The current review focuses on the role of PTPs in controlling CD4+ T cell activity in the intestinal mucosa and how disruption of PTP activity in CD4+ T cells can contribute to intestinal inflammation.Protein tyrosine phosphatases (PTPs) play a critical role in co-ordinating the signaling networks that maintain lymphocyte homeostasis and direct lymphocyte activation. By dephosphorylating tyrosine residues, PTPs have been shown to modulate enzyme activity and both mediate and disrupt protein-protein interactions. Through these molecular mechanisms, PTPs ultimately impact lymphocyte responses to environmental cues such as inflammatory cytokines and chemokines, as well as antigenic stimulation. Mouse models of acute and chronic intestinal inflammation have been shown to be exacerbated in the absence of PTPs such as PTPN2 and PTPN22. This increase in disease severity is due in part to hyper-activation of lymphocytes in the absence of PTP activity. In accordance, human PTPs have been linked to intestinal inflammation. Genome wide association studies (GWAS) identified several PTPs within risk loci for inflammatory bowel disease (IBD). Therapeutically targeting PTP substrates and their associated signaling pathways, such as those implicated in CD4 The gastrointestinal track is a large mucosal surface at which the host's immune system is juxtaposed with a dense microbial population and a diverse array of dietary antigens. Immune recognition of enteric antigens however, is minimized by physical compartmentalization. Bacteria and dietary products are retained within the gut lumen, while the host immune system is localized in the mucosal tissue. This physical separation is preserved by the intestinal mucosal barrier, which includes a mucin layer, a single epithelial cell lining sealed by tight and adherens junctions and a continuous secretion of anti-inflammatory soluble mediators Figure  1–5).–5.1–5).The mucosal barrier is not absolute however, and a small number of bacteria do translocate from the lumen to the underlying lamina propria. In such instances, a complex network of innate and adaptive immune cells impedes the spread of intestinal bacteria in a manner that limits tissue damage , 7. ThisPhysical compartmentalization and mucosal immunity therefore establish and maintain microbiome-host mutualism . The los+ T cell development and function. Examples include IL23R, JAK2, and STAT3  have been shown to induce remission in both CD and UC patients , Ustekinumab  and Infliximab   –33. It s+ T cells are classified into distinct subsets based on their inducing cytokines, transcription factor expression, and effector cytokine secretion. The initial classification of CD4+ T cells as TH1 IFNγ producers vs. TH2 IL-4 producers, has been broadened to include multiple additional subsets (H9 (IL-9), TH17 , TH22 (IL-22), T follicular helper TFH (IL-21) cells, as well as thymic-derived and peripherally-induced T regulatory cells   –40 , co-stimulatory molecules, and the T cell receptor (TCR), converge to induce gene expression profiles that drive CD4+ T cell activation and differentiation into distinct subsets  expressed on antigen presenting cells. Upon TCR engagement, Src-family kinases  are activated and phosphorylate tyrosine residues within the immune-receptor tyrosine-based activation motifs (ITAMs) in the TCR-associated CD3 and zeta chains –46. Phos+ T cell differentiation  cells are generated primarily from CD4+ Foxp3− T cells exposed to antigen under tolerogenic conditions or during homeostatic proliferation .–62.+ T c+ T cell activation and differentiation. The current review will examine the role of protein tyrosine phosphatases (PTP) in safeguarding this network, and how PTP deletion can perturb CD4+ T cell function and consequently contribute to intestinal inflammation.Thus a network of signaling pathways, heavily dependent on tyrosine phosphorylation, directs CD4The PTP family comprises a heterogeneous set of enzymes that were first defined by Tonks and colleagues by their capacity to dephosphorylate phospho-tyrosine residues and by their structurally related phosphatase catalytic domain , 64. PTPThe human PTP family members are divided into distinct classes based on their structural and biochemical properties Figure . The majMore recent studies characterized enzymes using alternate nucleophilic catalytic residues which were then added to the list of PTP family members. These two additional classes are the aspartic-based and histidine-based acid phosphatase enzymes. Thus, within this classification frame, Alonso and Pulido expanded the PTP gene superfamily to 125 members .Tyrosine phosphorylation is a reversible mechanism that promotes protein–protein interactions and propagates signaling cascades in all cell types. It therefore stands to reason that the PTPs targeting these phosphorylated amino acids play important roles in modulating the strength and duration with which a signaling pathway is activated. Moreover, given that PTPs are a heterogeneous gene family  it is not surprising that PTPs have been associated with many distinct pathologies in both animal models and in human disease . Of impoThe immune system relies heavily on the use of protein phosphorylation to recognize and transmit intra- and extracellular signals. Signaling cascades initiated at the cell surface by receptor kinases, or from the recruitment of cytoplasmic kinases such as the JAK and Src-like kinase families, translate into multiple immune cell responses like cellular adhesion, division, migration and others. Hence, from the list of over 100 classical Cys-based PTPs, Arimura and Yagi demonstrated that between 58 and 76 of them are expressed in various cell lineages of the immune system  and thatMultiple genome wide association studies (GWAS) have identified single nucleotide polymorphisms (SNPs) linked with IBD, in or near PTP coding units , 71. ExaPTPN22 is a non-receptor protein tyrosine phosphatase that is expressed primarily in hematopoietic cells . It has PTPN22 deficient mice exhibit a lymphoproliferative disease and accumulate memory-phenotype T cells with age, but do not display signs of spontaneous autoimmunity  tyrosine following TCR ligation and not due to changes to IL-2 or IL-15 receptor signaling  induced model, these studies attributed the increase in disease severity to changes in TLR4 signaling, type I interferon production and macrophage polarization , 95.PTPN2−/− CD4 and CD8 was identified as causing increased T cell activation due to hyper-phosphorylation of the activating tyrosine residue of Lck  presented higher expression of TH1 and TH17-related transcription factors and cytokines  in comparison to PTPN2 expressing CD patients. Such human studies solidify the important role of PTPN2 CD4+ T cell responses in the context of human IBD  is a cytoplasmic phosphatase expressed in all hematopoietic cell lineages throughout development and activation (me) and motheaten viable (mev) mice. These mice harbor mutations that result in undetectable and reduced PTPN6 expression respectively. Similar to PTPN2 deficient mice, me/me mice succumb to disease 2–3 weeks following birth . Similar to PTPN6, PTPN11 regulates signaling downstream of multiple surface receptors including growth factor receptors, cytokine receptors and integrins. Its broad tissue expression is in line with the embryonic lethality of PTPN11 knockout mouse at mid-gestation in –133.PTPN11 mutations in patients with multiple cancer types including leukemias, breast cancer and gastric cancer  differentiation is suppressed  mice were reconstituted with either WT or DUSP2−/− CD4+CD45RBhi T cells. In this model, an increase in body weight loss, severity of histopathology scoring, and colon thickening were observed following the transfer of DUSP2−/− T cells compared to controls. Such clinical signs were corroborated by a rise in the number of colon infiltrating CD4+ T cells expressing a TH17 gene signature. Moreover, such TH17 T cells in the inflamed tissue expressed elevated levels of pathogenic TH17-associated genes but not non-pathogenic genes  and is only increased minimally following PMA stimulation. The low levels of DUPS2 expression were found to be attributed to CpG methylation. Unfortunately, the levels of DUSP2 in colonic biopsies, or colonic T cells, were not quantified. As such, further evidence is required to definitely demonstrate that a reduction in T cell DUSP2 occurs in IBD patients and that this reduction impacts human T cell polarization in the disease state  signaling regulates multiple cellular responses to activation including T cell proliferation, cytokine production, survival and adhesion. CD4balance. –153.in vitro assays suggested a role for DUSP6 in the regulation of CD4+ T cell activation. Specifically, DUSP6 was shown to be upregulated following TLR4 stimulation and then restrained ERK activation and suppressed IFNγ production by TCR stimulation  that recognize and dephosphorylate the insulin receptor (IR). The regulation of IR activation by a PTP was hypothesized to decrease IR mediated signaling events, and subsequent entry of glucose into insulin receptive tissues. It was believed that a rise in the expression or activity of such a regulatory PTP would result in high levels of blood glucose in the circulation, which would then instigate type II diabetes. This hypothesis was confirmed by two seminal publications which described the PTP1B knockout mouse that presented with an increase in IR phosphorylation. These studies validated PTP1B as an outstanding candidate to be targeted for the treatment of type II diabetes , 159 andIn spite of all these reports, there are no small molecule inhibitors against PTP1B (or any PTP) that have been successfully developed beyond initial clinical trials. Multiple reviews have already examined the difficulties associated with developing PTP competitive inhibitors and this subject is beyond the scope of this review –163. In It is worthy to note that novel therapeutic strategies are also being developed to target PTPs. These include the forced dimerization and inhibition of receptors PTPs , 167, inex-vivo cell cultures. This allows for the modulation of PTP activity in a cell-type specific and temporary fashion. For example, we reported a protocol for dendritic cell (DC) vaccination that employs inhibitors of PTPN1-PTPN2 (Significant effort is also being made in the development of cell based therapies. Potential adverse effects that could be associated with systemically inhibiting PTPs are mitigated by using PTP inhibitors in N1-PTPN2 . This woFrom an IBD perspective, it is clear that inhibiting the PTPs presented above would most likely exacerbate disease by potentiating the effects of pro-inflammatory cytokines and promoting the differentiation of pathogenic T cells. Indeed this contention is supported by the fact that, biologics and small molecules that suppress cytokine receptor signaling have been clinically successful in IBD disease management. One example would be tofacitinib, a JAK inhibitor found to be effective in phase 2 and 3 trials in moderate to severe ulcerative colitis .Our understanding of PTP function in immune cells is expanding beyond cytokine receptor signaling, and our capacity to modulate cellular responses by titrating PTP expression is also evolving at a rapid pace. The cell-dependent context of positive or/and negative modulation bestowed by the nearly 80 PTPs expressed in immune cells, remains an exciting ground for study and clinical improvement. It remains to be seen if this renewed interest in PTP inhibitors would be applicable to inflammatory diseases such as UC and CD.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.


