Chronic postsurgical pain (CPSP) occurs in up to 50% of individuals after surgeries and 32% after hysterectomy, leading to major adverse effects on quality of life and socioeconomic burden. Little is known about whether and how large-scale neural networks being affected in CPSP, particularly with regard to the functional connectivity (FC) of insula which is known to be the hub of the intrinsic neural network playing a critical role in pain processing. Here, we sought to examine the dynamics of insular FC in the context of noxious stimuli in CPSP patients. To this aim, resting state fMRI data were acquired, before and after acute heat pain stimulation, from 11 individuals with chronic post-hysterectomy pain (CPHP) and 22 age-matched healthy controls (HCs) who had a hysterectomy but without chronic post-surgical pain. We examined whole-brain FC were mapped by seeding at the sensorimotor and chemosensory subfields of the insula and found significant group × stimulation interaction effects. Specifically, the HC group had increased FC between the left sensorimotor insula and right angular and middle occipital gyrus (MOG) and increased FC between the left chemosensory insula and bilateral angular and MOG following pain stimulation. In contrast, such pain stimulation related FC changes were absent in the CPHP group. Furthermore, higher insular FC at baseline and smaller increased insular FC after pain stimulation correlated with clinical pain scores in CPHP patients. Our findings suggest that CPSP is associated with altered dynamics of large-scale functional networks anchored in the insula. Chronic postsurgical pain (CPSP) is a potential adverse outcome after surgery. It has been described in patients after common surgical procedures and with an estimated incidence as high as 32% after hysterectomy  has become the primary tool to understand functional organizations in normal brain and altered large-scale functional networks in brain disorders  patients and controls. Here, two regions of insula were selected as the seeds to define the SN: sensorimotor and chemosensory regions. The sensorimotor part of insula has well-established linkage to somatosensory, motor and temporal cortices and is involved in sensory processing including pain  between October 2013 and September 2015. All participants completed pre-operative pain catastrophizing scale questionnaire  during which participants were instructed to remain still and keep their eyes on the fixation. Subjects were monitored throughout their time in the scanner for any potential adverse event.Participants were asked to cease pain medications other than paracetamol and nonsteroidal anti-inflammatory agents for 48 h prior to MRI. The participants then underwent neuroimaging sessions both before and after tonic heat stimulation, on a 3-T Siemens Skyra system  using a phased-array head coil. The session included the acquisition of high-resolution T1-weighted images and gradient-echo EPI of resting state fMRI pre and post heat stimulation  with a Peltier surface stimulator applied to the left lower thigh. A 44°C stimulation was chosen as the most appropriate one in terms of tolerance of participants during scanning. There were a 44 s break between the first resting state and pain stimulation and another interval of 50 s between the end of the pain stimulation and the second resting state scan. After a warning signal, the temperature rose (5°C/s) to 44°C and stayed at this level for 7 min. The participant then continuously rated the pain score every 30 s  using continuous visual analog scale  corrected . Four-month post-operative pain score difference (p < 0.001) and Pre-operative temporal summation score (p < 0.011) were also found to be significant between CPHPS and HCs. With regard to the head motions during fMRI scanning, we compared the absolute maximum displacement before and after heat stimulations between CPHPs and HCs using two-way ANOVA. We found no significant between-subject effects between CPHPs and HCs , as well as no significant within-subject effects of between pre and post-heat stimulation , on head motion parameters. Further, a test of interactions between the two factors was not significant  and right angular and middle occipital gyrus (MOG) increased after heat pain stimulation Figures . FC betwr2 = 0.565; p < 0.05, Figure r2 = 0.591; p < 0.05, Figure r2 = 0.467; p < 0.05, Figure r2 = 0.563; p < 0.05, Figure C, box plot of pre-stimulation FC in CPHP), we repeated the correlation analysis after excluding the outlying data point. The results remained significant . No association was found between FC and COVAS score during heat pain stimulation.At baseline, we found significant associations between FC and clinical pain-related scores in CPHP patient group, including FC between left sensorimotor insula and right MOG was positively related to 4-month post-operative pain scores (In the present study, we examined the insular FC in CPHP using resting state fMRI approach. Following heat pain stimulation, we observed increased insular FC in HC but not in CPHP group. Furthermore, FC abnormalities in CPHP group were correlated with their clinical severity that the smaller FC increment after heat stimulation was related to higher clinical pain-related scores. To our knowledge, this is the first study to look at intrinsic brain connectivity before and after provoked pain stimulation in patients presented with early CPSP. These findings suggest, at the level of large-scale network connectivity, CPHP patients may have lost the normal neural response toward acute pain stimulus, evidenced by the abnormalities in the insular processing after noxious input.We found increased FC between left chemosensory/sensorimotor insula and other cortical regions, including MOG and ANG, following pain stimulation in healthy subjects. The insula is an important cortical structure that is involved in somatosensory, autonomic, interoceptive, salience and cognitive processes Craig, . Prior wSpecifically, increased insular-MOG FC supports the involvement of the occipital region in pain processing or modulation. The occipital region has been implicated in the modulation of pain through activation of descending tract of dorsolateral funiculus. Involvement of the occipital cortex in descending inhibitory mechanism was well demonstrated (Reis et al., The angular gyrus is well known to be involved in a multitude of cognitive functions including attention, language and semantic memory (Binder et al., In our study, the increased SN connectivity following pain stimulation was not observed in CPHP, suggesting that the normal pain-induced neural response may be blunted or abolished in CPHP. Our findings support the idea that in CPHP, normal sensory processing pathway was dysregulated, result in altered brain functional circuit secondary to neuroplasticity. This is consistent with previous studies in which atypical insular responses have been demonstrated in several neuropsychiatric conditions, including schizophrenia (Palaniyappan and Liddle, Our study showed that in CPHP, the extent of FC increases toward provoked pain stimulus negatively correlated with higher pre-operative temporal summation score. This is consistent with the above findings that CPHP patients showed blunted FC response toward provoked pain. Temporal summation score is a dynamic test for central sensitization (Loeser and Treede, By identifying and quantifying insular dysfunctionality in the early stage of CPSP development, we can have a better understanding about neural mechanism of pain, and with the invention of effective therapeutic measures, we may possibly halt the progression of early to chronic CPSP. Further, insular FC changes and dysfunctionality can also potentially be used as a surrogate measure of pain score, thereby allow more objective monitoring of the therapeutic response of CPSP toward analgesia or other treatment.The present study had some limitations. Although only substance free participants were recruited, the study did not control for several comorbidities commonly associated with chronic pain condition such as depression and anxiety, which may potentially confound our findings. In view of the relatively small sample size, to reduce multiple comparisons the current study only focused on insular FC using a seed-based approach, which may not be able to provide a complete picture of whole-brain functional network changes. A more data-driven approach, such as whole brain FC analysis, would allow us to examine the involvements of other neural pathways in chronic pain modulations. Similarly, because of the small sample size, the findings from correlation analysis should be regarded as exploratory. Further work with large sample size is needed to address these issues.In conclusion, our findings highlight the insular FC abnormalities before and after heat pain stimulation in CPHP patients underlying their clinical symptoms. Future studies are needed to help stratify patients according to comorbidities and pain levels to characterize specific altered brain functional connectome underlying CPSP and develop such method to help intervention development.All 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.


