Dual aneurysms arising from the internal cerotic artery (ICA) is a very rare occurrence. Clinoid segment aneurysms (CSAs) are often seen at the carotid dural rings while cavernous carotid aneurysms (CCAs) are often a direct communication between the ICA and the cavernous sinus (CS). We present a case of complex concomitant occurrence of a CSA and a CCA complicated with delay aneurysmal rupture (DAR) resulting in carotid cavernous fistula (CCF) after our initial treatment of the patient with pipeline embolization devices (PLEDs)We present a 64-year old female who we admitted at our institution due to one-year history of double vision. Neurological examinations were unremarkable.Magnetic resonance imaging (MRI) and computer tomography (CT)-scan revealed dual aneurysms on the ICA. Digital subtracting angiogram (DSA) confirmed a small CSA and a large CCA on the right ICA.We treated both aneurysms with PLED and subsequently observed DAR of CCA as a complication.We successfully occluded the fistula with ONYX  via the trans-venous approach.PLED was the best endovascular treatment option though DAR was inevitable. Although the trans-arterial approach may be the gold standard for the managing of CCF, the complex nature of our case made us opt for trans-venous approach. The trans-venous route is very appropriate for fistulas with complex parent arteries. Anatomically, the clinoidal segment is the portion of the ICA that is located between the cavernous sinus (CS) and the subarachnoid space.,7 CCAs accounts for about 2% to 9% of all aneurysms in the brain.,9 These lesions often arise as a result of traumatic events although cases of idiopathic, infectious as well as iatrogenic have been reported.,10 They are usually asymptomatic during their initial development but become symptomatic when they grow into large and giant sizes. Their mass effects on adjacent cranial nerve often lead to diplopia, ophthalmoplegia, ptosis, pain as well as paresthesia. CCAs often have a low risk of causing major deformities as well as death except when they result into a fistula.Cavernous carotid aneurysms (CCAs) are often a direct communication between the ICA and the CS.,14 Studies have proven that, flow diverting devices (FDDs) treatment option often result in aneurysm rupture or parent vessel injury resulting in high flow shunts between the ICA and the CS, leading to CCF.,16 This often present as subarachnoid hemorrhage (SAH).,17 We present a complex occurrence of clinoid and cavernous segment aneurysms complicated with CCF as a result of delay aneurysm rapture (DAR) after our initial treating with pipeline embolization device (PLED).A carotid cavernous fistula (CCF) is an abnormal communication between the ICA and the CS as a result of trauma or rupture of an antecedent carotid segment aneurysm.2 and Computer tomography (CT)-scan revealed dual aneurysms on the ICA. A confirmatory digital subtracting angiogram (DSA) revealed a small CSA and a large cavernous segment aneurysm or CCA on the right ICA Fig.   A. The . Also, post-procedure Vaso-CT revealed excellent apposition of the stents. The PLED bridged aneurysmal necks in both aneurysms completely . The patient was put on daily aspirin 75 mg and clopidogrel 75 mg. She was discharged home 5 days after the endovascular procedure.We used a triaxial system comprising of a 7 French 90 cm cook long sheath, a 5 French 115 cm navien, and a marksman to provide support for the PLED implantation. A 4.5 mm × 35 mm PLED was successfully inserted across the necks of the 2 aneurysms without complications. The post-procedure angiogram showed obvious contrast retention in the large aneurysm sac, while no contrast was seen in the small  or cortical vein and superior ophthalmic vein (SOV), inferiorly via the pterygoid plexus (PP), inferior petrosal sinuses (IPS) and superior petrosal sinuses (SPS) Fig.   D and EAfter careful evaluation of the CCF and the implanted PLED, the utilization of coils and/or ONYX  to occlude the fistula via the trans-arterial approach was not possible. Therefore, we adopted the trans-venous approach. We advanced the microcatheter to the origin of right SOV Fig.   F and dPrior to his hospital discharge, a DSA was done to evaluate the residual cortical vein drainage. We observed a little decreased in blood flow in the cortical vein and a relieve of his symptomatology. Three months follow-up with DSA revealed occlusion of the CCF Fig.   I and J3,3 while CCAs are often a direct communication between the ICA and the CS.,3 Nevertheless, aneurysms at these segments often rupture resulting in high flow shunts between the ICA and the CS leading to CCF. Our case is a complex concomitant occurrence of a CSA and a CCA complicated DAR resulting in CCF after our initial treatment of the patient with PLED.Dual aneurysms arising from the ICA is a very rare occurrence. Clinoid segment aneurysms (CSAs) usually originates from the carotid dural rings,18 This segment of the ICA is inadequately comprehended due to its complex and variable anatomy. The name clinoidal segment was arrived at because the ICA is link to the anterior clinoidal process (ACP).,5 The ACP meticulously juxtaposed to the lateral wall of the ICA. This segment is just proximal to the usual location of the ophthalmic artery (OA) and superior hypophyseal artery (SHA). The OA derived is from the dorsomedial wall while the SHA is derived from the ventromedial wall.The clinoidal segment of the ICA is situated in between the CS and the subarachnoid space.,20 Nevertheless, Bouthillier et al observed same symptoms for aneurysms originating from the fifth cervical vertebra(C5). The complex and variable anatomical location of CSA often makes endovascular treatment very challenging. Meyer et al observed a complication rate of 10% during their endovascular management of CSA. The risk of rupture of CSAs with resultant subarachnoid hemorrhage (SAH) depends on their anatomic connections with the upper dural ring. We utilized PLED in management of our CSA. During our follow-ups, this segment of aneurysm did not present with a complication because of the small size of the aneurysm.Fischer observed that, aneurysms originating from the third cervical vertebra(C3) segment of the clinoidal artery often enlarge via the distal dural ring leading to either optic nerve compression or SAH.–10 The etiology of CCAs can be traumatic, infectious, or idiopathic. CCAs are often classified into high and low-risk aneurysm. The classification is based on precise anatomical location of the aneurysms. The potentials of rupture of CCAs as well as management options are founded on this classification system. CCAs are often asymptomatic and are detected incidentally during radiological evaluation. Nevertheless, symptomatic patients present with compressive symptoms on the 3rd, 4th, 5th, and 6th cranial nerves.,11,22 Unruptured CCAs often present with diplopia as well as pain due to mass effect. Unilateral headaches, retro-orbital pain as well as facial pain are the most reported pain syndromes.,9 Nevertheless, optic neuropathy, ocular sympathetic paresis, corneal hypesthesia, as well as trigeminal dysesthesias are most often also associated with CCAs.,10 Our patient presented with diplopia as his principal complain.On the other hand, CCAs constitutes about 2% to 9% of all aneurysms in the brain.,10 MRI and CT-scan were very helpful during our initial work-out leading to the substantive diagnosis of both the CSA and CCAs.DSA still remains the most valuable intracranial vascular imaging modality though the initial work out of their diagnosis usually starts with Magnetic resonance angiogram (MRA) and computed tomographic angiogram (CTA). Furthermore, 3D-DSA which is an advanced rotational cerebral angiography is very crucial in assessing specific anatomical classification as well as accurate image reconstructions of CCAs. Cure of CCAs often focus on either occlusion of the aneurysm sac or reconstruction of the aneurysmal walls. The most preferred treatment modality for small CCAs as well as those which ruptures into CS is endovascular coiling. Nevertheless, ICA occlusion is the most suitable treatment option for large and giant CCAs. In cases were ICA occlusion is not possible, selective coiling, bypass surgery prior to ICA occlusion, or conservative treatment are usually the possible alternatives.The principal treatment options for CCAs is either surgery or endovascular techniques.,11 On the other hand, direct microsurgical clipping of the aneurysm usually constitutes the reconstruction.,23 Moreover, reconstruction is often done via coil embolization with or without vascular reconstruction device, FDDs, or liquid embolic agents. We utilized PLED in the management of this segment of the aneurysm with DAR as the main complication which warranted a second endovascular procedure. Studies have confirmed that, CCAs can rupture resulting in CCF with occasionally SAH if the aneurysm extends through the dura or after endovascular procedures.,16 Our follow-ups after the initial operation was to detected any associated complications.Occlusive approaches may involve surgical ligation of parent artery or via endovascular procedures.,16 DAR or parent vessel injury can result in an anomalous connection between the ICA and the CS, initiating a CCF.,16 The cardinal presentation in our patient was proptosis and conjunctival congestion. This symptomatology prompted us on a complication as a result of our management.DAR is one of the rare outcomes following the management of ICAs with FDDs. Orbital anomalies such as proptosis, distended ophthalmic veins, protruding CS, as well as thickness of the extraocular muscles. Furthermore, MRI is very accurate in detecting intracerebral hemorrhage, ischemia as well as incidental injuries to the cranial nerves.MRI and CT-scan are very valuable in determining the accompanying cerebral parenchymal injury, edema, as well as orbital anomalies.,15 DSA is also very virtual in guiding endovascular management. We utilized DSA to assess the size and location of the fistula in our case. This imaging modality also aided in classifying the fistula as to direct or indirect. We also identified the outflow pathway from the CS, CS varix as well as high-risk hemodynamic anomalies like cortical venous drainage.Nevertheless, DSA is the gold standard imaging modality use in assessing and detecting CCF.,25 The gold standard treatment modality for CCFs is usually trans-arterial embolization. We successfully occluded the fistula with ONXY embolization agent via the trans-venous approach. We adopted the trans-venous approach because PLEDs where already inserted into both aneurysms via the artery route. Nevertheless, complications like thromboembolism as well as ischemic events are often associated with endovascular management of CCF. Other complications like edema, ocular anomalies, pseudoaneurysm formation, as well as changes in arterial flow leading to fetal hemorrhage has also been reported.,26 Two years follow-ups of the patient revealed no such reported complication above.Endovascular management of CCFs stereotypically involves either trans-arterial obliteration of the fistulous site or trans-venous embolization via the CS.4A complex concomitant occurrence of CSA and CCA complicated with a delay rupture of the CCA resulting in CCF after treatment with PLED is a very rare occurrence. PLED was the best endovascular treatment option though DAR was inevitable. Although the trans-arterial approach may be the gold standard treatment modality for managing CCF, the complex nature of our case made us opt for trans-venous approach. The trans-venous route is very appropriate for fistulas with complex parent arteries.Conceptualization: Wang Ting, Seidu A Richard, Zhang Changwei, Wang Chaohua, Xie Xiaodong.Data curation: Wang Ting, Seidu A Richard, Zhang Changwei, Wang Chaohua, Xie Xiaodong.Formal analysis: Wang Ting, Seidu A Richard, Zhang Changwei, Wang Chaohua, Xie Xiaodong.Investigation: Wang Chaohua, Xie Xiaodong.Methodology: Seidu A Richard, Zhang Changwei.Resources: Xie Xiaodong.Supervision: Zhang Changwei, Wang Chaohua.Writing – original draft: Seidu A Richard.Writing – review & editing: Wang Ting, Seidu A Richard, Zhang Changwei, Wang Chaohua, Xie Xiaodong.