TMEM106B as a primary robust driver of lung cancer metastasis. Ectopic expression of TMEM106B could significantly promote the synthesis of enlarged vesicular lysosomes that are laden with elevated levels of active cathepsins. In a TFEB-dependent manner, TMEM106B could modulate the expression of lysosomal genes of the coordinated lysosomal expression and regulation (CLEAR) pathway in lung cancer cells and patient samples. We also demonstrate that TMEM106B-induced lysosomes undergo calcium-dependent exocytosis, thereby releasing active lysosomal cathepsins necessary for TMEM106B-mediated cancer cell invasion and metastasis in vivo, which could be therapeutically prevented by pharmacological inhibition of cathepsins. Further, in TCGA LUAD data sets, 19% of patients show elevated expression of TMEM106B, which predicts for poor disease-free and overall-survival.Metastatic lung cancer is the leading cause of cancer-associated mortality worldwide, therefore necessitating novel approaches to identify specific genetic drivers for lung cancer progression and metastasis. We recently performed an in vivo gain-of-function genetic screen to identify driver genes of lung cancer metastasis. In the study reported here, we identify  TMEM106B in driving metastatic lung adenocarcinoma and suggest that TMEM106B-mediated secretion of cathespin impacts cell migration and invasion of lung cancer cells, increasing metastatic spreading.One of the major causes of cancer-related mortality is represented by metastatic lung cancer. Here the authors characterize the role of  The development of new therapeutic strategies requires a better understanding of the underlying “driver” genetic aberrations responsible for lung cancer metastasis, as such events represent new targets for drug intervention. To identify such driver events, we previously used function-based gain-of-function in vitro and in vivo genetic screens to successfully identify new oncogenes and their activating mutations in melanoma2, breast cancer3, and pancreatic cancer4 among others. Using a similar approach, we recently performed a robust, in vivo positive selection screen to identify individual drivers of lung cancer progression and metastasis5. For this, we performed a gain-of-function screen where we identified an enriched list of 217 putative driver genes in lung cancer by cross-species comparison of the genes overexpressed in autochthonous genetically engineered metastatic murine lung tumors and syngeneic lung cancer models, with human copy number amplifications documented by The Cancer Genome Atlas (TCGA). We used a mouse syngeneic lung cancer model, which was developed in our lab to elucidate the molecular mechanisms underlying epithelial‐to‐mesenchymal transition and metastasis. The model comprises of different cell lines isolated from lung tumors and metastases from genetically engineered KrasLA1/+;p53R172HΔG/+ mice, which vary in their metastatic potential when implanted in syngeneic mice8. Next, we generated a lentiviral unique-barcoded expression library, utilizing sequence-verified open reading frames (ORFs) of the candidate genes. Using a nonmetastatic syngeneic mouse lung cancer cell line (393P), we generated individually transduced stable overexpressing lines for each candidate gene. We transplanted pools of ORF expressing lines into syngeneic immune-competent 129sv mice, and observed primary tumor growth and occurrence of metastases in lungs and other organs. DNA extracted for primary tumors and metastases was used for barcode sequencing by next-generation sequencing. Metastasis drivers were identified upon enrichment of their specific ORF-associated barcodes in metastases, when compared to total barcode reads in input cells. Among others, we identified TMEM106B as one of the most robust drivers of lung cancer metastasis in vivo.Lung cancer is a deadly disease resulting in the highest lethality rates among all cancers. Like other cancers, metastasis is the primary cause of cancer-related mortality among lung cancer patientsTMEM106B is a single pass, type-II transmembrane protein reported to localize on cellular lysosomes. While the protein has not been thoroughly studied in the context of cancer, TMEM106B loss-of-function is known to be associated with frontotemporal lobar degeneration, and is also repressed in the brains of Alzheimer’s patients12. More recently it was reported that TMEM106B has a prominent role in regulating lysosome synthesis, size, trafficking, and localization14. It was also reported that overexpression of TMEM106B in neuronal cells resulted in the activation of the lysosomal stress signaling by translocating the transcription factor TFEB, a member of the MiTF family, into the nucleus and thus up-regulating the coordinated lysosomal expression and regulation (CLEAR) network genes14. The MiTF family of transcription factors has also been shown to directly regulate the expression of lysosomal hydrolases, primarily cathepsin K16.20. More recently, lysosome functions have been reported to be dramatically changed during cancer progression, where lysosomal volume, composition, and subcellular localization have been altered21. Lysosomes are reported to be routed toward the periphery of the cell under multiple stimuli, which are responsible for the onset of different pathological states. For example, during malignant transformation the peripheral juxtaposition of lysosomes with the plasma membrane and its contribution to cell invasion and migration makes this phenomenon of crucial importance for our research24. There is evidence that shows the elevated expression or activity of TFEB as a positive induction for lysosomal exocytosis. It has also been reported that increased production of lysosomal cathepsin B and L could act as signals for increased lysosome trafficking to the plasma membrane26. Upon localization at the pericellular space, the lysosome are fused to each other before fusing with the plasma membrane, usually as a result of intracellular calcium influx. This final fusion of the lysosomes to the plasma membrane results in the transfer of the luminal sialylated lysosomal membrane protein LAMP1 to the plasma membrane, and also the release of the complete lysosomal content into the extracellular matrix or media.Lysosomes are phospholipid bilayer membrane bound vesicles, ubiquitously present in all cell types and essential for the catabolic clearance and recycling of degraded proteins, macromolecules, dysfunctional organelles, and digestion of extracellular and foreign materials delivered to them by endocytosis, autophagy, and phagocytosis29. In our syngeneic mouse model, we have observed by proteomic profiling that the metastatic cells demonstrated a significantly high level of secreted cathepsins30, which might drive the hyperinvasive and metastatic phenotypes. Cathepsins secreted into the extracellular matrix (ECM) via lysosomal exocytosis or cell lysis, are the primary effectors of ECM degradation, dissolution of cell–cell adhesion molecules, and activation or initiation of cytokine and chemokine responses that are all critically contributory toward cancer cell invasion and metastasis35. Here, we have identified TMEM106B as a novel and specific driver of lung cancer metastasis. TMEM106B deregulates lysosome function by affecting lysosomal synthesis and exocytosis in lung cancer cells, thus resulting in increased release of lysosomal cathepsins into the extracellular matrix, leading to cellular invasion and metastasis. Therapeutic intervention of TMEM106B activity by inhibiting cathepsins in vivo in a metastatic syngeneic mice model with aloxistatin treatment successfully blocks TMEM106B-mediated metastasis.Cathepsins and other lysosomal hydrolases have been reported to be significantly overexpressed in a number of cancers leading to increased invasion of cancer cellsTMEM106B, whose associated barcode was present in 10 independent lung metastasis tissue cores from 5 different animals in the screen. To further validate TMEM106B as a driver of in vivo metastasis, 393P cells expressing the top five identified drivers and Mcherry (control) were implanted independently into the flanks of 129SV syngeneic mice, and observed for primary tumor growth and distant metastasis. Though primary tumor sizes for all the candidate lines were not significantly different from each other or control  and mean, 24.17  number of metastatic events, which were substantially higher than the other metastasis driver genes identified from our primary in vivo screen  injection into syngeneic mice. Out of the 217 genes screened, we identified 28 potent drivers of lung cancer metastasis (~12% of the screened genes) based on positive enrichment of their associated barcodes in lung metastases. Among the highest metastasis-enriched cDNAs was that encoding rol Fig. , TMEM106een Fig. . The metols Fig. . The metlls Fig. , which wlls Fig.  and enhalls Fig. .Fig. 1TMTMEM106B, its expression was stably inhibited by shRNAs targeting different segments of the TMEM106B mRNA in the highly metastatic KrasLA1/+;TP53R172HΔG (KP)36 mouse lung cancer cells, 344SQ and 344LN   with TMEM106B knockdown or Control (Scramble) were injected subcutaneously in syngeneic mice. Both groups formed comparable sized primary tumors by 7–8 weeks  Fig. . To testeks Fig. , but theons Fig. . These dTMEM106B is a single pass, type-II transmembrane protein reported to localize on lysosomes. We first sought to determine the localization and functional implication of TMEM106B expression by establishing both murine  and human (H157 and HCC827) cell lines with inducible expression of full-length, green fluorescent protein (GFP)-tagged TMEM106B. We observed that irrespective of the cell type, within 24–48 h of TMEM106B induction there was robust expression of the GFP-tagged TMEM106B  to monitor and quantitate intracellular cathepsin B activity in real time in live cells upon TMEM106B induction. We observed that in each of the different human and murine tumor cell models, upon TMEM106B induction, the enlarged lysosomes are loaded with active cathepsin B as evident from the enhanced red fluorescence in the lysosomal vesicles  or cells expressing TMEM106B -conjugated LAMP1 primary antibody, without permeabilization, to specifically stain the LAMP1 present on the cell surface due to fusion with the lysosomal membrane caused during exocytosis. We observed elevated surface staining of LAMP1 in TMEM106B induced cells compared to control  containing Ca2+. We observed that upon ionomycin-mediated lysosomal exocytosis, the conditioned PBS for the TMEM106B-expressing cells demonstrated significantly higher levels of activity for cathepsin B, D, H, and K, compared to ionomycin-treated conditioned PBS from GFP-expressing control cells (Vec), or DMSO-treated controls. The activities of the cathepsins were measured by specific fluorimetric activity assays, which were inhibited by the inclusion of specific inhibitors  cells and this invasive phenotype was completely abrogated when the assay was performed in the presence of a protease inhibitor cocktail  or TMEM106B were implanted into syngeneic mice. These mice were fed with doxycycline-containing feed. Half of the mice from either vector or TMEM106B-cell injected cohorts were treated with the irreversible cysteine protease inhibitor E64D , and the remaining half were dosed with the vehicle as control , which have been fully annotated for gene expression and contain associated follow-up data on patient outcome. According to this TCGA lung adenocarcinoma data set, containing 517 samples, 19% of the samples (n = 98) show either gene amplification or upregulation of the mRNA expression for TMEM106B  worsened disease-free survival and decrease in median