagnosis, bettering adherence to clinical guidelines, advancing the integration of novel research and clinical trials in clinical management, and improving patient outcomes . Prior sSince the COVID-19 pandemic, telemedicine has rapidly expanded across the globe to facilitate both more seamless interdisciplinary care and increased access to oncologic care, particularly within neuro-oncology . One revFurthermore, the ease of telemedicine platforms in enabling multidisciplinary discussions regardless of geographic location may help facilitate virtual tumor boards in the future . RecentlThe molecular pathological characterization of CNS tumors is becoming increasingly important in diagnosis and clinical management . PracticThe management of CNS metastases is an important component of oncologic care. It is optimal if this is integrated into the other aspects of the care of this patient population. This may be complex as some patients benefit from surgical, radiation, systemic, and/or CNS-delivered therapies delivered by a panoply of subspecialty providers. There are numerous models on how to best accomplish this high-quality multi-disciplinary care, with no single approach demonstrating definitive superiority over others. Considerations for implementation include resources, including sub-specialty clinical care providers with adequate availability and interest. We present a broad overview of how this care can be delivered.


Urology has always been at the forefront of surgical innovation which aims to improve patients outcomes and cost-effectiveness. This Special Issue of the Journal of Clinical Medicine aimed to publish contributions from distinguished authors who actively experience innovations in the field of urological surgery and aim to provide more solid scientific evidence.Certainly, the most promising field when looking at surgical innovation is robotic surgery with its numerous advantages, including low invasiveness, higher precision, a lower complication rate, and fast postoperative recovery.Regarding oncological robot-assisted surgery, Gandi C. and colleagues published a study comparing robot-assisted partial nephrectomy to the open technique in the scenario of an off-clamp approach, using propensity score matching and a new pentafecta outcome . No studThe advantages of robot-assisted surgery have also been reported in the field of radical cystectomy; however, published data are still limited. An evaluation of the learning curve of surgical procedures is of outmost importance, particularly for very complex procedures, such as radical cystectomy with intracorporal neobladder. Lombardo R. and colleagues aimed to assess the learning curve of this challenging procedure using a standardized learning curve assessment method (CUSUM method) and multiple outcomes in a single surgeon experience . While 2Robotic surgery is increasingly adopted to perform procedures which aim to treat benign diseases as well. This is the case of pyeloplasty. The surgical treatment of ureteropelvic junction obstruction has historically been performed with open dismembered pyeloplasty, according to the Anderson–Hynes technique, with a very high success rate. Minimally invasive approaches, and robotic surgery, in particular, offer several advantages. However, published data comparing the robotic versus the open approach are limited. Moretto S. and colleagues published a retrospective comparison between these two approaches evaluating multiple outcomes . While nRobot-assisted laparoscopic pyeloplasty is increasingly adopted for hydronephrosis in children as well, and Pakkasjärvi N. and colleagues sought to systematically summarize the published data on pediatric robot-assisted pyeloplasty to decipher the learning process by analyzing learning curves . They unWith an increasingly ageing population in western countries, research on surgical outcomes in elderly patients is of great interest. To address the research gap regarding the complications and impact on patients’ quality of life associated with different urinary diversions after radical cystectomy in older patients, Fuschi A. and colleagues compared perioperative outcomes in two groups of patients aged > 75 years with a high comorbidity state who underwent minimally invasive (laparoscopic or robot-assisted approach) radical cystectomy with an intracorporal ileal conduit (Bricker) or single-stoma ureterocutaneostomy [In recent years, the surgical treatment of benign prostatic hypertrophy (BPH) has been developed by a multitude of endoscopic and minimally invasive surgical techniques. In particular, to decrease the morbidity associated with the standard approach , a variety of lasers and laser-based procedures have been introduced, such as the thulium laser enucleation of the prostate (ThuLEP). Furthermore, several new procedures aim to preserve the ejaculatory function In this context, Trama F. and colleagues  performeIn the context of BPH laser surgery, Chen S-L and colleagues  performeIn conclusion, this Special Issue provides important new evidence in the field of urological surgery, including both endoscopic and minimally invasive procedures. The included publications represent a very valuable contribution to fill the gaps in knowledge on several aspects of urology. We hope that the results provided by the contributing authors may serve as an important resource for researchers and may encourage future studies.