LMNA are major structural and mechanotransductive components of the nuclear lamina. Previous reports have proposed lamin A as the isoform with the most dominant contributions to cellular mechanophenotype. Recently, expression of lamin C has also been shown to strongly correlate to cellular elastic and viscoelastic properties. Nevertheless, LMNA isoforms exist as part of a network that collectively provides structural integrity to the nucleus and their expression is ultimately regulated in a cell-specific manner. Thus, they have importance in mechanotransduction and structural integrity of the nucleus as well as potential candidates for biomarkers of whole-cell mechanophenotype. Therefore, a fuller discussion of lamin isoforms as mechanophenotypic biomarkers should compare both individual and ratiometric isoform contributions toward whole-cell mechanophenotype across different cell types. In this perspective, we discuss the distinctions between the mechanophenotypic correlations of individual and ratiometric lamins A:B1, C:B1, (A + C):B1, and C:A across cells from different lineages, demonstrating that the collective contribution of ratiometric lamin (A + C):B1 isoforms exhibited the strongest correlation to whole-cell stiffness. Additionally, we highlight the potential roles of lamin isoform ratios as indicators of mechanophenotypic change in differentiation and disease to demonstrate that the contributions of individual and collective lamin isoforms can occur as both static and dynamic biomarkers of mechanophenotype.Lamin A and lamin C isoforms of the gene  LMNA and include primarily lamin A and lamin C, although other minority isoforms and splice variants occur naturally as well . This result suggests that the lamin A isoform is more sensitive to mechanophenotypic changes than lamin C. Nevertheless, there is evidence to suggest this behavior holds for lamin A and C isoforms. In a previous study, we observed that disruption of the actin cytoskeleton using cytochalasin D resulted in a reduction in cell stiffness that was concomitant to 2.0- and 2.5-fold lower lamin C and A protein expression, respectively, in CytoD-stiff cells (LMNA mutations, where the trend in mechanosensitivity is reversed (While the individual lamin isoform-mechanophenotype correlations from our study suggest that lamin C is a better correlate of mechanophenotype, we also wanted to understand if these findings were also true at the lamin aggregate assembly and expression levels, since that is how these proteins would be found in living cells. To test this hypothesis, we explored the numerical interrelationships of lamin A, C, and A + C isoform expressions normalized to lamin B1 and whole-cell mechanophenotype. In our original study, protein expression level for each lamin was quantified using densitometry analysis of associated immunoblots . Averageff cells . Other sreversed . AltogetLMNA gene expression and nuclear stiffness can change drastically as stem cells differentiate, it has been hypothesized that changes in lamin isoform ratios could reveal aspects of lineage commitment, perhaps similarly to how cellular mechanical properties predict lineage differentiation potential in stem cell populations (LMNA is upregulated during osteogenesis but downregulated during adipogenesis (LMNA results in higher levels of lamin A and C in osteoblasts than in undifferentiated stem cells (LMNA mRNA transcripts, the lamin A isoform is expressed at higher levels than its lamin C counterpart in differentiated, stiff osteoblasts. Conversely, soft cells types, like differentiated neurons, express significantly lower levels of lamin A than lamin C and B1 (GFAP gene. While the LMNA gene itself is not mutated in this disease, brain tissues from mice models of Alexander disease exhibit higher lamin A and, specifically, higher lamin C expressions than normal brain tissues. These affected tissues expressing higher levels of lamin C were also found to be stiffer than those from wild-type mice (LMNA gene expression is upregulated in Alexander disease-mutated astrocytes, the fact that the lamin C isoform is expressed at a disproportionately higher level than lamin A suggests that the LMNA mRNA splicing in these GFAP-mutated cells favors lamin C expression over that of lamin A, even more than it already did in normal astrocytes (Since ulations . In the ogenesis . The uprem cells . DespiteC and B1 . Based oC and B1 . Upregulype mice . This fiype mice . While ttrocytes . Altogettrocytes , lamin iLMNA gene prevent the correct synthesis of mature lamin A and C proteins (LMNA gene end up affecting one isoform more than another (LMNA mRNA splice variants into progerin (In the context of disease, laminopathies can result when mutations introduced into the proteins . Since tproteins . In many another . In the  another . The res another . When th another . This ha another . Therefoprogerin . Lamin Cprogerin . Patientprogerin .LMNA gene expression is dysregulated. One of the phenotypic traits of malignant cancer cells is their ability to metastasize. It has been proposed and demonstrated that nuclei from metastatic cells can change their lamin A and C expression levels to migrate from tissues to the bloodstream and vice versa into other tissues without experiencing irreversible damage and rupture (LMNA variant expressed in advanced aged and overexpressed in HGPS patients, was inserted in melanoma cells, resulting in nuclear stiffening but reduced metastatic potential of the cells (LMNA gene expression and cell stiffness are reduced while gaining stem-like properties similar to tumor initiating cells (LMNA gene expression, and since total lamin A + C expression is strongly correlated to mechanophenotype, the mechanophenotype of the cells is also sensitive to these changes. Yet, the picture becomes more complicated as recent studies suggest that lamin A and C isoforms might be expressed differently in various cancer types (LMNA mRNA splice variants coding for lamin C isoforms increase while lamin A isoforms decrease (Other diseases like cancer can also occur when  rupture . This hyhe cells . In anotng cells . Altogeter types . In breadecrease . TherefoLamin A and C isoforms show great promise as biomarkers of mechanophenotype when considering their expression as ratios of the entire lamin meshwork. Analyzing lamin A and C isoform ratios could provide end-point analysis metrics of whole-cell mechanophenotype in the context of lineage and/or microenvironment. On the other hand, monitoring isoform ratio shifts throughout time could illustrate dynamic changes in mechanophenotype that serve as biological signatures of disease onset or progression and lineage commitment of differentiating cells. Therefore, future experiments should consider the joint contributions of lamin isoforms to mechanophenotype as well as any instances of isoform switching that could be useful to predict phenotypic changes.RG-C, KD, and ED contributed to this perspective, originating from work conducted by RG-C and ED. RG-C wrote the first draft of the manuscript, with KD and ED contributed to manuscript revisions. All authors approved the final version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Glioblastoma (GBM) is the most common type of primary brain tumor in adults. Despite recent advances in cancer therapy, including the breakthrough of immunotherapy, the prognosis of GBM patients remains dismal. One of the new promising ways to therapeutically tackle the immunosuppressive GBM microenvironment is the use of engineered viruses that kill tumor cells via direct oncolysis and via stimulation of antitumor immune responses. In this review, we focus on recently published results of phase I/II clinical trials with different oncolytic viruses and the new interesting findings in preclinical models. From syngeneic preclinical GBM models, it seems evident that oncolytic virus-mediated destruction of GBM tissue coupled with strong adjuvant effect, provided by the robust stimulation of innate antiviral immune responses and adaptive anti-tumor T cell responses, can be harnessed as potent immunotherapy against GBM. Although clinical testing of oncolytic viruses against GBM is at an early stage, the promising results from these trials give hope for the effective treatment of GBM in the near future. Glioblastoma (GBM) is the most common and most aggressive primary malignant brain tumor in humans . GBM canThe recent success of cancer immunotherapy using immune checkpoint inhibitors (CPIs) against many otherwise untreatable cancers has raised expectations that such approaches could also be successfully used against GBM. For example, combination of anti-PD-1, anti-TIM-3, and targeted radiation showed impressive results in the preclinical mouse GL261 glioma model . The draOncolytic virotherapy is based on utilizing replicating viruses that can selectively kill the infected cancer cells. The virus-induced cell death can occur through a variety of different mechanisms, most  of which can be immunogenic (reviewed by ). DuringOncolytic viruses can also be used to transfer therapeutic payloads to the tumor. Viruses armed with immunoregulatory inserts such as interleukin 12 (IL-12) and OX40 ligand are currently being tested in clinical trials . Other eThe oncolytic viruses used against GBM in current trials are listed in +/CD4+ T cell ratio [+ T cells can be found in patient GBM samples, they often display a PD-1+, LAG-3+, TIGIT+, CD39+, KLRG1−, and CD57− profile, which is indicative of an exhausted phenotype of these cells [The GBM microenvironment has proven to be a particularly challenging target for immunotherapeutic approaches. This can be largely attributed to the naturally isolated and tightly controlled brain microenvironment, which GBM thrives upon and skews even more immunosuppressive. The overall T cell infiltration in GBM is relatively poor and has a low CD8ll ratio . Althougse cells . GBM celse cells . In factse cells , making se cells . It mustse cells . In precse cells . While hse cells . This in+ T cells and had the potency to induce anti-glioma immunity [Together with a low mutational burden and high heterogeneity, the immune evasive factors present in GBM (reviewed by ) are likimmunity . Of noteimmunity . Adding immunity ,47,48,49immunity , while vimmunity . Glioma immunity . These c+, T-bet+ T cell infiltration together with a reduction of TIM-3 expression [The ability of oncolytic viruses to induce therapeutically relevant immune responses against GBM (at least in subsets of patients) has been demonstrated in clinical trials ,21,52. Ipression . One clapression . Pseudop+/CD8α+ DCs, also known as type-1 conventional DCs, which excel at cross presentation of TAAs to CD8+ T cells [Dendritic cells (DCs) are professional antigen presenting cells that are essential in the induction of adaptive immunity. Owing to their capability to activate T cells, DCs function in the interface between the innate and adaptive immune system. DC activation has been