We describe a rare case of multifocal extramedullary epidural neurosarcoidosis that presented with myelopathy without motor deficits and perform a literature review for previous cases of epidural neurosarcoidosis. A 46-year-old woman presented with lower back pain, urinary incontinence, gait disturbance, and sensory loss without motor deficits. Spine magnetic resonance imaging (MRI) showed multiple epidural lesions, the largest causing spinal cord compression at the T5 level. A computed tomography (CT)-guided biopsy of the dominant lesion showed noncaseating granulomas consistent with neurosarcoidosis. She was treated with a course of dexamethasone and discharged home after a 10-day hospital course. She was discharged home on oral prednisone taper over a four-month period. At her latest follow-up, she is neurologically intact and gainfully employed. This case demonstrates that certain cases of epidural neurosarcoidosis causing spinal cord compression may be treated with medical therapy alone in the absence of severe neurological deficits. Sarcoidosis is a highly mimetic disease process that can affect numerous organ systems. Although the classical association is with pulmonary disease, sarcoid may also affect the central nervous system in approximately 10% of cases. If the spinal cord is affected, the disease is typically intradural and standard management consists of corticosteroids and, in some cases, surgical decompression. Here we present a rare case of multifocal extradural neurosarcoidosis causing myelopathy with spinal cord compression. Her case was successfully managed with medical therapy alone and she achieved full recovery.We present here a case of a 46-year-old woman with a past medical history significant for a positive purified protein derivative (PPD) diagnosed in 1997 who presented to the emergency department complaining of severe low back pain. She reported that, as her pain worsened, she began to have difficulty walking, requiring a cane to ambulate. She also endorsed sensory loss below her knees, urinary incontinence, and chills without fever, however her incontinence was ostensibly related to a gynecological issue. She denied smoking, intravenous drug use, alcohol abuse, or recent travel. Her recent medical history was only significant for a mild cold several weeks ago. She presented to the emergency department for back pain two months prior and was discharged home after lumbar and sacral plain films were negative.Physical exam on this admission was significant for diminished sensation below the knees in non-dermatomal distribution without a sensory level, full strength throughout the upper and lower extremities, and an unsteady gait, corresponding to a Frankel grade of D. Hoffman’s sign was positive bilaterally and she was diffusely hyperreflexic. Rectal tone was intact and no cranial nerve deficits were noted.Vital signs were within normal range. Her initial labs revealed an elevated white blood cell count of 11.2 k/uL  with a left shift and erythrocyte sedimentation rate of 40 mm/h . Her C-reactive protein, basic metabolic panel, and liver function studies were within normal limits.Full spine magnetic resonance imaging (MRI) was performed that revealed three epidural lesions distributed throughout the thoracic and lumbar spine. The largest lesion was centered at the T5 vertebral body and extended from T4-T6 causing spinal cord compression with T2 signal changes Figure -1B. The CT-guided needle-core biopsy of the dominant lesion revealed noncaseating granulomatous inflammation without evidence of fungal or bacterial organisms, including acid fast bacteria, by gram, Fite, acid-fast bacilli (AFB) and Grocott-Methenamine Silver (GMS) stains, or of lymphoma or other neoplasms. Hematoxylin and eosin (H&E) stained sections show the granulomas as multiple nodules at low magnification Figure , composeThe patient was admitted to the intensive care unit under close neurological monitoring. A follow-up MRI was performed eight days after initial imaging and initiation of corticosteroid therapy to monitor disease response. It showed a significant reduction in the size of the posterior epidural masses centered at T5 and T8 Figure -5B. SpinAt follow-up the week after discharge, her back pain and gait improved significantly; Karnofsky Performance Status (KPS) was reported as 90. At her latest follow-up over 18 months after initial admission, she has gainful employment, ambulates without difficulty, and is neurologically intact.Overview of spinal neurosarcoidosisSarcoidosis is a common, chronic inflammatory disease of unknown etiology classically defined by noncaseating granulomas on histology , 2. NeurAlthough numerous immunosuppressive agents have been used as adjunctive agents in the management of neurosarcoidosis , corticosteroid therapy is the standard for medical care , 14. TheCases of epidural neurosarcoidosisOur review of the literature returned four cases of epidural neurosarcoidosis causing neurological deficits, each presenting as myelopathy without systemic manifestations of sarcoidosis. These confirmed cases of epidural neurosarcoidosis causing spinal cord compression required surgical intervention to forestall the disease , 15, 16.Barazi et al. described a 44-year-old woman who presented with sensory disturbance and mild lower extremity motor deficits whose imaging revealed a T2 hyperintense lesion with contrast enhancement at the thoracolumbar junction . WeissmaContrast with the present caseSeveral similarities are apparent between the above-mentioned cases and ours, such as presentation with myelopathic symptoms, no evidence of systemic disease, and an impressive response to treatment. Yet our case is unique in that the patient presented myelopathy secondary to multiple extradural lesions and did not require surgical intervention, despite the presence of spinal cord compression. Our patient’s full strength on presentation in the presence of minor sensory and gait disturbances (Frankel grade D) combined with a swift response to corticosteroid therapy allowed us to manage the case nonoperatively once pathology confirmed the diagnosis of neurosarcoidosis. It is critical to note that the patient’s neurological status remained stable during her admission and began to improve after steroid administration. In a scenario of refractory medical treatment and/or deteriorating neurological function, surgical decompression must be part of the treatment algorithm and hence close monitoring is imperative while the medical management is ongoing. However, our patient steadily improved on medical management, avoiding the need for surgical decompression.Future study of epidural neurosarcoidosis is certainly limited by the rarity of the disease, making large prospective study exceedingly difficult. As such, this case suggests that epidural neurosarcoidosis with spinal cord compression and without major neurological deficits may be successfully managed medically to achieve full recovery.


Circovirus sequences are frequently recovered in metagenomic investigations, indicating that these viruses are widespread, yet they remain relatively poorly understood. Endogenous circoviral elements (CVe) are DNA sequences derived from circoviruses that occur in vertebrate genomes. CVe are a useful source of information about the biology and evolution of circoviruses. In this study, we screened 362 vertebrate genome assemblies in silico to generate a catalog of CVe loci. We identified a total of 179 CVe sequences, most of which have not been reported previously. We show that these CVe loci reflect at least 19 distinct germline integration events. We determine the structure of CVe loci, identifying some that show evidence of potential functionalization. We also identify orthologous copies of CVe in snakes, fish, birds, and mammals, allowing us to add new calibrations to the timeline of circovirus evolution. Finally, we observed that some ancient CVe group robustly with contemporary circoviruses in phylogenies, with all sequences within these groups being derived from the same host class or order, implying a hitherto underappreciated stability in circovirus-host relationships. The openly available dataset constructed in this investigation provides new insights into circovirus evolution, and can be used to facilitate further studies of circoviruses and CVe.Circoviruses (family  Circovirus genomes are typically ∼2 kilobases (kb) in length and contain only two open reading frames (ORFs): one encoding a non-structural, replication-associated protein (Rep), and a second encoding the viral capsid (Cap). The family contains two genera: Circovirus and Cyclovirus, and many of the ssDNA viruses that have been identified in recent years fall into one of these two genera (Circoviruses (family o genera . Howevero genera .Endogenous circoviruses (CVe) provide an unconventional but useful source of information about circovirus distribution, diversity and evolution. These sequences are derived from the genomes of circoviruses that circulated millions of years ago, and became integrated into the host germline . Relativin silico to generate a comprehensive catalog of CVe. We used these data to: (i) extract information about the long-term evolution of circoviruses; (ii) generate an openly accessible data resource that can facilitate the further investigation of CVe and circoviruses.In this study, we screened vertebrate genomes 22.1in silico was performed using the database-integrated genome-screening tool (DIGS). The DIGS procedure used to identify CVe comprises two steps. In the first, a virtually translated circovirus probe sequence (e.g. a Cap or Rep protein sequence) is used to search a particular genome assembly file using the basic local alignment search tool (tBLASTn) program  for sequences homologous to circovirus proteins. Vertebrate genome assemblies and circovirus reference genomes were obtained from the NCBI genomes resource. Screening  program . In the We inferred the ancestral ORFs of CVe (and the number of stop codons and frameshifts interrupting these ORFs) via a combination of automated alignment and manual adjustment. Multiple sequence alignments were constructed using MUSCLE  and PAL22.2in silico screen, as well as a set of representative reference sequences for the Circovirus genus (Table S2). All of these sequences are linked to the appropriate auxiliary data; for the virus sequences, this includes information about the sample from which the sequence was obtained; for CVe, it includes the name of genome assembly and contig in which the CVe sequence was identified, and its coordinates and orientation within that contig.We used GLUE - an open, data-centric software environment specialized in capturing and processing virus genome sequence datasets  – to colCircovirus and the CVe recovered by our screen. Because each of these alignments is constrained to a standard reference sequence, alignments are linked to one another.The project also includes the key alignments constructed in this study, linked together using the GLUE ‘alignment tree’ data structure. These include: (i) ‘tip’ alignments in which all taxa are CVe that are known or putative orthologs of one another; (ii) a ‘root’ alignment constructed to represent proposed homologies between the genomes of representative viruses in the genus We applied a systematic approach to naming CVe. Each element was assigned a unique identifier (ID) constructed from a defined set of components. The first