Although pseudoporphyria closely resembles porphyria cutanea tarda (PCT) from a clinical and histologic standpoint, there are characteristically no laboratory abnormalities.Clinical history-taking is essential because patients with pseudoporphyria will often have a history of chronic renal disease, indoor tanning bed use, or recent exposure to phototoxic medications.A 71-year-old Filipino woman with ovarian cancer and a long-standing history of psoriasis treated with topical medications presented with a 1-year history of a pruritic, blistering rash that began shortly after beginning olaparib for the treatment of her ovarian cancer. The rash occurred intermittently on her face, chest, upper extremities, and lower extremities, with lesions beginning as erythematous macules that transformed into vesicles and eventually became firm papules. Each lesion cycle typically lasted one week before resolving. She reported minor itch relief with application of her psoriasis medications—clobetasol ointment 0.05% and tacrolimus ointment 0.1%, applied twice daily as needed. She denied any inciting factors or triggers with exception of starting olaparib therapy several weeks prior to the rash developing.Physical examination revealed scattered erythematous macules and firm papules present on the nose, right ankle, right dorsal aspect of the foot, and bilateral lower extremities. One erythematous vesicle was present on the left medial shin . PsoriatA, B) were consistent with a subepidermal bullous disorder, showing a subepidermal blister with sparse mononuclear cells and granulocytes, in addition to “caterpillar bodies” adherent to the undersurface of the epidermis. DIF test findings were positive for homogenous deposition of IgG and IgA in thick-walled blood vessels , all of which suggested a diagnosis of PCT vs pseudoporphyria.Results of the punch biopsy A, B were vessels A, B, allFurther workup to determine the diagnosis revealed total plasma porphyrin level within normal limits. Given the patient’s clinical presentation, histologic findings, and normal laboratory results, a diagnosis of pseudoporphyria was made. The temporal association of beginning olaparib therapy and development of the rash suggests drug-induced pseudoporphyria as the most likely explanation for this patient’s presentation. To further support this conclusion, the patient’s rash persisted for the duration of therapy with olaparib and resolved shortly after discontinuing the medication.Although considered rare in the United States, porphyria constitutes a group of 9 disorders characterized by abnormal functioning of the heme biosynthesis pathway. Each of these diseases involves a unique enzymatic abnormality that ultimately leads to the accumulation of porphyrins and other precursors of heme in the bloodstream. The accumulation of these chemicals leads to acute neurologic manifestations as seen in acute intermittent porphyria or phototoxicity as seen in PCT. In PCT, phototoxicity occurs due to the accumulation of porphyrin in the skin or dermal blood vessels and is characterized by the formation of vesicles or bullae on photoexposed skin.When a patient presents with clinical and histologic features consistent with PCT but without accompanying laboratory abnormalities, a diagnosis of pseudoporphyria is made. Histologically, the findings in pseudoporphyria are very similar to PCT and show a subepidermal blister with sparse inflammatory infiltrate. “Caterpillar bodies,” seen microscopically as linear eosinophilic globules comprising necrotic keratinocytes embedded in a thickened basement membrane zone adherent to the undersurface of the detached epidermis, are a characteristic finding in PCT. Results of DIF testing are similar in both conditions, with deposition of complement and immunoglobulins, particularly IgG, surrounding blood vessels.Because of the major overlap in presentation, differentiating between PCT and pseudoporphyria relies largely on laboratory testing and clinical history. Total plasma porphyrin levels will classically be elevated in patients with PCT, whereas plasma porphyrin abnormalities will be absent in pseudoporphyria. Although previous literature has documented pseudoporphyria in association with nonsteroidal anti-inflammatory drugs, antibiotics, diuretics, retinoids, and various systemic medications, a case of olaparib-associated pseudoporphyria is exceedingly rare.Olaparib is an oral PARP enzyme inhibitor that induces cell death in breast cancer gene (BRCA1/2) deficient tumor cells through the formation of double-stranded DNA breaks. This drug is indicated for use in patients with advanced ovarian cancer and certain BRCA-mutated ovarian cancers. Common adverse reactions of olaparib and other PARP inhibitors include hematologic toxicities, gastrointestinal adverse events, fatigue, and renal toxicity.This case shows the potential for phototoxic effects when taking olaparib and highlights the importance of discussing this potential adverse event with patients before initiating therapy. In addition, it is imperative for clinicians to counsel patients on the importance of sun protection with sunscreen, hats, and sun-protective clothing when taking these drugs.None.


Age-related neurodegenerative diseases, including Alzheimer’s disease (AD), Parkinson’s disease (PD), and amyotrophic lateral sclerosis (ALS), continue to be formidable challenges to the global medical community. As the global population ages, the prevalence of these diseases is also expected to increase. One of the most significant challenges in the fight against these diseases is the lack of effective cell-based models that can accurately recapitulate the complex pathophysiology observed in human patients. However, with recent advancements in stem cell technology, particularly human pluripotent stem cells (hPSCs), there is a beacon of hope.This Research Topic, titled “Cell-Based Neurodegenerative Disease Modeling,” brings together an array of studies that showcases the current strides in developing, improving, and validating cell-based neurodegenerative disease models. This is a collation of pioneering research articles and reviews from experts in the field, providing insights, methodologies, and challenges pertinent to the cellular models of these debilitating diseases.Lin et al. have investigated the potential of cellular therapy for ALS, a disease with unclear pathology and ineffective therapeutic modalities. They emphasized the immunomodulatory effects and potential protective measures of cellular therapy for motor neuron circuits in ALS while highlighting the ongoing debates on the safety and efficacy of such interventions.In a comprehensive review, Natalwala et al. have reported the development of an isogenic Research Topic method of pluripotent stem cell lines with elevated α-synuclein expression. Their findings reinforce the capability of pluripotent stem cell lines, even with increased α-synuclein levels, to efficiently differentiate into dopaminergic and cortical neurons. Such models have become invaluable tools for studying PD-associated early molecular changes.Further advancing our understanding of PD, a Lewy body disease, Sibuea et al. have studied the complex territory of maturation in stem cell-derived dopaminergic neuron. They have meticulously investigated the role of culture additives commonly used in maturation, like dbcAMP and TGFβ3, shedding light on their nuanced impact on the phenotype of midbrain dopaminergic neurons, thereby offering a fresh perspective on the current protocols.Yokota et al. have employed tyrosine hydroxylase reporter iPSC lines to elucidate the aberrations in ERMCS and mitochondrial Ca2+ flux in PRKN-mutant patient dopaminergic neurons. These findings emphasize the significance of studying the ERMCS to enhance our understanding of dopaminergic neuronal degeneration in specific patient populations.Drawing attention to the critical role of endoplasmic reticulum–mitochondrial contact sites (ERMCS) in neurodegenerative diseases, Moon et al. have holistically reviewed the intricate challenges associated with cell therapy for PD using hPSCs. By focusing on in vivo differentiation protocols and transplantation results, they underscored the potential of hPSC-derived ventral midbrain dopaminergic progenitors for PD treatment, while simultaneously focusing on current hurdles, such as lineage identification.This Research Topic of articles emphasizes the critical importance and urgency of refining and developing cell-based models, particularly those based on hPSCs, to emulate human neurodegenerative diseases. These models, as shown in this Research Topic, not only provide invaluable insights into disease pathogenesis, but also present an avenue for testing potential therapeutic interventions.In conclusion, as the gap between traditional animal and human cell models continues to widen, pioneering studies such as those presented here have become vital. I hope that this compilation will serve as a touchstone for future endeavors in neurodegenerative disease modeling and therapeutics.