shown to have an especially potent adjuvant effect in the promotion and development of antitumor immunity against GBM. For example, DC stimulation with Toll-like receptor 3 (TLR3) agonists enhances the anti-tumor immune response to anti-PD-1 therapy in the orthotopic GL261 glioma model . A poten T cells , are cruInterestingly, PVS-RIPO was also recently reported to be able to directly infect dendritic cells and macrophages . AlthougTaken together, the increasing amount of evidence suggests that oncolytic viruses can be used as potent immunotherapy against GBM. Given the immunologically cold GBM microenvironment, it is likely that potent “de novo” induction of antitumor T cell immunity is needed for effective therapy. In the context, oncolytic virotherapy can be seen to have an especially favorable effect, both disrupting the system by destruction of cancer cells and re-stimulating both the innate and adaptive immunity against the infected cancer cells.During recent years, the use of immune checkpoint inhibitors (CPIs) has shown great success in inducing long-term complete remissions in patients with otherwise refractory cancers . Since 2+ T cell responses against tumor cells, it can be expected that precursory oncolytic virotherapy would enhance the effectiveness of CPIs. The combination of checkpoint blockade with oncolytic virotherapy is also an attractive option because virus-induced inflammatory response in the tumor can lead to upregulation of PD1 on T cells and PD-L1 on tumor cells [CPIs work by blocking the negative regulators of T cell function, thereby sustaining T cell activity. Consequently, tumors with low T cell infiltration (such as GBM) are unlikely to get significant benefit from CPI therapy. In these cases, it would be important to induce T cell infiltration into the tumor prior to CPI therapy. As oncolytic viruses have a potent ability to induce CD8or cells . Taken t+ and CD8+ T cell infiltration in both infected and distant (non-infected) tumors, rendering them vulnerable to anti-CTLA-4 blockade [Many different viruses have been tested together with CPIs in different preclinical tumor models with encouraging results . For exablockade . As anotblockade . A notabblockade , VSV  and canSome viruses (such as SFV) have natural tropism to the central nervous system (CNS), which can be harnessed to achieve potent delivery of virus into the brain. At least, SFV ,48,71, vClassically, induction of virus neutralizing antibodies (NAbs) is considered to limit repeated virus injections or the use of virus strains that the patients’ immune system has previously encountered. Interestingly, studies by Berkeley et al. challenge this paradigm by showing that monocytes can uptake and internalize reovirus–NAb complexes (mediated by FcγRIII) and release functional replication competent reovirus to tumor . As GBM +/Ly6G+ and CD4+/FoxP3+ cells, but decreased Tc1 effector cells and CD11c+ DCs) [The first line of defense against viral infections is innate antiviral response, which is largely orchestrated by type-I interferons (IFN-I). IFN-Is are secreted by infected cells in response to PAMPs  detected by host cell pattern recognition receptors (PRRs). Generally speaking, the IFN-I response plays a major role in inducing an antiviral state  in the surrounding cells, thereby limiting viral spread. In addition, IFN-Is promote antigen presentation, natural killer (NK) cell function, and the development of antigen-specific adaptive immune responses . In the 1c+ DCs) . Also su1c+ DCs) . On the 1c+ DCs)  and cons1c+ DCs) . Autocri1c+ DCs) , probabl1c+ DCs) . Interes1c+ DCs) . As GBM Many of the currently used oncolytic viruses are either natural or the result of genetic engineering sensitive to the antiviral effect of IFN-Is. The rationale for use of such viruses is that cancer cells in many cases have dysfunctional IFN-I signaling . NotablyThe virus infection-induced release of IFN-I and other DAMPs attracts innate immune cells to eat and destroy the infected cells. While this is an important step in priming adaptive antitumor  immunity, the premature killing of infected cells can also lead to suboptimal virus spread in the tumor. For example, HSV therapy has been shown to increase infiltration of macrophages and microglia in the mouse GBM model . AlthougOn the basis of the findings in preclinical GBM models, temporal inhibition of innate responses in the early stage of viral therapy could lead to increased viral spread in the GBM microenvironment. This would in turn lead to more potent oncolysis and subsequent immunotherapeutic effect. Alternatively, viruses that can tolerate and escape the innate antiviral effect could be used (if safety can be assured). It is likely that preclinical GBM models do not perfectly recapitulate the innate immune microenvironment of human GBM, making direct translation of results difficult. In addition, human GBMs show high heterogeneity both within the same tumors and between different patients . BecauseThe use of viruses as oncolytic agents is not a particularly new idea. While the focus used to be on the direct oncolytic effect, the current working model of oncolytic virotherapy rather relies on utilizing viruses as immunotherapeutic agents. As oncolytic viruses can replicate in the target cells, they provide an intriguing possibility for one shot self-amplifying cancer therapy. On the other hand, it is also likely that repeated dosing could be beneficial  for the therapeutic effect through immunostimulatory effect as therapeutic vaccines. Intratumoral administration clearly has the benefit of delivering the virus directly to the tumor, thereby mostly evading virus neutralization by systemic antiviral antibodies, while the systemically delivered virus has the potential to break through the blood–brain barrier and reach tumor cells and sites not reachable by intratumoral injections. The effect on antitumor immune response from stimulating systemic antiviral immune response must be further studied before conclusions can be made. It is possible that combinations of intratumoral and intravenous doses of oncolytic viruses would produce synergistic therapeutic benefits by inducing both local and systemic immune responses. Direct side-by-side comparison of immune-related characteristics of the different viruses being developed and used in preclinical and clinical settings would also be valuable in order to evaluate the best candidates for GBM therapy. Nevertheless, recent phase I/II clinical trials demonstrate that the use of oncolytic viruses can significantly improve the survival of some GBM patients ,21,22. MThe addition of therapeutic payloads to oncolytic viruses to boost antitumor potency is an attractive option. The increased therapeutic effect of such an armed virus construct is naturally dependent on the efficient replication of the virus in the target cells. Robust replication-induced cell death can, however, lower the amount of transgene produced by the infected cell. However, adding genes or replacing viral genes with therapeutic ones can hamper viral replication potency. Therefore, any oncolytic virus construct that depends on delivery and expression of therapeutic genes must be carefully optimized. It must also be considered that excessive expression of immunoregulatory transgenes might have unwanted toxic side-effects . The GBM microenvironment presents a challenge for oncolytic virus delivery into the tumor, as well as effective viral replication and spread within the tumor B. AlthouMore work is needed to fully unlock the potential of viruses as effective anticancer agents. However, virus-based immunotherapy clearly shows promise in leading to curative treatment of currently incurable tumors such as GBM in the near future.


Delivery of genes, including plasmid DNAs, short interfering RNAs (siRNAs), and messenger RNAs (mRNAs), using artificial non-viral nanotherapeutics is a promising approach in cancer gene therapy. However, multiple physiological barriers upon systemic administration remain a key challenge in clinical translation of anti-cancer gene therapeutics. Besides extracellular barriers including sequestration of gene delivery nanoparticles from the bloodstream by resident organ-specific macrophages, and their poor extravasation and tissue penetration in tumors, overcoming intracellular barriers is also necessary for successful delivery of nucleic acids. Whereas for RNA delivery the endosomal barrier holds a key importance, transfer of DNA cargo additionally requires translocation into the nucleus. Better understanding of crossing membrane barriers by nucleic acid nanoformulations is essential to the improvement of current non-viral carriers. This review aims to summarize relevant literature on intracellular trafficking of non-viral nanoparticles and determine key factors toward surmounting intracellular barriers. Moreover, recent data allowed us to propose new interpretations of current hypotheses of endosomal escape mechanisms of nucleic acid nanoformulations. Cancer gene therapy remains a significant challenge due to numerous barriers limiting delivery of genetic cargo. In contrast to a vast majority of nanoformulated chemotherapeutic drugs, nanoparticles for nucleic acid delivery have to reach the specific intracellular compartment; either cytosol for siRNA and mRNA, or nucleus in case of DNA. The importance of overcoming these generally conserved (applicable to numerous cell types within the cancer milieu) intracellular barriers is increasing as additional genetic manipulation technologies, such as the CRISPR/Cas9 system, will all require delivery to specific intracellular compartments to be effective and clinically relevant.Despite advantages of viral vectors in terms of gene delivery efficacy, their use may cause immune responses and severe side effects  resultinThere are numerous reviews on the topic focusing mostly on recent advances in chemistry of nanocarriers for improving nucleic acid delivery without detailed description of the mechanisms . The purFigure 1). Internalization through phagocytic pathways occurs primarily in monocytes/macrophages, neutrophils, and dendritic cells . In particular, early endosomes lose tubular membrane structures which become the endocytic recycling compartment and provide retrograde transport of phospholipids and receptors to plasma membrane. Some fraction of internalized nanoparticles with genetic payload might be recycled by this route phosphate (BMP), also known as lysobisphosphatidic acid (LBPA), which along with ESCRT-III complex is involved into ILV formation P2], which regulates sorting of endocytic membranes for delivery to the trans-Golgi network .Upon uptake, endosomal vesicles undergo a maturation process driven mainly by phosphatidylinositols (PIs) in the cytosolic leaflet of the vesicle bilayer  and Rab is route , but thiis route . All theis route . MVBs arormation . Further network . This mer milieu . Transfor milieu , highly Figure 1).For delivery of