Haemophilus influenzae (NTHi) causes multiple diseases of the human airway and is a predominant bacterial pathogen of acute otitis media and otitis media in which treatment fails. NTHi utilizes a system of phase variable epigenetic regulation, termed the phasevarion, to facilitate adaptation and survival within multiple sites of the human host. The NTHi phasevarion influences numerous disease-relevant phenotypes such as biofilm formation, antibiotic resistance, and opsonization. We have previously identified an advantageous selection for a specific phasevarion status, which significantly affects severity and chronicity of experimental otitis media. In this study, we utilized pure cultures of NTHi variants in which modA was either locked ON or locked OFF, and thus modA was unable to phase vary. These locked variants were used to assess the progression of experimental otitis media and define the specific immune response induced by each subpopulation. Although the initial disease caused by each subpopulation was similar, the immune response elicited by each subpopulation was unique. The modA2 OFF variant induced significantly greater activation of macrophages both in vitro and within the middle ear during disease. In contrast, the modA2 ON variant induced a greater neutrophil extracellular trap response, which led to greater killing of the modA2 ON variant. These data suggest that not only does the NTHi phasevarion facilitate adaptation, but also allows the bacteria to alter immune responses during disease. Understanding these complex bacterial-host interactions and the regulation of bacterial factors responsible is critical to the development of better diagnostic, treatment, and preventative strategies for these bacterial pathogens.Non-typeable  Human associated bacteria must be able to endure a wide range of stresses in order to survive and persist within their human host. Stressors include changes in the microenvironment, such as temperature, pH, and nutrient availability, as well as host humoral and innate immune responses. Bacteria that reside within the airways experience additional stressors which include ciliary clearance, changes in humidity or gas concentrations, and the production of mucus. Bacteria have developed a range of tactics to adapt to and overcome these stressors. Furthermore, several mucosal associated human pathogens have developed a mechanism of bacterial epigenetic regulation, termed the phasevarion, which facilitates rapid, coordinated adaptation to microenvironmental fluctuations and host defenses  asymptomatically colonizes the nasopharynx of most of the human population, but can also cause multiple respiratory tract infections, which include otitis media, rhinosinusitis, conjunctivitis, bronchitis, and exacerbations in patients with chronic obstructive pulmonary disease (COPD) or cystic fibrosis (CF)  resulted in significantly greater disease severity within the middle ear compared to when there was no change in the overall population status. While the specific factors that result in this increased severity remain unknown, we propose that there may be a unique host immune response mounted against each discrete modA subpopulation. To test this hypothesis, herein we investigate how the two different modA2 subpopulations interface with the host and affect innate immune response using the chinchilla model of experimental otitis media. While all previous studies with this model used phase variable populations, which were able to switch modA2 status, in these studies we now use locked variants that are unable to phase vary or switch status. Our findings in the chinchilla are further compared with human innate immune cells.Initial studies into the phasevarion of NTHi found that within multiple diverse collections of clinical isolates, up to two-thirds of NTHi had an active phasevarion  to disease pathology, this work assessed disease induced by genetically modified variants of NTHi that are unable to phase vary, or switch status  and blindly assessed for signs of disease daily. Inoculums were confirmed by serial dilution and plating, and there was no significant difference in the number of bacteria delivered to each cohort at time of challenge. All animals had bilateral middle ear effusions by 24 h after challenge, as expected following direct inoculation of NTHi into the middle ear. Animals were euthanized 2 days after challenge, to assess early stages of disease, 5 days after challenge, to assess a developing disease, or 14 days after challenge near the peak of infection and assessed for overall signs of experimental disease  or middle ear mucosal samples (CFU/mg) of animals challenged with modA2 locked OFF compared to modA2 locked ON (modA2 status.Middle ear fluids (MEF) and middle ear mucosa with any adherent biomass (MEM) were collected and the viable host cells within each sample were enumerated. There was no difference in the cell density or total amount of sample collected between the cohorts at 2 days after challenge . The den variant . Togetheocked ON . These rmodA2 OFF vs. modA2 ON variants, there was no difference in the number of APCs in either the middle ear fluids or mucosal samples 2 days post-challenge  and live CD11b+ CD15+ granulocytes (neutrophils) in the samples collected from each animal study . During hallenge . Five damodA2 OFF or modA2 ON variants. Expression of pro-inflammatory cytokines  was determined for the bulk (mixed) cell populations collected from the middle ear fluids and mucosal biomass samples, with each sample assessed separately. There were no significant differences between cohorts in the expression of pro-inflammatory cytokines at the time points assessed   . These rmodA2 OFF or modA2 ON variants. Middle ear biomass from chinchillas infected with modA2 OFF or modA2 ON for 14 days were stained with hematoxylin and eosin (H&E)  . Abundanin (H&E) . No signodA2 OFF . To confmodA2 ON , we assessed this possibility by performing immunohistochemical analysis of unfixed tissues collected from the middle ear at day 14 post-infection. Interestingly, both strains of NTHi induced the formation of NET-like structures, but those structures were more abundant in the chinchillas challenged with modA2 ON . Animalslabeling , green, in vivo responses of innate immune cells to each variant were specific or a result of the larger inflammatory environment within the middle ear, we assessed neutrophil responses in vitro. Neutrophils were isolated from unchallenged, naïve chinchillas and co-cultured with NTHi modA2 locked populations at a MOI of 10 NTHi per neutrophil. Neutrophils were allowed to phagocytose the NTHi for 45 min and then incubated for 3 h to assess bacterial survival within the neutrophils. Significantly more viable modA2 OFF were recovered from within the chinchilla neutrophils compared to modA2 ON recovered   , left. PmodA2 locked OFF and modA2 locked ON variants. First, we assessed the ability of MDMs to recognize and kill each modA2 variant. NTHi locked variants were added to MDMs at a MOI of 10 and incubated for 16 h. After 16 h, significantly more viable modA2 ON remained compared to viable modA2 OFF  and human blood neutrophils to the odA2 OFF . Soluble variant . The inc variant , 3B. WhimodA2 ON variant is more susceptible to killing by neutrophil-derived reactive oxygen species than the modA2 OFF variant  and had aggregates of viable bacterial (shown in green) within and on the surface of the neutrophils (modA2 ON variant displayed long extracellular DNA strands (shown in blue) indicative of NETosis (modA2 ON bacteria (shown in green) were localized to the surface of some neutrophils  affects the progression and severity of experimental otitis media, and that a shift in phasevarion status within the middle ear results in significantly greater disease severity that when a shift does not occur  across multiple clinical NTHi collections and is also the most studied NTHi phasevarion isolate to date  and neutrophils at day 14 post infection, compared to those challenged with modA2 OFF. These differences may be due to subtle and yet-unidentified differences in the early stages of infection. It is important to note that only viable cells were included in the enumeration of immune cell populations by flow cytometry and that these numbers likely underrepresent the total immune cell infiltration, due to cell death pathways , and this may explain discrepancies between scarce counts of early recruiting neutrophils, which did not correlate to increased expression of MPO or ELANE in the middle ear of infected animals at 2 day post infection. Neutrophil extracellular traps (NETs) are an important part of the immune response to bacterial infection and are present within the infected middle ears of chinchillas and humans  contribute to the increased disease severity observed when modA2 status shifts and the host is thus exposed to both unique variants.Disease progression following challenge with either of the NTHi strain 723 modA2 OFF and modA2 ON variants each express a unique collection of virulence factors, we anticipated each may drive a unique immune response. To evaluate immune response and activation within the middle ear we first performed targeted studies on samples collected day 14 after challenge to determine gene expression of pro-inflammatory cytokines and neutrophil activation markers. No significant differences in expression were observed when we analyzed total RNA collected from within the middle ear, likely due to the tremendously heterogeneous nature of these samples . However, substantial differences in gene expression were observed when distinct viable immune cell populations were assessed, following fluorescently-activated-cell-sorting. APCs, which likely include mononuclear phagocytic cells, isolated from the middle ears of chinchillas challenged with modA2 OFF expressed significantly more pro-inflammatory cytokines than those that received modA2 ON. These results indicated that modA2 OFF, or a virulence factor expressed by this variant, induced greater activation or recognition by these phagocytic cells than did modA2 ON. We hypothesized that most of these APCs were macrophages, as neutrophils and macrophages constitute the majority of immune cells that infiltrate the middle ear early during otitis media  during a 16-h in vitro assay. Interestingly, the modA2 OFF variant was significantly more sensitive to killing by hMDMs than the modA2 ON variant, which may be due to greater activation of the macrophages in the presence of modA2 OFF compared to modA2 ON. Future work is necessary to fully investigate the mechanisms by which macrophages differentially recognized and kill each of these populations. Many proteins and saccharides on the outer surface of NTHi are known to affect opsonization and host immune response and may be regulated by ModA2 during disease  and neutrophil elastase (ELANE) compared to those challenged with modA2 OFF, suggesting that modA2 ON recruited more neutrophils to the middle ear. Catalase and peroxiredoxin-glutaredoxin enzymes produced by NTHi are protective against oxidative stressors and can promote bacterial persistence within NETs  than the modA2 ON variant, due to increased resistance to the antimicrobial effects of hydrogen peroxide , such as LOS or OMPs, than modA2 OFF, which could induce a differential activation of neutrophils. Several NTHi virulence factors can exhibit phase variable expression independent of the ModA2 regulon  weighing 500–800 g were obtained from Rauscher's Chinchilla Ranch  and acclimated in the vivarium for 7 days. Serum was collected from chinchillas prior to bacterial challenge to obtain baseline pre-immune serum. Chinchillas were cohorted based on sex and weight, and then challenged by transbullar inoculation with ~750 CFU