De novo mutations contribute to a large proportion of sporadic psychiatric and developmental disorders, yet the potential role of environmental carcinogens as drivers of causal de novo mutations in neurodevelopmental disorders is poorly studied.To explore environmental mutation vulnerability of disease-associated gene sets, we analyzed publicly available whole genome sequencing datasets of mutations in human induced pluripotent stem cell clonal lines exposed to 12 classes of environmental carcinogens, and human lung cancers from individuals living in highly polluted regions. We compared observed rates of exposure-induced mutations in disease-related gene sets with the expected rates of mutations based on control genes randomly sampled from the genome using exact binomial tests. To explore the role of sequence characteristics in mutation vulnerability, we modeled the effects of sequence length, gene expression, and percent GC content on mutation rates of entire genes and gene coding sequences using multivariate Quasi-Poisson regressions.We demonstrate that several mutagens, including radiation and polycyclic aromatic hydrocarbons, disproportionately mutate genes related to neurodevelopmental disorders including autism spectrum disorders, schizophrenia, and attention deficit hyperactivity disorder. Other disease genes including amyotrophic lateral sclerosis, Alzheimer’s disease, congenital heart disease, orofacial clefts, and coronary artery disease were generally not mutated more than expected. Longer sequence length was more strongly associated with elevated mutations in entire genes compared with mutations in coding sequences. Increased expression was associated with decreased coding sequence mutation rate, but not with the mutability of entire genes. Increased GC content was associated with increased coding sequence mutation rates but decreased mutation rates in entire genes.Our findings support the possibility that neurodevelopmental disorder genetic etiology is partially driven by a contribution of environment-induced germ line and somatic mutations. De novo mutations contribute to a large proportion of sporadic cases of ASD, schizophrenia, and intellectual disability  analysis on all genes which contained coding sequence (CDS) variants in PAH-treated iPSCs. GO analysis was performed using FUMA with ensembl version 92, protein coding genes set as the background, and a Bonferroni correction . In an aAutism spectrum disorder-implicated NDD genes tend to be longer than other genes . To visuTo further explore associations of length with mutability, we modeled the effects of sequence length, expression, and percent GC content on mutation rate using multivariate Quasi-Poisson regressions, with separate models for mutations in CDS and entire genes. Gene and CDS start and stop positions were obtained from GENCODE Release 38de novo mutations in individuals with neuropsychiatric diseases, including 42,607 ASD cases . Mutations per subclone increased linearly with the number of randomly sampled genes while the number of mutations per gene per subclone remained flat , confirmp-values from Monte Carlo empirical null distributions for PAH-treatment mutations  variants in PAH-treated iPSCs revealed enriched gene ontologies closely related to neurodevelopment: neuron projection, neuron part, and calcium ion binding . FurtherIn addition to the vulnerability of NDD genes to environmental mutagens uncovered here, genes associated with obesity and type 2 diabetes were mutated by chemical treatment more than expected. We therefore hypothesized that these genes might be linked to neurodevelopmental processes. To explore this possibility, we performed GO analysis on our list of obesity- and type 2 diabetes-associated genes, but found no evidence for enrichment of NDD processes .Quasi-Poisson regressions further supported a stronger role of sequence length in gene but not CDS mutation number. Controlling for expression and GC content, each interquartile range increase (IQR) in gene length was associated with a 1.104-fold increase in gene mutation rate (rate ratio (RR) = 1.104, 95% CI ; Quasi-Poisson regressions also showed significant effects of GC content and expression on gene and CDS mutability. Expression was not associated with mutability for genes   .de novo mutation has previously been hypothesized as a pathway linking environmental exposures to increased NDD risk, particularly ASD (de novo mutation rates, which in turn result in elevated incidence of neurodevelopmental disease. We are unaware of any studies employing this type of mediation approach for environmental exposures, although the mediating role of de novo mutations has been investigated for paternal age (Although arly ASD , this hyrnal age . In the in vivo. Because PAHs are a major air pollutant, we attempted to externally validate the results from PAH-exposed iPSCs by analyzing lung tumor mutations from humans living in highly polluted regions. However, future studies might better validate these results using animal models dosed with comparable levels of the environmental chemicals examined by de novo mutations.This work has several limitations. First, our findings that environmental chemicals may disproportionately mutate neurodevelopmental disease genes supports but is not an explicit test of the hypothesis described above. Second, the methods of gene set curation could bias our analyses comparing observed rates of exposure-induced mutations in disease gene sets with the mutation rate of control genes randomly sampled from the genome. To partially address this limitation, we created an online tool allowing researchers to query their own gene sets. Another limitation was our reliance on mutations called in cultured human iPSCs rather than www.environmentalmutation.com.Custom gene lists can be queried using the algorithm we generated at https://github.com/brennanhilton/environmental-mutation-calculator.Publicly available datasets were analyzed in this study. This data can be found here: Data are available from the primary sources cited. The SPARK gene variants are available to approved researchers through SFARI upon review. The code generated during this study are available on GitHub at: BB, JS, WC, and BP contributed to conception and design of the study. BB and SZ performed the statistical analysis. BB wrote the first draft of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.This work was supported by the NIH grants: P30ES009089, R21ES032913, and R24ES029489.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.