survival by 19 months, from 41.23 to 22.54 months   worse overall survival, with a decrease in median survival by 16 months, from 53.29 to 37.68 months  patients is not significantly associated with the KRAS mutational status, indicating that TMEM106B is an independent prognostic driver of lung cancer metastasis . We also observed that patients with elevated TFEB had no significant difference in disease outcome when compared to unaltered control patient group  Fig. . The patths Fig. . InteresTMEM106B in primary lung cancers lead to an increased nuclear translocation of TFEB. This results in a TFEB-dependent upregulation of lysosome genes of the CLEAR pathway, which are loaded in enlarged vesicular lysosomes. Upon intracellular calcium influx, these lysosomal vesicles are secreted by lysosomal exocytosis, releasing a host of lysosomal proteases, including a variety of cathepsins into the extracellular matrix. The release of these cathepsins and other hydrolases increases tumor cell invasion and metastasis , which has been shown to regulate not only lysosomal function, but also overall cellular metabolism. According to recent findings41, one of the master regulators of lysosome synthesis and function is the transcription factor TFEB, which transcriptionally regulates the biogenesis and function of active lysosomes and late endosomes. Most of the lysosomal genes contain a conserved palindromic motif named the CLEAR element in their promoter, which facilitates direct binding by TFEB and subsequent transcriptional regulation41. Accordingly, ectopic expression of TFEB results in an elevated number and increased luminal content of lysosomes, thus enhancing lysosomal catabolic activity41. We demonstrate that TMEM106B acts upstream of TFEB, with increased TMEM106B expression causing nuclear translocation of TFEB and resulting in alteration of lysosomal content and function. Interestingly, this role of TMEM106B appears to be operating independently from other stimuli that regulate TFEB nuclear translocation. These results suggest that TMEM106B expression in lung cancer cells results in increased lysosome size and enhanced activity, describing a new role for TMEM106B in regulating lysosome function in lung cancer metastasis. We also wanted to address whether TMEM106B could be directly regulated by TFEB. To test this, we first performed a motif search on the TMEM106B promoter (using JASPAR online tool: http://jaspar.genereg.net/), but were unable to identify any high scoring known TFEB binding motif on the TMEM106B promoter. This is consistent with published literature where TMEM106B was not identified as a TFEB transcriptional target46. Our work lays the foundation for future work to address the molecular mechanisms underlying the posttranslational changes of TFEB upon TMEM106B expression that result in its nuclear translocation.Dysfunctional lysosomes have been associated with several pathological disorders including inborn lysosome storage disorders, neuro-degenerative disorders like Alzheimer’s, Parkinson’s, Huntington’s, and frontotemporal disorder24. Elevated cathepsin activity and extracellular secretion/release by lysosomal exocytosis plays a crucial role in tumor progression and metastasis in several cancers through various mechanisms47. This is the first report that elaborates the novel connection between TMEM106B overexpression in lung cancer cells and elevated lysosome production. These lysosomes are actively secreted by lysosomal exocytosis in a calcium-dependent manner, which leads to increased secretion of lysosomal cathepsins in the ECM, facilitating enhanced invasion, migration, and metastasis. This study also invokes the need for further analysis to understand the exact molecular mechanism of anterograde trafficking of the enlarged lysosomes to the plasma membrane. We acknowledge the fact that cancer cells with elevated TMEM106B expression produce very large amounts of intracellular active cathepsins. As a result, there could be additional mechanisms that are co-activated and partially induce direct secretion of a fraction of the proteases from the trans-Golgi network to the plasma membrane, without being properly sorted into the lysosomes. This possibility needs further investigation that is beyond the scope of the current manuscript.Lysosomal exocytosis occurs due to a variety of stimuli, which are responsible for the onset of different pathological states. In cancer, the trafficking and fusion of lysosomes with the plasma membrane contribute to cell invasion and migration, making this phenomenon of crucial importance for our metastasis researchTMEM106B, where its expression in lung cancer cells stimulates the production of enlarged lysosomes and increased synthesis of lysosomal hydrolases that are packaged into the lysosomes. These lysosomes are actively secreted under conditions of calcium flux, releasing their cargo of proteases into the extracellular matrix and producing a hyperinvasive microenvironment. We also demonstrate that systemically impeding the activity of the proteolytic enzymes with a pharmacologic inhibitor could revert the metastatic phenotype caused by elevated expression of TMEM106B. Therefore, this experimental evidence suggests that targeting TMEM106B directly or its downstream functions could be developed into a novel therapeutic intervention strategy to prevent metastatic lung cancers.Our data here identify a novel function of TMEM106B cDNA was amplified by RT-PCR as a MfeI–XhoI fragment (primers as in Supplementary Table 48. Mouse TFEB cDNA was amplified by RT-PCR as a HindIII–BamHI fragment and cloned as a Mcherry fusion construct (primers as in Supplementary Table LAMP1 expressing plasmid construct was purchased from Addgene. Mouse TMEM106B shRNA construct was purchased from Open-Biosystems/GE-Dharmacon and Scramble control vectors were purchased from Thermo-Scientific. Human TMEM106B shRNAs (TRCN0000123314 and TRCN0000130047), and Scramble control vectors were purchased from Thermo-Scientific. siRNA for TFEB (SMARTpool:ON-TARGETplus) was purchased from Dharmacon/GE-Healthcare .Mouse 7. NCI-H157 cells were procured from ATCC and tested negative for mycoplasma.All cell lines were maintained in RPMI-1640 supplemented with 10% fetal bovine serum. DNA transfections were performed with Lipofectamine-2000 or Lipofectamine-LTX reagents , miRNA precursors were transfected at 50 nM final concentration for 96 h using Lipofectamin-2000 (Life technologies) and siRNAs were transfected at 25 nM final concentration using RNAi-Max  reagents as per manufacturer’s protocols. All mouse cell lines were generated in our lab and authenticated® Green PCR Master Mix (Life technologies). SYBR green qPCR analyses were normalized to expression of L32 . Taq-man assays (Life technologies) for miRNA qPCR analyses were normalized to miR-16. Western blots were performed with antibodies as listed  and invasion  assays were performed for 6 h (human cells) and 16 h (mouse cells) using standard protocol7. Spheroids were imaged and analyzed using an inverted microscope.Cells were seeded in a matrix comprising of matrigel  or a mixture of matrigel and collagen  and incubated for 6 days with regular replenishment with complete media containing 2% matrigel until formation of spheroidsAll animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee at The University of Texas M.D. Anderson Cancer Center. Cells were subcutaneously injected in the flanks of syngeneic 129/Sv mice of 8–10 weeks age and observed for tumor growth for a period of 4–8 weeks. Upon euthanasia, metastatic nodules on the surface of lung lobes were counted. Lung tissue was fixed in 10% Formalin and then processed for sectioning followed by H&E staining.TMEM106B inducible cells were subcutaneously injected in the flanks of syngeneic 129/Sv mice. Mice were fed with 625 mg/kg of doxycycline feed for a period of 4 weeks after 48 h of implantation of the cells. Both the vector control and TMEM106B mice cohorts were divided equally for treatment with either the cathepsin inhibitor E64D  or the vehicle as placebo controls. E64D was solubilized in complete DMSO at 60 mg/ml. For in vivo dosing, the solubilized E64D was suspended at a final concentration of 5% DMSO using a solution of 30% PEG-400 with 5% Tween-80 in water. Mice from respective cohorts were either treated with a daily dose of E64D at 25 mg/kg body weight, or similar volume of the vehicle with 5% DMSO (as placebo control), by oral gavage (P.O.) route. After 4 weeks of treatment mice were sacrificed and primary tumor volume and lung metastasis were scored. Primary tumors and internal organs were collected for RNA isolation and processed for biochemical assays to measure cathepsin activity.All animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee at The University of Texas M.D. Either vector GFP or Cells were grown on a glass bottom dishes and induced for expression of indicated genes. Induced cells were incubated with Lysotracker (100 nM) (Life technologies) for 1–2 h in a tissue culture incubator at normal culture conditions. Post staining cells were washed with PBS (twice) and counter stained with Hoechst33342 for 15 min followed by washing in PBS and incubated in imaging medium (Life technologies), before imaging in real time by confocal microscopy. Similarly, stained cells were trypsinized and staining intensity was quantified by flow cytometry or on a fluorescent plate reader (Labmate).Cells were grown on a glass bottom dishes and induced for expression of indicated genes. Induced cells were stained with Magic Red Cathepsin assay kit  for 1–2 h in a tissue culture incubator at normal culture conditions. Poststaining cells were washed with PBS (twice) and counter stained with Hoechst33342 for 15 min followed by washing in PBS and incubated in imaging medium (Life technologies), before imaging in real time by confocal microscopy. Similarly, stained cells were trypsinized and staining intensity was quantified on a fluorescent plate reader (Labmate) using specified excitation/emission filter as mentioned in the kit.Cathepsin activity in conditioned media or conditioned PBS after lysosomal secretion was performed with specific cathepsin activity assay kits , as per manufacturer’s protocol. Assay detection was performed with fluorescent plate reader (Labmate) at specified excitation/emission filter as mentioned in the kit.5 cells with 10 mM Ionomycin when suspended in 1 ml PBS in presence of 2 mM calcium chloride (Ca2+), which we referred as conditioned PBS. The conditioned PBS was assayed for activity of different cathepsins. To test whether active cathepsins released upon lysosomal exocytosis were essential to induce an invasive phenotype, Boyden chamber invasion assays were performed in presence or absence of protease inhibitors (Roche) or E64D (Selleckchem). Noninvasive 393P cells were suspended in conditioned PBS generated (as above) from 1 × 106 cells from different cells lines as indicated, in presence of 10 mM EGTA to chelate extra Ca2+ in the buffer during the invasion assay. These assays were completed and invaded cells were quantified as described before7.To assay for active cathepsins in the lysosomal compartment, lysosomal contents were secreted by inducing lysosomal exocytosis by treating a total of 5 × 10LAMP1 ) primary antibody, without permeabilization, to specifically stain the LAMP1 present on the cell surface. Coverslips were mounted in DAPI containing anti quench mounting solution and imaged by confocal microscopy. For quantification of staining, cells were trypsinized, fixed and stained as above and analyzed by flow cytometry.Cells grown on glass coverslips were first fixed using 4% paraformaldehyde for 10 min at 4 °C, followed by immune staining them for 1 h at 4 °C, using a PE conjugated Cell fractionations were performed using cell fractionation kit  following manufacturer’s protocol. Different fractions were estimated for protein content and western blot analysis was performed.TMEM106B or GFP vector inducible cells were seeded in triplicates, in a 24 well plate at varying cell numbers and induced for 24 h. Conditioned media was used to test for cytotoxicity by measuring LDH activity.LDH activity assay was performed with Pierce LDH cytotoxicity assay kit (Thermofisher scientific) using manufacture’s protocol. All relevant data, protocols and related information that are included in the article are available on demand from the authors.Supplementary InformationPeer Review File