DNA another essential barrier is the passage through the nuclear envelope. Nuclear entry of macromolecules occurs via channels of the nuclear pore complex (NPC) in a size-dependent manner. For example, macromolecules with size less than 9 nm (or 40 kDa molecular weight) are able to diffuse passively through the NPC. Import of larger molecules can occur through an energy- and signal- dependent active process . RegardiThus, delivery of genetic cargo to cytosol or nucleus is a complicated multi-step process affected by numerous factors. The success of nucleic acid delivery is a result of overcoming all mentioned barriers for which mechanisms and impacts are discussed below.Endocytic routes are the primary internalization mechanism for nucleic acid nanoformulations. Although non-endocytic mechanisms such as fusion of lipoplexes with the plasma membrane and pore formation on the cell membrane have been reported, the data confirming their involvement in nucleic acid delivery process are controversial, and direct proofs are still lacking . Since eνβ3 integrin . At the same time, a significant fraction of cargo stayed within endosomes as well as the lipid part of lipoplex   starting after 5–15 min of uptake by HeLa cells. Endosomal release of siRNA occurs instantly, and is followed by rapid (10–20 s) diffusion of siRNA throughout entire cytosol  .in vitro mimicking of lipid/DNA nanoparticle interactions with membranes containing the negatively charged phospholipid phosphatidylserine (PS) (jj phase) in endosomes and release of nucleic acids to the cytosol  . Accordi cytosol .2. However, they are also localized in the cytosolic leaflet of endosomal bilayers with PS, and their abundance is relatively low. An additional consideration is that MVBs are enriched with BMP/LBPA, an unconventional anionic phospholipid, which may play a primary role in endosomal release of genetic payload from lipoplexes.However, some questions still remain. For example, why lipoplexes discharge genetic payload in maturing endosomes, whereas the PS content in these compartments is as a minimum twofold less than in early endosomes ? Why a pin vitro that BMP/LBPA-containing liposomes are able to form ILVs at acidic intraluminal pH (pH-dependent) resembling MVBs , whereas PS flipping from cytosolic leaflet is thermodynamically restricted because of charged headgroups -based polyplexes escape from endosomes at approximately 30 min after uptake by HeLa cells. This process occurs instantly and is accompanied by partial release of PEI together with genetic payload. Discharge of genetic payload in case of polyplexes occurs from LAMP-1-positive vesicles corresponding to late endosomes/lysosomes . FurtherFigure 3A) responsible for osmotic swelling of endosomes, followed by the lysis of endocytic PEI-containing vesicles that supported the “proton sponge” hypothesis , whereas according to the “proton sponge” mechanism, pH should increase due to PEI buffering capacity  and PEI can permeabilize supported lipid bilayers and even cell plasma membranes   .Figure 3D). According to our model, both buffering capacity of polycation and its ability to interact with anionic lipids like BMP/LBPA and permeabilize endosomal membrane are important for endosomal escape.Our interpretation of these experimental data considers direct interaction of cationic part of polyplexes with negatively charged phospholipids, primarily with BMP/LBPA. Regarding buffering properties of the carriers, they could significantly facilitate endosomal escape. First, protonation of polycation may lead to more strong electrostatic interaction with negatively charged lipids, resulting in membrane permeabilization. Second, even though luminal pH does not increase, influx of chloride ions promotes osmotic swelling of endosomes. Upon reaching a critical value, osmotic pressure imparts mechanical impulse to nucleic acids and part of associated polycation leading to their discharge from endosome  composite is the oldest non-viral gene carrier; introduced in 1973 . CaP is ficacies . Probablvia thermal and non-thermal mechanisms  irradiation and control nucleic acid release chanisms , 2012. Achanisms . Local hchanisms . Howeverchanisms , which cCarbon nanotubes have a unique property of endocytosis-independent cell penetration due to the so-called “needle” mechanism and may completely avoid the endosomal barrier . Thus, tTwo other types of inorganic delivery vehicles, namely, silica-based  and supeTable 1). First, co-treatment of cells with lysosomotropic agents such as chloroquine, sucrose, polyvinylpyrrolidone (PVP) and others can be used -2-aminoethyl]aspartamide}-poly(L-lysine) for DNA complexation and providing long circulation, and dendrimeric phthalocyanine as the photosensitizer. Obtained micelles demonstrated 100-fold photoenhanced gene expression in HeLa cells and site-specific transfection of tumor cells . Numerous peptidic moieties have been proposed for this aim including fusogenic virus-derived  or synthg tissue  and Lewig tissue . In spitin vitro and in vivo s or PEI, facilitate endosomal release and significantly enhances transfection efficacy both  in vivo .N-acetylgalactosamine  moieties for targeting hepatocytes . The first approach considered covalent conjugation of DNA with a peptidic nuclear localization signal (NLS), responsible for active importin-mediated nuclear transfer of large cytoplasmic proteins. This approach improved nuclear transfer of plasmid DNA with attached SV40 viral NLS peptide in digitonin-permeabilized cells, but not in intact cells after microinjection of the modified DNA  was proposed to improve NPC-mediated nuclear uptake of DNA. This amphipathic alcohol temporary perturbed the barrier function of the NPCs and facilitated nuclear entry of dextrans and naked plasmid DNA. Moreover, TCHD treatment of cells enhanced lipoplex-mediated transfection (in vivo. It should be noted that the barrier function of NPC can be decreased by conjugation of gene delivery nanocarriers with a ligand moiety to the cytoplasmic glucocorticoid receptor. Interaction with this receptor induces NPC dilation up to 300 nm in diameter and nuclear translocation of ligand/receptor complex (trans-retinoic acid (ATRA) to cytoplasmic retinoic acid receptor also promotes nuclear translocation of ligand/receptor complex via a similar mechanism as in the case of glucocorticoid receptor. In spite of promising in vitro results for ATRA-targeted polyplex-based carriers  are able to penetrate the nucleus by passive diffusion through the NPC . As far fied DNA . The simfied DNA , but a lfied DNA . Anotherfied DNA . Howeversfection . However complex . The adv complex . Targeticarriers , in vivoterparts . Howeverterparts .It is commonly accepted that the primary way of foreign DNA entry is a passive entrapment during mitosis, when the nuclear envelope is disassembled starting from late prophase up to late anaphase, and its components (proteins and membranes) are associated with the endoplasmic reticulum . Thus, tMitosis-dependent transfection of cells may be significantly limited in cell lines with infrequent cell division. Remarkably, even without NLS and other nucleus-targeting sequences, lipoplex- or polyplex-formulated DNA can reach the nucleus in a mitosis-independent manner according to live-cell microscopy data . Some daIntracellular penetration of nucleic acid nanoformulations seems to be a relatively effective process, although several studies state that cellular uptake efficacy has a direct impact on the transfection process . Hence, lp and polyplexes Npp. The probabilities that transfected cells will get two types of DNA fragments in assumption of their release from different endosomal vesicles are 1 – 0.5Nlp-1 = 0.8 for lipoplexes, and 1 – 0.5Npp-1= 0.23 for polyplexes . Resulting in values of ppN = 1.4 and lp =N 3.3, indicating almost twofold higher efficiency of endosomal escape in case of lipoplexes, although this conclusion only applies to certain carriers (22 kDa linear PEI for polyplexes and Lipofectamine 2000 for lipoplexes) used in this study. Actually, between one and five release events per cell over some hours have been observed for polyplexes . PEGylation not only extends circulation time, but also improves diffusion-mediated permeation of tumor stroma due to minimization of electrostatic interactions with extracellular matrix (It should be noted that some aspects of nanoparticle tuning for r matrix . At the r matrix . Anotherr matrix . In extrr matrix , althougr matrix , as wellr matrix , are notin vivo application enhancing selectivity of tumor-targeted delivery, but potentially may impair nanoparticle penetration of tumor stroma due to “binding site barrier” effect detected for small targeted nanoparticles (Another requirement of nanocarriers for delivery of nucleic acids is efficient internalization, which besides positive surface charge, can be modulated by functionalization with a ligand moiety. This modification for improvement of non-viral vectors on a cellular level has minimal conflict with articles . For solarticles .in vitro use, very strict analysis of safety and biodegradability properties of nanocarriers should be carried out for clinical translation. Many types of cationic lipids or polycations commonly used for in vitro applications cannot be translated in vivo due to these issues. Additionally, modification of nanocarriers with virus- or bacteria-derived CPPs potentially holds immunogenicity limitations.Finally, as opposed to in vivo requirements and maintains relatively high efficacy of overcoming intracellular barriers. We believe that ongoing (As a result, in spite of a diversity of nucleic acid delivery systems, only a limited number fulfills these  ongoing  and futuDespite progress in studying mechanisms of cell transfection by non-viral vectors and elucidation of the impact of intracellular barriers on transfection efficacy, some important information is still lacking. For instance, it is still unknown how endosomal phospholipids interact with nucleic acid nanoformulations. Based on knowledge about endosome maturation process, we hypothesized that negatively charged BMP/LBPA might be an important participant of this process. Additionally, clinically relevant mitosis-independent mechanisms of DNA translocation into the nuclei of cancer cells are unknown, ineffective, and not well-managed. Probably, their identification and exploitation will significantly improve transfection efficacy of quiescent tumor cells with DNA-containing non-viral nanoformulations.Elucidation of transfection mechanisms is very important as it creates a strong basis for novel directions of nanocarrier improvement. We believe further analysis/understanding of endosomal escape and nuclear localization mechanisms will advance nucleic acid nanoformulations toward higher efficiency and enable clinical translation for numerous applications including cancer gene-based therapy.Both authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.