Isolated, ischemic scrotal lesions are an uncommon manifestation of systemic calciphylaxis. This highly morbid disease seen in patients with end-stage renal disease and other risk factors for small vessel calcification results in tissue necrosis and ulceration. Scrotal calciphylaxis is uncommon and difficult to diagnose in patients with comorbid conditions that cause isolated genital skin lesions, including prior systemic infections. We describe a 51-year-old male with end-stage renal disease on hemodialysis and recent history of bacteremia who developed isolated penile and scrotal ulceration due to calciphylaxis. The patient died two months after presentation despite multidisciplinary care. His physical exam was notable for scrotal duskiness, tenderness, and significant swelling, causing penile phimosis that buried his penis and urethra (A 51-year-old man presented to outpatient urology with progressive severe genital pain, scrotal ulceration and eschar, phimosis, and urinary retention. His medical history included type 2 diabetes mellitus, stage 5 chronic kidney disease initiated on dialysis three months prior to presentation, peripheral and coronary artery disease, and penile ulceration treated with topical ketoconazole cream and oral valacyclovir three weeks prior to presentation a. Six mo urethra b. His coEnterococcus faecium and Enterobacter cloacae. The patient was started on linezolid and meropenem per organism susceptibilities. Fluconazole was continued given concerns for candidal balanitis.Punch biopsy of the scrotum showed epidermal and dermal necrosis with vascular congestion and associated interstitial neutrophilic infiltrate but was negative for microorganisms and calcium. Tissue cultures were positive for Non-contrast CT imaging of the abdomen and pelvis  showed pOn the 11th day of his hospitalization, the patient's scrotal wound stabilized b. On day3,,Scrotal calciphylaxis is a rare complication of systemic calcific uremic arteriolopathy, a highly morbid disease seen in individuals with end-stage renal disease, co-occurring bone mineral disorders, and chronic inflammatory states.,,,7,11 Wound debridement to remove necrotic tissue can be offered to patients with the recognition that patients with calciphylaxis are more likely to develop non-healing wounds and therefore be poor surgical candidates. Finally, hyperbaric oxygen therapy can be offered to patients to increase the oxygenation of necrotic tissue.Calciphylaxis treatment is targeted at reversing the pathophysiologic processes that lead to microvessel calcification and tissue ischemia, caring for calciphylaxis related wounds, optimizing dialysis clearance, and addressing pain. In cases where pain does not respond to high-dose opioids, sciatic nerve cryoneurolysis or pudendal nerve blocks can be employed.It is important to note that while these treatments can be offered to patients, calciphylaxis continues to have a mortality rate between 45 and 80% within one year. Thus, clinicians should be prepared to coordinate with palliative care to engage in end-of-life discussions and treat severe pain caused by the disease.4This case shows the challenges of diagnosing and treating calciphylaxis given the inconclusive nature of diagnostic tools and lack of evidence-based, effective treatments. This underscores the importance of early clinical suspicion of the disease, even when patient presentation is atypical and diagnostic data is inconclusive.


Through the investigation of cell heterogeneity, microenvironment, function, and cellular interactions, spatial transcriptome technology can deeply explore the pathogenic mechanisms of cell-type-specific responses and spatial localization in neurological diseases. The present article overviews spatial transcriptome technologies based on microdissection, in situ hybridization, in situ sequencing, in situ capture, and live cell labeling. Each technology is described along with its methods, detection throughput, spatial resolution, benefits, and drawbacks. Furthermore, their applications in neurodegenerative disease, neuropsychiatric illness, stroke and epilepsy are outlined. This information can be used to understand disease mechanisms, pick therapeutic targets, and establish biomarkers.Spatial transcriptome technology acquires gene expression profiles while retaining spatial location information, it displays the gene expression properties of cells  These ofication . This tefication . Be thatTomography sequencing (Tomo-seq): Tomo-Seq, a spatially resolved transcriptomic method that combines classical histological sectioning of embryos or tissues with a highly sensitive RNA-sequencing technique : TSCS is a technology that combines LCM, laser-catapulting, whole-genome-amplification (WGA) and single cell DNA sequencing to measure genomic copy number profiles of single tumor cells while preserving their spatial information  were performed, the data was demultiplexed by cell library barcode and prepared to calculate genomic duplicate number profiles. At last, single-cell genomic information is mapped to spatial coordinates, not only for the genomic copy number profiles of individual tumor cells, but to depict the topographic organization of different clonal genotypes in the tissue sections Single-molecule fluorescence : Individual mRNA molecules in a fixed cell or tissue are imaged by hybridization with a few particularly labeled short DNA oligonucleotide probes which are complementary to the sequence of the target mRNA, and each probe are coupled to some fluorescent dyes. Imaging is then performed by recognizing the fluorescent signal displayed by the probes Sequential fluorescence : seqFISH is a temporal barcoding method that uses a limited number of fluorophores scaled exponentially with time to address the issue of fluorophore limitation on smFISH. Specifically, sequential rounds of hybridization, imaging, and probe stripping are performed on mRNA in fixed cells Multiplexed error-robustness fluorescence : The imaging method MERFISH can assess the spatial localization and copy number of thousands of mRNAs in a single cell utilizing combinatorial labeling and error-resistant barcodes Sequential fluorescence : seqFISH+ was further developed, which uses sequential hybridizations and standard confocal microscope imaging to achieve super-resolution imaging and multiplexing of 10,000 genes in single cells Fluorescent : FISSEQ is a non-targeted amplification detection method for gene expression profiling in fixed cells or tissues by converting mRNA into cross-linked cDNA amplicons and using SOLiD sequencing by ligation Barcode : Baristaseq is an improved version of padlock probe-based in situ barcode sequencing technology that combines Illumina sequencing by synthesis (SBS) with a five-fold increase