of NTHi in 300 μL of saline/bulla to induce experimental otitis media. At the time of challenge, the number of NTHi CFU per milliliter was confirmed by serial dilution and plating of inocula. At days 2, 5, and 14 after bacterial challenge, a pre-determined group of anesthetized chinchillas were euthanized, and the bullae were dissected from the skull. Left and right bullae were aseptically opened and imaged. Middle ear fluid and middle ear mucosa plus any adherent biomass were collected from the left ears for subsequent immunologic analyses. Fluids and mucosal samples from the right ears were collected and used for subsequent RNA isolation. The volume and weight of the respective samples were recorded at time of collection and before any processing.Adult chinchillas . Frozen sections (5 μm thick) were either processed for standard hematoxylin and eosin (H&E) staining or fixed and used for fluorescent immunohistochemistry (IHC). For IHC, the sections were probed with mouse anti-dsDNA antibodies (Abcam) and DyLight680-conjugated anti-GFP antibodies (ThermoFisher), the slides were washed twice and the anti-dsDNA antibodies were fluorescently labeled with AlexaFluor 488-conjugated goat anti-mouse IgG (H+L) secondary antibodies (ThermoFisher). Images were captured on a Zeiss Axio Observer inverted fluorescent microscope and processed with Zeiss Zen 2.6. For H&E stained section, images of replicate fields of view of multiple different sections were imaged on a Zeiss AxioScope 5 microscope with a 63x oil objective and captured on Zen 3.0 (Zeiss). Scrambled images were blindly scored by at least 3 independent reviewers to assess immune cell density and thickness of the strand-like structures using the rubric provided in To assess the structure and composition of the middle ear biomass chinchillas were challenged as described above using NTHi strain 723 Samples collected from the left ears were gently centrifuged and cell-free supernatants were snap frozen and stored. Cells were then resuspended in sterile saline and treated with DNAse and collagenase for 1 h and subsequently filtered with a 70 μM mesh, enumerated and divided for cell specific analysis. Washed cells were stained with the antibodies Alexa Fluor 700 anti-mouse/human CD11b clone M1/70 (Biolegend), biotin anti-human CD15 clone HI98 (Biolegend), BV 421 anti-human HLADR clone G46-6 (BD Horizon), FITC anti-mouse/human CD45R/B220 clone RA3-6B2 (Biolegend), APC-streptavidin (Biolegend), and incubated in ice-cold FACS buffer for 15 min, and analyzed on an LSR II cytofluorometer . Cells were gated according to size (SSC-A) and forward scatter (FSC-A) to obtain cell singlets and blue-fluorescent reactive dye L23105 (Life Technologies) was used to discard dead cells. Absolute cell numbers were calculated with the total cell count multiplied successively by the percentages for the appropriate gates obtained through flow cytometry.Following homogenization and enumeration, a fraction of cells collected on Day 14 were stained with the monoclonal antibodies anti-human BV421 HLADR, biotin anti-human CD15 and APC-streptavidin, then the cells were sorted on an Influx FACS  based on fluorescence profiles, as indicated. Due to the number of samples collected and sorter limits, cells from the fluid and mucosal samples of each ear were pooled prior to sorting at a ratio that reconstituted that present at time of collection. The resultant cell populations were homogenized in TRIzol reagent (ThermoFisher Scientific) and stored at −80°C for subsequent analysis.Quantitative real-time reverse transcriptase PCR was performed to determine the relative expression of select chinchilla genes. Custom TaqMan Gene Expression Assays were designed and validated for chinchilla genes of interest. DNA-free RNA was isolated and purified using TRIzol reagent according to manufacturer instructions. cDNA libraries were subsequently generated with the High-Capacity RAN-to-cDNA Kit (Applied Biosystems). Quantitative PCR was carried out with TaqMan Advanced 2x Master Mix on an ABI 7500 real-time thermocycler (Applied Biosystems). Relative expression (transcript) was calculated by the delta-delta-Ct method  from newly drawn blood according to the manufacturer directions. Fresh neutrophils were incubated with NTHi at a MOI of 10 NTHi per neutrophil for 45 min at 37°C to allow phagocytosis and internalization. Non-internalized bacteria were then removed and 5 × 105 cells per well a day prior to infection. The next day, MDMs were infected with NTHi at MOI of 10 for 45 min at 37°C. Extracellular bacteria were removed by washing the wells 3 times with PBS, and then cells were incubated at 37°C overnight. 0.1% Triton-X100 was added to release the intracellular bacteria and NTHi CFU were enumerated by performing serial dilutions on chocolate agar. Before addition of Triton-X100, a small aliquote of the cell culture medium was collected and total cytokines levels were measured with a Custom Cytometric Bead Array (BD Biosciences) analyzed on an Accuri C6 (BD Biosciences).Monocytes were differentiated to Monocyte-derived Macrophages (MDMs) by culturing them with RPMI media plus 10% FBS for 5 days, then MDMs were detached and seeded in 24 well plates at a density of 5 × 105 human neutrophils were seeded onto chambered coverglass for 30 min at 37°C, and then NTHi that constitutively express GFP (green fluorescent protein) were added at an MOI of 10 and incubated for an additional 3 h at 37°C. Cells were fixed for 30 min in 10% neutral buffered formalin, stained with fluorescent monoclonal antibodies against neutrophil elastase, triple rinsed with PBS and the DNA was stained with DAPI. No additional NTHi-specific staining was used as GFP fluorescence is maintained within intact NTHi cells following fixation. Decreased membrane integrity resulted in loss of GFP signal, which correlated with cell death assessed by bacterial viability staining. The samples were imaged on a LSM 800 laser scanning confocal microscopy with Airyscan detector and images were rendered with Zeiss Zen v2.5.A total of 105 human neutrophils were seeded onto coverslips for 30 min at 37°C, and then NTHi were added at a MOI of 10 and incubated for an additional 3 h at 37°C. The cells were then fixed with 2.5% glutaraldehyde in PBS buffer for 30 min, followed by a second 30 min fixation with 1% osmium tetroxide in PBS. The fixed samples were then triple washed in deionized water and dehydrated with graded ethanol . The ethanol was then replaced with hexamethyldisilazane (HMDS) and the samples were allowed to air dry. The coverslips were coated with 2.5 nM gold/palladium in an Emitech K550X sputter coater and examined on a Hitachi S-4800 field-emission scanning electron microscope. Images were captured at an accelerating voltage of 3–5 kV using a secondary electron detector and psuedocolored with Adobe Photoshop CC 2019. Human neutrophils were collected from 3 independent donors. A total of 10All statistics were performed with GraphPad Prism v 8.0, as indicated.The datasets generated for this study are available on request to the corresponding author.The animal study was reviewed and approved by Institutional Animal Care and Use Committee at the Research Institute at Nationwide Children's Hospital and the Medical College of Wisconsin.KB: conceptualization. FR-A, JR-R, and KB: methodology. FR-A, JR-R, and KB: investigation. KB and FR-A: writing—original draft. JR-R and SP-S: writing—review & editing. KB: funding acquisition. SP-S and KB: supervision. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer DH declared a past co-authorship with one of the authors KB to the handling editor.


The topic of which Cre-recombinase driver to use for gene targeting in pancreatic β cells comes up often around the bar at conferences. And these conversations last a long time. Challenges with the most commonly used tool for targeted gene recombination (Cre/Lox) plague the β cell field. With more than 20 lines generated to target these specialized endocrine cells, choice was never the problem.Ins2 promoter to drive Cre-recombinase (RIP-Cre25Mgn) was shown to be glucose-intolerant, even in the absence of a floxed gene target -Cre lines, which drive expression in the endocrine cells of adult mice when used in conjunction with an inducible Cre transgene. However, experiments with reporter mice demonstrate these Cre lines, along with other popular RIP (Ins2)-driven lines, have significant Cre expression in areas of the digestive and central nervous systems (Pdx1 and Ins2 promoters. Next up was the murine Ins1 gene promoter (MIP), which turns out to be much more specific to β cells   minigene, commonly added to constructs for its polyA tail and introns that increase transcription efficiency, can also express bioactive growth hormone . Expressinigene) . Althouginigene)  in this inigene)  and reduinigene) .Ins1-CreThor) and the other a tamoxifen-inducible Cre expresser (Ins1-CreERTThor)  of this new problem, potential reasons being the dose and/or method of tamoxifen administration, accessibility of the floxed target gene, or environmental influence from different mouse facilities. It seems that the field was faced, yet again, with another roadblock and a new mystery.Soon came 2 new mouse strains: 1 driving constitutive Cre expression in β cells (ERTThor) , both usEndocrinology, Mosleh et al  both for the constitutive and inducible β cell Cre-driving lines. Some floxed genes show no recombination at all, despite demonstrating high recombination efficiency using reporter lines. When compared to other Cre lines , the Ins1-Cre transgenes produce inconsistent and often milder phenotypes, and expression of Cre-recombinase in islets of these new lines was much lower. They attribute low Cre expression to increased methylation of CpG islands in the Ins1 promoter and transgene locus and conclude that hypermethylation leads to insufficient transgene expression, preventing efficient recombination of floxed genes.In the current issue of eh et al  shed lig Ins1 promoter knockin approach. Although targeting the endogenous Ins1 locus avoids random insertion off-target effects, is silencing inevitable? Is methylation specific for the Ins1 locus, or could it occur at all insulin genes? Decreased targeting efficiency is not yet reported for mouse lines using Ins2 or Pdx1 loci. Interestingly, recombination efficiency using the same mouse lines seems to differ greatly between research labs and across institutions. Perhaps methylation status is influenced by genetic background, housing conditions, diet, or other environmental factors, all of which may prove difficult to identify and control. One approach could be to mutate CpG islands in transgenes to prevent hypermethylation. But will this create new, unforeseen problems?This study brings up important considerations for the field. There are now many mouse lines using a similarIns1 promoter remains one of the best drivers of β cell–specific expression  recombination efficiency at the targeted locus and not rely on reporter mice or past publications to claim effectiveness.We believe that regardless of Cre driver chosen, including control groups expressing the Cre-transgene alone effectively controls for off-target effects. If recombination in other tissues is a concern, researchers could confirm β cell specificity of phenotypes using 2 separate Cre-drivers, Although this may seem like a “β cell–world problem,” issues with LoxP/Cre technology are likely widespread. The Mosleh study emphasizes the need for careful experimental design of focused gene knockouts in all cell types and broadens the understanding of ectopic gene regulation. How different cells react to genetic manipulation in unique ways has implications for gene-targeting approaches both in basic science and medicine.