High-dose methotrexate is used to treat a range of adult and childhood cancersincluding osteosarcoma. Significant neurotoxicity is reported in 1% to 4.5% ofpatients treated with high-dose methotrexate and can present in a wide varietyof symptoms. We present a case of a 14-year-old boy with a recent diagnosis ofosteosarcoma who presented to the emergency department with status epilepticus,altered mental status, and very high fever secondary to methotrexateneurotoxicity. We review current literature and discuss some controversiesrelated to this state. We also describe high fever as one of the possiblesymptoms associated with this condition and suggest using specific magneticresonance imaging sequence to uncover abnormal findings related to this state.Since high-dose methotrexate is not a rare treatment in this era, we believethat in addition to oncologists, emergency department and intensive careproviders should be aware of the potential role of methotrexate in causingsignificant neurotoxicity and include it in the differential diagnosis whentreating a patient presenting with new neurological symptoms in the setting ofrecent high-dose methotrexate treatment. Status epilepticus (SE) and altered mental status (AMS) are among the most commonemergency states encountered by emergency department (ED) and intensive careproviders. These states often require prompt evaluation and aggressive stabilizationdone in a timely manner and per the patient’s background and circumstancessurrounding his/her presentation. In some cases, the etiology of SE and AMS isobvious  while in other cases theetiology is more obscure, necessitating more thorough “digging” into the patienthistory and more use of ancillary tests.In this report, we present a case of a 14-year-old boy with a recent diagnosis ofosteosarcoma who presented to the ED with SE and AMS secondary to subacutemethotrexate (MTX) neurotoxicity 5 days after he finished his second dose ofintravenous high-dose MTX (HDMTX) treatment. We review current literature anddiscuss some controversies related to this state. We also describe high fever as oneof the possible symptoms associated with this condition and suggest using specificmagnetic resonance imaging (MRI) sequence to uncover abnormal findings related toMTX neurotoxicity.2, single dose, based on the American Osteosarcoma Study Group0331 [EURAMOS-1] protocol [ClinicalTrials.gov Identifier: NCT00134030]). Twenty-fourhours following HDMTX he had a generalized tonic-clonic seizure for the first timein his life. The seizure lasted 5 minutes and was stopped pharmacologically with asingle dose of midazolam. His electroencephalography was normal. MTX levels werefollowed and found to be in the nontoxic range . Seizures did not recur for 48 hoursand the patient was discharged home on levetiracetam treatment.A 14-year-old boy with a recent diagnosis of osteosarcoma of the right tibia withoutany known metastasis presented to the ED with SE and AMS. Five days prior to hispresentation the patient underwent his second course of HDMTX  that resolved completely as the patient stoppedseizing. His initial complete blood count showed leukocytosis and elevatedneutrophils  and C-reactiveprotein was mildly elevated . Head computed tomographywas normal without any focal findings. A subsequent brain MRI revealed subtlediffusion restriction in the posterior subcortical white matter, more prominent onthe left periventricular white matter, extending to the parietotemporal and centrumsemiovale areas. Signal changes were more prominent on the apparent diffusioncoefficient map than on the diffusion-weighted imaging ,eye opening, and moaning. His initial glucose and electrolytes, kidney, and liverfunction tests were all normal. Arterial blood gas revealed respiratory acidosis  in combination withhigh-dose steroids (dexamethasone). During his stay in the pediatric intensive careunit the patient remained seizure free, did not require hemodynamic or respiratorysupport, and continued to have persistent high fevers. Due to mild skin erythema andlimb swelling near his central venous catheter, the catheter was removed and thepatient completed 7 days of intravenous antibiotics for a “rule out” cellulitis.Repeated electroencephalography while obtunded, showed paroxysmal generalized deltawave activity with epileptiform activity in the temporal lobe consistent withencephalopathy. There was no improvement of the neurological examination until thefourth day of admission (~72 hours following presentation to the ED) when thepatient was able to open his eyes spontaneously and follow verbal commands. At thattime his neurologic examination was significant for central facial palsy, relativeweakness of his left upper and lower extremities (motor strength of 3-4/5), andanisocoria of his pupils (left > right). The patient underwent a repeated headcomputed tomography with angiography that did not show any signs for vascular strokeor diminished perfusion. The new neurological findings were attributed to MTXneurotoxicity versus Todd’s paresis. In the next few days, the patient recoveredalmost completely with only minimal residual neurological deficits . His fever subsided by day 4 andhe was transferred to the oncology floor for further observation.2 or higher, is used to treata range of adult and childhood cancers including acute lymphoblastic leukemia,central nervous system lymphomas, leptomeningeal metastases, andosteosarcoma.14High-dose MTX, defined as a dose of 500 mg/m5 Of note, neoplastic cells often have innate or acquire resistance to MTX,which may hamper MTX efficacy and can lead to treatment failure. At least 5mechanisms had been suggested to explain this phenomenon, one of which is decreasedMTX accumulation inside the cells due to impaired transporters function.6 In addition, several MTX transporters polymorphisms had been linked to higherplasma levels of MTX and worse prognosis.7MTX acts as an antimetabolite by interfering with the metabolism of folic acid. Itbinds to the intracellular enzyme dihydrofolate reductase with an affinity that isabout 1000-fold greater than folate. By doing so, MTX indirectly inhibits theconversion of dihydrofolate to tetrahydrofolate (THF), and since THF has anessential role in DNA synthesis, blockade of THF synthesis leads to inability ofcells to produce proteins and divide.812 With regard to HDMTXneurotoxicity, neurological symptoms are reported in 1% to 4.5% of patientsreceiving HDMTX within 2 weeks of initiation of treatment. Symptoms can follow anacute, subacute, or chronic course with variable manifestations includinghemiparesis or other stoke-like symptoms, ataxia, dysphasia, encephalopathy,seizures, headaches, and weakness.14 In cases of acute or subacuteencephalopathy, the most frequently seen neurotoxicity following HDMTX, clinicalsymptoms are often associated with leukoencephalopathy (white matter hyperintensityon T2-weighted and fluid attenuated inversion recovery MRI).15 Interestingly,leukoencephalopathy can be found in as many as 20% of asymptomatic patients treatedwith HDMTX.14 The pathogenesis of HDMTX-associated neurotoxicity is not clear, althoughseveral hypotheses have been proposed, including homocysteine toxicity, alteredfolate homeostasis, adenosine release, and/or direct neuronal damage byMTX.1618HDMTX therapy can cause significant toxicity, which can lead to substantial morbidityand mortality. Nephrotoxicity, hepatotoxicity, pulmonary toxicity, dermatologictoxicity, and neurotoxicity have all been described in association with MTXtreatment.18 and dextromethorphan (1-3 mg/kg), an N-methyl-D-asparate receptor antagonistthat inhibits homocysteine activity.20 The role of steroids in thissetting is not clear. HDMTX neurotoxicity with low blood levels of MTX should bedifferentiated from neurotoxicity associated with MTX overdose with toxic bloodlevels of MTX. In MTX overdose intrathecal administration of carboxypeptidase G2(CPDG2) is warranted. CPDG2 rapidly hydrolyzes MTX to inactive metabolites.21 Other treatments that had been suggested in cases of overdose arecerebrospinal fluid drainage, ventriculolumbar perfusion, systemic leucovorinadministration, and urine alkalization.22 There is no evidence thatCPDG2 and the other mentioned modalities have a role in neurotoxicity in the absenceof toxic blood levels of MTX.Currently there is no standard treatment for neurotoxicity related to HDMTX. Smallseries suggest neurological symptoms can be relieved by aminophylline (2.5 mg/kg)via competitive inhibition of adenosine14 followed 369 children with ALL who were treated with HDMTX of which 14exhibited MTX-related neurotoxicities. Most episodes were brief, and all but onepatient were successfully rechallenged with high-dose MTX and/or intrathecaladministration of MTX after resolution of symptoms. The authors concluded thatstopping MTX treatments following episodes of resolved neurotoxicity is unnecessary.14Acute and subacute encephalopathy related to HDMTX is usually transient, withrecovery occurring within 1 to 7 days of symptoms onset. Chronic encephalopathydevelops more slowly and may result in permanent neurological deficits. Whether torechallenge patients with MTX related neurotoxicity with repeated doses of HDMTX isa matter of debate. Bhojwani et alIn our case report, we describe a patient who presented to the ED with SE andAMS/encephalopathy who underwent an extensive evaluation leading to the diagnosis ofMTX neurotoxicity. Other important etiologies like brain metastases, brainhemorrhage, ischemic stroke, sepsis, and electrolytes abnormalities were excluded.For the most part his clinical course followed the known course of acute/subacuteneurotoxicity related to MTX as described above. The only exception was hisconsistent high fevers in the absence of clear source of infection (the redness andswelling of his left arm were only mild and not impressive enough to explain thehigh fevers he had) that resolved in parallel to the improvement in his neurologicalexamination. As far as we know this is the first report associating high fever withMTX neurotoxicity. We also suggest that using apparent diffusion coefficientsequence on MRI can potentially uncover abnormal signals that are not clearly andeasily seen on other sequences. Of note, possible polymorphisms of MTX transporterswere not tested in this case.HDMTX is not a rare treatment in this era. In addition to oncologists, ED and ICUproviders should be aware of its potential role in causing significant neurotoxicityand include it in the differential diagnosis when treating a patient presenting withnew neurological symptoms in the setting of recent HDMTX treatment.


Localized hepatobiliary tuberculosis (TB) is a rare disorder which can present with an obstructive jaundice mimicking other noninfectious causes such as cholangiocarcinoma. Here, we report a case of porta hepatis tuberculosis in 19-year-old female who presented with an obstructive jaundice, and her computed tomography (CT) of the abdomen revealed a hepatic hilar mass with radiological features mimicking a malignant biliary tumor. We also review the literature related to this disorder. Mycobacterium tuberculosis is a contiguous and potentially fatal disease that usually affects the lungs. However, it can be extra-pulmonary in 15-20% of the cases of which abdominal tuberculosis (TB) accounts for 11-15% and hepatic TB for less than 1% . Extra-pThe clinical and radiological manifestations of hepatobiliary TB can be mistaken for malignant lesions leading to erroneous clinical diagnosis and unnecessary surgical intervention. We report a case of localized TB of the porta hepatis in a 19-year-old female who presented with a clinical picture of an obstructive jaundice. CT abdomen and pelvis showed a hepatic hilar mass with features suggestive of a malignant lesion that was further evaluated with a liver magnetic resonance imaging (MRI). In addition to the redemonstration of the hepatic hilar mass, MRI revealed features of liver microabscesses which were not visible in the CT scan. The additional findings from the MRI pointed toward TB of the porta hepatis as an alternative differential diagnosis. Our case report highlights the importance of considering TB of the porta hepatis within the differential diagnosis of liver hilar mass in a young patient with obstructive jaundice, and to our knowledge it is the first case to demonstrate the usefulness of MRI as a problem-solving tool to suggest preinterventional diagnosis.A 19-year-old Omani female not known to have any significant medical history was referred to our hospital with a history of upper abdominal discomfort more localized to the epigastric region and associated with jaundice and dark urine. There was no history of fever or night sweat nor history of travel. On physical examination, the patient was jaundice; otherwise, the systemic examination was unremarkable.∗g/L). Liver function test revealed a picture of obstructive jaundice with a total bilirubin of 52 umil/L, Alkaline phosphatase 302 [iU] /L, and Alanine transaminase 457 [iU]/L. QuantiFERON-Tb gold test was positive.Complete blood count was within normal limits with a normal white blood count , probable TB diagnosis was made based on the histological and imaging findings, and the patient was treated for 6 months with Rifampicin 150 mg and Isoniazid 75 mg. The patient showed significant response to treatment with dramatic improvement of liver function test. A follow-up liver function test 3 months after starting anti-TB medication showed normalization of Alkaline phosphatase 91 [iU] /L along with improvement of total bilirubin of 24 umil/L and Alanine transaminase 211 [iU]/L. After completion of anti-TB medication, liver function test was back to normal with a total bilirubin of 17 umil/L, Alkaline phosphatase 58 [iU] /L, and Alanine transaminase 16 [iU]/L. A follow-up CT abdomen was performed 10 months later and showed complete resolution of porta hepatis mass and intrahepatic biliary dilatation along with significant regression of previously noted enlarged upper abdomen lymph nodes.Obstructive jaundice caused by tuberculosis is a rare disorder which can mimic other noninfectious causes and can be overlooked due to low incidence. Unlike diagnosis of pulmonary TB, establishing the diagnosis of extra-pulmonary tuberculosis can be challenging due to nonspecific clinical symptoms and radiological findings.Patient with abdominal TB can present with abdominal pain, low-grade fever, hepatosplenomegaly, and loss of weight and appetite , 9. BiliBiological clues to the presence of biliary TB are not specific. Patients with biliary TB can have an impaired liver function test with a cholestasis picture secondary to obstruction, mimicking other noninfectious causes such obstructive biliary stone, primary sclerosing cholangitis, and a malignant cholangiocarcinoma .Radiological diagnosis of biliary tuberculosis can be a challenging diagnosis with variable imaging findings ranging from bile duct thickening, biliary dilatation, and strictures to complete biliary obstruction . On ultrConcomitant radiological features of TB elsewhere such as pulmonary or nodal involvement can help to narrow the differential diagnosis. In one series, chest radiographic changes consistent with pulmonary TB were observed in 4 out of 14 patients with hepatobiliary TB . In our Magnetic resonance cholangiopancreaticography (MRCP) is noninvasive imaging modality which has better tissue characterization than the US and the CT. In hepatic tuberculosis, lesions appear hypointense on T1-weighted images and hypointense, isointense, or hyperintense lesions with a peripheral hypointense rim on T2-weighted images depending upon the stage of the disease . In our The definite diagnosis of hepatobiliary tuberculosis requires histopathologic evidence of caseating granuloma or demonstration of acid fast bacilli (AFB) on smear or culture of biopsy specimen. This can be achieve by obtaining a tissue sample by laparoscopy or endoscopic ultrasound with fine needle aspiration (FNA) . PositivLocalized hepatobiliary TB is a rare disorder which can mimic other noninfectious causes. The current case highlights the importance of considering TB of the porta hepatis within the differential diagnosis of liver hilar mass in a young patient with obstructive jaundice, and the usefulness of MRI for suggesting preinterventional diagnosis.