in detection efficiency : STARmap is a technology for 3D intact-tissue RNA sequencing, which integrates hydrogel-tissue chemistry (HTC), targeted signal amplification, and in situ sequencing Barcoded oligonucleotides ligated on RNA amplified for multiplexed and parallel : This mRNA detection technique by performing multiple parallel in situ analyses of barcoded oligonucleotides attached to mRNA without RT for spatially resolved, targeted, in situ mRNA identification of one or more targets (in situ hybridization (FISH) or sequenced in situ to obtain genetic information about the bases they encode. BOLORAMIS is more sensitive than non-targeting methods like FISSEQ, Slide-seq, and high-definition spatial transcriptomic (HDST) because it targets specific genes of interest in a highly multiplexed manner : Spatial transcriptomics is a technique for visualizing and quantifying mRNA at spatial resolution through barcode microarray slices. Tissues are placed on oligonucleotide microarrays slide (slide surfaces are shown as spots), and each array features contains unique positional barcode, unique molecular identifier (UMI), poly(d)T oligonucleotides and sequencing primer. Then the tissues are fixed, stained with H&E, and imaged. After permeabilization of tissue sections, poly(d)T captures mRNA and visualizes cDNA synthesis with fluorescently labeled nucleotides. After tissue is enzymatically removed, the cDNA containing UMI information and spatial barcodes is obtained. NGS is used to later create and sequence amplification-based sequencing libraries. For the purpose of visualization and analysis, the tissue images are aligned with array features, and the RNA-seq data is classified using spatial barcodes : Similar to Slide-seq, HDST uses spatial barcode bead arrays to capture mRNA from tissue sections for 2 μm resolution spatial gene expression analysis. 2,839,865 individually barcoded beads, each containing a different pool of spatial barcodes, unique molecular identifiers, and poly(d)T, are randomly placed into hexagonal arrays of 2 μm wells. The positions of the beads are then decoded through multiple rounds of sequential hybridization : DBiT-seq is a multi-omics sequencing technology based on microfluidic technology to introduce barcodes into tissue sections, therefore obtain transcriptomic and proteomic information at near single-cell resolution Transcriptome : TIVA is an ideal method for fluorescently labeling cells in living tissue, selectively activating and isolating mRNA from cells of interest, and retaining information about the spatial location of individual cells  onto the surface of living cells within intact tissues, linking scRNA-seq with spatial dimension and real-time phenotypic analysis  results in muscle denervation atrophy . TherefoA study used the spatial transcriptomics approach LCM-seq to analyze substantia nigra pars compacta (SNpc) and ventral tegmental area (VTA) dopamine neurons isolated individually from the postmortem brains of 18 individuals . The re-NL−G-F mouse model to investigate the connection between this pathological change and neurodegeneration  were the focus of a study , the hipneration . Early ineration , AD plaqneration . For exaneration , a risk neration  revealedNeuropsychiatric disorders: The spatial organization of the brain is fundamentally linked to its function. Based on cell type and density, the neocortex can be divided into six layers, and cells in different layers exhibit distinct patterns of gene expression  . Using tBurst discharges not only appear in animal models of brain tumors, but also in the peritumoral area of patients with malignant brain tumors during biopsy. In patients with malignant brain tumors, burst discharges in the peritumoral region may be a source of epileptic activity. In addition, growing evidence suggests that interactions between gliomas and neurons in the peritumoral region are necessary for the occurrence of epileptic discharges. A recent study established a rat glioma model and characterized it at the cellular and molecular levels in order to investigate the biological changes associated with the peritumoral region .A transcriptome analysis using LCM-seq in brain tissue sections of a rat glioma model identified 19 genes that were differentially expressed in the peritumoral region . Five ofSpatial transcriptomic technologies provide new insights to explore the molecular mechanisms of disease from the perspective of spatial heterogeneity and offer new means to discover potential biomarkers and predict disease progression, playing a huge application within the diagnosis and treatment of neurological diseases. Despite the expansion of spatial transcriptomic innovations, there is currently no single spatial transcriptomic technique suitable for all situations. These spatial transcriptomic methods still need to be enhanced for experimental studies. In general, these technologies have the following defects that need to be improved: 1) High-resolution profiles can be obtained using microdissection-based techniques, but they include laborious processes and are difficult to use. 2) Target mRNA can be detected using in situ hybridization techniques, but smFISH is limited by the amount of fluorophores, seqFISH, and MERFISH are expensive studies, and probe hybridization can be mistaken. 3) Technologies using in situ sequencing have a low detection throughput and can only perform targeted detection. 4) While increasing resolution and throughput, in situ capture technologies can only reach the level of a single cell. (4) Live cell-based spatial transcriptome technology is not entirely suitable to the study of human materials. In the Over all, future spatial transcriptome technologies must therefore be improved and enhanced. In order to fully describe and comprehend the microenvironment of brain tissue, the spatial transcriptome can combine with other spatial histology technologies like spatial proteomics to discover protein expression and localization patterns at the single-cell level. This will make it easier to show a complete and accurate brain cell atlas as anticipated from the Brain Initiative Cell Census Network (BICCN) and the Human Cell Atlas. In addition, multi-omics synthesis provides reliable information for investigating disease causation, finding therapeutic targets, and discovering new biomarkers. Secondly, combining spatial transcriptomic data with multimodal data, such as calcium imaging and/or optogenetic probing, can also produce interpretations of circuit activity that are specific to particular cell types. This can take full advantage of the spatial transcriptome to unravel the complex intercellular interactions, gene regulatory networks and subcellular structures under physiological and pathological conditions.