This mechanical hypersensitivity was characterised by significantly increased peak afferent firing rates and a decrease in the activation threshold of individual afferents. Bladder afferent hypersensitivity occurred in the absence of inflammation and changes in bladder muscle compliance, indicating a direct sensitisation of peripheral afferent endings. Bladder afferent mechanosensitive responses to distension returned to control levels by day 7 post-protamine sulfate treatment and remained at control levels at 28-days post-treatment. Here we demonstrate, contrary to the prevailing hypothesis, that increased bladder permeability alone does not induce chronic bladder afferent sensitisation. Whilst experimentally induced changes in bladder permeability are able to induce transient bladder afferent hypersensitivity in the absence of inflammation, highly regulated homeostatic mechanisms exist to rapidly repair the urothelial barrier and normalise bladder afferent mechanosensitivity. Together, these data suggest that additional pathophysiology is required to induce chronic bladder dysfunction.Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic urological condition characterised by urinary urgency, frequency and pelvic pain, that significantly impacts the quality of life for ∼5% of women. Bladder sensation is coordinated by primary afferent sensory neurons that innervate the bladder wall, translating bladder stretch into signals that travel to the brain via the spinal cord. Whilst the pathophysiology of IC/BPS remains unknown, an increase in the permeability of the bladder urothelium has been proposed as an initiating cause. Here we experimentally increased bladder permeability and tracked bladder afferent sensitivity for up to 28 days. We found that one day after increasing bladder epithelial permeability with  Interstitial cystitis/bladder pain syndrome (IC/BPS), is a common urological condition characterised by persistent or recurrent chronic pelvic pain that is often accompanied by urinary urgency and/or frequency . Whilst The bladder wall contains a dense network of afferent nerves found both in the detrusor smooth muscle, and in close proximity to the bladder lumen and urothelium . These aIn vivo bladder instillation of protamine sulfate is a commonly used method for experimentally inducing bladder permeability in the absence of overt bladder inflammation or significant urothelial damage that returns to normal over a period of 3-7 days  mucus layer, that block the movement of ions, solutes and toxic metabolites . In a he3-7 days .in vivo intrabladder infusion of protamine sulfate.It remains to be determined whether bladder permeability is part of the initiating pathophysiology of bladder hypersensitivity in IC/PBS patients or a downstream consequence of inflammation that further exacerbates the condition. In this study we sought to determine if chronic sensitisation of bladder afferents could be induced by specifically increasing urothelial permeability by using an in vivo procedures, mice were individually housed (1 mouse per cage) in individual ventilated cages. Mice had an average weight of 21–23 g on the day of experimentation. Ex vivo afferent recordings and myeloperoxidase (MPO) analysis were performed on separate mice at 16 weeks of age.12- to 16- week-old female C57BL/6J mice were used in this study. The South Australian Health and Medical Research Institute (SAHMRI) ethics committee approved all experiments involving animals . Mice were acquired from an in-house C57BL/6J breeding program  within SAHMRI’s specific and opportunistic pathogen-free animal care facility. Prior to bladder infusion with either protamine sulfate or vehicle , mice were group housed (5 mice per cage) in individual ventilated cages filled with chip coarse dust-free aspen bedding . Mice had free access to LabDiet JL Rat and Mouse/Auto6F chow  and autoclaved reverse osmosis water within a temperature-controlled environment of 22°C and a 12 hour light/12 hour dark cycle. Following 1) mice that received no treatment (Control: N = 5). (2) Mice that received intrabladder infusion of saline and used 1 day later (1-day post vehicle: N = 6). (3) Mice that received intrabladder infusion of protamine sulfate and used 1 day later (1-day post PS: N = 5). (4) Mice that received intrabladder infusion of protamine sulfate and used 7 days later (7 days post PS: N = 5). (5) Mice that received intrabladder infusion of protamine sulfate and used 28 days later (28 days post PS: N = 6).Five groups of mice were used in this study.  Krebs-bicarbonate solution  at 35°C. A catheter attached to a syringe pump was inserted into the bladder via the urethra to allow controlled distension at 100 μl/min with saline (0.9%). A second two-way catheter was inserted into the bladder dome, which is attached to a pressure transducer and an outflow tap, enabling the recording of intravesical pressure during bladder distension and the ability to passively drain the bladder after each distension. Pelvic afferents, isolated from nerve fibres between the pelvic ganglia and the spinal cord, were dissected into fine multiunit branches and inserted into a glass electrode. Multiunit afferent activity was detected by a Neurolog headstage , amplified, filtered  and captured by a computer via a power 1401 interface and Spike 2 software .Bladder afferent nerve recordings were performed using an cterised  on 16 weA single nerve bundle was isolated for each experiment (N = 1 per mouse) and inserted into the glass electrode during the dissection process. This nerve bundle was used for the entire experiment allowing multiunit afferent nerve recordings to be performed. Bladder distensions (100 μl/min) were performed from 0 to 50 mmHg at 10-minute intervals until the afferent response to distension was stable and reproducible.ex vivo afferent recordings was determined by the number of action potentials passing a pre-set threshold . This ensures only mechanosensitive afferents are recorded. Each afferent nerve contains a different number of single afferent units. In order to identify individual units, single unit analysis was performed offline by matching individual spike waveforms through linear interpolation using Spike 2 software. Baseline afferent sensitivity was determined by averaging the baseline nerve activity occurring between distensions over a 5 minute period. Single afferent units were characterised as previously described  and sonicated for 1 min. 20 μl of tissue homogenate was added to 200 μl of an o-dianisidine dihydrochloride (o-dianisidine) solution containing H2O2. Absorption data was recorded on a VERSAmax 20 plate reader (Molecular Devices) at 450 nm wavelength for 4 min with 30 sec interval-reading. All samples were run in triplicates and a blank was included.Bladders isolated from mice immediately following humane euthanasia with COnitrogen . Tissue ∗P< 0.05, ∗∗P < 0.01,∗∗∗P < 0.001. All data were analysed using GraphPad Prism 7  in a blinded manner. For bladder afferent responses to distension, compliance curves and multiunit and single unit data was found to be normally distributed and analysed using a two-way ANOVA with Tukey’s multiple comparisons. The number of single units per experiment, activation thresholds, peak afferent firing and MPO concentrations were analysed by one-way ANOVA with Tukey’s multiple comparisons. The specific tests used to analyse each data set and N numbers are indicated within the individual figure legends.Data are expressed as Mean ± SEM. N equals the number of animals, whilst n equals the number of individual afferents identified using single unit analysis. Differences were considered significant at a level of ex vivo recordings from bladder afferents  to bladder distension were actually reduced at 1-day post infusion . This paInterestingly in our study, and in contrast to the proposed hypothesis that changes in bladder permeability are an initiating factor in the development of IC/BPS , the blaOur data indicate that whilst changes in bladder permeability are able to induce transient bladder afferent hypersensitivity, highly regulated homeostatic mechanisms exist in a murine model to rapidly repair the urothelial barrier, and as such, it is unlikely that changes in permeability alone are the initiating factor in the development of bladder disorders such as IC/BPS.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by South Australian Health and Medical Research Animal Ethics Committee Project approval number: SAM234.in vivo bladder infusions. LG conducted ex vivo afferent recordings, analysis, made figures, and wrote the manuscript. AL performed MPO analysis. All authors provided feedback and revision of the final manuscript.LG, SMB, GH, and BG, conceived and designed research. EM provided essential technical assistance in bladder infusions. LG and AC performed 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.