component is the classifier ‘CVe’. The second is a composite of two distinct subcomponents separated by a period: the name of CVe group , and the second is a numeric ID that uniquely identifies the insertion. Orthologous copies in different species are given the same number, but are differentiated using the third component of the ID that uniquely identifies the species from which the sequence was obtained. In cases were a CVe element had expanded via duplication, an additional number was appended to the standard ID to discriminate individual duplicates.33.1), in 52 distinct species , and used to construct bootstrapped ML phylogenies . In gene3.2rep in the genome assembly of the inshore hagfish (Eptatretus burgeri). These sequences are relatively distinct from other circoviruses, and also showed relatively high genetic diversity relative to one another, forming three distinct groups in phylogenetic trees . Notably, the putative Rep polypeptides encoded by these sequences contained several in-frame indels relative to one another. Because such a pattern of variation is unlikely to arise through neutral accumulation of mutations in the germline, this suggests the occurrence of at least three distinct genome incorporation events, each involving distinct, but relatively closely related viruses. However, since we were unable to identify unambiguous genomic flanking sequences for any of these loci, their classification as CVe should for now be considered tentative.Extant vertebrates are divided into the jawed vertebrates (Gnathostomata) and jawless vertebrates (Agnatha). The Agnatha represent the most basal group of vertebrates and includes the hagfishes (myxinoids) and lampreys (petromyzontids). We identified seven sequences exhibiting homology to 3.3Barbus barbus) and European catfish (Silurus glanis), based on (i) the observation of viral particles in tissues, and the recovery of circovirus sequences from these tissues via nested PCR (Labeo rohita) (Actinopterygii) (Cyprinus carpio) and golden-line barbell (Sinocyclocheilus grahami) genomes - were orthologs of one another, indicating they were incorporated into the germline of cyprinid fish more than 39 million years ago  . We identerygii)  and S3. ears ago . These Crep in eight other species of ray-finned fish (Anguilla anguilla), approximately follows that of the host species, consistent with a single ancestral integration event > 200 million years ago  encoded a complete intact rep gene  (Rana catesbeiana) that partially overlaps that identified in Xenopus. Potentially, these sequences could be orthologs of one another, which would imply a minimum age of ∼204 MYA  . We iden∼204 MYA  Fig. 2)rep genes3.5Crotalus spp)  . We idenlus spp) . Examinalus spp) .3.6Egretta garzetta), white-throated tinamou (Tinamus guttatus), medium ground-finch (Geospiza fortis), and kea (Nestor notabilis) (Nestor notabilis)  . We identabilis)  (Table 2tabilis) )   . Identifngbirds)  Fig. 2)rep-derivTinamus guttatus) and little egret (Egretta garzetta)  and downy woodpecker (Picoides pubescens: order Piciformes) (In addition to identifying the previously reported CVe in the genomes of the white-throated tinamou (arzetta) , we ideniformes) . Both th3.7rep gene. However, the copy number of these elements has expanded subsequent to their incorporation into the germline, in some cases quite dramatically. The grouping of carnivore CVe in phylogenies  that grouped closely with circoviruses genome recovered from dogs (Mus musculus) ∼6–7 Mya, since it is absent from this species.A novel, relatively well-preserved rom dogs . This elChrysochloris asiaticus) matches to both cap and rep were identified. However, these occurred on distinct contigs and did not overlap. Furthermore, both CVe were relatively short and degraded, and were highly divergent relative to other CVe. CVe derived from cap were also identified in the genome of Hoffmann’s two-toed sloth (Choloepus hoffmanni) (In the cape golden mole (ffmanni) .Monodelphis domestica), an American marsupial  and the koala (Phascolarctos cinereus). Both these sequences derived from circovirus rep genes, and grouped together in phylogenetic trees . However, their placement relative to other taxa was not supported with confidence, reflecting their short and degraded nature. Several other short and degraded matches to Rep probes were identified in other mammalian species .Circovirus genus, spanning multiple geological eras  that encoded an intact rep gene that is predicted to express mRNA, suggesting it may have been functionalized in some manner  that are derived from viral polymerase or replication-associated genes, are expressed, and encode intact ORFs . These eCuriously, it is rare for more than one CVe to occur in the germline of any jawed vertebrate lineage. Carnivores are an obvious exception, since CVe have been amplified to relatively high copy number (10–20 copies) in several carnivore lineages , apparen5Circovirus have evolved and interacted with their hosts over the course of their evolution. The sequence-based resource implemented here can facilitate further characterisation of circovirus distribution, diversity and evolution as new CVe and circovirus sequence data become available.We identify the complete repertoire of CVe sequences in published vertebrate genome assemblies. Through comparative analysis of these sequences, we provide the most complete picture yet of how viruses in the genus


Drosophila melanogasteramos mutants that lack trichoid and basiconic sensilla, the single-walled sensilla in this species. This revealed 187 genes whose expression is enriched in these sensilla, including pickpocket ion channels and neuromodulator GPCRs that could mediate signaling pathways unique to single-walled sensilla. For our second approach, we computationally identified 141 antennal-enriched (AE) genes that are more than ten times as abundant in D. melanogaster antennae as in other tissues or whole-body extracts, and are thus likely to play a role in olfaction. We identified unambiguous orthologs of AE genes in the genomes of four distantly related insect species, and most identified orthologs were expressed in the antenna of these species. Further analysis revealed that nearly half of the 141 AE genes are localized specifically to either single or double-walled sensilla. Functional annotation suggests the AE genes include signaling molecules and enzymes that could be involved in odorant degradation. Together, these two resources provide a foundation for future studies investigating conserved mechanisms of odor signaling.Repellent odors are widely used to prevent insect-borne diseases, making it imperative to identify the conserved molecular underpinnings of their olfactory systems. Currently, little is known about the molecules supporting odor signaling beyond the odor receptors themselves. Most known molecules function in one of two classes of olfactory sensilla, single-walled or double-walled, which have differing morphology and odor response profiles. Here, we took two approaches to discover novel genes that contribute to insect olfaction in the periphery. We transcriptionally profiled  Vector borne diseases sicken hundreds of millions of people worldwide each year. Most infections are contracted through the bites of blood-feeding arthropods . The priDrosophila melanogaster, basiconics and trichoids are types of single-walled sensilla and coeloconics are double-walled sensilla . A small number of ORNs and non-neuronal auxiliary cells are housed within sensory hairs known as sensilla, and hundreds of these sensilla densely cover the primary insect olfactory organ, the antenna . In mostDrosophila melanogaster, OR receptors are expressed by ORNs in single-walled sensilla, and IR are receptors expressed in double-walled sensilla (Insects utilize a large number of receptors to detect and discriminate the immense number and variety of odors that exist in the environment. Research over the past two decades has characterized two large odor receptor families: Ionotropic Receptors (IRs) and Odorant Receptors (ORs) . In addisensilla . Recent Drosophila due to the number of available genetic tools that support molecular manipulations of specific receptors and neurons  were frozen in liquid nitrogen, and tissues were dissected into 1.5 ml Eppendorf tubes kept in liquid nitrogen. For RNA sequencing, each sample consisted of 300-400 antennae from approximately equal numbers of males and females. Analysis of the expression of known auditory organ genes found in the second antennal segment indicates that our samples predominantly contained antennal third (olfactory) segments as intended. Many of the most highly expressed genes in the second (auditory) segment of the antennae are not expressed in our CS dataset  (Canton-S and For quantitative reverse transcriptase PCR (qRT-PCR), tissue samples consisted of legs (∼50), antennae (∼200), entire bodies without heads and legs (∼10), and heads (∼10). Tissues were ground using disposable RNAse-free pestles and a QIAshredder column (Qiagen). Total RNA was extracted using RNeasy Micro Kits (Qiagen) for qRT-PCR and an RNeasy Mini Kit (Qiagen) for RNA-Seq. For both, the manufacturer’s RNeasy Mini Kit protocol was followed as it gave better yield and purity. To increase the yield, the RNeasy spin column was rolled horizontally during the RW1 and RPE steps to ensure no sample remained on the wall and cap of the RNeasy spin column, and RLT was gently warmed before use.amos flies and then cleared of genomic DNA using the DNAse in an iScript gDNA Clear cDNA Synthesis Kit (Bio-Rad). The six cleared RNA samples (∼0.5 µg each) were sent to the Center for Genome Innovation at the University of Connecticut for quality control, library preparation, and NextGen sequencing. Libraries were prepared using the Illumina mRNA sample prep kit for non-stranded RNA, and sequencing was carried out on an Illumina NextSeq 500 with 150 cycles to produce paired-end reads.Three independent antennal RNA samples were collected from Canton-S and Drosophila melanogaster genome and gene annotation file were downloaded from NCBI  (Raw reads were downloaded from BaseSpace Sequence Hub (Illumina). Quality control on the reads was performed using Sickle with the default parameters to produce trimmed reads . The refamos flies, HTSeq was used to obtain raw counts of reads mapping uniquely to each gene ; this was the same method as used previously for atonal analysis . Closer analysis revealed that for 68 of these genes, all wt samples and two amos samples had very low expression (average RPM 1.8), whereas there was some increased expression in amos sample 3 likely due to slight contamination by an additional tissue. Therefore, we considered the remaining 86 genes to be those that were truly upregulated in amos mutants . To be certain that this variability in amos sample 3 did not influence our identification of amos-depleted genes, we verified that the expression values of each of the 187 amos-depleted genes were similar in all three samples of a particular genotype.Using analogous criteria, we also identified 154 genes that were upregulated in We used Cufflinks to compare gene expression across tissues because this was similar to the data processing used for the ModEncode datasets  and whole bodies . Custom scripts written in Spyder3.2.3, a scientific Python development environment from the Anaconda navigator, were used to create two tables: 1) the 4,129 antennal genes, their