Echocardiography has become an indispensable tool for the study of heart performance, improving the monitoring of individuals with cardiac diseases. Diverse genetic factors associated with echocardiographic measures have been previously reported. The impact of several apoptotic genes in heart development identified in experimental models prompted us to assess their potential association with human cardiac function. This study aimed at investigating the possible association of variants of apoptotic genes with echocardiographic traits and to identify new genetic markers associated with cardiac function. Genome wide data from different studies were obtained from public repositories. After quality control and imputation, a meta-analysis of individual association study results was performed. Our results confirmed the role of caspases and other apoptosis related genes with cardiac phenotypes. Moreover, enrichment analysis showed an over-representation of genes, including some apoptotic regulators, associated with Alzheimer’s disease. We further explored this unexpected observation which was confirmed by genetic correlation analyses. Our findings show the association of apoptotic gene variants with echocardiographic indicators of heart function and reveal a novel potential genetic link between echocardiographic measures in healthy populations and cognitive decline later on in life. These findings may have important implications for preventative strategies combating Alzheimer’s disease. Pathological processes including cardiomyocyte cell death, inflammatory cell response and changes in interstitial tissue of the heart are factors leading to adverse remodelling and HF3.Echocardiographic assessment of cardiac structure offers prognostic information about cardiac conditions such as heart failure (HF)4, which involves Cyclophilin D5 and calpains6. By contrast, caspase proteins are now recognized as important factors for initial differentiation of stem cells to cardiomyocytes7, and its deficiency in vivo was shown to induce abnormal heart development9. In rodent cardiomyocytes, caspase-3 is involved in WNT signalling and myocyte growth11, contributing also to muscle-specific gene splicing by cleaving PTB12. In addition, apoptotic DNA nucleases were shown to play a role in the developmental process of C.elegans including the C.elegans Caspase-associated DNase (CAD), ENDOG and TATD orthologues13. Furthermore, ENDOG also contributes to the signalling pathways determining myocyte size through the control of reactive oxygen radicals (ROS)15. These facts lead us to hypothesize that caspases and the nucleases ENDOG and TATD play relevant functions in cardiomyocyte proliferation and maturation during development.A number of apoptotic genes have been investigated as potential targets to prevent cardiomyocyte death, but it is now increasingly evident that caspase-dependent cell death plays a minor if any role in adult myocyte loss17. Increased left ventricular mass (LVM) is a well-established predictor of adverse cardiovascular events and premature death19. End-Diastolic LV Internal Dimension (LVID) and LV Wall Thickness (LVWT) are other measures of left ventricular hypertrophy (LVH), usually a pathological compensatory mechanism of the LV overload which evolves towards progressive LV dysfunction and HF. The principal role of the left atrium (LA) is to modulate LV filling and cardiovascular performance, being LA enlargement and dysfunction also predictors of cardiovascular events, especially in patients with atrial fibrillation (AF)20. The risk of aortic aneurysm is strictly related to the diameter of the ascending aorta, and recent studies have shown high prevalence of aortic root (AROT) enlargement in the hypertensive population21. Using data from publicly available repositories, we aimed to explore the association between a selected group of candidate apoptosis-related genes and these echocardiographic phenotypes by means of meta-GWAS. Furthermore, we aimed to assess previously reported signals in our study datasets and performed an agnostic analysis to investigate relevant pathways revealed for each trait. Following the leading results of this analysis, we further explored the unsuspected genetic relationship between Alzheimer’s disease (AD) and these echocardiographic traits, by estimating their genetic correlation, and identifying common genetic determinants of these conditions.Recent genome wide association studies have been performed for evaluating comprehensive sets of echocardiographic traits in well characterized individuals included in large cohort studiesThis study is aimed at identified genetic variants associated with echocardiographic traits, using both a candidate gene approach and an agnostic approach in retrospective datasets available in public repositories.https://www.ncbi.nlm.nih.gov/gap) after requesting the appropriate permissions. In the case of a multi-ethnic study, only Caucasian samples after principal component analysis (PCA) were retained for analysis. A summary of the clinical characteristics of these populations is shown in Table The four cardiovascular datasets analysed in this study were downloaded from dbGAP  were retained for analysis. Demographic characteristics of these datasets are summarized in the Supplementary Table 22.Data from the most recent available echocardiographic examinations of each cohort were included in this study. The following five phenotypes were analysed: LVM (g), AROT (cm), LVID (cm), LA size LAS, cm), and LVWT (cm). The latter was defined as the sum of the End-Diastolic Thicknesses of the Posterior Wall (TPW) and End-Diastolic Thicknesses of the Interventricular Septum (TIS). LVM was calculated using the formula 0.8 [1.04{(LVID + TIS + TPW)3 −(LVID)3}] + 0.6The cardiovascular datasets included in this study were genotyped using different platforms: CARDIA and MESA were genotyped using the Affymetrix Genome-Wide Human 6.0 array, whereas the FHS was genotyped using the Affymetrix Human 500k array and the CHS cohort with the Illumina HumanCNV370-Duo v1.0.AD datasets were genotyped using the Illumina arrays Human 610‐Quad BeadChip , the HumanOmniExpress BeadChip , the Human660W-Quad (ADGC datasets 1 and 2) and the HumanHap300-Duo BeadChip (The Mayo study) or the Affymetrix 250k NspI (the Neocodex-Murcia study), 500k (the TGEN and GenADA studies) or 6.0 (ROSMAP study) arrays.23. At the genotype level, we removed single nucleotide polymorphisms (SNPs) with missing genotype rate > 5%, not in Hardy-Weinberg equilibrium (p < 10−6) and SNPs with minor allele frequency (MAF) < 1%. Duplicated and related individuals were identified and removed by means of Identity By State (IBS) estimates within and across studies.Prior to imputation, we first performed an extensive quality control excluding individuals with more than 3% missing genotypes, with excess autosomal heterozygosity (>0.35), those showing a discrepancy between genotypic and reported sex, as well as individuals of non-European ancestry based on PCA using SMARTPCA24. After imputation, SNPs with an R2 quality estimate lower than 0.3 were excluded from further analyses in accordance with software instructions.Genotype imputation is aimed at estimating unobserved genotypes using as reference known haplotypes from a well characterized population. Imputation was performed using the minimac3 algorithm and the SHAPEIT tool for haplotype phasing at the University of Michigan server using the HRC reference panel25 were fitted to investigate the association between genotypes and quantitative phenotypes, with age, sex, body mass index and the four principal components as covariates. For each phenotype, we obtained summary estimates of association across studies by using a fixed-effects model meta-analysis procedure implemented also in PLINK. For the genome wide SNP analysis, the conventional GWAS significance threshold was used (p = 5 × 10−8)26, whereas signals with p value < 10−5 were considered as suggestive of association and reported in the Supplementary Tables All analyses were performed in Caucasian populations only. Individuals with prevalent myocardial infarction (MI) or congestive heart failure (CHF) were excluded from the study. Linear regression models available from PLINK software27. All SNPs with MAF above 1% were used in these analyses. At each trait, genes were ranked according to the global p mean value.Gene-wise statistics were computed using MAGMA software, which takes into account physical distance and linkage disequilibrium (LD) between markers to estimate a summary gene p-value using a known approximation of the sampling distribution−3, which has been showed to have a good power for detecting relevant pathways and functions while maintaining type 1 error controlled27. For these analyses, we used the R packages Webgestalt  and enrichR29  with default parameters.Top results from MAGMA genome wide analyses were tested for over-representation of genes involved in known pathways, functions and diseases. For these enrichment analyses, we applied a moderated threshold of p < 1030. This method allows to identify pleiotropic gene effects associated with different diseases or disorders, providing a single measure of the proportion of shared genetic determinants. Furthermore, we obtained summary estimates of association across phenotypes performing unweighted meta-analysis of Fisher p-values.In order to explore genetic correlation between different traits we used a bivariate GREML analysis with GTCA softwareOverall, our study included data from 11,559 individuals with echocardiographic phenotypes from four different datasets Table . MAGMA sCASP8 and CASP10) and the apoptosis regulator protein CFLAR  with LVM. The same three genes were also linked to LVID with p < 0.05, along with the Fas receptor-associated adaptor FADD (Fas-associated protein with death domain) and BCL2 (B-cell lymphoma-2) genes. BCL2, FADD and TATDN1 (TatD DNase domain containing-1) showed a trend for association with AROT. We did not find evidence of association of any calpain family member with any of the analysed traits (data not shown).Because there is experimental evidence supporting the role of some apoptosis-related genes with cardiac development and disease, we first analysed the potential association of polymorphisms in a series of apoptosis-related genes with the cardiac phenotypes. Table −8) related with the analysed phenotypes, we observed several suggestive signals at the p < 10−5 level (Supplementary Tables −3) showed little overlap across phenotypes  than for the echocardiographic phenotypes (range: 0.17-0.36). Then, we looked for shared genetic loci across the genome between echocardiographic traits and AD using GREML analyses as suggested by the enrichment analyses. Specifically, we observed a positive correlation between AD and LAS , and negative correlations between AD and LVID , and AD and LVM ; of note, LVM and LVID are the most correlated echocardiographic traits   along with 12,730 