Physicians strive to improve the postsurgical experience and optimize patient-reported recovery outcome measures (PROMs) following elective cosmetic surgical procedures. Our previous pilot feasibility study demonstrated that twice daily postoperative topical body treatment with tripeptide and hexapeptide  reduced PROMs of swelling, induration, soft tissue fibrosis, and pain as well as improved visible and palpable skin quality. Evaluate whether adding a tripeptide/hexapeptide anhydrous gel  pre- and post-procedure to the existing postsurgical regimen of TFB significantly improves 6 PROMs in patients undergoing neck and body contouring cosmetic surgical procedures.Ten female patients underwent 15 neck and body contouring procedures and were blindly randomized to 1 of 2 topical treatment protocols (1 [TFB] and 2 [RSN/TFB]) pre- and post-procedure. Patient-reported scores of 5 skin parameters  and pain scores using the Visual Analog Scale were collected at 8 intervals for 12 weeks post-procedure.P < 0.05). All patients observed slower soft tissue recovery on the side that was treated with TFB alone and opted to break the code and use both topical treatments.The treatment side that used both topicals showed significantly reduced scores of edema, induration, and subcutaneous fibrous banding compared with the side that only used 1 topical, on days 5–7 and 10–14 (Patients had statistically significant improved patient-reported measures of skin edema, skin induration, and subcutaneous banding on the operated side that used both topicals. This conclusion was supported by results from gene expression, ultrasound, fibrometer analyses, and histology of skin biopsies that demonstrated extracellular remodeling and histological evidence of improved collagenesis and elastogenesis.2 The results of this recently published gene expression study demonstrated the molecular evidence for the clinical observation of accelerated post-procedure healing when participants used topical treatment with RSN and TFB,3 pre- and post-procedure. Other clinical studies have shown that wound bed preparation using topical RSN pre- and post-procedure accelerates healing from laser injury and provides evidence of extracellular matrix remodeling when used as preconditioning before aesthetic surgical procedures.5 TFB has also shown histologic evidence of remodeled extracellular matrix with regenerated collagen and elastin. The hexapeptide-11 component of TFB has been demonstrated to accelerate (upregulate) the process of autophagy, encouraging lipid droplet breakdown, and in vitro modeling shows macrophage recruitment to damaged fat cells with clinical trials confirming increased and hastened fat volume reduction.6As aesthetic surgeons who perform elective cosmetic procedures, we strive to optimize the patient experience and surgical outcomes. Our previous pilot feasibility study demonstrated that twice daily postoperative topical body treatment with tripeptide and hexapeptide (TFB) compared with the control group reduced patient-reported recovery outcome measures (PROMs) of swelling, induration, soft tissue fibrosis, and pain. Improved visible and palpable skin quality was also observed.The recent gene expression study demonstrated that pre- and post-procedural topical treatment with RSN and TFB stimulated extracellular remodeling and induced anti-inflammatory genes, leading to less post-procedural induration documented by ultrasound. The question that still remains is whether both products are necessary to improve PROMs and ensure patient compliance to use 2 separate products. This split neck and body, randomized, double-blinded study was designed to analyze whether adding a second topical treatment RSN  pre- and post-procedure improved 6 PROMs or whether a single topical treatment with TFB  would achieve the same improved post-procedural recovery.N = 1) and body contouring   involving fat reduction. Patients who met these criteria were not excluded. These 10 patients underwent 15 bilateral procedures: neck (n N = 8) .All 10 patients were provided 2 bottles labeled 1, which contained TransFORM Body Treatment with TriHex Technology (TFB) 6 oz, and 2, which contained Regenerating Skin Nectar with TriHex Technology (RSN) 4 oz. Each patient was instructed to use the bottle labeled topical treatment 1 (TFB) on both right and left sides of their neck or body to encompass the surgical site, for 10–14 days pre-procedure and for 12 weeks post-procedure. The 10 patients were also asked to select an unlabeled envelope out of a box that contained their randomized side (right side or left side). They were instructed to treat the side written in the envelope with topical treatment 2 (RSN) twice daily pre-procedure for 10–14 days and post-procedure for 12 weeks. Abdominoplasty patients were asked to treat abdominal and flank skin except for the skin that was to be excised. This planned skin excision area was drawn out at their preoperative visit to clarify skin that was not to be treated. Post-procedure, patients were instructed to use the topical in the same surgical areas and to include the skin at the incision. Application instructions per procedure are included in Patients were evaluated at 8 postoperative intervals: days 1–3, 5–7, 10–14, 21–25, 28–30, 35–40 and 42–50, and again at 12 weeks. At each post-procedure visit, the Visual Analog Scale (VAS) as a patient-reported outcome measure for pain was administered in person to the patient by the same medical assistant, and their score sheet for the right and the left side was placed into the patient’s study chart. The same medical assistant also asked each patient to score 5 skin parameters—ecchymosis, swelling, skin discoloration, induration, and subcutaneous banding of full stretch, for their right and left side, using a 0–4 scale . The medical assistant provided definitions of each skin parameter. Patients wrote down a score for the right and left sides for each of the 5 skin parameters. Score sheets for every visit were kept in each patient’s study chart. The study patients were deidentified and labeled 1–10, and their data sheets were entered onto a spreadsheet by an independent data entry person. At every post-procedure visit, each patient was given the option to proceed with the study, opt out, or break the code to use topical treatment 2 (RSN) on the contralateral side that they were treating with only topical treatment 1 (TFB). This study was conducted in accordance with the World Medical Association Declaration of Helsinki’s statement of ethical principles for medical research involving human patients, including research on identifiable human material and data. Full informed written consent was acquired from each patient in the study. t test) and nonparametric test  were used. Treatments were compared before and after the code was broken, that is, when patients opted to use both products. Skin parameters tested–skin discoloration, ecchymosis, edema, induration, and subcutaneous fibrous banding and pain (VAS).Ten females  completed the study. The following statistical analysis was performed: 15 procedures were analyzed computing the difference in skin parameters between TFB plus RSN vs TFB alone each day. Both parametric test  in which a scale was used to score 6 PROMs of their right and left sides. All patients requested to break the code because they encountered a difference in PROM from the side on which they used 2 topical treatments, topical treatment 1 (TFB) and topical treatment 2 (RSN). By day 14, all patients asked to use topical treatment 2 (RSN) on both sides, except patient 10, who asked to break code on day 26 to use topical treatment 2 (RSN).P value of the statistical tests. The results show that topical treatment 1 (TFB) plus topical treatment 2 (RSN) significantly reduced edema, induration, and subcutaneous fibrous banding compared with topical treatment 1 (TFB) alone on days 5–7 and days 10–14 (P < 0.05), before the code was broken, whereas reductions in skin discoloration, ecchymosis, and pain were not statistically significant. Please note that the results were similar when the data on broke days were excluded from the analysis (data not shown).8 together with the concept of the clearance of “waste products” related to surgery or metabolism, in this case, lipid droplets. It is hypothesized that lipid droplets create localized areas of inflammation, inflammasomes, which are not only large particles for macrophage digestion but also elaborate inflammatory mediators, which manifest as edema, induration, and in some cases localized fat necrosis. By preparing and remodeling the extracellular matrix before surgery8 and optimizing macrophage phagocytosis of lipid droplets,9 the clearance provided translates into improved skin induration and edema and lessened patient discomfort.The basis of the topical combination therapy devised in these cases relates to the advantages reported by preconditioning the skin before surgical procedures,3The 10 patients who underwent neck and body contouring procedures had statistically significantly improved patient-reported measures of skin edema, skin induration, and subcutaneous banding in the operated areas that underwent trauma from laser energy, mechanical liposuction, and skin undermining when they used the combination of topical treatment 1 (TFB) and topical treatment 2 (RSN) pre- and post-procedure. The skin parameters assessed that were treated with TFB and RSN as compared with TFB alone decreased before the code was broken, increased after the broken code, and settled at zero at month 3. This translated to a statistically significant decrease in edema, induration, and fibrous banding in the combination group up to the days that patients opted to use both products. This difference then dissipated (increased after broken code) and normalized at month 3. Specifically from a physiological perspective, the first 5–7 days would traditionally be the “clearance” phase of inflammation where proinflammatory particles are removed, and 10–14 days would be the inflammatory “switch off” phase where extracellular remodeling is producing new collagen and elastin. This would translate to a maximal decrease in induration and edema in the first 5–7 days with stabilization of the process and early fibrous banding at 10–14 days. These findings are consistent with the theory proposed above and with published gene marker and gene expression studies.2 Additional limitations include smaller sample size and lack of a control group that could have used 2 bland topicals.While this provides validation for the use of the 2 products in early recovery (14 days post-procedure), the broken codes do create limitations in analyzing the long-term impact of the use of the products. That noted, an additional liposuction study does present long-term improved outcomes with the use of these products in a split-body study.The 10 patients who underwent 15 neck and body contouring procedures that created trauma from laser energy, mechanical liposuction, and skin undermining had statistically significantly improved patient-reported outcome measures of skin edema, skin induration, and subcutaneous banding on the operated side that used the combination of topical treatments 1 (TFB) and 2 (RSN) 10–14 days pre-procedure and 12 weeks post-procedure. The data from the PROMs from this study support the use of combination treatment with both TFB and RSN to improve PROMs and their post-procedural experience.ojaa052_suppl_Supplementary_Appendix_1Click here for additional data file.