Asthma is an inflammatory lung disease that constitutes the most common noncommunicable chronic disease in childhood. Childhood asthma shows large heterogeneity regarding onset of disease, symptoms, severity, prognosis, and response to therapy.Evidence suggests that this variability is due to distinct pathophysiological mechanisms, which has led to an exhaustive research effort to understand and characterize these distinct entities currently designated as “endotypes.” Initially, studies focused on identifying specific groups using clinical variables yielding different “clinical phenotypes.” In addition, the identification of specific patterns based on inflammatory cell counts and cytokine data has resulted in “inflammatory endotypes.” More recently, an increasing number of molecular data from high-throughput technology (“omics” data) have allowed to investigate more complex “molecular endotypes.”A better definition and comprehension of childhood asthma heterogeneity is key for improving diagnosis and treatment. This review aims at summarizing the current knowledge on this topic and discusses some limitations in their application as well as recommendations for future studies. Asthma is the most common inflammatory disease of the airways in childhood being characterized by cough, wheeze, and shortness of breath. Scientific evidence indicates that asthma is a heterogeneous disease encompassing different pathophysiological mechanisms . This haBased on differences in the onset and progression of the disease, the severity of symptoms, frequency of exacerbations, inflammatory profiles, and response to therapy, childhood asthma is considered a heterogeneous disease . Early oThe heterogeneity in childhood asthma is due to different pathological mechanisms originating from the complex interplay between genetic, epigenetic, and environmental factors , 19. TwoGenetics also plays an important role in childhood asthma, where family history of asthma remains one of the most important risk factors , 25. NotThe inherent challenges of understanding heterogeneity in complex diseases are increased in childhood asthma due to differences in epidemiological definitions and a casual use of asthma severity and control classifications . Van WonDifferent approaches have been used to disentangle asthma heterogeneity, resulting in multiple phenotypes and endotypes. Phenotypes can be defined as a cluster or group that shares visible or measurable properties like demographic variables, symptom frequency, or physiological properties, whereas an endotype describes a specific biological mechanism which explains the disease . PrimariClinical phenotypes related to childhood asthma depend on the variables and methods used for identification, as well as the aim of the study. Pijnenburg and colleagues defined four domains where these phenotypes are most useful in clinical practice. These include diagnosis, pathophysiology, prediction, and response to therapy . Many stThe assessment of triggers and the persistence of wheezing symptoms have resulted in two phenotypes among preschool wheezing children . One is Among asthmatic children, two phenotypes have been identified based on the presence of atopy. Allergic asthma is characterized by allergen sensitization (specific IgE) and the presence of atopic diseases . Nonallergic asthma shows no allergic sensitization nor additional atopic diseases , 40. AllPrediction of asthma diagnosis in children is clinically relevant, and the description of longitudinal patterns of asthma-related traits results in phenotypes with different predictive values for asthma diagnosis. Sensitization is a key feature of allergic asthma, but the mechanisms underlying this relationship remain unclear. Recent studies investigated longitudinal trajectories of this trait and assessed their risk for an asthma diagnosis , 42, 43.The temporal trajectories of wheeze symptoms have also been investigated using hypothesis-driven or latent class analysis (LCA) methods. These studies consistently describe four distinct trajectories . In addition, studies using LCA algorithms report a fifth intermediate trajectory. Children following the late onset or the recurrent wheezing trajectories have an increased risk for lung function abnormalities and asthma diagnosis , 36, 44.Current treatment guidelines, such as the Global Initiative for Asthma (GINA), are based on the use of ICS; however, specific clinical sub-phenotypes are not included . AlthougTwo recent studies classified asthmatic children and adolescents from the Asthma Phenotypes in the Inner City (APIC) study into two groups: “difficult-to-control” (DTC) and “easy-to-control” (ETC) asthma. The DTC group included children requiring high doses of ICS over a 1-year period (6 visits) whereas the ETC children requiring low doses. It is important to mention that this group designation did not reflect asthma control , 48. KeyIn this section, different studies using clustering methods on specific children populations  are described, which aimed at identifying sub-phenotypes within these groups. When investigating severe asthmatic children, four distinct clusters were identified in a sample from the Severe Asthma Research Program (SARP) using a data-driven approach . Three cClustering methods have also been used to investigate a diverse school-aged asthmatic children population with differing severity and atopy levels. Zoratti and colleagues identified five distinct clusters in asthmatic children from the APIC study that covered all possible combinations of atopy and symptom severity . Three cAnother study took a similar approach and used these methods on recurrent wheezing children from the Childhood Asthma Prevention Study (CAPS) . These cThese unbiased methods have proven to be relevant for therapy response, where a differential response to treatment has been observed between different clusters. Howrylak and colleagues applied clustering methods in asthmatic children from the Childhood Asthma Management (CAMP) trial, a three-arm-randomized clinical trial comparing the treatment with budesonide, nedocromil, and placebo. Cluster identification was based on atopic burden, lung function, and exacerbation history . The oriFinally, asthma phenotypes have been almost exclusively described in cohorts from European countries and the