AD cases and controls both from internal and publicly available databases. First, we estimated the proportion of variance, as a proxy of trait heritability, explained by all SNPs in each one of these traits01) Fig. . The sig−3 threshold in the different meta-analyses performed, with a group of 30 genes consistently associated in all of them, including the CASP8/CASP10/CFLAR locus and the GABRR1 GABA receptor , teneurin (TENM2), calsyntenin (CLSTN3), or the cytoskeletal alpha-actin protein (ACTN2) for the diverse meta-analyses involving ventricular measures and AD.Based on these findings we performed a SNP-wise meta-analysis by pooling Fisher association p-values of two or more phenotypes aimed at identifying genes contributing most to both heart measures and AD. Thus, we combined in these meta-analyses p-values for LAS&AD, LVID&AD, LVM&AD, LAS&LVID&LVM&AD and LVID&LVM&AD and calculated gene-wise statistics Table –21. We otor Fig. . Consequses Fig. –25. We aOur study, based on data from 11,559 individuals free of cardiovascular disease, shows that variants affecting diverse genes involved in apoptosis regulation associate with echocardiographic phenotypes in humans. We have obtained these results by both hypothesis-driven and agnostic approaches. In addition, novel findings from the analysis of our GWAS also include previously unnoticed associations of variants in genes involved in cell proliferation, DNA replication and mRNA splicing with left ventricular morphology. Our data also suggest the existence of a set of genes, mainly related to apoptosis/inflammation signalling, whose variants are associated with both cardiac phenotype and AD.CFLAR (cFLIP) gene are strong predictors of LVM. Furthermore, our hypothesis-free approach found that caspase dependent pathways were overrepresented among top genes associated with the LVM phenotype. These results support our a priori hypothesis that the apoptotic signalling influences heart morphology with potential impact on heart performance. Our hypothesis was based in previous experimental work, including our own, showing that deficiency11 or overexpression32 of key apoptotic genes altered normal cardiomyocyte differentiation and heart development independently of cell death. Indeed, although these genes are best known for their role in regulating apoptotic cell death, experimental evidences show that the same genes also regulate myocyte proliferation, inflammation and hypertrophy in the heart35. Because cell death is not a major event during heart development, and based on the above experimental work, we suggest that the relationship between the apoptotic genes and the cardiac phenotype might involve non-apoptotic functions.Our hypothesis-driven analysis showed that caspases 8 and 10 and the regulatory SMG6, TSR1 and SRR genes and AROT17, but failed to detect statistically significant associations for other gene variants, possibly due to the limited power of this study. Given the strong influence of age in echocardiographic parameters, the diverse population structure of the study cohorts could also affect our power in the meta-analysis, with signals not replicating in populations largely differing in their median age. However, our analysis demonstrated genetic association between variants of genes previously shown to influence heart function and cardiac hypertrophy in experimental models, such as MLF136, which associates with LVM phenotype in our study, and KCNIP2 (KChIP2)39 and TRAF3IP241, which have also been associated with LVWT and LVM phenotypes respectively in humans and in this study as well. Also, from the top list of genes whose variants are associated to LIVD, ANKS6 has been associated with heart malformations42.In order to estimate the robustness of our GWAS analysis, we asked whether we had reproduced some signals already observed in previous genetic studies. Indeed, we found the already published link between 43, and has been shown to be involved in the regulation of cardiac function in rodent experimental models through interaction with fatty acids44. Therefore, our genetic results open the possibility that odorant receptors’ activity can influence cardiac function in humans. Interestingly, low OR expression has been found in the cortex of neuro-psychiatric patients45 and a genetic microduplication in the 11p15.4 region has been associated with familial intellectual disability and autism46.Unexpectedly, we found enrichment on AD related pathways for LAS, LVID and LVM that led us to explore comprehensively a possible link between AD and all these echocardiographic traits. Intriguingly, our results revealed for the first time a potential genetic link between AD and LAS, paired with a negative correlation between AD and either LVM or LVID. Interestingly, in line with our observation, recent reports found that LAS and LVID were independently associated to cognitive function in older adults, being predictors of cognitive decline after 14 years of follow-up48. The opposite direction of the correlation coefficients for atrial and ventricular measures could be related to the different development patterns of these chambers during embryogenesis49 and with the described strong association of left atrial size with long-term exposure to vascular risk factors, particularly high blood pressure and obesity. In fact, the CARDIA Brain MRI Substudy found association of higher left atrial volume in early adulthood with impairment of white matter integrity in midlife, but not for ventricular measures50.The GWAS analysis also showed association of variants of a group of odorant receptors genes located in the 11p15.4 chromosomic region coding  or in chromosome 16 (OR2C1) with LVWT and LVID. Expression of these genes in non-neural tissues is related to the control of different processes, including glucose and oxygen homeostasis or cell cycle control51. But our findings are compatible to a broader definition of- dementia which includes other diseases such vascular dementia, fronto-temporal dementia or Lewy Body diseases among others. In fact, the rule is the presence of multiple pathologies in the brain of people with dementia including pervasive vascular lesions52. The link between dementia and cardiac conditions is not well understood. The old concept of cardiogenic dementia was based on the high incidence of cardiac dysrhythmias observed in patients with dementia due to vascular causes53. However, the relation between coronary heart disease (CHD) or HF and AD in epidemiological studies remains controversial, with some studies showing an association with cognitive impairment and dementia56 whereas others found no association58. The fact that both conditions are competing risks complicates the study of their relationship. Lower cardiac index levels are related to lower cerebral blood flow in older adults free of CVD59, but individuals with cardiac conditions that did not result in premature death might include many individuals chronically exposed to brain hypoperfusion due to reduced cardiac output that adaptively decreased cerebrovascular resistance through arteriolar dilatation. This kind of antagonistic pleiotropy between these phenotypes has been previously suggested by Beeri et al. after observing that better cognitive performance was associated with worse cardiac functioning in very elderly subjects60.A recent GWAS by our group on AD clinical endophenotypes revealed that vascular processes were the main causal mechanisms in pure AD62. Population based studies have shown an inverted U-shaped association for LVM values and age, since they rise in adolescence and decline with increased age64. Furthermore, a U-shaped association between left ventricular ejection fraction (LVEF), a marker of systolic dysfunction, and abnormal cognitive decline has been reported, with increased dementia risk at the lowest and highest LVEF quintiles65.Moreover, whereas enlarged ventricular volume (LV hypertrophy) is a marker of diastolic dysfunction, LVM is also a marker of cardiovascular health, positively correlated with physical activity and cardiorespiratory fitness66, and the protein has been shown to be involved in amyloid related pathways and activated in both blood and brain cells from AD patients69. In our study, the GABA receptor GABRR1 was consistently associated in all meta-analyses involving heart traits and AD. Neurotransmission affects both cardiac and neuronal performance, and a few studies have examined synapse and neuron loss in AD brains and suggested that synaptic changes precede neuron loss71. In fact, GABA signalling is reduced in AD and other mental disorders such as dementia with Lewy bodies or frontotemporal lobar degeneration74. By contrast, augmented inhibitory GABAergic neurotransmission has been reported in animals models of HF and LVH through the involvement of the paraventricular nucleus of the hypothalamus76.To our knowledge, this is the first report analysing shared genetic factors between echocardiographic measures and AD. This method for estimating genome-wide pleiotropy has the advantage of being free of potential confounders determined by shared epidemiological risk factors such as high blood pressure or atherosclerosis. Our results show a negative genetic correlation for the ventricular measures LVID and LVM and AD, pointing to antagonist pleiotropic effects of shared genes by AD and LV cardiac measures, the main known functions of which are related to apoptosis  and neurotransmission (GABRR1). Interestingly, some CASP8 variants have been previously associated with ADIn summary, our GWAS data suggest the influence of gene variants affecting the apoptotic/inflammation signalling pathway on left ventricular morphology and cardiac function, uncover novel candidate gene variants regulating echocardiographic phenotypes and establish a genetic link between cardiac morphology alterations, mental illness and AD involving key genes in the regulation of apoptotic signalling that deserve functional assessment due to their diagnostic and therapeutic potential. These results should be replicated in larger datasets in order to confirm the observed pleiotropic effects for genes associated with echocardiographic traits.Supplementary informationSupplementary data