antennal expression, and their expression in other tissues, and 2) the 4,129 antennal genes, their antennal expression, and their expression in whole bodies over development. Genes with >10-fold higher expression in antennae compared to the ten tissue datasets and genes with >10-fold higher expression in antennae compared to seven whole body developmental time points were identified. The two gene datasets were then merged to identify 177 genes enriched in both. Automatic annotation using FlyBase determined that 141 of these genes encoded proteins, and these were considered the set of coding antennal-enriched (AE) genes for further analysis .There were 4,129 genes in CS wild-type antennae that are expressed >1 FPKM in each of the three samples and with an average expression of at least 10 FPKM. Expression levels of these genes in other tissues were obtained from datasets generated by the modENCODE consortium and downloaded from FlyBase  was used for the qRT-PCR assays. Gene expression in each tissue was measured relative to the expression of eIF1A in that sample. Each reaction was run on three to five independent tissue replicates. Differences between samples were analyzed with ANOVA followed by the Dunnett’s post hoc test using GraphPad Prism 7.Seven of the AE genes that had not been studied previously were selected for validation with qRT-PCR experiments. Equal amounts of RNA extracted from dissected fly tissues (200 ng per sample) were used to generate cDNA using the iScript gDNA Clear cDNA Synthesis Kit (Bio-Rad). The cDNA was used as a template for qRT-PCR with the SsoFast EvaGreen Supermix (Bio-Rad), similar to , Tribolium castaneum (beetles), Anopheles gambiae (mosquitos), and Apis mellifera (bees). These were selected because they represent a wide distribution along the insect phylogenetic tree, and because antennal transcriptomes for these species could be found in the NCBI SRA (Drosophila gene when used in a BLAST search of Drosophila melanogaster proteins.We searched for orthologs in other insect species for 96 of the AE genes. Known chemoreceptors (IRs/ORs/Grs) and OBPs were excluded because their conservation has been extensively researched in prior studies. The UniProt ID for each of the 96 genes was obtained using the Flybase gene symbol as a query in UniProtKB . Ortholohe query . Four inHarpegnathos saltator, SRX1164271; Tribolium castaneum, SRX757109; Apis mellifera, SRX518058; Anopheles gambiae, SRX765675) . Therefore, our analysis of the antennal dataset should not be affected.We note that after completing the study, we learned that the original paper for the amos-depleted dataset was determined using PANTHER 14.0 in AmiGO 2 with GO database release 2018-12-01  Canton-S flies to remove a nearby lethal mutation. We then carried out next generation transcriptional profiling on three sets of antennae from amos and wt flies . As expected, most genes are expressed at similar levels in the two genotypes . We considered these genes amos-depleted.To do this, we used RNA-Seq to compare antennal expression of genes in wild-type and sensilla  . The ten genes reduced with the greatest statistical significance included five genes previously shown to be localized to single-walled sensilla: three members of the OR family, Obp28a, and an enzyme Jhedup  terms . This is in part due to the enrichment of genes labeled with the molecular functions “olfactory receptor activity”  and “transmembrane signaling receptor activity” . Genes associated with the GO biological process term “sensory perception” were also highly enriched among the amos-depleted genes (33.3%) compared to 2.8% of FlyBase genes (P < 10−37).We then turned to Gene Ontology annotation to determine if particular categories of genes were enriched among the O) terms . The freO) terms  . Together OR and GR chemoreceptors represent ∼20% of the amos-depleted genes.Functional annotation of the se tools , Table 1amos-depleted genes have potential roles in neuronal signaling (+ channels (ENaCs). There are also seven GPCRs, including serotonin and acetylcholine receptors that are known to regulate presynaptic terminals in other neuronal systems  GstD11 and GstD8 were shown to be enriched in the antenna compared to other tissues . This set of genes included 12 non-coding RNAs, 17 biotransformation enzymes, four GPCRs, and five chitin-related genes in addition to others with diverse predicted functions. There were also four genes implicated in immune system activity and nine genes related to serine proteases and their inhibitors, which can also be involved in defense responses. This could indicate that the amos mutants are infected by microbes, consistent with their reduced fitness compared to wild-type flies. Other upregulated genes may be expressed in the ectopic mechanosensory bristles found on amos antennae   Brown e.Drosophila  protein-coding genes   to quantify the expression levels of antennal-enriched genes in tissues from wild-type flies. We investigated seven AE genes whose expression and function have never been directly examined. Each of these seven genes was detected at a significantly higher level in antennae compared to heads, legs, and bodies (n = 3-5) . Gene exDrosophila are also expressed in the antennae of distantly related insect species, which would be consistent with a conserved olfactory function. We chose to focus on the 96 genes that were not known odor receptors or OBPs, as the antennal expression of these gene families has been extensively studied in multiple insect species. We identified existing antennal RNA-Seq datasets on the NCBI Sequence Read Archive (SRA) from four insect species: mosquitoes , bees , ants , and beetles . These are a diverse set of insects with a nearest common ancestor over 300 million years ago . In each transcriptome analyzed, a greater fraction of the orthologs of random genes were not expressed than the fraction of AE genes , and we wondered if this might reflect contamination of the sample, and thereby bias our interpretation of the AE gene expression. We therefore identified a second Tribolium antennal dataset with a smaller fraction of randomly selected genes expressed. Again, the AE genes were more often expressed than the randomly selected genes . We note that the fraction of AE orthologs expressed in the Apis transcriptome was only slightly more than the fraction of expressed random orthologs, unlike the other three species. We therefore expanded our analysis to a fifth species, Bombyx mori. We found that the fraction of expressed AE orthologs was much greater than that of the randomly selected genes, similar to Anopheles, Harpegnathos, and Tribolium . Together, our data support the possibility that more AE gene orthologs are expressed in the antennal transcriptomes than would be expected by chance alone.Surprisingly few random genes were not expressed in Drosophila, we wondered whether some of the genes might function within signaling pathways specific to single or double-walled sensilla, similar to the odor receptor families themselves. Our first dataset revealed genes that are depleted in amos mutants and are therefore likely to be enriched in single-walled sensilla. We had previously generated a similar dataset for atonal flies, which selectively lack double-walled sensilla  of AE genes with no predicted function.lush, Snmp1, Amt, spineless and OR/IR odor receptors have established olfactory roles . In other systems, antisense RNAs are increasingly reported to regulate gene expression (Our study of amos flies indicate that many GRs are found within single-walled sensilla. It will be interesting to determine if they are co-expressed with OR receptors within ORNs, such as Or10a and Gr10a , or if there are unidentified ORNs that exclusively use these receptors such as those utilizing Gr21a/Gr63a (Recent reports indicate that several members of the gustatory receptor family are unexpectedly found in the antenna (B receptors (amos-depleted GPCRs such as hec, implicated in mating behavior, and Lgr1, responsive to hormones, could affect the known activation of some single-walled sensilla ORNs to mating pheromones ( Gαf, the kinases CG30274 and CG9541, and the phospholipase A2 CG42237, which could be components of intracellular signal transduction cascades. Five members of the pickpocket family of ligand-gated cation channels are also amos-depleted. Although the functions of most family members are poorly understood, some are gated by neuropeptides and other small molecules and could act as neuromodulators (We observed that many of the genes enriched in single-walled sensilla could comprise distinct molecular signaling pathways that underlie their unique olfactory functions. These include seven GPCRs that could serve to differentially regulate the activity of ORNs in response to neuromodulators or hormones (eceptors . Other aatonal or amos antennae. Additionally, there are a large number of such enzymes that are either amos-depleted or atonal-depleted, even if they are not antennal enriched. Together this indicates that different sensilla classes may utilize different degradation enzymes. One reason this might occur is sensilla may selectively express enzymes needed to metabolize odors their ORNs detect, with the segregation of enzymes for alcohols, esters, and long-chain hydrocarbon pheromones to single-walled sensilla and enzymes for amines and acids to double-walled sensilla (We found that a large number of AE genes encode enzymes with potential roles in odor degradation and that these are often depleted in either sensilla . If so, amos and atonal mutants, suggesting similar ciliary structures are found in both single- and double-walled sensilla. Further, antennal expression of ciliary gene orthologs is highly conserved in the four insect species examined.The structure of insect ORN outer dendrites is thought to resemble a modified cilium , but onlAlthough our approach sought to identify genes with exclusive roles in olfaction, it is clear that ciliary genes also contribute to the function of other ciliated cells and biological processes. Ciliary genes were most likely detected as antennal-enriched due to the dense tiling of olfactory sensilla on the antennal surface (Our study identified a large number of known olfactory genes and many more novel genes with potential roles in olfaction, but it is likely that some genes with relevance for olfaction were overlooked. By design, our computational approach discarded many genes that are important for olfaction and contribute to the physiology of other tissues. This may be the reason why we did not detect genes that are likely to maintain the transepithelial potential, an electrical potential that drives odor receptor transduction and likely relies on potassium ion movement (amos-depleted and atonal-depleted datasets, our identification of AE genes reveals new avenues for examining the mechanistic underpinnings of poorly understood processes that support faithful odor detection in the antenna.A key advantage of our approach is the identification of genes with potentially conserved roles in olfactory signaling. We discovered many AE genes with orthologs expressed in the antennae of all insects we examined, making them prime targets for future mechanistic investigations. When combined with information from our