USA. However, due to the increase in asthma incidence in South America, Asia, and Africa, studies identifying asthma phenotypes from these regions are emerging –53. One To sum up, many different clinical phenotypes have been identified, some of which are currently relevant to clinical practice like the STRA phenotype. In addition, many of the clinical phenotypes that were originally identified using hypothesis-driven methods are being confirmed and expanded by clustering analysis  Table . HoweverStudies aiming at identifying different inflammatory endotypes among asthmatic children use similar approaches as described before using data of cell counts from different tissues , sputum, biopsies, and serum), cytokine measurements, and specific inflammation biomarkers  . Evidenc+ and IL-13+ cells was observed [The most common and best-described endotype is the T2-high (eosinophilic), which is also present in the adult population. This endotype has been described in children with mild, moderate, and severe asthma and is characterized by a high degree of atopy, increased eosinophils (in sputum and serum), high levels of T2 cytokines , and early signs of airway remodeling , 64, 66.observed . Childreobserved . Thus, tobserved , 56. Twoobserved . The throbserved .The neutrophilic inflammatory endotype has been identified in children with STRA and is characterized by neutrophilic infiltration of the airways, where a Th1 or/and Th17 skewed immune profile has been implicated . AnderssThe non-T2 endotype (neutrophilic and paucigranulocytic) is discussed based on the current unclarity of (i) a novel mechanism underlying these specifics or (ii) just a steroid-driven result of immunosuppression. Solutions to this issue are difficult as characterization via inflammatory cells in the airways or sputum is primarily performed in severe asthmatics, generally requiring high doses of ICS. Importantly, a murine asthma model with low T2 cytokines, neutrophilic inflammation, and elevated levels of IL-17 and interferon (IFN)-γ with bronchial hyperresponsiveness has been developed and may help to elucidate its relevance . In summRecent studies are applying clustering methods to identify inflammatory endotypes using cell counts, cytokine data, and clinical parameters. Guiddir and colleagues performed a cluster analysis using clinical variables  and inflammatory cell counts (blood and BAL neutrophil and eosinophil counts) in children with recurrent wheeze (preschool age) or severe asthma from the Severe Asthma Molecular Phenotype (SAMP) study . They idRecent technological advances and lowering costs of high-throughput technologies from the “omics” fields are enabling asthma researchers to gather high amounts of molecular data from a large number of individuals , 73. ThiTranscriptomics constitutes the study of a defined RNA population from a cell or tissue that can comprise the whole transcriptome or a subset . This field has been intensively studied in relation to asthma in adults and children, reviewed elsewhere . HoweverAnother fast-evolving field is mass spectrometry (MS) omics, where a defined set of biomolecules  are quantified in a cell or tissue. Many studies have assessed the association between these molecules and asthma in children and adults, reviewed elsewhere . HoweverORMDL3 which has been associated with childhood asthma [Infant bronchiolitis is a risk factor for asthma diagnosis. Recent studies use different MS omics and clustering algorithms to identify endotypes in children suffering from acute bronchiolitis. These clusters were characterized, and their risk for developing asthma and recurrent wheeze was prospectively evaluated. Fujiogi and colleagues used lipidomics data from nasopharyngeal swabs and identified four endotypes, one characterized by high atopy and low levels of sphingolipids. Children in this cluster had the highest risk of developing recurrent wheeze and asthma. Interestingly, sphingolipid biosynthesis is regulated by the 17q21 locus gene d asthma . These rd asthma . In addid asthma .In summary, different phenotypes and endotypes have been described in asthmatic children and adults with potential use in clinical practice  . These sAlthough these phenotypes and endotypes have proven valuable for our understanding of asthma, several limitations hinder their inclusion into clinical practice yet, especially in children. One significant limitation is our poor understanding of their temporal stability. Most studies focusing on this topic use a cross-sectional design, thus lacking the assessment of longitudinal features. Many experts have criticized this, which led to the European Respiratory Society task force recommending in 2014 not to treat MTW and EVW phenotypes differently . ResearcValidation and replication of phenotypes and endotypes are also important, specifically for studies using clustering methods. These studies require inclusion of validation sets. Although many report cluster stability measures, only a few conducted proper validation . Of noteConsidering the high numbers of current studies using clustering methods, developing a guideline for these studies would help coordinate efforts across research groups. An interdisciplinary-based expert guideline aiming to include a set of standardized variables for cluster characterization, detailed clinical characterization, and study proceeding with carefully planned statistical strategies would greatly facilitate across-study comparisons and enable meta-analyses. Additional variables can still be included and presented as sensitivity analyses. Conrad and colleagues have proposed relevant features for this . FinallySeveral phenotypes and endotypes have emerged for childhood asthma, reflecting variability in clinical parameters, inflammatory profiles, and molecular mechanisms. Information regarding their temporal stability and reproducibility is lacking and can provide important addition for future studies. Recent advances in statistical methods and molecular techniques are currently applied, and more comprehensive analyses following a systems biology approach are expected. The identification of stable phenotypes and endotypes will change our understanding and the management of childhood asthma. This goal will be achieved by a careful combination of omics data with immunological functional assessment.