Sufficient pain control and rapid mobilisation after VATS are important to enhance recovery and prevent complications. Thoracic epidural analgesia (TEA) is the gold standard, but failure rates of 9–30% have been described. In addition, TEA reduces patient mobilisation and bladder function. Subpleural continuous analgesia (SCA) is a regional analgesic technique that is placed under direct thoracoscopic vision and is not associated with the mentioned disadvantages of TEA. The objective of this study was to assess surgical feasibility, pain control and patient satisfaction of SCA.n = 23). Pain scores  and patient satisfaction  were collected on postoperative day (POD) 0–3. Secondary outcomes were the period of urinary catheter use and period to full mobilisation.Observational pilot study in patients who underwent VATS pulmonary resection and received SCA  and was successful in all patients. Pain scores on POD 0–3 were 1.2 (SD 1.2), 2.0 (SD 1.9), 1.7 (SD 1.5) and 1.2 (SD 1.1) respectively. On POD 0–3 at least 79% of patients were satisfied or very satisfied on pain relief and mobilisation. The duration of subpleural continuous analgesia was 4 days . Urinary catheters were used zero days  and full mobilisation was achieved on POD 2 .Subpleural continuous analgesia in VATS pulmonary resection is feasible and provides adequate pain control and good patient satisfaction.This pilot study was not registered in a trial register. Sufficient pain control and rapid mobilisation after video-assisted thoracoscopic surgery (VATS) are important to improve recovery and prevent postoperative pulmonary complications . ThoraciThese disadvantages supported the development of other regional techniques for pain management. Single-shot thoracic paravertebral blockade, intercostal nerve blockade and serratus anterior blocks have been shown to be safe and effective for pain management after VATS , 7–9. ReSubpleural continuous analgesia (SCA) is a regional multilevel intercostal analgesic technique to control postoperative pain using a subpleural catheter placed under general anaesthesia and direct thoracoscopic vision. It does not influence mobility, bladder function or blood pressure. Jung et al. retrospectively compared SCA with intravenous patient controlled analgesia for pain management after VATS pulmonary resection and found average pain scores  higher than 3 on postoperative day (POD) 0–2 . The effSubpleural continuous analgesia is feasible, provides adequate pain control and has good patient satisfaction in patients after VATS pulmonary resection.We performed an observational pilot study in a Dutch teaching hospital . 23 consecutive patients who underwent VATS pulmonary resection and had an indication for TEA  between April and September 2018 were included and received SCA for pain management instead of TEA. The pain scores and postoperative outcomes of SCA patients were compared to a historical group to benchmark our results. Since this was an observational pilot study we were not aiming to prove non-inferiority or even superiority of either technique. The historical group contained 23 consecutive patients who underwent VATS pulmonary resection and received TEA between April and September 2017.Primary outcomes were surgical feasibility, pain control and patient satisfaction on postoperative day (POD) 0–3. Secondary outcomes were length of hospital stay, chest tube drainage and urinary catheter use, incidence of postoperative hypotension and degree of postoperative mobilisation.To assess surgical feasibility the duration of placement (minutes) and success of placement were collected. A numeric rating scale (NRS 0–10) was used to assess the level of pain in rest, while coughing and while moving. In addition, the necessity for intravenous and oral analgesics and the presence of postoperative nausea and vomiting were registered. Patient satisfaction on pain relief and mobilisation was measured on a 5-point Likert scale .For secondary outcomes we collected length of postoperative hospital stay (days), length of chest tube drainage (days), the length of urinary catheter use (days) and the incidence of postoperative complications . To examine hypotension we collected the lowest reported blood pressure in the morning, afternoon and evening on POD 0–3. Postoperative mobility was scored as the maximum achieved score on a non-validated 6-point scale . Pain control, patient satisfaction and mobility scores were assessed on the recovery room (only pain scores), at the evening after surgery and in the morning, afternoon and evening on POD day 1 and 2 and in the morning and evening on POD 3. In order to describe the use of SCA we collected details on the SCA catheter placement ) and possible complications in the postoperative period . Finally, we collected the following data from the electronical medical records: age, gender, length, weight, blood pressure at the pre-operative visit, American Society of Anaesthesiologists (ASA)-classification, indication for surgery , surgical details .In the historical TEA group prospectively collected pain scores on POD 1–3 were available. The baseline characteristics and postoperative outcomes data were collected retrospectively from the electronical medical records.Results were reported according to the Strengthening the Reporting of Observational studies in Epidemiology guidelines for observational studies . The meaPerforation of the parietal pleura during the placement of the subpleural catheter could have led to a decreased analgesic dose around the intercostal nerves due to leakage of analgesics into the pleural cavity. Therefore, we divided the SCA patients in a perforation and no perforation group and compared baseline characteristics, surgical details and pain scores between these groups.p = .05. All calculations and statistical analysis were done using the Statistical Package for Social Sciences, version 22.0 .Descriptive data were presented as means (with standard deviation (SD) and/or range) or medians (with interquartile range (IQR) and/or range) depending on  distribution of data. Categorical data were presented as counts and percentages. Categorical data were compared by the Chi-square test, whereas numerical data were compared by the unpaired T-test or Mann-Whitney U test depending on  distribution of data. We calculated 95% confidence intervals (95%-CI) around proportions using the Wilson interval . All perAll patients received general anaesthesia using propofol, sufentanil and rocuronium for induction and either propofol or sevoflurane for maintenance of anaesthesia (at the discretion of the anaesthesiologist). After surgery patients received additional analgesics according to the analgesic pain relief ladder; oral paracetamol (standard dose of 4000 mg per 24 h) with additional nonsteroidal anti-inflammatory drugs  and if necessary morphine or morphine equivalent .The multi-orifice 25 cm length On-Q local anaesthetic infiltrating catheter  was placed intraoperatively under direct thoracoscopic vision by one of two lung surgeons . After general anaesthesia and endotracheal intubation the patient was positioned in the lateral decubitus position with the affected side up. Access to perform was achieved by either uni- or multiportal incision(s). The pain catheter was inserted dorsally and at least two costal levels caudally of the most caudal placed incision which is generally made in the 8th intercostal space. After skin incision we searched for the subpleural space using blunt dissection under direct thoracoscopic vision Fig. , image 1n = 4) or conventional (n = 19) VATS. 18 Patients (78%) underwent an anatomical resection  and in the remaining five patients a large wedge was resected. We did a lobe-specific mediastinal lymph node dissection in 16 patients (70%). All patients received one chest tube for postoperative drainage , 74% males) were included for analysis. The primary surgical technique was either uniportal  and was successful in all patients. The mean pain score in rest on the recovery room was 1.1 (SD 1.2) and on the evening after surgery it was 3.1 (SD 2.0). On POD 1–3 the mean pain scores in rest were 2.0 (SD 1.9), 1.7 (SD 1.5) and 1.2 (SD 1.1) respectively. The mean pain score in rest on POD 1–3 together was 1.6 (SD 1.3)  with a period of chest tube drainage of 2 days . The period of urinary catheter use was 0 days . At the pre-operative visit none of the patients suffered from hypotension with a mean MAP of 100 mmHg (SD 12). Hypotension during the first three postoperative days was seen in only one patient (5%). SCA patients were able to walk to the bathroom without help on median POD 1 . Walking around the ward without help was median possible on POD 2 . Postoperative complications occurred in seven patients (30%) (Table n = 11) and the pleural perforation group (n = 12) in left or right sided surgery, duration of the placement of the pain catheter, mobilisation and pain scores in rest, while coughing or while moving on POD 0–3.The duration of subpleural continuous analgesia was 4 days . One patient reported deafness of the skin around the location where the catheter was inserted. Leakage of local anaesthetic from the skin insertion place of the pain catheter occurred in 6 patients (23%). We saw no other SCA-catheter related complications or ropivacaine related systemic side effects. Minor perforation of the parietal pleura during placement of the catheter occurred in 12 patients (52%). None of these patient had abundant drain production and no additional interventions were required to repair the perforations. We found no significant differences between the no pleural perforation group (p = 0.01) (Table p = .78) (Table p = .04), while the pre-operative MAP was comparable between groups . The length of hospital stay  and chest tube drainage  were similar to SCA patients. The period of urinary catheter use was 2 days  which was significantly longer compared to patients with SCA . Postoperative complications occurred in seven patients (30%) with SCA and six patients (26%) with TEA (p = .74)  Table . Signifi8) Table . HypotenSufficient pain control after VATS enables adequate breathing and early mobilisation hereby reducing postoperative complications and enhance recovery. Our results suggest that subpleural continuous analgesia in VATS pulmonary resection is feasible and provides adequate pain management and good patient satisfaction.To the best of our knowledge no consensus on optimal postoperative pain management after VATS is achieved in current literature. A review of enhanced recovery after thoracic surgery (ERATS) protocols strengthened the lack of unambiguity. The five included protocols all used different techniques for postoperative pain management: oral, intravenous, intercostal, paravertebral and epidural anaesthesia . The useUrinary catheters could be another cause of restricted postoperative mobilisation. Its use combined with TEA is however common practice in most hospitals. A prospective study showed significant post-void residuals in patients with thoracic epidural analgesia . In a raSufficient pain management allows patients to mobilize. Insufficient pain control by epidural or local analgesia could be compensated by additional oral or intravenous analgesics. However, since opioids and NSAIDs have certain disadvantages it is important to minimalize the use of these drugs. SCA patients had a higher opioid consumption on POD 1 and 2. These results are in concordance with current literature where slightly higher additional analgesic use is described in continuous local analgesia groups versus TEA-groups in thoracic surgery , 24. HowPlacement of the SCA catheter was easy and safe. Placement of the catheter took an average of 11 min resulting in a non-significant prolonged operation time in SCA patients. We noticed a very steep learning curve since the placement of the first 12 catheters took an average of 14 min versus 8 min in the last 11 catheters.The main benefits of SCA compared to TEA and other regional analgesic techniques  were fast placement under direct thoracoscopic vision and no need of additional equipment or specialized staff (beside the catheter). In addition, patients benefit from the placement of the analgesic catheter under general anaesthesia compared to possible stressful awake placement of a thoracic epidural catheter or awake percutaneous regional analgesic techniques . Lastly,The main limitations of our study were the observational character and the small sample size. This may have led to an underestimation of differences among TEA and SCA due to insufficient power. Since the primary objective was feasibility, pain control and patients satisfaction of SCA the results of the comparison with the historical group should be interpreted with care.The results and limitations of our study and the lack of consensus in current literature strengthen the need for prospective, randomised research on the most (cost-)effective technique of postoperative pain management after VATS pulmonary resection. Results of future research may aide in optimizing patient care and improve ERATS protocols.Subpleural continuous analgesia in VATS pulmonary resection is feasible and provides adequate pain control and good patient satisfaction.