Sarcomas are malignant soft tissue and bone tumours affecting adults, adolescents and children. They represent a morphologically heterogeneous class of tumours and some entities lack defining histopathological features. Therefore, the diagnosis of sarcomas is burdened with a high inter-observer variability and misclassification rate. Here, we demonstrate classification of soft tissue and bone tumours using a machine learning classifier algorithm based on array-generated DNA methylation data. This sarcoma classifier is trained using a dataset of 1077 methylation profiles from comprehensively pre-characterized cases comprising 62 tumour methylation classes constituting a broad range of soft tissue and bone sarcoma subtypes across the entire age spectrum. The performance is validated in a cohort of 428 sarcomatous tumours, of which 322 cases were classified by the sarcoma classifier. Our results demonstrate the potential of the DNA methylation-based sarcoma classification for research and future diagnostic applications. Sarcomas are morphologically heterogeneous tumours rendering their classification challenging. Here the authors developed a classifier using DNA methylation data from several soft tissue and bone sarcoma subtypes, which has the potential to improve classification for research and clinical purposes. A high inter-observer variability among pathologists is reflected in considerable discrepancy rates between primary institutions and specialized referral centres with access to comprehensive molecular testing4. Pathologists often rely on the determination of tumour specific molecular alterations if available4. While the determination of characteristic molecular alterations most often consisting of translocations that generate gene fusions has become a diagnostic standard for many sarcoma types, approximately half of the sarcoma entities lack unequivocal molecular hallmarks1. Even in some cases defined by specific gene fusions, it may not be possible to identify adequately the fusion by FISH or RNA-based methods for a variety of technical and specimen-related limitations. Novel approaches are needed to fill these diagnostic gaps5.Sarcomas are a heterogeneous group of tumours, which pose challenges to pathologists. Many entities lack unequivocal morphologic or molecular hallmarks and the overall rarity of sarcomas result in a widespread lack of experience6. In cancer, DNA methylation patterns reflect both the cell type of origin, as well as acquired changes during tumour formation7. Profiling of human brain tumours has demonstrated entity-specific methylation signatures and has led to the identification of several novel and clinically relevant subtypes13. On this basis, a comprehensive brain tumour classifier has been developed15. Recently, we have extended the principle of methylation-based tumour profiling to small blue round cell sarcomas evading a definite histological diagnosis, thereby resolving these cases into established sarcoma entities16. Further, DNA methylation-based profiling showed diagnostic potential for soft tissue and bone sarcoma subtyping22. In this work, we aimed at developing a DNA methylation-based classification tool for soft tissue and bone sarcomas representing a broad range of subtypes and age groups.DNA methylation is a key epigenetic mark and plays important roles in normal development and disease23 thereby identifying groups of tumours sharing methylation patterns (methylation classes). To minimize potential clustering artefacts at least seven cases were required for defining a methylation class, which empirically proved sufficient for training a classifier and allowed prediction15. Unsupervised clustering, respecting the minimal number of seven cases per group, led to the designation of 62 tumour methylation classes belonging altogether to 54 histological types, and three non-neoplastic control methylation classes MYOD1 alteration, plexiform neurofibroma, dedifferentiated chordoma and small blue round cell tumours with either BCOR alteration or CIC alteration. Three methylation classes combined WHO entities (category 3). The methylation class angioleiomyoma/myopericytoma and the methylation class atypical fibroxanthoma/pleomorphic dermal sarcoma each combined two entities, while the methylation class undifferentiated sarcoma contained undifferentiated (pleomorphic) sarcoma, myxofibrosarcoma and a fraction of pleomorphic liposarcoma, thereby providing further evidence that these sarcoma subtypes probably fall into a morphologic continuum of a single entity as suggested by previous genetic-based studies26. Two methylation classes point towards novel entities not yet defined by the WHO (category 4)19. The methylation class SARC (RMS-like) was identified in sarcomatous CNS tumours with various morphologic patterns not matching established tumour categories. Unifying features of cases mapping to this class are rhabdomyoblast-like cells and DICER1 mutations13. Methylation class SARC (MPNST-like) was reported as a subset of malignant peripheral nerve sheath tumours19. Cases assigning to SARC (MPNST-like) present similar to MPNST, however, retain trimethylation at histone 3 lysine 27 (H3K27me3). In addition, based on 28 non-neoplastic tissue specimens methylation classes were established for non-neoplastic skeletal muscle, reactive soft tissue and leukocytes. Supplementary Data Based on 1077 tumour cases, methylation classes were assigned to four categories relating to the WHO classification Fig. . Categor27. Cross-validation, an internal performance metric15, of the sarcoma classifier provided an estimated error rate of 1.95% for raw scores and a discriminating power of 99.9% by area under receiver operating characteristic curve analysis. The low rate of misclassifications demonstrates the discriminating power of the classifier algorithm  plots from all sarcomas of the reference cohort as described14. Frequently encountered alterations include MDM2 amplification for well-/dedifferentiated liposarcomas, MYC amplification for radiation induced angiosarcoma or segmental chromosomal deletions on chromosome 22q encompassing SMARCB1 for rhabdoid tumours. While these alterations often are characteristic for distinct sarcoma entities, they usually are not pathognomonic because of their occasional occurrence also in other entities. However, in combination with methylation profiles, CNV plots frequently add to the diagnostic decision process. The frequency of chromosomal or subchromosomal numerical alterations within the methylation classes/entities can be depicted by summary CNV plots  block. The validation set included sarcomas enrolled in the INFORM, NCT-MASTER, PPT and MNP2.0 studies30. Rare sarcoma entities have not been over-represented. However, availability determined inclusion resulting in over-representation of high-grade sarcomas in the validation set.All samples of the reference and validation set are from individual/different patients. All cases of the reference set had undergone rigorous morphological examination by pathologists specialized in diagnosing sarcomas and also tumour-type specific molecular testing for identification of the relevant alterations, whenever possible. For each specimen, we aimed at a tumour cell content of ≥70%, with the caveat that microscopically estimated tumour cell percentage is prone to being relatively imprecise. However, determining tumour cell content by random forest regression demonstrated that this goal was not reached for many samplesTo exclude low-quality samples from the cohort, the on-chip quality metrics of all samples were checked and compared to a set of 7,500 pairs of IDAT-files. In addition, for each sample, an overall noise-level was computed using the R package conumee version 1.6.0. Samples showing low quality values ranging in the 10th percentile for at least one of the sample controls  and showing an overall noise level greater than 3, were excluded from this study.All computational analyses were performed in R version 3.4.4 . Raw signal intensities were obtained from IDAT-files using the minfi Bioconductor package version 1.24.0. Illumina EPIC and 450k samples were merged to a combined dataset by selecting the intersection of probes present on both arrays . Each sample was individually normalized by performing a background correction (shifting of the 5th percentile of negative control probe intensities to 0) and a dye-bias correction  for both colour channels. Subsequently, a correction for the type of material tissue (FFPE/frozen) and array (450k/EPIC) was performed by fitting univariate, linear models to the log2-transformed intensity values . The methylated and unmethylated signals were corrected individually. Beta-values were calculated from the retransformed intensities using an offset of 100 (as recommended by Illumina).n = 11,551), removal of probes containing a single-nucleotide polymorphism (dbSNP132 Common) within five base pairs of and including the targeted CpG-site (n = 7998), probes not mapping uniquely to the human reference genome (hg19) allowing for one mismatch , and 450k array probes not included on the EPIC array. In total, 428,230 probes were kept for downstream analysis.Before further analysis was undertaken, the following filtering criteria were applied: removal of probes targeting the X and Y chromosomes  using 3000 iterations and a perplexity value of 30. In addition, to assess the stability of the resulting projection, we repeated the t-SNE 500 times for subsamples of 90% of the data, sampled without replacement.Unsupervised hierarchical clustering was performed using the 20,000 most variably methylated CpG sites across the dataset according to median absolute deviation, Euclidean distance and Ward’s linkage method.14 the Random Forest27 algorithm (R package randomForest version 4.6-12) was applied to generate 10,000 binary decision trees, incorporating genome-wide information from all 1077 reference samples of the 65 methylation classes. We used the 10.000 CpGs with highest variable importance. In addition, to address unequal class size we performed downsampling as described14. The distribution of these CpGs position within the gene region and their regulatory feature group are indicated  on the likelihood to prevent overfitting, as well as to stabilize estimation in situations in which classes are perfectly separable. Independent Random Forest scores are needed to fit this model, that is, the scores need to be generated by a Random Forest classifier that was not trained using the same samples, otherwise the Random Forest scores would be systematically biased and not comparable to scores of unseen cases. As such, Random Forest scores generated by the threefold cross-validation are used. To validate the class predictions generated by using the recalibrated scores of the calibration model, a nested threefold cross-validation loop is incorporated into the main threefold cross-validation that validates the Random Forest classifier15. Within each cross-validation run this nested threefold cross-validation is applied to generate independent Random Forest scores, which are then used to train a calibration model. The predicted Random Forest scores resulting from predicting the one-third test data of the outer cross-validation loop are then recalibrated by applying the calibration model that was fitted on the Random Forest scores generated during the nested cross-validation.To obtain classifier scores that are comparable between classes and that are improved estimates of the certainty of individual predictions, we performed a classification score recalibration by mapping the original scores to more accurate class probabilities15. By tuning the calibration model in this way we can regulate the amount of calibration so that the scores perform well at the prespecified common threshold of 0.9. This allows us to establish a common threshold for all forthcoming updates of the proposed classifier, which facilitates the communication with clinicians. A scheme summarizing the classifier algorithm steps is provided in Supplementary Fig. To determine the optimal amount of L2-penalization for a calibration model a parameter tuning is performed using a resampling approach. To this end, each time a calibration model is fitted using raw RF scores from training data to calibrate raw RF scores from test data, 500 random data sets are generated by sampling 70% of the raw scores training data without replacement. For each of these random data sets, L2-multinomial logistic regression models were fitted applying a range of reasonable penalization parameters lambda. The remaining 30% scores were then calibrated by these models and maximum of the calibrated scores over all methylation classes was used to generate class predictions. Then a new binary class was defined, that is, predictions in agreement with the actual true class were considered ‘classifiable’ and predictions not in agreement were labelled ‘non-classifiable’. This new binary variable and the accompanying maximum score over all class scores was then analysed by a receiver operator characteristics (ROC), i.e. calculating the Youden index (Specificity + Sensitivity − 1) for all possible thresholds. The final lambda was then determined such that the average Youden index over all resampling iterations at the prespecified cut-off threshold of 0.9 is maximalMisclassification errors mainly occurred within seven groups of histologically and biologically closely related tumour methylation classes. Therefore, we defined three ‘methylation class families’ (MCF) encompassing these seven tumour groups. Calibrated MCF score were calculated by summing up the calibrated class scores within one MCF.38. For the illustrations, the predictions obtained with the method ‘ABSOLUTE’ were used.The estimated tumour purity for all reference cases was computed using the R package RF_Purify as describedhttps://github.com/dstichel/conumee). Summary copy number profiles were created by summarizing these data in the respective set of reference cases for each methylation class.Copy number alterations of genomic segments were inferred from the methylation array data based on the R-package conumee after additional baseline correction (42 and total RNA sequencing from FFPE material43, whenever necessary.Cases enrolled in INFORM and NCT MASTER were subjected to total RNA and whole-exome sequencing; cases enrolled in MNP2.0 and PTT2.0 were subjected to a customized gene panel NGSFurther information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Reporting Summary