Rapid, easy and early pancreatic cancer diagnosis and therapeutic follow up continue to necessitate an increasing attention towards the development of effective treatment strategies for this lethal disease. The non invasive quantitative assessment of pancreatic heterogeneity is limited. Here, we report the development of a preclinical imaging protocol using ultrasonography and shear wave technology in an experimental in situ pancreatic cancer model to measure the evolution of pancreatic rigidity.Methods: Intrapancreatic tumors were genetically induced by mutated Kras and p53 in KPC mice. We evaluated the feasiblity of a live imaging protocol by assessing pancreas evolution with Aixplorer technology accross 36 weeks. Lethality induced by in situ pancreatic cancer was heterogeneous in time.Results: The developed method successfully detected tumor mass from 26 weeks onwards at minimal 0.029 cm3 size. Elastography measurements using shear wave methodology had a wide detection range from 4.7kPa to 166.1kPa. Protumorigenic mutations induced a significant decrease of the rigidity of pancreatic tissue before tumors developed in correlation with the detection of senescent marker p16-positive cells. An intratumoral increased rigidity was quantified and found surprisingly heterogeneous. Tumors also presented a huge inter-individual heterogeneity in their rigidity parameters; tumors with low and high rigidity at detection evolve very heterogeneously in their rigidity parameters, as well as in their volume. Increase in rigidity in tumors detected by ultrafast elasticity imaging coincided with detection of tumors by echography and with the detection of the inflammatory protumoral systemic condition by non invasive follow-up and of collagen fibers by post-processing tumoral IHC analysis.Conclusion: Our promising results indicate the potential of the shear wave elastography to support individualization of diagnosis in this most aggressive disease. Heterogeneity is not simply a function of the cancer cells themselves, but encompasses also the manner in which cancer cells interact with other cells of the tumor microenvironment, also called stroma. This, in turn, impacts the cancer response to treatment therapies, which is different in each individual. As a consequence, treatment choice and its efficiency evaluation should be individualized and tested as such in preclinical settings, to help clinicians predict the best therapeutic option in the shorter timeframe possible, and then to assess the variablity of response of the newly developed therapeutic strategies Pancreatic ductal adenocarcinoma (PDAC) is a dismal disease without effective therapeutic option except surgery. This disease is the most lethal of the common cancers, and is projected by 2030 to be the second highest cause of death due to cancer. The European Union has the highest incidence of pancreatic cancer in the world; incidence is increasing in France, and throughout European latin countries 4Ultrasound echography (US) is an inexpensive, non-invasive method of diagnostic or treatment evaluation that can be performed easily and repeatedly. However, US images do not quantitatively measure the changes of the physical characteristics of the tumors, possibly indicative of the changes of tumor physiopathology, neither measure the physical changes occuring in pancreatic parenchyma before the detection of the tumors, which could help to diagnose earlier Two dimentional shear wave elastography (2D SWE) as opposed to point shear wave elastography (point SWE) is a recent live ultrafast imaging method which allows a longitudinal follow-up of tissue rigidity in time and space in situ intra-abdominal tumors, and in particular of pancreatic tumors is emerging  model of colon cancer 23. As opposed to point measurements by other technologies, the real‐time imaging by 2D-SWE allows in theory the assessement of the stability of the measurement and quantification of an average value of rigidity in a large region of interest for higher reliability.Tissue stiffness affects tumor growth, invasion, metastasis and treatment. Currently, several elastography procedures exist and measure tissue stiffness We aimed to further investigate the role of ultra fast shear wave elastography as a complement to B mode echography using a spontaneous complex mouse model of PDAC, so as to set a standard of this physiologically relevant quantitative imaging of pancreatic cancer development. Here, we report on multimodal echographic and elastographic non-invasive imaging in GEM with endogenous aggressive PDAC to demonstrate that its intratumoral and interindividual heterogeneity in rigidity is associated with different tumoral evolution.+/- and LSL-KrasG12D/+ and LSL-Trp53R172H/+ mice, called hereafter KPC) mice were used as a model of locally advanced PDAC that exhibits a typical human-like morphology with abundant desmoplasia, moderate to poor epithelial differentiation and a highly aggressive clinical course   Figure B, values) Figure D. To con) Figure E. The in) Figure B,E,F. As) Figure G; the av) Figure A. We con) Figure A,B,E. Th) Figure H.in situ tissular rigidity.Total area, intra-pancreatic area of the pancreas, kidney cortex measurements lead to reproducible and consistent quantification of normal in situ developed tumors. We performed tumor detection using B-mode imaging in a cohorte of KPC mice, and longitudinally analyzed body weights and elastography in pancreas with SWE mode , two in the lower rigidity group , three with high rigidity values . Blood perfusion is known to influence the rigidity values We next questioned if the measure of pancreatic tissular rigidity could detect the microscopic changes in the pancreatic parenchyma which occur before tumor development, possibly helping in an earlier detection of tumors. Indeed, pancreatic oncogenesis develops from preneoplastic lesions, encompassing acino-ductal metaplastic lesions (ADM), pancreatic intraepithelial neoplasia (panIN) or cysts (mucinous cystic neoplasm (MCN), intra-ductal papillary mucinous neoplasm (IPMN). Very frequently (> 90% of cases), and at a very early stage (from low grade panIN), the activating mutation of the Kras oncogene is found. These lesions are found in KPC mice. We thus longitudinally analyzed body weights and elastography in pancreas with SWE mode, while tumor detection was performed using B-mode imaging Figure C. We comUltra fast shear-wave elastography allows the quantification of a pretumoral stage in a reproductive manner and demonstrates in a non invasive manner the increase in mechanical rigidity in tumors.We then aimed to discover the origin of the baseline modification of elasticity induced by protumoral mutations in the pancreas prior tumor development Figure , by analFinally, we searched whether this modification in rigidity of the tissue could be correlated with changes in stainings of F4/80 macrophages, in picro-sirius red positive collagen fibers, or in circulating white blood cell parameters, inflammation being usually associated with senescence in pretumoral condition. For the latter, we analyzed blood counts in the cohorts of mice defined above in Figure Microscopic tissular modifications in the pretumoral tissue where the cancer originates, tissue also described as the pretumoral niche, relate to elastographic measurement modifications. Further modifications of rigidity in tumors is associated with changes in the tissular composition . Measure of apparent tissular rigidity by SWE appears as an efficient mode to non-invasively qualify and quantify tissular composition parameters in both pretumoral niche and in locally advanced primary tumors of the pancreas .Advances in therapeutic approaches and molecular subtyping of PDAC The pancreas is a small organ located deep into the body and is strongly affected by aortic pulsation, which complicates the imaging possibilities In vitro data show that cancer cells present an increased elasticity possibly due to changes in their cytoskeleton properties Surprisingly, in our model, pretumoral niche was associated with a significant baseline lower rigidity in the pancreatic parenchyma. Our data so far show that elastography detects earlier signs of tumoral development induced by focal oncogenic mutations. These signs appear to be early pancreatic inflammatory parenchyma (as assessed by increased senescence), translated as a decrease in tissue rigidity. in situThe biomechanical properties of a tissue in terms of elasticity, relatively measured as a rigidity parameter in Pa vary markedly between organs and tissues, and are inherently related to tissue function and content. Increased expression of enzymes modifying extracellular matrix (ECM) by stromal cells results in increased collagen linearization and tissue rigidity in pre-malignant breast tissue Finally, survival of patients with PDAC does not correlate with total fibrillar collagen content within the tumor, but is significantly linked to the localized increased thickness of collagen fibers directly adjacent to PDAC epithelium In conclusion, early signs of tumor development include changes in the mechanical properties of tissues. As tumors metastasize, some cancer cells within stiffen their microenvironment by increasing the production and cross-linking of collagen. This local rise in rigidity helps the metastatic cells migrate and invade other regions of the body, this occur in coordination with the recruitment of protumoral immune cells. Our data pave the road for the development of novel preclinically and clinically relevant methodologies to assess tumor initiation and tumor - environment heterotypic dialog in pancreatic cancer.G12D and LSL-p53R172H knockin , Pdx1-cre  strains were interbred on mixed background (CD1/SV129/C57Bl6) to obtain compound mutant LSL-KrasG12D;LSL-p53R172H;Pdx1-Cre (named KPC). Littermates not expressing Cre as well as Pdx1-Cre of the same age were used as control. All procedures and animal housing are conformed to the regulatory standards and were approved by the Ethical committee according to European legislation translated to French Law as Décret 2013-118 1st of February 2013 (APAFIS 3601-2015121622062840). Genotyping was performed using primers as described in The LSL-Kras™ SL22-7lab (Supersonic Imagine)with preset called "standard" or "optimized" (Supplementary Table c2) in the studied region of interest. Measurement of the shear wave speed results in qualitative and quantitative estimates of apparent tissue elasticity. Tumor volume is calculated as follows: Width2 x Length x 0.5. Mean detection time of tumor is of 29 weeks; hence this time point was considered as T0 for the Ctrl cohorte. Rigidity values corresponds to the mean of E measured one to six times at each time point. During post-processing analysis, images in the pretumoral of Ctrl pancreas with grey zones were excluded, because values are repeated in time. Some tumors presented limited areas (<5-10%) with grey zones, images which could not be improved by changing the angle of imaging; they were not excluded.KPC and control mice (Ctrl) are imaged once a week. The probe SuperLinearHematoxylin eosin stainings were conducted using standard methods on formalin-fixed, paraffin-embedded tissues. All pancreata were analyzed in blinded fashion. Pancreas were fixed in 10% neutral buffered formalin and embedded in paraffin. For histopathological analysis, pancreata were serially sectioned (4 µm) and every 10 sections stained with hematoxylin and eosin. Histopathological scoring of pancreatic lesions was performed using serial H&E-stained sections . Picro sirius red staining was performed using the manufacturer condition (AbCAM#150681), with minor modifications corresponding to a 30 minute only incubation with the staining reagent.Immunostainings were conducted using standard methods on formalin-fixed, paraffin-embedded tissues. Antigen retrieval and antibody dilution  was carried out as described in table below followed by AEC or DAB incubation prior secondary antibody  or BA-4001 ).www.lasec.cuhk.edu.hk/.../power_calculator_14_may_2014.xls).Experimental data provided at least 3 experimental replicates. Statistical analyses were performed with GraphPad Prism using t-test, or parametric Mann-Whitney tests: * p < 0.05, ** p < 0.01, *** p < 0.001. Non-significant (ns) if p ≥ 0.05. Correlation analysis was performed using Pearson r test. Statistical relevance of the cohort size was determined using a Power Calculation test (