Cervical spondylosis is a common age-related disorder that results in pain, radiculopathy, and myelopathy. A retrospective chart and radiograph review of a 50-year-old male who underwent surgical treatment for correction of cervical radiculopathy was performed. Immediately after surgery the patient reported complete relief of his preoperative arm pain. In two weeks, he had recovered full strength and sensation. Six months postoperatively, the patient reported relief of all pain and complete recovery of strength and sensation. Anterior cervical discectomy and fusion (ACDF) with an open architecture titanium implant was successfully utilized to improve cervical radiculopathy. This technique increases the likelihood of fusion and improved patient outcome. The objective of the report is to highlight the treatment of cervical radiculopathy through anterior cervical discectomy and fusion with a 3D printed titanium alloy with an arched design and large porous openings. This is one of the first reports using this interbody device in a multilevel procedure. The resulting symptoms include pain, radiculopathy, and myelopathy. First line therapy is conservative; however, patients are surgical candidates once they have failed conservative therapy. The mainstay surgical treatment for cervical radiculopathy is anterior cervical discectomy and fusion (ACDF), which is currently the most commonly performed surgical procedure for degenerative cervical spine disease [The most common cause of neural dysfunction in the cervical spine is attributed to spondylosis. This age-related change can result in symptoms directly through disc herniation, osteophyte formation, and a hypertrophied ligament compressing the neuraxis . The res disease . ACDF is disease . Increases in osteogenic cell differentiation have been demonstrated through this modification by increasing total protein and alkaline phosphatase levels [The implants used in ACDF have undergone dramatic changes since Cloward, Robinson, and Smith introduced the anterior approach using a dowel graft in the 1950s. Cage fusion technology was first introduced in 1988 by Bagby and it too has undergone rapid changes . Currente levels . Cage tee levels . This paA 50-year-old male presented with a two-month history of progressive right arm weakness and pain. The initial presenting symptom was a stiff neck with intermittent low-grade neck pain that developed into severe right shoulder pain. Several weeks after the onset of pain, he noticed diffuse right arm weakness and loss of muscle tone that persisted. Upon presentation, he reported painful paraesthesia in C6 dermatomal distribution. Motor examination found diffuse weakness in the right arm: shoulder abduction 4/5, elbow flexion 4/5, elbow extension 5/5, finger flexion 4/5. He was hyperreflexive in all extremities.Magnetic resonance imaging (MRI) of the cervical spine Figure  showed mC4-5 and C5-6 anterior cervical discectomy and fusion with placement of a titanium interbody  and titanium plate and screws  was performed.Postoperative courseImmediately postoperatively the patient had relief of arm pain and improvements in strength and sensation. This was maintained for nine months after surgery. Two weeks postoperatively, the patient’s symptoms and neurologic function improved drastically. Greater right arm strength was noted with 5/5 strength in upper and lower extremities bilaterally. Right shoulder abduction was 5/5 and external rotation 4+. Six months postoperatively, the patient reported relief of all pain and complete recovery of strength and sensation. At the nine month follow-up, the patient remained completely asymptomatic. A lateral X-ray at four months Figure  showed iThe goals of anterior cervical discectomy and fusion are to achieve neural decompression, maintain cervical lordosis, provide segmental stabilization, and eliminate symptoms of radiculopathy. Interbody spacers are created using a variety of materials and in different designs -11. FurtThis was one of the first operations in which an open matrix cervical interbody device was used in a multilevel procedure. There are advantages of using a roughened titanium surface as opposed to others such as smooth titanium alloy or poly-ether-ether-ketone (PEEK). PEEK does not integrate well with the surrounding bone and instead may form a fibrous connective interface -17. A 20Interbody devices have undergone enormous evolution since their inception in the 1950s and have improved clinical outcomes of anterior cervical discectomy and fusion (ACDF). ACDF is currently the most common surgical intervention for degenerative cervical disease. It is a very well-tolerated operation that continues to see improvement through advancements in research and technology. Our case report illustrates ACDF in a patient, using a porous titanium interbody device that stimulates osteogenesis and enhances fusion, with a remarkable improvement of symptoms.


NOD2 variants are strongly associated with development of CD, and NOD2 is part of the innate immune response to bacteria. This study aimed to identify differences in fecal microbiota in CD patients and non-IBD controls stratified by NOD2 genotype.Current models of Crohn’s disease (CD) describe an inappropriate immune response to gut microbiota in genetically susceptible individuals. NOD2 genotype were identified from patients in previous UK IBD genetics studies and the Cambridge bioresource (genotyped/phenotyped volunteers). Individuals with known CD-associated NOD2 mutations were matched to those with wild-type genotype. We obtained fecal samples from patients in clinical remission with low fecal calprotectin (<250 µg/g) and controls without gastrointestinal disease. After extracting DNA, the V1-2 region of 16S rRNA genes were polymerase chain reaction (PCR)-amplified and sequenced. Analysis was undertaken using the mothur package. Volatile organic compounds (VOC) were also measured.Patients with CD and non-IBD controls of known Ruminococcaceae, Rikenellaceae, and Christensenellaceae and an increase in Enterobacteriaceae. No significant differences could be identified in microbiota by NOD2 genotype, but fecal butanoic acid was higher in Crohn’s patients carrying NOD2 mutations.Ninety-one individuals were in the primary analysis . Comparing CD with nonIBD controls, there were reductions in bacterial diversity, NOD2 genotype and fecal microbiota, we identified no significant genotype-microbiota associations. This suggests that the changes associated with NOD2 genotype might only be seen at the mucosal level, or that environmental factors and prior inflammation are the predominant determinant of the observed dysbiosis in gut microbiota.In this well-controlled study of  Recent research also has emphasized the functional aspect of the gut microbiota through measurement of microbial metabolites such as the volatile organic compounds (VOC) present in feces.4The precise etiology of Crohn’s disease (CD) remains unknown. However, the key pathogenic process involves an inappropriate immune response that results in bowel inflammation and damage. The targets of this response are thought to be antigens derived from constituents of the microbiota, a view supported by the benefits of altering the microbiota5–7 For some loci, the disease gene and associated point mutations are known . NOD2 has the largest effect, and a large recent subphenotype-genotype analysis has confirmed that NOD2 is strongly associated in particular with ileal CD.8 Viewed alongside other functionally interrelated genes that have been associated with CD , an impaired capability of the host to regulate microbial constituents consistently emerges as a major common theme.The last few years has seen rapid advances in the genetics of IBD as a result of large cohort genome-wide association studies (GWAS) of cases and controls. Over 200 IBD susceptibility loci have now been reported.9 Variants of NOD2 associated with CD are mutated in the ligand recognition domain and fail to induce autophagy on MDP triggering, which results in aberrant bacterial handling and antigen presentation in these cells.10–12 NOD2 possesses other antibacterial effects, including the ability to prime human dendritic cells (DCs) to promote T-helper 17 (Th17) responses (via NOD2-induced-expression of IL-23 and IL-1)13 ,and the ability to induce antimicrobial peptide defensins in the intestine.14 If expression of CD-variant NOD2 leads to dysregulated bacterial destruction within the cells in which it is expressed, bacteria may persist abnormally in the mucosa and activate tissue inflammation in these sites.NOD2 is a cytosolic pattern recognition receptor (PRR) that is a key player in immunity to intracellular bacteria and inflammatory responses. NOD2 recognizes muramyldipeptide (MDP), a ubiquitous component of bacterial cells walls, and its stimulation leads to induction of autophagy in human cells.NOD2 but remain healthy, which reinforces the role for coexistent genetic or environmental factors in initiation of CD.16 The association of defective antibacterial mechanisms with CD-associated polymorphisms in NOD2 suggest that the presence of these variants may influence the nature of the microbiota over time. This in turn might either lead to a critical dysbiotic state being reached, or the presence of specific microbes emerging to initiate the cycle of inflammation observed in disease. For example, altered release of antibacterial peptides from variant-NOD2-expressing Paneth cells, defective Th17 responses, or defective autophagic bacterial processing in the gut mucosa could change gut bacterial burden or species diversity.However, approximately 11%–14% of white Europeans are heterozygous and 0.4%–0.9% homozygous or compound-heterozygous for CD-risk-variant NOD2 mutations. Human studies to date have been limited in scope due to small numbers of individuals homozygous for NOD2 mutations without accurate matching of controls. Frank and colleagues revisited the dataset from their index 2007 study on the microbiota in IBD, stratifying patients retrospectively for NOD2 and ATG16L1 genotype. Due to its retrospective design, this study is severely constrained by limited power; despite this, they observed clear shifts in microbial composition as a result of genotype.17 The aim of the current study was to prospectively define the role of NOD2 genotype in influencing the nature of the host microbiota in health and in CD.Little is known of the nature of the microbiota in the presence of NOD2 genotype were identified from the UK IBD genetics consortium , G908R (rs2066845), or L1007fs (rs2066847)]19 as measured using genotyping arrays for the original genetics studies in which they had been involved (Affymetrix GeneChip 50020 and ImmunoChip6). Patients were recruited if they were deemed by their treating physician to be in clinical remission. Each NOD2-mutant patient was matched to a homozygous NOD2-wild-type patient. Exclusion criteria for CD patients included antibiotics within the months before recruitment, active CD , and presence of an ileostomy. For all CD patients, a household control was approached . Healthy volunteers stratified by the same NOD2 variants were recruited from the Cambridge BioResource.21 The Cambridge BioResource is a panel of around 16,000 volunteers, both with and without health conditions, who have previously submitted DNA for genotyping. Participants can be approached for studies on the basis of genotype and phenotypic characteristics. Volunteers from the BioResource had no known gastrointestinal