We describe a case of a 71-year-old male with an isolated recurrence of urothelial carcinoma in an ileal neobladder without involvement of the upper urinary tract or urethra. He was diagnosed with high grade urothelial carcinoma involving a bladder diverticulum with associated carcinoma in situ. He underwent a radical cystectomy and orthotopic Studer ileal neobladder. On routine follow-up, 11 years following cystectomy, voided urine cytology was positive for high grade urothelial carcinoma. Further workup revealed normal upper urinary tracts, normal urethra, and a solitary lesion at the left anteroinferior wall of the neobladder. He subsequently underwent resection of the neobladder and conversion to an ileal conduit with pathology confirming the diagnosis of high grade urothelial carcinoma. Isolated recurrence of urothelial carcinoma within a neobladder without involvement of the upper urinary tract or urethra is rare. No guidelines exist regarding its management. Herein we present our management as well as the current literature published on this topic. The orthotopic ileal neobladder has become the preferred method of continent urinary diversion following radical cystectomy for bladder cancer. Urothelial carcinoma recurrence has been widely reported involving the urethra –3, upperA 71-year-old gentleman who was diagnosed at age of 59 with high grade urothelial carcinoma involving a bladder diverticulum with associated carcinoma in situ underwent a radical cystectomy, prostatectomy, extended lymphadenectomy, and orthotopic Studer ileal neobladder. Pathology confirmed a high grade urothelial carcinoma arising in a right-sided diverticulum with extension into the perivesical fat and associated carcinoma in situ. There was no evidence of extension into the urethra or ureters and all surgical margins were negative. None of the 40 lymph nodes removed showed carcinoma (pathologic stage pT3aN0Mx). He underwent adjuvant chemotherapy , but only tolerated 2 cycles before discontinuing due to side effects. He continued to have surveillance imaging and follow-up every 6-12 months.On routine follow-up, 11 years following cystectomy, voided urine cytology was positive for high grade urothelial carcinoma. Follow-up CT imaging showed normal upper tracts and interval development of focal mural thickening involving the left anteroinferior neobladder . He undeHe subsequently underwent resection of the orthotopic neobladder and conversion to an ileal conduit. Intraoperatively, the mass involved the neobladder with possible extension into the pelvic sidewall and pubic bone periosteum. All gross disease was resected. Frozen section margins from pubic bone periosteum and urethra were negative. On gross examination of the final pathologic specimen, a 4.2cm x 2.3cm exophytic, friable mass was noted protruding into the neobladder lumen. Upon sectioning, this mass was noted to have variable depths of invasion, the highest being 9mm through the mucosa, submucosa, and serosa with involvement of the surrounding adipose tissue. Final pathology confirmed high grade urothelial carcinoma involving the neobladder and extending into the surrounding fat (Figures An isolated recurrence of urothelial carcinoma in an ileal neobladder without involvement of the upper tract or urethra is rare. To our knowledge, only 3 case reports have been previously published in the literature  9–11]. –11. 9–11The mechanism of local recurrence in the mucosa of the ileal neobladder is unclear. The development of recurrent solitary or multifocal tumors in the urinary tract is a well-known characteristic of urothelial cell carcinoma. After urinary diversion, recurrences have been reported to occur in the urethra –3, upperWe report a rare case of urothelial carcinoma recurrence without involvement of upper tracts, ureteroileal anastomosis, or urethra, in an orthotopic ileal neobladder 11 years after radical cystectomy. Our patient underwent resection of the neobladder and conversion to an ileal conduit. At his most recent follow-up visit 3 months later, he continued to be without evidence of recurrence or metastasis. No guidelines exist regarding the management of urothelial carcinoma recurrence. Tumor recurrences have been reported up to 12 years from initial cystectomy, making it imperative that these patients have long-term follow-up.


Chromosome organization poses a remarkable physical problem with many biological consequences: How can molecular interactions between proteins at the nanometer scale organize micron-long chromatinized DNA molecules, insulating or facilitating interactions between specific genomic elements? The mechanism of active loop extrusion holds great promise for explaining interphase and mitotic chromosome folding, yet remains difficult to assay directly. We discuss predictions from our polymer models of loop extrusion with barrier elements and review recent experimental studies that provide strong support for loop extrusion, focusing on perturbations to CTCF and cohesin assayed via Hi-C in interphase. Finally, we discuss a likely molecular mechanism of loop extrusion by structural maintenance of chromosomes complexes. Functionally, TADs are believed to demarcate coherent cis neighborhoods of gene-regulatory activity and hence are crucial for development  , often e corners , grids oboundary  velocity along the chromatin fiber (kb/sec)separation between LEFs (kb)permeability of the extrusion barriers (probability)processivity (kb), which indicates the average size of a loop that a LEF would extrude if left unobstructed. Motivated by observations of CTCF motif orientations at TAD boundaries and at peaks . Barriers can be modeled as either halting LEFs as long as the blocking factor is present, or stalling them until LEF dissociation , as a function of genomic separation, s. Local compaction is seen as a region of P(s) with a shallow slope (~100–500 kb), which we refer to as the shoulder  the shoulder ; decompaP(s) curve. This differentiates our predictions for CTCF depletion from those for cohesin depletion.For the depletion of site-specific extrusion barriers, as imposed by CTCF, our simulations also predict the loss of TADs and associated Hi-C peaks . HoweverP(s), as opposed to how it recedes in the case of cohesin depletion. Macroscopically, sufficient compaction would cause chromosomes to condense into a prophase-like state with a cohesin-rich central scaffold.For the depletion of a cohesin unloading factor, like Wapl, our model predicts that the consequent increased processivity and number of LEFs would lead to several phenotypes : (i) peaCrucially, our model predicts that the loss of cohesin loop extruders and the loss of CTCF extrusion barriers should both lead to the loss of TADs and Hi-C peaks, yet in completely distinct fashions. Furthermore, increased processivity of cohesin extruders is predicted to manifest distinct phenotypes on Hi-C maps and macroscopic chromosome organization.Whereas perturbing CTCF and cohesin dynamics is crucial for testing predictions of loop extrusion, depletion of such essential complexes poses many experimental challenges. For CTCF, cells begin dying after ~4 days of stringent depletion . For cohP(s) shoulder   and acutshoulder . Decompashoulder . We noteshoulder  saw limiA corollary of the Nipbl depletion result is that cohesin must be constantly loaded on chromatin to maintain TADs and associated corner peaks. Consistently, TADs and Hi-C peaks are both rapidly lost upon AID-mediated degradation of Rad21 (<3 h ) and reeFuture studies will be useful to dissect the dynamics of the processes and the potential role of Nipbl beyond that of a loader . In partP(s) curve did not change, implying that although demarcation of contact-insulating boundaries in Hi-C maps was lost, the same degree of chromatin compaction was maintained. In support of the dynamic exchange of LEFs in our model, the effect of CTCF depletion was fully reversible following a 2-day wash-off period  curve and, (v) the emergence of prophase-like vermicelli chromatids via imaging  and in the emergence of vermicelli.Depletion of another component of the cohesin unloading machinery, Pds5A and Pds5B (Pds5A/B), led to many of the same phenotypes . HoweverCollectively, the congruence of both Hi-C and imaging experiments following the perturbation of CTCF and cohesin dynamics strongly supports the role of loop extrusion in interphase. Future simulations and experiments will be valuable for probing the consequences of multiple simultaneous perturbations .track consistently in cis along chromatin, over large distances (up to tens-of-thousands of nucleosomes) without falling off. Moreover, the substrate, chromatin, is highly disordered due to nucleosomes and other DNA-bound proteins, likely posing a greater challenge than tracking along microtubules performed by cytoplasmic motors. Here we discuss how recent single molecule experiments argue that loop extrusion likely occurs via an active process, driven by molecular motors. Although many of these observations were made with condensin and bacterial SMCs, they illustrate that loop-extrusion is a plausible mechanism of action for the whole family of SMC proteins, including cohesin.Although providing strong support for chromosome folding by loop extension in vivo, the studies discussed above do not directly probe the molecular details of loop extrusion. Molecularly realizing the process of loop extrusion presents a considerable challenge, namely, that the protein complexes performing loop extrusion need to Recently,  showed tThe high structural homology of cohesin to condensin makes it likely that the same physical mechanism would govern its processive motion, in addition to its established role of mediating sister chromatin cohesion . Indeed,Although strongly supporting the loop extrusion mechanism, the single-molecule experiments leave open several questions of how loop extrusion can work in vivo:How can SMCs translocate on chromatinized rather than naked DNA?How can translocation result in loop extrusion?Is the measured speed of translocation sufficient to generate TADs and peaks?Do cells have sufficient ATP budgets to support extrusion during interphase?In particular, it remains to be understood how SMC complexes can translocate on chromatin fibers rather than naked DNA. Translocations while maintaining constant contact with DNA may not always be possible due to the complexity of chromatin fiber and abundance of DNA-bound proteins. Although the size of an SMC complex (~50 nm) exceeds that of a single nucleosome (~10 nm), nucleosomes would constitute challenging obstacles for SMC translocation if maintaining constant contact with DNA is required for translocation.walking mechanism would allow translocation where obstacles such as nucleosomes and other DNA-bound proteins can be passed over, avoiding disruptions of the underlying nucleosomal array. During each step of the walking process, one SMC head can remain DNA-bound, whereas the other hops forward and rebinds nearby DNA  a single translocating motor attached to a chromatin anchor; (2) two connected motors translocating in opposite directions; (3) a single motor that switches between two chromatin fiber substrates . These aThe measured rates of stepping and step sizes for condensin  agree weWe note that pushing by RNA Pol II alone, at its reported velocities, would be too slow (~1.5–3 kb/min ). The ob5 ATP/sec). This constitutes <0.02% of the 109 ATP/sec production rate by a fibroblast (A simple estimate shows that the energy burden of ATP consumption by loop-extruding cohesins in interphase is negligible as compared to ATP production in a mammalian cell. Again using 2 ATP per sec per SMC complex , and thebroblast . Thus thWhile in proofs, a paper  appearedAlthough the key role of molecular motors in the cytoplasm is broadly appreciated , there ihttp://mirnylab.mit.edu/projects/emerging-evidence-for-loop-extrusion.Hi-C maps and videos are available at Supplemental MethodsSupplemental VideoTable S1