The imaging spectrum of pulmonary metastases varies greatly, with solid and partly cavitating nodules being the most common. When imaging the oncology patient, specifically follow-up imaging post-treatment, the radiological aim is to assess for disease regression and thus treatment response, usually with resolution of these nodules. We report an interesting case series of a patient with primary endometrial carcinoma presenting with pulmonary metastases. This imaging series eloquently depicts the temporal evolution of the metastatic solid pulmonary nodules to cavitating nodules and finally to thin-walled cysts. Baseline imaging in this scenario is vital to exclude pre-existing cystic lung disease. The progression of solid pulmonary metastases to simple cysts is an uncommon therapy-related consequence, but an important entity to recognise, not only as an indicator of good treatment response, but also to evaluate for potential life-threatening complications such as spontaneous pneumothoraces. The radiological imaging spectrum of pulmonary metastases is vast and may range from solid nodules to lesions mimicking pulmonary cysts. The histology of the primary tumour is often helpful, particularly in the setting of squamous cell carcinomas, where primary and secondary pulmonary lesions often appear to be cavitating.We report an interesting case series of patient KM, a 64-year-old female who had presented with a 4-month history of post-menopausal vaginal bleeding. Her co-morbidities included senescent hypertension and cardiac disease, stable on double agent anti-hypertensives. She was also a social smoker. An initial pipelle biopsy sent for microscopy revealed moderately differentiated carcinoma in the endometrial tissue which stained positive for vimentin and oestrogen receptors, but negative for carcinoembryonic antigen (CEA). This was consistent with endometrial adenocarcinoma. She subsequently presented to radiology for staging computed tomography (CT) scan post-surgery . Her post-operative recovery was volatile with ureteric reimplantation required after iatrogenic injury, several urinary tract infections and renal complications. Patient KM’s initial CT abdomen/pelvis, with imaging through the lung bases, revealed multiple bilateral solid pulmonary nodules of moderate size, in keeping with Stage 4 disease. The distribution was random with no associated calcifications, septal thickening or pleural effusions. She received single agent palliative chemotherapy with Paclitaxel 280 mg, a taxane-based chemotherapy agent targeted for the treatment of endometrial adenocarcinomas. The usually used combination treatment of a cisplatin was not utilised owing to her ongoing renal dysfunction.On dedicated chest imaging, approximately a month after initial staging and initiation of treatment, several of the previously noted solid pulmonary nodules now demonstrated discrete central lucencies, in keeping with cavitation. 1 Chemotherapy was reserved for patients with recurrent or disseminated disease, especially in the setting of failed hormone therapy.1 The use of taxane-based chemotherapy agents has been documented as being highly effective in the treatment of adenocarcinomas.2 Together with platinum agents, such as cisplatin, it is often the first-line chemotherapy agent used. In the documented literature, a patient known with primary adenocarcinoma of the lung, who was treated with Docetaxel, demonstrated secondary cavitatory change of the pulmonary metastases with bilateral spontaneous pneumothoraces.2 Docetaxel is also a member of the taxane family of chemotherapy agents. Paclitaxel, the drug of choice in patient KM’s case, demonstrated good treatment responses in advanced/recurrent disease.1Prior to a decade ago, primary treatment for patients with uterine cancer was radiotherapy based.3 Pulmonary metastases via haematogenous or lymphatic spread is usual, with cavitating or cystic appearing pulmonary metastases considered a subgroup.4 This makes the distinction from incidental non-malignant lung pathology much harder.5 The clinical circumstances and course of disease, that is, an acute onset versus protracted illness, are important considerations when evaluating cystic and cavitating lung lesions.3The lung is the most common location for metastatic lesions from a non-pulmonary primary malignancy.6 On imaging, it appears as a rounded lucent lesion and is usually thin-walled.6 In comparison, a cavity is a lucent lesion within an area of consolidation or within a nodule.6 A nodule is classified as a rounded opacity measuring up to 3 cm, and may appear poorly or well-defined.6According to the Fleischner glossary of terms, a cyst is defined as a ‘round circumscribed space surrounded by an epithelial wall of variable thickness’.7 Upper lobe and centrally located lesions are more likely to cavitate.7 Whilst squamous cell carcinomas are renowned for cavitating pulmonary lesions, other possible primaries include sarcomas, and gastrointestinal, bladder or pancreatic malignancies.5 It has been well-documented that certain chemotherapy agents are known to induce cavitation.1–3 Several postulated mechanisms included tumour necrosis or insinuation of air with a check-valve mechanism into the tumour.1–3 Spontaneous pneumothoraces have also been reported in previous literature, citing increased intrathoracic pressures following emetic sequelae of chemotherapy or possible bronchopleural fistulae formation from tumour lysis/necrosis, as aetiologies.2 Rupture of sub-pleural bullae or emphysematous bullae have also been postulated.8 Such patients were subsequently treated with closed chest tube insertion and chemical pleurodesis.2Cavitation of solid pulmonary nodules may be spontaneous or therapy-induced, from either chemotherapy or radiotherapy.The evolution of solid pulmonary metastases to cavitating and then cystic lesions, as a consequence of therapy, is a fairly rare entity albeit an important one to consider. The possibility of spontaneous pneumothorax is an uncommon but significant complication, which requires interval clinical and imaging evaluation to preclude preventable patient morbidity and mortality.


Despite great efforts in stroke research, disability and recurrence rates in ischaemic stroke remain unacceptably high. To address this issue, one potential target for novel therapeutics is the glycoprotein von Willebrand factor (vWF), which increases in thrombogenicity especially under high shear rates as it bridges between vascular sub-endothelial collagen and platelets. The rationale for vWF as a potential target in stroke comes from four bodies of evidence. (1) Animal models which recapitulate the pathogenesis of stroke and validate the concept of targeting vWF for stroke prevention and the use of the vWF cleavage enzyme ADAMTS13 in acute stroke treatment. (2) Extensive epidemiologic data establishing the prognostic role of vWF in the clinical setting showing that high vWF levels are associated with an increased risk of first stroke, stroke recurrence or stroke-associated mortality. As such, vWF levels may be a suitable marker for further risk stratification to potentially fine-tune current risk prediction models which are mainly based on clinical and imaging data. (3) Genetic studies showing an association between vWF levels and stroke risk on genomic levels. Finally, (4) studies of patients with primary disorders of excess or deficiency of function in the vWF axis  which demonstrate the crucial role of vWF in atherothrombosis. Therapeutic inhibition of VWF by novel agents appears particularly promising for secondary prevention of stroke recurrence in specific sub-groups of patients such as those suffering from large artery atherosclerosis, as designated according to the TOAST classification. Stroke remains among the top three diseases causing death and disability worldwide.While mortality rates are decreasing in both high- and low-income countries, morbidity rates in stroke survivors are still increasing over time.Besides enormous personal burden, costs in the United States for post-stroke care are estimated to be about $100 billion each year, including direct and indirect costs.Many promising options for prevention and treatment of ischaemic stroke have been tested, including invasive endovascular techniques and conservative medical management strategies, but only a limited number of treatments have gained approval by the Food and Drug Administration and the European Medicines Agency for either stroke prevention and/or treatment within the last decade.Development of pharmacotherapy for ischaemic stroke indications continues to lag behind than that for myocardial infarction. Especially in regard to anti-platelet therapy, many drugs with a favourable benefit:risk ratio in coronary disease have not proven beneficial in cerebrovascular disease, where the intrinsic susceptibility to treatment-induced bleeding is seemingly increased. These include: heparin, the GPIIb/IIIa inhibitors abciximab and tirofiban,Improved understanding of ischaemic stroke pathogenesis at the molecular level and the identification of high-risk sub-types of stroke should enable development of novel and specific treatment strategies. In recent years, von Willebrand factor (vWF) has been consistently implicated in the epidemiologic literature as a risk factor for ischaemic stroke. Its role in platelet thrombogenesis at sites of arterial vascular injury in the presence of high shear force, and functional ‘silence’ in other vascular beds in the absence of shear force, makes it a logical target for primary and secondary stroke prevention, and possibly also for stroke treatment.This review summarizes available evidence for the rationale for vWF inhibition in ischaemic stroke in the light of disadvantages of currently used pharmacotherapy, suggests how these may be addressed by targeting the GPIb-vWF axis and discusses stroke populations that may benefit from vWF-targeted therapy. Animal studies that improved understanding of the impact of vWF on stroke have recently been thoroughly reviewed by Denorme and De MeyerIschaemic brain injury arises in conditions of insufficient blood supply. This results from thrombosis, emboli or systemic hypo-perfusion, which together account for approximately 80% of all strokes. Specifically, large artery atherosclerosis . VWF protects FVIII from proteolytic inactivation by activated protein C and S.Endothelium-derived vWF is synthesized mainly within endothelial cells. After several post-translational modifications including cleavage of a propeptide, both mature vWF and the propeptide are stored in Weibel–Palade bodies.Thrombus initiation by