The primary goal of this study is to elucidate how the basic properties of MSC derived EVs can be exploited for function-specific activity in regenerative medicine. Our first important observation is that, MSC EVs possess a common mechanism of endocytosis across multiple cell types. Second, altering the MSC state by inducing differentiation into multiple lineages did not affect the exosomal properties or endocytosis but triggered the expression of lineage-specific genes and proteins in vitro and in vivo respectively. Overall, the results presented in this study show a common mechanism of endocytosis for MSC EVs across different cell types and the feasibility to generate functionally enhanced EVs by modifications to parental MSCs.Mesenchymal stem cells (MSCs) are multipotent cells with regenerative and immunomodulatory properties. Several aspects of MSC function have been attributed to the paracrine effects of MSC derived extracellular vesicles (EVs). Although MSC EVs show great promise for regenerative medicine applications, insights into their uptake mechanisms by different target cells and the ability to control MSC EV properties for defined function  Human mesenchymal stem cells (HMSCs) are multipotent somatic stem cells that can be isolated from a variety of tissues such as the bone marrow, adipose tissue, and dental pulp. The regenerative, protective, and anti-inflammatory properties of HMSCs especially bone marrow derived HMSCs are well documented  and makeMany existing tissue-engineering approaches focus on delivery of selected proteins  or nucleic acids to host or implanted stem cells to achieve lineage specific differentiation. A variety of techniques ranging from exogenous addition of growth factors and controlled release devices [reviewed in ] to utilEVs are nano-vesicles (40–150 nm) secreted by cells to facilitate intracellular communication . As thesRecent evidence suggests that several of the beneficial effects of human mesenchymal stem cell (HMSC) therapy can be attributed to paracrine effects of the HMSC secretome . More spThe paracrine aspect of HMSC function involves the directed uptake of HMSC derived EVs by target cells. Further, the multilineage differentiation potential of HMSCs suggests that lineage specific function could be reflected as lineage specific exosomal effects on naïve target cells. Harnessing the fundamental mechanistic features of EV-mediated signaling can be turned into an application-specific tool to direct lineage specific tissue repair/regeneration and disease treatment. With these goals in mind, the present study characterizes basic mechanistic aspects of HMSC EV function.Human bone marrow derived primary HMSCs (HMSCs) were purchased from ATCC and Lonza. Over the course of this study, HMSCs from at least three individual lots spanning at least three donors were utilized. These cells were cultured in αMEM (Gibco) containing 20% fetal bovine serum , 1% L-Glutamine (Gibco), and 1% antibiotic-antimycotic solution (Gibco). For induction of differentiation of HMSCs into osteogenic , chondro6 HMSCs. This equated to a stock concentration of 10,000 particles/µl as determined by nanoparticle tracking analysis (NTA).EVs were isolated from the culture medium as per our previously published and standardized protocols . BrieflyThe isolated EVs were characterized for number and size distribution and presence of membrane markers by NTA, immunoblotting, and transmission electron microscopy (TEM) as per established standards . For NTAFor immunoblotting, exosomal proteins were isolated in radioimmunoprecipitation assay (RIPA) buffer and 10–20 µg of EV protein isolate was resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto nitrocellulose membranes and probed with primary rabbit anti-CD63  and mouse anti-CD9 , mouse anti-BMP2  antibodies and near infrared dye conjugated secondary antibodies  as per previously published protocols . The bloFor transmission electron microscopy (TEM), 10 µl of 1/10 dilutions of the EV suspensions were placed on to carbon fomvar coated nickel TEM grids and incubated for 1 h followed by fixing with 4% formalin, washing with double deionized water and air drying. For immunogold labeling of CD63, the EV containing grids were blocked in PBS with 5% BSA, incubated with CD63 antibody  followed by washing and incubation with 10 nm gold tagged secondary antibody . The grids were then washed and air-dried. All the grids were imaged using a Joel JEM3010 TEM.For all the experiments described in this study, EVs isolated from one lot of HMSCs were used for functional experiments on the same lot of HMSCs.For endocytosis experiments, HMSC EVs were fluorescently labeled using the ExoGlow green labeling kit (System Biosciences) that labels the exosomal proteins fluorescently as per our previously published and standardized protocols . The EVsFor quantitative experiments HMSC cells were plated on to 96 well tissue culture plates at a concentration of 10,000 cells per well and incubated for 18 h to facilitate cell attachment. The cells were then incubated with increasing amounts of fluorescently labeled HMSC EVs for 2 h at 37°C. The cells were washed with PBS and fixed in neutral buffered 4% paraformaldehyde. The fluorescence from the endocytosed EVs was measured using a BioTek Synergy2 96 well plate reader equipped with the appropriate filter sets to measure green fluorescence. The results were plotted as mean (+/− SD) normalized fluorescence intensities  as a function of dosage (n = 6 per group).For quantitative endocytosis blocking experiments, the cells were plated in 96 well plates as described previously or in 12 well culture plates  and prior to EV treatment, were pre-treated with the blocking agents for 1 h as per our previously published protocols . Cell suFor qualitative endocytosis experiments, 50,000 cells (HMSCs) were plated on coverslips placed in 12 well tissue culture dishes. Fluorescently labeled EVs at 2x saturation volume were then added with/without inhibitors as described above and incubated for 2 h in the presence/absence of blocking agents as described above. The cells were then washed, fixed in 4% neutral buffered paraformaldehyde, permeablized, and counter stained using mouse monoclonal anti-tubulin antibody , rabbit polyclonal anti-caveolin1 antibody , or rabbit polyclonal anti-clathrin antibody  followed by treatment with TRITC labeled anti mouse/rabbit secondary antibody. The coverslips were then mounted using mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI) (Vector Labs) to label the nuclei and imaged using a Zeiss LSM 710 Meta confocal microscope.in vitro differentiation experiments, naïve HMSCs  were embedded in type I collagen sponges in quadruplicates. Clinical grade collagen sponges (Zimmer collagen tape) were used for these experiments. 2x saturation volume of the different EVs  were then added to the cells and incubated for 72 h. The saturation volume was determined by the quantitative dose dependence endocytosis experiment described in the previous section. The saturation was reached at 20 µl of standardized EV suspension per 10,000 HMSCs. NTA was used to measure the amount of EVs and this amounted an average of 10,000 EV particles/µl of standardized EV suspension from HMSCs. 1x108 EV particles was used per group in this experiment. Untreated cells received PBS treatment of equal volume. Post-72 h, RNA was isolated from the embedded HMSCs followed by cDNA synthesis and qPCR for selected marker genes for osteogenic, chondrogenic, and adipogenic differentiation as per our previously published protocols and primer sequences  or Sprague Dawley rats  as per protocols and procedures approved by the University of Illinois animal care committee (ACC). All animals were housed in appropriate cages in temperature and humidity-controlled facilities. Food and water were made available ad libitum.All in vivo in an immunocompromised mouse subcutaneous implantation model. Briefly, 1x106 HMSCs were seeded on to a 1 cm x 1 cm square of clinical grade collagen tape (Zimmer) with 2x saturation volume (approximately 4x108 EVs) of respective control (naïve HMSC EV) or experimental EV  suspension and implanted within the subcutaneous pocket bilaterally on the back of immunocompromised mice. The mice were anesthetized by intraperitoneal injection of ketamine (80–100 mg/kg)/xylazine (10 mg/kg). A 1.5 cm incision was made on the back along the midline and the control or experimental scaffolds were placed bilaterally within the subcutaneous pocket. All experiments were performed in quadruplicate. Four weeks post-implantation, the animals were sacrificed by carbon dioxide asphyxiation followed by cervical dislocation. The scaffolds were extracted, fixed in neutral buffered 4% paraformaldehyde, embedded in paraffin, and sectioned in to 5 µm sections. The sections were then immunostained fluorescently for marker proteins as per previously published protocols , the EV concentration for HMSCs was approximately 1x108 particles/ml of the EV suspension. Osteogenic, chondrogenic, and adipogenic differentiation of HMSCs yielded EVs with a similar average size, but an altered distribution of EV sizes  are involved in the endocytosis of glioblastoma cell derived exosomes . HSPGs a the EVs . We ther the EVs .Depending on the target cell type, exosomes can be endocytosed by either clathrin or caveolin mediated endocytosis . We perfFollowing these observations on HMSC EV endocytosis, we next evaluated if a change in cell state would affect the endocytosis of lineage specified, HMSC derived EVs. HMSCs were first differentiated along the osteogenic, chondrogenic, and adipogenic lineages. EVs isolated from these cells were harvested and evaluated for dose dependent and saturable endocytosis. To test if HMSC EVs possess a common endocytic mechanism across different cell types, we evaluated the endocytosis of naïve HMSC derived EVs in two additional cell types namely: J774A.1 monocyte-like cells and TIGK gingival keratinocytes. Results presented in Undifferentiated HMSCs in 3D cultures were incubated with EVs isolated from naïve and differentiated HMSCs for 72 h. Osteogenic, chondrogenic, and adipogenic EVs induced a significant increase in the expression levels of respective lineage specific marker genes with respect to untreated controls  and osteocalcin (OCN) were analyzed. Results presented in in vitro and in vivo. The verification of the in vitro results in vivo validated the lineage specificity of the EVs and additionally, also provided evidence that the effect that was observed after a few days in vitro translates into a long-term effect over a period of 4 weeks in vivo.Collectively, these results indicate that EVs isolated from differentiating HMSCs can induce lineage-specific phenotypic changes in naïve HMSCs Regenerative strategies require the recruiting and instructing of cells to form new tissues. HMSC EVs are of current interest because they demonstrate immunomodulatory and regenerative potential that may rival the use of HMSCs or growth factors in regenerative medicine . FurtherIn this study, we have provided insights into some of the basic properties of HMSC derived EVs and how they may be utilized and exploited for improving tissue engineering strategies. We began by investigating HMSC EV endocytosis. Identification of the endocytic mechanism can provide valuable information to target EVs for therapeutic delivery. With respect to EV endocytosis, the clathrin pathway, caveolar pathway, phagocytosis, and even macropinocytosis have all been implicated in endocytosis of EVs . We obsevia the cell surface HSPGs. We chose these two cell types as representative cells for hematopoietic and ectodermal cells respectively. Further studies with other representative cell types belonging to multiple germ layers is required to conclusively establish one common pathway across all cell types. However, this important first observation identifies a pathway that can be targeted to enhance the endocytic efficiency of therapeutic EVs. For example, the HIV TAT peptide and poly arginine repeats containing peptide sequences have been implicated to be endocytosed by target cells via HSPGs and in addition, HSPGs have been implicated as an accessible target receptor for delivery of biological cues to enable disease treatment as well as tissue regeneration .in vivo experiments and edited the manuscript. SR conceptualized the original hypothesis, directed C-CH and RN, performed the imaging analyses, and wrote the manuscript.C-CH is the first author and designed and performed most of the experiments. MK performed the experiments with the monocytes in LC’s laboratory. RN was the technician that assisted C-CH in his experiments. LD’s laboratory provided the keratinocytes and performed the endocytosis experiments using them. PG performed the This work was funded by NIH R01DE027404 and the Osteology foundation advanced researcher award to SR and PG (Award # 17-106) and NIH R01GM50875 to LD.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.