A 40-year-old female with a history of chronic alcohol use disorder presented with an acute intractable left-sided headache for three days and progressively worsening unsteady gait requiring a wheelchair to ambulate. The patient had a history of chronic alcoholism since 2019 but reported abstinence since September 2021. One month after quitting alcohol, she experienced a sudden deterioration in bilateral extremity neuropathy, forgetfulness, difficulty writing, and severe mood swings, which continued to worsen until her presentation in July 2022. Laboratory tests, including complete blood count and electrolyte levels, were within normal ranges. A previous MRI performed during the investigation for alcoholic neuropathy a few months before she quit drinking showed no abnormalities. However, a subsequent MRI during work-up for the current acute symptoms revealed significant signal abnormalities involving the central pons, bilateral cerebral peduncles, and medullary pyramids, consistent with chronic central pontine myelinolysis (CPM) with extrapontine myelinolysis (EPM) extending into the peduncles. The patient received treatment with folate and multivitamins and was scheduled for outpatient follow-up with physical therapy for rehabilitation. This case highlights CPM as a consequence of alcohol withdrawal and emphasizes the importance of timely diagnosis and appropriate management in such patients. Osmotic demyelination syndrome (ODS) is a non-inflammatory demyelination disorder that encompasses both extrapontine myelinosis (EPM) and central pontine myelinolysis (CPM). It was initially described in 1959 . The undA 40-year-old female presented at the emergency department with a persistent acute left-sided headache that had been unresponsive to medication for a duration of three days. The headache was sharp and severe accompanied by photophobia and nausea, with no radiation or other eye symptoms. Further history revealed a progressive deterioration of symptoms over the past 10 months, including difficulty writing, increased forgetfulness and gait disturbances. Initially, the patient required a walker for mobility due to lower limb weakness and gait disturbances, but upon admission, she relied on a wheelchair. Her medical history included chronic alcohol use disorder, alcoholic neuropathy, and alcoholic liver cirrhosis. Although she had been a chronic alcoholic since 2019, she reported discontinuing alcohol consumption in September 2021. There had been no hospital admissions since quitting alcohol until her presentation in July 2022. She mentioned an increased appetite after cessation and consumed more than usual for the first few weeks.Prior to the onset of her current symptoms, she exhibited mild neuropathy in both lower extremities attributed to her alcoholism, characterized by lower limb weakness. However, she reported a sudden worsening of neuropathy in both extremities one week after abruptly stopping her alcohol intake. Over the following months, her symptoms progressed and worsened. During the physical examination, the patient exhibited 3/5 motor strength in her lower extremities, without any concurrent fasciculations or sensory or cranial nerve deficits. However, due to her wheelchair-bound condition, an assessment of her gait could not be performed. Laboratory tests, including complete blood count, creatinine, and electrolyte levels, were all within normal limits, except for a mild decrease in potassium to 3.3, as shown in Table A computed tomography (CT) scan of the head Figure  revealedThere were also scattered areas of reduced density in the subcortical white matter Figure , includiTo further evaluate the condition, a contrast-based magnetic resonance imaging (MRI) scan of the brain was ordered. The MRI Figure  showed hThe patient was diagnosed with central pontine myelinolysis and extra pontine myelinolysis resulting from alcohol withdrawal. Central pontine myelinolysis (CPM) and extrapontine myelinolysis (EPM) are rare neurological disorders that can occur during alcohol withdrawal. Although the exact pathogenesis of CPM and EPM remains unclear, several studies suggest that cytotoxic edema is a central factor in CPM, while vasogenic edema plays a significant role in EPM -5.CPM is characterized by the loss of myelin in the central region of the basis pons, primarily resulting from the rapid correction of hyponatremia -6. In caEPM is characterized by the loss of myelin in the brainstem, cerebellum, and other areas outside the pons . It can Chronic alcoholism is a recognized risk factor for developing both CPM and EPM -5. AlcohAlthough there have been reports implicating causes other than hyponatremia in the development of CPM and EPM, a few cases have suggested a potential association with alcohol withdrawal and refeeding syndrome -7.Refeeding syndrome is a condition characterized by dangerous shifts in electrolytes and fluids in malnourished patients who receive artificial feeding, either parenterally or enterally. This syndrome can lead to metabolic and hormonal changes that give rise to clinical complications. One of the primary manifestations of refeeding syndrome is the development of hypophosphatemia. Additionally, changes in glucose, thiamine, protein, and fat metabolism can occur, contributing to neurological complications. In the case of our patient, who engaged in binge drinking for the past two years and abruptly ceased in September 2022, she reported a significant increase in appetite and consumed more food than usual in the following week. One week later, she experienced a deterioration in bilateral neuropathy and the onset of weakness and forgetfulness. While no abnormal changes in her electrolytes were observed upon admission, we cannot exclude the possibility of refeeding syndrome as a potential cause of her subsequent development of CPM, considering her presentation to the hospital 10 months later.The primary treatment for CPM and EPM is supportive care, with the prognosis dependent on the severity of the neurological symptoms -6. Due tIt is essential to include CPM and EPM in the differential diagnosis of patients who manifest new onset psychiatric or neurological symptoms after alcohol withdrawal. Further research is necessary to understand the pathophysiology and risk factors for CPM and EPM induced by alcohol withdrawal.