vWF–GPIb interactions is a crucial step, because it promotes platelet-rolling on the injured vessel wall and further engagement of platelet receptors and their substrates. One of these platelet receptors is GPIIbIIIa, which is not only capable of binding to fibrinogen, but also to vWF itself. However, while vWF–GPIb interactions are substantial for thrombus initiation, vWF-collagen and vWF-GPIIbIIIa mainly contribute to thrombus growth.vWF has also been linked to numerous pathological processes including angiogenesis, cell proliferation and inflammation. Several mechanisms are involved in vWF-mediated inflammation, specifically platelet interactions and the GPIb–vWF axis. Direct interactions at low shear rates between vWF and leukocytes, as well as vWF-induced increased vascular permeability, diapedesis of leukocytes and accumulation of vWF in the extra-vascular space all promote inflammation.Inflammation plays a major role in atherothrombosis, highlighting its importance in ischaemic stroke. Arteriosclerosis is a localized inflammatory process in which increased amounts of ultra-large vWF strings are released from Weibel–Palade bodies under shear stress and attached to the inflamed endothelial surface. Cholesterol-induced atherosclerotic plaques were shown to contain high levels of Weibel–Palade bodies and vWF deposits..It is conceivable that pharmacological vWF inhibition may limit or even prevent development of atherothrombosis at vascular sites of high shear stress, but further studies are needed to conclusively define the potential of vWF as therapeutic target in the prevention or treatment of arteriosclerosis per seAs vWF is critically involved in platelet aggregation and thrombus formation upon endothelial activation, it has been studied intensively as a potential risk factor for stroke and stroke-related outcome.An association between vWF levels, the risk of first-ever ischaemic stroke,Several laboratory tests are available for assessing vWF activity and vWF quantity . MeasurvWF can be measured quantitatively by either vWF:Ag or vWF:pp. As mature vWF and vWF propeptide differ in their kinetics, the assessment of both helps in determining vWF secretion, clearance and survival. The half-life of vWF propeptide is 2 to 3 hours and therefore shorter than the 8 to 10 hours half-life of mature vWF.Supportive evidence for a causative role of vWF in stroke comes from animal models, which have consistently shown that deficiency in vWF is protective against cerebral ischaemia.Numerous case–control studies that determined vWF levels at the time of ischaemic stroke showed a significant increase in vWF as compared with controls.Markers closely associated with vWF, such as ADAMTS13 and FVIII, are also significantly different between stroke patients and controls; e.g., higher FVIII levelsStill, low ADAMTS13 activity might be an independent risk factor for stroke occurrence as shown by a large observational case–control study in young women with stroke in which the ADAMTS13 measurements were performed in the very late phase after stroke. The retrospective odds ratio (OR) for first-ever stroke comparing the lowest quartile to the highest quartile of convalescent ADAMTS13 activity was 3.1 (1.6–5.8), even after adjustment for several risk factors.Evidence for the causative role of vWF in stroke comes from large longitudinal studies that investigated stroke-naïve patients and analysed their stroke prevalence with respect to baseline vWF levels and ADMTS13 activity . Smith In a much larger sample size, the ‘Atherosclerosis Risk in Communities study’ showed a positive association between baseline vWF levels and the risk of stroke occurrence. In this prospective study that followed more than 14,000 patients free of cardiovascular disease for up to 9 years, the risk of stroke in patients in the highest vWF quartile was 1.7  times higher than in those in the lowest vWF quartile. Likewise, in an observational case–control study of patients with first time stroke, the risk of stroke increased by 80% with every quartile of vWF determined at the time of ischaemic event.Further supporting data come from the ‘Rotterdam study’, another prospective population-based cohort study that followed more than 6,000 patients, who were free from stroke at baseline over a median follow-up time of more than 10 years .Increasing vWF levels were associated with a significant increase in the risk of stroke . In a subsequent analysis of the Rotterdam study population, Sonneveld et al were the first to perform a combined analysis for vWF-antigen and ADAMTS13 activity in a longitudinal study. Patients with high vWF Antigen levels (≥ 75th percentile) along with low ADAMTS13 activity (≤ 25th percentile) were at a significantly increased risk for ischaemic stroke with a HR of 1.71 .Taken together, these data support the rationale that not only vWF levels, but also the vWF-modifying enzyme ADAMTS13 plays an important role in the development of first-ever stroke. Therefore, screening patients free of stroke at preventive check-ups could help to identify high-risk patients, stratifying them for risk of future stroke based on their vWF levels and/or ADAMTS13 activity.Recent genome wide association studies identified several gene loci linked to sub-types of ischaemic stroke,vWF plasma levels are strongly heritableGenetic determinants and vWF levels were investigated with respect to overall stroke risk in the large prospective Rotterdam study. However, no genetic predictor of stroke risk was found while higher genetic scores correlated with vWF levels.Data for the impact of vWF levels on stroke outcome and the risk for stroke recurrence came from observational and case–control studies. vWF levels were measured at the time of first ischaemic event and therefore no clear assumption can be made if vWF determines post-stroke morbidity and mortality as well as recurrence or only predicts outcome in stroke patients.In this context, Carter et al found that 3-year mortality from stroke increases with vWF activity ranging from 16% in those with levels < 135%  to 52% in patients with vWF levels > 243%.p = 0.0007; HR, 1.26) and fully adjusted  models. Tools to predict early stroke recurrence integrate only clinical and/or imaging information, but accuracy might improve taking laboratory parameters into account.With respect to stroke recurrence, few data are available. Williams et al showed that elevated vWF levels increase the risk for stroke recurrence in both unadjusted (defined above) sub-type of stroke compared with other sub-types have also been reported by Sonneveld et al in a large observational study. These authors additionally found a strong association between vessel calcification volume in the aortic arch and carotid arteries and plasma vWF levels at the time of ischaemic event in 925 patients.vWF levels are significantly higher in large- than in small-vessel disease.Therefore, these findings suggest that the LAA sub-type of stroke could be particularly sensitive to a vWF blocking strategy. This is plausible considering the key role of vWF in shear stress-dependent haemostasis at the site of vessel stenosis and plaques.To date, the only Food and Drug Administration-approved medication for acute ischaemic stroke is recombinant tissue plasminogen activator (rtPA), based on the results from the National Institute of Neurological Disorders and Stroke (NINDS) landmark study published in 1995.More recently, mechanical endovascular interventions have been introduced into the standard of care for acute stroke management, and in 2015, the current guidelines for acute stroke treatment were updated and mechanical treatment with or without rtPA is recommended. Stent retrievers are recommended in addition to rtPA in internal carotid artery or middle cerebral artery-M1 occlusion.Intravenous thrombolysis is only administered to 5.2%Although there is some evidence that mechanical thrombectomy is beneficial in a time window of up to 12 hours after symptom onset, i.e., when rtPA is not further indicated, definitive data are still lacking. However, a recent randomized controlled trial showed that thrombectomy was still beneficial if performed 6 to 24 hours after stroke onset in patients with a mismatch between infarct size and clinical disability. Thirty-three per cent more patients achieved functional independence by 90 days  in the arm receiving thrombectomy plus standard medical care compared with those in the arm receiving standard medical care alone. As the sample size was rather small with 206 enrolled patients, larger trials confirming this benefit are still warranted.In patients with myocardial infarction and pulmonary embolism who receive thrombolysis with rtPA, the risk for symptomatic intra-cranial bleeding is 1 to 1.5%.However, in the limited number of patients who receive thrombolytics, early recanalization 2 hours after the start of rtPA treatment is achieved in approximately 50% of patients,Besides clarity concerning the beneficial time window for mechanical clot retrieval, definitive evidence concerning the best endovascular treatment option is still lacking. To date, mechanical stent-retrievers are the most promising devices.Furthermore, there are only limited data for combined treatment with mechanical thrombectomy and concomitant anti-platelet agents. In a prospective study, administration of tirofiban in addition to endovascular thrombectomy was safe and associated with lower odds of death.Besides modification of traditional risk factors for stroke, such as hypertension, diabetes mellitus, smoking, dyslipidaemia and physical inactivity,p = 0.043) over aspirin for the intention-to-treat population regarding the primary combined end point of ischaemic stroke, myocardial infarction or vascular death.p = 0.26).Two previous trials, however, reported greater benefit for secondary stroke risk reduction with aspirin plus extended-release dipyridamole than with aspirin aloneWhile data from animal studies showed a possible neuroprotective effect of aspirin with or without dipyridamole,Short-term dual anti-platelet therapy with aspirin and clopidogrel for 90 days is indicated only in high-risk patients with symptomatic intra-cranial large artery disease, as these patients are known to have a high risk of early stroke recurrence after first-ever transient ischaemic attack. Aspirin and clopidogrel in combination, however, should not be used for long-term stroke prevention because of a lack of superiority compared with clopidogrel alone together with a substantially increased risk of major bleeding events.The critical issue in secondary stroke prevention is finding the right balance between sufficient anti-platelet effects and a low bleeding risk. Recent data focusing on major bleeding events showed that the cumulative 3-year major bleeding risk for stroke patients on anti-platelet therapy was overall 23%. Risk for ischaemic events despite anti-platelet therapy was even above 60%.One reason for the limited potency of aspirin and clopidogrel in secondary stroke prevention may be mediated by haemostatic mechanisms of platelet activation and thrombus formation that are independent from platelets' cyclooxygenase and adenosine diphosphate receptor pathways. Twenty years ago, it was already demonstrated that shear-induced platelet aggregation mediated by exogenous or platelet-derived vWF cannot be inhibited by aspirin in vitro.p < 0.001), which likely contributes to the resistance to the inhibitory effects