At the beginning of this century, the Human Genome Project produced the firstdrafts of the human genome sequence. Following this, large-scale functionalgenomics studies were initiated to understand the molecular basis underlying thetranslation of the instructions encoded in the genome into the biological traitsof organisms. Instrumental in the ensuing revolution in functional genomics werethe rapid advances in massively parallel sequencing technologies as well as thedevelopment of a wide diversity of protocols that make use of these technologiesto understand cellular behavior at the molecular level. Here, we review recentadvances in functional genomic methods, discuss some of their currentcapabilities and limitations, and briefly sketch future directions within thefield. Progress in genomics during the last decade has been largely dependent on thedevelopment of methods for massively parallel sequencing of large numbers (up to afew hundred million per experiment in some cases) of short DNA sequences, where asingle sequence read is about 50 to 150 base pairs. These methods—oftenreferred to as next-generation sequencing (NGS)—are used for genomesequencing, but their high throughput also makes them very attractive for functionalgenomics assays, where the primary goal is to annotate genome regions with theirbiological function. This often requires quantitative estimates, which typically arederived from read counts. A myriad of experimental protocols and the accompanyingbioinformatic pipelines have been designed that leverage the high-throughputcapabilities of NGS to monitor a wide variety of molecular phenotypes reflectingvery diverse genomic functions.1, have largely been substituted by sequencing-based methods. Our review thusfocuses on these. We also discuss recently developed methodologies for large-scaleperturbation experiments that can probe the functionality of genomic loci in amassively parallel fashion. We discuss the limitations of short read length, effortstoward developing long read sequencing technologies, and novel approaches to analyzethe behavior of single cells and cells in the context of their tissue of origin. Weconclude by discussing the role of large-scale international consortia and futuredirections in functional genomics.In this review, we summarize the protocols that are most frequently applied infunctional genomics. Although oligonucleotide array-based methods for genotyping andgene expression are still widely used for large population screens because of thelow cost and straightforward bioinformatics, genome tiling arrays, which wereinstrumental in the emergence of functional genomics 2. The number of cells from which the DNA is extracted is important, as too fewcells may cause excessive digestion, leading to reads mapping to closed chromatinregions, whereas an excess of cells can cause insufficient digestion, creating longfragments that may be difficult to sequence 3. The methylation status of DNA can be assayed by using endonuclease digestionby methylation-dependent restriction enzymes, by using bisulfite treatment toconvert unmethylated cytosine to uracil (which is recognized as thymine duringsequencing), or by using methylation-dependent antibodies to immunoprecipitatemethylated chromatin followed by sequencing (ChIP-seq) 4. False positives may occur if unmethylated cytosines fail to convert touracil and are interpreted as methylated cytosines. It is essential to minimize DNAdegradation during bisulfite treatment, as DNA fragmentation will hamper polymerasechain reaction (PCR) amplification. Tet-assisted bisulfite sequencing 5 can be used to resolve 5-methylcytosine and 5-hydroxymethylcytosine, both ofwhich are found in mammalian genomes 7 but are indistinguishable in traditional bisulfite sequencing. ChIP-seq-basedapproaches do not give single-nucleotide resolution of DNA methylation and areaffected by genomic variations in CpG density.Chromatin can be divided into the transcriptionally active open euchromatin and thetranscriptionally silent heterochromatin or closed chromatin. The ATAC-seq (assayfor transposase-accessible chromatin using sequencing) protocol uses transposasesacting on accessible chromatin to create DNA fragments, which then are sequenced toidentify open genomic regions in a particular cell type or condition 8 or the binding affinities of transcription factors to genomic regions 10. Improvements to the ChIP-seq protocol have allowed greater resolution whilereducing the number of cells required 12. The use of a sensitive but specific antibody is essential for obtaininghigh-quality ChIP-seq data. Genomic assays typically produce an “epigenomicprofile” along the genome (by mapping reads obtained by the assay to thegenome). To combine and integrate epigenomic profiles produced by different assays, segmentationalgorithms are employed , in contrast, specifically sequences the5′ end of transcripts to pinpoint the transcription start site and thereforethe promoter region of each transcript 18. CAGE uses a random oligonucleotide as the primer for the reversetranscription reaction, instead of an oligo-dT primer against the poly(A)-tail,allowing both poly(A)+ and poly(A)− transcripts—such as certain longnon-coding RNA (lncRNAs)—to be profiled. Specific RNA fractions can beselected for inclusion in the sequencing libraries depending on their size, thechemical group at their 5′ end, the presence or absence of a poly(A) tail, orsubcellular localization in the cytoplasm, nucleoplasm, or chromatin fraction 21. These fractions may correspond to different classes of RNA or differentstages in the pathway of RNA synthesis and post-processing. Protocols to assess theRNA-interacting partners include ribosome profiling 22 to identify mRNAs undergoing translation as well as various protocols basedon high-throughput sequencing of RNA after cross-linking and immunoprecipitation(CLIP-seq and its variants) 23 to find transcripts bound to a specific protein. RNA modifications 24 such as methylation of adenosine nucleotides 25 can be identified by methylation-dependent immunoprecipitation of RNAfollowed by sequencing 27.Quantitative profiling of the transcriptional output of the genome relies onsequencing libraries of cDNA fragments derived from RNA (RNA-seq) 28. Performing ligation of the 5′ adapter after reverse transcriptionallows ligation to the 5′ end of the cDNA, avoiding biases due to thepresence of any modifications at the 5′ end of the short RNA molecule 29. To avoid biases introduced by oligonucleotide adapter ligation to the3′ end, polyadenylation of the short RNA may be used instead. However, adisadvantage of this approach is that it does not allow the identification of theexact 3′ end of the short RNA, which is unfortunate, as addition and removalof nucleotides—in particular, adenosines—to the 3′ end ofmiRNAs are prevalent and may play a regulatory role in miRNA stabilization anddegradation 32.Profiling of short non-coding RNAs—in particular, microRNAs(miRNAs)—typically relies on the ligation of oligonucleotide adapters to the3′ and 5′ end of the short RNA followed by reverse transcription andPCR amplification making use of primer sequences in the adapters 33, which are produced by chemical cross-linking, DNA fragmentation, proximityligation, and high-throughput sequencing of ligated fragments, have shown thatchromatin is organized in topologically associating domains, each consisting ofchromosome regions that frequently interact with each other, and chromatin loopingin which distal regulatory regions such as enhancers and promoters come in closephysical proximity to each other. ChIA-PET  34 combines proximity ligation of DNA fragments with chromatinimmunoprecipitation of specific proteins to globally detect the chromatininteractions in which they participate.The 3D structure of the genome in the nucleus provides the physical architecture forexpression regulation. Hi-C libraries 36 may mediate distal interactions 37, identifying the chromatin binding sites of regulatory RNAs is of centralimportance. Chromatin isolation by RNA purification (ChIRP-seq) 38 uses tiling oligonucleotides to pull down a specific lncRNA together with theprotein and DNA bound to it, followed by sequencing of the DNA component todetermine the exact binding positions of the lncRNA to chromatin. For unbiasedmapping of RNA–chromatin interactions, on a genome-wide scale, severalprotocols related to Hi-C have been developed that rely on proximity ligation of DNAto RNA using a bivalent oligonucleotide linker molecule: MARGI (mappingRNA–genome interactions) 39, global RNA interactions with DNA by deep sequencing (GRID-seq) 40, and chromatin-associated RNA sequencing (ChAR-seq) 41.As transcripts produced at regulatory sites Although chromatin features and transcriptional activity at a genomic region can beindicative of a biological role, ultimately the function of a genomic region isdefined by its contribution to the phenotype. Perturbation studies, in whichactivation or inhibition of specific genomic regions or their products is followedby phenotypic or molecular assays of the cellular response, can be used to elucidatethe functional role of genomic loci.42 to edit or otherwise modify the genome at specific sites. Whereas programmingzinc finger nucleases 43 and transcription activator-like effector nucleases (TALENs) 44 to target specific genomic sites required extensive protein engineering thatlimited the scalability of these approaches, CRISPR/Cas9 recognizes and cleaves aparticular DNA site using only a single guide RNA, which can easily bemass-produced. Nucleotide insertions and deletions introduced near the double-strandbreak by non-homologous end-joining can abrogate the expression or function ofprotein-coding genes but may not be sufficient to fully abolish the function oflncRNAs. Therefore, dual-CRISPR systems have been developed that use a pair of guideRNAs to generate double-strand breaks on both sides of a gene of interest, followedby non-homologous end-joining to repair the lesion, resulting in a full deletion ofthe gene 46.Recent developments in perturbation technologies now allow genomic perturbationexperiments to be highly multiplexed, with thousands of knockdowns or activationsoccurring in parallel in a population of cells. High-throughput perturbationexperiments have become feasible in particular because of the development of theCRISPR/Cas9 technology 47, tight binding of CRISPR-dCas9 to DNA prevents binding of the transcriptionalmachinery, effectively shutting down transcript production at the targeted site.Improvements to the knockdown efficiency were achieved by fusing aKrüppel-associated box (KRAB) repressor domain to Cas9 48, inducing histone modification changes associated with repression ofexpression. Similarly, dCas9 fused to a transactivating domain can be used to inducegene expression at specific sites 48. Epigenome editing can be performed by fusing dCas9 with epigenome writers orerasers  49.The applicability of this technology can be extended beyond genome editing byreplacing the endonuclease Cas9 with catalytically inactive Cas9 , which binds to specific genomic sites without inducing cleavage. In CRISPRinterference (CRISPRi) 50.Second-generation CRISPR editing tools allow the conversion of specific nucleotidesat the genomic target site without inducing a double-strand break  51. CRISPRi-mediated knockdowns followed by single-cell genome-wide expressionprofiling (Perturb-seq) 53 can be employed to obtain detailed information on the global transcriptomeresponse to the perturbation and to observe cell state changes that are not easilyassessed in a straightforward cellular assay.Large-scale CRISPR-based screening relies on designing guide RNAs in large quantitiesand introducing them, usually using a lentiviral vector, into a population of cellsat a sufficiently low