TTN, are emerging as a common cause of myopathies. The diagnosis of a TTN-related myopathy is, however, often not straightforward due to clinico-pathological overlap with other myopathies and the prevalence of TTN variants in control populations. Here, we present a combined clinico-pathological, genetic and biophysical approach to the diagnosis of TTN-related myopathies and the pathogenicity ascertainment of TTN missense variants. We identified 30 patients with a primary TTN-related congenital myopathy (CM) and two truncating variants, or one truncating and one missense TTN variant, or homozygous for one TTN missense variant. We found that TTN-related myopathies show considerable overlap with other myopathies but are strongly suggested by a combination of certain clinico-pathological features. Presentation was typically at birth with the clinical course characterized by variable progression of weakness, contractures, scoliosis and respiratory symptoms but sparing of extraocular muscles. Cardiac involvement depended on the variant position. Our biophysical analyses demonstrated that missense mutations associated with CMs are strongly destabilizing and exert their effect when expressed on a truncating background or in homozygosity. We hypothesise that destabilizing TTN missense mutations phenocopy truncating variants and are a key pathogenic feature of recessive titinopathies that might be amenable to therapeutic intervention.Mutations in the sarcomeric protein titin, encoded by The online version contains supplementary material available at 10.1007/s00401-020-02257-0. TTN gene, which assembles the primary ~ 100 kb transcript from 364 exons in a 300 kb gene  utations . It is pTTN pathogenic variants previously reported , demonsent 4 in  (includeThe impact of TMD/LGMD2J-linked mutations in Ig-169 (M10) and HMERF-linked mutations in Fn3-119 have been attributed to misfolding and/or thermal destabilisation of the domains . AlongsiIt is striking that structural “mirror images” of five of the twelve missense variants in our cohort have previously been linked to titinopathies. Analogous to Fn3-119 being a hotspot for HMERF-linked mutations, particular residue positions within titin domains may be hotspots for titinopathies, at least in trans with truncating variants; this observation may be of importance for the interpretation of missense variants identified in future.TTN truncating/missense variants is very similar to that found in patients carrying two recessive truncating variants, raising the question how these recessive TTN variants impact on the titin protein in vivo [TTN missense variants on proteostasis could be a key pathogenic feature of recessive titinopathies, a hypothesis supported by the observations of variant titin fragments co-localising with p62/SQSTM1 in C2C12 and HEK293 cells. We hypothesise that TTN missense variants can derail proteostasis by inducing local cleavage or accelerated protein turnover, causing disintegration of titin  or cleaD/LGMD2J . AlteratTTN-related myopathies, and emphasizes the need for a combined clinico-pathological, genetic and molecular approach to optimize diagnostic accuracy. Rare missense variants should be experimentally assessed at the protein domain level, with thermal destabilisation by more than approximately 15 degrees or complete misfolding/unfolding of the expressed domain supporting a pathogenic diagnosis for the variant when inherited in trans with a second TTN truncating or destabilising missense variant. These criteria should also prove helpful to assess the possible impact of suspicious missense variants in cardiomyopathies.The present study offers clear strategies addressing the challenges of an accurate assessment of patients with suspected Supplementary file1 (XLSX 19 KB)Supplementary file2 (PDF 20076 KB)Supplementary file3 (XLSX 82 KB)Below is the link to the electronic supplementary material.


Euthanization of animals was performed at 3 and 6 months after surgery. Tissues harvested were subjected to macroscopic and radiographic assessments. Specimens were then stained for histological analysis. Both control and experimental animals were capable of inducing the neoformation of fibrous connective tissue at both time points where superior amounts of tissue formation and mineralization were detected for experimental groups  and MOE-modified HA-TCP (at 3 mos)). Histological results, however, revealed that mature bone formation was only observed for specimens fabricated with MOE-modified HA-TCP in a time-dependent manner. The present study has successfully demonstrated the in vivo utility of experimental biphasic bioceramics modified by MOE in an ectopic grafting sheep model. Promising osteoconductive and osteoinductive properties must be further developed and confirmed by subsequent research.Novel biomaterials capable of accelerating the healing process of skeletal tissues are urgently needed in dentistry. The present in vivo study assessed the osteoconductive and osteoinductive properties of experimental biphasic bioceramics (HA-TCP) modified or not by a nacre extract  in a sheep model. Fabrication of MOE involved mixing ground nacre  with glacial ethanoic acid  for 72 hours using external magnetic stirring (25°C). Nonreactive carriers  pertaining to the control (empty) or experimental groups (HA-TCP or MOE-modified HA-TCP) were implanted intramuscularly into the abdominal segment of the torso in sheep ( It is estimated that billions of dollars are spent every year all over the world for the placement of bone defect repairs , 2. In tβ-tricalcium phosphate (TCP) [Autografts are typically regarded as the gold standard for bone repair because of their high levels of histocompatibility and the presence of a 3-dimensional porous matrix containing essential osteoconductive components  . These tte (TCP) , and lowte (TCP) . More rete (TCP) . The finThe Animal Use Ethics Committee of the Positivo University reviewed and approved the execution of the present randomized and controlled in vivo study .Perna perna (brown) mussels. After being thawed, mussels were washed (30 sec each) under copious irrigation with fresh water (25°C) and were air-dried for no less than 24 hours. Mussels' shells were then reduced into a fine powder using a pestle for bone  and a Teflon planetary grinding jar . Pulverized particles of nacre (0.05 g) were then mixed with glacial ethanoic acid  using external magnetic stirring for 72 hours . Experimental MOE was then centrifuged at 4,000 rpm  and the supernatant was carefully aspirated using a calibrated pipette.Our laboratory has previously reported the methodology used for the fabrication of the experimental MOE used in the present study . A localn = 8, 2 yrs. of age, ≈45 kg, http://www.randomizer.org). Nonreactive carriers  containing GenPhos HA-TCP  were then implanted intramuscularly at each animal's torso abdominal segment. Empty sterile nonreactive carriers served as the control . Animals of each experimental group (euthanized either 3 or 6 months) received 3 tubes  as shown in Female sheep  were performed on the epidermis and dermis to allow access to the muscular fascia (Longissimus dorsi) region. Individual intramuscular incisions (1.0 cm/each) were performed perpendicularly to the vertebral column to allow access for the placement of nonreactive carriers containing the materials investigated within the muscular tissue. Vicryl 5-0 absorbable  was used for the deep suture of the muscle and fascia using the interrupted technique. Nonabsorbable nylon 5-0  was used for the suture of the external dermis using a continuous technique. Anti-inflammatory and antibiotic drugs  were intramuscularly administered to animals 3 and 5 days after surgery.Sheep were identified with earrings following common practices typically used in animal breeding. The present study was conducted with the approval of the Animal Care Committee and followed the guidelines established by European Community for the handling, care, and utilization of experimental animals (DE 86/609/CEE) . AnimalsEuthanization of animals was performed (at 3 or 6 months) using a previously published protocol . Sodium http://imagej.nih.gov/ij/).Histology-ready specimens were then assessed for their macroscopic features using a semiprofessional digital camera  and digital image analysis software  performed with traditional X-ray equipment  connected to a digital sensor .μm/slice) in their longitudinal plane, as commonly reported in histological studies. Individual slices were treated with hematoxylin and eosin (HE) and Masson's trichrome (MT). An optical microscope  coupled with a digital camera  was then used to image stained slices. Digital images were then processed by a single and previously trained operator using Photoshop CS4  and ImageJ .Trichloroacetic acid 5%  was used (for 40 days) to demineralize the specimens. Specimens were inserted into blocks of paraffin and were serially sliced  with a confidence level of 95%.Mean values from the macroscopic , radiographic (radiopacity levels), and microscopic assessments (mature bone areas) were analyzed for normality using the Shapiro–Wilk test. Since a normal distribution was observed, results obtained were then statistically analyzed using Student's The results of the macroscopic analysis, at 3 and 6 months, were reported in the present study in terms of representative digital images Figures  and horip = 0.034). Histological results (at 3 and 6 mos) are displayed in Figures  or experimental  groups were associated with the lowest  and highest  amounts of fibrotic or bone-like tissues, respectively. Specimens of experimental group 6  displayed intermediate levels of tissue growth when compared to the remaining groups investigated. The radiographic assessment (radiopaque areas) resulted in means of 0.219 ± 0.037 in 2 for groups 1 and 4, and 0.695 ± 0.005 in 2 for groups 2, 3, 5, and 6 Figures  where it Figures . FiguresThe present controlled and randomized in vivo study had the objective of comprehensively characterizing the osteoconductive and osteoinductive functionalities of unaltered or MOE-modified GenPhos HA-TCP as materials to promote the guided deposition of bone tissues. The animal model used in the present study was selected because it provides physiological and biochemical environments that, comparable to those observed in humans , are rapp > 0.05) amounts of tissues were formed independently of the monitoring time considered (either at 3 or 6 mos). The results from the radiographic analysis shown in Figures The reason for the selection of nonreactive polymer carriers was based on results reported previously by our group , 25, wheThe utilization of MOE as a promoter of bone growth was supported by this material's proven biocompatibility, low antigenicity properties, and growth factors' concentration  that are considered adequate for human utilization , 28. Prep = 0.0003). The results reported in The macroscopic results presented in Figures Pinctada oyster), have demonstrated that nacre can also modulate the differentiation of stem cells into osteoclasts that were able to resorb the material, and can be used to partially explain the results presented in Figures A study investigating the in vitro biomineralization of human osteoblasts has demonstrated that nacre is capable of attracting and activating osteoblasts , therebyThe present study has successfully reported the experimental association of biphasic bioceramics modified by a marine organic extract from nacre. The comprehensive assessment approach used, suggests that experimental materials investigated (MOE-modified HA-TCP) displayed promising in vivo osteoconductive and osteoinductive functionalities in an ectopic sheep model. It is anticipated that after complete development and characterization, experimental materials reported will display active osteogenic properties. The utilization of these materials is expected to be translated into shorter healing times, materials able to heal critical-sized defects, decreased bone repair costs, and patients displaying improved quality of life. Despite the promising results reported, additional research is necessary to further develop the functionalities described and to confirm the findings of the present study.