of aspirin under high shear rates.In a recent longitudinal observational study investigating platelet function by means of multiple electrode aggregometry in 624 stroke patients on anti-platelet therapy, 11% of patients with a stroke while on aspirin had high on-treatment platelet reactivity, and treatment with clopidogrel was accompanied by even higher proportions of unresponsiveness (36%), independent of the etiologic sub-type of stroke.p < 0.001) was confirmed by a recent meta-analysis, which analysed 15 studies with 4,762 stroke or transient ischaemic attack patients treated with clopidogrel.n = 4,009, 84%). Another very recent study investigated the impact of glycaemic control on clopidogrel responsiveness in Chinese patients. Data showed that treatment with clopidogrel was beneficial only in non-carriers of CYP2C19 loss-of-function alleles who maintained good glycaemic control.Clopidogrel's efficacy in preventing stroke likely depends on CYP2C19 genetic variants, which determine conversion of the inactive pro-drug to its active metabolite. Clopidogrel in addition to aspirin reduced the risk of recurrent stroke only in patients who did not carry CYP2C19 loss-of-function alleles in a large trial involving almost 3,000 patients with minor ischaemic stroke or transient ischaemic attack.Several novel pharmacological approaches have been tested to overcome the difficulties of current stroke therapy, but with ambiguous results. Desmoteplase and de-fibrogenating agents were drug candidates promising to overcome the issue of rtPA-resistant thrombi which showed encouraging results in terms of reperfusion rates and disability, but still failed due to an increase in intra-cerebral bleeding.As the GPIb-vWF axis is haemostatically important, primarily under high shear conditions, physiological haemostasis under low or moderate shear conditions should theoretically not be impaired by vWF blockade. This is in agreement with available safety data on serious bleeding events from pre-clinical and clinical studies investigating vWF inhibitors in healthy volunteersRecently, the large COMPASS trial, including more than 27,000 patients, showed a risk reduction for stroke when rivaroxaban was combined with aspirin versus aspirin alone, while rivaroxaban alone was not significantly better.vWF might not only be an attractive target in acute stroke treatment but also in secondary stroke prevention, particularly if aimed at a sub-population at high risk of stroke recurrence that could especially benefit from a more potent anti-platelet therapy with a better risk profile.While multiple agents representing several drug classes have proven benefit in coronary artery disease, including heparin, GPIIb/IIIa inhibitors, P2Y12-receptor antagonists, such as ticagrelor and prasugrel, as well as the thrombin-receptor antagonist vorapaxar, none of these drugs have improved a benefit:risk ratio for stroke prevention superior to aspirin.n = 3,906) suggest that Asian patients or those suffering from LAA could potentially benefit from the more potent platelet inhibition by ticagrelor.The use of ticagrelor, a more potent third-generation adenosine diphosphate-receptor blocker, was not superior to aspirin in reducing the rate of stroke recurrence, myocardial infarction or death within 90 days in a large prospective trial involving 13,199 patients with a non-severe ischaemic stroke or high-risk transient ischaemic attack, and thus is currently not recommended.Prasugrel is contraindicated in patients with a history of cerebrovascular events based on results from the TRITON-TIMI trial. This study compared prasugrel to clopidogrel in more than 13,600 patients with acute coronary syndrome and found a significant harm from prasugrel among those with a history of stroke or transient ischaemic attack.Likewise, the thrombin-receptor antagonist vorapaxar is contraindicated in patients with prior stroke, because it increases the risk of intra-cranial haemorrhage in this population The use of dual anti-platelet therapy with clopidogrel and aspirin in secondary stroke prevention is considered beneficial only in the first 90 days because it was shown to cause harm in the form of major bleeding thereafter.long-termprevention of stroke recurrence. Aspirin reduced the risk of early recurrent stroke (i.e. in the first 6 weeks from the index event) in a recent longitudinal study investigating individual patient data from all available controlled trials on use of aspirin versus control in secondary prevention after transient ischaemic attack or ischaemic stroke, in contrast, its use had no effect on the 12-week risk of stroke recurrence.These data highlight the need for novel anti-thrombotic strategies that are safer and more effective than available anti-platelet agents, especially regardingThere are three potential targets that mediate vWF-dependent thrombus formation. These are vWF-GPIb, vWF-collagen and vWF-GPIIbIIIa. However, animal models showed, that while collagen- and GPIb-binding are essential in stroke development, GPIIbIIIa interactions are without any relevance.Nano-bodies are fragments of antibodies with lower molecular mass and size, which enable better permeability and quicker elimination. Furthermore, nano-bodies are less likely to cause cytotoxicity triggered by complement activation, as they lack the Fc-region.Another approach to target specific molecules is facilitated by aptamer technology. Aptamers are molecules composed of single stranded nucleic acids folded into a specific tertiary structure, that specifically bind to their target, e.g., proteins, enzymes, viruses or receptors.As vWF–platelet interactions are promoted by arterial shear stress, it is conceivable that patients with conditions, in which vWF likely plays a key role in thrombus formation, would in particular stand to gain benefit from therapeutic vWF blockade. This may include patients with stenotic LAA, those who have undergone carotid stenting, patients with persistently elevated vWF levels after an ischaemic event and those unresponsive to fibrinolytic or anti-platelet therapy.Whether vWF is independently associated with non-responsiveness to conventional anti-platelet drugs across the heterogeneous population of ischaemic stroke patients or is rather a major contributing factor in specific stroke sub-types or in the presence of distinct co-morbidities is as yet unclear and needs further investigation.The proportion of stroke patients unresponsive to aspirin and clopidogrel measured by whole blood aggregometry, however, is substantial.Future studies need to define populations that are prone to developing vWF-mediated stroke and identify stroke sub-types that are sensitive to vWF inhibition to further refine the strategy for the clinical development of such agents.Early re-canalization and vessel re-opening is vital to achieving good outcome after ischaemic stroke. Attempts at vessel re-canalization by thrombolytics, however, fail in the large majority of patients.Lytic susceptibility depends on the specific structural aspects of the clot. vWF-rich thrombi formed under shear stress were shown to be resistant to conventional recombinant tissue-type plasminogen activator in both stroke and myocardial infarction.In patients with 50 to 99% stenosis of a large intra-cranial artery, the annual rate of ischaemic stroke ranges between 6 and 15% despite standard medical treatment with current anti-platelet agents.Following results from a previous meta-analysis comparing incidence and sub-types of stroke between Chinese and European patients, the prevalence of LAA is significantly higher in Chinese patients.Increasing insights into vWF-mediated platelet–endothelium interactions at the site of arteriosclerotic lesions suggest that patients with intra-cranial major artery arteriosclerosis could possibly benefit from (add-on) treatment with a vWF-blocking agent. Plaque-associated thrombus formation is critically dependent on blood-borne vWF, offering the opportunity for a vWF-targeting approach.The significance of vWF in human atherogenesis, however, remains under investigation. High levels of vWF in LAA patients,In animal models of atherosclerosis, both the inhibition and deletion of vWF successfully reduced inflammation, plaque size and platelet adhesion,Lifelong administration of aspirin is recommended after carotid endarterectomy. To date, endarterectomy with stenting is recommended only in patients with symptomatic extra-cranial internal carotid artery atherosclerotic disease without total occlusion. Restenosis, however, may occur in up to 12% of patients within the first 24 months.In patients who undergo carotid artery stenting, dual anti-platelet treatment is commonly administered for 6 weeks to limit the risk for thromboembolic complications and restenosis.Endovascular procedures induce damage and activation of the endothelial layer with subsequent release of inflammatory markers including vWF release,The occurrence of stent restenosis was significantly associated with persistently elevated vWF levels in a prospective study investigating the relation between vWF plasma levels and restenosis after carotid artery stenting within the first 6 months.The rationale for why Asian individuals in particular might benefit from an anti-vWF approach is based upon several pieces of evidence. In China, approximately 2.4 million people suffer from stroke each year,This disproportionally high rate of intra-cranial atherosclerosis, the high prevalence of ischaemic stroke overall and the large percentage of LAA stroke sub-typesThe rationale for vWF as a potential target in stroke comes from four bodies of evidence: animal models, that help us to understand the pathogenesis of stroke and give rise to the concept of vWF-targeting strategies; epidemiologic data demonstrating that plasma levels of vWF predict the occurrence and recurrence of stroke; genetic studies that establish an association between vWF levels and stroke risk on genomic levels; and lessons learned from patients with vWF disorders that showed the crucial role of vWF in atherothrombosis and stroke. However, randomized clinical trials with vWF inhibitors are needed to ultimately demonstrate the relevance of blocking vWF for the prevention or treatment of ischaemic stroke in this patient population.Conceivably, identification of vWF-rich thrombi with high-resolution imaging techniques could help to stratify patients susceptible for an anti-vWF approach in the acute setting. Individualized acute stroke treatment could improve re-canalization rates with the maintenance of acceptable bleeding rates.Also, for primary and secondary stroke prevention, targeting high-risk populations will likely improve the benefit:risk ratio. Therefore, sub-populations of patients need yet to be defined that might best respond to vWF inhibition. The contemporary understanding of stroke as a clinically and biologically diverse entity led to the consideration of a more individualized approach to pharmacotherapy and endovascular interventions for care of patients with stroke, based on risk stratification and careful differentiation between patient sub-groups. However, patients' risk stratification should probably not only be based on pathophysiologic mechanism but also on the genetic determinants and biomarkers that reflect increased risk. Specifically, patients with a high degree of LAA and CYP2C19 loss of function alleles carriers might be ideal candidates for a vWF targeting approach.