diagnosis and had not taken antibiotics in the preceeding 3 months. All study participants had fecal calprotectin (FC) measured by a standard ELISA . CD patients with FC >250 µg/g and controls (household or BioResource) with FC >50 µg/g were excluded from further analysis.Individuals with CD of known nsortium . Patient22 were collected using a patient questionnaire, interrogation of the medical record, and use of previous phenotype information recorded on the IBD cohorts as part of a rephenotyping exercise.8 Probiotic and medication use were documented at the time of sampling. Participants with missing data were excluded from the analysis of that specific datapoint.Clinical data including medical and surgical history, smoking status, medication history, antibiotic use, probiotic use, weight, height, Montreal disease location, and behavior23 nor the VOC profile (unpublished data). Each sample was thoroughly mixed and aliquots were transferred into lysing matrix E tubes  for subsequent DNA extraction, head space vials  for (VOC) analysis, and universal containers for fecal calprotectin analysis. Samples were stored at −80 °C before shipping to a central processing laboratory in Aberdeen, UK, where DNA was extracted within 1 month of collection.Fecal samples were collected from each study participant using the Fisher Fecal Commode Collection Kit. The collection container was held in the toilet bowl using the supplied trivet, and whole fecal samples collected without contamination by urine. Samples were kept cold using phase-change refrigerant gel packs and processed within 24 hours of collection. This short period of storage is not expected to significantly influence molecular estimation of microbial community composition,NOD2 variants listed above using TaqMan assays . Where a genotype was not determined, the allelic discrimination plots were examined manually to ensure homozygotes for the minor allele had not been missed. For the purposes of analysis of the household controls, missing genotypes were inferred to be wild-type genotypes.Household controls had not been previously genotyped. Saliva samples were acquired using Oragene kits . DNA was extracted following the manufacturer’s protocol and was genotyped for the 3 CD-associated Ethical Considerations:Ethical approval was granted by the North of Scotland Research Ethics Committee (reference 12/NS/0050). All participants provided written consent.24For each fecal sample, an approximately 400 mg aliquot was placed in a lysing matrix E tube and 978 µl of sodium phosphate buffer and 122 µl MT buffer were added to each tube and vortex mixed. This then was processed using the FastDNA SPIN kit for Soil following the manufacturer’s instructions  as described previously.25 The primers were designed with the Illumina adapter sequences already included and with 1 of 200 barcode sequences included in the 338R reverse primer, thus avoiding the need for a separate step to add the adapter sequences and barcode. Twenty cycles of polymerase chain reaction (PCR) amplification were performed using the Q5 polymerase kit following the manufacturer’s instructions . Postamplification, samples were quantified using a Qubit fluorometer  and then pooled to obtain equimolar concentrations.The V1-V2 region of the 16S rRNA gene was amplified using 27F and 338R primers.Sequencing was performed using an Illumina MiSeq sequencer using Illumina V2 chemistry and paired-end 2 × 250 base pair reads. Initial sequence data processing was performed in the Illumina MiSeq Reporter to demultiplex samples and strip adapters and primers and sequence data were exported in the FASTQ format.26 following the MiSeq SOP.27The 16S rRNA gene sequence data were further processed using mothur28 Community structures were compared using trees generated using Jaccard and Yue Clayton distance metrics after subsampling to 3943 reads per sample. The trees were then plotted graphically using the Interactive Tree of Life.30 Trees were compared using the parsimony command within mothur. Subsequent statistical analysis was done in R 3.2.2 . Microbial diversity was assessed using inverse Simpson and compared using a Mann-Whitney U test.31 Comparisons at the family, genus, and operational taxonomic unit (OTU) level were done using Mann-Whitney U tests for binary comparisons and corrected using Holm’s method.32 The 12 most abundant families were selected for plotting graphically.Alignment and classification were done against the SILVA v119 reference set.33 Briefly, gas chromatography-mass spectroscopy (GCMS) was used to quantify the metabolites in the headspace gas taken from vials containing an aliquot of participants’ feces. The raw GCMS data were processed using AMDIS . Compounds detected in fewer than 20% of the study population were filtered out. The resultant ion intensity data were log transformed and the limma package used to facilitate running multiple linear models including disease status and NOD2 genotype as covariates.34P values were corrected using Holm’s method.VOC data were generated using previously described methodology.NOD2 mutant), 30 bioresource volunteers (58% NOD2 mutant), and 24 household controls. All were of white European ethnicity. There were no differences in phenotype within the CD patients by NOD2 status . Hierarchical clustering using the Jaccard metric demonstrated clustering by CD versus controls in either cohort (P < 0.001), but not by NOD2 genotype .There was a significant reduction in diversity  between CD cases and both Bioresource and household controls (in cases ). The CDRuminococcaceae, Rikenellaceae, and Christensenellaceae , and an increase in Enterobacteriaceae (P < 0.001 corrected) in samples from CD patients vs controls  −2.11], piperidinone [logFC −2.43], butanone [logFC −2.19], and acetone [logFC −3.90],NOD2 mutations, there was a single significant association after correction for multiple testing using the Holm’s method with butanoic acid . On further examination, this VOC was noted to be less abundant specifically in patients with CD with wild-type NOD2 (Linear models were constructed for logype NOD2 .NOD2 genotype and the fecal microbiota in human participants stratified by NOD2 genotype. It further confirms previously identified shifts in gut microbiota in CD patients when compared to non-IBD controls, notably a reduction in obligate anaerobic lineages in tandem with an increase in the facultatively anaerobic Enterobacteriaceae family. These changes have previously been described in both inflamed and uninflamed tissue and in both fecal and mucosal samples.35–38 However, no significant differences in fecal microbiota were seen when analysed by NOD2-status, at any of the taxonomic levels assessed. The present study also includes VOC data and demonstrates the value of having a means to assess the functional aspects of the gut microbiota, and we were able to demonstrate higher butanoic acid concentrations in CD patients with NOD2 mutations than those without.This prospective study examines the relationship between NOD2 genotype and gut microbiota. Both Rehman et al and Mondot et al showed reductions in diversity and changes in specific taxa when comparing wild-type and NOD2 knockout mice.40 However, more recently, Shanahan et al conducted experiments where the knockout and wild-type mice were cohoused and failed to demonstrate a NOD2 genotype-specific effect on gut microbiota. They concluded that the cage environment was more important than genotype. Carmody et al went further and looked at the relative impact of genotype and diet on the gut microbiota in mice; they demonstrated dominant effects of diet, regardless of the underlying host genetics.41 Nonetheless, Nabhani et al found NOD2-related differences in mucosal microbiota even when NOD2-knockout and wild-type mouse embryos were mixed and transferred to wild-type surrogates and were subsequently cohoused.42Earlier animal studies have shown an association between NOD2 on intestinal microbiota. Knights et al reported results from cohorts comprising a total of 474 individuals with IBD, though not stratified by NOD2 status.43 They identified an association between 6 causal NOD2 variants and increased Enterobacteriaceae measured in intestinal biopsies. Of note, they were able to identify similar patterns in ulcerative colitis patients with NOD2 mutations, suggesting that the observed effect is not just one of disease phenotype. However, in a network of associations between bacterial taxa, host, and environmental factors, the effect of NOD2 genotype was only modest compared to antibiotic usage, immunosuppressants, biopsy location, and cohort of origin. Li et al reported differences in intestinal biopsy microbial profile related to NOD2 genotype alongside disease phenotype, with an increase in the C. coccoides-E. rectales group in patients with ileal CDcarrying a risk NOD2 allele.44 More recently, Imhann et al reported an interaction between an IBD genetics risk score that included NOD2 variants and the fecal microbiota, although the impact of NOD2 on its own was not described.45In humans, others have previously reported an effect of NOD2 genotype, with close matching of the phenotypic characteristics across genotypes. Establishing the causal relationship between the gut microbiota and IBD remains challenging; intestinal inflammation is well established as a cause of dysbiosis.46 The study excluded participants with either clinical or biomarker evidence of active disease, reducing the possibility of confounding by disease activity. Although the use of patients in remission will have removed one source of variability, it is also possible that the effects of NOD2 are manifest during active disease. With regards to limitations, this study explores only the changes in gut microbiota in the fecal contents, which are unlikely to fully reflect changes at the mucosal level, particularly in the terminal ileum where one might expect NOD2 to exert its strongest effect. This reflects the difficulty in accessing colonoscopic biopsy samples in a cohort of non-IBD controls and patients in remission. Although only a single VOC was significantly different by NOD2 status, this suggests a possibile difference in metabolically active bacteria not well-represented in fecal samples. The patient cohort also had well-established disease, with a history of surgical resection in most participants. This may reflect a higher risk of surgical resection in patients with NOD2 mutations, noting that the wild-type controls were matched using this phenotype among others. Shotgun metagenomic analysis might have facilitated detection of differences at the species or gene level between cohorts that could be missed with 16S rRNA taxonomic analysis.Strengths of this present study include the use of patients and nonIBD controls of known NOD2 genotype and ileal-associated bacteria, ideally using either cohorts again stratified by genotype or very large cohorts to generate adequate numbers of individuals carrying 2 disease-associated mutations. Large cohort studies also offer the opportunity to perform more extensive genotype-microbiota-phenotype analyses, which should lead to a better understanding of these complex interactions.This study confirms associations between altered fecal microbiota and Crohn’s disease, but failed to identify any differences in microbiota between individuals stratified by NOD2 genotype. Future studies should explore the relationship between Inflammatory Bowel Diseases online.Supplementary data are available at