LMNB1 and LMNB2 have been much less clearly defined.Lamins are the major component of nuclear lamina, maintaining structural integrity of the nucleus. Lamin A/C variants are well established to cause a spectrum of disorders ranging from myopathies to progeria, termed laminopathies. Phenotypes resulting from variants in We investigated exome and genome sequencing from the Deciphering Developmental Disorders Study and the 100,000 Genomes Project to identify novel microcephaly genes.Starting from a cohort of patients with extreme microcephaly, 13 individuals with heterozygous variants in the two human B-type lamins were identified. Recurrent variants were established to be de novo in nine cases and shown to affect highly conserved residues within the lamin ɑ-helical rod domain, likely disrupting interactions required for higher-order assembly of lamin filaments.LMNB1 and LMNB2 as a genetic cause of primary microcephaly, implicating a major structural component of the nuclear envelope in its etiology and defining a new form of laminopathy. The distinct nature of this lamin B–associated phenotype highlights the strikingly different developmental requirements for lamin paralogs and suggests a novel mechanism for primary microcephaly warranting future investigation.We identify dominant pathogenic variants in  Consequently, as well as maintaining structural integrity of the nucleus, they influence chromatin organization, DNA transcription, repair, and replication.1The nuclear lamina is a protein structure that lines the inner nuclear membrane and provides structural support to the nucleus.1Vertebrate cells express two classes of lamins, A and B, grouped based on sequence homology. Lamins A and C (A-types) are splice isoforms encoded by the same gene, while the B-type lamins are the products of different genes.LMNA variants, collectively termed laminopathies. Four major disease categories have been described with overlapping features: striated muscle diseases, lipodystrophy syndromes, peripheral neuropathies, and accelerated aging  disorders.2Over the past two decades many disorders have been linked to LMNA, few reports have associated human disease with variants in B-type lamin genes. No pathogenic single-nucleotide variants in LMNB1 have been reported, although genomic duplications incorporating LMNB1 cause adult-onset leukodystrophy (MIM 169500).3 For LMNB2, a homozygous missense variant in a family with progressive myoclonic epilepsy and ataxia has been described.4 Enrichment of heterozygous LMNB2 variants in acquired partial lipodystrophy patients has also been reported.5In contrast to 6 Many genes associated with centrosome and/or mitotic spindle function have been described, suggesting primary microcephaly to be a disorder of neural stem cell mitosis.7Microcephaly is a reduction in head size, defined by a head circumference at least three standard deviations below the mean for age and gender. The underlying reduction in brain volume has many etiologies: chromosomal, environmental, as well as single-gene syndromic and nonsyndromic causes. Primary microcephaly represents a recognizable monogenic form of microcephaly in which brain size is markedly reduced in the absence of other malformations and/or significant neurological deficits aside from intellectual disability.LMNB1 and LMNB2, implicating the nuclear lamina in the etiology of microcephaly.Here we report the identification of recurrent heterozygous variants in Ethical approval was obtained as follows: Scottish Multicentre Research Ethics Committee (05/MRE00/74); Deciphering Developmental Disorders (DDD) Study ; 100,000 Genomes Project (100kGP), East of England–Cambridge South REC (14/EE/1112). Informed written consent obtained for all participating families. Parents provided written authorization for publication of clinical photographs.8 De novo variants were identified using VASE . For 100kGP, variants and Human Phenotype Ontology (HPO) terms were extracted from the Genomics England research environment.DDD Study exome sequencing and variant calling were performed as previously described.For de novo variant calling criteria, structural modeling, and immunofluorescence experiment methodology, see Supplementary LMNB1 . This identified two individuals P1 and P2) with de novo variants in  with de LMNB1 variants were identified in seven individuals  and one individual from the 100kGP study (P12) with the same variant in LMNB2   where it disrupts protofilament assembly in vitro and causes nuclear aggregates of lamin in vivo.12Evidence for pathogenicity can also be drawn from the observation that all mutated codons are conserved in lamin A/C paralogs. Indeed, three of the LMNB variants  correspond to residues in LMNA that are altered in lamin A/C associated disorders.13 Cells expressing the LMNB1/B2 variants frequently contained nuclear aggregates and/or significantly altered nuclear morphology in comparison to cells expressing wild-type GFP-LMNB1/2 , there were neuronal migration defects resulting in abnormal layering of the cerebral cortex,18 suggesting potential discordance with the human phenotype and neuroimaging findings. This may be accounted for however by a different mechanism in human cases where variants are heterozygous rather than biallelic null. The pathogenic variants reported here are most likely dominant negative rather than haploinsufficient given that microcephaly is not present in the Lmnb1+/− mouse. The recurrent nature of the pathogenic variants at specific residues and absence of truncating variants favors gain of function or dominant negative mechanisms. Furthermore, substitutions in corresponding residues in lamin A/C do not destabilize the protein, rather disrupt lamin filament formation,19 a common scenario by which point variants in multimeric proteins can act in a dominant negative manner to perturb macromolecular complexes.20The LMNB1/B2 microcephaly phenotype is distinct from previously described lamin A/C laminopathies, pointing to different cellular and developmental roles for lamin B proteins, despite all lamins acting as major structural components of the nuclear envelope. The human microcephaly is in keeping with the previously reported mouse knockout model for 1 So it is striking that pathogenic variants in both cause primary microcephaly, suggesting an etiology in which a shared function is disrupted. Many primary microcephaly proteins are involved in mitosis, and often encode centrosomal or spindle components that when mutated result in altered spindle orientations disrupting symmetric cell division dynamics in neural progenitors.7 This reduces neuronal cell number generated during neurogenesis. B-type lamins associate with the mitotic spindle21 with defects in spindle orientation in neuronal precursor cells reported in Lmnb1 and Lmnb2 knockout mice,17 suggesting this could be the mechanism underlying microcephaly. Other mitotic processes may also be perturbed, given the involvement of LMNs in nuclear envelope reassembly. However, the potential for LMNB1/2 variants to alter the physical properties of lamin dimers and filaments  or with a progressive microcephaly/encephalopathy.27 As well, nuclear–cytoplasmic transport has been demonstrated to be impaired,27 so the microcephaly in nuclear pore disorders could have a different mechanistic basis.Notably, several genes encoding components of the nuclear pore have also been implicated in microcephaly. While nuclear pores are also important components of the nuclear envelope, these are generally clinically distinct disorders, often associated with nephrotic syndrome (Galloway–Mowat syndromeLMNB1 and LMNB2 as causes of primary microcephaly, implicating the nuclear lamina in its etiology and defining a novel form of laminopathy. Future in vitro and in vivo studies will be important to provide further evidence for causality of the variants reported here, and to shed light on how they cause a laminopathy distinct from those previously described.In conclusion, our findings establish heterozygous variants in Supplementary information