multiplicity of infection, such that most cells will receiveeither zero or one guide RNA; puromycin selection is applied to exclude cells thatwere not infected. Typically, up to 10 guide RNAs are used for a single locus,allowing checks for reproducibility and off-target effects due to inadvertentbinding of guide RNAs elsewhere in the genome. During cell culture, the proportionof cells containing guide RNAs that inhibit the expression of genes essential forcell growth will decrease. Therefore, genes involved in cell growth can beidentified by quantifying, using NGS, the relative enrichment of each guide RNAafter culturing 54.For functional genomics, perturbation technologies that target DNA have one inherentdrawback: it is unclear whether any observed phenotype is due to the inhibition ofregulatory DNA elements at the targeted locus or to any RNA product originatingthere. This ambiguity can be avoided by targeting specific transcripts directlyusing locked nucleotide antisense oligonucleotides (ASO gapmers) or by using smallinterfering RNAs (siRNAs) or small hairpin RNAs (shRNAs). Target RNAs bound by anASO gapmer are recognized and cleaved by endonuclease RNase H, followed bydegradation of the RNA. In contrast, the RNA interference (RNAi) pathway relies onprocessing of siRNAs and shRNAs by Dicer and uptake into the RNA-induced silencing(RISC) complex, which binds to the target RNA, causing its cleavage by Argonaute anddegradation by cellular exonucleases. The relative performance of RNAi or RNaseH-mediated knockdown depends in part on the cellular localization of the targetedtranscripts in either the nucleus or the cytoplasm 55. The second makes use of the ability to detect changes in an electrical fieldthat are induced when a single nucleotide in the DNA molecule passes through a porestructure 56. Long read technologies have been used for “de novo” genomesequencing 57, resequencing of the human genome 58, and phased diploid genome assembly 59. They have also been used for transcript characterization 60, since short reads are unable to fully resolve the exonic structure ofalternative transcript isoforms, which usually share a substantial fraction of thesequence. One advantage of long read technologies is that they are able to directlysequence RNA molecules 61, eliminating the biases induced during the generation of the cDNA librariesinherent to short read NGS.Reconstruction of the full-length sequence of long DNA or RNA molecules from NGSsequence reads is challenging because of their short read length. Two maintechnologies are being developed for long read sequencing . The first relies on the abilityto detect the incorporation of a single nucleotide in an elongating DNA chain, whichis synthesized by a DNA polymerase attached to the template DNA molecule to besequenced 62 and long 63 read sequencing.Because of the large dynamic range of genome activity , unbiased sequencing of samples to interrogate function genome-wide maybe ineffective in uncovering sites within the genome with weak activity. Therefore,depending on the purpose, NGS analysis may be restricted to targeted genomic locicaptured using oligonucleotide probes. Capture has been performed, for instance, toidentify and characterize the exonic structure of lowly abundant transcripts using both short 65. Traced back to single-molecule fluorescence in situhybridization (FISH) 66, these methods have dramatically increased throughput by using massivelyparallel sequencing. For instance, histological sections have been positioned onarrayed reverse transcription primers with unique positional barcodes to maintainthe two-dimensional positional information in subsequent RNA-seq analysis 67.Protocols to prepare libraries for NGS typically require a fair amount of inputmaterial (RNA or chromatin), necessitating large numbers of cells to produce asingle library. If the samples are obtained from heterogeneous biological sources,such as tissues and organs, they are likely to contain multiple distinct cell types,which interact in a highly structured anatomical environment. Bulk sequencing ofthese samples reflects an average across the populations of cells used and is unableto resolve the underlying cell types and their position within the tissue sample.Recently, spatially resolved omics methods have been developed with the aim ofperforming functional genomics assays while retaining positional information 68. Genome sequencing of single cells has been used to identify genomic changesin tumor samples and to track their evolution 74.Population averages, such as obtained from bulk tissue sequencing, may furtherobscure the biological reality of cellular heterogeneity even among cells of thesame type. For example, genes that appear to be lowly expressed on the basis oftheir population average may be highly expressed in a few cells in the populationand exert their biological role only there. To capture the heterogeneity among cellsin a population, newer protocols have been developed that dramatically lower theamount of input material required, allowing profiling of individual cells 4 cells in parallel perexperiment, depending on the protocol used; typical coverages are 1,000 to 6,000genes per cell in case of primary cells and 5,000 to 10,000 genes for cell lines 75. In plate-based methods such as STRT (single-cell tagged reversetranscription) 76, Smart-seq 77, and Smart-seq2 78, 50 to 500 single cells are placed in individual wells in 96-well plates, andcell lysis and reverse transcription occur in each well. Alternatively, amicrofluidic chip can be used for cell placement and reactions 79.Single-cell RNA profiling can assay 50 to 10 81, a unique barcode is applied to individual cells in the initial stages in theprotocol. As the barcode is incorporated in the cDNA during reverse transcription,the cDNAs originating from different cells are distinguishable and can be pooled forPCR amplification. To further increase throughput and reproducibility, in massivelyparallel single-cell RNA-seq, cells are sorted by fluorescence-activated cellsorting (FACS) into more homogeneous subpopulations in a 384-well plate beforesingle-cell sequencing 82. As a poly-T primer is typically used for reverse transcription toselectively include poly(A)-tailed transcripts into the library while avoidingribosomal RNA, other untailed transcripts such as some lncRNAs may be missed.Alternative strategies to reduce the ribosomal RNA component include duplex-specificnuclease treatment 83 and using a template-switching reaction to enrich for 5′-cappedtranscripts 84. Droplet-based isolation of single cells 86 enabled massive parallelization with numbers of cells in the range of 10,000or more, albeit at lower sensitivity.Cell selection is biased by the cellular size and condition, which affects theprobability of a cell to be placed successfully in a well. In pooled approaches suchas CEL-seq 87.As an alternative to single-cell analysis, deconvolution methods can be employed toestimate the proportions of constituent cell types in bulk tissue transcriptomicsamples on the basis of the expression of known cell type marker genes 88 and therefore genome comparisons are, in general, less helpful to annotatethem. For instance, whereas 80% of human protein-coding genes can be detected inmouse, fewer than 10% of lncRNAs are conserved between the two species 89. Comparative transcriptomic studies help to further understand to what extentthe patterns of gene expression and the regulatory networks are conserved acrossspecies 92. Particular attention has been paid to comparisons between human and mousegiven the unique role of the mouse as a model of human biology 94. While gene expression levels averaged over RNA samples are correlatedbetween human and mouse 89, substantial differences are found when comparing the transcriptome ofhomologous cell types 97. Overall, conservation of the patterns of expression between human and mouseseems to largely depend on the gene. The expression of some genes variesconsiderably across species and little across organs and that of other genes variesmore across organs than across species. The mouse is a particularly good model forthe biology of these genes in humans 98.Comparative genomics methods have also been widely used to identify and cataloguefunctional regions in genomes. The underlying assumption is that functional regionsare more conserved through evolution than genomic regions devoid of function. Genomesequence comparisons are widely used, in particular, to annotate protein-codinggenes in newly sequenced genomes. However, while protein coding genes are understrong selective pressure, other functional regions, such as lncRNA genes andregulatory regions, have levels of conservation that are often barelydistinguishable from background conservation 103. These projects have not only generated data resources that are widely usedby the scientific community but also defined “de facto” communitystandards for experimental protocols and bioinformatic pipelines, placing genomicsin general and functional genomics in particular within the reach of individuallaboratories. As a result of advances in experimental and computational methods,functional genomics assays are rapidly expanding beyond cell lines and tissuesamples, which were mostly profiled in these projects, to complex organs,conditions, primary cells, and even single cells. Indeed, the Human Cell Atlasconsortium has formed to characterize each individual cell type in the human body 104. Functional genomics assays are also being increasingly performed acrossmultiple individuals within a species, allowing investigators to specifically linkgenetic variants to molecular phenotypes, as well as across different species. Mostof these analyses to date have focused on the identification of single-nucleotidepolymorphisms (SNPs) that affect gene expression (expression quantitative traitloci) 105 but other molecular phenotypes, such as methylation and histonemodifications, have also been monitored 106. Moving toward personalized genomics, the EN-TEx pilot project is currentlycarrying out multiple functional genomics assays in multiple tissues extracted froma few donors 107.Massively parallel sequencing has been at the core of most large-scale functionalgenomic projects to date 108 and neuropathology 109. The GTEx project 103 has produced high-resolution histological images for 20,000 human tissuesamples, together with matching genome and transcriptome sequencing data. Methods toidentify SNPs associated with image-derived organ phenotypes such as neuroanatomicaltraits  have been developed, and an upcoming challenge is to relate molecularphenotypes, as monitored by functional genomics, to organ phenotypes, as monitoredby imaging technologies.How these molecular phenotypes propagate in turn through intermediate levels oforganizational complexity  determines how geneticvariation ultimately impacts the phenotypic traits of organisms. Thanks to advancesin digital imaging, data on intermediate phenotypes, such as tissues and organs,have recently been made available with associated genomics and molecular data inareas as diverse as cancer invivo multi-omics characterization of all of the cells within amulticellular organism—without interfering with the system. In the pathtoward this ideal, less-invasive protocols and protocols to probe the multiplefacets of genome function at single-cell resolution  will certainly be developed,as will methods able to simultaneously integrate data monitoring the behavior ofmillions of cells. The computational challenges associated with the exponentialgrowth of data, driven by genomic technologies, will be enormous. New algorithms aswell as new developments in hardware that overcome the limitations of currenttechnological solutions may need to be developed.We believe that we are still in the infancy of genomic technologies and there isample opportunity for progress. The ultimate goal is the real-time